miR-4734 Targets BMP7 to Accelerate Non-Small Cell Lung Cancer Progression and Serves as a Poor Prognostic Biomarker

miR-4734 Targets BMP7 to Accelerate Non-Small Cell Lung Cancer Progression and Serves as a Poor Prognostic Biomarker | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article miR-4734 Targets BMP7 to Accelerate Non-Small Cell Lung Cancer Progression and Serves as a Poor Prognostic Biomarker Yiming Hu, Kai Yu, Yingqiao Yang This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8644164/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background NSCLC is a leading cause of cancer mortality worldwide, highlighting the need for non-invasive biomarkers. Circulating miRNAs are promising candidates due to their stability in serum. Methods This study measured serum levels of miR-4734 using qRT-PCR in NSCLC patients and healthy controls. The diagnostic ability was assessed through ROC analysis, while the prognostic significance was evaluated with Kaplan–Meier survival curves and Cox regression. Functional assays, including CCK-8, ELISA, Transwell, and tube formation tests, were conducted to examine the role of miR-4734 in cell proliferation and angiogenesis in NSCLC and endothelial cells. BMP7 was identified as a direct target of miR-4734 via bioinformatic predictions, dual-luciferase reporter assays, and rescue experiments. Results Serum miR-4734 was significantly elevated in patients with NSCLC compared to healthy controls, demonstrating moderate diagnostic accuracy with an AUC of 0.787 (95% CI: 0.721–0.853). High levels of miR-4734 were linked to advanced TNM stage and shorter overall survival. Moreover, miR-4734 served as an independent adverse prognostic factor, with an HR of 2.103 (95% CI: 1.091–4.051). Functionally, miR-4734 encouraged cell proliferation in NSCLC, increased the secretion of pro-angiogenic factors, and boosted endothelial cell migration and tube formation. Mechanistically, miR-4734 directly targeted and inhibited BMP7, and the oncogenic effects of miR-4734 were partly reversed when BMP7 was overexpressed. Conclusion Serum miR-4734 serves as a potential non-invasive biomarker for diagnosing and predicting NSCLC outcomes. It promotes tumor growth by increasing proliferation and angiogenesis through directly inhibiting BMP7. Angiogenesis BMP7 miR-4734 NSCLC Prognosis Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Lung cancer remains the leading cause of cancer-related mortality worldwide, with non-small cell lung cancer (NSCLC) affecting over 80% of lung cancer individuals [1, 2].Despite considerable advances in therapeutic strategies, the overall prognosis of NSCLC individuals remains dismal, primarily owing to the lack of effective approaches for early detection and accurate prognostic evaluation[3, 4]. Thus, the identification of novel biomarkers and the elucidation of their underlying molecular mechanisms are crucial to improving clinical management and clinical outcomes. MicroRNAs (miRNAs) have emerged as pivotal regulators of tumorigenesis and promising circulating biomarkers due to their stability and accessibility[5]. Beyond single miRNA entities, current research increasingly focuses on miRNA signatures derived from extracellular vesicles and the construction of multi-miRNA panels to enhance diagnostic specificity and sensitivity. While the roles of miRNAs like miR-21 and miR-145 in NSCLC are well-characterized [6-8], the clinical significance and functional mechanisms of miR-4734—despite its documented oncogenic role in other cancers—are entirely unknown in the NSCLC context, representing a significant knowledge gap [9, 10]. Bone morphogenetic protein 7 (BMP7), a member of the TGF-β superfamily, exhibits context-dependent roles in cancer, influencing processes such as epithelial-mesenchymal transition (EMT) in lung cancer [11, 12]. However, its prognostic value and regulatory network in NSCLC are not fully understood. Notably, whether any miRNA can modulate NSCLC progression by directly targeting BMP7 remains an unexplored avenue of research. To address these gaps, the present study was designed to investigate the role of miR-4734 in NSCLC. Serum miR-4734 was first identified as a differentially expressed candidate via bioinformatics screening, with its upregulation, diagnostic potential, and association with poor prognosis further validated in clinical cohorts. Furthermore, miR-4734 was demonstrated to promote NSCLC cell proliferation and angiogenesis through the secretion of pro-angiogenic factors. Mechanistically, BMP7 was identified as a direct and functional target of miR-4734, and rescue experiments confirmed that the miR-4734/BMP7 axis critically regulates these oncogenic phenotypes. Collectively, these findings unveil miR-4734 as a novel prognostic biomarker and a promoter of tumor angiogenesis via BMP7 inhibition, thereby providing new insights into NSCLC pathogenesis and potential therapeutic targeting. Materials and methods Clinical Sample Collection This study retrospectively analyzed serum samples from 100 patients with pathologically confirmed NSCLC and 100 healthy controls, with strict adherence to ethical guidelines and the protection of participant welfare. All samples were collected at the First Affiliated Hospital of Hubei University of Science and Technology between October 2022 and October 2024. De-identified samples were utilized following broad informed consent obtained from all participants. This study protocol was reviewed and approved by the Ethics Committee of Xianning Central Hospital, and all procedures conformed to the principles outlined in the Declaration of Helsinki. To ensure cohort homogeneity, NSCLC patients with concurrent malignant tumors, severe cardiovascular/cerebrovascular diseases, or autoimmune disorders were excluded from the study. Bioinformatics Analysis Serum differentially expressed miRNAs in NSCLC patients vs. healthy controls were screened via ExomiRHub. miR-4734 target genes were predicted using TargetScan, miRWalk, and miRDB, with the intersection set as candidate targets. GO enrichment analysis (BP, MF, CC) of candidate targets was conducted on Metascape. Lung cancer-specific highly expressed targets were further filtered via DepMap, and their correlation with NSCLC survival was analyzed using GEPIA2. miR-4734 binding sites in the 3'UTR of BMP7 were predicted via TargetScan. Cell Culture and Transfection Human NSCLC cell lines (A549/H1299/H23: adenocarcinoma; SK-MES-1/H226: squamous cell carcinoma; H460: large cell carcinoma) and BEAS-2B (normal bronchial epithelium) were from Shanghai Cell Bank (CAS); HUVECs were from ATCC (USA). Cells were cultured in 10% FBS/100 U/mL penicillin/100 μg/mL streptomycin-supplemented medium: RPMI-1640 for NSCLC/BEAS-2B, DMEM for HUVECs; 37°C/5% CO₂ humidified incubator. Log-phase cells were seeded in 6/96-well plates 1d pre-transfection; 60%–70% confluent cells were transfected with miR-4734 mimic/inhibitor/NC, BMP7-OE/empty vector (Lipofectamine 3000, Thermo Fisher), and experiments were performed 24h post-transfection. Quantitative Real-Time PCR (qRT-PCR) Total RNA was extracted via TRIzol reagent and reverse-transcribed into cDNA per kit instructions. qRT-PCR used U6 (miR-4734) and GAPDH (BMP7 mRNA) as internal controls. Relative expression: 2⁻ΔΔCt method. Primers: Sangon Biotech (Shanghai). CCK-8 Assay for Cell Viability and Proliferation Detection Transfected cells were seeded in 96-well plates; 48 h later, 10 μL CCK-8 was added, incubated for 1 h, and OD₄₅₀ measured for cell viability. Conditioned medium (CM) from transfected NSCLC cells was collected. HUVECs seeded in 96-well plates had the medium replaced with CM; CCK-8 assays measured OD₄₅₀ to assess HUVEC proliferation. ELISA for Cytokine Secretion Detection Transfected NSCLC cells were seeded in 6-well plates and cultured for 48 h; supernatants were collected and centrifuged to remove impurities. VEGF, bFGF, and Ang-2 secretion levels were quantified via ELISA kits per manufacturer instructions. OD values were measured at the optimal wavelength using a microplate reader, and concentrations were calculated from standard curves. Transwell Migration Assay Matrigel was diluted 1:8 with serum-free medium, coated evenly on Transwell upper chambers, and solidified at 37°C for 30 min. CM was collected from transfected NSCLC cells. HUVECs were seeded into upper chambers, with 600 μL CM added to lower chambers; incubation proceeded at 37°C/5% CO₂ for 24 h. Non-migrated cells were wiped off; migrated cells were fixed with 4% paraformaldehyde, stained with 0.1% crystal violet, rinsed, and air-dried. Migrated cells were counted in 5 random microscopic fields, and the mean was calculated. Matrigel Tube Formation Assay Matrigel was diluted 1:1 with serum-free medium; 50 μL of the mixture was coated per 96-well plate and solidified at 37°C for 30 min. CM was collected from transfected NSCLC cells. HUVECs were seeded into Matrigel-precoated wells, with 100 μL CM added per well. Plates were incubated at 37°C/5% CO₂ for 6 h. Tube length and branch number were quantified in 5 random microscopic fields using ImageJ. Dual-Luciferase Reporter Gene Assay Wild-type BMP7 3'UTR (WT-BMP7 3'UTR) and mutant-type BMP7 3'UTR (MUT-BMP7 3'UTR) reporter gene vectors were constructed; site-directed mutagenesis of the miR-4734 binding sites was performed in the mutant vectors. A549, SK-MES-1, and H460 cells were seeded into 24-well plates; when cell confluence reached 60%, the cells were co-transfected with WT-BMP7 3'UTR or MUT-BMP7 3'UTR vector plus miR-4734 mimic or NC. At 48 h post-transfection, firefly luciferase activity and renilla luciferase activity were measured strictly following the manufacturer’s instructions of the dual-luciferase reporter assay kit. Renilla luciferase activity was used as an internal reference to calculate the relative luciferase activity. Statistical Analysis Data analysis was performed using SPSS 22.0. An independent samples t-test was used for two-group comparisons, and a one-way ANOVA with an LSD post hoc test for multiple-group comparisons. Spearman correlation analysis assessed the association between miR-4734 expression and clinicopathological parameters. Kaplan-Meier survival curves and the log-rank test were used for survival analysis. The Cox proportional hazards regression model (variables: age, gender, smoking history, chronic bronchial disease history, clinical stage, pathological subtype, advanced TNM stage, miR-4734 expression) identified independent prognostic factors. Two-tailed P < 0.05 was considered statistically significant. Results miR-4734 Is Highly Expressed in NSCLC Serum and Exhibits Favorable Diagnostic Value as Well as Correlation with Poor Prognosis Serum miRNA expression profiles from NSCLC and healthy controls were screened via the ExomiRHub database. Among 2525 differentially expressed miRNAs, 12 upregulated and 22 downregulated candidate miRNAs were finally identified based on the screening criteria. Notably, miR-4734 was significantly upregulated (Log₂ FC = 1.16, Adj. p-value = 1.35, Ave Expr = 12.55), and no previous studies have established a direct association between miR-4734 and NSCLC. Thus, it was selected as the core target for subsequent investigations (Figure 1A). The baseline characteristics of the 100 NSCLC individuals and 100 healthy controls enrolled in this study are summarized in Table 1. The two groups were well-matched in terms of age and gender. However, significant differences were observed in smoking history and the prevalence of chronic bronchial disease, with both being more common in the NSCLC group. As anticipated, the serum level of miR‑4734 was significantly higher in NSCLC individuals compared to healthy controls (Figure 1B). Further evaluation of its diagnostic efficacy via receiver operating characteristic (ROC) curve analysis revealed that the area under the curve (AUC) of miR-4734 for distinguishing NSCLC from healthy controls was 0.787 (95% CI: 0.721–0.853), indicating a moderate diagnostic value for NSCLC (Figure 1C). Correlation analysis between miR-4734 expression and clinicopathological characteristics demonstrated no significant association with tumor pathological subtypes, including squamous cell carcinoma (SCC), adenocarcinoma (ADC), large cell carcinoma (LCC), and adenosquamous carcinoma (ASC) (Figure 1D). However, miR-4734 expression was significantly positively correlated with TNM stage and clinical stage, suggesting that miR-4734 expression gradually increases with disease progression (Figure 1E–1F). Survival analysis showed that individuals in the miR-4734 high-expression group had significantly shorter overall survival than those in the low-expression group (Figure 1G). Multivariate Cox proportional hazards regression was performed to identify independent prognostic factors. The model included age, gender, smoking history, history of chronic bronchial disease, clinical stage, pathological subtype, and advanced TNM stage. After adjusting for these covariates, high serum miR-4734 expression remained an independent predictor of poor overall survival. In conclusion, miR-4734 is highly expressed in the serum of individuals with NSCLC, and it not only exhibits potential diagnostic value but also is closely associated with poor prognosis in this population. Table 1 Baseline characteristics of NSCLC individuals and healthy controls Characteristic NSCLC Patients n=100 Healthy Controls n=100 P value Demographics Age (years), mean ± SD 60.5 ± 10.6 60.6 ± 10.5 0.940 Gender, n (%) 0.305 Male 60 (60.0) 67 (67.0) Female 40 (40.0) 33 (33.0) Smoking history, n (%) <0.001 Never 31 (31.0) 68 (68.0) Ever/Current 69 (69.0) 32 (32.0) History of chronic bronchial disease, n (%) <0.001 Yes 59 (59.0) 20 (20.0) No 41 (41.0) 80 (80.0) Clinical Features Histological subtype, n (%) SCC 28 (28.0) - ADC 56 (56.0) - LCC 8 (8.0) - ASC 8 (8.0) - TNM Stage (AJCC 8th), n (%) I-II 36 (36.0) - III-IV 64 (64.0) - Serum Biomarker Level miR-4734 (relative expression), median (IQR) 2.71 (1.70-3.31) 1.7 (1.26-2.14) <0.001 Footnotes: Abbreviations: IQR, interquartile range; AJCC, American Joint Committee on Cancer; SCC, squamous cell carcinoma; ADC, adenocarcinoma; LCC, large cell carcinoma; ASC, adenosquamous carcinoma. Data are presented as mean ± standard deviation (SD), number (percentage), or median (interquartile range, IQR). Continuous variables were compared using the independent samples t-test (Age) or the Mann-Whitney U test (miR-4734). Categorical variables were compared using the Chi-square test. Figure 1. High serum miR-4734 expression correlates with diagnostic efficacy and poor prognosis in NSCLC. (A) Schematic diagram of the screening workflow for NSCLC-associated miRNAs. (B) Serum miR-4734 expression levels in NSCLC individuals and healthy individuals detected by qRT-PCR. (C) Diagnostic efficacy of miR-4734 for distinguishing NSCLC individuals from healthy individuals analyzed via ROC curve. (D–F) Associations between miR-4734 expression and NSCLC pathological subtypes, TNM stage, and clinical stage analyzed by Pearson correlation analysis. (G) Relationship between miR-4734 expression and individuals’ overall survival analyzed using Kaplan-Meier survival curves. The difference between groups was assessed using the log‑rank test. The HR and 95% CI were derived from a multivariate Cox proportional hazards model adjusted for key clinicopathological variables High Expression of miR-4734 in NSCLC Cells Promotes Cell Proliferation To explore the functional role of miR-4734 in NSCLC cells, in vitro experiments were performed. First, miR-4734 expression levels were detected in multiple NSCLC cell lines and normal bronchial epithelial cells (BEAS-2B). As shown in Figure 2A, miR-4734 expression was significantly upregulated in NSCLC cell lines (A549, H23, H1299, H226, SK-MES-1, and H460) compared with BEAS-2B cells. For subsequent functional analyses, three NSCLC cell lines representing distinct pathological subtypes were selected: A549, SK-MES-1, and H460, which are widely used as representative models for respective NSCLC subtypes in preclinical studies [13, 14]. Transfection efficiency was verified by qRT-PCR. The results demonstrated that transfection with miR-4734 mimic significantly increased miR-4734 expression in all three cell lines, while transfection with miR-4734 inhibitor remarkably downregulated its expression (Figure 2B-2D), confirming the validity of the transfection system. The CCK-8 assay was used to evaluate cell viability and assess the impact of miR-4734 on NSCLC cell proliferation. Overexpression of miR-4734 significantly enhanced the proliferative capacity of NSCLC cells (Figure 2E–2G), whereas inhibition of miR-4734 led to a marked reduction in cell viability. Collectively, these findings indicate that miR-4734 is highly expressed in NSCLC and exerts a pro-proliferative effect by promoting the viability of NSCLC cells. Figure 2. High miR-4734 expression correlates with enhanced proliferation in NSCLC cells. (A) The relative expression level of miR-4734 in normal bronchial epithelial cell line BEAS-2B and NSCLC cell lines (A549, H23, H1299, H226, SK-MES-1, H460) was detected by qRT-PCR. Compared with the BEAS-2B group, * P < 0.05, ** P < 0.01. (B–D) NC mimic, miR-4734 mimic, NC inhibitor or miR-4734 inhibitor were transfected into A549, SK-MES-1 and H460 cells, respectively, and the transfection efficiency was verified by qRT-PCR. Compared with the respective control group, ** P < 0.01, *** P < 0.001. (E–G) The CCK-8 assay was used to detect the cell viability changes of the above cells after corresponding treatments. Compared with the respective control group, * P < 0.05, *** P < 0.001. All experiments were independently repeated at least three times. miR-4734 Enhances Angiogenic Capacity of NSCLC by Upregulating Pro-Angiogenic Factors To elucidate the underlying mechanism of miR-4734, its potential target genes were predicted by integrating TargetScan, miRWalk, and miRDB databases, and 38 high-confidence candidate target genes were screened out (Figure 3A). GO enrichment analysis revealed that these target genes were mainly involved in biological processes including myocardial tissue development, hematopoietic regulation and transmembrane receptor signaling pathway (Figure 3B), suggesting that miR-4734 may modulate pathological processes such as angiogenesis. At both mRNA and protein levels, miR-4734 mimic significantly upregulated the expression and secretion of pro-angiogenic factors, including VEGF, bFGF and Ang-2; in contrast, miR-4734 inhibition markedly reduced the levels of these factors, indicating that miR-4734 positively regulates the expression of pro-angiogenic factors (Figure 3C-3D, Supplementary Figure S1A-S1D). Furthermore, CM assays were performed to investigate how the miR-4734-modulated tumor microenvironment affects HUVEC functions. Cell proliferation assays revealed that, compared with the control group, CM from miR-4734 mimic-transfected cells significantly promoted HUVEC proliferation at 72 h, whereas CM from miR-4734 inhibitor-transfected cells markedly inhibited this process (Figure 3E, Supplementary Figure S1E–S1F). Cell migration assays showed that CM from miR-4734 mimic-transfected cells significantly increased the number of migrated HUVECs, while CM from miR-4734 inhibitor-transfected cells led to a marked reduction in migration (Figure 3F, Supplementary Figure S1G–S1H). Tube formation assays further demonstrated that, relative to the control group, the miR-4734 mimic CM group significantly increased HUVEC tube length and branch number; conversely, the miR-4734 inhibitor CM group caused a marked decrease in both parameters (Figure 3G, Supplementary Figure S1I–S1J). Collectively, these findings indicate that miR-4734 enhances the angiogenic capacity of NSCLC cells by upregulating pro-angiogenic factors such as VEGF, bFGF and Ang-2. Figure 3. miR-4734 enhances NSCLC cell angiogenic capacity by upregulating pro-angiogenic factors. (A) Target genes of miR‑4734 were predicted with TargetScan, miRDB and miRWalk, and overlapping results were shown in a Venn diagram. (B) GO functional enrichment analysis of candidate target genes, displaying the main biological processes they are involved in. (C) The relative mRNA expression levels of VEGF, bFGF and Ang‑2 in A549 cells (transfected with NC mimic, miR‑4734 mimic, NC inhibitor or miR‑4734 inhibitor) were detected by qRT‑PCR. Compared with the control group, *** P < 0.001. (D) The secretion levels of VEGF, bFGF and Ang‑2 in the culture medium of A549 cells from the above treatment groups were measured by ELISA. Compared with the control group, * P < 0.05, ** P < 0.01, *** P < 0.001. (E) The effect of CM from A549 cells in different treatment groups on HUVEC proliferation was detected by CCK‑8 assay. Compared with the control group, *** P < 0.001. (F) The number of migrated HUVECs (treated with CM from different treatment groups) was counted by cell migration assay. Compared with the control group, *** P < 0.001. (G) The effect of CM from different treatment groups on HUVEC tube length and branch number was analyzed by tube formation assay. Compared with the control group, * P < 0.05 for branch number; *** P < 0.001 for tube length. All experiments were independently repeated at least three times. Supplementary Figure S 1 . miR-4734 enhances NSCLC cell angiogenic capacity by upregulating pro-angiogenic factors. (A-B) The relative mRNA expression levels of VEGF, bFGF and Ang-2 in SK-MES-1/H460 cells (transfected with different reagents) were detected by qRT-PCR. Compared with the control group, ** P < 0.01, *** P < 0.001. (C-D) The secretion levels of VEGF, bFGF and Ang-2 in the CM of the above cells were measured by ELISA. Compared with the control group, ** P < 0.01, *** P < 0.001. (E-F) The effect of CM-SK-MES-1/CM-H460 (from different treatment groups) on HUVEC proliferation was detected by CCK-8 assay. Compared with the control group, * P < 0.05, *** P < 0.001. (G-H) The number of migrated HUVECs treated with the above CM was counted by Transwell assay. Compared with the control group, * P < 0.05, *** P < 0.001. (I-J) The effects of the above CM on HUVEC tube length and branch number were analyzed by tube formation assay. Compared with the control group, ** P < 0.01, *** P < 0.001 for tube length; # P < 0.05, ## P < 0.01, ### P < 0.001 for branch number. All experiments were independently repeated at least three times. miR-4734 Targets BMP7 and Represses Its Expression To further characterize the candidates, the 38 predicted target genes were subjected to lung cancer-specific expression screening via the DepMap database, which ultimately identified 7 genes highly expressed in lung cancer. Among these genes, BMP7, GRIN1 and OPRL1 showed statistically significant differential expression (Figure 4A, Supplementary Figure S2A–S2F). Subsequently, the GEPIA2 database was used to analyze correlations between these genes and overall survival (OS) as well as disease-free survival (DFS) in individuals with NSCLC. Results demonstrated that BMP7 was significantly overexpressed in lung cancer cells; while no significant correlation with OS was observed, its high expression was markedly associated with prolonged DFS (Figure 4B, C). In contrast, GRIN1 and OPRL1 had no significant correlations with either OS or DFS (Supplementary Figure S2G–S2J). To explore the potential targeting relationship between miR-4734 and BMP7, TargetScan was employed to predict their binding sites, revealing complementary pairing within the seed region (Figure 4D). This interaction was then validated using a dual-luciferase reporter assay. In A549 and SK-MES-1 cells, miR-4734 mimic significantly suppressed the reporter activity of wild-type BMP7 3'UTR, whereas no obvious effect was noted on the mutant vector, confirming that BMP7 is a direct target gene of miR-4734 (Figure 4E). However, no significant inhibition was detected in H460 cells, suggesting this regulatory relationship may exhibit cell-type specificity. Clinical sample analysis revealed a significant negative correlation between serum miR-4734 expression levels and BMP7 protein concentrations in individuals with NSCLC (Figure 4F). Cellular experiments further confirmed that miR-4734 mimic downregulated BMP7 mRNA levels, while miR-4734 inhibition upregulated its expression (Figure 4G-4H). Collectively, these findings indicate that miR-4734 negatively regulates BMP7 at the post-transcriptional level. Figure 4. miR-4734 targets BMP7 and represses its expression. (A) BMP7 expression levels in lung cancer cell lines, analyzed using the DepMap database. (B–C) Correlations between BMP7 expression and individual OS as well as DFS, analyzed via the GEPIA2 database. (D) Schematic diagram of the predicted complementary pairing between miR-4734 and the seed region of BMP7 3′UTR, generated by TargetScan. (E) Validation of the targeting effect of miR-4734 on BMP7 via dual-luciferase reporter assay in A549, SK-MES-1 and H460 cells. (F) BMP7 protein concentration in the serum of NSCLC individuals was measured by ELISA, and its correlation with serum miR-4734 expression level was analyzed. (G–H) Changes in BMP7 mRNA expression in A549 and SK-MES-1 cells were detected by qRT‑PCR after transfection with NC mimic, miR‑4734 mimic, NC inhibitor, or miR‑4734 inhibitor. Compared with the control group, * P < 0.05, ** P < 0.01, *** P < 0.001; ns, not significant. All experiments were independently repeated at least three times. Supplementary Figure S 2. Lung cancer-specific expression and survival correlation analysis of candidate target genes. (A-F) Expression profiles of GRIN1, BATF, CRISPLD2, OPRL1, TENM4 and TNN in lung cancer cell lines, analyzed using the DepMap database. (G-J) Correlations of GRIN1 and OPRL1 expression with OS and DFS in NSCLC individuals, evaluated via the GEPIA2 database. miR-4734 Promotes NSCLC Cell Proliferation and Angiogenesis by Inhibiting BMP7 To investigate the role of miR-4734 targeting BMP7 in lung cancer cells, rescue experiments were performed. The results showed that miR-4734 mimic significantly promoted the secretion of VEGF, bFGF and Ang-2 in the supernatants of A549 and SK-MES-1 cells, whereas co-expression of miR-4734 mimic and BMP7-OE partially reversed this effect, with their secretion levels remaining higher than those in the BMP7-OE group (Figure 5A–5B). Cell proliferation assays demonstrated that CM from miR-4734 mimic-transfected cells significantly enhanced HUVEC proliferation, and this effect was also partially abrogated by BMP7-OE (Figure 5C–5D). Further Transwell migration assays revealed that CM from miR-4734 mimic-transfected cells markedly increased HUVEC migratory capacity, and this promotive effect was partially reversed by BMP7-OE as well (Figure 5E–5F). Additionally, HUVEC tube formation assays confirmed that CM from miR-4734 mimic-transfected cells significantly elevated angiogenesis-related indices (migrated cell number, branch number and tube length), and this effect was partially inhibited by BMP7-OE (Figure 5G–5J). Collectively, these results indicated that BMP7 could partially antagonize the positive regulatory effects of miR-4734 on pro-angiogenic factor secretion and vascular endothelial cell functions, but failed to completely reverse such effects. In conclusion, miR-4734 enhances the proliferation of NSCLC cells and their mediated angiogenic capacity by targeting and inhibiting BMP7, thereby facilitating tumor progression. Figure 5. miR-4734 promotes NSCLC cell proliferation and angiogenesis by inhibiting BMP7. (A-B) The secretion levels of VEGF, bFGF and Ang-2 in A549 and SK-MES-1 cells (from different treatment groups) were measured. Compared with the control group, * P < 0.05, ** P < 0.01, *** P < 0.001. (C-D) The effect of CM from A549 and SK-MES-1 cells on HUVEC proliferation was detected by CCK-8 assay. Compared with the control group, * P < 0.05, ** P < 0.01, *** P < 0.001. (E-F) The number of migrated HUVECs (treated with CM from A549 and SK-MES-1 cells) was counted by Transwell migration assay. Compared with the control group, * P < 0.05, ** P < 0.01, *** P < 0.001. (G-J) The indices of HUVEC tube formation after treatment with CM from A549 and SK-MES-1 cells were quantified. Compared with the control group, * P < 0.05, ** P < 0.01, *** P < 0.001. All experiments were independently repeated at least three times. Discussion Improving the prognosis of NSCLC relies on early diagnosis and precision therapy, both of which depend on the discovery of specific molecular biomarkers [15]. Endowed with favorable serum stability and tumor-specific expression, microRNAs serve as ideal diagnostic biomarkers [16, 17]. This study employs an integrated approach encompassing bioinformatic screening, clinical validation, and in vitro experiments to demonstrate significant upregulation of serum miR‑4734 in individuals with NSCLC. Their favorable diagnostic efficacy underscores the potential of serum miR‑4734 as a diagnostic biomarker for NSCLC. The NSCLC cohort had a higher prevalence of established risk factors, including smoking and chronic bronchial disease. In the multivariate Cox model, however, neither factor retained independent prognostic significance, suggesting that their influence on survival may be mediated through other variables in the model, such as tumor stage or miR‑4734 expression. Critically, even after rigorous adjustment for these and other clinicopathological confounders—most notably advanced TNM stage—high serum miR‑4734 expression remained a strong and independent predictor of poor outcome. This finding indicates that miR‑4734 is not merely a surrogate for smoking-related inflammation but is directly implicated in NSCLC aggressiveness, providing prognostic value additive to standard parameters. This pro-oncogenic role aligns with reported functions of miR‑4734 in hepatocellular carcinoma and breast cancer, suggesting a conserved tumor-promoting mechanism across malignancies [9, 18]. At the cellular level, miR-4734 enhances NSCLC cell viability and upregulates VEGF, bFGF and Ang-2 secretion, thereby promoting HUVEC proliferation, migration and tube formation. This observation is consistent with previous reports that miRNAs such as miR-126 modulate angiogenic factor secretion to drive tumor angiogenesis [19, 20]. However, rescue experiments revealed a complex and context-dependent regulation of miR-4734. While miR-4734 directly targets BMP7 to suppress its expression in A549 and SK-MES-1 cells, this targeting relationship is absent in H460 cells, despite the consistent anti-proliferative effect of miR-4734 across all tested cell lines. This clear dissociation between phenotype and mechanism in H460 cells definitively reveals that miR-4734 can operate through BMP7-independent pathways. Such cell-type specificity reflects the intrinsic molecular heterogeneity among NSCLC subtypes. Distinct genetic backgrounds, such as the unique mutation profiles (e.g., TP53, KRAS) in H460 compared to other cells, could alter miRNA-target accessibility through epigenetic modifications or differential expression of RNA-binding proteins [21, 22]. This mechanistic divergence underscores that the same oncogenic miRNA may engage distinct downstream target networks to fulfill its function in different cellular contexts, a crucial consideration for developing subtype-specific therapeutic strategies [23]. Intriguingly, even in cells where the miR-4734/BMP7 axis is functional, overexpression of BMP7 only partially reverses the pro-angiogenic and pro-proliferative effects induced by miR-4734. This "partial rescue" phenomenon should not be viewed as a limitation but rather as a crucial clue pointing to the complexity of miRNA-mediated regulation. It aligns with the well-established paradigm that a single miRNA often exerts its biological effects by regulating multiple target genes in parallel [24, 25].Therefore, miR-4734 likely functions as a core regulatory node within a broader cooperative network. Beyond BMP7, it may simultaneously target other members of the TGF-β superfamily (e.g., BMP2, BMP4) or key signaling components, or regulate additional pathways crosstalking with the BMP signaling pathway (e.g., PI3K/AKT, MAPK/ERK); these co-regulated pro-tumor pathways synergistically drive tumor progression [26, 27]. This study has several limitations that should be acknowledged. First, the translational relevance of the findings is constrained by the study design. The conclusions are primarily based on serum samples and in vitro models, lacking validation in matched tumor tissues and in vivo systems. While serum miR-4734 shows promise as a biomarker, its direct role within the tumor microenvironment and its functional impact on tumor growth and metastasis in vivo remain to be established [28].Second, the mechanistic understanding of miR-4734 remains partial and context-dependent. BMP7 was validated as a direct target in A549 and SK-MES-1 cells; however, this specific interaction was not observed in H460 cells, where miR-4734 still exerted a clear pro-proliferative effect. This indicates the existence of robust, BMP7-independent pathways. Furthermore, the partial phenotypic rescue upon BMP7 overexpression even in responsive cells suggests that miR-4734 functions as a node within a broader regulatory network. Therefore, a comprehensive mapping of its direct targets—for instance, through techniques like AGO2-CLIP-seq—is essential to fully elucidate its oncogenic network and to assess its potential as a subtype-specific therapeutic target [29, 30]. This study establishes serum miR-4734 as a novel diagnostic and prognostic biomarker for NSCLC, while elucidating its oncogenic mechanism via targeting BMP7. Clinically, elevated serum miR‑4734 not only aids in distinguishing NSCLC individuals from healthy individuals but also independently predicts advanced disease stage and unfavorable survival outcomes. Mechanistically, the results demonstrate that miR-4734 promotes tumor proliferation and angiogenesis by directly suppressing BMP7, thereby driving NSCLC progression. Abbreviations IQR interquartile range AJCC American Joint Committee on Cancer SCC squamous cell carcinoma ADC adenocarcinoma LCC large cell carcinoma ASC adenosquamous carcinoma. Declarations Acknowledgments We sincerely thank all participants for their involvement in this study. We are grateful to the Department of Pulmonary Critical Care Medicine of Xianning Central Hospital (the First Affiliated Hospital of Hubei University of Science and Technology) for its support, as well as the National Experimental General Practice Education Demonstration Center of Xianning Medical College, Hubei University of Science and Technology for providing experimental support and technical guidance during the study. Funding This research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors. Author contribution s All authors contributed to the study. Conception and design were performed by Yiming Hu. Administrative support was provided by Yingqiao Yang. Provision of study materials or patients was conducted by Yiming Hu and Kai Yu. Collection and assembly of data were performed by Kai Yu. Data analysis and interpretation were carried out by Yiming Hu and Yingqiao Yang. The manuscript was written by all authors, and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript. Yiming Hu and Kai Yu contributed equally to this work. Competing interests Yiming Hu declares that he has no conflict of interest. Kai Yu declares that he has no conflict of interest. Yingqiao Yang declares that she has no conflict of interest. Ethical approval The study protocol was reviewed and approved by the Ethics Committee of Xianning Central Hospital (Approval No.: HKSY-YL-YJ-2022-036). All procedures adhered to the ethical principles of the Declaration of Helsinki. Consent to participate: The use of de-identified samples for secondary research was covered in the broad written informed consent obtained from all participants, which permitted future use of their anonymized data/specimens. All participants provided informed consent after full understanding of the research purpose. Consent to publish: Consent for publication was obtained for every individual person’s data included in the study; this permission is also explicitly covered in the broad written informed consent obtained from all participants. Clinical trial registration This study is a secondary analysis of de-identified samples and does not involve interventional clinical trial procedures. Therefore, a clinical trial number is not applicable. Data availability The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request. References Garinet S, Wang P, Mansuet-Lupo A, et al. Updated prognostic factors in localized NSCLC. Cancers (Basel). 2022;14. 10.1097/MD.0000000000032899 . Suster DI, Mino-Kenudson M. Molecular pathology of primary non-small cell lung cancer. Arch Med Res. 2020;51:784–98. 10.1016/j.arcmed.2020.08.004 . Alduais Y, Zhang H, Fan F, et al. Non-small cell lung cancer (NSCLC): a review of risk factors, diagnosis, and treatment. Med (Baltim). 2023;102:e32899. 10.1097/MD.0000000000032899 . Penault-Llorca F, Socinski MA. Emerging molecular testing paradigms in non-small cell lung cancer management—current perspectives and recommendations. Oncologist. 2025;30. 10.1093/oncolo/oyae357 . Babaei K, Shams S, Keymoradzadeh A, et al. An insight of microRNAs performance in carcinogenesis and tumorigenesis; an overview of cancer therapy. Life Sci. 2020;240:117077. 10.1016/j.lfs.2019.117077 . Soliman SE, Abdelaleem AH, Alhanafy AM, et al. Circulating miR-21-5p and miR-126-3p: diagnostic, prognostic value, and multivariate analysis in non-small-cell lung cancer. Mol Biol Rep. 2021;48:2543–52. 10.1007/s11033-021-06302-3 . Zhang Z, Huang Y, Li J, et al. Antitumor activity of anti-miR-21 delivered through lipid nanoparticles. Adv Healthc Mater. 2023;12:e2202412. 10.1002/adhm.202202412 . Tahmasebi S, Amani D, Adcock IM, et al. Harnessing miR-145 in NSCLC: mechanistic roles, diagnostic-prognostic utility, and therapeutic potential. Cancer Cell Int. 2025. 10.1186/s12935-025-04091-4 . Du F, Yuan P, Zhao Z, et al. A miRNA-based signature predicts development of disease recurrence in HER2 positive breast cancer after adjuvant trastuzumab-based treatment. Sci Rep. 2016;6:33825. 10.1038/srep33825 . Michael D, Feldmesser E, Gonen C, et al. miR-4734 conditionally suppresses ER stress-associated proinflammatory responses. FEBS Lett. 2023;597:1233–45. 10.1002/1873-3468.14548 . Shen W, Pang H, Xin B, et al. [Corrigendum] Biological effects of BMP7 on small-cell lung cancer cells and its bone metastasis. Int J Oncol. 2025;66. 10.3892/ijo.2018.4469 . Prieto TG, Baldavira CM, Machado-Rugolo J, et al. Pulmonary neuroendocrine neoplasms overexpressing epithelial-mesenchymal transition mechanical barriers genes lack immune-suppressive response and present an increased risk of metastasis. 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Oncol Rep. 2023;49. 10.3892/or.2022.8445 . Chen QY, Jiao DM, Zhu Y, et al. Identification of carcinogenic potential-associated molecular mechanisms in CD133 + A549 cells based on microRNA profiles. Tumour Biol. 2016;37:521–30. 10.1007/s13277-015-3675-9 . Nammian P, Razban V, Tabei S, et al. MicroRNA-126: dual role in angiogenesis dependent diseases. Curr Pharm Des. 2020;26:4883–93. 10.2174/1381612826666200504120737 . Liu L, Yuan L, Huang D, et al. miR-126 regulates the progression of epithelial ovarian cancer in vitro and in vivo by targeting VEGF-A. Int J Oncol. 2020;57:825–34. 10.3892/ijo.2020.5082 . Li Y, Xia L, Tan K, et al. N6-methyladenosine co-transcriptionally directs the demethylation of histone H3K9me2. Nat Genet. 2020;52:870–7. 10.1038/s41588-020-0677-3 . Dagogo-Jack I, Shaw AT. Tumour heterogeneity and resistance to cancer therapies. Nat Rev Clin Oncol. 2018;15:81–94. 10.1038/nrclinonc.2017.166 . Mitschka S, Mayr C. Context-specific regulation and function of mRNA alternative polyadenylation. Nat Rev Mol Cell Biol. 2022;23:779–96. 10.1038/s41580-022-00507-5 . Wen XQ, Qian XL, Sun HK, et al. MicroRNAs: multifaceted regulators of colorectal cancer metastasis and clinical applications. Onco Targets Ther. 2020;13:10851–66. 10.2147/OTT.S265580 . Xu J, Zhang L, Zheng J. Inhibition of bone morphogenetic protein 2 suppresses the stemness maintenance of cancer stem cells in hepatocellular carcinoma via the MAPK/ERK pathway. Cancer Manag Res. 2021;13:773–85. 10.2147/CMAR.S281969 . Guo J, Guo M, Zheng J. Inhibition of bone morphogenetic protein 2 suppresses the stemness maintenance of cancer stem cells in hepatocellular carcinoma via the MAPK/ERK pathway. Cancer Manag Res. 2021;13:773–85. 10.2147/CMAR.S281969 . Hong T, Rui-min C, Xiao-yan L, et al. Differences of CD4 + T lymphocyte miRNA gene expression in acute coronary artery syndrome (ACS) patients and the effects of rosuvastatin on its expressions. Jiefangjun Yixue Zazhi. 2014;39:99. Karkampouna S, La Manna F, Benjak A, et al. Patient-derived xenografts and organoids model therapy response in prostate cancer. Nat Commun. 2021;12:1117. 10.1038/s41467-021-21300-6 . Porter DF, Garg RM, Meyers RM, et al. Analyzing RNA-protein interactions by cross-link rates and CLIP-seq libraries. Curr Protoc Mol Biol. 2023;3:e659. 10.1002/cpz1.659 . Rykova EY, Ershov NI, Degtyareva AO, et al. The search for and functional analysis of genetic variants in microRNA-binding sites using massively parallel reporter assay. Bull Exp Biol Med. 2024;176:595–8. 10.1007/s10517-024-06074-3 . Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8644164","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":585451446,"identity":"7b9085cf-a1af-4d8c-ae38-18d0e0fa1dd6","order_by":0,"name":"Yiming Hu","email":"","orcid":"","institution":"The First Affiliated Hospital of Hubei University of Science and Technology","correspondingAuthor":false,"prefix":"","firstName":"Yiming","middleName":"","lastName":"Hu","suffix":""},{"id":585451447,"identity":"98446b68-0f24-4d02-8678-2ff5f1a0836f","order_by":1,"name":"Kai Yu","email":"","orcid":"","institution":"The First Affiliated Hospital of Hubei University of Science and Technology","correspondingAuthor":false,"prefix":"","firstName":"Kai","middleName":"","lastName":"Yu","suffix":""},{"id":585451448,"identity":"40d11403-e7c4-4ef5-9145-6cad7d148978","order_by":2,"name":"Yingqiao Yang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABCElEQVRIiWNgGAWjYNACAyBmBuKEiho5Nvb2AyRoeXDmmDEfz5kE4i1jfNjCnDhPwsEAv/nHzx5+zVNwx67vOPPDB4kNbOltEgwJDD8qtuHWciYvzZrH4FnyzMNsxgaJO2Ry26QbDzD2nLmNW8uBHDNjHoPDyQaHGcwkEs+w5bbJHEhgZmzDo+X8G5gW9m8SiW3M6WwSCQb4tdzIMX4M1GJncJjHDKQlgaAWyRtvzBjnGBxOkDzMU2yQcOaYYRswkA/i8wvf+RzjD2/+HLbnO39848MfFTXy8u3tBx/8qMCtReEAA5sUDwNDYsMBJNEDOFSDgXwDA/PHHwwM9viVjYJRMApGwYgGALKrX3SGcOHlAAAAAElFTkSuQmCC","orcid":"","institution":"Hubei University of Science and Technology","correspondingAuthor":true,"prefix":"","firstName":"Yingqiao","middleName":"","lastName":"Yang","suffix":""}],"badges":[],"createdAt":"2026-01-20 02:53:49","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8644164/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8644164/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":102209792,"identity":"33f58a3d-3b83-415c-8c9f-ea4f8cc0fe07","added_by":"auto","created_at":"2026-02-09 12:14:18","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":111798,"visible":true,"origin":"","legend":"\u003cp\u003eHigh serum miR-4734 expression correlates with diagnostic efficacy and poor prognosis in NSCLC. (A) Schematic diagram of the screening workflow for NSCLC-associated miRNAs. (B) Serum miR-4734 expression levels in NSCLC individuals and healthy individuals detected by qRT-PCR. (C) Diagnostic efficacy of miR-4734 for distinguishing NSCLC individuals from healthy individuals analyzed via ROC curve. (D–F) Associations between miR-4734 expression and NSCLC pathological subtypes, TNM stage, and clinical stage analyzed by Pearson correlation analysis. (G) Relationship between miR-4734 expression and individuals’ overall survival analyzed using Kaplan-Meier survival curves. The difference between groups was assessed using the log‑rank test. The HR and 95% CI were derived from a multivariate Cox proportional hazards model adjusted for key clinicopathological variables\u003c/p\u003e","description":"","filename":"Picture1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8644164/v1/8b5140a9c463901fce43035f.jpg"},{"id":102209788,"identity":"7c5f799b-51dd-47fc-a217-9befb24fc08a","added_by":"auto","created_at":"2026-02-09 12:14:18","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":122491,"visible":true,"origin":"","legend":"\u003cp\u003eHigh miR-4734 expression correlates with enhanced proliferation in NSCLC cells. (A) The relative expression level of miR-4734 in normal bronchial epithelial cell line BEAS-2B and NSCLC cell lines (A549, H23, H1299, H226, SK-MES-1, H460) was detected by qRT-PCR. Compared with the BEAS-2B group, *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01. (B–D) NC mimic, miR-4734 mimic, NC inhibitor or miR-4734 inhibitor were transfected into A549, SK-MES-1 and H460 cells, respectively, and the transfection efficiency was verified by qRT-PCR. Compared with the respective control group, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001. (E–G) The CCK-8 assay was used to detect the cell viability changes of the above cells after corresponding treatments. Compared with the respective control group, *\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.05, ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001. All experiments were independently repeated at least three times.\u003c/p\u003e","description":"","filename":"Picture2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8644164/v1/1b8e274c684eda75b93f89dd.jpg"},{"id":102209793,"identity":"b4e0aaef-4b45-4030-b8a9-558dcda97e9c","added_by":"auto","created_at":"2026-02-09 12:14:18","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":124960,"visible":true,"origin":"","legend":"\u003cp\u003emiR-4734 enhances NSCLC cell angiogenic capacity by upregulating pro-angiogenic factors. (A) Target genes of miR‑4734 were predicted with TargetScan, miRDB and miRWalk, and overlapping results were shown in a Venn diagram. (B) GO functional enrichment analysis of candidate target genes, displaying the main biological processes they are involved in. (C) The relative mRNA expression levels of VEGF, bFGF and Ang‑2 in A549 cells (transfected with NC mimic, miR‑4734 mimic, NC inhibitor or miR‑4734 inhibitor) were detected by qRT‑PCR. Compared with the control group, ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001. (D) The secretion levels of VEGF, bFGF and Ang‑2 in the culture medium of A549 cells from the above treatment groups were measured by ELISA. Compared with the control group, *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.001. (E) The effect of CM from A549 cells in different treatment groups on HUVEC proliferation was detected by CCK‑8 assay. Compared with the control group, ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001. (F) The number of migrated HUVECs (treated with CM from different treatment groups) was counted by cell migration assay. Compared with the control group, ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001. (G) The effect of CM from different treatment groups on HUVEC tube length and branch number was analyzed by tube formation assay. Compared with the control group, *\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.05 for branch number; ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001 for tube length. All experiments were independently repeated at least three times.\u003c/p\u003e","description":"","filename":"Picture3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8644164/v1/157ce01d945bf915f41a40da.jpg"},{"id":102209791,"identity":"59e025c4-3ae1-466d-a8fd-22c7798070c6","added_by":"auto","created_at":"2026-02-09 12:14:18","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":152070,"visible":true,"origin":"","legend":"\u003cp\u003emiR-4734 targets BMP7 and represses its expression. (A) BMP7 expression levels in lung cancer cell lines, analyzed using the DepMap database. (B–C) Correlations between BMP7 expression and individual OS as well as DFS, analyzed via the GEPIA2 database. (D) Schematic diagram of the predicted complementary pairing between miR-4734 and the seed region of BMP7 3′UTR, generated by TargetScan. (E) Validation of the targeting effect of miR-4734 on BMP7 via dual-luciferase reporter assay in A549, SK-MES-1 and H460 cells. (F) BMP7 protein concentration in the serum of NSCLC individuals was measured by ELISA, and its correlation with serum miR-4734 expression level was analyzed. (G–H) Changes in BMP7 mRNA expression in A549 and SK-MES-1 cells were detected by qRT‑PCR after transfection with NC mimic, miR‑4734 mimic, NC inhibitor, or miR‑4734 inhibitor. Compared with the control group, *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001; ns, not significant. All experiments were independently repeated at least threetimes.\u003c/p\u003e","description":"","filename":"Picture4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8644164/v1/b3681f8c2751dd465c82e8f2.jpg"},{"id":102209789,"identity":"1ec26de4-ee16-44cf-a6d2-2e0f637e4c94","added_by":"auto","created_at":"2026-02-09 12:14:18","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":167950,"visible":true,"origin":"","legend":"\u003cp\u003emiR-4734 promotes NSCLC cell proliferation and angiogenesis by inhibiting BMP7. (A-B) The secretion levels of VEGF, bFGF and Ang-2 in A549 and SK-MES-1 cells (from different treatment groups) were measured. Compared with the control group, *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.01, ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001. (C-D) The effect of CM from A549 and SK-MES-1 cells on HUVEC proliferation was detected by CCK-8 assay. Compared with the control group, *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.001. (E-F) The number of migrated HUVECs (treated with CM from A549 and SK-MES-1 cells) was counted by Transwell migration assay. Compared with the control group, *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001. (G-J) The indices of HUVEC tube formation after treatment with CM from A549 and SK-MES-1 cells were quantified. Compared with the control group, *\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001. All experiments were independently repeated at least three times.\u003c/p\u003e","description":"","filename":"Picture5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8644164/v1/8cea0a0290af289add4a7275.jpg"},{"id":105912942,"identity":"e9add758-3b16-43df-9525-95a1b6dc29be","added_by":"auto","created_at":"2026-04-01 11:03:34","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1642465,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8644164/v1/a3a1ebc3-4857-426a-b9f1-c6bd8efba53d.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"miR-4734 Targets BMP7 to Accelerate Non-Small Cell Lung Cancer Progression and Serves as a Poor Prognostic Biomarker","fulltext":[{"header":"Introduction","content":"\u003cp\u003eLung cancer remains the leading cause of cancer-related mortality worldwide, with non-small cell lung cancer (NSCLC) affecting over 80% of lung cancer individuals [1, 2].Despite considerable advances in therapeutic strategies, the overall prognosis of NSCLC individuals remains dismal, primarily owing to the lack of effective approaches for early detection and accurate prognostic evaluation[3, 4]. Thus, the identification of novel biomarkers and the elucidation of their underlying molecular mechanisms are crucial to improving clinical management and clinical outcomes.\u003c/p\u003e\n\u003cp\u003eMicroRNAs (miRNAs) have emerged as pivotal regulators of tumorigenesis and promising circulating biomarkers due to their stability and accessibility[5]. Beyond single miRNA entities, current research increasingly focuses on miRNA signatures derived from extracellular vesicles and the construction of multi-miRNA panels to enhance diagnostic specificity and sensitivity. While the roles of miRNAs like miR-21 and miR-145 in NSCLC are well-characterized [6-8], the clinical significance and functional mechanisms of miR-4734—despite its documented oncogenic role in other cancers—are entirely unknown in the NSCLC context, representing a significant knowledge gap [9, 10].\u003c/p\u003e\n\u003cp\u003eBone morphogenetic protein 7 (BMP7), a member of the TGF-β superfamily, exhibits context-dependent roles in cancer, influencing processes such as epithelial-mesenchymal transition (EMT) in lung cancer [11, 12]. However, its prognostic value and regulatory network in NSCLC are not fully understood. Notably, whether any miRNA can modulate NSCLC progression by directly targeting BMP7 remains an unexplored avenue of research.\u003c/p\u003e\n\u003cp\u003eTo address these gaps, the present study was designed to investigate the role of miR-4734 in NSCLC. Serum miR-4734 was first identified as a differentially expressed candidate via bioinformatics screening, with its upregulation, diagnostic potential, and association with poor prognosis further validated in clinical cohorts. Furthermore, miR-4734 was demonstrated to promote NSCLC cell proliferation and angiogenesis through the secretion of pro-angiogenic factors. Mechanistically, BMP7 was identified as a direct and functional target of miR-4734, and rescue experiments confirmed that the miR-4734/BMP7 axis critically regulates these oncogenic phenotypes. Collectively, these findings unveil miR-4734 as a novel prognostic biomarker and a promoter of tumor angiogenesis via BMP7 inhibition, thereby providing new insights into NSCLC pathogenesis and potential therapeutic targeting.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cp\u003e\u003cstrong\u003e\u003cem\u003eClinical Sample Collection\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study retrospectively analyzed serum samples from 100 patients with pathologically confirmed NSCLC and 100 healthy controls, with strict adherence to ethical guidelines and the protection of participant welfare. All samples were collected at the First Affiliated Hospital of Hubei University of Science and Technology between October 2022 and October 2024. De-identified samples were utilized following broad informed consent obtained from all participants. This study protocol was reviewed and approved by the Ethics Committee of Xianning Central Hospital, and all procedures conformed to the principles outlined in the Declaration of Helsinki. To ensure cohort homogeneity, NSCLC patients with concurrent malignant tumors, severe cardiovascular/cerebrovascular diseases, or autoimmune disorders were excluded from the study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eBioinformatics Analysis\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSerum differentially expressed miRNAs in NSCLC patients vs. healthy controls were screened via ExomiRHub. miR-4734 target genes were predicted using TargetScan, miRWalk, and miRDB, with the intersection set as candidate targets. GO enrichment analysis (BP, MF, CC) of candidate targets was conducted on Metascape. Lung cancer-specific highly expressed targets were further filtered via DepMap, and their correlation with NSCLC survival was analyzed using GEPIA2. miR-4734 binding sites in the 3'UTR of BMP7 were predicted via TargetScan.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eCell Culture and Transfection\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHuman NSCLC cell lines (A549/H1299/H23: adenocarcinoma; SK-MES-1/H226: squamous cell carcinoma; H460: large cell carcinoma) and BEAS-2B (normal bronchial epithelium) were from Shanghai Cell Bank (CAS); HUVECs were from ATCC (USA).\u003c/p\u003e\n\u003cp\u003eCells were cultured in 10% FBS/100 U/mL penicillin/100 μg/mL streptomycin-supplemented medium: RPMI-1640 for NSCLC/BEAS-2B, DMEM for HUVECs; 37°C/5% CO₂ humidified incubator. Log-phase cells were seeded in 6/96-well plates 1d pre-transfection; 60%–70% confluent cells were transfected with miR-4734 mimic/inhibitor/NC, BMP7-OE/empty vector (Lipofectamine 3000, Thermo Fisher), and experiments were performed 24h post-transfection.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eQuantitative Real-Time PCR (qRT-PCR)\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTotal RNA was extracted via TRIzol reagent and reverse-transcribed into cDNA per kit instructions. qRT-PCR used U6 (miR-4734) and GAPDH (BMP7 mRNA) as internal controls. Relative expression: 2⁻ΔΔCt method. Primers: Sangon Biotech (Shanghai).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eCCK-8 Assay for Cell Viability and Proliferation Detection\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTransfected cells were seeded in 96-well plates; 48 h later, 10 μL CCK-8 was added, incubated for 1 h, and OD₄₅₀ measured for cell viability. Conditioned medium (CM) from transfected NSCLC cells was collected. HUVECs seeded in 96-well plates had the medium replaced with CM; CCK-8 assays measured OD₄₅₀ to assess HUVEC proliferation.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eELISA for Cytokine Secretion Detection\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTransfected NSCLC cells were seeded in 6-well plates and cultured for 48 h; supernatants were collected and centrifuged to remove impurities. VEGF, bFGF, and Ang-2 secretion levels were quantified via ELISA kits per manufacturer instructions. OD values were measured at the optimal wavelength using a microplate reader, and concentrations were calculated from standard curves.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eTranswell Migration Assay\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMatrigel was diluted 1:8 with serum-free medium, coated evenly on Transwell upper chambers, and solidified at 37°C for 30 min. CM was collected from transfected NSCLC cells. HUVECs were seeded into upper chambers, with 600 μL CM added to lower chambers; incubation proceeded at 37°C/5% CO₂ for 24 h. Non-migrated cells were wiped off; migrated cells were fixed with 4% paraformaldehyde, stained with 0.1% crystal violet, rinsed, and air-dried. Migrated cells were counted in 5 random microscopic fields, and the mean was calculated.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eMatrigel Tube Formation Assay\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMatrigel was diluted 1:1 with serum-free medium; 50 μL of the mixture was coated per 96-well plate and solidified at 37°C for 30 min. CM was collected from transfected NSCLC cells. HUVECs were seeded into Matrigel-precoated wells, with 100 μL CM added per well. Plates were incubated at 37°C/5% CO₂ for 6 h. Tube length and branch number were quantified in 5 random microscopic fields using ImageJ.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eDual-Luciferase Reporter Gene Assay\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWild-type BMP7 3'UTR (WT-BMP7 3'UTR) and mutant-type BMP7 3'UTR (MUT-BMP7 3'UTR) reporter gene vectors were constructed; site-directed mutagenesis of the miR-4734 binding sites was performed in the mutant vectors. A549, SK-MES-1, and H460 cells were seeded into 24-well plates; when cell confluence reached 60%, the cells were co-transfected with WT-BMP7 3'UTR or MUT-BMP7 3'UTR vector plus miR-4734 mimic or NC. At 48 h post-transfection, firefly luciferase activity and renilla luciferase activity were measured strictly following the manufacturer’s instructions of the dual-luciferase reporter assay kit. Renilla luciferase activity was used as an internal reference to calculate the relative luciferase activity.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eStatistical Analysis\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData analysis was performed using SPSS 22.0. An independent samples t-test was used for two-group comparisons, and a one-way ANOVA with an LSD post hoc test for multiple-group comparisons. Spearman correlation analysis assessed the association between miR-4734 expression and clinicopathological parameters. Kaplan-Meier survival curves and the log-rank test were used for survival analysis. The Cox proportional hazards regression model (variables: age, gender, smoking history, chronic bronchial disease history, clinical stage, pathological subtype, advanced TNM stage, miR-4734 expression) identified independent prognostic factors. Two-tailed \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05 was considered statistically significant.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003e\u003cem\u003emiR-4734 Is Highly Expressed in NSCLC Serum and Exhibits Favorable Diagnostic Value as Well as Correlation with Poor Prognosis\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSerum miRNA expression profiles from NSCLC and healthy controls were screened via the ExomiRHub database. Among 2525 differentially expressed miRNAs, 12 upregulated and 22 downregulated candidate miRNAs were finally identified based on the screening criteria. Notably, miR-4734 was significantly upregulated (Log₂ FC = 1.16, Adj. p-value = 1.35, Ave Expr = 12.55), and no previous studies have established a direct association between miR-4734 and NSCLC. Thus, it was selected as the core target for subsequent investigations (Figure 1A).\u003c/p\u003e\n\u003cp\u003eThe baseline characteristics of the 100 NSCLC individuals and 100 healthy controls enrolled in this study are summarized in Table 1. The two groups were well-matched in terms of age and gender. However, significant differences were observed in smoking history and the prevalence of chronic bronchial disease, with both being more common in the NSCLC group. As anticipated, the serum level of miR‑4734 was significantly higher in NSCLC individuals compared to healthy controls (Figure 1B). Further evaluation of its diagnostic efficacy via receiver operating characteristic (ROC) curve analysis revealed that the area under the curve (AUC) of miR-4734 for distinguishing NSCLC from healthy controls was 0.787 (95% CI: 0.721\u0026ndash;0.853), indicating a moderate diagnostic value for NSCLC (Figure 1C). Correlation analysis between miR-4734 expression and clinicopathological characteristics demonstrated no significant association with tumor pathological subtypes, including squamous cell carcinoma (SCC), adenocarcinoma (ADC), large cell carcinoma (LCC), and adenosquamous carcinoma (ASC) (Figure 1D). However, miR-4734 expression was significantly positively correlated with TNM stage and clinical stage, suggesting that miR-4734 expression gradually increases with disease progression (Figure 1E\u0026ndash;1F). Survival analysis showed that individuals in the miR-4734 high-expression group had significantly shorter overall survival than those in the low-expression group (Figure 1G). Multivariate Cox proportional hazards regression was performed to identify independent prognostic factors. The model included age, gender, smoking history, history of chronic bronchial disease, clinical stage, pathological subtype, and advanced TNM stage. After adjusting for these covariates, high serum miR-4734 expression remained an independent predictor of poor overall survival.\u003c/p\u003e\n\u003cp\u003eIn conclusion, miR-4734 is highly expressed in the serum of individuals with NSCLC, and it not only exhibits potential diagnostic value but also is closely associated with poor prognosis in this population.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 1\u0026nbsp;Baseline characteristics of NSCLC individuals and healthy controls\u003c/strong\u003e\u003c/p\u003e\n\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" width=\"105%\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 36px;\"\u003e\n \u003cp\u003eCharacteristic\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 21px;\"\u003e\n \u003cp\u003eNSCLC Patients\u0026nbsp;\u003c/p\u003e\n \u003cp\u003en=100\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 22px;\"\u003e\n \u003cp\u003eHealthy Controls n=100\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 19px;\"\u003e\n \u003cp\u003e\u003cem\u003eP\u0026nbsp;\u003c/em\u003evalue\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 36px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eDemographics\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 21px;\"\u003e\u0026nbsp;\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 22px;\"\u003e\u0026nbsp;\u003cp\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 19px;\"\u003e\u0026nbsp;\u003cp\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 36px;\"\u003e\n \u003cp\u003eAge (years), mean \u0026plusmn; SD\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 21px;\"\u003e\n \u003cp\u003e60.5 \u0026plusmn; 10.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 22px;\"\u003e\n \u003cp\u003e60.6 \u0026plusmn; 10.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 19px;\"\u003e\n \u003cp\u003e0.940\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 36px;\"\u003e\n \u003cp\u003eGender, n (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 21px;\"\u003e\u0026nbsp;\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 22px;\"\u003e\u0026nbsp;\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 19px;\"\u003e\n \u003cp\u003e0.305\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 36px;\"\u003e\n \u003cp\u003eMale\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 21px;\"\u003e\n \u003cp\u003e60 (60.0)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 22px;\"\u003e\n \u003cp\u003e67 (67.0)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 19px;\"\u003e\u0026nbsp;\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 36px;\"\u003e\n \u003cp\u003eFemale\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 21px;\"\u003e\n \u003cp\u003e40 (40.0)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 22px;\"\u003e\n \u003cp\u003e33 (33.0)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 19px;\"\u003e\u0026nbsp;\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 36px;\"\u003e\n \u003cp\u003eSmoking history, n (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 21px;\"\u003e\u0026nbsp;\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 22px;\"\u003e\u0026nbsp;\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 19px;\"\u003e\n \u003cp\u003e\u0026lt;0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 36px;\"\u003e\n \u003cp\u003eNever\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 21px;\"\u003e\n \u003cp\u003e31 (31.0)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 22px;\"\u003e\n \u003cp\u003e68 (68.0)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 19px;\"\u003e\u0026nbsp;\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 36px;\"\u003e\n \u003cp\u003eEver/Current\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 21px;\"\u003e\n \u003cp\u003e69 (69.0)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 22px;\"\u003e\n \u003cp\u003e32 (32.0)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 19px;\"\u003e\u0026nbsp;\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 36px;\"\u003e\n \u003cp\u003eHistory of chronic bronchial disease, n (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 21px;\"\u003e\u0026nbsp;\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 22px;\"\u003e\u0026nbsp;\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 19px;\"\u003e\n \u003cp\u003e\u0026lt;0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 36px;\"\u003e\n \u003cp\u003eYes\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 21px;\"\u003e\n \u003cp\u003e59 (59.0)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 22px;\"\u003e\n \u003cp\u003e20 (20.0)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 19px;\"\u003e\u0026nbsp;\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 36px;\"\u003e\n \u003cp\u003eNo\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 21px;\"\u003e\n \u003cp\u003e41 (41.0)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 22px;\"\u003e\n \u003cp\u003e80 (80.0)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 19px;\"\u003e\u0026nbsp;\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 36px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eClinical Features\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 21px;\"\u003e\u0026nbsp;\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 22px;\"\u003e\u0026nbsp;\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 19px;\"\u003e\u0026nbsp;\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 36px;\"\u003e\n \u003cp\u003eHistological subtype, n (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 21px;\"\u003e\u0026nbsp;\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 22px;\"\u003e\u0026nbsp;\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 19px;\"\u003e\u0026nbsp;\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 36px;\"\u003e\n \u003cp\u003eSCC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 21px;\"\u003e\n \u003cp\u003e28 (28.0)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 22px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 19px;\"\u003e\u0026nbsp;\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 36px;\"\u003e\n \u003cp\u003eADC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 21px;\"\u003e\n \u003cp\u003e56 (56.0)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 22px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 19px;\"\u003e\u0026nbsp;\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 36px;\"\u003e\n \u003cp\u003eLCC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 21px;\"\u003e\n \u003cp\u003e8 (8.0)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 22px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 19px;\"\u003e\u0026nbsp;\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 36px;\"\u003e\n \u003cp\u003eASC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 21px;\"\u003e\n \u003cp\u003e8 (8.0)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 22px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 19px;\"\u003e\u0026nbsp;\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 36px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eTNM Stage (AJCC 8th), n (%)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 21px;\"\u003e\u0026nbsp;\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 22px;\"\u003e\u0026nbsp;\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 19px;\"\u003e\u0026nbsp;\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 36px;\"\u003e\n \u003cp\u003eI-II\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 21px;\"\u003e\n \u003cp\u003e36 (36.0)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 22px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 19px;\"\u003e\u0026nbsp;\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 36px;\"\u003e\n \u003cp\u003eIII-IV\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 21px;\"\u003e\n \u003cp\u003e64 (64.0)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 22px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 19px;\"\u003e\u0026nbsp;\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 36px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eSerum Biomarker Level\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 21px;\"\u003e\u0026nbsp;\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 22px;\"\u003e\u0026nbsp;\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 19px;\"\u003e\u0026nbsp;\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 36px;\"\u003e\n \u003cp\u003emiR-4734 (relative expression), median (IQR)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 21px;\"\u003e\n \u003cp\u003e2.71 (1.70-3.31)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 22px;\"\u003e\n \u003cp\u003e1.7 (1.26-2.14)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 19px;\"\u003e\n \u003cp\u003e\u0026lt;0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cstrong\u003eFootnotes:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAbbreviations: IQR, interquartile range; AJCC, American Joint Committee on Cancer; SCC, squamous cell carcinoma; ADC, adenocarcinoma; LCC, large cell carcinoma; ASC, adenosquamous carcinoma.\u003c/p\u003e\n\u003cp\u003eData are presented as mean \u0026plusmn; standard deviation (SD), number (percentage), or median (interquartile range, IQR). Continuous variables were compared using the independent samples t-test (Age) or the Mann-Whitney U test (miR-4734). Categorical variables were compared using the Chi-square test.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFigure 1.\u003c/strong\u003e High serum miR-4734 expression correlates with diagnostic efficacy and poor prognosis in NSCLC. (A) Schematic diagram of the screening workflow for NSCLC-associated miRNAs. (B) Serum miR-4734 expression levels in NSCLC individuals and healthy individuals detected by qRT-PCR. (C) Diagnostic efficacy of miR-4734 for distinguishing NSCLC individuals from healthy individuals analyzed via ROC curve. (D\u0026ndash;F) Associations between miR-4734 expression and NSCLC pathological subtypes, TNM stage, and clinical stage analyzed by Pearson correlation analysis. (G) Relationship between miR-4734 expression and individuals\u0026rsquo; overall survival analyzed using Kaplan-Meier survival curves. The difference between groups was assessed using the log‑rank test. The HR and 95% CI were derived from a multivariate Cox proportional hazards model adjusted for key clinicopathological variables\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eHigh Expression of miR-4734 in NSCLC Cells Promotes Cell Proliferation\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo explore the functional role of miR-4734 in NSCLC cells, in vitro experiments were performed. First, miR-4734 expression levels were detected in multiple NSCLC cell lines and normal bronchial epithelial cells (BEAS-2B). As shown in Figure 2A, miR-4734 expression was significantly upregulated in NSCLC cell lines (A549, H23, H1299, H226, SK-MES-1, and H460) compared with BEAS-2B cells.\u003c/p\u003e\n\u003cp\u003eFor subsequent functional analyses, three NSCLC cell lines representing distinct pathological subtypes were selected: A549, SK-MES-1, and H460, which are widely used as representative models for respective NSCLC subtypes in preclinical studies [13, 14]. Transfection efficiency was verified by qRT-PCR. The results demonstrated that transfection with miR-4734 mimic significantly increased miR-4734 expression in all three cell lines, while transfection with miR-4734 inhibitor remarkably downregulated its expression (Figure 2B-2D), confirming the validity of the transfection system. The CCK-8 assay was used to evaluate cell viability and assess the impact of miR-4734 on NSCLC cell proliferation. Overexpression of miR-4734 significantly enhanced the proliferative capacity of NSCLC cells (Figure 2E\u0026ndash;2G), whereas inhibition of miR-4734 led to a marked reduction in cell viability.\u003c/p\u003e\n\u003cp\u003eCollectively, these findings indicate that miR-4734 is highly expressed in NSCLC and exerts a pro-proliferative effect by promoting the viability of NSCLC cells.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFigure 2.\u003c/strong\u003e High miR-4734 expression correlates with enhanced proliferation in NSCLC cells. (A) The relative expression level of miR-4734 in normal bronchial epithelial cell line BEAS-2B and NSCLC cell lines (A549, H23, H1299, H226, SK-MES-1, H460) was detected by qRT-PCR. Compared with the BEAS-2B group, *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01. (B\u0026ndash;D) NC mimic, miR-4734 mimic, NC inhibitor or miR-4734 inhibitor were transfected into A549, SK-MES-1 and H460 cells, respectively, and the transfection efficiency was verified by qRT-PCR. Compared with the respective control group, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001. (E\u0026ndash;G) The CCK-8 assay was used to detect the cell viability changes of the above cells after corresponding treatments. Compared with the respective control group, *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001. All experiments were independently repeated at least three times.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003emiR-4734 Enhances Angiogenic Capacity of NSCLC by Upregulating Pro-Angiogenic Factors\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo elucidate the underlying mechanism of miR-4734, its potential target genes were predicted by integrating TargetScan, miRWalk, and miRDB databases, and 38 high-confidence candidate target genes were screened out (Figure 3A). GO enrichment analysis revealed that these target genes were mainly involved in biological processes including myocardial tissue development, hematopoietic regulation and transmembrane receptor signaling pathway (Figure 3B), suggesting that miR-4734 may modulate pathological processes such as angiogenesis.\u003c/p\u003e\n\u003cp\u003eAt both mRNA and protein levels, miR-4734 mimic significantly upregulated the expression and secretion of pro-angiogenic factors, including VEGF, bFGF and Ang-2; in contrast, miR-4734 inhibition markedly reduced the levels of these factors, indicating that miR-4734 positively regulates the expression of pro-angiogenic factors (Figure 3C-3D, Supplementary Figure S1A-S1D).\u003c/p\u003e\n\u003cp\u003eFurthermore, CM assays were performed to investigate how the miR-4734-modulated tumor microenvironment affects HUVEC functions. Cell proliferation assays revealed that, compared with the control group, CM from miR-4734 mimic-transfected cells significantly promoted HUVEC proliferation at 72 h, whereas CM from miR-4734 inhibitor-transfected cells markedly inhibited this process (Figure 3E, Supplementary Figure S1E\u0026ndash;S1F). Cell migration assays showed that CM from miR-4734 mimic-transfected cells significantly increased the number of migrated HUVECs, while CM from miR-4734 inhibitor-transfected cells led to a marked reduction in migration (Figure 3F, Supplementary Figure S1G\u0026ndash;S1H). Tube formation assays further demonstrated that, relative to the control group, the miR-4734 mimic CM group significantly increased HUVEC tube length and branch number; conversely, the miR-4734 inhibitor CM group caused a marked decrease in both parameters (Figure 3G, Supplementary Figure S1I\u0026ndash;S1J).\u003c/p\u003e\n\u003cp\u003eCollectively, these findings indicate that miR-4734 enhances the angiogenic capacity of NSCLC cells by upregulating pro-angiogenic factors such as VEGF, bFGF and Ang-2.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFigure 3.\u0026nbsp;\u003c/strong\u003emiR-4734 enhances NSCLC cell angiogenic capacity by upregulating pro-angiogenic factors. (A) Target genes of miR‑4734 were predicted with TargetScan, miRDB and miRWalk, and overlapping results were shown in a Venn diagram. (B) GO functional enrichment analysis of candidate target genes, displaying the main biological processes they are involved in. (C) The relative mRNA expression levels of VEGF, bFGF and Ang‑2 in A549 cells (transfected with NC mimic, miR‑4734 mimic, NC inhibitor or miR‑4734 inhibitor) were detected by qRT‑PCR. Compared with the control group, ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001. (D) The secretion levels of VEGF, bFGF and Ang‑2 in the culture medium of A549 cells from the above treatment groups were measured by ELISA. Compared with the control group, *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001. (E) The effect of CM from A549 cells in different treatment groups on HUVEC proliferation was detected by CCK‑8 assay. Compared with the control group, ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001. (F) The number of migrated HUVECs (treated with CM from different treatment groups) was counted by cell migration assay. Compared with the control group, ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001. (G) The effect of CM from different treatment groups on HUVEC tube length and branch number was analyzed by tube formation assay. Compared with the control group, *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05 for branch number; ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001 for tube length. All experiments were independently repeated at least three times.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSupplementary\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;Figure\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eS\u003c/strong\u003e\u003cstrong\u003e1\u003c/strong\u003e\u003cstrong\u003e.\u003c/strong\u003e miR-4734 enhances NSCLC cell angiogenic capacity by upregulating pro-angiogenic factors. (A-B) The relative mRNA expression levels of VEGF, bFGF and Ang-2 in SK-MES-1/H460 cells (transfected with different reagents) were detected by qRT-PCR. Compared with the control group, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001. (C-D) The secretion levels of VEGF, bFGF and Ang-2 in the CM of the above cells were measured by ELISA. Compared with the control group, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001. (E-F) The effect of CM-SK-MES-1/CM-H460 (from different treatment groups) on HUVEC proliferation was detected by CCK-8 assay. Compared with the control group, *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001. (G-H) The number of migrated HUVECs treated with the above CM was counted by Transwell assay. Compared with the control group, *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001. (I-J) The effects of the above CM on HUVEC tube length and branch number were analyzed by tube formation assay. Compared with the control group, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001 for tube length; \u003csup\u003e#\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, \u003csup\u003e##\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, \u003csup\u003e###\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001 for branch number. All experiments were independently repeated at least three times.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003emiR-4734 Targets BMP7 and Represses Its Expression\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo further characterize the candidates, the 38 predicted target genes were subjected to lung cancer-specific expression screening via the DepMap database, which ultimately identified 7 genes highly expressed in lung cancer. Among these genes, BMP7, GRIN1 and OPRL1 showed statistically significant differential expression (Figure 4A, Supplementary Figure S2A\u0026ndash;S2F). Subsequently, the GEPIA2 database was used to analyze correlations between these genes and overall survival (OS) as well as disease-free survival (DFS) in individuals with NSCLC. Results demonstrated that BMP7 was significantly overexpressed in lung cancer cells; while no significant correlation with OS was observed, its high expression was markedly associated with prolonged DFS (Figure 4B, C). In contrast, GRIN1 and OPRL1 had no significant correlations with either OS or DFS (Supplementary Figure S2G\u0026ndash;S2J).\u003c/p\u003e\n\u003cp\u003eTo explore the potential targeting relationship between miR-4734 and BMP7, TargetScan was employed to predict their binding sites, revealing complementary pairing within the seed region (Figure 4D). This interaction was then validated using a dual-luciferase reporter assay. In A549 and SK-MES-1 cells, miR-4734 mimic significantly suppressed the reporter activity of wild-type BMP7 3\u0026apos;UTR, whereas no obvious effect was noted on the mutant vector, confirming that BMP7 is a direct target gene of miR-4734 (Figure 4E). However, no significant inhibition was detected in H460 cells, suggesting this regulatory relationship may exhibit cell-type specificity.\u003c/p\u003e\n\u003cp\u003eClinical sample analysis revealed a significant negative correlation between serum miR-4734 expression levels and BMP7 protein concentrations in individuals with NSCLC (Figure 4F). Cellular experiments further confirmed that miR-4734 mimic downregulated BMP7 mRNA levels, while miR-4734 inhibition upregulated its expression (Figure 4G-4H). Collectively, these findings indicate that miR-4734 negatively regulates BMP7 at the post-transcriptional level.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFigure 4.\u0026nbsp;\u003c/strong\u003emiR-4734 targets BMP7 and represses its expression. (A) BMP7 expression levels in lung cancer cell lines, analyzed using the DepMap database. (B\u0026ndash;C) Correlations between BMP7 expression and individual OS as well as DFS, analyzed via the GEPIA2 database. (D) Schematic diagram of the predicted complementary pairing between miR-4734 and the seed region of BMP7 3\u0026prime;UTR, generated by TargetScan. (E) Validation of the targeting effect of miR-4734 on BMP7 via dual-luciferase reporter assay in A549, SK-MES-1 and H460 cells. (F) BMP7 protein concentration in the serum of NSCLC individuals was measured by ELISA, and its correlation with serum miR-4734 expression level was analyzed. (G\u0026ndash;H) Changes in BMP7 mRNA expression in A549 and SK-MES-1 cells were detected by qRT‑PCR after transfection with NC mimic, miR‑4734 mimic, NC inhibitor, or miR‑4734 inhibitor. Compared with the control group, *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001; ns, not significant. All experiments were independently repeated at least three times.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSupplementary Figure\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eS\u003c/strong\u003e\u003cstrong\u003e2.\u0026nbsp;\u003c/strong\u003eLung cancer-specific expression and survival correlation analysis of candidate target genes. (A-F) Expression profiles of GRIN1, BATF, CRISPLD2, OPRL1, TENM4 and TNN in lung cancer cell lines, analyzed using the DepMap database. (G-J) Correlations of GRIN1 and OPRL1 expression with OS and DFS in NSCLC individuals, evaluated via the GEPIA2 database.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003emiR-4734 Promotes NSCLC Cell Proliferation and Angiogenesis by Inhibiting BMP7\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo investigate the role of miR-4734 targeting BMP7 in lung cancer cells, rescue experiments were performed. The results showed that miR-4734 mimic significantly promoted the secretion of VEGF, bFGF and Ang-2 in the supernatants of A549 and SK-MES-1 cells, whereas co-expression of miR-4734 mimic and BMP7-OE partially reversed this effect, with their secretion levels remaining higher than those in the BMP7-OE group (Figure 5A\u0026ndash;5B). Cell proliferation assays demonstrated that CM from miR-4734 mimic-transfected cells significantly enhanced HUVEC proliferation, and this effect was also partially abrogated by BMP7-OE (Figure 5C\u0026ndash;5D). Further Transwell migration assays revealed that CM from miR-4734 mimic-transfected cells markedly increased HUVEC migratory capacity, and this promotive effect was partially reversed by BMP7-OE as well (Figure 5E\u0026ndash;5F). Additionally, HUVEC tube formation assays confirmed that CM from miR-4734 mimic-transfected cells significantly elevated angiogenesis-related indices (migrated cell number, branch number and tube length), and this effect was partially inhibited by BMP7-OE (Figure 5G\u0026ndash;5J). Collectively, these results indicated that BMP7 could partially antagonize the positive regulatory effects of miR-4734 on pro-angiogenic factor secretion and vascular endothelial cell functions, but failed to completely reverse such effects.\u003c/p\u003e\n\u003cp\u003eIn conclusion, miR-4734 enhances the proliferation of NSCLC cells and their mediated angiogenic capacity by targeting and inhibiting BMP7, thereby facilitating tumor progression.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFigure 5.\u0026nbsp;\u003c/strong\u003emiR-4734 promotes NSCLC cell proliferation and angiogenesis by inhibiting BMP7. (A-B) The secretion levels of VEGF, bFGF and Ang-2 in A549 and SK-MES-1 cells (from different treatment groups) were measured. Compared with the control group, *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001. (C-D) The effect of CM from A549 and SK-MES-1 cells on HUVEC proliferation was detected by CCK-8 assay. Compared with the control group, *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001. (E-F) The number of migrated HUVECs (treated with CM from A549 and SK-MES-1 cells) was counted by Transwell migration assay. Compared with the control group, *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001. (G-J) The indices of HUVEC tube formation after treatment with CM from A549 and SK-MES-1 cells were quantified. Compared with the control group, *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001. All experiments were independently repeated at least three times.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eImproving the prognosis of NSCLC relies on early diagnosis and precision therapy, both of which depend on the discovery of specific molecular biomarkers\u0026nbsp;[15]. Endowed with favorable serum stability and tumor-specific expression, microRNAs serve as ideal diagnostic biomarkers\u0026nbsp;[16, 17]. This study employs an integrated approach encompassing bioinformatic screening, clinical validation, and in vitro experiments to demonstrate significant upregulation of serum miR‑4734 in individuals with NSCLC. Their favorable diagnostic efficacy underscores the potential of serum miR‑4734 as a diagnostic biomarker for NSCLC.\u003c/p\u003e\n\u003cp\u003eThe NSCLC cohort had a higher prevalence of established risk factors, including smoking and chronic bronchial disease. In the multivariate Cox model, however, neither factor retained independent prognostic significance, suggesting that their influence on survival may be mediated through other variables in the model, such as tumor stage or miR‑4734 expression. Critically, even after rigorous adjustment for these and other clinicopathological confounders—most notably advanced TNM stage—high serum miR‑4734 expression remained a strong and independent predictor of poor outcome. This finding indicates that miR‑4734 is not merely a surrogate for smoking-related inflammation but is directly implicated in NSCLC aggressiveness, providing prognostic value additive to standard parameters. This pro-oncogenic role aligns with reported functions of miR‑4734 in hepatocellular carcinoma and breast cancer, suggesting a conserved tumor-promoting mechanism across malignancies\u0026nbsp;[9, 18].\u003c/p\u003e\n\u003cp\u003eAt the cellular level, miR-4734 enhances NSCLC cell viability and upregulates VEGF, bFGF and Ang-2 secretion, thereby promoting HUVEC proliferation, migration and tube formation. This observation is consistent with previous reports that miRNAs such as miR-126 modulate angiogenic factor secretion to drive tumor angiogenesis\u0026nbsp;[19, 20]. However, rescue experiments revealed a complex and context-dependent regulation of miR-4734. While miR-4734 directly targets BMP7 to suppress its expression in A549 and SK-MES-1 cells, this targeting relationship is absent in H460 cells, despite the consistent anti-proliferative effect of miR-4734 across all tested cell lines. This clear dissociation between phenotype and mechanism in H460 cells definitively reveals that miR-4734 can operate through BMP7-independent pathways. Such cell-type specificity reflects the intrinsic molecular heterogeneity among NSCLC subtypes. Distinct genetic backgrounds, such as the unique mutation profiles (e.g., TP53, KRAS) in H460 compared to other cells, could alter miRNA-target accessibility through epigenetic modifications or differential expression of RNA-binding proteins\u0026nbsp;[21, 22]. This mechanistic divergence underscores that the same oncogenic miRNA may engage distinct downstream target networks to fulfill its function in different cellular contexts, a crucial consideration for developing subtype-specific therapeutic strategies\u0026nbsp;[23].\u003c/p\u003e\n\u003cp\u003eIntriguingly, even in cells where the miR-4734/BMP7 axis is functional, overexpression of BMP7 only partially reverses the pro-angiogenic and pro-proliferative effects induced by miR-4734. This \"partial rescue\" phenomenon should not be viewed as a limitation but rather as a crucial clue pointing to the complexity of miRNA-mediated regulation. It aligns with the well-established paradigm that a single miRNA often exerts its biological effects by regulating multiple target genes in parallel\u0026nbsp;[24, 25].Therefore, miR-4734 likely functions as a core regulatory node within a broader cooperative network. Beyond BMP7, it may simultaneously target other members of the TGF-β superfamily (e.g., BMP2, BMP4) or key signaling components, or regulate additional pathways crosstalking with the BMP signaling pathway (e.g., PI3K/AKT, MAPK/ERK); these co-regulated pro-tumor pathways synergistically drive tumor progression\u0026nbsp;[26, 27].\u003c/p\u003e\n\u003cp\u003eThis study has several limitations that should be acknowledged. First, the translational relevance of the findings is constrained by the study design. The conclusions are primarily based on serum samples and in vitro models, lacking validation in matched tumor tissues and in vivo systems. While serum miR-4734 shows promise as a biomarker, its direct role within the tumor microenvironment and its functional impact on tumor growth and metastasis in vivo remain to be established [28].Second, the mechanistic understanding of miR-4734 remains partial and context-dependent. BMP7 was validated as a direct target in A549 and SK-MES-1 cells; however, this specific interaction was not observed in H460 cells, where miR-4734 still exerted a clear pro-proliferative effect. This indicates the existence of robust, BMP7-independent pathways. Furthermore, the partial phenotypic rescue upon BMP7 overexpression even in responsive cells suggests that miR-4734 functions as a node within a broader regulatory network. Therefore, a comprehensive mapping of its direct targets—for instance, through techniques like AGO2-CLIP-seq—is essential to fully elucidate its oncogenic network and to assess its potential as a subtype-specific therapeutic target\u0026nbsp;[29, 30].\u003c/p\u003e\n\u003cp\u003eThis study establishes serum miR-4734 as a novel diagnostic and prognostic biomarker for NSCLC, while elucidating its oncogenic mechanism via targeting BMP7. Clinically, elevated serum miR‑4734 not only aids in distinguishing NSCLC individuals from healthy individuals but also independently predicts advanced disease stage and unfavorable survival outcomes. Mechanistically, the results demonstrate that miR-4734 promotes tumor proliferation and angiogenesis by directly suppressing BMP7, thereby driving NSCLC progression.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eIQR\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003einterquartile range\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eAJCC\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eAmerican Joint Committee on Cancer\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eSCC\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003esquamous cell carcinoma\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eADC\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eadenocarcinoma\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eLCC\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003elarge cell carcinoma\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eASC\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eadenosquamous carcinoma.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe sincerely thank all participants for their involvement in this study. We are grateful to the Department of Pulmonary Critical Care Medicine of Xianning Central Hospital (the First Affiliated Hospital of Hubei University of Science and Technology) for its support, as well as the National Experimental General Practice Education Demonstration Center of Xianning Medical College, Hubei University of Science and Technology for providing experimental support and technical guidance during the study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contribution\u003c/strong\u003e\u003cstrong\u003es\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors contributed to the study. Conception and design were performed by Yiming Hu. Administrative support was provided by Yingqiao Yang. Provision of study materials or patients was conducted by Yiming Hu and Kai Yu. Collection and assembly of data were performed by Kai Yu. Data analysis and interpretation were carried out by Yiming Hu and Yingqiao Yang. The manuscript was written by all authors, and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript. Yiming Hu and Kai Yu contributed equally to this work.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eYiming Hu declares that he has no conflict of interest.\u003c/p\u003e\n\u003cp\u003eKai Yu declares that he has no conflict of interest.\u003c/p\u003e\n\u003cp\u003eYingqiao Yang declares that she has no conflict of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe study protocol was reviewed and approved by the Ethics Committee of Xianning Central Hospital (Approval No.: HKSY-YL-YJ-2022-036). All procedures adhered to the ethical principles of the Declaration of Helsinki.\u003c/p\u003e\n\u003cp\u003eConsent to participate: The use of de-identified samples for secondary research was covered in the broad written informed consent obtained from all participants, which permitted future use of their anonymized data/specimens. All participants provided informed consent after full understanding of the research purpose.\u003c/p\u003e\n\u003cp\u003eConsent to publish: Consent for publication was obtained for every individual person’s data included in the study; this permission is also explicitly covered in the broad written informed consent obtained from all participants.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eClinical trial registration\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study is a secondary analysis of de-identified samples and does not involve interventional clinical trial procedures. Therefore, a clinical trial number is not applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eGarinet S, Wang P, Mansuet-Lupo A, et al. Updated prognostic factors in localized NSCLC. Cancers (Basel). 2022;14. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1097/MD.0000000000032899\u003c/span\u003e\u003cspan address=\"10.1097/MD.0000000000032899\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSuster DI, Mino-Kenudson M. Molecular pathology of primary non-small cell lung cancer. 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Bull Exp Biol Med. 2024;176:595\u0026ndash;8. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1007/s10517-024-06074-3\u003c/span\u003e\u003cspan address=\"10.1007/s10517-024-06074-3\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Angiogenesis, BMP7, miR-4734, NSCLC, Prognosis","lastPublishedDoi":"10.21203/rs.3.rs-8644164/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8644164/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eNSCLC is a leading cause of cancer mortality worldwide, highlighting the need for non-invasive biomarkers. Circulating miRNAs are promising candidates due to their stability in serum.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eThis study measured serum levels of miR-4734 using qRT-PCR in NSCLC patients and healthy controls. The diagnostic ability was assessed through ROC analysis, while the prognostic significance was evaluated with Kaplan\u0026ndash;Meier survival curves and Cox regression. Functional assays, including CCK-8, ELISA, Transwell, and tube formation tests, were conducted to examine the role of miR-4734 in cell proliferation and angiogenesis in NSCLC and endothelial cells. BMP7 was identified as a direct target of miR-4734 via bioinformatic predictions, dual-luciferase reporter assays, and rescue experiments.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eSerum miR-4734 was significantly elevated in patients with NSCLC compared to healthy controls, demonstrating moderate diagnostic accuracy with an AUC of 0.787 (95% CI: 0.721\u0026ndash;0.853). High levels of miR-4734 were linked to advanced TNM stage and shorter overall survival. Moreover, miR-4734 served as an independent adverse prognostic factor, with an HR of 2.103 (95% CI: 1.091\u0026ndash;4.051). Functionally, miR-4734 encouraged cell proliferation in NSCLC, increased the secretion of pro-angiogenic factors, and boosted endothelial cell migration and tube formation. Mechanistically, miR-4734 directly targeted and inhibited BMP7, and the oncogenic effects of miR-4734 were partly reversed when BMP7 was overexpressed.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003eSerum miR-4734 serves as a potential non-invasive biomarker for diagnosing and predicting NSCLC outcomes. It promotes tumor growth by increasing proliferation and angiogenesis through directly inhibiting BMP7.\u003c/p\u003e","manuscriptTitle":"miR-4734 Targets BMP7 to Accelerate Non-Small Cell Lung Cancer Progression and Serves as a Poor Prognostic Biomarker","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-02-09 12:14:13","doi":"10.21203/rs.3.rs-8644164/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"d6907698-2c23-4649-88de-717e86bb3aa1","owner":[],"postedDate":"February 9th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-04-01T10:43:50+00:00","versionOfRecord":[],"versionCreatedAt":"2026-02-09 12:14:13","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8644164","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8644164","identity":"rs-8644164","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}