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Expression of miR-1271-5p in renal cell carcinoma and its effect on proliferation and apoptosis of A-498 cells
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Abstract:
Objective: To investigate the expression of miR-1271-5p in renal cell carcinoma（RCC）tissues and cell lines and its effect on the proliferation and apoptosis of RCC A-498 cell line. Methods: Pathologically confirmed RCC tissues and para-cancerous tissues were collected. Real-time fluorescent quantitative PCR（qPCR）was used to analyze the expression of miR-1271-5p in collected RCC tissues and RCC cell lines (ACHN, A498, HK-2, 786-O, CaKi-1) as well as human embryonic kidney cell line HEK293. miR-1271-5p (experiment group) and miR-NC (control group) were transfected into A-498 cells, spectively. Bioinformatics predicts that DOCK1 is a possible target gene for miR-1271-5p. Double luciferase reporter gene vector of DOCK1 gene 3 'UTR (both wild and mutant type)were constructed and the luciferase activity was detected. The expression of DOCK1 mRNA was detected by qPCR. Western blotting was used to analyze the protein expressions of DOCK1, p-ERK, p-AKT, Bcl-2 and Bax in two groups of cells. MTS assay and colony formation assay were performed to detect cell viability and proliferation. Cell apoptosis was analyzed by flow cytometry. Results：Compared with para-cancerous tissue and human embryonic kidney cells, the expression of miR-1271-5p was significantly decreased in RCC tissues and cell lines (P<0.01). Double luciferase reporter gene system showed that DOCK1 is a target gene of miR-1271-5p (P<0.01). Compared with miR-NC transfected cells, the expression of DOCK1 mRNA in A-498 cells transfected with miR-1271-5p was significantly decreased (P<0.01); the protein expressions of DOCK1, p-ERK, p-AKT and Bcl-2 were significantly down-regulated（P<0.05）while the expression of Bax protein was significantly up-regulated (P<0.05); The viability and colony formation of A-498 cells was significantly decreased (all P<0.01), while apoptosis was ignificantly increased (P<0.01). Conclusion: miR-1271-5p was downregulated in RCC tissues and cell lines. miR-1271-5p significantly inhibited the proliferation and induced apoptosis of RCC A-489 cell by interfering with the expression of DOCK1 gene. This provides a theoretical basis for miR-1271-5p as a promise molecular target in RCC treatment.
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Project Supported:
Project supported by the Natural Science Foundation of Hubei Province（No.2017CFB176）
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