沉默MAL2对乳腺癌细胞增殖和药物敏感性的影响

Effects of silencing MAL2 on proliferation and drug sensitivity of breast cancer cells

目的探讨沉默髓鞘和淋巴细胞蛋白2(MAL2)基因对乳腺癌MDA-MB-231和MCF-7细胞增殖和药物敏感性的影响,同时研究乳腺癌细胞对阿霉素(DOX)和顺铂(DDP)的耐药机制。方法利用数据库分析MAL2基因在正常组织、癌旁组织和乳腺癌组织中的表达及与预后的相关性;采用蛋白印迹(Western blot)法检测MAL2在人正常乳腺上皮细胞MCF-10A和乳腺癌MDA-MB-231、MCF-7及MDA-MB-468细胞中的蛋白表达;利用转染干扰序列沉默乳腺癌MDA-MB-231和MCF-7细胞中MAL2的表达,根据序列不同分为对照组(siRNA-NC组)和实验组(siRNA-MAL2-1组、siRNA-MAL2-2组及siRNA-MAL2-3组);选取沉默效果最好的实验组(siRNA-MAL2-3组)序列进行慢病毒构建及实验,沉默乳腺癌MDA-MB-231和MCF-7细胞中MAL2的表达,分为sh-NC组(序列同siRNA-NC组)和sh-MAL2组(序列同siRNA-MAL2-3组),采用Western blot法检测MAL2的蛋白表达,CCK8法和平板克隆形成实验检测细胞的增殖能力;CCK8法、流式细胞术及Western blot检测沉默MAL2基因的表达后乳腺癌细胞对DOX和DDP的敏感性;Western blot检测p-AKT/m-TOR通路相关蛋白[磷酸化蛋白激酶B(p-AKT)、雷帕霉素靶蛋白(m-TOR)、磷酸化雷帕霉素靶蛋白(p-mTOR)]的表达情况。结果生物信息学分析显示MAL2在乳腺癌组织中高表达、且其高表达和患者预后不良相关(P<0.05),MAL2在乳腺癌细胞系中高表达(P<0.01);与siRNA-NC组相比,siRNA-MAL2-3组的MAL2蛋白沉默效果较好(P<0.01);感染慢病毒实验结果表明,与sh-NC组比较,sh-MAL2组MAL2表达被抑制(P<0.05);与sh-NC组相比,sh-MAL2组细胞增殖能力无变化(P>0.05)、对DOX和DDP的敏感性增强(P<0.05)、p-AKT和p-mTOR蛋白表达下调(P<0.05)。结论沉默MAL2对乳腺癌细胞增殖能力没有影响,但可促进乳腺癌细胞对DOX和DDP的敏感性,其机制可能与AKT/m-TOR通路相关蛋白被抑制有关。

Objective To investigate the effects of Myelin and lymphocyte protein 2(MAL2)gene silencing on proliferation and drug sensitivity of MDA-MB-231 and MCF-7 in breast cancer cells,in order to explore the mechanism of drug resistance[to Doxorubicin(Dox)and Cisplatin(DDP)].Methods The expression of MAL2 gene in healthy tissues,paracancerous tissues and breast cancer tissues and its correlation with prognosis were analyzed using database.Western blot was used to detect the protein expression of MAL2 in human normal breast epithelial cells MCF-10A and breast cancer cells MDA-MB-231,MCF-7 and MDA-MB-468.MAL2 in MDA-MB-231 and MCF-7 cells was silenced by interference sequence transfection and lentivirus infection.The control group was of the same sequence with siRNA-NC group while the experimental groups belong to the sequence of siRNA-MAL2-1,siRNA-MAL2-2,and siRNA-MAL2-3 groups.The expression of MAL2 of MDA-MB-231 and MCF-7 cells in breast cancer was silenced and divided into sh-NC group(constructed from the sequence of siRNA-NC group)and sh-MAL2 group(constructed from the sequence of siRNA-MAL2-3 group).Western blot was used to detect the expression of MAL2 protein.The proliferative ability of cells was detected by CCK8 assay and cloning formation assay.The effect of breast cancer cells in silencing MAL2 gene expression on the sensitivity of doxorubicin(DOX)and cisplatin(DDP)was detected by CCK8,Flow cytometry(FCM)and Western blot.Western blot was also used to detect the expression of AKT/m-TOR pathway related proteins[phosphorylated protein kinase B(p-AKT),mammalian target of rapamycin(m-TOR),phospho-mammalian target of rapamycin(p-mTOR)].Results Bioinformatics analysis showed that MAL2 was highly expressed in breast cancer tissues,and its high expression was closely correlated with poor prognosis of patients with breast cancer(P<0.05).MAL2 was highly expressed in breast cancer cell lines(P<0.01).Compared with the siRNA-NC group,the siRNA-MAL2-3 group had better silencing effect of MAL2 protein(P<0.01).The lentivirus infection showed that MAL2 expression was inhibited in sh-MAL2 group,compared with sh-NC group(P<0.05).Compared with sh-NC group,the cell proliferation capacity of sh-MAL2 group had no change(P>0.05),the sensitivity of sh-MAL2 group to DOX and DDP increased(P<0.05),and the protein expression of P-Akt and P-mtor in sh-MAL2 group was down-regulated(P<0.05).Conclusion MAL2 silencing has no effect on the proliferation of breast cancer cells,but can promote their sensitivity to DOX and DDP.The mechanism may be associated with the inhibition of AKT/m-TOR pathway related proteins.