The Role And Molecular Mechanism Of Long Noncoding RNA In Predicting Tumor Recurrence And Patient Survival After Liver Transplantation For Hepatocellular Carcinoma

Hepatocellular carcinoma (HCC) is one of the most prevalent cancers worldwide. China alone accounts for55%of the world’s cases each year, due to high incidence of chronic hepatitis B virus infection and liver cirrhosis. Currently, liver transplantation (LT) has been considered as a curative treatment since it provides complete tumor resection and correction of impaired liver function. However, the long-term survival of patients following LT remains unsatisfactory, because of the high frequency of recurrence and metastasis. Therefore, a better understanding of the molecular mechanisms underlying HCC recurrence is highly desirable.Part I Identification of long noncoding RNAs in explant HCC tissuesAims:Using RNA-Seq approach, we especially focused on cancer specific long noncoding RNAs (lncRNAs) in explant HCC tissues, which provided us a comprehensive understanding of the heterogeneity of the genome and the biological mechanisms of hepatocellular carcinoma.Methods:This study enrolled ten HCC patients who received liver transplant. The patient’s pathological data and other clinical data were completed. RNA-seq was performed with RNA samples extracted from tumor tissues and adjacent non-tumorous tissues by solexa sequencing technology. Then the data was analyzed by bioinformatics.Results:Seventeen differentially expressed lncRNAs (annotated) between the tumor tissues and adjacent non-tumorous tissues were identified based on the following criteria:Fold Change≥2-fold, FDR≤0.05and upregulated or downregulated in more than50%of the samples. Most of them were annotated to the regions on chromosome with loss of heterozygosity or amplification.Conclusions:Seventeen aberrantly expressed lncRNAs would be validated and studied about their expression patterns and functions in HCC, which could provide us new ideas and methods for the prevention of HCC recurrence after liver transplantation. Part II RNA-seq identified lncRNA HOTTIP was associated with tumor recurrence and poor outcome after liver transplantation for hepatocellular carcinomaAims:Recurrence after LT for HCC has been viewed as a progressive multistep process involving multiple genes. It has been reported that lncRNAs played key roles in tumor development and metastasis. Based on our preliminary RNA-seq data, we identified a new HCC related lncRNA HOTTIR This study was aimed to evaluate the expression patterns, biological effects and prognostic significance of HOTTIP in HCC. Methods:We examined the expression of HOTTIP by quantitative real time PCR, and analyzed the correlation between HOTTIP level and tumor recurrence after LT. The effects of HOTTIP depletion in HCC cells were determined in viability, apoptosis and invasion assay. Changes in gene expression induced by HOTTIP were identified by whole human genome expression microarray and immunoblot analyses.Results:HOTTIP was overexpressed in HCC cell lines (Huh7、MHCC97H、MHCC97L) and tumor tissues. Patients with HOTTIP high expression in HCCs showed a significantly shorter overall survival and higher cumulative recurrence rates after LT, compared with HOTTIP low expression group. Furthermore, in patients exceeding the Milan criteria, those with a high expression level of HOTTIP revealed a significantly higher cumulative recurrence rates. Multivariate analysis indicated that high expression of HOTTIP was an independent prognostic factor for overall survival and cumulative recurrence. In vitro studies, down-regulated HOTTIP resulted in decreased cell invasion ability, and sensitized doxorubicin, etoposide and oxaliplatin chemotherapy. Microarray and bioinformatics analysis revealed that HOTTIP modulated the WNT signal pathway involved in tumor metastasis. Depletion of HOTTIP significantly decreased the expression of noncanonical Wnt-5a in HCC.Conclusions:HOTTIP plays a critical oncogenic role in controlling HCC metastasis. HOTTIP may regulate tumor cell apoptosis, invasion and chemo-sensitivity through Wnt-5a pathway. In addition, HOTTIP could serve as a novel biomarker in predicting tumor recurrence and prognosis of HCC after LT. PARTⅢ Long noncoding RNA HOTAIR promotes cell migration and invasion via down-regulation of RNA binding motif protein38and predicts tumor recurrence in hepatocellular carcinoma patients.Aims:The long noncoding RNAs exerted a critical role in epigenetic modification. The aim of the present study is to examine the biological functions of HOTAIR in hepatocellular carcinoma (HCC), and uncover epigenetic mechanism in promoting HCC cell migration and invasion.Methods:We examined the expression of HOTAIR in HCC samples using quantitative real time PCR and analyzed its correlation with clinical parameters and prognosis in HCC patients that have undergone liver transplantation (LT). Suppression of HOTAIR using siRNA was performed to explore its roles in tumor progression. Changes in gene expression induced by HOTAIR were identified by whole human genome expression microarray and immunoblot analyses.Results:The expression level of HOTAIR was upregulated in HCC cell lines. siRNA suppression of HOTAIR in a liver cancer cell line reduced cell viability and cell invasion, sensitized TNF-a induced apoptosis, and increased the chemotherapeutic sensitivity of cancer cells to cisplatin and doxorubicin. Then, we profiled its gene expression pattern by microarray analysis of HOTAIR loss in BEL-7402HCC cell line. Microarray data and bioinformatics analysis indicated that several RNA binding proteins were involved in this biological process. HOTAIR suppression using RNAi strategy increased the expression levels of RNA binding motif protein38(RBM38). In addition, HOTAIR could promote migration and invasion of HCC cells by inhibiting RBM38, which indicated critical roles of HOTAIR and RBM38in HCC progression. In the clinical sample studies, the expression level of HOTAIR in cancer tissues was significantly higher than in adjacent noncancerous tissues. Patients with HOTAIR high expression in HCCs showed significantly shorter overall survival and higher cumulative recurrence rates after LT. Cox multivariate analysis indicated high expression level of HOTAIR in HCC was an independent prognostic factor for predicting HCC recurrence in LT patients. Furthermore, in patients exceeding the Milan criteria, those with a high expression level of HOTAIR revealed a significantly shorter recurrence-free survival and overall survival.Conclusions:HOTAIR exerted regulatory functions in various biological processes in cancer cells, such as apoptosis, chemo-sensitivity, mobility and invasion. Moreover, HOTAIR might regulate HCC cells migration and invasion through RBM38. In addition, HOTAIR had potential clinical value in predicting tumor recurrence and prognosis of HCC after LT. PART Ⅳ Long noncoding RNA PCAT-1predicting tumor recurrence and patient survival after liver transplantation for hepatocellular carcinomaAims:Tumor recurrence remains a critical issue in liver transplantation (LT) for hepatocellular carcinoma (HCC) patients. However, there is a lack of effective biomarker for predicting recurrence and patient survival. The aim of the present study is to investigate the expression status of long noncoding RNA PCAT-1, and its clinical significance as well as biological role in HCC. Methods:We examined the expression of PCAT-1in184HCC explant liver samples between2003and2011using quantitative real-time PCR, and analyzed its correlation with clinicopathological features and prognosis in those had undergone LT. Suppression of PCAT-1using siRNA in BEL-7402was performed to explore its roles in cancer progression.Results:PCAT-1expression levels were up-regulated in cancerous tissues than in corresponding noncancerous tissues. No significant correlations with any single clinicopathological parameters were observed, including tumor number, tumor size, preoperative AFP level, or histopathological grading. High expression level of PCAT-1was an independent prognostic factor for predicting HCC recurrence after LT. Furthermore, in patients within Milan criteria, those with a high expression level of PCAT-1revealed a significantly shorter disease free survival and overall survival. Moreover, BEL-7402liver cancer cell line transfected PCAT-1siRNA significantly reduced cell viability, cell invasion, and sensitized HCC cells to apoptosis that was triggered by either hypoxia, serum starvation, or chemotherapeutic drugs.Conclusions:The high expression level of PCAT-1in HCC could be a candidate biomarker for predicting tumor recurrence in HCC patients after LT, and may serve as a potential therapeutic target. Combination of Milan criteria and PCAT-1expression level may improve the stratification of HCC.