The Study Of The Function And Mechanism Of Hsa＿circ＿0034762 In Lung Adenocarcinoma

Background:Lung cancer is the most common malignancy in China and even in the world,as well as the leading cause of tumor-related deaths,posing a great threat to the health of all mankind.Lung adenocarcinoma is one of the most common pathological types of lung cancer.Although lung cancer has experienced rapid development in diagnosis and treatment in recent years,its five-year survival rate is still low due to the concealment of early symptoms,the tolerance to radiotherapy and chemotherapy,and the drug resistance to targeted therapies.Therefore,it is important to explore the pathogenesis and developmental processes of lung cancer.Circular RNA(CircRNA),as a special type of non-coding RNA,plays an important role in the occurrence and development of malignant tumors.CircRNAs can increase the expression of downstream proteins of miRNA(microRNA)by acting as miRNA sponge,thus regulating the processes of many diseases.Currently,studies on lung cancer-related CircRNAs remain scarce.Therefore,the main purpose of this study is to explore the expression of circular RNA hsa＿circ＿0034762 in lung adenocarcinoma,and also,to first verify its functions and the underlying mechanisms.Methods:1.The RNA chip data was used to identify 81 CircRNAs whose expression were downregulated in lung cancer.We then selected 10 CircRNAs with the most significant differences of expression level for verification by PCR.Finally,hsa＿circ＿0034762 were singled out to verify its expressions in lung adenocarcinoma(LUAD)cells by PCR again.2.We constructed the overexpressed plasmids of hsa＿circ＿0034762,afterwards,liposome transfection technology was used to upregulated hsa＿circ＿0034762 in H1299 and HCC827 cells.3.We performed PCR experiment to verify the expression of hsa＿circ＿0034762 in H1299 and HCC827 cells after transfection.4.CCK-8 and clone-formation assays were performed to verify the effects of hsa＿circ＿0034762 on the proliferation and colony-formation ability in H1299 and HCC827 cells after transfection.5.Transwell assay and wound healing assay was performed to verify the effects of hsa＿circ＿0034762 on migration in H1 299 and HCC827 cells.6.The apoptosis experiment was performed to verify the effects of hsa＿circ＿0034762 in H1299 cells.7.Online databases were used to predict the target miRNAs and KEGG analysis was performed to obtain protein interactions.8.The PCR experiment was performed to verify the expression of has-miR-140-3p.2.9.The western blot assay was performed to preliminary verify the underlying mechanism of hsa＿circ＿0034762.Results1.In general,hsa＿circ＿0034762 was downregulated in lung adenocarcinoma cells.2.The results of PCR suggested that hsa＿circ＿0034762 was successfully over-expressed in H1299 and HCC827 cells.3.CCK-8 assays and clone-formation assays suggested that the proliferation and clone-formation ability of H1299 and HCC827 cells were inhibited after upregulation of hsa＿circ＿0034762.4.The results of transwell assays and wound healing assays indicated that the migration and wound healing ability of H1 299 and HCC827 cells were inhibited after upregu-lation of hsa＿circ＿0034762.5.Apoptosis experiments indicated that the apoptosis rates of H1299 cells were increased compared with the control group.6.The online database showed that there might be 40 miRNAs combine with hsa＿circ＿0034762,among which has-miR-140-3p.2 might inhibit the expression of BAK.7.The results of PCR experiments suggested that the expression of has-miR-140-3p was decreased.However,the expression of MAPKBP1 did not show any significant alterations.8.The results of western blot assays showed that the expressions of BAK and cleaved Caspase-3 were increased,and the PARP protein was split into two fragments.CONCLUSIONS:CircRNA hsa＿circ＿0034762 was downregulated in LUAD cell lines.The proliferation,migration,and clone-formation abilities were inhibited after upregulation of hsa＿circ＿0034762 in H1299 and HCC827 cell lines.Furthermore,the apoptosis rates of H1299 cells were increased after upregulation of hsa＿circ＿0034762.The above results suggested that hsa＿circ＿0034762 might play a role similar to that of tumor suppressor genes.In general,hsa＿circ＿0034762 may promote the expression of BAK through sponging has-miR-140-3p.2,thereby promoting a series of processes of LUAD cell apoptosis.