Mechanism of microRNA150 in regulating invasion and metastasis of gastric cancer by targeting Ras-interacting protein 1 / 中华消化杂志

ABSTRACT

Objective To investigate the effects and mechanism of microRNA150 (miRNA150) on proliferation, invasion and metastasis of gastric cancer.Methods From January 2015 to June 2016, 45 surgical specimens were collected.The expression of miRNA150 and Ras-interacting protein 1(RASIP1) at miRNA level in gastric cancer tissues and paracancerous tissues were quantified by quantitative real-time fluorescent reverse transcriptase-polymerase chain reaction (qRT-PCR).The correlation between miRNA150 and the biological features of gastric cancer as well as RASIP1 expression was analyzed.Gastric cancer cell line SGC-7901 was cultured and transfected with pcDNA3.1-miRNA150 expression plasmids.The effect of miRNA150 over-expression on the proliferation of SGC-7901 cells was determined by 3-(4,5-dimethyl-2-thiazolyl)-2,5-dipheayl 2-H-tetrazolium bromide (MTT) assay.And the effect of miRNA150 over-expression on the invasion and metastasis of SGC-7901 cells was detected by Transwell assay.The potential target gene of miRNA150 was analyzed by bioinformatics software and dual-luciferase reporter assay system.The effect of miRNA150 over-expression on RASIP1 expression in SGC-7901 cells was tested by qRT-PCR and Western blotting.Analysis of variance and t test were used to compare normal distribution data.And the Mann-Whitney rank sum test was used to compare skewed distribution data.Spearman assay was used for correlation analysis.Results The median level of miRNA150 in gastric cancer tissue was higher than that of paracancerous tissues (3.85, 0.26 to 7.92 vs 1.98, 0.19 to 5.66), and the difference was statistically significant (U=466.22,P<0.05).The median level of RASIP1 mRNA in gastric cancer tissue (1.65, 0.13 to 3.59) was lower than that of paracancerous tissues (2.96, 0.59 to 6.08), and the difference was statistically significant (U=522.31,P<0.05).The results of correlation analysis indicated that RASIP1 expression level was negatively correlated with miRNA150 expression (r=-0.589, P=0.008).The RASIP1 expression at mRNA level was negatively correlated with miRNA150 expression (r=-0.614, P=0.004).The dual-luciferase reporter assay showed RASIP1 was the target gene of miRNA150.The miRNA150 expression level was related with tumor size, TNM staging and lymph node metastasis(χ2=5.81, 6.00 and 10.04,all P<0.05).The results of MTT assay showed that after SGC-7901 cells cultured for 24 hours, the A value of pcDNA3.1-miRNA150 plasmid transfected cells was higher than that of the untransfected SGC-7901 cells (0.51±0.04 vs 0.79±0.03), and the difference was statistically significant (t=4.745, P<0.05).The results of Transwell assay indicated that there were more invasive and metastatic cells in pcDNA3.1-miRNA150 plasmid transfected cells.The results of qRT-PCR showed that the relative levels of RASIP1 mRNA in control group, pcDNA3.1-miRNA150 plasmid transfected cells and pcDNA3.1 empty plasmid transfected cells were 1.00±0.02, 0.51±0.03 and 1.08±0.03, respectively.The RASIP1 mRNA level in pcDNA3.1-miRNA150 plasmid transfected cells was lower than untransfected and pcDNA3.1 empty plasmid transfected cells, and the differences were statistically significant (t=3.940, 4.120, both P<0.05).miRNA150 could negtively regulate the RASIP1 protein expression and promote the proliferation and invasion of gastric cacer cells.Conclusions Over-expression of miRNA150 induced invasion and metastasis of gastric cancer by down-regulating RASIP1 expression.miRNA150 may be a novel biomarker for the diagnosis and treatment of tumor metastasis.