Vitamin D3 Inhibits Proliferation Of Liver Cancer Cells By Promoting The Expression Of MiR-15a-5p

Backgrounds and purposes:Liver cancer is a malignant tumor which seriously threats human health.The morbidity and mortality of liver cancer have always been at the forefront of malignant tumors.Previous study has shown that vitamin D3(VD3)can play an anti-tumor role by inhibiting the growth of cancer cells,promoting apoptosis,anti-angiogenesis,and repressing invasion and metastasis of tumor cells.The biological regulation mechanism involved needs further study.MiRNAs are a class of non-coding small RNAs of 18-24 bp in length that play function at the post-transcriptional level by regulating downstream target genes.Existing studies have shown that the expression of some miRNAs in gastrointestinal tumors was regulated by 1,25(OH)2D3(1-alpha,25-dihydroxyvitaminD3),the active form of vitamin D3.Our previous studies have found that miR-203a-3p,miR-214-3p,miR-15a-5p played the role of tumor suppressor genes in a variety of tumors,so we first detect whether the expression of these three miRNAs is regulated by 1,25(OH)2D3.The results suggested that 1,25(OH)2D3 can regulate the expression of miR-15a-5p.Bioinformatics analysis shows that miR-15a-5p has a good target binding relationship with E2F3.In this study,we apply qRT-PCR experiments to detect the relationship of 1,25(OH)2D3 and miR-15a-5p.Double luciferase reporter assay is applied to detect the relationship between miR-15a-5p and E2F3.MTT,flow cytometry and transwell assays are performed to analyze the regulation mechanism of VD3-miR-15a-5p-E2F3 axis in the development of liver cancer.Methods:1.MTT,flow cytometry and transwell were used to analyze the effect of 1,25(OH)2D3 on the cell proliferation,cell apoptosis and cell migration of Hep3B and HepG2 cells.Then,we used qRT-PCR to detect the expression levels of miR-203a-3p,miR-214-3p,miR-15a-5p in 1,25(OH)2D3 treated liver cancer cells.2.qRT-PCR was used to detect the expression level of miR-15a-5p in liver cancer and its corresponding normal samples,and the expression level of miR-15a-5p in liver cancer cell lines Hep3B,HepG2 and normal liver cells HL-7702.3.We synthesized miR-15a-5p mimics and inhibitor.MTT,cell colony,flow cytometry,transwell and Western Blot were performed after miR-15a-5p mimics or inhibitor was transfected into Hep3B and HepG2 cells to detect the effect of miR-15a-5p on the cell proliferation,colony formation,cell cycle,cell apoptosis and migration of Hep3B and HepG2 cells.4.E2F3 was predicted as the miR-15a-5p target gene by bioinformatics.Dual luciferase reporter assay and Western Blot were used to detect the targeting relationship between miR-15a-5p and E2F3.5.qRT-PCR was used to detect the expression level of E2F3 in liver cancer and its corresponding normal tissue samples.We silenced the expression of E2F3 by interfering RNA.We performed MTT,cell colony,flow cytometry,transwell and Western Blot assay to detect the effect of silencing E2F3 on the cell proliferation,colony formation,cell cycle,cell apoptosis and migration of Hep3B and HepG2 cells.6.The HepG2 cells infected with LV-ctrl and LV-miR-15a were inoculated under the inguinal skin of nude mice,and the tumor formation experiment in the nude mice was used to verify the effect of miR-15a on tumor formation in vivo.Results:1.Through MTT and cell migration assay,we found that 1,25(OH)2D3 inhibited the proliferation and migration of Hep3B and HepG2 cells.Cell apoptosis assay showed that 1,25(OH)2D3 induced the apoptosis of HepG2 cells.qRT-PCR showed that the expression level of miR-15a-5p significantly increased in Hep3B and HepG2 cells treated with 1,25(OH)2D3.2.miR-15a-5p was decreased in liver cancer tissues and liver cancer cell lines(Hep3B and HepG2)compared with their respective control.3.Overexpression of miR-15a-5p suppressed proliferation,colony formation and migration ability,and caused G1 phase cell cycle arrest,and increased apoptosis in Hep3B and HepG2 cells;Inhibition the expression of miR-15a-5p increased cells proliferation and colony formation ability in Hep3B and HepG2 cells.Inhibition the expression of miR-15a-5p increased the migration ability of Hep3B cells.4.The dual fluorescent reporter gene assay showed that E2F3 was a target gene of miR-15a-5p.5.qRT-PCR showed that E2F3 was highly expressed in liver cancer tissues compared with normal tissues.We silenced the expression of E2F3 by synthesizing si-E2F3.Silencing E2F3 inhibited proliferation,colony formation and migration,and caused G1 phase cell cycle arrest,and promoted apoptosis of liver cancer cell.6.Tumor formation in nude mice showed the weight of the tumors in the LV-miR-15a group was significantly lighter than that of the control group.Conclusion:1.1,25(OH)2D3 inhibited proliferation and migration of Hep3B and HepG2 cells.1,25(OH)2D3 promoted apoptosis of HepG2 cells.1,25(OH)2D3 increased the expression of miR-15a-5p in Hep3B and HepG2 cells.2.miR-15a-5p was significantly decreased in liver cancer tissues and liver cancer cell lines compared with their respective control.3.miR-15a-5p inhibited proliferation and migration,and promoted apoptosis of liver cancer cells by targeting E2F3.4.Silencing E2F3 inhibited proliferation and migration,and promoted apoptosis of liver cancer cells.5.miR-15a inhibited the growth of tumors in vivo.