LncRNA AC130710通过miR-129-5P/WNT4轴促进子宫内膜癌细胞增殖和上皮间质转化

目的探讨LncRNA AC130710通过miR-129-5P/WNT4轴对子宫内膜癌细胞（HEC-1A细胞）增殖、凋亡及上皮间质转化（EMT）的影响及机制研究。 方法通过实时荧光定量PCR检测LncRNA AC130710、miR-129-5P和WNT4在子宫内膜癌细胞（HEC-1A细胞）和人子宫内膜上皮细胞（HEEC）中的表达。细胞分别转染（1）siRNA NC、AC130710 siRNA、WNT4 siRNA；（2）inhibitor NC、miR-129-5P inhibitor；（3）pcDNA-3.1 （+）+mimics NC、pcDNA-AC130710+mimics NC、pcDNA-3.1 （+）+miR-129-5P mimics、pcDNA-AC130710+miR-129-5P mimics。MTT实验检测LncRNA AC130710、miR-129-5P和WNT4的表达对HEC-1A细胞增殖能力的影响；Western blot检测LncRNA AC130710、miR-129-5P和WNT4的表达对HEC-1A细胞凋亡相关蛋白B淋巴细胞瘤-2基因相关蛋白X （Bax）、剪切的半胱氨酰天冬氨酸特异性蛋白酶-3 （cleaved caspase-3）、cleaved caspase-9和B淋巴细胞瘤-2基因（Bcl-2）表达的影响；Western blot检测LncRNA AC130710、miR-129-5P和WNT4的表达对HEC-1A细胞EMT的影响。miRanda和双荧光素酶报告基因实验分析LncRNA AC130710和miR-129-5P之间的关系，TargetScan数据库分析miR-129-5P与WNT4的相关性，双荧光素酶报告基因检测miR-129-5P与WNT4的相互作用；RT-qPCR法检测LncRNA AC130710通过miR-129-5P对WNT4表达的影响。两组间比较采用独立样本t检验，多组间比较采用单因素方差分析，两两比较采用LSD-t检验。 结果与HEEC细胞比较，HEC-1A细胞中AC130710表达水平（1.86±0.21比0.85±0.06）、WNT4表达水平（1.88±0.26比1.08±0.12）升高；HEC-1A细胞中miR-129-5P表达水平（0.89±0.16比1.76±0.08）降低。与转染siRNA NC比较，转染AC130710 siRNA细胞内Bax、cleaved caspase-3、cleaved caspase-9、E-cadherin蛋白相对表达水平（1.37±0.14比0.84±0.21），（1.08±0.16比0.37±0.07），（1.26±0.24比0.39±0.06），（1.87±0.17比1.32±0.26）]上升，Bcl-2、N-cadherin、Snail和Vimentin蛋白相对表达水平（0.38±0.08比1.18±0.14），（0.36±0.04比0.85±0.24），（0.35±0.09比1.12±0.18），（0.42±0.10比1.26±0.27）]下降；与转染inhibitor NC比较，转染miR-129-5P inhibitor细胞的Bcl-2、N-cadherin、Snail和Vimentin蛋白相对表达水平（0.98±0.07比0.65±0.08），（1.39±0.15比0.68±0.09），（0.95±0.08比0.42±0.06），（1.16±0.16比0.54±0.02）]上升，Bax、cleaved caspase-3、cleaved caspase-9、E-cadherin蛋白相对表达水平（0.27±0.09比0.85±0.13），（0.48±0.05比1.16±0.28），（0.52±0.14比1.19±0.15），（0.43±0.09比1.08±0.26）]下降；与转染siRNA NC比较，转染WNT4 siRNA细胞的Bcl-2、N-cadherin、Snail和Vimentin蛋白相对表达水平（0.23±0.08比0.84±0.12），（0.28±0.09比1.14±0.17），（0.42±0.23比1.06±0.15），（0.35±0.08比1.13±0.08）]降低，Bax、cleaved caspase-3、cleaved caspase-9、E-cadherin蛋白相对表达水平（0.96±0.12比0.42±0.08），（1.13±0.25比0.45±0.06），（1.54±0.23比0.72±0.12），（1.87±0.24比1.26±0.18）]上升。 结论LncRNA AC130710可通过miR-129-5P/WNT4轴调控子宫内膜癌HEC-1A细胞增殖、凋亡及EMT。

LncRNA AC130710 promotes endometrial cancer cell proliferation and epithelial mesenchymal transformation through miR-129-5p/WNT4 axis

ObjectiveTo investigate the effect of LncRNA AC130710 on proliferation, apoptosis and epithelial mesenchymal transformation (EMT) of endometrial cancer cells (HEC-1A cells) through miR-129-5p/WNT4 axis and its mechanism. MethodsThe expressions of LncRNA AC130710, miR-129-5P and WNT4 in HEC-1A and HEEC cells were detected by real-time quantitative PCR (RT-qPCR) , then the cells were transfected with (1) siRNA NC, AC130710 siRNA and WNT4 siRNA; (2) inhibitor NC, miR-129-5p inhibitor; (3) pcDNA-3.1 (+) + mimics NC, pcDNA-AC130710+ mimics NC, pcDNA-3.1 (+) +miR-129-5p mimics, pcDNA-AC130710+ miR-129-5p mimics. The effects of LncRNA AC130710, miR-129-5P and WNT4 expressions on the proliferation ability of HEC-1A cells were detected by MTT experiment. Western blot was used to detect the effects of LncRNA AC130710, miR-129-5p and WNT4 on the expression of apoptosis-related proteins Bax,cleaved-caspase-3, cleaved-caspase-9 and Bcl-2 in HEC-1A cells. Western blot assay was used to detect the effects of LncRNA AC130710, miR-129-5P and WNT4 expressions on EMT of HEC-1A cells. The targeted relationship between LncRNA AC130710 and miR-129-5P was analyzed by miRanda and double luciferase reporter gene assay. The correlation between miR-129-5P and WNT4 was analyzed by the TargetScan database, and the interaction between miR-129-5P and WNT4 was detected by the double luciferase reporter gene. RT-qPCR detected the effect of LncRNA AC130710 on WNT4 expression through miR-129-5P. ResultsCompared with HEEC cells, AC130710 expression was increased in HEC-1A cells (1.86±0.21 vs 0.85±0.06) . The expression level of miR-129-5p in HEC-1A cells was decreased (0.89±0.16 vs 1.76±0.08) ; Compared with HEEC cells, the expression of WNT4 in HEC-1A cells was increased (1.88±0.26 vs 1.08±0.12) . Compared with the siRNA NC transfection group, the relative expression levels of Bax (1.37±0.14 vs 0.84±0.21) , cleaved caspase-3 (1.08±0.16 vs 0.37±0.07) , cleaved caspase-9 (1.26±0.24 vs 0.39±0.06) , E-cadherin (1.87±0.17 vs 1.32±0.26) in AC130710 siRNA transfection group increased, the relative expression levels of Bcl-2 (0.38±0.08 vs 1.18±0.14) , N-cadherin (0.36±0.04 vs 0.85±0.24) , Vimentin (0.35±0.09 vs 1.12±0.18) and Snail (0.42±0.10 vs 1.26±0.27) decreased. LncRNA AC130710 3'UTR has a direct targeting relationship with miR-129-5P. Compared with the inhibitor NC transfection group, the relative expression levels of Bcl-2 (0.98±0.07 vs 0.65±0.08) , N-cadherin (1.39±0.15 vs 0.68±0.09) , Vimentin (1.16±0.16 vs 0.54±0.02) and Snail (0.95±0.08 vs 0.42±0.06) protein increased, the relative expression levels of Bax (0.27±0.09 vs 0.85±0.13) , cleaved caspase-9 (0.52±0.14 vs 1.19±0.15) , cleaved caspase-3 (0.48±0.05 vs 1.16±0.28) and E-cadherin (0.43±0.09 vs 1.08±0.26) proteins decreased. LncRNA AC130710 overexpression promoted HEC-1A cell proliferation and EMT through miR-129-5P. miR-129-5P specifically binds to the WNT4 3'UTR. Compared with the siRNA NC transfected group, the proliferation ability of WNT4 siRNA relative expression level of Bcl-2 (0.23±0.08 vs 0.84±0.12) , N-cadherin (0.28±0.09 vs 1.14±0.17) , Vimentin (0.35±0.08 vs 1.13±0.08) and Snail (0.42±0.23 vs 1.06±0.15) protein decreased. The relative expression level of Bax (0.96±0.12 vs 0.42±0.08) , cleaved caspase-3 (1.13±0.25 vs 0.45±0.06) , cleaved caspase-9 (1.54±0.23 vs 0.72±0.12) , and E-cadherin (1.87±0.24 vs 1.26±0.18) increased. Downregulation of LncRNA AC130710 inhibits WNT4 expression via miR-129-5P. ConclusionLncRNA AC130710 may regulate the proliferation, apoptosis and EMT of endometrial cancer cells (HEC-1A cells) by miR-129-5P/WNT4 axis.