The Role Of MiR-224 And Its Related Mechanism In Gastric Cancer

Gastric cancer,a kind of malignant tumor derived from gastric epithelial cells,which is one of the most common digestive tract malignant tumors with poor clinical outcome and high mortality.Further study in depth is very important to promote the development of gastric cancer molecular biological mechanism and find a more effective treatment strategy of gastric cancer.In this study,we will talk about the expression of miR-224 in gastric carcinoma,the effect on the biological behavior of gastric cancer cells of miR-224 and the related molecular mechanism based on gastric cancer tissue,gastric cancer cell line and nude mouse tumorigenic model.The experiment is divided into three parts:1.Expression of miR-224 in gastric carcinoma and gastric cancer cell lines and its clinical significance Micro RNA(miRNA)is a kind of endogenous non-coding small RNA molecule,which plays a very important role in the regulation of eukaryotic gene expression.Studies have shown that some of the miRNAs in gastric cancer tissues are abnormal and participate in the pathogenesis of gastric cancer growth and metastasis.In this study,we collected gastric cancer tissues and adjacent tissues from patients with gastric cancer surgery from January 2010 to December 2012,and differentially expressed miRNAs were detected by Deep sequencing.The results showed that 30 miRNAs(such as miR-188,miR-490,etc.)were down-regulated in gastric cancer tissues,and 32 miRNAs(such as miR-224,miR-212,etc.)was up-regulated compared with adjacent tissues.Among them,the expression of miR-224 in gastric carcinoma was the most significant.The expression of miR-224 in gastric cancer tissues was also significantly higher than that in adjacent tissues(P <0.01).The expression level of miR-224 in five kinds of gastric cancer cell lines(HS-746 T,MKN-45,HGC-27,SNU-5 and BGC-823)was significantly higher than that in normal cells(GES-1)(P <0.001).To investigate the clinical significance of miR-224 expression abnormality,we analyzed the clinical data of patients.The results showed that the expression of miR-224 in gastric carcinoma was positively correlated with tumor size and lymph node metastasis(P <0.05),and the survival time of gastric cancer patients with high expression of miR-224 was significantly shorter than that of patients with low expression of miR-224.The results suggest that miR-224 expression in gastric cancer tissue is higher than the adjacent tissue,can be used as a biological marker of gastric cancer progression,and its increased expression suggests poor prognosis of gastric cancer.2.miR-224 promotes the proliferation of gastric cancer cells and inhibits the apoptosis of gastric cancer cells The first part of the study showed that miR-224 was highly expressed in gastric cancer and related with poor prognosis of gastric cancer,but the effect of miR-224 on the biological behavior of gastric cancer cells was not reported.Therefore,this part of the study is to further explore the proliferation,metastasis and anti-apoptosis role of miR-224 in gastric cancer cells(HS-746 T and BGC-823).The effect of miR-224 on the proliferation of gastric cancer cells was detected by CCK8.The results showed that the proliferation of gastric cancer cells treated with miR-224 inhibitor was decreased(p <0.05)compared with the control group,and miR-224(miR-224 Mimics)increased gastric cancer cell proliferation(p <0.05).Flow cytometry was used to detect the cell cycle.The miR-224 inhibitor(miR-224 inhibitors)could increase the proportion of gastric cancer cells in G1 phase,decrease the S phase cells(p <0.05),and induce the increasing of gastric cancer cell apoptosis(P <0.01).The expression of PCNA,Cyclin-D1 and Cyclin-D2 were down-regulated in the gastric cancer cells treated with miR-224 inhibitor and the expression of P21 and P27 were up-regulated by Western blot and QRT-PCR(P<0.05).Immunofluorescence assay also showed that transfection with miR-224 inhibitor attenuated the expression of Cyclin-D1 and CDK4 in gastric cancer cells.HS-746 T cells untreated or transfected with miR-224 were subcutaneously inoculated into nude mice to observe the tumor growth rate ofnude mice.It was observed that the tumor growth rate of the nude mice bearing the low expression of miR-224 was lower(p <0.001),and the average weight was smaller(p <0.001)compared with the control group.The results of this study show that miR-224 can promote the proliferation and anti-apoptotic ability of gastric cancer cells and promote the growth of gastric carcinoma.so it is a potential method that inhibiting the expression of miR-224 for the treatment of gastric cancer.3.miR-224 promotes the proliferation,metastasis anti-apoptotic ability of gastric cancer by targeting m TOR To explain the carcinogenesis molecular mechanism of miR-224,we searched the potential targets of miR-224 by the Pictar,miRBase and Target Scan databases and found that the 3'UTR of the key regulatory protein of apoptosis and proliferationm TOR was associated with miR-224 gene Site.The luciferase reporter assay confirmed that miR-224 could bind to this site to regulate luciferase expression.The expression of m TOR m RNA and protein in gastric cancer cells(HS-746 T and BGC-823)transfected with miR-224 mimics was increased by Western blot and QRT-PCR(p <0.05),and the expression of m TOR was decreased in gastric cancer cells treated with miR-224 inhibitors(p <0.05).Transwell experiments and cell colony experiments showed that miR-224 could promote the migration,invasion and in vitro colonization of gastric cancer cells,The results showed that miR-224 inhibitor treatment or knockout m TOR could significantly decrease the invasion,invasion and colony formation ability of gastric cancer cells.The expression of m TOR and Bcl2 was attenuated by miR-224 inhibitor,and the expression of Bax and Caspase 3/9 was enhanced by Western blot and QRT-PCR in cells.Immunofluorescence staining showed that miR-224 and miR-224 inhibitor respectively up-regulated and down-regulated the expression of m TOR and Caspase 3 in gastric cancer cells.Immunohistochemical staining of tumor tissue in nude mice showed that miR-224 inhibitor could increase the number of positive cells in Tunel cells and the number of Ki67 positive cells.Western blot showed that miR-224 inhibitor could down-regulate the expression of m TOR,Bcl2,PCNA,Cyclin-D1 and Cyclin-D2 in tumor tissues andup-regulate the expression of Bax,Caspase 3/9,P21 and P27.The above results show that m TOR is a direct target of miR-224.miR-224 promotes the expression of anti-apoptotic and proliferating protein and inhibits the expression of pro-apoptotic protein by m TOR,which promotes the growth,metastasis and Anti-apoptotic ability of gastric cancer.miR-224 / m TOR signal axis can be used as a new drug target,which is expected to provide a new way for gastric cancer clinical precision treatment.