| Background:Hepatocellular carcinomahas become the fifth most common type of cancer worldwide and the third leading cause of cancer-related deaths.Due to the complex pathophysiological process of hepatocellular carcinoma,the hidden clinical manifestations,the low specificity of biochemical indicators,and the poor sensitivity,it will have a major effect on improving the survival rate of liver cancer patients by thoroughly clarifying the molecular events in the process of liver cancer occurrence and development and early diagnosis.Alpha-fetoprotein is currently the most effective biomarker for diagnosing HCC,but in 30-40% of HCC patients,alphafetoprotein expression is at a normal level,resulting in some patients still missing the diagnosis.As a result,scholars have been exploring new diagnostic markers and therapeutic targets for HCC.Cyclic RNA is one of the non-coding RNAs and has been a hot topic in recent years;its diagnostic,therapeutic and prognostic value in cancer development has gradually been demonstrated.In 2019,the only research report at home and abroad found that the expression of cyclic RNA Hsa＿circ＿0008583 in HCC tissue was upregulated,suggesting that it may be involved in the occurrence and development of HCC,but what is its specific biological function? How is expression at the HCC cell level? And what is its role and mechanism of action on the development of HCC? These issues have not been reported.We are ready to start here to reveal this rarely reported new mechanism of liver cancer.We find from the Circ Interactome database that the tumor suppressor gene miR-1301-3p in HCC can bind to hsa＿circ＿0008583.Through the Starbase 2.0database,the 3-UTR region of the onco-promoting gene METTL3,which is a kind of m6 A methyl transferase in HCC,can also bind to miR-1301-3p.Salmena L et al.proposed the competitive endogenous RNA(ce RNA)hypothesis in 2011,in which all types of RNA transcripts bind to each other by competing with shared miRNA binding sites.The ce RNA hypothesis is one of the key theories explaining gene expression regulation and biological function.Therefore,we proposed the following scientific hypothesis: hsa＿circ＿0008583 competitively bind miR-1301-3p with METTL3 through ce RNA mechanism,thereby weakening the silence effect of MIR-1301-3p on METTL3in-directly promoting the expression of METTL3,upregulating the level of RNA methylation,and finally inducing the proliferation,migration and invasion of HCC cells and inhibiting their apoptosis,promoting the occurrence and development of liver cancer.Objective:(1)To study the expression level of hsa＿circ＿0008583 in HCC tissues and cells,to determine whether hsa＿circ＿0008583 were upregulated in HCC as reported;To explore the effects of hsa＿circ＿0008583 on the proliferation,apoptosis,migration and invasion of HCC cells,and to further determine its role in HCC cells;(2)To verify whether hsa＿circ＿0008583 also plays an important role in promoting HCC tumor growth in vivo by nude mouse tumorigenesis experiments;(3)To determine whether hsa＿circ＿0008583 promotes the development of HCC by regulating the miR-1301-3p/METTL3 signaling pathway according to the ce RNA mechanism.Methods:Firstly,the total RNA in HCC tissues and cells was extracted and q RT-PCR was performed to determine the expression of hsa＿circ＿0008583 in HCC tissues and cells;then,by transfection of recombinant plasmid pc DNA-hsa＿circ＿0008583 into Hep3 B cells,the expression of hsa＿circ＿0008583 in Hep3 B cells was upregulated,and the expression efficiency was detected by q RT-PCR;si-hsa＿circ＿0008583 was transfected to Huh-7 cells for down-regulating the expression of hsa＿circ＿0008583,and the silencing efficiency was detected by q RT-PCR.Cell proliferation was detected by CCK8,apoptosis was detected by flow cytometry,and cell migration and invasion were detected by Transwell experiments.Secondly,we used si RNA interference technology and Cell transfection method to gain the Huh-7 cell line with stable expression hsa＿circ＿0008583 knockout,and the si-has＿circ＿0008583 lentivirus was chemically synthesized.IN the nude mouse oncogenic experiments,we injected Huh-7 cells into nude mice and observed the volume and weight of the three groups of nude tumors during multiple time periods.Then,we observed.the effect of hsa＿circ＿0008583 after knockout on the tumorigenic capacity of liver cancer cells.Lastly,hsa＿circ＿0008583 WT,hsa＿circ＿0008583 Mut,METTL3 3’UTR WT,METTL3 3’UTR Mut,miR-1301-3p mimics,miR-1301-3p inhibitors,si-METTL3,si-hsa＿circ＿0008583,pc DNA-hsa＿circ＿0008583 and various negative controls were transfected to Hep3 B or Huh-7 cells respectively,and the luciferase activity of the corresponding group was detected by the luciferase reporter gene kit,which was in order to determine the target relationship and binding site between the hsa＿circ＿0008583 and miR-1301-3p,miR-1301-3p and METTL3.Cell proliferation was detected by CCK8.Cell apoptosis was detected by Flow cytometry and cell migration and invasion was detected by the Transwell experiment.Outcome:(1)The expression of hsa＿circ＿0008583 in HCC tissue was significantly increased(n = 12)compared with adjacent normaltissues(n = 12);the expression of hsa＿circ＿0008583 in HCC cells was significantly enhanced(p < 0.01)compared with hsa＿circ＿0008583 expression in normal hepatocytes;in addition,Hep3 B cells were significantly upregulated due to transfection of hsa＿circ＿0008583overexpressed plasmids,so the m RNA expression level of hsa＿circ＿0008583 was significantly upregulated.The proliferation of Hep3 B cells increased significantly,and the migration and invasion ability of Hep3 B cells was enhanced.Conversely,apoptosis of Hep3 B cells in hsa＿circ＿0008583 group was significantly reduced(p <0.01).(2)The tumor volume of lv-si-hsa＿circ＿0008583 nude mice was significantly reduced compared with that of LV-NC group and Ctrl group;in addition,the tumor weight of LV-si-hsa＿circ＿0008583 nude mice was also significantly lower than that of LV-NC group and Ctrl group(p < 0.01).(3)Compared with the mimics-NC group,the luciferase activity of miR-1301-3p mimics was significantly downregulated with hep3 B cells co-transfected by hsa＿circ＿0008583 WT,however,there was no significant change in luciferase activity of miR-1301-3p mimic with hep3 B cells co-transfected by hsa＿circ＿0008583 Mut;compared with the mimics-NC group.The luciferase activity of hep3 B cells co-transfected by miR-1301-3p-mimics and METTL3 WT was significantly downregulated;however,there was no significant change in luciferase activity in Hep3 B cells co-transfected by miR-1301-3p-mimics and METTL3 Mut(p< 0.01);in addition,transfection of miR-1301-3p-mimics could reverse the promotion of hsa＿circ＿0008583 overexpression on the proliferation,migration and invasion of liver cancer cells.Transfection of si-METTL3 can reverse the promotion effect of hsa＿circ＿0008583 overexpression on proliferation,migration and invasion of hepatocellular carcinoma cells,and reverse the inhibitory effect of hsa＿circ＿0008583 overexpression on apoptosis of hepatocellular carcinoma cells.Transfection of miR-1301-3p-inhibitor reverses the inhibition of hsa＿circ＿0008583 knockout expression on proliferation,migration,and invasion of hepatocellular carcinoma cells,and reverses hsa＿circ＿0008583 the effect of knockdown expression on apoptosis of hepatocellular carcinoma cells(p< 0.01).Conclusion:The content of hsa＿circ＿0008583 in HCC tissues and cells was significantly increased,and hsa＿circ＿0008583 could promote the invasion,migration and proliferation of HCC cells,inhibit the apoptosis of HCC cells and promote the growth of HCC tumors in vivo.Hsa＿circ＿0008583 binds with miR-1301-3p via competing to METTL3 through ce RNA regulation mechanism.As a result,the silencing effect of miR-1301-3p on METTL3 expression was weakend,the expression of METTL3 indirectly was increased,than the level of m6 A methylation was increased;thereby hsa＿circ＿0008583 promoted the invasion,proliferation and migration of HCC cells,and inhibited the apoptosis of HCC cells. |
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