miR-203逆转结直肠癌细胞对紫杉醇耐药及作用机制研究

REVERSE EFFECT OF MIR-203 ON PACLITAXEL RESISTANCE IN COLORECTAL CANCER CELLS AND ITS MECHANISM

目的探讨miR-203对紫杉醇耐药结直肠癌细胞紫杉醇敏感性的影响及其作用机制。方法利用RT-qPCR检测结直肠癌紫杉醇耐药细胞(HT-29/Taxol)及其亲代细胞(HT-29)中miR-203及SIK2表达水平;利用LipofectamineTM 2000将Pre-miR-203、siRNA-SIK2、pre-miR-203与SIK2的过表达组合载体及相关对照转染至HT-29/Taxol细胞;采用MTT法测定各转染组HT-29/Taxol细胞对紫杉醇的敏感性;Target Scan预测miR-203的潜在靶基因,双荧光素酶实验进行验证;免疫印迹实验(Western blot)测定转染PremiR-203后SIK2蛋白表达水平。结果 HT-29/Taxol细胞中miR-203表达显著下调(P<0.05),SIK2表达显著上调(P<0.01);过表达miR-203及敲低SIK2表达显著增加了HT-29/Taxol细胞对紫杉醇的敏感性(P<0.05);与pre-miR-203单独转染相比,pre-miR-203与SIK2共转染降低了HT-29/Taxol细胞对紫杉醇的敏感性(P<0.05);SIK2是miR-203的靶基因,过表达miR-203显著抑制了SIK2蛋白表达(P<0.05),以上差异均有统计学意义。结论结直肠癌紫杉醇耐药细胞中miR-203表达下调,SIK2表达上调;过表达miR-203及敲低SIK2表达显著提高了HT-29/Taxol细胞对紫杉醇的敏感性;miR-203可能通过靶向抑制SIK2表达影响结直肠癌细胞对紫杉醇的敏感性。

Objective To investigate the effect of miR-203 on paclitaxel sensitivity in paclitaxel-resistant colorectal cancer cells and its mechanism.Methods RT-qPCR was used to determine the expression of miR-203 and SIK2 in human colorectal cancer paclitaxel-resistant cells(HT-29/Taoxol) and its parent cells(HT-29).LipofectamineTM 2000 was used to transfect pre-miR-203,siRNA-SIK2,pre-miR-203 and SIK2 overexpression combination vector and related control into HT-29/Taxol cells;The sensitivity of HT-29/Taxol cells to paclitaxel in transfected groups was determined by MTT assay;Target Scan predicted the potential Target genes of miR-203,which was verified by dual luciferase assay;Western blot assay was used to determine the protein expression level of SIK2 after transfection of pre-miR-203.Results The expression of miR-203 in HT-29/Taxol cells was significantly down-regulated(P<0.05),and the expression of SIK2 was significantly up-regulated(P<0.01).Overexpression of miR-203 and knockdown of SIK2 expression significantly increased the sensitivity of HT-29/Taxol cells to paclitaxel(P<0.05).Compared with pre-miR-203 alone, the sensitivity of HT-29/Taxol cells to paclitaxel was decreased by co-transfection with pre-miR-203 and SIK2(P<0.05).SIK2 is the target gene of miR-203,and the overexpression of miR-203 significantly inhibited the expression of SIK2 protein(P<0.05),the above differences were statistically significant.Conclusion MiR-203 expression was down-regulated and SIK2 expression was up-regulated in paclitaxel-resistant CRC cells.Overexpression of miR-203 and knockdown of SIK2 expression significantly increased the sensitivity of HT-29/Taxol cells to paclitaxel.MiR-203 may affect the sensitivity of colorectal cancer cells to paclitaxel by targeting the inhibition of SIK2 expression.