Mechanisms of LARP7 in regulating the progression of non-small cell lung cancer

Introduction

Lung cancer is the leading cause of cancer-related deaths worldwide. Each year, 2.21 million people are diagnosed with lung cancer, accounting for about 12.20% of all newly diagnosed cancers. It is estimated that about 1.8 million people died of lung cancer annually from 2020 to 2022 in China, accounting for 18.15% of all cancer deaths (1). Approximately 75% of patients succumb within five years after diagnosis. The high intratumoral heterogeneity (ITH) and the complexity of drug-resistant cancer cells further complicate treatment (2-4). Therefore, it is important to explore the mechanisms underlying the occurrence of lung cancer and identify the potential therapeutic targets.

RNA-binding proteins (RBPs) are intracellular proteins that bind to RNA, forming ribonucleoprotein complexes that are responsible for regulating, as well as directly participating in, biological processes such as variable splicing, methylation modification, stabilization, localization, translation, silencing and degradation of RNAs. It is reported that binding of RBPs to RNA is involved in nearly every aspect of post-transcriptional regulation. Many studies in recent years have revealed that abnormalities in RBPs are closely associated with the development of cancers (5-7). High-throughput bioinformatics analyses based on thousands of tumor-normal sample pairs from The Cancer Genome Atlas (TCGA) have also revealed that the expression of RBPs is dysregulated in different types of malignancies (8-10).

La ribonucleoprotein domain family member 7 (LARP7) is an RBP of the La-related proteins (LARPs) family. The proteins of LARPs family are of ancient origin and have been discovered in all eukaryotic organisms except Plasmodium, which actively participate in the processing of RNA in cellular cytoplasm and nucleus (11). For instance, LARP7 forms the 7SK small nuclear ribonucleoprotein (snRNP) complex with 7SK RNA, HEXIM1/2, and positive transcription elongation factor-b (P-TEFb), which is responsible for isolating the positive transcription factor P-TEFb in inactive state. When stimulated by transcription, the 7SK snRNP complex releases P-TEFb, which in turn acts as an important regulator of RNA polymerase II activity and promotes polymerase II-mediated messenger RNA (mRNA) synthesis (12-14). In addition, LARP7 is capable of recruiting splicing factors to the C-terminal region, and consequently affects the alternative splicing of RNA, resulting in various biological events such as the formation of circular RNA (15).

Back in 2002, the analysis of genetic mutations in microsatellite instability (MSI) gastric cancers showed that LARP7 shift mutations occurred frequently; notably, two different microsatellites in the LARP7 gene were frequently mutated in cancer (16-18). Cheng et al. evaluated the expression of LARP7 mRNA in 18 gastric cancer patients and found that LARP7 mRNA expression was significantly reduced in all cases. Studies in breast cancers showed that the reduction of LARP7 was closely related to the aggressiveness of breast tumors, and LARP7 knockdown promoted epithelial-mesenchymal transition (EMT) of breast cancer cells (19). Further analysis in LARP7 knockout models also suggested that LARP7 promoted cell motility and metastatic ability, and up-regulated gastric carcinogenesis by negatively regulating P-TEFb. At the transcriptional level, the knockdown of LARP7 was accompanied by the enhancement of RNA polymerase II activity, resulting in promoting the transcriptional extension of genes (20). On the other hand, overexpression of LARP7 inhibited the growth, proliferation, migration, and invasion of lung cancer cells. These studies suggest that LARP7 may act as a tumor suppressor to affect the metastasis and consequent spread of tumors (21). We have followed up several non-small cell lung cancer (NSCLC) patients in The First Affiliated Hospital of Anhui Medical University, and found that the prognoses of patients with low LARP7 were poorer than those with higher LARP7 expression, and thus we hypothesize that LARP7 acts as a tumor suppressor to regulate lung cancer progression. Therefore, this study was conducted to verify the hypothesis and further explore possible underlying biological mechanisms. We present this article in accordance with the MDAR and ARRIVE reporting checklists (available at https://jtd.amegroups.com/article/view/10.21037/jtd-2026-0579/rc).

Methods

Bioinformatics analysis

Data from large databases including TCGA, Gene Expression Omnibus (GEO), and European Genome-phenome Archive (EGA) were used for the bioinformatics analysis of LARP7 expression patterns in NSCLC tumors and controls. GEPIA2 (http://gepia2.cancer-pku.cn/#index) is an online tool for TCGA gene expression and survival analysis, which was used in this study to analyze LARP7 expression patterns between NSCLC tumor groups and control groups (22). Kaplan-Meier Plotter (http://kmplot.com/analysis/) is an online platform specializing in survival analysis, utilizing data from multiple large databases. This website offers the most comprehensive and authoritative survival analysis services, exploring and validating survival biomarkers through meta-analysis. In this study, this platform was utilized to analyze NSCLC data from GEO and TCGA, and explore the relationship between LARP7 gene expression and prognosis of patients (22).

Clinical analysis of LARP7 in NSCLC patients

The study was conducted in accordance with the Declaration of Helsinki and its subsequent amendments. This study was approved by the Clinical Medical Ethics Committee of The First Affiliated Hospital of Anhui Medical University (No. 5101096), and written informed consent was obtained from all participants. A protocol was prepared before the study without registration.

NSCLC patients were included in The First Affiliated Hospital of Anhui Medical University between June 2015 and June 2020. The inclusion criteria for the subjects were as follows: (I) pathologically confirmed NSCLC patients; (II) specimens of both primary lung cancer tissues and adjacent non-cancerous tissues were available; and (III) follow-up data were available. The exclusion criteria were as follows: (I) incomplete clinical or pathological data; (II) presence of synchronous or metachronous primary malignancies; and (III) incapable of adhering to follow-up protocols for any reason.

NSCLC patients were categorized into high- and low-expression groups according to the RNA level of LARP7, including 483 cases of adenocarcinoma, 466 cases of squamous cell carcinoma, and normal tissue samples. Specifically, patients with the LARP7 RNA level ≥ median were categorized in the high-expression group, while the ones with LARP7 RNA level < median were categorized in the low-expression group. The patients were followed up regularly every 1–3 months within the first year, then followed up 3–6 months in the second year, and then switched to every half year thereafter. Follow-up examinations included physical examinations, lung cancer tumor markers, and imaging examinations (including chest CT, cranial MRI, neck and abdominal ultrasound or CT, and bone scintigraphy). Suspicious lymph node enlargement by ultrasound was further investigated by CT, MRI, PET/CT, and/or cytology for confirmation. Data of treatments and recurrence statuses of patients were acquired via the Hospital Outpatient and Inpatient System, and additional information was obtained through regular phone follow-ups. Survival data were censored on December 31, 2021. Log-rank tests by R software (Version 4.4.1) and RStudio were used to analyze the survival time.

Cell culture

A549/H1299 cells were purchased from Procell Life Science & Technology Co., Ltd. (Wuhan, China). All the cell lines were cultured in MEM medium with 10% fetal bovine serum (FBS), 100 µg/mL streptomycin, and 100 U/mL penicillin at 37 ℃, 5% CO_2. The cells were passaged every 2–3 days, and cells in the logarithmic growth phase were harvested for further experiments.

Cell transfection

PLVX-puro was used as the vector for construction of the overexpression plasmid, with the primer sequences of LARP7 as follows: forward: CTTGGTACCGAGCTCGGATCCGCCACCatgatccctaacatagaaggaat; reverse: GAAGGGCCCTCTAGACTCGAGatcatattcagaaaatcttatatgt. The linearized vector was obtained by digestion with restriction endonucleases. The target gene fragment was prepared by polymerase chain reaction (PCR) amplification. During primer design, homologous recombination sequences were added to the 5’ ends of the primers to ensure exact matching of the 5’ and 3’ ends of the amplification products with the ends of the linearized cloning vector. The linearized vector and the amplified target gene fragment were combined to set up a recombinant reaction system for in vitro ligation. The recombinant products were then used for transformation, and single clones were selected for PCR identification. Positive clones were sequenced and analyzed; the correct clones were expanded, and high-purity plasmids were obtained for subsequent experiments.

A549/H1299 cells were transfected with plasmid (PLVX-puro) to construct the LARP7-overexpressing lung cancer cells (LARP7 group) and empty vector control (Ctrl group) lung cancer cells. Forty-eight hours prior to transfection, cells in the logarithmic growth phase were trypsinized, counted, and then the cells were seeded at a density of 10,000 per well in 6-well plates and cultured until they reached 50–60% confluence. The cells were then seeded into culture dishes and incubated at 37 ℃, 5% CO₂ for 48 h. After reaching 70–80% confluence, the cell culture medium was replaced with fresh basal medium, and cell transfection was performed using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s protocol. Transfected cells were harvested 48 h later for reverse transcription quantitative PCR (RT-qPCR) and Western blot analysis.

RT-qPCR

TRIzol reagent (Invitrogen) was used to extract RNA from A549/H1299 cells. PrimeScript RT reagent Kit (Takara, Shiga, Japan) and SYBR Green One-Step RT-qPCR Kit (Takara) were used for the RT-qPCR. Data were normalized by glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and the 2^−ΔΔCt method was used for quantification. The full-length sequence of the candidate gene was retrieved from NCBI. Primers were designed using Primer Premier 5.0; sequences are listed in Table S1.

Cell proliferation assay

A549/H1299 cells in logarithmic growth phase were collected, adjusted to 5×10⁴/mL, and incubated for 24 h (37 ℃, 5% CO₂). Then 10 µL CCK-8 (Dojindo Laboratories, Japan) solution was added to each well, and the cells were incubated for 2 h. Afterwards, the optical density (OD) value at the wavelength of 450 nm was measured by the microplate reader.

Wound healing assay

A549/H1299 cells in logarithmic growth phase were seeded at a density of 2×10⁵ cells per well in a 6-well plate and incubated until they reached 90% confluency. A sterilized pipette tip was used to make a vertical scratch to the monolayer, and PBS was used to wash off the floating cells gently. The cells were cultured in serum-free medium; the pictures of the wound were taken under microscopy at 24 h, and the migration distance was assessed using ImageJ. The wound healing rate was then calculated.

Transwell assay

Matrix gel (50 µL; Corning, Corning, NY, USA) was added to the upper chamber of Transwell system, which was placed in a 24-well plate and incubated until solidification. Cell suspension at a density of 1–10 ×10⁵/mL was added to the upper chamber, and 600 µL complete medium was added to the lower chamber. The cells were incubated for 24 h. The upper chamber of Transwell system was obtained, the redundant matrix gel was carefully removed by a moistened cotton swab, and then the cells attached to the lower layer of the chamber membrane were fixed with 4% neutral paraformaldehyde for 15 minutes, followed by rinsing with PBS. The chamber was dried and then immersed in 0.1% crystal violet for 15–20 minutes, and the stained cells were observed under microscope.

Colony formation assay

A549/H1299 cells were re-suspended with fresh medium, and the density was adjusted to 600 cells/well. Then the cells were evenly seeded into 6-well plates and cultured in complete culture medium for 15–20 consecutive days. The culture was terminated when the cells in all groups formed clusters and appeared as obvious colonies. The cells were fixed with 4% paraformaldehyde for 15 min, immersed in crystal violet for 15–20 min for staining, and then the cell colonies were observed and counted.

Flow cytometry

A549/H1299 cells were cultured under 37 ℃ and 5% CO₂ conditions for 48 h, and then harvested and washed twice with ice-cold PBS. Approximately 2×10⁵ cells were then resuspended in 100 µL binding buffer and gently mixed. Subsequently, the mixture was incubated at room temperature in dark for 15 minutes. After incubation, the cells were stained with 10 µL Annexin V-PE and 7-AAD, followed by immediate analysis using flow cytometry (Invitrogen Bigfoot). Cells with only Annexin V-PE positive were considered as early apoptotic cells, while cells positive for both Annexin V-PE and 7-AAD were late apoptotic or necrotic cells. In this study, both early and late apoptotic cells were summarized and indicated as apoptotic cells.

Western blot

A549/H1299 cells were seeded at a density of 1×10⁵ cells per well in 6-well plates and allowed to grow until they reached 80% confluence. After 48 h of incubation with 1-NP, the cells were collected for protein extraction. To extract the proteins, each well was supplemented with 100–200 µL of RIPA strong lysis buffer containing 1 mM phosphatase inhibitor and 1 mM PMSF, and the cells were lysed at 4 ℃. The protein of cells was quantified by BCA method, and then western blot was performed using anti-LARP7 mAb (Proteintech, China) and anti-β-Actin mAb (Cell Signaling Technology, Danvers, MA, USA). The protein levels were quantified by the gel-imaging system (Tianen Technology, Shanghai, China).

Tumor xenografts

Animal experiments were performed under a project licence (No. 20210229) granted by the Animal Ethics Committee of Anhui Medical University, in compliance with the national guidelines for the care and use of animals (23). Ten male BALB/c nude mice (4 weeks old) were purchased from SLAC (Shanghai, China). The mice were acclimated for 7 days at the Experimental Animal Centre and then using a computer-based random order generator were divided into two groups. A549-OE-LARP7 cells and A549-Ctrl cells at logarithmic growth stage were obtained and suspended in 150 µL DMEM (without FBS or penicillin/streptomycin) to obtain the density of 1×10⁷/L. For each mouse, 0.2 mL suspension was injected subcutaneously at the axillary region of the right forelimb. Subsequently, the mice underwent continuous monitoring of body weight and tumor volume for 25 days and testing order was randomized daily (the researchers were not blinded). White globular protrusions with a diameter of about 5 mm were visible in the axilla of the injection site 5–7 days later. After 30 days, the mice were euthanized and tumors were obtained for analysis.

Immunohistochemistry

Antigen repair was performed with 3% H₂O₂, incubated for 10 min at room temperature in dark, using EDTA-Tris solution. After normal serum closure, the primary antibody (1:200) was added for incubation overnight. Sections were deparaffinized by baking at 60 ℃, then sequentially treated with xylene, ethanol, and distilled water. Endogenous peroxidase activity was blocked with 3% H₂O₂. After washing with PBST, antigen retrieval was performed using microwave heating in boiling EDTA-Tris solution. Following additional PBST washes, sections were blocked with normal serum and incubated with primary antibodies (1:200) overnight at 4 ℃. After washing, HRP-conjugated secondary antibodies (1:500) were added and incubated for 2 h. Sections were then stained with DAB, counterstained with hematoxylin, and mounted for microscopy.

Single-Cell RNA-sequencing

Total RNA (≥1 µg) was used for library preparation by the KCTM Stranded mRNA Library Prep Kit for Illumina. The mRNA was initially enriched using Oligo(dT) beads, followed by fragmentation and reverse transcription into cDNA. The cDNA was then purified, repaired, and amplified to construct the sequencing library. The final library was subjected to quality control by initial quantification (Qubit 2.0), insert size verification (Agilent 2100 bioanalyzer, Agilent Technologies, Santa Clara, CA, USA), and RT-qPCR to ensure the effective concentration exceeded 2 nM. Sequencing of the qualified RNA samples was performed on the Illumina NovaSeq 6000 platform (Illumina, San Diego, CA, USA), utilizing fluorescent dNTPs and DNA polymerase to translate fluorescence signals into base sequences.

Statistical analysis

R software (Version 4.4.1) with RStudio, as well as SPSS 23.0 software was used for the statistical analysis in this study. All experiments were performed in triplicate. Data were expressed as mean ± standard deviation (SD), and compared by Student’s t-test or one-way analysis of variance (ANOVA), with P<0.05 considered statistically significant. Survival curves were analyzed using Kaplan-Meier method with Log-rank test. Differentially expressed genes (DEGs) were defined with a false discovery rate (FDR) <0.05 and a fold change (FC) ≥2 or ≤0.5.

Results

LARP7 expression in NSCLC patients

We analyzed the data of LARP7 gene expression in NSCLC using the TCGA database, which included 483 lung adenocarcinoma (LUAD) and 466 lung squamous carcinoma (LUSC) specimens, as well as normal tissue samples. The analysis revealed that LARP7 expression was significantly lower in both LUAD and LUSC compared to normal controls (Figure 1A). In addition, we selected samples from six lung cancer patients from the Random Medical Anhui Provincial Hospital Cancer Tissue Bank. Our results confirmed that the expression of LARP7 in lung cancer tissues was significantly lower compared to adjacent non-cancerous tissues (Figure 1B,1C). Immunohistochemical analysis further corroborated these findings, showing a significant reduction in LARP7 expression in lung cancer tissues relative to matched adjacent tissues (Figure 1D).

Figure 1 Transcriptome analysis of LARP7 and its relationship with survival in NSCLC. (A) Bar plot showing the expression pattern of LARP7 in LUAD and LUSC from TCGA. (B) The expression of LARP7 in NSCLC samples from The First Affiliated Hospital of Anhui Medical University (FAHAMU). (C) Histogram of normalized level of LARP7 protein expression, comparing with the normal tissue group. (D) IHC images of LARP7 of lung tumor samples (left) and IHC score of lung tumor samples (right). (E) Overall survival analysis of LARP7 in lung cancer (n=949) from TCGA. (F) Survival analysis of LARP7 in lung cancer from The First Affiliated Hospital of Anhui Medical University. *, P<0.05; **, P<0.01; ***, P<0.001. HR, hazard ratio; IHC, immunohistochemistry; LC, lung cancer; LUAD, lung adenocarcinoma; LUSC, lung squamous cell carcinoma; NSCLC, non-small cell lung cancer; TCGA, The Cancer Genome Atlas.

To investigate the impact of LARP7 on prognosis, we utilized GEPIA2 and Kaplan-Meier survival analysis based on public databases. The survival analysis indicated that patients with low LARP7 expression had significantly poorer prognosis compared to those with high LARP7 expression [P=0.00082, hazard ratio (HR) =0.79] (Figure 1E). Additionally, we performed a follow-up study on 56 NSCLC patients and confirmed that patients with high LARP7 expression had a significantly better survival rate compared to those with low LARP7 expression (Figure 1F).

Effects of LARP7 overexpression on bio-behaviors of lung cancer cells

The experimental group (LARP7-OE) consisted of lung cancer cells transfected with LARP7 overexpression plasmids, while the control group (Ctrl) contained cells transfected with empty vector plasmids. To confirm LARP7 overexpression, RNA was extracted from both groups, and RT-qPCR was conducted to measure LARP7 gene expression. Results showed a significantly higher level of LARP7 expression in the experimental group compared to the control group (P<0.05) (Figure 2A). Subsequent Western blot analysis also revealed a significant increase in LARP7 protein levels in the experimental group (P<0.05) (Figure 2B), confirming successful overexpression of LARP7.

Figure 2 Effect of overexpression of LARP7 on proliferation of A549 and H1299 cells. (A) RT-qPCR results of the gene expression level of LARP7 in LARP7-OE and Ctrl groups. (B) Western blot results of the expression level of LARP7 in LARP7 and Ctrl groups. (C) The proliferation level of the LARP7 and Ctrl cancer cells was detected by CCK-8 assay. (D) Colony forming ability of LARP7 and Ctrl cancer cells was detected by plate cloning assay (staining method). ***, P<0.001. CCK-8, Cell Counting Kit-8; OD, optical density; OE, overexpression; RT-qPCR, reverse transcription quantitative polymerase chain reaction.

To assess the impact of LARP7 on the proliferative capabilities of lung cancer cells, cell viability and OD values were monitored for five consecutive days using the CCK-8 assay. The findings indicated that LARP7 overexpression suppressed the proliferative capacity of A549 and H1299 lung cancer cells (Figure 2C). Additionally, the plate cloning assay revealed a substantially lower number of colonies formed by A549 and H1299 cells in the LARP7-OE group compared to the Ctrl group (P<0.001) (Figure 2D).

Wound healing assay was performed to investigate the effect of LARP7 overexpression on the migration ability of lung cancer cells, and the results revealed that LARP7-overexpressing lung cancer cells exhibited a reduced scratch healing ability compared to the control cells, indicating significantly impaired migration capability (Figure 3A).

Figure 3 LARP7 overexpression affects the migration and invasion ability of lung cancer cells. (A) Wound healing assay showed the migration ability of LARP7 and Ctrl cancer cells (scale bar). (B) Transwell experiment showed the invasion ability of LARP7 and Ctrl cancer cells (staining method, scale bar). Unpaired t-test was used unless otherwise stated. *, P<0.05; **, P<0.01; ***, P<0.001.

Regarding cell invasion ability, the results showed a significant reduction in the number of LARP7-overexpressing cells that had migrated through the chambers, demonstrating a decrease in invasion ability (Figure 3B). The wound healing assay and Transwell assay confirmed that LARP7 overexpression inhibited lung cancer cell migration and invasion in vitro.

Effect of LARP7 overexpression on apoptosis of lung cancer cells

Flow cytometry with Annexin V-PE/7-AAD staining indicated that the apoptosis rates (early and late) of A549 and H1299 cells were significantly increased in the LARP7-overexpressing group compared to the Ctrl group, with statistically significant differences (P<0.001). These results demonstrate that LARP7 overexpression enhances apoptosis in lung cancer cells in vitro (Figure 4).

Figure 4 LARP7 overexpression affects the apoptosis level of lung cancer cells detected by flow cytometry. ***, P<0.001.

Xenograft assay

At 25 days of the xenograft experiment, mice were sacrificed and the tumors were excised and photographed (Figure 5A). Additionally, measurements of tumour volume and weight at 25 days of xenografting revealed that tumors in the A549-OE-LARP7 group were significantly smaller in both volume and weight compared to the A549-Ctrl group (P<0.01) (Figure 5B,5C). Immunohistochemical analysis was subsequently conducted on the xenograft tumors. The results revealed that the LARP7 expression levels in tumor tissues of the A549-OE-LARP7 group were significantly elevated compared to those in the A549-Ctrl group. Ki-67, a marker of tumor proliferation, was used to assess the degree of malignant proliferation within the tumor tissues. Notably, elevated LARP7 expression in tumor tissues correlated with reduced Ki-67 expression, indicating a diminished proliferative capacity of the tumors. Conversely, tumor tissues with low LARP7 expression exhibited higher Ki-67 levels, signifying enhanced proliferative activity (P<0.001) (Figure 5D). Collectively, these experiments substantiate the inhibitory effects of high LARP7 expression on lung cancer tissues in vivo.

Figure 5 Inhibitory effect of LARP7 on tumor growth in vivo. (A) The tumor formation in nude mice was evaluated in the control group (A549-Ctrl) and the LARP7 overexpression group (A549-OE-LARP7). (B) Comparison of tumor-forming tumor volume between A549-Ctrl and A549-OE-LARP7 groups in nude mice. (C) Comparison of tumor-forming tumor weight between A549-Ctrl and A549-OE-LARP7 groups in nude mice. (D) Comparison of LARP7 and Ki-67 expression levels in tumor tissues between A549-Ctrl and A549-OE-LARP7 groups (staining, scale bar: ). ***, P<0.001; ****, P<0.0001.

LARP7 overexpression on gene expression profiles

The paired-end sequencing of LARP7 overexpression cells was performed using the Illumina Novaseq 6000 platform, yielding an average of 71,075,022 raw reads per sample, which were then filtered to 70,343,398 high-quality clean reads (Table S2). Mapping with TopHat2 to the human GRCh38 genome showed a 97.10–97.44% mapping rate, with 97.66–98.00% being uniquely mapped reads, predominantly located in CDS regions (56.35–57.88%) and 3’UTR regions (Table S3). This study used FPKM to evaluate gene expression levels, counting the number of genes with FPKM >0 and FPKM ≥1 based on the FPKM formula (Table S4).

Differential gene expression between LARP7 overexpression and control groups was analyzed using edgeR software, with DEGs defined by FDR<0.05 and FC≥2 or ≤1/2. The results revealed 1,805 significantly DEGs, including 310 upregulated and 1,495 downregulated genes, between the two groups (Figure 6A). The principal component analysis (PCA) also revealed significant differences in gene expression profiles between the overexpression group and the control group (P<0.05) (Figure S1). To further investigate the regulatory mechanisms of LARP7, Gene Ontology (GO) enrichment analysis on the 1,805 DEGs induced by LARP7 overexpression was performed, and the top ten pathways were shown in Figure 6B. GO analysis of the 310 upregulated genes revealed enrichment in biological processes such as synaptic transmission, coagulation, signal transduction, and cell adhesion, while the 1,495 downregulated genes were enriched in pathways related to cytokine-mediated signaling, viral defense responses, and immune responses. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis indicated that downregulated genes were involved in cytokine receptor interactions and immune responses, whereas upregulated genes were enriched in pathways related to water reabsorption, cGMP-PKG signaling, and retinol metabolism (Figure 6C). RT-qPCR validation confirmed the downregulation of adenosine deaminase acting on RNA (ADAR), fibronectin 1 (FN1), and other DEGs in LARP7-overexpressing A549 and H1299 cells, consistent with sequencing results, with ADAR and FN1 showing significant downregulation in both cell lines (P<0.05) (Figure 6D).

Figure 6 Impact of LARP7 overexpression on gene expression profiles in lung cancer cells. (A) Volcano plot showing all DEGs between LARP7 and Ctrl samples (left). Hierarchical clustering heatmap displaying expression levels of all DEGs (right). (B) GO analysis of DEGs. Bubble diagram illustrating the most enriched GO biological process results for up-regulated and down-regulated DEGs. (C) KEGG analysis of DEGs. Histogram depicting the most enriched KEGG pathways for up-regulated and down-regulated DEGs. (D) Validation of DEGs expression using RT-qPCR in LARP7 overexpression conditions. Bar plot demonstrating the expression pattern and statistical differences of DEGs. *, P<0.05; **, P<0.01; ***, P<0.001. DEGs, differentially expressed genes; GO, Gene Ontology; KEGG, Kyoto Encyclopedia of Genes and Genomes; RT-qPCR, reverse transcription quantitative polymerase chain reaction.

LARP7 overexpression on alternative splicing

Unique mapped reads from RNA-seq data were used to analyze alternative splicing, revealing 267,266 exons across six samples, covering 72.76% of annotated exons (Table S5). TopHat2 identified 437,841 splice junctions, including 180,520 known and 257,321 novel junctions, which were then analyzed for splicing patterns including ES, A5SS, A3SS, IntronR, MXE, and others (Tables S6-S9). Comparison of splicing levels between samples using t-test identified 2,178 significant regulated alternative splicing events (RASEs), with predominant types being cassette Exon, ES, A5SS, and A3SS (Figure 7A). GO and KEGG pathway analyses of these RASEs revealed significant enrichment in pathways related to cell proliferation, apoptosis, and various metabolic processes (Figure 7B,7C). Upon performing an overlap analysis between DEGs and regulated alternative splicing genes (RASGs), 143 genes were found to overlap (Figure S2). Notably, the expression levels and alternative splicing of FN1 and ADAR genes exhibited significant differences between the LARP7 overexpression group and the Ctrl group, with statistical significance, (P<0.05) (Figure S3). Validation through RT-qPCR confirmed increased expression of FN1 and ADAR splice variants in LARP7-overexpressing A549 and H1299 cells (Figure 7D).

Figure 7 Impact of LARP7 overexpression on alternative splicing in lung cancer cells. (A) Bar plot showing the number of alternative splicing events regulated by LARP7, classified into nine types. (B) GO analysis of LARP7-RASGs. Bubble plot displaying the top ten enriched GO biological process terms for RASGs. (C) KEGG analysis of LARP7-RASGs. Histogram illustrating the most enriched KEGG pathways for RASGs. (D) Validation of RASGs expression using RT-qPCR in LARP7 overexpression conditions. Bar plot demonstrating the expression pattern and statistical differences of RASGs. *, P<0.05; **, P<0.01; ***, P<0.001. GO, Gene Ontology; KEGG, Kyoto Encyclopedia of Genes and Genomes; RASGs, regulated alternative splicing genes; RT-qPCR, reverse transcription quantitative polymerase chain reaction.

Discussion

This study investigates the tumor-suppressive role of the RBP LARP7 in NSCLC by examining clinical data, cellular models, and animal experiments. RNA-seq analysis revealed that LARP7 expression is significantly lower in tumor tissues compared to normal controls. Additionally, lower LARP7 expression correlates with poorer prognosis (P=0.00082, HR =0.79). Validation using single-center NSCLC data showed consistent results, with LARP7 levels significantly reduced in tumor tissues of six randomly selected patient pairs. Follow-up of 56 NSCLC patients confirmed that lower LARP7 expression is associated with worse outcomes. To further explore the role of LARP7, we established LARP7-overexpressing NSCLC cell lines, which demonstrated reduced cell proliferation, migration, and invasion, and increased apoptosis, aligning with previous findings (24). In vivo experiments with LARP7-overexpressing cells in nude mice showed reduced tumor growth and size. Immunohistochemistry revealed higher LARP7 expression and lower Ki-67 levels in tumors from LARP7-overexpressing cells, indicating decreased proliferative capacity. Overall, our results confirm that LARP7 inhibits NSCLC both in vitro and in vivo.

LARP7, a member of the LARPs family of RBPs, predominantly resides in the nucleus and inhibits RNA polymerase II activity. Additionally, LARP7 recruits splicing factors to its C-terminal domain, thereby influencing alternative splicing (25). Numerous studies have confirmed that LARP7 plays a role in regulating various malignancies, including lung cancer. Research by Cheng et al. demonstrated that LARP7 negatively regulates P-TEFb binding and stabilization within the 7SK RNA complex. The P-TEFb complex, composed of cyclin-dependent kinase 9 (CDK9) and transcription elongation factor B, polypeptide 1 (CycT1), is a key promoter of mRNA elongation and processing and is associated with proliferation and dedifferentiation in several cancers. Cheng’s study assessed LARP7 mRNA levels in 18 gastric cancer patients and found that LARP7 expression was significantly lower compared to adjacent normal tissues and healthy controls. Knocking out LARP7 resulted in a 72% reduction in 7SK RNA complex in gastric mucosal epithelial cells, with a 23% decrease in cell proliferation and a 22% decrease in migration. These findings suggest that LARP7 downregulation promotes gastric cancer progression by negatively regulating P-TEFb (20).

Zhang et al. found that levels of LARP7 protein were significantly higher in cancer tissue than in peritumoral normal tissue (26). Further analysis revealed that decreased LARP7 levels were associated with aggressive tumor characteristics, including larger tumor size, poorer differentiation, and metastasis. Importantly, higher LARP7 levels were associated with better overall survival and disease-free survival. Functional studies showed that LARP7 knockdown promoted EMT and malignancy in non-invasive breast cancer cell lines. Downregulated LARP7 and 7SK snRNA resulted in the redistribution of P-TEFb to active super elongation complexes, leading to increased transcription of EMT-related factors (including snail family transcriptional repressor 2, Forkhead Box C2, Zinc finger E-box-binding homeobox 2, and twist family BHLH transcription factor 1), thereby promoting EMT, invasion, and metastasis in breast cancer (19).

Li et al. reported that LARP7 expression correlates with prognosis and chemotherapy resistance in breast cancer; our study demonstrates that LARP7 similarly functions as a tumor suppressor in NSCLC through the regulation of alternative splicing. RNA sequencing analysis identified numerous exon skipping and alternative splice site events mediated by LARP7, with target genes enriched in RNA binding, mRNA splicing, ubiquitin-mediated proteolysis, and cell cycle regulation. These results suggest that LARP7 exerts its antitumor effects through conserved post-transcriptional mechanisms, yet with distinct tissue-specific splicing targets (23).

Regarding lung cancer, research on LARP7 is limited. LARP7 knockout enhanced cell motility and migration, with increased RNA polymerase II activity and gene transcription elongation (24). Knockdown of LARP7 inhibited the expression of the CDK1 complex and arrested cells in the G2/M phase (26). These results indicate that LARP7 promotes gene transcription within cells, leading to the upregulation of genes associated with increased tumor cell metastasis and affecting lung cancer malignancy. Based on our previous studies, this study utilizes RNA-seq technology to analyze genomic data from LARP7-overexpressing lung cancer cells to further explore the mechanisms by which LARP7 regulates lung cancer progression.

Gene differential expression and alternative splicing are closely related to tumor development (27). In this study, we analyzed genomic data from lung cancer cells with LARP7 overexpression and control groups to explore the impact of LARP7 on gene expression and alternative splicing. The results revealed that LARP7 significantly alters gene differential expression and alternative splicing. Analysis of DEGs revealed that LARP7 overexpression induced significant gene expression changes. In our analysis of DEGs, LARP7 was found to cause significant differential expression in 1,805 genes, predominantly downregulating 1,495 genes. The downregulated genes are primarily enriched in immune-inflammatory pathways, such as cytokine-mediated signaling, type I interferon-mediated signaling, viral defense response, viral response, negative regulation of viral genome replication, innate immune response, inflammatory response, interferon-γ-mediated signaling, and T cell-mediated cytotoxicity. Conversely, the upregulated genes are associated with biological processes such as synaptic transmission, coagulation, signal transduction, intracellular signaling, nervous system development, cell adhesion, extracellular matrix organization, small molecule metabolism, and transmembrane transport.

It is evident that LARP7 primarily downregulates immune-inflammatory genes in lung cancer. Chronic inflammation has long been recognized as a hallmark of cancer, as it plays a role in both initiating and maintaining tumor progression. Current research indicates that the intensity and duration of inflammation are the results of two interacting processes: the activation of inflammatory responses and the production of pro-inflammatory mediators, along with the resolution of inflammation mediated by various factors aimed at suppressing inflammation and restoring tissue homeostasis. Modulating inflammatory mediators can influence tumor development (28). Chronic inflammation is closely related to lung cancer, which can be triggered by asbestos, silica dust, and cigarette smoke. The immune system’s inability to clear these substances leads to persistent inflammation, contributing to tumor development, metastasis, and growth. Early inflammation can directly cause DNA damage and mutations or trigger oxidative stress through cytochrome P-450 enzymes or flavin monooxygenases, resulting in protein and DNA damage (29). In our study, significant downregulation of immune-related genes such as beta-2 microglobulin (B2M) and bone marrow stromal antigen 2 (BST2) was observed in the LARP7 overexpression group. B2M, a component of the human leukocyte antigen class I (HLA-I) complex, plays a role in immune surveillance. Pereira and colleagues found that 5% of 230 lung cancer patients carried somatic mutations in B2M, most of which impaired the proper formation of the HLA-I complex. Genes involved in HLA-I complex maturation, such as calreticulin (CALR), protein disulfide isomerase family A member 3 (PDIA3), and transporter associated with antigen processing 1 (TAP1), were also altered in lung cancer. Gene expression microarray studies revealed that restored B2M levels upregulated Interferon alpha/Interferon gamma (IFNα/IFNγ) targets in lung cancer cells. Additionally, one-third of lung cancers lack the HLA-I complex, which correlates with reduced CD8⁺ T cell infiltration. B2M and HLA-I protein levels are associated with PD-L1 levels. These findings suggest that B2M inactivation, along with alterations in CALR, PDIA3, TAP1, and HLA-I complex downregulation, contributes to lung cancer development (30).

Another immune-related gene, BST2, is recognized as an oncogenic driver and immunomodulatory hub in various malignancies (31). BST2 contributes to papillary thyroid microcarcinoma (PTMC) progression by promoting human lymphatic endothelial cells proliferation and migration (32). BST2 overexpression is also observed in liver cancer tissues and is linked to adverse clinical outcomes and prognosis, serving as an independent poor prognostic factor (33). Overall, immune inflammation plays a crucial role in tumor development. LARP7 may further regulate tumor-associated immune inflammation by modulating immune-related gene expression, thus influencing lung cancer progression. In summary, immune inflammation plays a crucial role in the initiation and progression of tumors. LARP7 may regulate tumor-related immune inflammation by modulating the differential expression of immune-related genes, thereby influencing lung cancer progression. However, it is important to note that further experimental evidence is required to support and verify the direct impact of LARP7 on immune inflammatory pathways.

We further analyzed the changes in alternative splicing induced by LARP7 overexpression and identified 2,178 significantly different RASEs. The corresponding RASGs were primarily enriched in pathways related to positive regulation of the I-κB kinase (IKK)/nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) cascade, cell division, cell cycle, DNA repair, mitotic cell cycle, DNA replication, negative regulation of apoptotic signaling, negative regulation of type I interferon production, apoptosis, and G1/S transition in the mitotic cell cycle. These RASGs are highly enriched in biological processes related to cell proliferation and apoptosis. Cell proliferation and apoptosis are well-known to be closely related to tumorigenesis and cancer progression. Zhou et al. performed a retrospective analysis of NSCLC samples and found that the expression level of miR-135a is closely associated with NSCLC occurrence and progression. Further in vitro experiments demonstrated that miR-135a suppresses cell proliferation, migration, invasion, and tumor angiogenesis in NSCLC through targeting insulin-like growth factor 1 (IGF-1) via the IGF-1/PI3K/Akt signaling pathway, while promoting apoptosis (34).

Deng et al. discovered that long non-coding RNA HOXA transcript at the distal tip (HOTTIP) is overexpressed in lung cancer tissues. Knockdown of HOTTIP led to decreased lung cancer cell proliferation and increased apoptosis, and in animal experiments, the tumor volume of HOTTIP knockdown mice was significantly smaller than that of the control group (35). Liu et al. showed that miR-335 expression is decreased in NSCLC tumors compared to adjacent non-cancerous tissues, while Transformer 2 beta (Tra2b) expression is increased. Clinical data indicated that increased Tra2b expression and decreased miR-335 expression are significantly associated with poor patient prognosis. In vitro experiments demonstrated that miR-335 overexpression inhibits growth, invasion, and migration of A549 lung cancer cells by targeting Tra2b. Conversely, inhibiting miR-335 or overexpressing Tra2b stimulates A549 lung cancer cell growth, invasion, and migration in vitro (36). Overall, regulating cell proliferation and apoptosis can impact tumor development and outcome. In our study, we constructed LARP7-overexpressing lung cancer cell lines and observed a significant decrease in proliferation, migration, and invasion abilities, along with increased apoptosis levels. In animal experiments, the volume and weight of tumors in LARP7-overexpressing mice were significantly smaller than those in the control group, indicating that LARP7 expression levels affect tumor proliferation and apoptosis. Therefore, LARP7 may regulate lung cancer progression by modulating relevant RASGs and further influencing tumor proliferation and apoptosis.

In this study, overexpression of LARP7 in lung cancer cells resulted in significant differential expression of 1,805 genes. Based on gene expression levels, ratio values, and literature review, genes closely associated with lung cancer, such as ADAR, FN1, interferon-induced protein 6 (IFI6), interferon-induced protein 27 (IFI27), B2M, BST2, histone cluster 3, H2a (HIST3H2A), Solute Carrier Family 45, Member 1 (SLC45A1), and Rho GTPase Activating Protein 42 (ARHGAP42), were identified. RT-qPCR validation showed that in LARP7-overexpressing A549 cells, ADAR, FN1, IFI27, B2M, BST2, HIST3H2A, and SLC45A1 were significantly downregulated. In LARP7-overexpressing H1299 cells, ADAR, FN1, IFI6, B2M, BST2, HIST3H2A, SLC45A1, and ARHGAP42 were also notably downregulated (Figure 6D), which is consistent with the sequencing results, confirming the impact of LARP7 on the differential expression of these genes. Alternative splicing is a process where genes produce different splice variants by rejoining RNA segments, resulting in multiple protein isoforms from a single gene. It is a widespread cellular process that expands the transcriptome through differential exon usage. However, with the advent of transcriptome sequencing and patient database access, significant changes in alternative splicing in cancer have garnered increasing attention (37). Studies have shown that alternative splicing contributes to cancer by altering the expression of key enzyme isoforms involved in cell cycle arrest, metabolism, and cell signaling (38-40). Additionally, alternative splicing can mimic gene mutation-induced loss of function by reducing the expression of full-length proteins. For instance, in acute myeloid leukemia (AML), abnormal inclusion of exon 9b in the enhancer of zeste homolog 2 (EZH2) gene leads to premature termination codons and reduced EZH2 protein expression (41). About 5% of AML patients have EZH2 gene loss-of-function mutations, but the EZH2 variant including exon 9b lacks additional EZH2 mutations, suggesting that alternative splicing can also cause gene function loss beyond gene mutations. Beyond specific gene splicing changes associated with cancer development, advances in RNA-seq technology, including increased sequencing depth and improved data analysis tools, have revealed extensive variability in splicing patterns across many cancers (42). Notably, alternative splicing is more prevalent in genes involved in apoptosis, metabolism, and gene expression, including known oncogenes and tumor suppressors, contributing to tumor progression (43). In this study, LARP7 overexpression induced significant alternative splicing changes in 2,178 genes. As previously mentioned, RASGs are highly enriched in biological processes related to cell proliferation and apoptosis, suggesting that LARP7 may regulate tumor progression by modulating alternative splicing of relevant genes. Overlap analysis and literature review identified FN1 and ADAR as genes closely associated with lung cancer. RT-qPCR validation showed that in LARP7-overexpressing A549 and H1299 cells, the expression levels of FN1 and ADAR transcripts were significantly higher than in control groups (P<0.05), further confirming the effect of LARP7 overexpression on FN1 and ADAR gene splicing.

Fibronectin (FN) is a glycoprotein widely expressed across various cell types, essential for cell adhesion, migration, and extracellular matrix formation (44). FN1, a member of the FN family, plays a key role in processes such as cell adhesion, migration, and cytoskeletal regulation, and is implicated in several diseases, including tumors. Studies have shown that FN1 is aberrantly expressed in tumor tissues and is crucial for tumor development, metastasis, and response to chemotherapy (45). In NSCLC, FN1 is overexpressed and functions as an autocrine/paracrine mitogenic factor. It interacts with integrins, heparin, collagen/gelatin, and fibrin, activating signaling pathways like extracellular signal-regulated kinase (ERK), Rho kinase, and mammalian target of rapamycin, mTOR (Akt/mTOR), thus promoting cell growth, adhesion, migration, and invasion (46).

Notably, FN1 and ADAR have been found to play significant roles in the progression and drug resistance of NSCLC (47). FN1 contributes to drug resistance by interacting with integrin-β1 and activating the Wnt/β-catenin pathway (48), while ADAR, through its RNA editing activity, affects gene expression and contributes to resistance against various treatments, including epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI) (49). Its involvement in NSCLC progression is evident, yet the specific mechanisms by which LARP7 regulates these factors require further investigation.

The findings on the role of LARP7 in NSCLC offer a clue for clinical management. For instance, LARP7 can serve as a novel biomarker for prognosis and personalized treatment, and modulating LARP7 may enhance current NSCLC treatments. However, it should be noted that the findings of this study must be further validated before clinical translation. As a tumor suppressor gene, LARP7 provides a new perspective for the clinical management of NSCLC, by upregulating of which along with inhibiting known oncogenes could lead to a more comprehensive treatment approach. The findings of this study are aligned with recent trends emphasizing the importance of both pathways in effective cancer treatments. Future studies should validate LARP7 as a biomarker and explore its interactions with other oncogenic pathways. Preclinical assessments in animal models will pave the way for clinical trials.

Conclusions

This study identified significant differential expression of the RBP LARP7 between lung cancer and normal tissues through public database analysis. LARP7 was found to be downregulated in lung cancer tissues, and its expression level negatively correlated with lung cancer prognosis. This finding was validated using samples from our hospital, suggesting that LARP7 may serve as a potential molecular target for clinical treatment and prognosis evaluation in lung cancer. Further investigation into the underlying mechanisms of LARP7 revealed its inhibitory effects on lung cancer cell proliferation, migration, and invasion, while promoting apoptosis in vitro. In vivo experiments showed that LARP7 overexpression suppressed tumor growth. Genomic data analysis indicated that LARP7 induces notable changes in gene expression and ASEs, primarily affecting immune response, inflammation, cell proliferation, and apoptosis pathways. This suggests that LARP7 may regulate lung cancer progression through these mechanisms. Based on gene expression levels, ratio values, and an overlap analysis of DEGs and RASGs, we identified FN1 and ADAR as genes with significant expression and splicing differences between the LARP7 overexpression and control groups, closely linked to lung cancer and deserving further investigation. The potential applications of LARP7 in lung cancer merit multi-center, in-depth exploration to pave new avenues for clinical drug development. Although our findings support the therapeutic potential of LARP7 restoration in NSCLC, clinical translation is hampered by its identity as a nuclear RBP, which precludes conventional small-molecule targeting and necessitates complex gene or protein replacement strategies with efficient nuclear delivery. Future efforts should focus on developing tumor-specific delivery platforms and predictive biomarkers to overcome these barriers.

Acknowledgments

We’d like to thank the Platform of Environmental Exposure and Life Health Research in Anhui Medical University and The First Affiliated Hospital of Anhui Medical University.

Footnote

Reporting Checklist: The authors have completed the MDAR and ARRIVE reporting checklists (Available at https://jtd.amegroups.com/article/view/10.21037/jtd-2026-0579/rc).

Data Sharing Statement: Available at https://jtd.amegroups.com/article/view/10.21037/jtd-2026-0579/dss

Peer Review File: Available at https://jtd.amegroups.com/article/view/10.21037/jtd-2026-0579/prf

Funding: This work was supported by the Natural Science Foundation of Anhui Province (No. 1708085MH220), the Basic-Clinical Cooperative Research Promotion Project of Anhui Medical University (No. 2019xkjT025) and the Student’s Platform for Innovation and Entrepreneurship Training Program of China (No. 2414010038).

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://jtd.amegroups.com/article/view/10.21037/jtd-2026-0579/coif). All authors report that this research was supported by the Natural Science Foundation of Anhui Province (No. 1708085MH220), the Basic-Clinical Cooperative Research Promotion Project of Anhui Medical University (No. 2019xkjT025) and the Student’s Platform for Innovation and Entrepreneurship Training Program of China (No. 2414010038). The authors have no other conflicts of interest to declare.

Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. The study was conducted in accordance with the Declaration of Helsinki and its subsequent amendments. The study was approved by the Clinical Medical Ethics Committee of The First Affiliated Hospital of Anhui Medical University (No. 5101096), and written informed consent was obtained from all participants. Experiments were performed under a project licence (No. 20210229) granted by the Animal Ethics Committee of Anhui Medical University, in compliance with the national guidelines for the care and use of animals.

Open Access Statement: This is an Open Access article distributed in accordance with the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License (CC BY-NC-ND 4.0), which permits the non-commercial replication and distribution of the article with the strict proviso that no changes or edits are made and the original work is properly cited (including links to both the formal publication through the relevant DOI and the license). See: https://creativecommons.org/licenses/by-nc-nd/4.0/.

References

1. Liang X, Liu M, Zhang L, et al. Global trends of incidence of lung cancer. China Cancer 2022;31:683-92.
2. Nooreldeen R, Bach H. Current and Future Development in Lung Cancer Diagnosis. Int J Mol Sci 2021;22:8661. [Crossref] [PubMed]
3. Svoboda E. Artificial intelligence is improving the detection of lung cancer. Nature 2020;587:S20-2. [Crossref] [PubMed]
4. Ling S, Hu Z, Yang Z, et al. Extremely high genetic diversity in a single tumor points to prevalence of non-Darwinian cell evolution. Proc Natl Acad Sci U S A 2015;112:E6496-505. [Crossref] [PubMed]
5. Sánchez-Jiménez C, Ludeña MD, Izquierdo JM. T-cell intracellular antigens function as tumor suppressor genes. Cell Death Dis 2015;6:e1669. [Crossref] [PubMed]
6. Wurth L, Papasaikas P, Olmeda D, et al. UNR/CSDE1 Drives a Post-transcriptional Program to Promote Melanoma Invasion and Metastasis. Cancer Cell 2016;30:694-707. [Crossref] [PubMed]
7. Hopkins TG, Mura M, Al-Ashtal HA, et al. The RNA-binding protein LARP1 is a post-transcriptional regulator of survival and tumorigenesis in ovarian cancer. Nucleic Acids Res 2016;44:1227-46. [Crossref] [PubMed]
8. Sebestyén E, Singh B, Miñana B, et al. Large-scale analysis of genome and transcriptome alterations in multiple tumors unveils novel cancer-relevant splicing networks. Genome Res 2016;26:732-44. [Crossref] [PubMed]
9. Zhang B, Babu KR, Lim CY, et al. A comprehensive expression landscape of RNA-binding proteins (RBPs) across 16 human cancer types. RNA Biol 2020;17:211-26. [Crossref] [PubMed]
10. Wang J, Liu Q, Shyr Y. Dysregulated transcription across diverse cancer types reveals the importance of RNA-binding protein in carcinogenesis. BMC Genomics 2015;16:S5. [Crossref] [PubMed]
11. Kao TL, Huang YC, Chen YH, et al. LARP3, LARP7, and MePCE are involved in the early stage of human telomerase RNA biogenesis. Nat Commun 2024;15:5955. [Crossref] [PubMed]
12. Hasler D, Meister G, Fischer U. Stabilize and connect: the role of LARP7 in nuclear non-coding RNA metabolism. RNA Biol 2021;18:290-303. [Crossref] [PubMed]
13. Dock-Bregeon AC, Lewis KA, Conte MR. The La-related proteins: structures and interactions of a versatile superfamily of RNA-binding proteins. RNA Biol 2021;18:178-93. [Crossref] [PubMed]
14. Yang Y, Liu S, Egloff S, et al. Structural basis of RNA conformational switching in the transcriptional regulator 7SK RNP. Mol Cell 2022;82:1724-1736.e7. [Crossref] [PubMed]
15. Barboric M, Lenasi T, Chen H, et al. 7SK snRNP/P-TEFb couples transcription elongation with alternative splicing and is essential for vertebrate development. Proc Natl Acad Sci U S A 2009;106:7798-803. [Crossref] [PubMed]
16. Kondelin J, Gylfe AE, Lundgren S, et al. Comprehensive Evaluation of Protein Coding Mononucleotide Microsatellites in Microsatellite-Unstable Colorectal Cancer. Cancer Res 2017;77:4078-88. [Crossref] [PubMed]
17. Fujimoto A, Fujita M, Hasegawa T, et al. Comprehensive analysis of indels in whole-genome microsatellite regions and microsatellite instability across 21 cancer types. Genome Res 2020;30:334-46. [Crossref] [PubMed]
18. Mori Y, Sato F, Selaru FM, et al. Instabilotyping reveals unique mutational spectra in microsatellite-unstable gastric cancers. Cancer Res 2002;62:3641-5.
19. Ji X, Lu H, Zhou Q, et al. LARP7 suppresses P-TEFb activity to inhibit breast cancer progression and metastasis. Elife 2014;3:e02907. [Crossref] [PubMed]
20. Cheng Y, Jin Z, Agarwal R, et al. LARP7 is a potential tumor suppressor gene in gastric cancer. Lab Invest 2012;92:1013-9. [Crossref] [PubMed]
21. Sui X, Sui Y, Wang Y. LARP7 in papillary thyroid carcinoma induces NIS expression through suppression of the SHH signaling pathway. Mol Med Rep 2018;17:7521-8. [Crossref] [PubMed]
22. Mori Y, Sato F, Selaru FM, et al. Instabilotyping reveals unique mutational spectra in microsatellite-unstable gastric cancers. Cancer Res 2002;62:3641-5.
23. Brown MJ, Symonowicz C, Medina LV, et al. Culture of Care: Organizational Responsibilities. In: Weichbrod RH, Thompson GA, Norton JN, eds. Management of Animal Care and Use Programs in Research, Education, and Testing. 2nd ed. Boca Raton (FL): CRC Press/Taylor & Francis; 2018.11-26.
24. Markert A, Grimm M, Martinez J, et al. The La-related protein LARP7 is a component of the 7SK ribonucleoprotein and affects transcription of cellular and viral polymerase II genes. EMBO Rep 2008;9:569-75. [Crossref] [PubMed]
25. Barboric M, Lenasi T, Chen H, et al. 7SK snRNP/P-TEFb couples transcription elongation with alternative splicing and is essential for vertebrate development. Proc Natl Acad Sci U S A 2009;106:7798-803. [Crossref] [PubMed]
26. Zhang F, Yan P, Yu H, et al. L ARP7 Is a BRCA1 Ubiquitinase Substrate and Regulates Genome Stability and Tumorigenesis. Cell Rep 2020;32:107974. [Crossref] [PubMed]
27. Chen B, Xu X, Lin DD, et al. KRT18 Modulates Alternative Splicing of Genes Involved in Proliferation and Apoptosis Processes in Both Gastric Cancer Cells and Clinical Samples. Front Genet 2021;12:635429. [Crossref] [PubMed]
28. Khusnurrokhman G, Wati FF. Tumor-promoting inflammation in lung cancer: A literature review. Ann Med Surg (Lond) 2022;79:104022. [Crossref] [PubMed]
29. Liotti F, Marotta M, Melillo RM, et al. The Impact of Resolution of Inflammation on Tumor Microenvironment: Exploring New Ways to Control Cancer Progression. Cancers (Basel) 2022;14:3333. [Crossref] [PubMed]
30. Pereira C, Gimenez-Xavier P, Pros E, et al. Genomic Profiling of Patient-Derived Xenografts for Lung Cancer Identifies B2M Inactivation Impairing Immunorecognition. Clin Cancer Res 2017;23:3203-13. [Crossref] [PubMed]
31. Zheng C, Wang J, Zhou Y, et al. IFNα-induced BST2(+) tumor-associated macrophages facilitate immunosuppression and tumor growth in pancreatic cancer by ERK-CXCL7 signaling. Cell Rep 2024;43:114088. [Crossref] [PubMed]
32. Cao Z, Wang Y, Wu J, et al. Serum small extracellular vesicles-derived BST2 as a biomarker for papillary thyroid microcarcinoma promotes lymph node metastasis. Cancer Gene Ther 2025;32:38-50. [Crossref] [PubMed]
33. Xu X, Wang Y, Xue F, et al. BST2 Promotes Tumor Growth via Multiple Pathways in Hepatocellular Carcinoma. Cancer Invest 2020;38:329-37. [Crossref] [PubMed]
34. Zhou Y, Li S, Li J, et al. Effect of microRNA-135a on Cell Proliferation, Migration, Invasion, Apoptosis and Tumor Angiogenesis Through the IGF-1/PI3K/Akt Signaling Pathway in Non-Small Cell Lung Cancer. Cell Physiol Biochem 2017;42:1431-46. [Crossref] [PubMed]
35. Deng HP, Chen L, Fan T, et al. Long non-coding RNA HOTTIP promotes tumor growth and inhibits cell apoptosis in lung cancer. Cell Mol Biol (Noisy-le-grand) 2015;61:34-40.
36. Liu J, Bian T, Feng J, et al. miR-335 inhibited cell proliferation of lung cancer cells by target Tra2β. Cancer Sci 2018;109:289-96. [Crossref] [PubMed]
37. Cherry S, Lynch KW. Alternative splicing and cancer: insights, opportunities, and challenges from an expanding view of the transcriptome. Genes Dev 2020;34:1005-16. [Crossref] [PubMed]
38. Hasler D, Meduri R, Bąk M, et al. The Alazami Syndrome-Associated Protein LARP7 Guides U6 Small Nuclear RNA Modification and Contributes to Splicing Robustness. Mol Cell 2020;77:1014-1031.e13. [Crossref] [PubMed]
39. Isaac R, Vinik Y, Mikl M, et al. A seven-transmembrane protein-TM7SF3, resides in nuclear speckles and regulates alternative splicing. iScience 2022;25:105270. [Crossref] [PubMed]
40. Anczuków O, Krainer AR. Splicing-factor alterations in cancers. RNA 2016;22:1285-301. [Crossref] [PubMed]
41. Rahman MA, Lin KT, Bradley RK, et al. Recurrent SRSF2 mutations in MDS affect both splicing and NMD. Genes Dev 2020;34:413-27. [Crossref] [PubMed]
42. PCAWG Transcriptome Core Group. Genomic basis for RNA alterations in cancer. Nature 2020;578:129-36. [Crossref] [PubMed]
43. Phillips JW, Pan Y, Tsai BL, et al. Pathway-guided analysis identifies Myc-dependent alternative pre-mRNA splicing in aggressive prostate cancers. Proc Natl Acad Sci U S A 2020;117:5269-79. [Crossref] [PubMed]
44. Wang J, Li R, Li M, et al. Fibronectin and colorectal cancer: signaling pathways and clinical implications. J Recept Signal Transduct Res 2021;41:313-20. [Crossref] [PubMed]
45. Wang X, Tan M, Huang H, et al. Hsa_circ_0000285 contributes to gastric cancer progression by upregulating FN1 through the inhibition of miR-1278. J Clin Lab Anal 2022;36:e24475. [Crossref] [PubMed]
46. Ritzenthaler JD, Han S, Roman J. Stimulation of lung carcinoma cell growth by fibronectin-integrin signalling. Mol Biosyst 2008;4:1160-9. [Crossref] [PubMed]
47. He S, Shi J, Zhou H, et al. Lnc-ABCA12-8 confers acquired resistance to gefitinib in non-small cell lung cancer by regulating the alternative splicing of fibronectin 1 in the IIICS region. Cancer Gene Ther 2022;29:1686-96. [Crossref] [PubMed]
48. Qin S, Zhang B, Xiao G, et al. Fibronectin protects lung cancer cells against docetaxel-induced apoptosis by promoting Src and caspase-8 phosphorylation. Tumour Biol 2016;37:13509-20. [Crossref] [PubMed]
49. Hua H, Zeng J, Xing H, et al. The RNA editing enzyme ADAR modulated by the rs1127317 genetic variant diminishes EGFR-TKIs efficiency in advanced lung adenocarcinoma. Life Sci 2022;296:120408. [Crossref] [PubMed]