长链非编码RNA-MALAT1调控miR-22/snail轴对胃癌增殖、迁移及侵袭的影响

Effects of long non-coding RNA MALAT1 on the proliferation，migration，and invasion of gastric cancer through regulating the miR-22/snail axis

摘要:

目的：探讨长链非编码RNA MALAT1（long non-coding RNA MALAT1，lnc RNA MALAT1）调控miR-22/snail轴对胃癌增殖、迁移及侵袭的影响。方法：采用siRNA干扰技术抑制MALAT1表达，同时过表达miR-22和Snail；采用RT-PCR检测MALAT1，miR-22和snail在胃癌细胞系中的表达水平（n=3）；采用Western blot检测NC组、si-MALAT1组中snail蛋白的表达水平（n=3）；采用CCK-8法在12、24、48、72 h检测NC组、si-MALAT1组、miR-22组、miR-22+snail组、si-MALAT1+miR-22组中细胞吸光度值（n=3）；采用划痕实验检测NC组、si-MALAT1组、miR-22组迁移速度（n=3）；采用Transwell检测NC组、si-MALAT1组、miR-22组、miR-22+snail组、si-MALAT1+miR-22组穿过膜细胞的个数（n=3）；采用双荧光素酶检测NC+pMIR-MALAT1-WT组、NC+pMIR-MALAT1-MUT组、miR-22+pMIR-MALAT1-WT组、miR-22+pMIR- MALAT1-MUT、NC+ pMIR-Snail-WT组、NC+pMIR-Snail-MUT组、miR-22+pMIR-Snail-WT组、miR-22+pMIR-Snail-MUT组的荧光活性（n=3）。两独立样本间的比较采用t检验，多组数据之间的比较采用单因素方差进行分析，涉及时间因素时采用重复测量方差分析。结果：MALAT1在胃癌细胞系中的表达水平明显高于正常胃黏膜上皮细胞（均P=0.000）；低表达MALAT1后，AGS和SGC-7901增殖能力（P1=0.001，P2=0.005），迁移能力（P1=0.018，P2=0.007），侵袭能力（P1=0.024，P2=0.015）均降低；同时双荧光素酶结果显示MALAT1能够靶向结合miR-22（P=0.001），miR-22能够靶向结合snail（P=0.001）；miR-22在胃癌细胞中的表达水平明显低于正常胃黏膜上皮细胞（均P<0.05）；过表达miR-22后，AGS和SGC-7901增殖能力（P1=0.004，P2=0.009），迁移能力（P1=0.034，P2=0.005），侵袭能力（P1=0.037，P2=0.011）均降低；在低表达MALAT1和过表达miR-22后能够更进一步增加低表达MALAT1后对AGS和SGC-7901的增殖（P1=0.022，P2=0.006）和侵袭（P1=0.024，P2=0.015）的抑制作用；同时过表达miR-22和snail后能够反转miR-22对AGS和SGC-7901的增殖（P1=0.003，P2=0.004）和侵袭（P1=0.015，P2=0.01）的抑制作用。结论：MALAT1在胃癌细胞中明显高表达，并能通过调控miR-22/snail轴促进胃癌增殖、迁移及侵袭。

Abstract:

Objective：To investigate the effects of long non-coding RNA MALAT1 on the proliferation，migration，and invasion of gas－tric cancer through regulating the miR-22/snail axis. Methods：The expression of MALAT1 was inhibited by siRNA technology，and miR-22 and snail were overexpressed；real-time PCR was used to determine the expression levels of MALAT1，miR-22，and snail in gastric cancer cell lines（n=3）；Western blot was used to determine the expression level of snail protein in NC group and si-MALAT1 group（n=3）；CCK8 assay was used to determine the cell absorbance values in NC group，si-MALAT1 group，miR-22 group，miR-22+snail group，and si-MALAT1+miR-22 group at 12 h，24 h，48 h，and 72 h（n=3）；wound healing assay was used to determine the mi－gration rate in NC group，si-MALAT1 group，and miR-22 group（n= 3）；transwell assay was used to determine the number of trans－membrane cells in NC group，si-MALAT1 group，miR-22 group，miR-22+snail group，and si-MALAT1+miR-22 group（n=3）；dual luciferase assay was used to determine the fluorescence activity in NC+pMIR-MALAT1-WT group，NC+pMIR-MALAT1-MUT group，miR-22+pMIR-MALAT1-WT group，miR-22+pMIR-MALAT1-MUT group，NC+pMIR-Snail-WT group，NC+pMIR-Snail-MUT group，miR-22+pMIR-Snail-WT group，and miR-22+pMIR-Snail-MUT group（n=3）. The t-test was used for comparison between two independent samples；one-way analysis of variance was used for comparison between multiple sets of data，and repeated measures analysis of variance was used for time-associated fac－tors. Results：The expression level of MALAT1 in gastric cancer cell lines was significantly higher than that in normal gastric mucosal epithelial cells（all P=0.000）；after underexpression of MALAT1，AGS and SGC-7901 had significantly decreased prolifer－ative ability，migration ability，and invasion ability（P1=0.001，0.018，and 0.024，respectively；P2=0.005，0.007，and 0.015，respectively）；meanwhile，the dual luciferase assay results showed that MALAT1 could targetedly bind to miR-22，and miR-22 could targetedly bind to snail（both P=0.001）. The expression level of miR-22 in gastric cancer cells was significantly lower than that in normal gastric mucosal epithelial cells（P<0.05）；after overexpression of miR-22，AGS and SGC-7901 had significantly decreased proliferative ability，migration ability，and invasive ability（P1=0.004，0.034，and 0.037，respectively；P2=0.009，0.005，and 0.011，respectively）. Concomi－tant underexpression of MALAT1 and overexpression of miR-22 could further enhance the inhibitory effect on the proliferation and invasion of AGS and SGC-7901 after underexpression of MALAT1（P1=0.022 and 0.024，respectively；P2=0.006 and 0.015，respectively）；concomitant overexpression of miR-22 and snail could reverse the inhibitory effect of miR-22 on the proliferation and invasion of AGS and SGC-7901（P1=0.003，and 0.015，respectively；P2=0.004 and 0.01，respectively）. Conclusion：There is significantly overex－pressed MALAT1 in gastric cancer cells，which can facilitate the proliferation，migration，and invasion of gastric cancer by regulating the miR-22/snail axis.