| Objective:Liver cancer accounts for about 90% worldwide,and most liver cancers worldwide occur in the Asia-Pacific region.Hepatitis B virus(HBV)infection is the main risk factor for the development of liver cancer,and about 50% of liver cancers could be attributed to HBV infection.Meta-analyses showed that people with hepatitis B virus infection are 15-20 times more likely to develop liver cancer than non-infected people.Circular RNA(circRNA)was defined as non-coding RNA,which was characterized by its special covalent closed loop structure.The expression pattern of circRNA was complex,showing tissue/stage specific expression.Compared with non-tumor tissues,a large number of circRNAs are significantly up-regulated or down-regulated in tumor tissues.In previous studies,it was found that circ＿0027089was highly expressed in hepatitis B-related liver cancer tissues,but its role in hepatitis B-related liver cancer has not been reported yet.Studies had found that circRNA could competitively inhibit miRNA expression and biological functions by acting as a molecular sponge or competitive endogenous RNA(ce RNAs).The online prediction website(circBank)found that circ＿0027089 had a binding site with miR-136-5p.Studies reported that miR-136-5p was down-regulated in liver cancer tissues,but its role in hepatitis B-related liver cancer tissues remains to be explored.We guess that circ＿0027089 functions through miR-136-5p.Through further analysis,the online prediction website(star Base)predicted that miR-136-5p had a binding site with NACC1,and the high expression of NACC1 accelerates the progression of liver cancer,so it was speculated whether circ＿0027089 by regulating the miR-136-5p/NACC1 axis to regulate the development of hepatitis B-related liver cancer,it aims to provide a new direction for the treatment of hepatitis B-related liver cancer.Methods:The qPCR method was used to detect HBV DNA levels in the liver cancer cell lines HepG2,HepG2.2.15,HepAD38,and HepAD38(tet-off).The expressions of circ＿0027089,miR-136-5p and NACC1 in the Control group,HBV-and HBV+ were detected by qRT-PCR and Western blot respectively.qRT-PCR and Western blot were used to detect the expression of circ＿0027089,miR-136-5p,NACC1 in hepatitis B-related liver cancer cells HepG2.2.15 and HepAD38(tet-off).The Pearson method was used to detect the correlation between circ＿0027089 and miR-136-5p expression,the correlation between miR-136-5p and NACC1 expression,and the correlation between circ＿0027089 and NACC1 expression in liver cancer tissues(HBV+).Taking HepG2.2.15 and HepAD38(tet-off)cells as the research objects,experimental groups: Vector group,pCD5-circ＿0027089 group,si-NC group,si-circ＿0027089#1 group,si-circ＿0027089#2 group,si-circ＿0027089#2+anti-miR-NC group,si-circ＿0027089#2+anti-miR-136-5p group,miR-NC group,miR-136-5p group,miR-136-5p+pc DNA group,miR-136-5p +NACC1 group.The CCK8 experiment was used to detect cell viability.The plate clone formation experiment was used to detect the cell clone formation rate.Flow cytometry was used to detect cell apoptosis rate and cell cycle.A kit was used to detect the activity of Caspase3/7.The scratch test was used to detect the healing rate of cell scratches.Transwell chamber experiment was used to detect cell invasion ability.The dual luciferase reporter gene experiment meta-RIP experiment was used to detect the targeting relationship between circ＿0027089 and miR-136-5p,and the targeting relationship between miR-136-5p and NACC1.sh-NC and sh-circ＿0027089 were respectively transfected into HepG2.2.15 cells and subcutaneously injected into nude mice to establish a nude mouse model of hepatitis B-related liver cancer.After 27 days of culture,the nude mice were killed by de-neck method,and the transplanted tumor tissue was taken out and tested separately Weight and volume of transplanted tumor tissue.qRT-PCR and Western blot were used to detect the expression levels of circ＿0027089,miR-136-5p and NACC1 in transplanted tumor tissues.Results:1.The HBV DNA level in HepG2.2.15 cells was significantly higher than that in HepG2 cells(P<0.05),and the HBV DNA level in HepAD38(tet-off)cells was significantly higher than that in HepAD38 cells(P<0.05).2.Compared with the Control group,the expression level of circ＿0027089 and NACC1 in liver cancer tissue(HBV-)and liver cancer tissue(HBV+)increased(P<0.05),and the expression level of miR-136-5p decreased(P < 0.05),and there was a statistically significant difference between liver cancer tissue(HBV-)and liver cancer tissue(HBV+).Compared with human liver immortalized cells THLE-2,the expression levels of circ＿0027089 and NACC1 in HepG2.2.15 and HepAD38(tet-off)cells are increased(P <0.05),the expression level of miR-136-5p decreased(P<0.05).3.Compared with the Vector group,the cell viability of the pCD5-circ＿0027089 group increased(P< 0.05),the rate of clone formation and scratch healing increased(P < 0.05),the proportion of S-phase cells increased(P<0.05),and invasion Cell number increased(P<0.05),but apoptosis rate and Caspase3/7 activity decreased(P<0.05),G0/G1 phase cell ratio decreased(P < 0.05).4.Compared with the si-NC group,the expression level of circ＿0027089 in the si-circ＿0027089#1 group and si-circ＿0027089#2 group decreased(P < 0.05),and the expression level of circ＿0027089 in the si-circ＿0027089#2 group was relatively low,so it was chosen si-circ＿0027089#2 Perform follow-up experiments.5.Compared with the si-NC group,the cell viability of the si-circ＿0027089#2 group was reduced(P<0.05),the rate of clone formation and scratch healing was reduced(P<0.05),and the proportion of S-phase cells was reduced(P < 0.05),the number of invasive cells was significantly reduced(P<0.05),while the rate of apoptosis and Caspase3/7 activity increased(P<0.05),and the ratio of cells in G0/G1 phase increased(P<0.05).6.The dual luciferase reporter gene experiment and RIP experiment confirmed that circ＿0027089 could target and regulate miR-136-5p,and circ＿0027089 was negatively correlated with miR-136-5p(r=-0.5564,P=0.0009).7.Compared with the si-circ＿0027089#2+anti-miR-NC group,the cell viability of the si-circ＿0027089#2+anti-miR-136-5p group increased(P<0.05),the clone formation rate and the scratch healing rate increased(P<0.05),the proportion of S-phase cells increased(P < 0.05),the number of invasive cells increased(P < 0.05),while the apoptosis rate and Caspase3/7 activity decreased(P < 0.05),the ratio of cells in G0/G1 phase Decrease(P<0.05).8.The dual luciferase reporter gene experiment and RIP experiment confirmed that miR-136-5p could target and regulate NACC1,and miR-136-5p is negatively correlated with NACC1(r=-0.6363,P < 0.001).9.Compared with the miR-NC group,the cell viability of the miR-136-5p group was reduced(P<0.05),the rate of clone formation and scratch healing was reduced(P<0.05),and the proportion of cells in the S phase decreased(P<0.05),the number of invasive cells decreased(P < 0.05),while the rate of apoptosis and Caspase3/7activity increased(P<0.05),and the ratio of cells in G0/G1 phase increased(P<0.05).10.Compared with the miR-136-5p+pc DNA group,the cell viability of the miR-136-5p+NACC1 group was increased(P<0.05),the clone formation rate and the scratch healing rate were increased(P < 0.05),the proportion of cells in the S phase increased(P<0.05),the number of invasive cells increased(P<0.05),while the apoptosis rate and Caspase3/7 activity decreased(P < 0.05),and the ratio of G0/G1 cells decreased(P < 0.05).11.Circ＿0027089 was positively correlated with NACC1(r=0.7232,P<0.0001).Compared with the si-NC group,the expression of NACC1 in the si-circ＿0027089#2 group decreased(P < 0.05).Compared with si-circ＿0027089#2+anti-Compared with the miR-NC group,the expression of NACC1 in the si-circ＿0027089#2+anti-miR-136-5p group was increased(P<0.05).12.Compared with the sh-NC group,the volume and weight of transplanted tumors in nude mice in the sh-circ＿0027089 group were significantly reduced(P<0.05),the expression levels of circ＿0027089 and NACC1 were reduced(P < 0.05),and the expression levels of miR-136-5p increased(P<0.05).Conclusion : 1.Knockdown of circ＿0027089 could significantly reduce the proliferation,clone formation,migration and invasion ability of hepatitis B-related liver cancer cells,and could induce cell cycle arrest in G0/G1 phase,and can also induce cell apoptosis.2.The overexpression of miR-136-5p could inhibit the proliferation,colony formation,migration and invasion of hepatitis B-related liver cancer cells,and can induce cell cycle arrest,and could also promote cell apoptosis,while the overexpression of NACC1 could significantly antagonize the effect of miR-136-5p overexpression on the biological behavior of hepatitis B-related liver cancer cells.3.Knockdown of circ＿0027089 could inhibit the growth of hepatitis B-related liver cancer xenografts in nude mice by regulating the miR-136-5p/NACC1 molecular axis.4.circ＿0027089 could up-regulate the expression of NACC1 by negatively regulating the expression of miR-136-5p to participate in the occurrence and development of hepatitis B-related liver cancer. |
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