| Background and objective:Hepatocellular carcinoma(HCC)is the most common type of liver cancer,with high incidence rate and high mortality rate.HCC itself is a multi-step,multi-stage disease,involving complex regulatory mechanisms.The abnormal expressions of a great number of genes play important roles in the progress of HCC.Organic anion transporter polypeptide 1B3(OATP1B3)is a membrane transport protein expressed in normal liver tissues,which can transport hormones,drugs and other endogenous and exogenous substances.A large number of studies have reported that the expression of OATP1B3 in pancreatic cancer,colorectal cancer,cholangiocarcinoma,breast cancer,prostate cancer,gastric cancer and other malignant tumors can be used as a prognostic biomarker,and participate in the progress of some tumors.In HCC,the expression level of OATP1B3 was significantly correlated with tumor differentiation,suggesting that the differential expression of OATP1B3 may be closely related to the progression of HCC.Therefore,this study will explore the relationship between the expression of OATP1B3 in HCC,clinicopathological factors,and prognosis,as well as the effect and mechanism of OATP1B3 on the biological function of HCC cells.Methods:1.Kaplan-Meier method was used to analyze the correlation between OATP1B3 mRNA and disease-free survival rate and overall survival rate of 302 HCC patients from TCGA database.Immunohistochemical staining was used to detect the expression of OATP1B3 in131 tumor tissues of HCC patients and 89 adjacent normal tissues.qRT-PCR was used to detect the expression of OATP1B3 mRNA in 30 pairs of fresh HCC and matched normal tissues.Western blot was used to detect the protein expression level of OATP1B3 in 34 pairs of fresh HCC and matched normal tissues.The ?2 test was applied to analyze the correlation between OATP1B3 expression and the clinical parameters of HCC patients.The prognostic value of OATP1B3 in HCC patients was estimated by Kaplan-Meier survival analysis.The Cox stepwise proportional hazards model was used to analyze the independent prognostic factors affecting the survival of HCC patients.2.MHCC97-H,SMMC-7721,Huh7,Hep G2 and L02 cells were cultured in vitro.The expression of OATP1B3 mRNA was detected by qRT-PCR.SMMC-7721 cells with high expression level of OATP1B3 mRNA were selected to construct the lentiviral interference model of OATP1B3,and the overexpression model of OATP1B3 plasmid was constructed in Huh7 cells with low expression level of OATP1B3 mRNA.The interference and overexpression efficiency of OATP1B3 were verified by qRT-PCR and Western blot.MTT and clone formation assays were performed to observe the effect of OATP1B3 on the proliferation of hepatoma cells.Transwell and wound healing assay were used to observe the effect of OATP1B3 on invasion and migration of hepatoma cells.Flow cytometry assays were used to detect the effect of OATP1B3 on apoptosis and cell cycle of hepatoma cells.3.Hep G2 cells with stable high expression of OATP1B3 and SMMC7721 cells with stable low expression of OATP1B3 were constructed.Western blot was used to detect the effects of OATP1B3 on the expression of Epithelial-mesenchymal transition(EMT)related markers E-cadherin,N-cadherin and vimentin,and the activation of Wnt/?-catenin/c-Myc pathway.Hep G2 and SMMC7721 cells were injected into the liver capsule of nude mice to establish the orthotopic liver cancer implantation model.After 35 days,the liver tumor metastasis of nude mice was observed by MRI.The survival time of nude mice in each group was recorded.After the nude mice died,the liver was dissected and removed for general observation of liver surface.Finally,the liver tumor tissues of nude mice were sectioned,stained with HE and observed under microscope.Results:1.According to the analysis of TCGA database,the overall survival rate and disease free survival rate of HCC patients with high expression of OATP1B3 mRNA were significantly higher than those with low expression(63.9% vs 26.6%,P = 0.005;27.6% vs 13.0%,P =0.017).Immunohistochemical staining showed that the expression of OATP1B3 in HCC tissues(59.5%,78/131)was significantly down-regulated compared with adjacent normal tissues(25.8%,23/89)(P < 0.0001).The expression level of OATP1B3 was significantly correlated with tumor size,recurrence,tumor differentiation and TNM stage(P < 0.05 for each),but not with age,gender,tumor capsule,HBs Ag,cirrhosis,tumor number,vascular invasion and serum AFP level.The overall survival rate and disease free survival rate of HCC patients with high expression of OATP1B3 were significantly higher than those with low expression of OATP1B3(33.0% vs 12.9%,P = 0.001;18.8% vs 5.3%,P < 0.0001).Cox proportional hazard model analysis showed that OATP1B3,vascular invasion and TNM stage were independent prognostic factors affecting overall survival rate of HCC patients(P < 0.05);OATP1B3 and TNM stage were independent prognostic factors influencing disease free survival rate of HCC patients(P < 0.05).2.The expression of OATP1B3 mRNA in L02,SMMC-7721,MHCC97-H,Huh7 and Hep G2 cells decreased in turn.SMMC7721 cells with stable low expression of OATP1B3 and Huh7 cells with stable high expression of OATP1B3 were constructed.The results of MTT and clone formation assays showed that up-regulation of OATP1B3 could inhibit the proliferation and clone formation rate of HCC cells,and down-regulation of OATP1B3 could promote the proliferation and clonal formation rate of HCC cells in a time-dependent manner.Transwell migration and invasion assays showed that up-regulation of OATP1B3 could inhibit the migration and invasion of HCC cells,and down-regulation of OATP1B3 could promote the migration and invasion of HCC cells.Wound healing assay results showed that up-regulation of OATP1B3 could reduce the migration and motility of HCC cells,and down-regulation of OATP1B3 could promote the migration and motility of HCC cells.The results of apoptosis showed that up-regulation of OATP1B3 could promote the apoptosis of HCC cells,and down-regulation of OATP1B3 could inhibit the apoptosis of HCC cells.Cell cycle analysis showed that up-regulation of OATP1B3 could shorten the S phase of HCC cells,and down-regulation of OATP1B3 could prolong S phase of HCC cells.3.Hep G2 and SMMC-7721 cells were transfected with lentivirus to establish stable overexpression and interference cell models of OATP1B3,respectively.After up regulating the expression of OATP1B3,the expression of E-cadherin,Vimentin,N-cadherin,?-catenin and c-Myc were decreased.After down regulating the expression of OATP1B3,the expression of E-cadherin,Vimentin,N-cadherin,?-catenin and c-Myc were increased.MRI examination and survival time statistics of nude mice showed that after up regulating the expression of OATP1B3,the tumor invasion and metastasis ability was inhibited,the number of liver metastases in nude mice was less,and the survival time was longer;after down regulating the expression of OATP1B3,the tumor invasion and metastasis ability was enhanced,and the nude mice had more liver metastases and shorter survival time.Finally,a large number of tumor nests could be seen in histopathological sections of each group.Conclusion:1.The expression of OATP1B3 was down regulated in HCC,and its expression level was significantly correlated with tumor size,recurrence,tumor differentiation and TNM stage.Combined with the analysis results of TCGA database,OATP1B3 could be used as a tumor biomarker for prognosis judgment of HCC patients.2.OATP1B3 can inhibit the proliferation,invasion and metastasis of HCC cells,promote apoptosis and shorten the proportion of S phase.3.OATP1B3 may inhibit the process of EMT through Wnt/?-catenin/c-Myc pathway.In nude mice,OATP1B3 can inhibit the invasion and metastasis of orthotopic implantation liver tumor.In conclusion,OATP1B3 may play an anti-tumor role in HCC through a variety of ways,and can be used as a potential tumor biomarker for prognosis of HCC patients. |
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