MiR-150-5p靶向HIF1α调控结直肠癌细胞西妥昔单抗耐药的相关机制研究

Relevant Mechanism of MiR-150-5p Targeting HIF1αto Regulate Cetuximab Resistance in Colorectal Cancer Cells

目的探讨miR-150-5p调控结直肠癌西妥昔单抗敏感性可能的机制。方法将miR-150-5p的模拟物、miR-150-5p的对照组分别转染到结直肠癌细胞株HTC8细胞中,然后加入300 mg/L的西妥昔单抗。首先通过RT-qPCR检测转染之后miR-150-5p在各组的表达水平,然后运用CCK-8分别比较HTC8细胞在转染miR-150-5p模拟组、转染miR-150-5p对照组以及西妥昔单抗+miR-150-5p模拟物组的细胞增殖情况。RT-qPCR检测各组缺氧诱导因子(hypoxia inducible factor-1 alpha,HIF-1α)的mRNA水平,Western blot检测各组HIF1α的蛋白水平。流式细胞术检测各组细胞的凋亡情况。双荧光素酶报告基因实验验证miR-150-5p与HIF-1α之间作用。结果过表达miR-150-5p加西妥昔单抗处理组显著抑制细胞株HTC8的增殖(P<0.05)并促进其凋亡(P<0.05),进一步降低HIF1αmRNA和蛋白的表达(P<0.05),双荧光素酶报告基因实验证明HIF-1α是miR-150-5p的直接作用靶点。结论MiR-150-5p可能通过靶向HIF-1α调控HTC8细胞的增殖与凋亡,同时miR-150-5p可增加结肠癌细胞对西妥昔单抗的敏感性。

Objective To investigate the possible mechanism of miR-150-5p in regulating the sensitivity of cetuximab in colorectal cancer.Methods The miR-150-5p mimic and miR-150-5p control group were transfected into colorectal cancer cell line HTC8 cells,respectively,and then 300 mg/L cetuximab was added.First,the expression level of miR-150-5p in each group after transfection was detected by RT-qPCR.Then,CCK-8 was used to compare the proliferation of HTC8 cells in the transfected miR-150-5p mimic group,the transfected miR-150-5p control group and the cetuximab+miR-150-5p mimic group.RT-qPCR was used to detect the mRNA levels of hypoxia inducible factor-1 alpha(HIF-1α),and Western blot was used to detect the protein level of HIF1α.Flow cytometry was used to detect the apoptosis of cells.The interaction between miR-150-5p and HIF-1αwas verified by dual luciferase reporter gene experiment.Results Over-expression of miR-150-5p plus cetuximab treatment group significantly inhibited the proliferation of cell line HTC8(P<0.05)and promoted its apoptosis(P<0.05),further reducing the expression of HIF1αmRNA and protein(P<0.05).The dual luciferase reporter gene experiments proved that HIF-1αwas a direct target of miR-150-5p.Conclusion MiR-150-5p may regulate the proliferation and apoptosis of HTC8 cells by targeting HIF-1α.Meanwhile,miR-150-5p could increase the sensitivity of colon cancer cells to cetuximab.