环状RNA MYLK对子宫内膜癌细胞生长凋亡和侵袭及上皮间质转换的影响

Effects of circRNAMYLK on growth,apoptosis,invasion and epithelial-mesenchymal transformation of endometrial cancer cells

目的探讨环状RNA MYLK干扰对子宫内膜癌HEC-1A细胞增殖、凋亡、侵袭和上皮间质转化(EMT)的作用及对小鼠异种移植瘤生长的影响。方法MYLK-shRNAs转染HEC-1A细胞,构建MYLK干扰细胞模型。将转染后的HEC-1A细胞皮下注射裸鼠,建立移植瘤裸鼠模型。体外实验分为对照组、shRNA-NC组、circMYLK-shRNA1组、circMYLK-shRNA2组和circMYLK-shRNA3组,小鼠实验取shRNA-NC组和circMYLK-shRNA1组。RT-qPCR检测MLYK的表达并筛选出干扰效率高的干扰链;克隆形成实验检测细胞克隆形成情况;流式细胞术检测细胞凋亡;Transwell实验检测细胞侵袭;Western Blot检测EMT相关蛋白E-cadherin、N-cadherin、Vimentin的表达;免疫组化检测肿瘤组织的Ki67和Vimentin的表达。结果三种shRNAs干扰MYLK,shRNA1干扰效率最佳,用于后续实验。与对照组比较,circMYLK-shRNA1组的克隆形成率显著降低(P<0.05),细胞凋亡率显著升高(P<0.05),侵袭细胞数量显著减少(P<0.05),E-cadherin的表达显著上调(P<0.05),N-cadherin和Vimentin的表达显著下调(P<0.05)。体内实验中,与shRNA-NC组比较,circMYLK-shRNA1组肿瘤体积显著减小(P<0.05),肿瘤重量显著减轻(P<0.05),组织MYLK的表达显著下调(P<0.05),组织Ki67和Vimentin的表达显著下调(P<0.05)。结论MYLK干扰抑制了子宫内膜癌HEC-1A细胞的增殖、凋亡、侵袭和上皮间质转化,也阻碍了小鼠异种移植瘤的生长。

Objective To investigate the effects of circRNAs MYLK interference on the proliferation,apoptosis,invasion and epithelial-mesenchymal transformation(EMT)of endometrial carcinoma HEC-1A cells and on the growth of xenograft tumors in mice.Methods MYLK-shRNAs were transfected into HEC-1A cells and MYLK interference cell model was established.The transfected HEC-1A cells were subcutaneously injected into nude mice to establish a xenograft model.In vitro experiments was grouped as Control,shRNA-NC,circMYLK-shRNA1,circMYLK-shRNA2 and circMYLK-shRNA3;in vivo experiment was grouped as shRNA-NC and circMYLK-shRNA1.The expression of MLYK was detected by RT-qPCR to verify the most effective silencing sequence.The clone formation experiment detected the cell clone formation;Flow cytometry was used to detect apoptosis.Cell invasion was detected by Transwell assay.Expression of EmT-related proteins E-cadherin,N-cadherin and Vimentin were detected by Western Blot.Immunohistochemistry was used to detect the expression of Ki67 and Vimentin in tumor tissues.Results Three shRNAs interfered with MYLK and shRNA1 with the best efficiency.Compared with the control group,the clone formation rate of MYLK-shrNA1 group was significantly lower(P<0.05).The apoptosis rate was significantly increased(P<0.05).The number of invaded cells was significantly reduced(P<0.05).E-cadherin expression was significantly up-regulated(P<0.05),while N-cadherin and Vimentin expression was significantly down-regulated(P<0.05).In vivo,compared with shRNA-NC,MYLK-shRNA1 significantly reduced tumor volume(P<0.05)and tumor weight(P<0.05).The expression of MYLK was significantly down-regulated(P<0.05).The expression of Ki67 and Vimentin in tissues was significantly down-regulated(P<0.05).Conclusion MYLK interference inhibited the proliferation,apoptosis,invasion and epithelial-mesenchymal transformation of endometrial carcinoma HEC-1A cells,and also hindered the growth of xenograft tumors in mice.