Antiviral Therapy For Patients With Intrahepatic Cholangiocarcinoma And The Role Of Long Noncoding RNA On Tumor Progression

PART Ⅰ: Antiviral therapy for patients with intrahepatic cholangiocarcinomaBackground and aims: Hepatitis B virus(HBV)infection is an important risk factor for intrahepatic cholangiocarcinoma(ICC).This study aimed to evaluate the effect of antiviral therapy(AVT)on surgical prognosis of ICC patients with HBV infection.Methods: The clinicopathological data of 928 ICC patients with HBV infection who underwent hepatectomy at the Eastern Hepatobiliary Surgery Hospital and the Mengchao Hepatobiliary Surgery Hospital between 2006 and 2011 were retrospectively reviewed.Detailed records of viral reactivation,tumor recurrence,cancer-specific survival(CSS)and overall survival(OS)were obtained.To adjust the potential covariates,survival rates were analyzed using the time-dependent Cox regression model.Results: The postoperative viral reactivation rates for patients who received preoperative AVT,who did not receive preoperative AVT with a low,or a high HBV-DNA level(< or ≥ 2000IU/mL)were 3.3%,8.3% and 15.7%,respectively(P < 0.001).A high viral level and viral reactivation were independent risk factors of recurrence(hazard ratio [HR]: 1.22 and 1.34),CSS(HR: 1.36 and 1.46)and OS(HR: 1.23 and 1.36).Five-year recurrence,CSS and OS for AVT patients(70.5%,46.9% and 43.0%)were better compared with patients who did not receive AVT and had a high viral level(86.5%,20.9% and 20.5%,all P <0.001);the differences in recurrence,CSS and OS were minimal compared with noAVT patients with a low viral level(71.7%,35.5% and 33.5%,P =0.057,0.051 and 0.060).Compared with no-AVT patients with a high viral level,patients who initiated AVT either before or after surgery had better long-term outcomes(HR: 0.44 and 0.54 for recurrence;0.38 and 0.57 for CSS;0.46 and 0.54 for OS).Conclusions: Viral reactivation was associated with worse prognosis after liver resection for HBV-infected ICC patients.AVT decreased viral reactivation and prolonged long-term survival for patients with a high viral level.PART Ⅱ: Regulation role and molecular mechanism of long noncoding RNA in the progression of intrahepatic cholangiocarcinomaBackground and aims: The incidence and mortality of intrahepatic cholangiocarcinoma(ICC)has increased in the past decades,which made this disease a research hotspot.Liver resection is the only curative treatment for ICC,but the tumor recurrence rate is high and the long-term prognosis is unsatisfactory.Because the onset of ICC is concealed and there are no obvious clinical symptoms in the early stage,most patients are found with late tumor stage and lose the opportunity of operation.Long noncoding RNA(lnc RNA)is longer than 200 bp and has no or weak protein coding ability.Increasing evidences suggest that lnc RNA is widely involved in cell processes,including epigenetic characteristics and gene expression regulation,but few studies have been conducted in ICC.This study will explore the regulation role and molecular mechanism of lnc RNA in the progression of ICC,in order to find new diagnostic markers and therapeutic targets.Methods: The differentially expressed lnc RNA in ICC and adjacent non-tumor tissues were identified by microarray detection and verified by real-time quantitative PCR.High content screening was used to find proliferation-related functional positive target genes.The relationship between the expression of target genes and the clinicopathological characteristics and surgical prognosis of patients was analyzed by large sample validation.Knockdown or overexpression of the target gene in RBE cell line were used to examine its effect on cell apoptosis,cell cycle and cloning ability.Stable knockdown of the target gene in QBC939 cell lines was used to evaluate its role in tumor growth in nude mice.Subcellular localization of the target gene was identified by RNA fluorescence in situ hybridization and nuclear-cytoplasmic isolation PCR.Bioinformatics predicted targeted micro RNAs and its downstream target genes,and the interaction between them was verified by dual-luciferase reporter assay.Results: Seven pairs of ICC tumor and adjacent non-tumor tissues were used for microarray detection,and 113 lnc RNAs were found to be highly expressed in tumor tissue.Through high content screening,we found a proliferation-related functional positive lnc RNA and named it URICC(up regulated in intrahepatic cholangiocarcinoma).Data of 116 ICC patients was reviewed to analyze the relationship between URICC expression and clinicopathological characteristics.Compared with patients with low expression of URICC,patients with high expression of URICC had higher serum CA19-9 level(58.8 vs.27.2 U/m L,P = 0.030),larger tumor size(7.0 vs.6.1 cm,P = 0.019),higher proportion of multiple tumor and marcovascular invasion(50.0% vs.25.9%,P = 0.007;25.9% vs.8.6%,P = 0.014,respectively).A high URICC level was independent risk factor of tumor recurrence(hazard ratio [HR]: 1.974,P = 0.020)and overall survival(HR: 1.796,P = 0.047).Interference of URICC can block cell cycle in G2/M phase,promote cell apoptosis and inhibit clone formation.In addition,in vivo experiments showed that inhibition of URICC expression can weaken the subcutaneous tumorigenicity of QBC939 cells in nude mice.Dual-luciferase reporter assay demonstrated that URICC can eliminate the inhibition of MCL-1 by targeting mi R-320 a through competitive endogenous RNA(ce RNA)mechanism,thus avoiding mitochondrial-dependent cell apoptosis.Conclusions: Lnc RNA URICC is highly expressed in ICC tissues and RBE cell lines.High expression of URICC is significantly associated with poor prognosis of patients underwent liver resection.Mechanismly,URICC can target mi R-320 a and lead to the restoration of MCL-1 expression,thus promoting the progress of ICC,which made URICC a potential therapeutic target and tumor marker.