The Role And Mechanism Of MiR-146a On Gastric Cancer

Objective:To investigate the role of miRNA-146a in gastric cancer and explore the target genes of miR-146a related to its role in gastric cancer.Methods:Quantitative real-time polymerase chain reaction (qRT-PCR) was used to detect the levels of miR-146a expression in60cases of gastric cancer tissues and corresponding adjacent normal tissues. The correlation between miR-146a expression patterns in human gastric cancer tissues with clinicopathological features and overall survival was assessed. The levels of miR-146a in gastric cancer cell lines MKN-45.SGC-7901, MGC-803and HGC-27were also detected. Overexpress miR-146a in MKN-45and SGC-7901cells and knockdowm miR-146a in MGC-803and HGC-27cells with lentiviral particles. The proliferation of gastric cancer cell lines were examed by CCK-8assays and colony formation assays."Scratch wound healing" and Transwell with or without matrigel was used to investigated the impact of miR-146a on migratory and invasive abilities of gastric cancer cell lines. Nude mouse lung metastasis model with tail vein injection in vivo further verify the role of miR-146a in metastasis of gastric cancer. Target gene prediction software combined with dual fluorescent reporter gene system explored target genes of miR-146a involving tumor metastasis. qRT-PCR and Western blot studied the mechanism of miR-146a acting on its target gene. Overexpress WASF2in miR-146a lowexpression gastric cancer cells while knockdown WASF2in miR-146a overexpression gasteic cancer cells. Transwell assay detected the effect of WASF2on miR-146a overexpression or knockout gastric cancer cells. The WASF2levels of60cases of gastric cancer tissues and corresponding adjacent normal tissues were detected by immunohistochemistry and the relationship between WASF2levels and the clinicopathological parameters were analyzed. The correlation beteen WASF2levels and miR-146a levels in gastrc cancer tissues were also assayed.Results:1. miR-146a expression was significantly lower in gastric cancer tissues than in corresponding adjacent normal tissues. The relative median expression level of miR-146a in gastric cancer tissues was0.162±0.019and that in corresponding adjacent normal tissues was0.823±0.072.The differences were significant with a P value minor than0.01.2. mir-146a levels in gastric cancer tissues correlated with TNM stages and lymph node metastasis. The lower of miR-146a levels, the later of TNM stage is(P=0.018); The higher of miR-146a levels, the lower probabilities of lymph node metastasis is(P <0.001).3. miR-146a levels in gastric cancer cell lines MKN-45and SGC-7901were relative lower while in MGC-803and HGC-27cells were higher. Overexpress miR-146a in MKN-45and SGC-7901cells and knockdown miR-146a in MGC-803and HGC-27cells for the subsequent cell biological-behavior assays.4. The CCK-8assay showed that the introduction of miR-146a caused no significant effect on the gastric cancer cell growth curves. Colony formation assay revealed that neither overexpression nor knockdown of miR-146a in gastric cancer cell lines had effect on their colony formation rates which involves the groups dependence and proliferative capacities.5."scratch wound healing"assay showed that miR-146a can reduce the migration of gastric cancer cells. Scratches of MKN-45and SGC-7901cells transfected with miR-146a were still distant24hours later while the control group obvious narrowered. Tranwell without matrigel results showed that overexpression of miR-146a significantly inhibited migratory numbers of MKN-45cells and SGC-7901cells, with P values equal to0.0037and minor0.0001respectively. Knockdown of miR-146a enhanced migratory numbers of MGC-803and HGC-27cells dramatically with P values minor0.0001and equal to0.0039respectively.6. Tranwell with matrigel assay suggested that overexpression of miR-146a significantly inhibited invasive numbers of MKN-45cells and SGC-7901cells, with P values equal to0.0007and minor0.0001respectively. Knockdown of miR-146a enhanced invasive numbers of MGC-803and HGC-27cells dramatically with P values equal to0.0045and equal to0.0015respectively.7. Nude mouse lung metastasis model with tail vein injection with MKN-45-miR-146a and control cells showed that miR-146a can inhibit the metastasis of gastric cancer in vivo. The number of lung metastatic nodules decreased significantly in MKN-45-miR-146a group compared to the control group under a fluorescence microscope. The number of lung metastatic nodules were8士2and20士3respectively(P=0.0182).8. Target gene predictive software combined with dual fluorescent reporter gene system results verified that WASF2is one of the metastasis target genes of miR-146a. miR-146a could reduce forty-five percent relative fluorescence value of WASF23’UTR cloning vector psiCHECK2.9. qRT-PCR and Western blot detected the mRNA and protein levels of WASF2in gastric cell lines with miR-146a overexpression or knockdown. The results cleared that the mechanism miR-146a regulating WASF2is the inhibition of the posttranscription of WASF2.10. Complementation tests showed that overexpression of WASF2can reverse the inhibition of miR-146a on MKN-45cells’migration and invasion, while knockdown of WASF2reversing the enhance of MKN-45cells’ migration and invasion by miR-146a inhibitor.11. Immunohistochemistry was used to detect the levels of WASF2in60cases of gastric cancer tissues and corresponding adjacent normal tissues. The levels of WASF2in gastric cancer tissues were higher than corresponding adjacent normal tissues. The differences were statistically significant (P<0.001). And the WASF2expression levels were lymph node metastasis-associated. The higher of WASF2levels, the more probable of lymph node metastasis are(P=0.013).12. Analysis of the correlation between miR-146a and WASF2levels in gastric cancer tissues showed that they were negative correlated. The median levels of miR-146a in WASF2lower group was0.227±0.035, while in WASF2higher group was0.118±0.017(P=0.0309).Conclusion:miR-146a expression was significantly lower in gastric cancer tissues than in corresponding adjacent normal tissues. miR-146a levels in gastric cancer correlated with TNM stages and lymph node metastasis. miR-146a acts as a tumor suppressor gene in gastric cancer and inhibits the migration and invasion of gastric cancer. The mechanism behind is downregulating WASF2, one of its target genes, which mediated elongated movement of cancer cells. The miR-146a/WASF2axis revealed a new mechanism about gastric cancer metastasis and represents as a new potential therapeutic target for gastric cancer.