Screening And Mechanism Research On The LncRNAs Involved In Hepatocellular Carcinoma Metastasis

Primary liver cancer is one of the most common malignancies in the world,especially in Asia and Africa,the incidence of liver cancer was significantly higher than other regions,and liver cancer mortality is in the second place among malignant tumors.Hepatocellular carcinoma(HCC)is a primary malignant neoplasm derived from hepatocytes,accounting for about 80% of all liver cancers.Therefore,the study of hepatocellular carcinoma-related oncogene or tumor suppressor gene,and the mechanism underlying cancer metastasis are important for hepatocellular carcinoma prevention and treatment.At present,clinical resection and liver transplantation are still the most effective treatment for hepatocellular carcinoma.However,due to the rapid progression and malignancy of liver cancer,most patients are in the advanced stage at the time of diagnosis and miss the best time for surgery.Hence,it limits the extensive application of surgical treatment.On the other hand,a number of patients are prone to relapse due to cancer metastasis even after surgery,leading to unfavorable outcomes.For these patients,the surgical treatment may not be the best option.Like all the malignancies,the occurrence and metastasis of hepatocellular carcinoma is a multi-gene,multi-step process involving complex biological events.Until now,there is an urgent need to discover novel diagnosis markers and therapeutic targets.Most of the previous studies have been focused on protein-coding genes' dysregulation or mutation to explore the molecular mechanisms or biomarkers of HCC.In recent years,recent advances in technologies,such as tiling arrays and RNA deep sequencing(RNA-seq),have made it possible to survey the transcriptomes of many organisms to an unprecedented degree.The discovery of long non-coding RNAs(lnc RNAs)has changed our view of the complexity of human genomes and transcriptomes.Accumulating reports suggest that aberrant lnc RNA expression may be a major contributor to tumorigenesis and progression,including liver cancer.Since there is a lack of direct screening of lnc RNAs in metastatic cancer and non-metastatic cancer in HCC,the biological functions of lnc RNAs in liver cancer metastasis are still obscure.Therefore,the present study applied a high-throughput strategy to directly identify lnc RNAs associated with hepatocellular carcinoma metastasis and to explore their functions and possible clinical significance.In the first chapter,the human lnc RNA + m RNA microarray of Capital Bio Co.,Ltd.was applied to investigate the differentially expressed lnc RNAs between metastatic HCC and nonmetastatic HCC tissues using 3 pairs of metastatic hepatocellular carcinoma tissues/adjacent tissues and 3 pairs of metastatic hepatocellular carcinoma tissues/adjacent tissues.After screening,the differentially expressed lnc RNAs were confirmed by using q RT-PCR and reanalyzed in in 44 patients' tissue and the adjacent tissue.The microarray results showed that there are 275 dysregulated lnc RNAs between metastatic and non-metastatic HCC tissues,among which 179 lnc RNAs were upregulated,76 lnc RNAs were downregulated.Interestingly,we identified that HOTAIR was down-regulated in HCC tissues,contrary to a previous study.Moreover,further analysis revealed that the expression level of HOTAIR has no correlation with metastasis and TNM staging of HCC patients.Since no available data can be obtained from the public database The Cancer Genome Atlas(TCGA),we believe that the biological roles of HOTAIR in HCC need to be re-examined.More importantly,we uncovered a dysregulated lnc RNA,a pseudogene-encoded lnc RNA named GOLGA2P10 Transcript1,which showed a significant correlation with clinical features of HCC patients.Furthermore,the clinical data in TCGA database shows that its upregulation was associated with shorter survival time in HCC patients,suggesting that it may be used as a prognostic marker.In the second chapter,the biological function of GOLGA2P10 were explored at the cellular and molecular level,showing that GOLGA2P10 could promote the proliferation and migration in SMMC-7721 and L02 cells but had no effect in Hep G2 cells,in which CTNNB1 is mutated.To investigate the underlying mechanism,we first used RT-PCR and FISH to study the localization and distribution of GOLGA2P10 in the SMMC-7721 cell.We found that GOLGA2P10 was mainly located in the nucleus.Then we used the m RNA microarray to screen the downstream genes and signaling pathways relating to GOLGA2P10.Our data showed that GORGA2P10 overexpression may activates Wnt signaling cascades in SMMC-7721 and L02 via promoting FZD3 and FZD6 expression.While in Hep G2 cells,GOLGA2P10 overexpression had no obvious effect in activating Wnt signaling pathway due to the mutation of CTNNB1 that lacking the Ser/Thr mitif which regulating its degradation.Notably,we found that GOLGA2P10 overexpression in SMMC-7721 cells could upregulate MMP7 expression,and promote c-myc and its downstream target gene GLS1,an important regulator for cellular metablism(lipid metabolism,glutamine metabolism).To investigate the mechanism of GOLGA2P10 upregulating FZD3 and FZD6,GO analysis,IPA analysis,cell biology and molecular biology experiments were performed.We hypothesized that GOLGA2P10 may interact with transcription factor CTBP1 and bind to the enhancer or promoter region of FZD3 and FZD6.However,RIP,CHIP and IP experiments are needed for verification.The third chapter aims to clarify the molecular mechanism of HOXD-AS1,a known lnc RNA we previously demonstrated to promote the metastasis of HCC.Our previous data confirmed that HOXD-AS1 was upregulated in metastatic HCC and its overexpression strongly accelerates HCC cell growth and metastasis in vitro and in vivo.Herein,we found that HOXDAS1 can function as a ce RNA,which inhibit mi R-19 a expression and upregulate ARHGAP11 A expression,a mi R-19 a target gene,consequently.We also confirmed that ARHGAP11 A was up-regulated in metastatic HCC samples,and its expression of was positively correlated with HOXD-AS1(r2 = 0.3393).The Survival analysis also showed that patients with higher ARHGAP11 A expression had shorter survival time and poor prognosis.The present investigation strongly indicates that HOXD-AS1 is an oncogenic lnc RNA that promotes HCC metastasis and that the pro-metastatic phenotype can partially be attributed to the HOXDAS1/mi R19a/ARHGAP11 A signaling axis.