Effect of miR-145 targeting Six1 on invasion of gastric cancer and its molecular mechanism
10.3760/cma.j.issn.1673-422X.2018.08.005
- VernacularTitle:miR-145靶向Six1对胃癌侵袭的影响及分子机制
- Author:
Li JIANG
1
;
Hui LI
;
Guiying ZHANG
;
Yibing MU
;
Haitao LIU
Author Information
1. 410006,长沙市第四医院消化内科
- Keywords:
Stomach neoplasms;
MicroRNAs;
Neoplasm invasiveness;
Six1
- From:
Journal of International Oncology
2018;45(8):470-477
- CountryChina
- Language:Chinese
-
Abstract:
Objective To investigate the expressions of microRNA-145 (miR-145) and Six1 in gastric cancer tissues and gastric cancer cell lines,and to investigate the effect of miR-145 targeting Six1 on invasion of human gastric cancer cells and its molecular mechanism.Methods Sixty patients with advanced gastric cancer confirmed by gastroscopy and pathology in the Fourth Hospital of Changsha were selected,from January to November,2017.The expressions of miR-145 mRNA and Six1 in 60 cases of gastric cancer and their matched adjacent tissues were detected by real-time fluorescence quantitative PCR (qRT-PCR) and their correlations were analyzed.miR-145 mimics were transfected into MKN-45,BGC-823 and SGC-7901 gastric cancer cell lines.The relative levels of miR-145 mRNA in three gastric cancer cell lines were detected by qRT-PCR.The SGC-7901 gastric cancer cell lines with the highest expression level of miR-145 mRNA were selected for subsequent experiments.The mimic group (transfected with miR-145 mimic),NC group (transfected with miR-145 negative control) and empty carrier group (no addition of any oligonucleotides) were set up.The invasive ability of gastric cancer cells was detected by Transwell test.The ability of angiogenesis was verified by microtubule formation experiment.The expression levels of downstream proteins of the gastric cancer cell lines SGC-7901 such as Six1,E-cadherin and vimentin were detected by Western blotting method.The MirTrap system and the luciferase report test verified whether miR-145 targeted the Six1 gene.Results The expressions of miR-145 mRNA in gastric cancer tissues and in para cancerous tissues were 0.579 ± 0.086 and 1.009 ±0.121,respectively.The difference was statistically significant (t =-22.498,P ＜0.001).The expressions of Six1 were 2.516 ± 0.208 and 1.041 ± 0.227,respectively.The difference was statistically significant (t =37.119,P ＜ 0.001).The expressions of miR-145 mRNA and Six1 were negatively correlated (r =-0.728,P ＜0.001).After overexpression of miR-145,in the mimic,NC and empty carrier groups,the numbers of cells passing through the membrane were 48.550 ± 3.716,82.800 ± 3.797,87.467 ± 8.023,respectively.The difference was statistically significant (F =87.789,P ＜ 0.001).There were significant differences between the mimic and NC groups and between the mimic and empty carrier groups (both P ＜0.001).In the mimic,NC and empty carrier groups,the numbers of tube formation of human umbilical vein endothelial cells were 24.333 ± 1.211,34.167 ±2.041,36.500 ±3.209,respectively.The difference was statistically significant (F =47.103,P ＜ 0.001).There were significant differences between the mimic and NC groups and between the mimic and empty carrier groups (both P ＜ 0.001).In the mimic,NC and empty carrier groups,the expression levels of Six1 in gastric cancer SGC-7901 cells were 1.392 ±0.072,2.426 ±0.099,2.371 ±0.079,respectively.The difference was statistically significant (F =289.517,P ＜ 0.001).There were significant differences between the mimic and NC groups and between the mimic and empty carrier groups (both P ＜0.001).In the mimic,NC and empty carrier groups,the expression levels of E-cadherin were 4.001 ±0.132,2.714 ±0.181,2.653 ±0.218,respectively.The difference was statistically significant (F =106.572,P ＜0.001).There were significant differences between the mimic and NC groups and between the mimic and empty carrier groups (both P ＜ 0.001).In the mimic,NC and empty carrier groups,the expression levels of vimentin were 1.634 ± 0.132,3.349 ± 0.102,3.501 ± 0.185,respectively.The difference was statistically significant (F =389.032,P ＜0.001).There were significant differences between the mimic and NC groups and between the mimic and empty carrier groups (both P ＜ 0.001).MirTrap system verified that the target Six1 mRNA levels in the mimic,NC and empty carrier groups were 1.101 ± 0.097,0.582 ± 0.037,0.573 ± 0.032,respectively.The difference was statistically significant (F =138.922,P ＜ 0.001).There were significant differences between the mimic and NC groups and between the mimic and empty carrier groups (both P ＜0.001).Luciferase reporter assay showed that the double fluorescence ratios of Six1 wild type recombinant vector group were 7.324 ± 0.415,10.755 ± 0.481,10.430 ± 0.309 in the mimic,NC and empty carrier groups respectively.The difference was statistically significant (F =129.345,P ＜ 0.001).There were significant differences between the mimic and NC groups and between the mimic and empty carrier groups (both P ＜0.001).The double fluorescence ratios of Six1 mutant recombinant vector group were 10.938 ± 0.091,11.077 ±0.126,11.028 ±0.205 in the mimic,NC and empty carrier groups respectively,and the difference was not statistically significant (F =1.318,P =0.297).The MirTrap system and the luciferase report test showed that miR-145 was targeted to the Six1 gene.Conclusion The expressions of miR-145 mRNA and Six1 in gastric cancer and para cancerous tissues were different and negatively correlated.miR-145 inhibited the invasion,angiogenesis and epithelial mesenchymal transition of gastric cancer cells by targeting Six1 gene.
