[Mechanism of miR-199a-5p prevents migration and invasion of cholangiocarcinoma by targeting hypoxia-inducible factor 1α].

Objective: To investigate the expression of miR-199a-5p in intrahepatic cholangiocarcinoma (ICC) and its regulatory mechanism in tumor invasion and metastasis. Methods: Tumor tissues and adjacent normal tissues from 38 ICC patients (aged 40-80 years, including 14 males) were collected at the First Affiliated Hospital of Nanhua University from January 2020 to April 2023. Differential genes and target genes were screened using transcriptome sequencing and multi-database bioinformatics analysis. Transcriptome sequencing was performed to detect the expression of miR-199a-5p and hypoxia-inducible factor 1α (HIF-1α). At the same time, clinical and pathological data such as patients' TNM staging and pathological differentiation were collected to analyze the correlation between the expression levels of miR-199a-5p and HIF-1α and clinicopathological features. Transcriptome sequencing was used to detect the RNA expression of miR-199a-5p and HIF-1α in tumor tissues and adjacent non-cancerous tissues of ICC patients. Immunohistochemistry was employed to detect HIF-1α expression in the tissues. Human normal bile duct epithelial cells, HuCC-T1 and RBE cell lines were cultured and used. The targeting relationship between miR-199a-5p and HIF-1α was verified by dual-luciferase reporter assay. The expression levels of miR-199a-5p and HIF-1α at the cellular level were detected using real-time quantitative PCR (qRT-PCR). RBE cells were divided into three groups through transfection using Lipofectamine 3000 reagent: control group, miR-199a-5p mimic group, and miR-199a-5p inhibitor group. The transfection efficiency and HIF-1α mRNA expression levels were assessed by qRT-PCR. Western blot analysis was used to detect the protein expression levels of HIF-1α, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), proliferating cell nuclear antigen (PCNA), matrix metalloproteinase 9 (MMP9), and Cleaved caspase3. Cell migration and invasion abilities were assessed using wound healing and Transwell assays. HIF-1α knockdown cell models were established in RBE cells by transfecting them with HIF-1α-specific small interfering RNA (si-HIF-1α), with a negative control group (si-NC) set up simultaneously. After verifying the knockdown efficiency by qRT-PCR and Western blotting, changes in biological behavior of cholangiocarcinoma cells following HIF-1α suppression were observed by examining the expression of proliferation-related protein PCNA, performing wound healing assays, and conducting Transwell migration and invasion assays. This was aimed at clarifying the central role of HIF-1α in cholangiocarcinoma migration and invasion, further supporting that miR-199a-5p exerts its tumor-suppressive effect by targeting and inhibiting HIF-1α. All experimental data were repeated three times. Results: Transcriptome sequencing and multi-database analysis identified miR-199a-5p and HIF-1α as targets. Dual-luciferase reporter assays confirmed that miR-199a-5p directly targets the 3'-UTR of HIF-1α. Transcriptome sequencing tissue expression analysis showed that compared with adjacent non-cancerous tissue, the expression of miR-199a-5p was decreased in ICC (FPKM: 1.24±0.31 vs 3.87±0.56), while the expression of HIF-1α was significantly increased (FPKM: 18.76±6.23 vs 56.15±8.42) (both P<0.05). Immunohistochemistry results showed high expression of HIF-1α in ICC. Analysis of clinical pathological features indicated that ICC patients in the low miR-199a-5p expression group and the high HIF-1α expression group both had later TNM staging and poorer pathological differentiation (both P<0.05). Mechanistically, overexpression of miR-199a-5p downregulated PCNA and MMP9 and upregulated Cleaved caspase 3, while inhibition of miR-199a-5p led to the opposite changes (all P<0.05). Overexpression of miR-199a-5p inhibited the expression of HIF-1α and suppressed cell migration and invasion, whereas inhibition of miR-199a-5p produced the opposite effects (all P<0.05). After knocking off HIF-1α, both the number of migrating and invading cells decreased (both P<0.05). Conclusion: miR-199a-5p is downregulated in ICC and suppresses tumor migration and invasion by directly targeting HIF-1α and regulating downstream molecules involved in proliferation, invasion and apoptosis.