MRPL51 expression is associated with poor prognosis and regulates lung adenocarcinoma progression via neurotensin

Introduction

According to the latest statistics from the International Agency for Research on Cancer (IASCL), lung cancer was the most commonly diagnosed cancer worldwide in 2022, with approximately 2.5 million new cases and 1.8 million deaths (1). Non-small cell lung cancer (NSCLC) is the predominant subtype of lung cancer, accounting for approximately 85% of all cases. Among NSCLC subtypes, adenocarcinoma is the most common, comprising approximately 40% of cases, with a notable increasing trend observed over the past few decades (2). The treatment of lung adenocarcinoma (LUAD) involves various strategies, including surgery, chemotherapy, targeted therapy, and immunotherapy. These approaches are typically tailored to the individual patient, with clinical staging and overall health status being key factors (3). In recent years, targeted therapies aimed at regulating key growth and survival pathways in cancer cells have substantially improved the treatment outlook for patients with LUAD. However, approximately half of the patients are unable to benefit from these therapies due to the limitations in available therapeutic targets. Moreover, for specific targets such as KRAS and HER2, targeted therapies are currently recommended only as second-line treatment options (4). Therefore, there is a critical need to implement new strategies that can identify reliable biomarkers and to conduct comprehensive translational research to broaden the therapeutic options available for cancer treatment.

Mitochondrial dysfunction and its involvement in apoptotic pathways have been well-documented in cancer (5,6). In line with their critical role in tumorigenesis, mitochondria have emerged as promising targets for cancer therapy (7,8). Mitochondrial ribosomal proteins (MRPs), as essential components of mitochondrial function and structure, have been implicated in the regulation of apoptosis and the progression of cancer (9). For instance, MRPS29 has been shown to participate in the mitochondrial apoptotic pathway, triggering apoptosis through mechanisms that are independent of both Fas induction and the tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) pathway (10). MRPL52 is significantly upregulated in human breast cancer and enhances the hypoxia-induced epithelial-mesenchymal transition (EMT), migration, and invasion capabilities of breast cancer cells through activation of the ROS/Notch1/Snail signaling pathway. Moreover, its overexpression is strongly associated with aggressive clinicopathological characteristics and an elevated risk of metastasis in patients with breast cancer (11). However, the specific mechanisms underlying the role of MRPs in LUAD remain poorly understood. Through bioinformatics analysis combined and subsequent validation in lung cancer cell models, Maiuthed et al. identified multiple genes exhibiting more than a five-fold upregulation in lung cancer, among which MRPL51 was prominently identified (12). The MRPL51 gene, located on chromosome 12p13.31, encodes the protein components of the large mitochondrial ribosome subunit (13). Current research on MRPL51 remains relatively limited, although existing evidence suggests its potential role as a downstream target of the transcription factor Forkhead Box M1(FOXM1) (14). Notably, the details of the molecular mechanisms underlying its involvement in LUAD pathogenesis remain poorly characterized, and thus further investigation to clarify its tumorigenic regulatory pathways is warranted.

This study integrates data from The Cancer Genome Atlas (TCGA) and Genotype-Tissue Expression (GTEx) tumor-related databases with clinical samples of LUAD to analyze the expression of MRPL51 and its correlation with clinical characteristics. Subsequently, MRPL51 overexpression and knockout models were established in the A549 LUAD cell line. The gene function of MRPL51 was examined both in vitro and in vivo, with cellular functional assays, molecular biology techniques, and nude mouse models being employed. Finally, next-generation sequencing (NGS) and bioinformatics analysis were performed to identify the signaling pathways and key molecules associated with MRPL51. We present this article in accordance with the ARRIVE and MDAR reporting checklists (available at https://jtd.amegroups.com/article/view/10.21037/jtd-2025-1774/rc).

Methods

Data acquisition and processing

Data of patients with LUAD were obtained from an online database (www.kmplot.com). All patients with cancer in the database were identified from the Gene Expression Profiling Interactive Analysis [TCGA (https://portal.gdc.cancer.gov), or the GTEx (https://www.gtexportal.org/]. The database, which was established using gene expression data and survival information from 585 patients with LUAD, was used to establish the clinical relevance of MRPL51 expression to the survival times of patients with LUAD, after biased arrays were excluded. The expression values for MRPL51 and clinical data from those samples were extracted and used for the survival analysis. The samples were split into high and low groups of MRPL51 expression. Hazard ratios (HRs) and the log-rank P value were calculated according to the formulas. The HR is the ratio of the hazard rates that correspond to the conditions described by two levels of an explanatory variable in survival analysis. A P value <0.05 was considered to be statistically significant.

Identification of differentially expressed genes (DEGs)

Differential gene expression analysis was performed via the “limma” package in R software (The R Software of Statistical Computing). DEGs were identified between the MRPL51 knockout group, overexpression group, and their respective control groups. The thresholds for DEG selection were set as |log fold change (FC)| >1 (logFC >1 or logFC <–1) and a P value <0.05.

Survival analysis

For the analysis of online databases, the Kaplan-Meier (KM) plotter (https://kmplot.com/analysis/) was used to generate survival curves and to evaluate the prognostic value of MRPL51. For clinical patient cohorts, follow-up data, including patient outcomes, were collected via regular telephone interviews, and corresponding survival curves were subsequently plotted.

Transcriptome sequencing

Total RNA was extracted from cells and subsequently sent to Novogene Co., Ltd (Beijing, China) for whole transcriptome sequencing. The transcriptome sequencing was performed with the Illumina sequencing platform (Illumina, San Diego, CA, USA), with a sequencing depth of 8 Gb.

Cell culture and treatment

The A549, BEAS-2B, Calu-3, NCI-H1975, and PC-9 cell lines were purchased from BNCC (Beijing, China), and HEK293T cells were obtained from the American Type Culture Collection (ATCC; Manassa, VI, USA). HEK293T, A549, BEAS-2B, Calu-3, NCI-H1975, and PC-9 cells were authenticated via short tandem repeatprofiling, tested for mycoplasma contamination, and grown in DMEM, F-12-K or Opti-MEM (Gibco, Thermo Fisher Scientific, Waltham, MA, USA), supplemented with 10% fetal bovine serum (FBS) in the presence of antibiotics in a humidified 5% CO₂ incubator at 37 ℃.

Immunohistochemistry (IHC)

All tissue samples were collected from 100 consecutive patients who were diagnosed with LUAD and who underwent surgical resection at the Department of Thoracic Surgery, Sir Run Run Shaw Hospital, Zhejiang University School of Medicine, during a study period spanning from January 2015 to December 2016. Primary antibody against MRPL51 (ab243821; Abcam, Cambridge, UK) was diluted at 1:200 for IHC analysis. This study was conducted in accordance with the Declaration of Helsinki and its subsequent amendments. This study was approved by the Ethics Review Committee of Sir Run Run Shaw Hospital, Zhejiang University School of Medicine (approval No. 2024; research ethics review No. 1179). Individual consent for this retrospective analysis was waived.

The chromogenic visualization in IHC was achieved via a polymer-based detection system (PV60001; ZSGB-BIO, Beijing, China) containing 3,3’-diaminobenzidine (DAB) substrate and secondary antibody polymer complexes. The IHC results were independently evaluated by two experienced pathologists using a semiquantitative scoring system. The immunoreactivity score (IRS) was calculated as the product of staining intensity and the percentage of positive cells. Staining intensity was graded on a four-tier scale as follows: negative staining, 0 point; weak positivity (light yellow), 1 point; moderate positivity (brownish yellow), 2 points; and strong positivity (dark brown), 3 points. The percentage of positive cells was categorized into four grades as follows: ≤25%, 1 point; 26–50%, 2 points; 51–75%, 3 points; and >75%, 4 points. The expression levels of MRPL51 in cancerous tissues and adjacent noncancerous tissues were statistically compared via the chi-squared test. Furthermore, the correlation between MRPL51 expression and clinicopathological parameters of patients was analyzed with the same statistical method.

Western blotting

Cellular proteins were extracted with radioimmunoprecipitation assay buffer (RIPA) on ice according to standard protocols. Sodium dodecyl sulfate (4–20%)-polyacrylamide gel electrophoresis (SDS-PAGE) was employed to disassociate and separate all the proteins according to their molecular masses. SDS-PAGE gels were transferred onto polyvinylidene fluoride (PVDF) membranes. Total protein was identified via Western blotting with anti-MRPL51 (1:1,000; Abcam) and anti-β-actin (1:10,000; Proteintech, Rosemont, IL, USA). After incubation with the second antibodies (goat anti-rabbit; 1:5,000), proteins were identified with an enhanced chemiluminescence (ECL) detection kit (Millipore, Burlington, MA, USA) in accordance with the manufacturer’s guidelines.

Quantitative reverse-transcription polymerase chain reaction (qRT-PCR)

Total RNA was extracted with a RNA-Quick Purification Kit (YEASEN Science, Shanghai, China) and quantified via NanoDrop spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). Next, complement DNA (cDNA) was generated with Hifair V one-step RT-gDNA digestion SuperMix for quantitative polymerase chain reaction (qPCR) (Yeasen Biotechnology, Shanghai, China). After the synthesized cDNA was mixed with SYBR Green Master Mix (Accurate Biology, USA) and various sets of gene-specific primers, qRT-PCR was performed with a CFX96 real-time PCR system (Thermo Fisher Scientific). The primer sequences included those used for action [forward (F): 5'-CCTGGACTTCGAGCAAGAGATGG-3'; reverse (R): 5'-CAGGAAGGAAGGCTGGAAGAGTC-3'], MRPL51 (F: 5'-AGGGCCATGTTCGGAGTGTAT-3'; R: 5'-CCCTTTCCAACCTCGAAGCC-3'], and neurotensin (NTS) (F: 5'-GCATGCTACTCCTGGCTTTCA-3'; R: 5'-CTCAGCTGGGCTGTTCAAAT-3').

Transwell cell inverted invasion assay

Cell invasion assays were conducted using 24-well plates with 8-µm-pore Transwell chambers (Corning Inc., Corning, NY, USA). The lower chamber was filled with culture medium containing 10% FBS. A549 cells were seeded at a density of 1×10⁵ cells/well into the upper chamber filled with Matrigel (Corning Inc.). One day after seeding, the cells on the surface of the bottom layer were stained with 1% crystal violet dye, and cell counting was performed after treatment with paraformaldehyde.

Wound-healing assay

A549 cells were seeded at 2×10⁵ cells/mL, and a wound was established with a one-time with a 1-mm-thick pipette tip. Cell migration rate was quantified and compared via measurements and calculations of the cell-covered area via ImageJ (US National Institutes of Health, Bethesda, MD, USA) and Photoshop software (Adobe, San Jose, CA, USA).

Cell Counting Kit-8 (CCK-8) assay

The CCK-8 assay was performed with a CCK-8 kit (Yeasen Biotechnology). Cells were resuspended and seeded into 96-well plates at a density of 2×10³ cells per well for culturing. All plates were incubated at 37 ℃ for 1 hour. Absorbance was measured at optical density (OD) of 450 nm with an enzyme-linked immunosorbent assay (ELISA) plate reader (Bio-Rad Laboratories, Hercules, CA, USA).

In vivo tumor xenograft assay

Animal experiments were performed under a project license (No. ZJU20240096) approved by the Ethics Committee of Zhejiang University, in compliance with the institutional guidelines for the care and use of animals.

Cells were detached with 0.25% trypsin (Solarbio Science & Technology Co., Ltd., Beijing, China) and resuspended in phosphate-buffered saline (PBS). A total of 1 mL of the cell suspension was subcutaneously injected into the flanks of 6-week-old male BALB/c nude mice (Strain NO. D000521, GemPharmatech Co., Ltd., Jiangsu, China). The mice were randomly divided into control and MRPL51-knockout groups. After 7 weeks, all mice were killed, and tumors were isolated. A study protocol was prepared prior to the research and has been registered at http://114.255.48.20.

Flow cytometry assay

Cell cycle analysis was performed with propidium iodide (PI) staining followed by flow cytometry (Beckman Coulter, Brea, CA, USA). Cells were washed with PBS and resuspended in 1× binding buffer. Data analysis was conducted via FlowJo software (Flowjo, Ashland, OR, USA).

Lentivirus-based plasmid preparation and transfection

Lentivirus-based CRISPR/Cas9 transfer plasmids targeting human MRPL51 (gene access no. NM_016497.4) were obtained from Beyotime Biotechnology Co., Ltd. (Nantong, China). The target sequence of pLenti-MRPL51-sgRNA (pLenti vector carrying MRPL51-targeting single guide RNA) was 5'-GCCTACCTGCCCCGGATAAG-3'. For the overexpression of MRPL51, PLVX-puro plasmid (Tskingke Biological Technology Inc., Beijing, China) was used.

Statistical analysis

Statistical analysis was performed with SPSS software (IBM Corp., Armonk, NY, USA), and statistical significance was set at a P value <0.05. For sample sizes smaller than 50, the Shapiro-Wilk test was used to assess the normality of the data. For normally distributed continuous variables, data are presented as the mean ± standard deviation (x¯±s), and differences between groups were compared with independent samples t-tests. Categorical variables were analyzed with the Chi-squared test. Survival curves were evaluated with the log-rank test to assess differences.

Results

MRPL51 overexpressed in LUAD and correlated with poor clinical pathological features

We used the Gene Expression Profiling Interactive Analysis (GEPIA) database to investigate the expression profile of MRPL51 across various malignant tumors and their corresponding normal tissues. The results indicated that MRPL51 messenger RNA (mRNA) expression was significantly upregulated in LUAD (Figure 1A). Furthermore, we analyzed MRPL51 expression in 526 LUAD samples and 486 unmatched normal samples using data from TCGA and GTEx databases. The results demonstrated that MRPL51 expression was significantly upregulated in LUAD tissues as compared to normal tissues (P<0.001; Figure 1B).

Figure 1 Expression of MRPL51 in LUAD. (A) Analysis of MRPL51 expression across multiple cancer types via the GEPIA database revealed that MRPL51 is significantly upregulated in LUAD as compared to normal tissues; (B) MRPL51 expression was assessed in LUAD tissues in comparison to unmatched adjacent normal lung tissues from TCGA and GTEx database; (C) MRPL51 expression profiles across distinct pathological stages of LUAD in the TCGA; (D) correlation between MRPL51 expression and lymph node metastasis in LUAD based on TCGA database analysis. AJCC, American Joint Committee on Cancer; CPM, counts per million; FC, fold change; GEPIA, Gene Expression Profiling Interactive Analysis; GTEx, Genotype-Tissue Expression; LUAD, lung adenocarcinoma; N, regional lymph node; TCGA, The Cancer Genome Atlas.

To clarify the relationship between MRPL51 expression and pathological stage, we classified the LUAD samples into four groups based on stages I–IV of the American Joint Committee on Cancer (AJCC) staging system. Comparative analysis revealed that MRPL51 expression was significantly higher in stage II, III, and IV LUAD tissues than in stage I tissues, with statistically significant differences. However, no significant differences in MRPL51 expression were observed between stages II, III, and IV (Figure 1C). Additionally, MRPL51 expression was significantly higher in LUAD tissues with lymph node metastasis than in those without metastasis (P=0.01; Figure 1D). IHC analysis revealed a significant disparity in MRPL51 protein expression between LUAD tissues and adjacent noncancerous tissues. Specifically, positive MRPL51 expression was detected in 72% (72/100) of LUAD specimens, whereas only 32% (32/100) of adjacent noncancerous tissues exhibited MRPL51 positivity. Statistical analysis demonstrated that MRPL51 protein expression levels were significantly elevated in LUAD tissues compared to their adjacent counterparts (P=0.001; Figure 2 and Table 1). We categorized patients into two distinct groups based on the median MRPL51 expression level: the high-MRPL51 expression group and the low-MRPL51 expression group. Comprehensive clinicopathological data were subjected to statistical analysis via the Pearson Chi-squared test. The analysis revealed significant associations between elevated MRPL51 expression levels and several key prognostic factors, including T stage, N stage, and AJCC stage. However, no statistically significant correlations were observed between MRPL51 expression levels and patient age or gender (Table 2). These findings provide strong evidence supporting the association between increased MRPL51 expression and adverse clinicopathological characteristics in patients with LUAD.

Figure 2 Representative immunohistochemical staining of MRPL51 in adjacent non-tumor tissues and lung adenocarcinoma tissues. Magnification 40× and 200×.

High expression of MRPL51 was associated with poor prognosis in patients with LUAD

To investigate the impact of MRPL51 expression on overall survival (OS) in patients with LUAD, we stratified tissue samples from TCGA and GTEx databases into high-expression and low-expression groups based on the median MRPL51 expression level. KM survival analysis was then performed with the corresponding clinical data (Figure 3A). The results indicated that patients with low MRPL51 expression (n=252) had a significantly longer OS compared to those with high MRPL51 expression (n=252), with a statistically significant difference (HR =1.573; log-rank P=0.003).

Figure 3 Association of MRPL51 expression with patient prognosis in LUAD. OS based on (A) TCGA-LUAD datasets; (B) DFS in patients with low and high MRPL51 expression; (C) OS in patients with low and high MRPL51 expression. DFS, disease-free survival; HR, hazard ratio; LUAD, lung adenocarcinoma; OS, overall survival; TCGA, The Cancer Genome Atlas.

We conducted a comprehensive 5-year follow-up study involving 100 patients who underwent surgical resection for LUAD, systematically collecting data on tumor recurrence and survival outcomes. To evaluate the prognostic significance of MRPL51 expression, we performed KM survival analysis comparing patients stratified into high MRPL51 expression (n=49) and low MRPL51 expression (n=51) groups (Figure 3B,3C). The analysis revealed that patients with low MRPL51 expression exhibited significantly longer disease-free survival (DFS) compared to those with high MRPL51 expression (HR =2.617; P=0.012). Although a potential tendency toward improved OS was observed in the low-MRPL51 expression group, this difference did not reach statistical significance (HR =1.992, P=0.14).

MRPL51 promoted the growth of LUAD cells in vitro

The baseline expression levels of MRPL51 were assessed via Western blot analysis in the normal human alveolar epithelial cell line BEAS-2B and the LUAD cell lines A549, Calu-3, H1975, and PC-9. The results revealed elevated MRPL51 expression in LUAD cell lines as compared to the normal cell line (Figure 4A). For MRPL51-knockout and-overexpression experiments, the A549 cell line was used. The successful generation of MRPL51-knockout and -overexpression cell models was confirmed through Western blot and qRT-PCR analyses (Figure 4B,4C).

Figure 4 MRPL51 promoted the proliferation of LUAD cells. (A) Western blot analysis of MRPL51 expression in normal cell lines and LUAD cell lines (mean ± SD, n=3); (B) qRT-PCR analysis of MRPL51 knockdown and overexpression efficiency; (C) Western blot analysis of MRPL51 knockdown and overexpression efficiency (mean ± SD, n=3); (D) CCK-8 assay was performed to assess the effects of MRPL51 knockdown and overexpression on the growth rate of LUAD cells (A549); (E) flow cytometry analysis of the cell cycle changes in A549 cells following MRPL51 knockdown and overexpression. Representative fluorescence histograms illustrating the impact on cell cycle distribution in A549 cells; (F) bar graph showing the percentages of LUAD cells in the G1, S, and G2 phases; (G) Western blot analysis of CDK2 and cyclin A protein levels in MRPL51-knockout and-overexpression A549 cells (mean ± SD, n=3). *, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001. CCK8, Cell Counting Kit-8; LUAD, lung adenocarcinoma; OE, overexpression; qRT-PCR, quantitative real-time polymerase chain reaction; RMSD, root mean square deviation; SD, standard deviation.

The proliferative capacity of A549 cells across different treatment groups was evaluated via CCK-8 assays. Following calibration of the OD values on the initial day of measurement, the results demonstrated that the proliferation rate of cells with MRPL51 gene knockout was significantly reduced as compared to control cells. Conversely, overexpression of MRPL51 led to a moderate increase in cellular proliferation (Figure 4D). These findings suggest that MRPL51 plays a role in promoting the proliferation of LUAD cells.

To further investigate the role of MRPL51 in cell cycle regulation, PI staining coupled with flow cytometry was employed to quantitatively assess the distribution of cells across various cell cycle phases. The analysis revealed that MRPL51 knockout significantly increased the proportion of cells in the S phase. In contrast, MRPL51 overexpression did not induce significant alterations in cell cycle distribution as compared to the control condition (Figure 4E,4F). To corroborate the flow cytometry findings, Western blot analysis was conducted to evaluate the expression levels of key regulatory proteins implicated in S-phase progression. Consistent with the flow cytometry results, MRPL51 knockout was associated with a notable downregulation of cyclin A and CDK2, both of which are critical regulators of S-phase progression (Figure 4G).

MRPL51 promoted the invasion and migration of LUAD cells

To assess the impact of MRPL51 on the malignant behavior of lung cancer cells, a series of functional assays were performed. Wound healing assays demonstrated that MRPL51 knockout significantly impaired the migratory capacity of lung cancer cells, whereas MRPL51 overexpression did not notably enhance cell migration (Figure 5A). Transwell invasion assays revealed that MRPL51 depletion markedly reduced the number of cells penetrating the extracellular matrix, while overexpression of MRPL51 did not significantly increase the invasive capacity (Figure 5B,5C). These findings suggest that inhibition of MRPL51 expression attenuates the migratory and invasive potential of lung cancer cells. Furthermore, colony formation assays were conducted to evaluate the effect of MRPL51 on the clonogenic ability of lung cancer cells. The results showed that MRPL51 knockout led to a significant reduction in the number of A549 cell colonies, while MRPL51 overexpression did not induce a notable increase in colony formation (Figure 5D,5E), indicating that MRPL51 suppression diminishes the clonogenic potential of lung cancer cells. Collectively, these results demonstrate that MRPL51 plays a critical role in promoting the key malignant biological behaviors of LUAD cells, including migration, invasion, and clonogenic growth.

Figure 5 Knockdown of MRPL51 suppressed the invasion and migration in LUAD cells. (A) Scratch assay evaluated the effect of MRPL51 expression on the migratory ability of A549 cells (magnification 10×); (B,C) Transwell assay evaluated the effect of MRPL51 expression on cell invasive ability (crystal violet staining; magnification 40×); (D,E) colony formation assay evaluated the effect of MRPL51 expression on cell clonogenic ability (crystal violet staining; magnification 40×). ***, P<0.001. LUAD, lung adenocarcinoma; OE, overexpression.

MRPL51 knockout attenuated tumor growth kinetics in vivo

The in vitro experiments in our study demonstrated that MRPL51 significantly enhanced the biological capabilities of LUAD cells, including proliferation, migration, and invasion. To further investigate whether MRPL51 exhibits similar oncogenic properties in vivo and considering budgetary constraints, we established an animal model by subcutaneously inoculating A549 cells with MRPL51 knockout and control cells into the right axilla of nude mice, respectively.

The tumor growth dynamics were monitored and analyzed. Notably, MRPL51 knockout significantly inhibited tumor growth progression. Statistically significant differences in tumor volume between the control and knockout groups were observed from the fifth week after implantation, with the control group exhibiting a tumor volume of 278.63±136.65 mm³ and the knockout group a significantly reduced tumor volume of 72.59±47.77 mm³ (Figure 6A). The collected data followed a normal distribution (Shapiro-Wilk test). This significant difference in tumor growth persisted until the experimental endpoint, demonstrating the critical role of MRPL51 in promoting LUAD progression in vivo.

Figure 6 Knockdown of MRPL51 suppressed LUAD cells growth in vivo. MRPL51-knockout and control A549 cells were injected subcutaneously into nude mice. The mice were euthanized, and tumors were excised. (A) At specified time points, tumor size was measured with a caliper; (B) measurement of tumor weight in xenograft models following MRPL51 knockout; (C) representative H&E staining and immunohistochemical analysis of Ki-67 expression in tumor tissues (magnification 10× and 40×). Data are expressed as the mean ± standard deviation. n=5. *, P<0.05; ***, P<0.001; ****, P<0.0001. H&E, hematoxylin and eosin; LUAD, lung adenocarcinoma.

Following the euthanasia of the mice, subcutaneous tumors were harvested through blunt dissection and meticulously cleared of surrounding connective tissues. Tumor mass was precisely measured using an analytical balance. Quantitative analysis revealed that control group tumors weighed 0.49±0.17 g, whereas MRPL51-knockout group tumors exhibited a significantly reduced weight of 0.18±0.13 g. The collected data adhered to a normal distribution (Shapiro-Wilk test P=0.31), and statistical analysis demonstrated a significant difference in tumor weight between the two groups (P<0.001; Figure 6B). These findings provide compelling evidence that MRPL51 knockout substantially diminishes tumor growth in vivo, as reflected by the reduced tumor mass.

To determine whether the observed reduction in tumor growth was associated with antiproliferative effects, the IHC analysis of proliferation markers was performed. Specifically, Ki-67 staining was conducted on resected xenograft tumor tissues from each experimental group. Compared to the control group, the MRPL51-knockout group exhibited a significant decrease in the number of nuclei positively stained for Ki-67 (Figure 6C), indicating reduced proliferative activity. Representative hematoxylin and eosin (H&E) staining was also included to confirm the histological identification of tumor tissues. These data collectively demonstrate that MRPL51 knockout effectively suppresses the proliferation of LUAD in vivo, further supporting its role as a potential therapeutic target in LUAD progression.

Transcriptome sequencing identified NTS as a downstream molecule of MRPL51

To elucidate the molecular mechanisms underlying MRPL51’s role in LUAD progression, we performed high-throughput sequencing on MRPL51 knockout, MRPL overexpression, and the corresponding control cell lines. The primary objective was to identify the critical downstream molecules and signaling pathways regulated by MRPL51. Following sequencing, raw data underwent rigorous normalization processing, yielding fragments per kilobase of transcript per million mapped reads (FPKM) values for gene expression quantification.

Subsequent principal component analysis (PCA) revealed distinct clustering patterns between the experimental groups (Figure 7A). Notably, intergroup samples exhibited significant dispersion, whereas intragroup samples demonstrated tight clustering. This clear separation pattern between groups suggests the existence of DEGs associated with MRPL51 modulation, warranting further comprehensive differential expression analysis.

Figure 7 MRPL51 coexpression genes were identified following NGS analysis. (A) PCA plot of the analyzed data; (B) Venn diagram illustrating the overlap between downregulated genes in the MRPL51-knockout group compared to the knockout control group and upregulated genes in the overexpression group compared to the OE control group; (C) Venn diagram illustrating the overlap between upregulated genes in the MRPL51 knockout group compared to the knockout control group and downregulated genes in the MRPL51 overexpression group compared to the overexpression control group; (D) volcano plot of differentially expressed genes between the MRPL51 knockout group and the knockout control group; (E) volcano plot of differentially expressed genes between the MRPL51 overexpression group and the overexpression control group. Red indicates a positive correlation, and blue indicates a negative correlation. NGS, next-generation sequencing; NTS, neurotensin; OE, overexpression; PCA, principal component analysis.

To clarify the gene expression alterations associated with MRPL51 manipulation, we employed a comparative transcriptomic analysis. Venn diagram analysis was performed to identify overlapping and differential genes between the following comparisons: downregulated genes in the MRPL51-knockout group relative to the knockout control group and the upregulated genes in the MRPL51-overexpression group relative to the overexpression control group. This analysis revealed four genes shared between these two sets (Figure 7B). In contrast, no overlapping genes were identified between upregulated genes in the knockout group and downregulated genes in the overexpression group (Figure 7C).

Volcano plots were constructed to visualize the DEGs, with red, blue, and gray dots representing upregulated, downregulated, and nonsignificant genes, respectively. In the MRPL51-knockout group, 156 genes were upregulated, and 100 genes were downregulated compared to the knockout control group. Meanwhile, the MRPL51-overexpression group had 62 upregulated genes and 22 downregulated genes relative to the overexpression control group. Notably, bioinformatics analysis identified NTS as a co-expressed factor of MRPL51. NTS expression was significantly decreased upon MRPL51 knockout but markedly increased following MRPL51 overexpression (Figure 7D,7E). The identification of overlapping genes suggests that MRPL51 may regulate these genes through shared molecular mechanisms, thereby influencing fundamental cellular processes. These findings provide valuable insights into the transcriptional networks governed by MRPL51 and its potential role in modulating cell function.

To validate the sequencing data, we performed qRT-PCR and Western blot analyses in A549 cells. The results demonstrated that MRPL51 knockout led to significant downregulation of both NTS mRNA and protein expression levels. Conversely, MRPL51 overexpression resulted in marked upregulation of NTS mRNA and protein expression, which was consistent with the sequencing data. However, no significant changes were observed in the protein expression level of NTSR1, the receptor associated with NTS (Figure 8A,8B).

Figure 8 NTS contributed to MRPL51-mediated regulation of cellular proliferation and invasion. (A) Expression levels of NTS mRNA in A549 cells following MRPL51 knockdown; (B) expression levels of NTS protein and its receptor NTSR1 protein in A549 cells following MRPL51 knockdown (mean ± SD, n=3); (C) CCK8 assay demonstrated that exogenous NTS rescues the suppression of cell proliferation induced by MRPL51 knockout; (D,E) Transwell assay demonstrated that exogenous NTS rescues the suppression of cell invasion induced by MRPL51 knockout (crystal violet staining; magnification 40×). ***, P<0.001; ****, P<0.0001. CCK8, Cell Counting Kit-8; mRNA, messenger RNA; NTS, neurotensin; OE, overexpression; SD, standard deviation.

In lung cancer cells, NTS promotes primary tumor growth and extensive lymph node metastasis through autocrine and paracrine regulatory circuits (15,16). We hypothesize that NTS may function as a downstream effector of MRPL51, with MRPL51 modulating cellular functions via NTS expression. To clarify the relationship between MRPL51 expression and cancer progression, we designed experiments using exogenous NTS treatment. Rescue experiments were performed by supplementing MRPL51-knockout cells with 100 nM of exogenous NTS. The results demonstrated that 100 nM of NTS significantly reversed the inhibitory effect of MRPL51 knockout on A549 cell proliferation, as determined by cell viability assays (Figure 8C). Notably, the same concentration of NTS did not affect the proliferation of knockout CTRL A549 cells. Furthermore, in Transwell migration assays, exogenous NTS effectively rescued the suppressed invasive capacity induced by MRPL51 knockout (Figure 8D,8E), suggesting that the MRPL51-NTS axis plays a crucial role in regulating cancer cell migration.

To characterize the functional and compositional characteristics of DEGs, we performed Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis on the identified DEGs. The analysis revealed that DEGs between the MRPL51-knockout group and the control group were predominantly enriched in several inflammation- and metabolism-related signaling pathways, including the nuclear factor kappa light-chain-enhancer of activated B cells (NF-κB) signaling pathway, TNF signaling pathway, and phosphatidylinositol 3-kinase/protein kinase B (PI3K/AKT) signaling pathway (Figure 9A). In contrast, DEGs from the MRPL5-overexpression group compared to its control group were primarily enriched in the hypoxia-inducible factor 1 alpha (HIF-1α) pathway (Figure 9B).

Figure 9 Significantly enriched KEGG pathways and GO annotations associated with MRPL51. (A) KEGG pathway enrichment analysis of differentially expressed genes between the MRPL51-knockout group and the knockout control group; (B) KEGG pathway enrichment analysis of differentially expressed genes between the MRPL5-overexpression group and the overexpression control group; (C) PI3K/AKT signaling pathway and cell cycle signaling pathway; (D) GO annotation analysis of differentially expressed genes between the MRPL51-knockout group and the knockout control group; (E) GO annotation analysis of differentially expressed genes between the MRPL51-overexpression group and the overexpression control group. AKT, protein kinase B; GO, Gene Ontology; GSEA, gene set enrichment analysis; KEGG, Kyoto Encyclopedia of Genes and Genomes; PI3K, phosphoinositide 3-kinase.

Gene set enrichment analysis (GSEA) results demonstrated that the cell cycle signaling pathway was downregulated in the MRPL51-knockout group as compared to the control group. The normalized enrichment score (NES) for the PI3K/AKT signaling pathway was 1.30, while the cell cycle signaling pathway showed an NES of –1.63 (Figure 9C).

Gene Ontology (GO) analysis revealed that genes co-expressed with MRPL19 were significantly enriched in the biological process of ion-gated channel regulation (Figure 9D,9E).

Upon binding to its high-affinity receptor NTSR1, NTS transactivates epidermal growth factor receptor (EGFR), resulting in increased tyrosine phosphorylation of EGFR. The phosphorylated EGFR subsequently activates multiple downstream pathways, including the PI3K/AKT signaling pathway (16,17). Based on the gene functional enrichment analysis, we further validated, through Western blot experiments, that MRPL51 can influence the activation of downstream pathways through NTS. The results demonstrated that knockdown of MRPL51 expression led to a reduction in NTS levels, accompanied by the decreased phosphorylation of EGFR at Tyr1173 and diminished the expression of phosphorylated PI3K and AKT. Notably, the supplementation of the cell culture medium with 100 nM of exogenous NTS partially restored the expression levels of phosphorylated EGFR Tyr1173, PI3K, and AKT. Importantly, exogenous administration of NTS did not affect the expression level of MRPL51 (Figure 10). These findings suggest that MRPL51 potentially regulates the malignant biological behaviors of LUAD through modulation of the NTS-mediated EGFR/PI3K/AKT signaling axis.

Figure 10 Knockout of MRPL51 inhibited the downstream signaling axis of NTS. Western blot analysis was performed to detect the downstream proteins of NTS, including the phosphorylation of EGFR at the Tyr1173 site and phosphorylated PI3K and AKT, as well as the total expression levels of EGFR, PI3K, and AKT. Knockout of MRPL51 inhibited the EGFR-PI3K-AKT signaling axis, which was subsequently reactivated following exogenous supplementation of NTS (mean ± SD, n=3). *, P<0.05; **, P<0.01; ****, P<0.0001; AKT, protein kinase B; EGFR, epidermal growth factor receptor; NTS, neurotensin; PI3K, phosphoinositide 3-kinase; SD, standard deviation; Tyr, tyrosine.

Discussion

Lung cancer ranks first in both incidence and mortality among males worldwide and ranks second in both parameters among females, accounting for 18.7% of all cancer-related deaths globally (1,18). As the most prevalent subtype of lung cancer, LUAD has demonstrated a rising incidence in recent decades (3). In recent years, targeted therapies have been developed to modulate critical growth, and survival pathways in cancer cells have significantly advanced the treatment landscape of LUAD, resulting in a notable extension of survival in some patients with advanced-stage lung cancer. However, more than half of the patient population remains excluded from this treatment due to target limitations. Additionally, the increasing prevalence of drug resistance and adverse reactions associated with existing targeted therapies has become increasingly evident. Consequently, the identification of additional potential therapeutic targets has emerged as a critical focus in the advancement of targeted therapy.

MRPs are essential components for the structural and functional integrity of mitochondrial complexes. Throughout evolution, mammalian mitochondria have acquired novel MRP genes to compensate for the loss of ribosomal RNA. In mammals, more than 80 MRPs have been identified, and these genes do not constitute a single gene family but rather a group of similarly named proteins that function within mitochondria. The expression levels of many MRP genes have been found to be altered in various types of cancer, and these changes are associated with clinical features of certain malignancies (19,20).

This study demonstrates that MRPL51 is upregulated during the progression of LUAD and is associated with poor prognosis in patients with LUAD. IHC analysis of clinical samples from patients confirmed the hypothesis that the high expression of MRPL51 is associated with poorer prognosis in these patients. Analysis of clinical patient data suggests that the expression of MRPL51 is associated with the T stage, AJCC stage, and the presence of lymph node metastasis prior to surgery in the tumor tissue of patients. Further data on 5-year postoperative recurrence and survival rates were collected, and survival curves were constructed, followed by log-rank test analysis. The analysis demonstrated that the group with a high expression of MRPL51 exhibited significantly worse 5-year DFS as compared to the low-expression group. The results of this study indicate that MRPL51 expression may play a role in regulating tumor cell proliferation and metastasis and is associated with poor prognosis. Therefore, MRPL51 may serve as a prognostic marker for LUAD.

To further investigate the role of MRPL51 in LUAD cells, we knocked out MRPL51 in the A549 LUAD cell line using the CRISPR/Cas9 system and established an MRPL51-overexpression model to study the impact of MRPL51 expression modulation on the biological behavior of LUAD cells. In the A549 cell line, MRPL51 knockout significantly inhibited malignant behaviors such as proliferation and invasion, while MRPL51 overexpression partially enhanced the proliferative capacity of LUAD cells. Our cell functional assays further validated the results from bioinformatics analysis. Thus, we speculate that MRPL51 expression is associated with the malignant biological behaviors of lung cancer.

To further simulate the tumor growth environment in vivo, we established a subcutaneous xenograft tumor model of LUAD in nude mice. Starting from the 5th week, a statistically significant difference in tumor growth rates was observed between the two groups. At the 7th week, the mice were euthanized, and the subcutaneous tumors were excised and weighed. H&E staining confirmed that the subcutaneous tumors were adenocarcinoma. Compared to the control group, the MRPL51-knockdown group exhibited negative expression of proliferation-associated proteins within the tumor cell nuclei, indicating a reduction in cellular proliferative capacity.

Given that the mechanistic role of MRPL51 in LUAD remains largely unexplored, we aimed to identify downstream effectors and associated pathways of MRPL51 through NGS and bioinformatics analysis, thereby elucidating the biological significance of MRPL51 in LUAD. Following the construction of a volcano plot for DEGs, we identified a significant positive correlation between NTS and MRPL51 expression. Notably, NTS exhibited the most pronounced downregulation upon MRPL51 knockout, whereas its expression was markedly upregulated following MRPL51 overexpression. NTS, a neuropeptide composed of 13 amino acids, is extensively distributed throughout the central nervous system and peripheral tissues, contributing significantly to a wide range of physiological and pathophysiological pathways. In peripheral tissues, NTS achieves its multifaceted biological effects mainly through activation of three distinct NTS receptors (NTSR1, NTSR2, and NTSR3) (15,21). In NSCLC, the NTS receptor NTSR1 is highly expressed and exhibits the highest affinity for NTS. In a study involving IHC staining of tumor samples from 139 patients, positive expression of NTS was detected in 60.4% of the samples, while positive expression of NTSR1 was observed in 59.7%. Additionally, concurrent positive expression of both NTS and NTSR1 was identified in 38.8% of the cases (22). Furthermore, evaluating the expression levels of NTS/NTR1 in malignant tumors can provide predictive insights into their responsiveness to EGFR tyrosine kinase inhibitor (EGFR-TKI) therapy. This suggests that malignant tumors with high NTS/NTSR1 expression may exhibit a more favorable therapeutic response to EGFR-TKI treatment (17).

To further elucidate the biological role of MRPL51 LUAD, we performed comprehensive functional annotations, including GO analysis, KEGG pathway enrichment, and GSEA, on the co-expressed genes of MRPL51. GO annotation analysis revealed that genes co-expressed with MRPL19 were significantly enriched in the biological process of ion-gated channel regulation. KEGG pathway analysis revealed that MRPL51 knockout significantly perturbed multiple neural-related pathways, inflammation-associated pathways, and NF-κB and PI3K-AKT signaling pathways, while the overexpression of MRPL51 induced alterations in the HIF-1α pathway. GSEA revealed significant alterations in signaling pathways between MRPL51-knockdown and control groups. Notably, the cell cycle pathway exhibited substantial inhibition in MRPL51-depleted cells, as evidenced by a negative NES of −1.63. The PI3K-AKT signaling pathway demonstrated positive enrichment with an NES of 1.30. In lung cancer cells, the regulatory mechanisms governing the cell cycle are frequently dysregulated, leading to uncontrolled cellular proliferation (23). Based on integrated analysis of cell cycle assays and Western blot results, we hypothesize that MRPL51 knockdown induces cell cycle arrest at the S phase in lung cancer cells. These findings suggest that the observed overexpression of MRPL51 in lung cancer cells may facilitate S-to-G2 phase transition by promoting DNA replication, consequently enhancing cellular proliferative capacity.

Previous studies have reported that when NTS binds to its receptor NTSR1 through autocrine and paracrine mechanisms, it can enhance the tyrosine phosphorylation levels of the EGFR. Phosphorylated EGFR subsequently activates downstream signaling pathways, including the PI3K/AKT pathway. This cascade of events ultimately promotes the growth of primary tumors and the occurrence of extensive lymph node metastasis (15,16). Under physiological conditions, the PI3K/AKT signaling pathway is activated by growth factors and cytokines, mediating diverse metabolic responses, particularly glycolysis, to support cellular growth and survival. However, this pathway represents one of the most frequently dysregulated molecular cascades in human malignancies, contributing significantly to tumorigenesis and cancer progression (24). Therefore, we speculate that MRPL51 may regulate the downstream EGFR/PI3K/AKT signaling axis through NTS.

EGFR, a member of the transmembrane receptor tyrosine kinase family, transduces growth factor signals from the extracellular environment to the intracellular compartment (25). Upon ligand binding, EGFR can form homodimers or heterodimers with HER2. Following dimerization, the tyrosine kinase activity of EGFR is activated, leading to autophosphorylation of tyrosine residues either intramolecularly or between adjacent molecules. This facilitates the interaction with adaptor molecules, thereby coupling the receptor to downstream signaling pathways. The autophosphorylation sites of EGFR serve as binding sites for multiple downstream signaling molecules, one of which is PI3K.

The activation of the PI3K/AKT signaling pathway in cancer cells drives extensive metabolic reprogramming through the upregulation of glucose transporters and glycolytic enzymes. This regulatory mechanism profoundly influences multiple metabolic processes, including the pentose phosphate pathway, mitochondrial oxidative phosphorylation, de novo lipid synthesis, and redox homeostasis, thereby simultaneously fulfilling the catabolic and anabolic requirements essential for tumor cell proliferation and survival (26). Concurrently, the activation of this pathway confers multiple oncogenic properties to tumor cells, including sustained proliferative signaling, evasion of apoptosis, enhanced tissue invasion and metastatic potential, and promotion of angiogenesis. These hallmarks of cancer are intricately associated with the initiation and progression of NSCLC, highlighting the critical role of this signaling cascade in oncogenesis (27). Western blot analysis revealed a significant downregulation of phosphorylated EGFR, PI3K, and AKT upon MRPL51 knockout. Notably, exogenous administration of NTS reactivated the EGFR-PI3K-AKT signaling axis. These findings suggest that MRPL51 potentially modulates the malignant biological behaviors of LUAD through the NTS/EGFR/PI3K/AKT signaling pathway.

This study involved several limitations that should be addressed. First, the restricted sample size and limited clinical case availability might have introduced potential bias in the statistical outcomes. Second, the biological functional experiments employed a narrow selection of cell lines, which could compromise the generalizability of the findings. Third, the mechanistic pathways identified through transcriptome sequencing require additional validation, and the precise regulatory mechanism by which MRPL51 modulates NTS expression remains to be fully characterized.

Conclusions

This study demonstrates that MRPL51 expression is significantly upregulated in LUAD tissues and is closely associated with unfavorable clinicopathological features, advanced tumor stage, and poor postoperative survival prognosis. In the A549 LUAD cell line, CRISPR-mediated knockout of MRPL51 markedly suppressed malignant behaviors, including proliferation and invasion, whereas MRPL51 overexpression partially enhanced the proliferative capacity. Mechanistically, MRPL51 promotes the malignant biological behaviors of LUAD by upregulating NTS expression and subsequently activating the downstream EGFR/PI3K/AKT signaling axis. These findings indicate MRPL51 to be a critical regulator of LUAD progression and suggest its potential as a therapeutic target for clinical intervention.

Acknowledgments

None.

Footnote

Reporting Checklist: The authors have completed the ARRIVE and MDAR reporting checklists. Available at https://jtd.amegroups.com/article/view/10.21037/jtd-2025-1774/rc

Data Sharing Statement: Available at https://jtd.amegroups.com/article/view/10.21037/jtd-2025-1774/dss

Peer Review File: Available at https://jtd.amegroups.com/article/view/10.21037/jtd-2025-1774/prf

Funding: This work was supported by the “Leading Goose” Research and Development Program of Zhejiang (No. 2025C02057), and the National Natural Science Foundation of China (No. 82372773).

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://jtd.amegroups.com/article/view/10.21037/jtd-2025-1774/coif). All authors reports that this work was supported by the “Leading Goose” Research and Development Program of Zhejiang (No. 2025C02057), and the National Natural Science Foundation of China (No. 82372773). 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. This study was conducted in accordance with the Declaration of Helsinki and its subsequent amendments. This study was approved by the Ethics Review Committee of Sir Run Run Shaw Hospital, Zhejiang University School of Medicine (approval No. 2024; research ethics review No. 1179). Individual consent for this retrospective analysis was waived. Animal experiments were performed under a project license (No. ZJU20240096) approved by the Ethics Committee of Zhejiang University, in compliance with the institutional 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/.

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