MiR-144-3p调控E2F8对肾透明细胞癌增殖、凋亡的影响

Effect of MiR-144-3p Regulation of E2F8 on Proliferation and Apoptosis in Renal Clear Cell Carcinoma

目的研究miR-144-3p对肾透明细胞癌(KIRC)增殖、凋亡的影响并探究其分子机制.方法检测肾小管上皮细胞HK2和KIRC细胞系786-O、ACHN、OS-RC-2中miR-144-3p、E2F8 mRNA和E2F8蛋白的表达差异;检测KIRC组织和癌旁组织中miR-144-3p和E2F8 mRNA的表达差异.采用荧光素酶报告实验验证E2F8 mRNA和miR-144-3p的互作关系.分别在786-O细胞中过表达miR-144-3p和沉默E2F8,采用WB检测增殖、凋亡相关蛋白的表达;同时在786-O细胞中过表达miR-144-3p和过表达E2F8,并检测增殖、凋亡相关蛋白的表达;分别采用CCK8法和流式细胞术检测上述转染细胞的增殖和凋亡.结果与HK2细胞相比,KIRC细胞系786-O、ACHN、OS-RC-2中miR-144-3p表达水平较低(P<0.001),与E2F8表达水平呈负相关;与癌旁组织相比,KIRC组织中miR-144-3p表达水平较低(P<0.001),E2F8表达水平较高(P<0.01).过表达miR-144-3p可以抑制786-O细胞增殖并促进其细胞凋亡,沉默E2F8也可以达到相同性质的效果.过表达E2 F8可以逆转过表达miR-144-3p对786-O细胞增殖和凋亡的影响.荧光素酶报告实验证实miR-144-3p靶向调控基因E2F8.结论miR-144-3p可以通过调控基因E2F8抑制KIRC肿瘤细胞786-O的增殖和促进其细胞凋亡.

Objective To explore the effect of miR-144-3p on proliferation and apoptosis in kidney renal clear cell carcinoma(KIRC)and its molecular mechanisms.Methods The expression of miR-144-3p,E2F8 mRNA and E2F8 mRNA protein was measured in renal tubular epithelial cells(HK2)and KIRC cell lines(E2F786-O,ACHN and OS-RC-2).In addition,the expression of miR-144-3p and E2F8 mRNA was measured in KIRC and paracancerous tissues.The interaction between E2F8 mRNA and miR-144-3p was verified by luciferase assay.In 786-O cells,miR-144-3p was overexpressed,E2F8 was silenced,or both miR-144-3p and E2F8 were overexpressed.The expression of proliferation-and apoptosis-related proteins was detected by Western blot.Furthermore,the proliferation and apoptosis of the transfected cells were detected by CCK8 assay and flow cytometry,respectively.Results The expression of miR-144-3p in HK2 cells was higher than that in 786-O,ACHN or OS-RC-2 cells(P<0.001),and its expression was negatively correlated with the expression of E2F8.Compared with the adjacent normal tissues,miR-144-3p expression decreased and E2F8 expression increased in KIRC(P<0.001).Both miR-144-3p overexpression and E2F8 silence inhibited the proliferation and promoted the apoptosis in 786-O cells.Overexpression of E2F8 reversed the effect of miR-144-3p overexpression on proliferation and apoptosis of 786-O cells.Luciferase reporter assay confirmed that miR-144-3p directly targeted E2F8 gene.Conclusion miR-144-3p can inhibit the proliferation and promote the apoptosis in KIRC 786-O cells by regulating E2F8 gene.