Study On The Biological Role And Mechanism Of Hsa＿circ＿0001610 In Gastric Cancer

Objective : To explore the biological role and preliminary mechanism of hsa＿circ＿0001610 in gastric cancer through in vivo and in vitro experiments,enriching the research data for the study of the regulatory network on the progression of gastric cancer;providing novel strategies and sufficient theoretical basis for the expansion of gastric cancer molecular types and the molecular targeting therapy of gastric cancer.Methods:First of all,the research was approved by the ethics committee of Inner Mongolia People’s Hospital,and all of the enrolled patients signed the Informed Consent.RNA was extracted according to the routine protocol.Two kinds of human gastric cancer cell line AGS and MKN45 were used;and the human normal gastric mucosa cell line GES-1 was used as the control cells in the cellular experiments.Hsa＿circ＿0001610 from gastric cancer cells were identified by Sanger sequencing and Rnase R digestion.The expression of hsa＿circ＿0001610 in gastric cancer tissues and cell lines were detected by q RT-PCR.Instantaneous hsa＿circ＿0001610 knockdown gastric cancer cell lines AGS and MKN-45 were constructed by transfection of small interfering RNA(si RNA),and the knockdown efficiency of hsa＿circ＿0001610 was verified by q RT-PCR.The AGS and MKN-45 cell lines of hsa＿circ＿0001610knockdown group(si1610 group)and control group(si Ctrl group)were constructed.Incucyte S3 real-time dynamic cell imaging analysis system was used to monitor and analyze the differences in proliferation and invasion ability of AGS and MKN-45 cells between the si1610 and si Ctrl groups.Soft AGAR method was used to observe the difference in clonal formation ability of AGS and MKN-45 cells between si1610 and si Ctrl groups.Flow cytometry was used to detect and analyze the apoptosis of AGS and MKN-45 cells in si1610 and si Ctrl groups,and western blot was used to detect the expression of apoptosis-related proteins Bcl-x L and Caspase3 in AGS and MKN-45 cells in si1610 and si Ctrl groups.NOD/SCID mice were subcutaneously injected with MKN-45 cells from si1610 and si Ctrl groups,respectively.The mice were sacrificed after fed and monitored for four weeks.Tumors were dissected to observe and analyze the differences in tumor volume and weight between the two groups.Results:The expression of hsa＿circ＿0001610 in gastric cancer tissues was significantly higher than that in the normal tissues(p<0.05).Compared with normal gastric mucosa cell GES-1,the expression of hsa＿circ＿0001610 was increased in gastric cancer cells MKN-45,AGS,MKN-74,MGC-803 and BGC-823(p<0.01).AGS and MKN-45 cells with hsa＿circ＿0001610 were instantaneously knockdown were successfully constructed.The knockdown efficiency of hsa＿circ＿0001610 was more than 50% after transfection with si1610 for 24 h,48h and 72 h,compared with si Ctrl transfection group,detected by q RT-PCR.(p<0.0001).Incucyte S3 analysis showed that,compared with si Ctrl group,the proliferation,invasion and clonal formation of AGS and MKN-45 cells in si1610 groups were decreased(p<0.05).Flow cytometry results showed that compared with si Ctrl groups,the cell apoptosis number of AGS and MKN-45 in si1610 groups were significantly increased.Western blot results showed that compared with the si Ctrl groups,the expression of Bcl-x L protein in AGS and MKN-45 in the si1610 groups were decreased,and the expression of Cleaved Caspase3 protein was increased.The results of subcutaneous tumor-forming experiment from NOD/SCID mice showed that compared with si Ctrl group,the volume and weight of tumors in si1610 group were distinctly decreased(p<0.001).Conclusions:Hsa＿circ＿0001610 is overexpressed in human gastric cancer tissues and gastric cancer cells.Hsa＿circ＿0001610 promoted the progression of gastric cancer.Knockdown of hsa＿circ＿0001610 inhibited the proliferation,invasion and clone formation of gastric cancer cells,and promoted the apoptosis of gastric cancer cells.Hsa＿circ＿0001610 may be a novel potential biotarget for gastric cancer.