FOXO4过表达慢病毒载体构建及顺铂耐药稳定细胞株的建立

Construction of FOXO4 overexpressing lentivirus vector and establishment of cisplatin resistance stable cell line

目的构建稳定过表达叉头框基因O4(FOXO4)的顺铂耐药细胞株MDA-MB-231/DDP,探讨FOXO4在三阴性乳腺癌(TNBC)细胞顺铂耐药中的作用,为改善TNBC顺铂耐药提供理论基础。方法通过药物剂量递增法诱导乳腺癌细胞MDA-MB-231获得顺铂耐药性,CCK-8法确定细胞的耐药指数。将目的基因FOXO4定向插入载体质粒GV492中构建重组慢病毒表达载体LV-FOXO4-OERNA。通过预实验选取助感液类型、确定感染的最佳转染复数值(MOI)以及嘌呤霉素筛选的最佳浓度。将重组慢病毒表达载体感染MDA-MB-231/DDP细胞,嘌呤霉素最佳浓度筛选FOXO4过表达细胞株(FOXO4-OERNA组)。qRT-PCR和Western blotting检测目的基因表达情况,测定不同组IC 50确定细胞对顺铂敏感性。结果成功建立乳腺癌顺铂耐药细胞株(耐药指数=5.231)。与亲本细胞株相比,MDA-MB-231/DDP耐药细胞株FOXO4 mRNA表达水平显著降低(P<0.05)。通过基因测序证明,序列与预计序列一致,重组慢病毒表达载体构建成功,通过嘌呤霉素成功筛选出FOXO4稳定表达株。通过过表达FOXO4可以降低MDA-MB-231细胞对顺铂的耐药性,FOXO4-OERNA组耐药指数(RI=3.063),逆转指数达到1.708。结论通过药物剂量递增法可建立MDA-MB-231顺铂耐药细胞株,过表达FOXO4可逆转MDA-MB-231/DDP细胞对顺铂的耐药性。

Objective A cisplatin-resistant cell line,MDA-MB-231/DDP and stably overexpressing forkhead box gene 4(FOXO4)was constructed to explore the role of FOXO4 in cisplatin resistance of triple-negative breast cancer(TNBC)cells and to provide a cellular model in targeting FOXO4 for chemotherapy resistance.Methods Cisplatin resistance was obtained by induction of MDA-MB-231 in breast cancer cells by drug dose escalation method,and the resistance index of the cells were determined by CCK-8 method.The recombinant lentiviral expression vector LV-FOXO4-OERNA was constructed by inserting the target gene FOXO4 directionally into the vector plasmid GV492.Pre-experiments were performed to select the type of helper solution,determine the optimal MOI value for infection,and the optimal concentration for puromycin screening.The recombinant lentiviral expression vector was infected with MDA-MB-231/DDP cells,and the optimal concentration of puromycin was screened for FOXO4 overexpression cell lines(FOXO4-OERNA group).The expression of target genes were detected by qRT-PCR and Western blotting,and IC 50 of different groups were determined to determine the sensitivity of cells to cisplatin.Results A breast cancer cisplatin-resistant cell line(resistance index=5.231)was successfully established.The level of FOXO4 mRNA expression was significantly lower in the MDA-MB-231/DDP resistant cell line compared to the parental cell line(P<0.05).It was proved by gene sequencing that the sequence was consistent with expected sequence;the recombinant lentiviral expression vector was successfully constructed;and the FOXO4 stable expression strain was successfully screened by puromycin.The resistance of MDA-MB-231 cells to cisplatin could be reduced by overexpression of FOXO4,and the resistance index(RI=3.063)of FOXO4-OERNA group reached 1.708 with reversal index.Conclusion MDA-MB-231 cisplatin-resistant cell lines could be established by drug dose escalation,and overexpression of FOXO4 could reverse the cisplatin resistance of MDA-MB-231/DDP cells.