The Role And Regulation Mechanisms Of LINC00319 In Tumor Growth And Metastasis In Gastric Cancer

Background and objective:Gastric cancer is one of fourth most common malignancies in the world,which has brought great threat to human heath.The incidence and mortality of gastric cancer in China rank second among all kinds of tumors.The pathogenesis of gastric cancer is very complex,which can be induced by various environmental factors and many other ways.Surgery is the most effective method for the treatment of gastric cancer,and early diagnosis can improve the survival rate of patients with gastric cancer.For the limitations of the treatment of gastric cancer,the complexity of the pathogenesis of gastric cancer and many inducements,it is necessary and urgent to elucidate and reveal the pathogenesis of gastric cancer.Long non-coding RNAs(lncRNAs),subclass of non-coding RNAs which express across the mammalian genome,have substantial influence on tumorigenesis and progression of tumors via the mechanism of competing endogenous RNA(ceRNA)of microRNAs(miRNAs).LINC00319 plays a role in enhancing proliferation and invasion of lung cancer cells by suppressing miR-32.Our previous bioinformatics analysis and RT-qPCR experiments showed that LINC00319 was highly expressed in gastric cancer gene expression chip,gastric cancer tissue and gastric cancer cells.Combining with our bioinformatics prediction that LINC00319 target adenylate cyclase 3(ADCY3),we speculated that LINC00319 might play important roles in gastric cancer.Therefore,this study is to figure out the role and regulation mechaisms of LINC00319 in tumor growth and metastasis in gastric cancer,to verify whether LINC00319 sponges the miR-335-5p targeting ADCY3 to regulate the expression of ADCY3 and participates in the growth and metastasis of gastric cancer.This paper will provide a new theoretical basis and direction for the prevention and treatment of gastric cancer.Materials and Methods:1.Expression pattern of LINC00319 in gastric cancer(1)Based on gene expression microarray data related to gastric cancer obtained from the comprehensive gene expression database(https://www.ncbi.nlm.nih.gov/geo/),differential expression genes and long non-coding RNA related to gastric cancer were screened,and the targeted miRNA to these differential expression genes and long non-coding RNA were predectied by bioinformation analysis.(2)RT-qPCR was carried out the determine LINC00319 and miR-335-5p expression in 11 gastric cancer tissues and paired non-tumor tissues.(3)RT-qPCR was carried out the determine LINC00319 and miR-335-5p expression in both GC cell lines(BGC-823,MGc-803 and SGC-7901)and normal gastric epithelial cell lines(GES-1 and RGM-1).(4)The cellular localization of LINC00319 was dectected using fluorescence in situ hybridization(FISH).2.The interation mode of of LINC00319(1)The interaction between LINC00319 and miR-335-5p,and between miR-335-5p and ADCY3 were detected by dual luciferase reporter analysis and RNA pull down.(2)The mRNA expressions of ADCY3 and LINC00319 in response to the treatment of LINC00319 and si-LINC00319 were dected by RT-qPCR.3.The role of LINC00319 in the growth and metastasis of gastric cancer(1)After overexpression of LINC00319 in gastric cancer cells,the mRNA and protein expression levels of CDK4,MTA1 and GAS1 were detected by RT-qPCR and Western blot.Next,EDU analysis,flow cytometry and scratch test were used to detect cell proliferation,apoptosis and migration.(2)We transplanted the gastric cancer cell line transfected with LINC00319 and the non-transfected control cell line into nude mice to detect the tumor weight,tumor volume,liver metastasis rate,ascites rate,ascites volume and microvessel density.4.The regulation mechanism of LINC00319 on the growth and metastasis of gastric cancer(1)We detected the proliferation and migration of gastric cancer cells with miR-335-5p knockdown or ADCY3 overexpression.The mRNA and protein expression levels of ADCY3,CDK4,MTA1 and GAS1 were detected by RT-qPCR and Western blot.EDU,flow cytometry and scratch test were used to test cell proliferation,migration and apoptosis.(2)We transplanted the gastric cancer cell line with miR-335-5p knockdown or ADCY3 overexpression into nude mice to detect the tumor weight,tumor volume,liver metastasis rate,ascites rate,ascites volume and microvessel density.Results:1.Expression pattern of LINC00319 in gastric cancer(1)LINC00319 and ADCY3 were overexpressed in gene expression chips related to gastric cancer,and miR-335-5p is the co-targeted miRNA of LINC00319 andADCY3.(2)Comparing to non-tumor tissues,LINC00319 overexpression and miR-335-5p inhibition were found in gastric cancer tissues.(3)Comparing to normal gastric epithelial cell lines,LINC00319 overexpression and miR-335-5p inhibition were found in gastric cancer cell lines.(4)LINC01239 was expressed in the cytoplasm.2.The interation mode of of LINC00319(1)LINC00319 interacts with miR-335-5p,and miR-335-5p interacts with ADCY3.(2)Comparng to the blank and NC groups,the LINC00319 group had elevated LINC00319 and ADCY3 expression,while the si-LINC00319 group showed the opposite effect.3.The role of LINC00319 in the growth and metastasis of gastric cancer(1)Comparing to the blank group,the mRNA and protein levels of CDK4,MTA1 increased,while the mRNA and protein level of gas1 decreased in the LINC00319 group.Comparing to the blank group,the cell proliferation and migration were enhanced,while the cell apoptosis rate was decreased in the LINC00319 group.(2)Comparing to the blank group,the tumor weight,tumor volume,liver metastasis rate,ascites rate,ascites volume and MVD increased significantly.4.The regulation mechanism of LINC00319 on the growth and metastasis of gastric cancer(1)Comparing to the blank group,the miR-335-5p expression decreased in the miR-335-5p inhibitor group,while increased in the miR-335-5p mimic+ADCY3 vector group.Comparing to the blank group,the mRNA and protein levels of CDK4,MTA1 increased,while the mRNA and protein level of gas1 decreased in the miR-335-5p inhibitor group and the ADCY3 vector group.Comparing to the blank group,the cell proliferation and migration were enhanced and the percentage of apoptotic cells decreased in the miR-335-5p inhibitor group and the ADCY3 vector group.(2)Comparing to the blank group,the tumor weight,tumor volume,liver metastasis rate,ascites rate,ascites volumeand MVD increased in the miR-335-5p inhibitor group and the ADCY3 vector group.Conclusion:1.High expression of LINC00319 and ADCY3 and low expression of microRNA-335-5p were observed in gastric cancer.Bioinformatics analysis predicted that LINC00319 could participate in ADCY3 regulation through competitive binding with miR-335-5p.2.Overexpression of LINC00319 promotes the proliferation and migration of gastric cancer cells,but inhibits cell apoptosis,and promotes the growth and migration of tumors in nude mice.3.LINC00319 promotes the expression of target gene ADCY 3 by sponging microRNA-35-5p.4.MiR-335-5p inhibits the proliferation and migration of gastric cancer cells by inhibiting the expression of ADCY3,and inhibits the growth and migration of tumors in nude mice.