| Objective:Primary liver cancer is the sixth most common malignant tumor in the world in terms of incidence rate and the third in terms of mortality.Most of them are hepatocellular carcinoma(75%～85%).Although surgery can effectively treat hepatocellular carcinoma,70%～80%of liver cancer patients are in the middle and late stages when they come to the hospital without the opportunity of radical surgery.At the same time,the existing targeted drug therapy has the dilemma of few targets and large individual differences in efficacy.Therefore,it is necessary to continue to explore the molecular mechanism of the occurrence and development of hepatocellular carcinoma,and find new biomarkers and therapeutic targets.Because of its unique biological characteristics,circular RNA can participate in the regulation of tumor progression through a variety of mechanisms,and may become a new molecular marker for clinical cancer diagnosis and treatment.The purpose of this study is to find new circ RNA molecules of hepatocellular carcinoma by searching GEO database,and the target circ RNA were verified by clinical hepatocellular carcinoma tissue specimens and hepatocellular carcinoma cell lines.The effects of target circ RNA on hepatocellular carcinoma proliferation,migration,invasion and epithelial-mesenchymal transformation were investigated by cell functional and animal models.The target miRNA and target m RNA of target circ RNA were predicted by biological database,and the mechanism of circ RNA/target miRNA/target m RNA in hepatocellular carcinoma was elucidated by cell functional experiments,in order to provide new biomarkers and potential therapeutic targets for the diagnosis and treatment of hepatocellular carcinoma.Methods:(1)GEO database was searched with the search formula"(circ RNA OR circular RNA)AND hepatocellular carcinoma",and the circ RNA microarray data of HCC and normal tissues were selected.Differential expression circ RNA was screened by GEO2R online tool,and the target circ RNA(hsa＿circ＿0066568)was confirmed by clinical specimens and cancer cells.hsa＿circ＿0066568 real-time PCR method was established.83 pairs of hepatocellular carcinoma tissues and adjacent tissues were collected from the First Affiliated Hospital of Changsha Medical College,and the expression level of hsa＿circ＿0066568 was detected by qRT-PCR.χ~2 test was used to investigate the relationship between hsa＿circ＿0066568 expression level and clinicopathologic features of patients with hepatocellular carcinoma,and Cox regression analysis was used to investigate the relationship between hsa＿circ＿0066568 expression level and prognosis of patients with hepatocellular carcinoma.The sequence composition of hsa＿circ＿0066568 was obtained by querying circbank database.The stability of hsa＿circ＿0066568 was measured by RNase R treatment of hepatocellular cancer cell line,and the half-life of the cancer cell line determined by actinomycin D treatment.The expression level of hsa＿circ＿0066568 was detected by cytoplasmic separation of hepatocellular carcinoma cells and the localization of hsa＿circ＿0066568was confirmed.(2)hsa＿circ＿0066568 overexpression/knockdown expression lentiviral plasmid was constructed,and after packaging,the lentivirus infected Huh7 cells and Hep G2 cells.qRT-PCR was used to verify the efficiency of hsa＿circ＿0066568 overexpression and down-expression.CCK8 was used to detect the proliferation ability of cancer cells.Transwell assay was used to detect the migration and invasion ability of cancer cells,and Western blotting was used to detect the expression of EMT-related proteins.hsa＿circ＿0066568 stable overexpression cell line Huh7 were constructed.The tumor size was observed after subcutaneous injection in nude mice,and the tumor tissues were sacrificed at the 27en day for HE staining.(3)Target miRNAs of hsa＿circ＿0066568 predicted by Circ Interactome and circ Bank database,and the expression of miRNAs in Huh7 and Hep G2 cell lines with normal or knockdown hsa＿circ＿0066568,and miR-935 was identified as the target miRNA.Huh7 cells were transfectedwithsi-NC,si-hsa＿circ＿0066568and si-hsa＿circ＿0066568+miR-935 inhibitor,respectively.qRT-PCR was used to detect the expression of miR-935 in cells to test the transfection efficiency.Potential target m RNA of miR-935 were predicted from Targetscan,miRDB and miRTar Base websites,and FZD6 with strong evidence binding was selected.The expression of FZD6 m RNA and protein in Huh7 cells with normal or knockdown miR-935 expression to verify evidence binding.Hep G2 cells were transfected with Vector,OE-hsa＿circ＿0066568 and OE-hsa＿circ＿0066568+si-FZD6,respectively,and the expression of FZD6 m RNA was detected by qRT PCR to verify the transfection efficiency.CCK8 was used to detect cell proliferation,Transwell assay to detect cell invasion,and Western blotting was used to detect the expression of EMT-related proteins.Results:(1)The GSE155949 dataset was retrieved from GEO database,and 2339 differentially expressed circrnas were analyzed by GEO2R online tool.The expression of hsa＿circ＿0066568 in HCC tissues was significantly higher than that in adjacent tissues by qRT-PCR verified(P<0.05).The expression of hsa＿circ＿0066568 in 83 HCC tissues was significantly higher than that in adjacent tissues(P<0.05),and the proportion of cases with high expression of hsa＿circ＿0066568 was 79.5%(66/83).The proportion of hsa＿circ＿0066568 high expression in HCC cases with BCLC stage C,TNM stage III,HBV infection,liver cirrhosis,AFP≥400ng/ml or vascular invasion was higher than that in HCC cases with BCLC stage A/B,TNM stage I/II,no HBV infection,no liver cirrhosis,AFP<400ng/ml or no vascular invasion,respectively(P<0.05).The 3-year cumulative survival rate of cases with low hsa＿circ＿0066568expression(70.6%)was higher than that of cases with high hsa＿circ＿0066568 expression(30.3%).High level of hsa＿circ＿0066568(HR=2.555,95%CI:1.520-4.295)was one of the risk factors for prognosis of cases with hepatocellular carcinoma(P<0.05).hsa＿circ＿0066568 is formed by cyclification of exons 6,7 and 8 of ROBO1 gene[chr3(p12.3)]located on the short arm of human chromosome 3.Its abundance does not decrease significantly after RNase R treatment,and its half-life exceeds 16 hours,mainly localized in the cytoplasm of cells.(2)After transfection of si＿hsa＿circ＿0066568/OE＿hsa＿circ＿0066568,the expression level of hsa＿circ＿0066568 in Huh7 and Hep G2 cells was significantly decreased/increased(P<0.05).But the m RNA expression level of maternal gene ROBO1 had no significant change(P>0.05).Compared with si-NC control group,the growth activity,the number of migrating and invading cells,and the expression of proteins in interstitial cells of Huh7 and Hep G2 cells in hsa＿circ＿0066568 knockdown were decreased,while the expression of proteins in epithelial cells increased(P<0.05).Compared with Vector control group,the growth activity,the number of migrating and invading cells,and the expression of proteins in interstitial cells of Huh7 and Hep G2 cells in overexpression hsa＿circ＿0066568 were increased,while the expression of proteins in epithelial cells decreased(P<0.05).After 27 days of tumor formation,the tumor volume of Huh7-circ66568 group was significantly larger than that of MGC-EV group(P<0.05).(3)Circ Interactome and circ Bank database jointly predicted that miR-935 and miR-1183 were miRNAs adsorbated by hsa＿circ＿0066568,and the binding sites were located in exon 6 and exon 7 respectively.After knockdown of hsa＿circ＿0066568,the expressions of both hsa＿circ＿0066568 and miR-935 were up-regulated in Huh7 and Hep G2cells,and the up-regulated level of miR-935 was higher(P<0.05).The expression of miR-935 in HCC tissues was lower than that in adjacent tissues,and miR-935 was negatively correlated with hsa＿circ＿0066568(P<0.05).The miR-935 binding sequence on the miRNA reaction element of hsa＿circ＿0066568 was"GUAACUG".The abundance of hsa＿circ＿0066568 and miR-935 in AGO2 antibody group was higher than that in Ig G antibody group(P<0.05).Compared with si-NC group,the growth activity,the number of migrating and invading cells,and the expression of proteins in interstitial cells of Huh7 cells after hsa＿circ＿0066568 knockdown were decreased,while the expression of proteins in epithelial cells increased(P<0.05).Compared with si-hsa＿circ＿0066568 group,the growth activity,the number of migrating and invading cells,and the protein expression of interstitial cells were increased in si-hsa＿circ＿0066568+miR-935 inhibitor group,while the protein expression of epithelial cells was decreased(P<0.05).Twenty-one candidate target genes of miR-935 were predicted by Target Scan,miRDB and miRTar Base databases,and FZD6 was the preferred target gene of miR-935.Knockdown of hsa＿circ＿0066568 can down-regulated the m RNA expression of FZD6 in Hep G2 cells,and knockdown of miR-935can up-regulated the m RNA expression of FZD6 in Hep G2 cells(P<0.05).The expression level of FZD6 m RNA in HCC tissues was higher than that in adjacent tissues,and the overall survival rate of HCC cases with low expression of FZD6 m RNA was better than that in high expression group(P<0.05).Compared with Vector group,the growth activity,the number of migrating and invading cells,and the protein expression of interstitial cells after hsa＿circ＿0066568 overexpression were increased,while the expression of proteins in epithelial cells decreased(P<0.05).Compared with OE-hsa＿circ＿0066568 group,the growth activity,the number of migrating and invading cells,and the protein expression of interstitial cells were decreased in OE-hsa＿circ＿0066568+si-FZD6 group,while the expression of proteins in epithelial cells was increased(P<0.05).Conclusions:(1)hsa＿circ＿0066568 is abnormally high expressed in hepatocellular carcinoma,which is closely related to the pathological stage and vascular invasion of hepatocellular carcinoma patients,and the prognosis of hepatocellular carcinoma patients with high level of hsa＿circ＿0066568 is worse.(2)hsa＿circ＿0066568 was cycled from exons6,7 and 8 of ROBO1 gene located on the short arm of human chromosome 3[chr3(p12.3)],which had a ring structure and high stability,and was mainly located in the cytoplasm.(3)hsa＿circ＿0066568overexpression can promote the proliferation,migration,invasion and epithelial mesenchymal transformation of hepatocellular carcinoma cells,and promote the growth of transplanted tumor in nude mice in vivo.(4)hsa＿circ＿0066568 sponge can adsorb miR-935,miR-935 target to bind FZD6,and hsa＿circ＿0066568 can be used as miRNA molecular sponge to regulate miR-935/FZD6 axis and then promote the progression of hepatocellular carcinoma.58 figures,11 tables and 197 references. |
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