Mechanisms Involved In Long Noncoding RNA Gastric Cancer-related LncRNA1 Mediating Gastric Malignancy

Objective The purpose of this study is to discover and analyze the key regulatory molecules and their regulatory mechanisms in the proliferation and metastasis of gastric cancer cells from the perspective of long non-coding RNAs,so as to find new tumor markers and interventional molecules,to improve the early diagnosis rate of gastric cancer and reduce the metastasis rate,and do some useful exploration for the future precise treatment of gastric cancer.Methods The expression of long non-coding RNAs(lncRNAs)in gastric cancer tissues was detected by microarray.According to the results,we searched for differentially expressed lncRNAs in gastric cancer and adjacent tissues.Eight highly expressed lncRNAs were further detected by real-time fluorescence quantitative polymerase chain reaction(qPCR)in a variety of gastric cancer cell lines and tissue samples to determine the target lncRNA.The effects of lncRNA on malignant biological behavior of gastric cancer cells were detected by MTT incorporation and EdU uptake assay,Annexin V-PI double staining by flow cytometry,tranwell assay,subcutaneous tumor and tail vein injection in nude mice to establish lung metastasis model.Effect of target lncRNA on tumorigenicity and metastasis of gastric cancer cell line BGC-823 cells were detected.Further,bioinformatics analysis predicts that miRNAs(microRNAs,miRNAs)may interact with the lncRNA.The microRNAs regulated by the lncRNA were identified by dual luciferase reportor assay(DLR)and RNA enrichment assay.Combined with bioinformatics analysis,the target proteins of predicted microRNAs were analyzed by expression analysis,binding analysis and biological function detection.Subsequently,Western blotting and immunohistochemistry(IHC)were used to verify the expression levels of target proteins in both animal and clinical samples.Finally,the correlation among GCRL1,microRNAs and target genes in gastric cancer was analyzed.Results Firstly,the expression of lncRNA RP11-290F20.3 in gastric cancer was high in several gastric cancer cell lines and most gastric cancer tissue samples.The mechanism of action of lncRNA RP11-290F20.3 has not been reported in any literature till now,so it was named gastric cancer-related lncRNA 1(GCRL1).Cell proliferation test and transwell test showed that GCRL1 could significantly promote the proliferation,migration and infiltration of gastric cancer cells,while silent GCRL1 could significantly inhibit the proliferation and migration of gastric cancer cells.Subcutaneous tumor test and lung metastasis model in nude mice showed that inhibition of GCRL1 could inhibit tumor growth and metastasis potential in vivo.Bioinformatics prediction,DLR and RNA enrichment experiments showed that GCRL1 could target the expression of micRNA-885-3p.MTT,EdU,transwell and in vivo experiments in nude mice showed that miRNA-885-3p could inhibit the proliferation,migration and infiltration of gastric cancer cells.DLR assay showed that miRNA-885-3p could bind directly to the 3’-UTR region of CDK4 mRNA.Western blotting assay showed that miRNA-885-3p could regulate the level of CDK4 protein and affect the expression of CDK4 at post-transcriptional level.Western blotting and immunohistochemical staining of tumor-bearing tissues and clinical samples in vivo showed the expression of CDK4 was decreased along with the decreased expression of GCRL1,while the expression of CDK4 was decreased in the tissues with high expression of miRNA885-3p.These in vitro and in vivo experiments have confirmed that GCRL1 regulates the expression of CDK4 through miRNA-885-3p,and GCRL1 can enhance the proliferation of gastric cancer cells and promote the occurrence of gastric cancer.The expression levels of GCRL1 and CDK4 were positively correlated in gastric cancer tissues,while the expression of microRNAs-885-3p was negatively correlated with that of CDK4.Conclusions The expression levels of lnc-RP11-290F20.3(Gastric Cancer Related LncRNA 1,GCRL1)in gastric cancer cells and tissues are significantly increased.Inquiry into the function and mechanism of GCRL1 shows that: Firstly,GCRL1 can significantly promote the proliferation,migration and infiltration of gastric cancer cells,but has no obvious effect on the apoptosis of gastric cancer cells.The subcutaneous tumor-bearing experiment and the lung metastasis model in nude mice show that GCRL1 suppression could inhibit the growth and metastasis potential of tumors in animals.Secondly,GCRL1 can target and regulate the expression of microRNA-885-3p.Thirdly,miRNA-885-3p could significantly inhibit the proliferation and migration of gastric cancer cells in vitro and in vivo.Fourthly,miRNA-885-3p can directly bind to the 3’-UTR region of CDK4 gene,and both binding sites in the 3’-UTR region play a role in this regulation.Western blotting of cell and animal tumor-bearing tissue samples also showed that microRNA-885-3p could regulate the level of CDK4 protein,thus affecting the expression of CDK4 at post-transcriptional level.Fifthly,the high expression of CDK4 not only enhanced the proliferation,but also enhanced the infiltration potential of gastric cancer cells,which was consistent with the role of GCRL1.Sixthly,GCRL1 regulates CDK4 through miRNA-885-3p as a new regulatory axis,can promote cell proliferation and metastasis and thus promote the development of gastric cancer.