小分子干扰RNA抑制TNC基因的表达并逆转卵巢癌紫杉醇耐药

目的 研究小分子干扰RNA(siRNA)介导的RNA干扰(RNAi)对卵巢癌紫杉醇耐药细胞TNC基因表达的影响,探讨TNC基因在紫杉醇耐药中的功能.方法 用脂质体转染剂LipofectamineTM2000将针对TNC基因特异性合成的siRNA转染SKOV3/TAX30细胞,分别在转染后24、48、72 h收集细胞,检测各组细胞TNC mRNA和TNC蛋白的表达水平.siRNA转染SKOV3/TAX30细胞后,采用MTT法检测细胞对紫杉醇的半数抑制浓度(IC50).Western Blot检测SKOV3/TAX30及SKOV3/TAX300两种耐药细胞Wnt信号通路中关键蛋白β-catenin及下游Cyclin D、E-cadherin蛋白的表达.结果 siRNA转染SKOV3/TAX30细胞24、48、72 h后,TNC mRNA和TNC蛋白表达水平均下降,SKOV3/TAX30细胞对紫杉醇药物的IC50下降.TNC表达升高的耐药细胞中,Wnt信号通路中关键蛋白β-catenin及下游Cyclin D蛋白表达上调,E-cadherin蛋白表达下调.结论 针对TNC基因特异性合成的siRNA可激发RNAi介导的TNC沉默,并且逆转了紫杉醇化疗耐药,TNC表达上调与紫杉醇耐药相关,TNC可能通过Wnt信号通路的激活参与了化疗耐药.

Inhibition of TNC gene expression and reversal of drug resistance via small interference RNA in paclitaxel resistant cell line

Objective To study the effect of small interference RNA (siRNA)-mediated RNAi on TNC gene expres-sion and to explore the function of the TNC gene in paclitaxel resistant cells. Method Specifically synthetic siRNA was transfected into SKOV3/TAX30 cells using LipofectamineTM2000. Cells were collected at 24, 48 and 72 h after transfec-tion. TNC mRNA and TNC protein expression in cells were measured. IC50 of paclitaxel resistant cells was measured us-ing MTT assay. The expression of the critical proteinβ-catenin and the downstream proteins Cyclin D and E-cadherin in the Wnt signaling pathway was measured using Western Blot in SKOV3/TAX30 and SKOV3/TAX300 cells. Result In 24, 48 and 72 h after the transfection of synthetic siRNA into SKOV3/TAX30 cells, the TNC gene demonstrated de-creased mRNA and protein levels. SKOV3/TAX30 cells demonstrated reduced IC50 in paclitaxel resistant cells. Among drug resistant cells that had overexpressed TNC, the level ofβ-catenin and Cyclin D increased while the level of E-cad-herin decreased. Conclusion Synthetic siRNA can be applied to activate the silence of the RNAi-mediated TNC gene and reverse chemo-resistance. Overexpression of TNC was related to paclitaxel resistance of ovarian cancer cells. TNC may modulate drug resistance by activating the Wnt signaling pathway.