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中华细胞与干细胞杂志(电子版) ›› 2022, Vol. 12 ›› Issue (04) : 206 -214. doi: 10.3877/cma.j.issn.2095-1221.2022.04.003

论著

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
梁芳1, 刘广申2, 徐艳3,†()   
  1. 1. 410000　长沙，湖南省长沙市第四医院妇产科
    2. 410000　长沙，湖南省长沙市妇幼保健院妇产科
  • 收稿日期:2021-05-19 出版日期:2022-08-01
  • 通信作者: 徐艳

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

Fang Liang1, Guangshen Liu2, Yan Xu3,†()   

  1. 1. Changsha Maternal and Child Health Care Hospital Obstetrics and Gynecology, Changsha 410000, China
    3. Department of Gynecology, the Fourth Hospital of Changsha, Changsha 410000, China
  • Received:2021-05-19 Published:2022-08-01
  • Corresponding author: Yan Xu
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引用本文：

梁芳, 刘广申, 徐艳. 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[J]. 中华细胞与干细胞杂志(电子版), 2022, 12(04): 206-214.

Fang Liang, Guangshen Liu, Yan Xu. LncRNA AC130710 promotes endometrial cancer cell proliferation and epithelial mesenchymal transformation through miR-129-5p/WNT4 axis[J]. Chinese Journal of Cell and Stem Cell(Electronic Edition), 2022, 12(04): 206-214.

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。

关键词: Lnc RNA, miR-129-5P, WNT4, HEC-1A, 细胞增殖, EMT
Objective

To 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.

Methods

The 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.

Results

Compared 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.

Conclusion

LncRNA AC130710 may regulate the proliferation, apoptosis and EMT of endometrial cancer cells (HEC-1A cells) by miR-129-5P/WNT4 axis.

Key words: Lnc RNA, miR-129-5P, WNT4, HEC-1A, Cells proliferation, EMT
表1 引物序列信息
基因 序列（5’-3’） 预期扩增产物长度（bp）
LncRNA AC130710 上游GGGGGCTTGTTTCTTTGTGTAG 246
  下游CATTCGGCTCTGACCTCTCAC  
miR-129-5P 上游ACCCAGTGCGATTTGTCA 223
  下游ACTGTACTGGAAGATGGACC  
U6 上游ACGAATACCGGCGTGAGAAA 412
  下游TCGTGAAAGACCGCAGCAAA  
WNT4 上游ATGTTCTAGGCCATTACGACA 187
  下游GCAGCACCTTCAGCACTCT  
GAPDH 上游CAAGGACCTCTACGCCAACAC 258
  下游TGGAGGCGCGATGATCTT  
表2 检测下调AC130710对细胞活力、凋亡蛋白和EMT相关蛋白表达的影响（±s）
分组 24h细胞活力（%） 48h细胞活力（%） 72h细胞活力（%） Bcl-2 Bax cleaved caspase-3
siRNA NC 58.26±2.12 70.23±2.43 90.14±1.78 1.18±0.14 0.84±0.21 0.37±0.07
AC130710 siRNA 42.16±1.75 59.24±1.82 68.53±2.65 0.38±0.08 1.37±0.14 1.08±0.16
t值 12.168 13.256 12.289 20.152 22.353 19.682
P值 0.011 0.024 0.017 0.005 0.005 0.002
分组 cleaved caspase-9 E-cadherin N-cadherin Snail Vimentin
siRNA NC 0.39±0.06 1.32±0.26 0.85±0.24 1.12±0.18 1.26±0.27
AC130710 siRNA 1.26±0.24 1.87±0.17 0.36±0.04 0.35±0.09 0.42±0.10
t值 23.582 15.435 20.253 23.452 22.856
P值 0.007 0.001 0.005 0.007 0.006
图1 Western blot检测下调LncRNA AC130710对HEC-1A细胞内Bax、Bcl-2、cleaved caspase-3、cleaved caspase-9表达及EMT的影响
表3 不同细胞组双荧光素酶活性检测（±s）
分组 实验次数 WT-AC130710 MUT-AC130710
mimics NC 3 1.37±0.23 1.36±0.23
miR-129-5P mimics 3 0.47±0.08 1.28±0.15
t值   22.674 2.696
P值   0.005 0.062
图2 预测LncRNA AC130710与miR-129-5P之间的结合位点
表4 检测下调miR-129-5P对细胞活力、凋亡蛋白和EMT相关蛋白表达的影响（±s）
分组 实验次数 24h细胞活力（%） 48h细胞活力（%） 72h细胞活力（%） Bcl-2 Bax cleaved caspase-3
inhibitor NC 3 42.68±1.29 63.52±2.26 70.18±1.95 0.65±0.08 0.85±0.13 1.16±0.28
miR-129-5P inhibitor 3 59.62±2.47 78.53±3.14 82.64±3.52 0.98±0.07 0.27±0.08 0.48±0.05
t值   14.352 12.178 13.256 21.532 23.625 20.483
P值   0.026 0.018 0.024 0.006 0.007 0.006
分组 实验次数 cleaved caspase-9 E-cadherin N-cadherin Snail Vimentin
inhibitor NC 3 1.19±0.15 1.08±0.26 0.68±0.09 0.42±0.06 0.54±0.02
miR-129-5P inhibitor 3 0.52±0.14 0.43±0.09 1.39±0.15 0.95±0.08 1.16±0.16
t值   26.854 25.482 23.327 19.685 24.153
P值   < 0.01 0.009 0.007 0.005 0.008
图3 Western blot实验检测下调miR-129-5P对HEC-1A细胞内Bax、Bcl-2、cleaved caspase-3、cleaved caspase-9及EMT的影响
表5 检测LncRNA AC130710通过miR-129-5P对细胞活力、凋亡蛋白和EMT相关蛋白表达的影响（±s）
分组 实验次数 细胞活力（%） Bcl-2 Bax cleaved caspase-3
pcDNA-3.1（+）+mimics NC 3 58.24±1.16 0.95±0.13 0.75±0.06 1.15±0.26
pcDNA-AC130710+mimics NC 3 86.29±2.36 2.15±0.22 0.32±0.04 0.23±0.02
pcDNA-3.1（+）+miR-129-5P mimics 3 37.42±3.12 0.25±0.03 1.32±0.14 2.25±0.12
pcDNA-AC130710+miR-129-5P mimics 3 60.48±2.52 0.98±0.12 0.78±0.08 1.36±0.24
t值   12.786 25.692 27.526 24.356
P值   0.019 0.008 0.009 0.008
分组 实验次数 cleaved caspase-9 E-cadherin N-cadherin Snail Vimentin
pcDNA-3.1（+）+mimics NC 3 0.97±0.05 1.08±0.15 1.18±0.26 0.53±0.12 0.72±0.17
pcDNA-AC130710+mimics NC 3 0.21±0.06 0.46±0.07 2.25±0.34 1.38±0.14 1.42±0.18
pcDNA-3.1（+）+miR-129-5P mimics 3 1.72±0.18 2.18±0.24 0.18±0.06 0.15±0.02 0.38±0.15
pcDNA-AC130710+miR-129-5P mimics 3 1.24±0.19 1.02±0.21 1.29±0.32 0.73±0.21 0.82±0.13
t值   22.157 20.058 23.643 24.353 22.852
P值   0.006 0.006 0.007 0.008 0.007
图4 Western blot实验检测下调miR-129-5P对HEC-1A细胞内Bax、Bcl-2、cleaved caspase-3、cleaved caspase-9表达及EMT的影响注：1为pcDNA-3.1 （+）+ mimics NC；2为pcDNA-AC130710+mimics NC；3为pcDNA-3.1（+）+miR-129-5P mimics；4为pcDNA-AC130710+miR-129-5P mimics
图5 预测miR-129-5P与WNT4之间的结合位点
表6 检测下调WNT4对细胞活力、凋亡蛋白和EMT相关蛋白表达的影响（±s）
分组 实验次数 24h细胞活力（%） 48h细胞活力（%） 72h细胞活力（%） Bax Bcl-2
siRNA NC 3 58.67±1.26 70.26±3.14 80.29±2.45 0.42±0.08 0.84±0.12
WNT4 siRNA 3 49.25±1.68 59.21±2.28 70.12±3.62 0.96±0.12 0.23±0.08
t值   14.352 12.178 13.256 22.436 23.654
P值   0.021 0.018 0.019 0.003 0.004
分组 实验次数 cleaved caspase-3 cleaved caspase-9 E-cadherin N-cadherin Snail Vimentin
siRNA NC 3 0.45±0.06 0.72±0.12 1.26±0.18 1.14±0.17 1.06±0.15 1.13±0.08
WNT4 siRNA 3 1.13±0.25 1.54±0.23 1.87±0.24 0.28±0.09 0.42±0.23 0.35±0.08
t值   20.152 28.743 17.532 29.627 23.752 28.154
P值   0.002 0.008 0.002 0.009 0.005 0.007
图6 Western blot检测下调WNT4对HEC-1A细胞内Bax、Bcl-2、cleaved caspase-3、cleaved caspase-9表达及EMT的影响
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