干扰LncRNA MLK7-AS1对宫颈癌顺铂耐药细胞增殖及凋亡的影响

The effect of interfering with LncRNA MLK7-AS1 on the proliferation and apoptosis of cervical cancer cell lines resistant to cisplatin

目的探讨干扰LncRNA MLK7-AS1对宫颈癌顺铂耐药细胞增殖、凋亡的影响及其可能作用机制。方法采用低浓度加量持续诱导法建立宫颈癌顺铂耐药细胞SiHa/cDDP,qRT-PCR法检测宫颈癌细胞中MLK7-AS1、miR-143表达量;si-NC、si-MLK7-AS1、si-MLK7-AS1+NC、si-MLK7-AS1+miR-143 inhibitor分别转染入SiHa/cDDP细胞,加含顺铂的培养液(4μg/mL)培养48h。MTT实验与平板克隆形成实验检测增殖能力;流式细胞术检测凋亡率;双荧光素酶报告实验检测MLK7-AS1与miR-143的靶向关系。结果与ECT1/E6E7细胞比较,SiHa细胞、SiHa/cDDP细胞中MLK7-AS1表达水平升高(P<0.05),miR-143表达水平降低(P<0.05)。与SiHa细胞比较,SiHa/cDDP细胞中MLK7-AS1表达水平升高(P<0.05),miR-143表达水平降低(P<0.05)。与转染si-NC比较,转染si-MLK7-AS1可明显提高miR-143表达水平、凋亡率(P<0.05),降低细胞存活率(P<0.05),减少克隆形成数(P<0.05)。双荧光素酶报告实验证实MLK7-AS1能靶向调控miR-143。共转染si-MLK7-AS1与miR-143 inhibitor可明显提高细胞存活率(P<0.05),增强克隆形成能力(P<0.05),降低凋亡率(P<0.05)。结论干扰MLK7-AS1表达可通过上调miR-143而抑制宫颈癌顺铂耐药细胞增殖及诱导细胞凋亡进而增强细胞对顺铂的敏感性。

Objective:To explore the effect of interfering with LncRNA MLK7-AS1 on the proliferation and apoptosis of cisplatin-resistant cervical cancer cells and its possible mechanism.Methods:The low concentration and continuous induction method was used to establish the cisplatin-resistant cervical cancer cell SiHa/cDDP.qRT-PCR method was used to detect the expressions of MLK7-AS1 and miR-143 in cervical cancer cells.si-NC,si-MLK7-AS1,si-MLK7-AS1 and NC,si-MLK7-AS1 and miR-143 inhibitor were respectively transfected into SiHa/cDDP cells and then added with cisplatin-containing culture medium(4μg/mL)cultivate for 48h.MTT experiment and plate clone formation experiment were used to detect cell proliferation ability.Flow cytometry was used to detect the apoptosis rate.The dual luciferase reporter experiment was used to detect the targeting relationship between MLK7-AS1 and miR-143.Results:Compared with ECT1/E6E7 cells,the expression level of MLK7-AS1 in SiHa cells and SiHa/cDDP cells was increased(P<0.05),and the expression level of miR-143 was decreased(P<0.05).Compared with SiHa cells,the expression level of MLK7-AS1 in SiHa/cDDP cells was increased(P<0.05),and the expression level of miR-143 was decreased(P<0.05).Compared with transfection of si-NC,transfection of si-MLK7-AS1 could significantly increase the expression level of miR-143,apoptosis rate(P<0.05),reduce cell survival rate(P<0.05),and reduce the number of colonies formed(P<0.05).The dual luciferase report experiment confirmed that MLK7-AS1 could target miR-143.Co-transfection of si-MLK7-AS1 and miR-143 inhibitor could significantly increase cell survival rate(P<0.05),enhance clone formation ability(P<0.05),reduce apoptosis rate(P<0.05).Conclusion:Interfering with the expression of MLK7-AS1 could inhibit the proliferation of cisplatin-resistant cervical cancer cells and induce apoptosis by up-regulating miR-143,thereby enhancing the sensitivity of cells to cisplatin.