Expression And Clinical Significance Of Hsa＿circ＿0084606 And Its Parental Gene ASPH And Potential Regulatory Protein U2AF65 In Hepatocellular Carcinoma

Objective(s):1.The aim of this study was to evaluate the m RNA expression and clinical significance of hsa＿circ＿0084606 and its parental gene ASPH and potential regulatory protein U2AF65 in hepatocellular carcinoma tissues and their adjacent liver tissues,and the correlation between hsa＿circ＿0084606 and U2AF65 protein expression in HCC tissues;2.To determine whether preoperative neoadjuvant therapy affects the expression of hsa＿circ＿0084606 in HCC tissues;3.To investigate the regulation of hsa＿circ＿0084606 expression on the proliferation of hepatocellular carcinoma cells by in vitro cell assay.Methods:1.By collecting 49 fresh HCC tissues and adjacent liver tissues from patients after hepatobiliary and pancreatic surgery in our hospital(39 patients received no preoperative treatment and 10 received neoadjuvant therapy before surgery),29 HCC paraffin tissue samples without preoperative treatment were collected from the Department of Pathology.2.The m RNA expression of hsa＿circ＿0084606,ASPH and U2AF65 in 39 HCC patients without preoperative treatment and their adjacent liver tissues were analyzed by RT-q PCR;the expression of hsa＿circ＿0084606 in 10 HCC patients with preoperative neoadjuvant therapy and their adjacent liver tissues were detected by RT-q PCR.The effect of preoperative neoadjuvant therapy on hsa＿circ＿0084606 expression in HCC tissues and adjacent non-cancerous liver tissues was analyzed by subgroups.3.To investigate the relationship between the expression of hsa＿circ＿0084606,ASPH and U2AF65 in HCC tissues and the clinical data of patients;to analyze the correlation between the expression levels of hsa＿circ＿0084606 and the expression of ASPH and U2AF65,respectively.4.The expression of U2AF65 protein in 29 HCC patients without preoperative treatment was detected by immunohistochemistry,and the relationship between the expression of hsa＿circ＿0084606 and the expression of U2AF65 protein was analyzed.5.Finally,the expression of hsa＿circ＿0084606 in different HCC cell lines were detected by RT-q PCR,and PLC and Hep 3B cells were used as the study subjects to detect the proliferation ability of low and overexpression hsa＿circ＿0084606 in regulating HCC cells using CCK-8 kit.Results:1.Collect 49 cases of fresh HCC tissues after operation in the Department of Hepatobiliary and Pancreatic Surgery of our hospital,of which 39 cases did not receive any treatment before operation and 10 cases received preoperative neoadjuvant therapy;in the Department of Pathology of our hospital,only 29 cases of HCC tissues met the experimental requirements(10 patients had no tumor or loss of paraffin blocks).2.In HCC patients without preoperative treatment,the expression of hsa＿circ＿0084606,ASPH and U2AF65 in HCC tissues were higher than those in adjacent liver tissues(P < 0.05).In 10 HCC patients treated with preoperative neoadjuvant therapy,the expression of hsa＿circ＿0084606 was lower in cancer tissues than in adjacent liver tissues,but it was statistically insignificant(P > 0.05).Subgroup analysis showed that neoadjuvant therapy significantly affected hsa＿circ＿0084606 expression in HCC cancer tissues(P < 0.05).3.The expression of hsa＿circ＿0084606 was correlated with the expression of MVI and Ki-67(20%)protein in 39 HCC tissues(P < 0.05);in addition,the m RNA expression of ASPH was correlated with the presence or absence of hepatitis B history and preoperative AFP level(400 ug/L)in HCC patients(P < 0.05);and the m RNA expression of U2AF65 was correlated with the Ki-67(20%)protein expression level in HCC patients(P < 0.05).Correlation analysis showed that hsa＿circ＿0084606 expression were positively correlated with RNA expression of ASPH(r = 0.3940,P < 0.05)and U2AF65(r = 0.3400,P < 0.05),respectively.4.By immunohistochemical score,0 of 29 HCC patients without preoperative treatment were negative(-),12 were weakly positive(+),16 were positive(+ +),and 1 was strongly positive(+ + +)for U2AF65 protein in cancer tissues.hsa＿circ＿0084606 expression were correlated with U2AF65 protein expression levels(P < 0.05).5.In vitro cell experiments,hsa＿circ＿0084606 was significantly highly expressed in hepatoma cell lines(Hep G2,Huh-7,Hep 3B,HCC LM3,PLC)compared with normal hepatocytes L02(P < 0.05).Compared with the control group,the expression of hsa＿circ＿0084606 was significantly up-regulated in PLC and Hep 3B cells overexpressing hsa＿circ＿0084606(P < 0.05);hsa＿circ＿0084606 could not be knocked down in PLC and Hep 3B cells at present.CCK-8 assay showed that the proliferation ability of PLC and Hep 3B cells overexpressing hsa＿circ＿0084606 was significantly enhanced(P < 0.05).Conclusion(s):1.Circular RNA hsa＿circ＿0084606 and its parental gene ASPH and potential regulatory protein U2AF65 were expressed at higher m RNA levels in HCC tissues than in adjacent liver tissues.And highly expressed hsa＿circ＿0084606 was closely related to MVI and Ki-67(20%)in HCC patients,which may be a malignant biological feature that promotes HCC proliferation.2.Preoperative neoadjuvant therapy can reduce the expression of hsa＿circ＿0084606 in HCC tissues,suggesting that hsa＿circ＿0084606 may be used as a target to evaluate the efficacy of neoadjuvant therapy for HCC.3.Hsa＿circ＿0084606 is closely related to the expression of U2AF65 protein,and its molecular mechanism may be related to the regulation of RNA-binding protein.4.Overexpressed hsa＿circ＿0084606 enhanced the proliferation of hepatocellular carcinoma cells.