miR-1251-5p inhibits tumorigenesis and metastasis in renal cell carcinoma by targeting MTHFD2

Introduction

Renal cell carcinoma (RCC) is the 14th most frequently diagnosed cancer worldwide, with more than 400,000 new cases in 2020. In US, it is the sixth most common cancer in males and ninth in females in 2023 (1-3). About 70–80% of RCC patients have clear cell histology (ccRCC), and 30% patients developed metastatic ccRCC, which has a poor prognosis (4). For patients with early stage RCC, surgical resection is the standard treatment. Radiofrequency ablation, cryoablation, and stereotactic ablative body radiotherapy can also be used for localized RCC, particularly in small tumors (2,5-7). For patients with advanced stage RCC, systemic therapy is the main treatment modality, including immune checkpoint inhibitors, tyrosine kinase inhibitors, cell therapy, and other strategies (4,8,9). However, due to drug resistance, low response rate, and other factors, the 5-year survival rate is only about 12% in RCC patients who have metastasis, which brings a heavy tumor burden to the society (10). Because the genetics and environment determine the occurrence and development of RCC, it is essential to find more biomarkers at the molecular levels.

microRNAs (miRNAs) are small non-coding RNAs with ~22 nucleotides. They play important roles in many biological activities through silencing the expression of target genes. The disorder of miRNAs has been clarified to be closely related with multiple diseases such as cancer, neurological, cardiovascular, and developmental diseases (11-14). miRNAs have been reported playing key roles in the occurrence and development of RCC (15). Du et al. showed that miR-30a-5p was regulated by LINC00926, and it can inhibit the expression of SRY-box transcription factor 4 (SOX4), and the disorder of this axis can promote the progression of RCC (16). Another study showed that miR-182-5p/cytochrome P450 family 1 subfamily B member 1 (CYP1B1) axis was regulated by circPPAP2B and related to the metastasis of ccRCC (17). miR-1251-5p was abnormal expressed in multiple cancers, it was up-expressed in ovarian cancer, and hepatocellular carcinoma (18,19). In ovarian cancer, miR-1251-5p functioned as an oncogene to suppress tubulin-specific chaperone C (TBCC) and α/β-tubulin expression. Han et al. showed that miR-1251-5p drove hepatocellular carcinoma cell proliferation, migration and invasion by targeting A-kinase anchor protein 12 (AKAP12). miR-1251-5p was down-expressed in pancreatic cancer, and ccRCC (20,21). It is spongified by circ_0001666 in pancreatic cancer and targets SOX4 to promote epithelial-mesenchymal transition (EMT) of pancreatic cancer. In ccRCC, it has been reported that proliferation, migration, and immune escape can be inhibited by targeting neuronal pentraxin 2 (NPTX2) (21).

In the present study, we determined the expression of miR-1251-5p in RCC through data from The Cancer Genome Atlas (TCGA) and cell lines. mirDIP and starBase database were used to predict potential target genes of miR-1251-5p. Finally, the function of miR-1251-5p and its target genes were determined. We present this article in accordance with the MDAR reporting checklist (available at https://tau.amegroups.com/article/view/10.21037/tau-2025-1-935/rc).

Methods

Cell culture and transfection

Renal normal cell lines HK2 (human, GNHu47) and RCC cell lines 786-O (human, TCHu186), Caki-1 (human, TCHu135) and ACHN (human, TCHu199) were purchased from the cell bank of the Chinese Academy of Sciences (Shanghai, China) and cultured in Dulbecco’s Modified Eagle’s Medium (DMEM, Gibco, Grand Island, USA) supplemented with 10% fetal bovine serum (FBS; Gibco, USA). Incubation was done at 37 ℃ with 5% CO₂. miR-1251-5p mimic, siRNAs and plasmids were purchased from GenePharma (Shanghai, China). The sequences were listed as follows: miR-1251-5p mimics: 5’-ACUCUAGCUGCCAAAGGCGCU-3’; siMTHFD2: 5’-GCGAGAAUCCUGCAAGUCATT-3’. Transfection of cells was performed using Lipofectamine 3000 (Thermo Fisher Scientific, Waltham, USA).

Quantitative real-time polymerase chain reaction (qRT-PCR)

Total RNA was extracted from cells by using Trizol (Invitrogen, Carlsbad, USA). Nanodrop (Thermo Fisher Scientific) measured the RNA concentration, and cDNA was obtained based on reverse transcription reagent kit (GENESEED, Guangzhou, China). qRT-PCR was performed by Applied Biosystems 7500 Real-Time PCR Detection Systems (Thermo Fisher Scientific, USA). SYBR supermix (Bio-Rad, Hercules, USA) was used to detect relative expression of miRNA and mRNA. U6 was the control for miRNA, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was the control for mRNA. The relative expression ratio of miRNA/mRNA (2^−ΔΔCt) was obtained. △△CT = (CT of miRNA/mRNA in RCC − CT of U6/GAPDH in RCC) − (CT of miRNA/mRNA in HK2 − CT of U6/GAPDH in HK2). Sequences of primers for qRT-PCR are listed in Table S1.

Western blotting (WB)

Proteins were obtained by lysis of cells using Racial and Identity Profiling Act (RIPA) lysis buffer (FUDEBIO, Hangzhou, China), and total protein concentration was determined by bicinchoninic acid (BCA) protein kit (FUDEBIO, Hangzhou, China). Proteins of different molecular weights were separated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels and transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, Burlington, USA). The membranes were blocked with fat-free milk, marker was used as a reference to trim the membranes appropriately according to the molecular weight of the target protein, and then membranes were incubated with antibody (1:1,000) for 12 hours at 4 ℃. After incubation of the membranes with horseradish peroxidase (HRP)-labeled secondary antibody (1:5,000, Beyotime Biotech, Shanghai, China) at room temperature for 1 hour, immunoassays were performed using electrochemiluminescence (ECL) detection solution (FUDEBIO) and combined with a ChemiDoc touch imaging system (Bio-Rad, USA) to acquire the results of WB. The antibodies used in this research were: anti-methylenetetrahydrofolate dehydrogenase 2 (anti-MTHFD2; ab307428, Abcam, Cambridge, UK); anti-GAPDH (ab8245, Abcam, Cambridge, UK).

Cell proliferation and colony formation

For proliferation testing, 786-O and Caki-1 cells were seeded into 96-well plates after transfection. Cells were subjected to Cell Counting Kit-8 (CCK-8) reagent (Sigma-Aldrich, Burlington, USA) at 10 µL per well and then incubated at 37 ℃ for 2 hours while kept away from light. Cells proliferation was measured at 0, 24, 48, 72 and 96 hours after seeding. For clonogenicity, cells were seeded into 6-well plates and the medium was changed every three days. Cells were cultured in 37 ℃ for 14 days and stained with crystal violet.

Transwell and wound healing assay

For Transwell assay, 786-O and Caki-1 cells were seeded into the inserts of 24-well transwell chambers (Corning, Corning, USA) after transfection with the serum-free medium. The medium in the lower compartment was added 20% FBS. And then incubated at 37 ℃ for 48 hours, cells in the lower compartment were fixed and stained. The number of cells migrating to the lower layer can be obtained by light microscopy (Olympus, Tokyo, Japan). For wound healing assay, 786-O and Caki-1 cells were seeded in 6-well plates after transfection. When the cells filled the plate, they were scratched with a micropipette tip and incubated at 37 ℃ for 12 hours in the serum-free medium. The distance of cell migration in the wound healing assay could be observed by light microscopy.

Dual-luciferase reporter assay

The Dual-Luciferase Reporter assay was used for analysis of the interaction of miRNA and mRNA. The original or mutated sequences of about 200 adjacent positions on both sides of the binding site will be inserted into MTHFD2 vector, and the wild type (wt) or mutant type (mut) reporter plasmids (Promega, Madison, USA) were respectively constructed. 293T cells were selected to be co-transfected with wt/mut reporter plasmids and miRNA mimics/miRNA negative control (NC). The Firefly luciferase activity and Renilla luciferase were measured by Reporter Assay System Kit (Promega, USA). Finally, SynergyMx M5 (Molecular Devices, San Jose, USA) detected the intensity of signals.

Bioinformatics analysis

• GEO (https://www.ncbi.nlm.nih.gov/geo/): the database GSE61741 was used for analysis of expression of miRNAs in blood of RCC patients.
• MethPrimer (http://www.urogene.org/methprimer/): the database was used for analysis of cytosine-phosphate-guanine (CpG) island in miRNA transcript sequence (22).
• StarBase (http://starbase.sysu.edu.cn/): the database was used to predict the correlation of miRNA and target gene, the downstream genes corresponding to the base sequence of miRNA, the relationship between gene expression and clinical prognosis, and the correlation between two genes (23).
• GEPIA (http://gepia.cancer-pku.cn/): the database was used to analyze the effect of target gene on overall survival and progression-free survival of RCC patients (24).
• LinkedOmics (http://www.linkedomics.org/login.php): the database was used for analyzing the correlation between miRNA and gene, or between two genes (25).
• UALCAN (http://ualcan.path.uab.edu/analysis.html): the database was used in the analysis of expression of target genes in different tumor tissues and different clinical stages, and the relationship between gene expression and clinical prognosis (26).
• mirDIP (http://ophid.utoronto.ca/mirDIP/): the database was used for the forecast of the summary target genes of miRNA (27).
• DAVID (https://david.ncifcrf.gov/summary.jsp): the database was used to Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis of genes (28). The study was conducted in accordance with the Declaration of Helsinki and its subsequent amendments.

Statistical analysis

All experiments in this study were performed in 3 replicates, data was analyzed by using GraphPad Prism 5, the final results were presented as averages ± standard deviation (SD). Data was mainly compared by two-tailed student t-test and one-way analysis of variance (ANOVA). The two-tailed student t-test was used to compare the two experimental groups, and the one-way ANOVA was used to compare more than two experimental groups. P<0.05 was considered statistically significant.

Results

miR-1251-5p is down-regulated in RCC

Firstly, we analyzed the expression of miR-1251-5p in the TCGA database and RCC cell lines. The results showed that the expression of miR-1251-5p in RCC tissues compared to normal renal tissues showed a significantly down-regulation (Figure 1A). We further analyzed the survival rate of 253 RCC patients with high expression of miR-1251-5p and 253 RCC patients with low expression in the TCGA database. The results showed that the overall survival rate of RCC patients with high expression of miR-1251-5p was higher (Figure 1B). Meanwhile, with the progression of lymph node metastasis and tumor grade, expression of miR-1251-5p decreased significantly (Figure S1A,S1B). Using GSE61741, we found that the expression of miR-1251-5p in blood samples of RCC patients was significantly downregulated (Figure 1C). Compared with normal human renal cell line HK-2, human RCC cell lines 786-O, Caki-1 and ACHN showed significant down-regulation of miR-1251-5p (Figure 1D). The results of MethPrimer showed that no CpG island was found in the region around 4,000 bp before and after the miR-1251-5p transcript (Figure 1E,1F), suggesting that the abnormal expression of miR-1251-5p in RCC was less likely to be caused by over-methylation of CpG islands in the promoter region.

Figure 1 Expression of miR-1251-5p in RCC. (A) Expression of miR-1251-5p in RCC in TCGA database; (B) overall survival of miR-1251-5p in RCC in TCGA database. Patients were divided into high (red line, n=253) and low (blue line, n=253) miR-1251-5p expression groups using the median expression level as the cutoff. P<0.01, log-rank test. (C) Volcano plot of DE-miRNAs in GSE61741. Grey dots represent miRNAs which are not differentially expressed in the RCC group; red dots and green dots represent miRNAs that are upregulated and downregulated significantly in RCC group; (D) expression of miR-1251-5p in RCC cell and normal renal cell lines examined by RT-qPCR; (E) the distribution of CpG islands in the 4,000 bp region in front of the miR-1251-5p transcript; (F) the distribution of CpG islands in the 4,000 bp region behind the miR-1251-5p transcript. **, P<0.01; ***, P<0.001. The experiments were independently repeated three times. CpG, cytosine-phosphate-guanine; DE, differentially expressed; FC, fold change; RCC, renal cell carcinoma; RPM, reads per million mapped reads; RT-qPCR, quantitative real-time polymerase chain reaction; TCGA, The Cancer Genome Atlas.

miR-1251-5p inhibits RCC proliferation and migration

To investigate the role of miR-1251-5p in RCC, miR-1251-5p was over-expressed in 786-O and Caki-1 cell lines transfected with miRNA mimics (Figure S2A). Over-expression of miR-1251-5p could inhibit the proliferation of RCC cell lines using CCK-8 and plate clone formation (Figure 2A-2C). Transwell and wound healing assay demonstrated that the ability of RCC cell migration was inhibited with the treatment of miR-1251-5p mimics (Figure 2D-2F, Figure S2B-S2D). Data from TCGA indicated that the expression of miR-1251-5p was significantly correlated with the expression of important factors in EMT such as cadherin 1 (CDH1, also known as E-cadherin, E-cad), EMT-related transcription factor SNAIL family transcriptional repressor 2 (SNAIL2, also known as Slug) and matrix metallopeptidase 9 (MMP9) in RCC tissues (Figure 2G,2H).

Figure 2 miR-1251-5p inhibits RCC proliferation and migration. (A,B) Cell viability was evaluated by CCK-8 in 786-O and Caki-1 cell lines treated with miR-1251-5p mimics/mimics NC; (C) cell viability was evaluated by plate clone formation assay stained with crystal violet in 786-O and Caki-1 cell lines treated with miR-1251-5p mimics/mimics NC; (D) cell migration was evaluated by Transwell stained with crystal violet in 786-O and Caki-1 cell lines treated with miR-1251-5p mimics/mimics NC (magnification: 100×); (E,F) cell migration was evaluated by wound healing assay in 786-O and Caki-1 cell lines treated with miR-1251-5p mimics/mimics NC; (G) correlation of miR-1251-5p and CDH1, SNAI2, MMP9 in RCC in starBase database; (H) correlation of miR-1251-5p and CDH1, SNAI2, MMP9 in RCC in LinkedOmics database. *, P<0.05; ***, P<0.001. The experiments were independently repeated three times. CCK-8, Cell Counting Kit-8; NC, negative control; RCC, renal cell carcinoma.

miR-1251-5p targets MTHFD2 in RCC

miRNAs mainly function through target genes. We used mirDIP and starBase encori database to predict the key target genes of miR-1251-5p in RCC (Figure 3A). Target genes existing simultaneously in two databases were selected for KEGG and GO-biological process (BP) enrichment analysis. KEGG enrichment analysis showed that the predicted target genes were closely related to tumor pathways such as Hippo, hypoxia inducible factor-1 (HIF-1) and AMP-activated protein kinase (AMPK) (Figure 3B). GO-BP results indicated that the predicted target genes of miR-1251-5p participate in cell proliferation, adhesion, and WNT signaling pathway (Figure S3A). Then, the correlation between miR-1251-5p and predicted target genes was analyzed using starBase and LinkedOmics. Results from both two databases showed that the expression of miR-1251-5p and MTHFD2 showed significant negative correlation in RCC (Figure 3C, Figure S3B). To determine the regulation between miR-1251-5p and MTHFD2, we found the expression of MTHFD2 was markedly decreased on mRNA level after over-expressing miR-1251-5p (Figure 3D). A significant decrease in the relative luciferase activities was observed when MTHFD2-3’UTR-wildtypes were co-transfected with miR-1251-5p mimic. The results showed that miR-1251-5p could interact with MTHFD2-3’UTR and exert a suppression effect. When the binding site was mutated, the suppressive effect was removed (Figure 3E). These results showed that MTHFD2 is target gene of miR-1251-5p.

Figure 3 miR-1251-5p targets MTHFD2 in RCC. (A) Venn diagram of target genes of miR-1251-5p predicted by mirDIP and ENCORI database; (B) KEGG pathway enrichment analysis for 326 target genes; (C) correlation of miR-1251-5p and MTHFD2 in RCC in starBase database; (D) mRNA level of MTHFD2 in 786-O and Caki-1 cell lines treated with miR-1251-5p mimics/mimics NC was examined by RT-qPCR; (E) sequence prediction of miR-1251-5p and MTHFD2. Relative luciferase activity of the binding of miR-1251-5p and MTHFD2. **, P<0.01; ***, P<0.001. The experiments were independently repeated three times. FPKM, fragments per kilobase of transcript per million mapped reads; KEGG, Kyoto Encyclopedia of Genes and Genomes; NC, negative control; RCC, renal cell carcinoma; RPM, reads per million mapped reads; RT-qPCR, quantitative real-time polymerase chain reaction.

MTHFD2 is up-regulated in RCC

Then, we explored the expression and function of MTHFD2 in RCC. It played a very important role in tumorigenesis and development, and it was generally highly expressed in most tumors (Figure S4A). The expression of MTHFD2 in RCC tissues was increased significantly using TCGA database (Figure 4A). The expression of MTHFD2 in normal renal tissues, renal cancer tissues without lymph node metastasis and those with lymph node metastasis had been continuously increasing (Figure 4B). Meanwhile, the expression of MTHFD2 was increased with the improvement of tumor grade and stage of RCC (Figure S4B,S4C). The overall survival rate of RCC patients with high expression of MTHFD2 was significantly lower (Figure 4C). The similar trend was observed in progression-free survival in RCC patients (Figure 4D). According to these results, MTHFD2 is highly expressed in RCC and is associated with poor prognosis.

Figure 4 Expression of MTHFD2 in RCC. (A) Expression of miR-1251-5p in RCC in TCGA database; (B) the expression of MTHFD2 in different lymph node metastasis stages in TCGA database; (C) overall survival of MTHFD2 in RCC in TCGA database; (D) disease-free survival of MTHFD2 in RCC in TCGA database. FPKM, fragments per kilobase of transcript per million mapped reads; HR, hazard ratio; RCC, renal cell carcinoma; TCGA, The Cancer Genome Atlas; TPM, transcripts per million.

MTHFD2 promotes RCC proliferation and migration

To investigate the role of MTHFD2 in the RCC development, siRNAs targeting MTHFD2 were transfected (Figure 5A). Down-regulation of MTHFD2 could inhibit the ability of RCC proliferation using CCK-8 and plate clone formation (Figure 5B-5D, Figure S5A). The results of the Transwell and wound healing assay demonstrated that the ability of RCC cell migration was inhibited with the down-expression of MTHFD2 (Figure 5E-5G, Figure S5B,S5C). We also found that the expression of MTHFD2 was significantly correlated with the expression of CDH1, SNAI2, and MMP9 (Figure S5D,S5E).

Figure 5 MTHFD2 promotes RCC proliferation and migration. (A) mRNA level of MTHFD2 in 786-O and Caki-1 cell lines treated with siMTHFD2/siNC was examined by RT-qPCR; (B,C) cell viability was evaluated by CCK-8 in 786-O and Caki-1 cell lines treated with siMTHFD2/siNC; (D) cell viability was evaluated by plate clone formation assay stained with crystal violet in 786-O and Caki-1 cell lines treated with siMTHFD2/siNC; (E) cell migration was evaluated by Transwell stained with crystal violet in 786-O and Caki-1 cell lines treated with siMTHFD2/siNC (magnification: 100×); (F,G) cell migration was evaluated by wound healing assay in 786-O and Caki-1 cell lines treated with siMTHFD2/siNC. ***, P<0.001. The experiments were independently repeated three times. CCK-8, Cell Counting Kit-8; RCC, renal cell carcinoma; RT-qPCR, quantitative real-time polymerase chain reaction.

Overexpression of MTHFD2 blocks the suppression of RCC by miR-1251-5p

To further test our hypothesis, miR-1251-5p and MTHFD2 were over-expressed simultaneously (Figure 6A). The results of CCK-8 and plate clone formation experiments revealed that miR-1251-5p can inhibit proliferation and clonogenicity, while MTHFD2 could block this effect (Figure 6B,6C, Figure S6A). The results of the transwell assay showed that compared with the cells co-transfected with miR-1251-5p mimic and vector, the number of migrating cells co-transfected with miR-1251-5p mimic and oeMTHFD2 was significantly increased (Figure 6D, Figure S6B). A similar trend could be observed in the wound healing assays (Figure 6E, Figure S6C). These results suggested that miR-1251-5p could inhibit the proliferation and migration of RCC by targeting MTHFD2.

Figure 6 Overexpression of MTHFD2 blocks the suppression of RCC by miR-1251-5p. (A) Protein level of MTHFD2 in 786-O and Caki-1 cell lines treated with oeMTHFD2 and miR-1251-5p mimics simultaneously was examined by Western blotting; (B) cell viability was evaluated by CCK-8 in 786-O and Caki-1 cell lines treated with oeMTHFD2 and miR-1251-5p mimics simultaneously; (C) cell viability was evaluated by plate clone formation assay stained with crystal violet in 786-O and Caki-1 cell lines treated with oeMTHFD2 and miR-1251-5p mimics simultaneously; (D) cell migration was evaluated by Transwell stained with crystal violet in 786-O and Caki-1 cell lines treated with oeMTHFD2 and miR-1251-5p mimics simultaneously (magnification: 100×); (E) cell migration was evaluated by wound healing assay in 786-O and Caki-1 cell lines treated with oeMTHFD2 and miR-1251-5p mimics simultaneously. ***, P<0.001. The experiments were independently repeated three times. CCK-8, Cell Counting Kit-8; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; NC, negative control; RCC, renal cell carcinoma.

Discussion

Many studies have shown that the abnormal expression of miRNAs is closely related with the occurrence and development of RCC. In current study, we found that miR-1251-5p was down-expressed in RCC, which is consistent with previous study (21). Our research also showed that miR-1251-5p functions by targeting MTHFD2, and inhibiting MTHFD2 could suppress the progression of RCC. The roles of MTHFD2 in RCC have been clarified in previous studies. Over-expression of MTHFD2 has been suggested as prognostic indicator for RCC (29,30). As a key enzyme in folate metabolism, MTHFD2 can function through multiple pathways. It is one of the key enzymes involved in one-carbon metabolism, which is essential for the biosynthesis of thymidine and purines, maintenance of amino acids and REDOX homeostasis (31). It was found to form a complex with poly (ADP-ribose) polymerase 3 (PARP3), which functions in non-homologous end-joining (NHEJ) (32). There is relatively little research on the mechanism of MTHFD2 in RCC. Nathanael et al. showed MTHFD2 was significantly elevated in human RCC tissues, MTHFD2 and HIF-2α form a positive feedforward loop, then promote metabolic reprograming and tumor growth (33). Sunitinib is a first-line targeted therapy against RCC, Liu et al. showed that MTHFD2 can enhance cMYC O-GlcNAcylation and promote sunitinib resistance in RCC (34). These results are consistent with our study.

miRNAs can be found in serum and other body fluids, and they can serve as biomarkers for multiple diseases including RCC (35). Martina et al. found that circulating miR-378 and miR-451 in serum are biomarkers for RCC [combination of miR-378 and miR-451, sensitivity 81%, specificity 83%, area under the curve (AUC) =0.86] (36). Another research also showed that the combination of serum miR-122-5p and miR-206 is a non-invasive prognostic biomarker for RCC, high miR-122-5p and miR-206 serum levels were associated with poor prognosis (37). There are two studies about the value of miR-1251-5p in predicting the prognosis of ccRCC and pancreatic cancer. It showed a good predictability (38,39). However, these studies only used bioinformatics analysis. So, our future research will focus on diagnostic values of miR-1251-5p/MTHFD2 axis in RCC using serum or other body fluid samples.

miRNA-based therapeutics can modulate target gene expression accurately (40). There are also some clinical trials showing the potential of miRNAs against cancers. MRX34, a liposomal mimic of miR-34a, has been using in clinical trials in multiple solid tumors. Though the trials were terminated because of immune-related serious adverse events, they still provided the necessary proof-of-concept for the efficacy of miRNA-based anticancer therapy (41,42). TargomiRs are minicells loaded with miR-16-based mimic, Nico et al. found that TargomiRs showed acceptable safety profile and early signs of activity in malignant pleural mesothelioma patients, which suggested the value of further research (43). There is no study about miR-1251-5p-based therapy strategy so far. Our research showed the potential of miR-1251-5p/MTHFD2 axis in treating RCC. So, future research will focus on precision treatment system based on miR-1251-5p.

There are also several other limitations in our study. We have determined the role of miR-1251-5p/MTHFD2 in RCC through bioinformatics analysis and in vitro experiments. However, we have not clarified the function of this axis using animal models and clinical samples. Future studies should therefore aim to investigate the roles of miR-1251-5p/MTHFD2 axis in RCC using more models further.

Conclusions

In a word, our study confirmed the key role of miR-1251-5p/MTHFD2 in RCC, targeting this axis may be a novel strategy against RCC (Figure 7).

Figure 7 miR-1251-5p inhibits the occurrence and development of RCC by targeting MTHFD2. EMT, epithelial-mesenchymal transition; RCC, renal cell carcinoma.

Acknowledgments

None.

Footnote

Reporting Checklist: The authors have completed the MDAR reporting checklist. Available at https://tau.amegroups.com/article/view/10.21037/tau-2025-1-935/rc

Data Sharing Statement: Available at https://tau.amegroups.com/article/view/10.21037/tau-2025-1-935/dss

Peer Review File: Available at https://tau.amegroups.com/article/view/10.21037/tau-2025-1-935/prf

Funding: This work was supported by National Natural Science Foundation of China (grant Nos. 82303071 and 82400766).

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://tau.amegroups.com/article/view/10.21037/tau-2025-1-935/coif). The authors have no conflicts of interest to declare.

Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. The study was conducted in accordance with the Declaration of Helsinki and its subsequent amendments.

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

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