| BACKGROUND&OBJECTIVEHeptocellular carcinoma?HCC?is one of the most common cancer,and of which the world's morbidity is continuing in a rise.It is with characters of concealed onset,high malignant degree and being difficult to treat.The 5-year-survival rate of HCC after diagnosis is less than 10%and its cancer related death ranks the second in the world.According to the statistics,the HCC consists the second most common cause of cancer-related mortality in some urbans while has become the top cause in some rural areas in China.The pathological mechanism of HCC is very complicated,involving viral infection,gene recombination,inflammation,cell reparing,differentiation and regeneration etc.Based on current studies,during HCC,many important oncogenes,like ?-catenin,Axinl,PI3K and K-ras,are activated,while many tumor-suppressor genes,like P53,Rb1,CDKN2A,IGF2R and PTEN,are inactivated,resulting in the abnormality of many signal pathways like WNT,MAPK,NF-?B.The pathogenesis and development of HCC is a very complicated process with which the definite molecular mechanism has not been fully clarified due to the complexity of HCC pathogenesis.Early stage of HCC can be effectively treated through hepatectomy.However,due to the concealed early clinical symptoms and lacking of specific biochemical indicators,HCC is often diagnosed at an advanced stage,leading to a dismal prognosis and missing the best opportunity of surgery.It is urgent to find new ways for diagnosis and treatment.Advanced study of the molecular mechanisms of HCC is necessary for exploring new diagnostic and therapeutic target.Chronic infection with hepatitis B virus?HBV?serves as the major factor for HCC,which accounts for 52%of all HCC[1]HBV x protein?HBx?,encoded by HBV X gene,has been verified to act as a multifunctional oncogenic factor in the development of HBV-related HCC,including promoting cell cycle progression,inactivating negative growth regulators,regulating apoptosis and inhibiting nucleotide excision repair of damaged cellular DNA.However,the molecular mechanisms underlying HBx protein-mediated tumorigenesis have not been entirely clarified.In recent years,technological advances in high-throughput sequencing have enabled us to identify numerous non-coding RNAs?ncRNAs?.Apart from protein coding genes,some non-coding RNAs are now thought to function as oncogenes or tumor suppressors,participating in the pathogenesis and development of cancer.Long ncRNAs?lncRNAs?are broadly defined as transcribed RNA molecules greater than 200 nt in length and lacking an open reading frame or containing high density terminator,thus it cannot be translated into protein.Basically,lncRNAs appear to be involved in all aspects of gene regulation,including epigenetic regulation,transcriptional regulation,post-transcriptional regulation and so on.Through such gene regulation,lncRNAs are involved in a wide range of biological processes with spatial-temporal characteristics,including proliferation,cell cycle,apoptosis,differentiation etc.The role lncRNAs play in cancer development and progression has aroused wide concern.Some HCC related lncRNAs have been found.While the function and mechanism of molecular regulation mediated by lncRNA in HCC largely remains unknown when compared with microRNA?miRNA?.It's urgent to find out HCC related IncRNAs and further explore their functions and mechanisms.Recently,a microarray analysis carried out by Magkoufopoulou,C.et al.[2]revealed that quercetin can down-regulation IncRNA DBH-AS1 expression?NCBI Accession NO.NR102735;UCSC ID uc031tfk.1?in HepG2 cells.Quercetin is regarded as the prototype of a naturally-occurring chemopreventive agent for various cancers and is reported to be able to possess anti-proliferative and antioxidant activities in hepatocytes.Importantly,Hsieh,A.et al found that quercetin effectively repressed HBx-mediated upregulation of several key oncogenes.However,whether IncRNA DBH-AS1 is involved in the development of HBV-related HCC has not been elucidated so far.In our study,we aim to elucidate the function and mechanism of DBH-AS1 from three different aspects:clinical significance analysis,cell experiment and animal in vivo essay.The study can be devided into three parts as below:Part1 Expression of IncRNA DBH-AS1 in HCC tissues and its clinical significanceOBJECTIVETo figure out the expression of DBH-AS1 in HCC tissues and infer its functions in HCC development through the correlation analysis between DBH-AS1 expression level and clinicopathological characteristics.METHODSWe collected 45 HCC tissues and corresponding clinical data.There were 31 HBsAg test positive cases and 14 HBsAg test negative cases.Real-Time PCR was used to detect the expression of DBH-AS1 and HBx.SPSS 13.0 software was applied to the analysis of the correlation between IncRNA DBH-AS 1 expression levels and clinicopathological characteristics,thus to preliminarily infer the direction of the research on DBH-AS1's functions and mechanisms.RESULTSReal-Time PCR showed that,in the HCC clinical tissue specimen of 45 cases,the expression level of DBH-AS1 in HBsAg positive cases was higher than HBsAg negative cases?x2=4.132,P=0.042?.Statistical analysis revealed that high DBH-AS1 expression level was associated with tumor size??2=8.006,P=0.005?.While we did not find any correlation between DBH-AS1 expression levels and other clinico-pathological features.CONCLUSIONOur study found for the first time that the DBH-AS1 expression level is positively correlated with the expression level of HBx and also related to the HBV infection and tumor size.Part 2 The in vivo and in vitro biological functional study of DBH-AS1 OBJECTIVETo identify the impact of DBH-AS1 has on the biological behaviors of HCC,like cell growth,proliferation,invasion and migration,etc.METHODS1.Real-Time PCR was used to detect the expression level of DBH-AS1 of five different HCC cell lines?SK-Hepl,HepG2,Hep3B,SMMC-7721,MHCC97H?and immortal heptocyte cell lines?LO2,QSG7701?.Choose two cell lines which are with lower DBH-AS 1 expression level among the five HCC cell lines to construct DBH-AS1 stably overexpressed cell strain.Choose DBH-AS 1 with highest expressed HCC cell lines to construct DBH-AS1 stably knocked down cell strain.2.To explore the function of DBH-AS1 in vitro and in vivo,DBH-AS1 stably up-regulated and down-regulated cell lines were established by lentiviral vector infection system?LV-DBH-AS1/sh-DBH-AS1?with green fluorescent protein?GFP?tag as well as the corresponding negative control cells?NC?.Then use Real-Time PCR combined with GFP expression to evaluate the infection efficiency.3.EdU,CCK-8 and plate colony formation assay were used to evaluate the effect of DBH-AS1 on proliferation ability.Cell cycle distribution was detected by flow cytometry method?FCM?.To observe the effect of DBH-AS1 on tumorigenesis in vivo,the DBH-AS1 over-expressed SMMC-7721 cells and NC group cells were implanted subcutaneously to nude mice.RESULTS1.Real-Time PCR results indicate that DBH-AS1 expression is higher in HCC cell lines than in immortal hepatocyte cell lines.SK-Hepl and Hep3B cells have higher DBH-AS1 expression levels than SMMC-7721 and HepG2 cells2.We successfully constructed DBH-AS1 up-regulated stable cell lines and DBH-AS1 down-regulated stable cell lines.The efficiency of infection were confirmed by Real-Time PCR,indicating satisfied up or down regulation of DBH-AS1 compared with negative control cells.3.HepG2,SMMC-7721 showed a significantly increased proliferation abilities after DBH-AS1 over expressed,while Hep3B showed a significantly decreased proliferation abilities after DBH-AS1 down regulated,compared with the NC cells,as determined by EdU assay,CCK8 assay and plate colony formation assay.FCM assay indicated that over expression DBH-AS1 promotes cell cycle from G1 phase to S or G2 phase.The in vivo assay also showed stronger tumor growth ability after over expressing DBH-AS 1.CONCLUSIONThe in vivo and in vitro assay indicates that DBH-AS1 plays an important role in the proliferation and growth of HCC,which might be achieved through promoting the cell cycle progression.Part 3 The molecular mechanism of DBH-AS1 in regulating HCC cell proliferationOBJECTIVETo find out the mechanisms of DBH-AS1 of promoting cell proliferation and the upstream regulators of DBH-AS1.METHODS1.Real-Time PCR was used to detect the expression of key genes related to cell growth,cell proliferation,cell cycle in the DBH-AS1 overexpression or knockdown group and negative control?NC?group cells.2.Western blot was adopted to evaluate the activating level of key proteins?ERK?p38?JNK?in MAPK pathway in test groups and NC groups.3.To find out the upstream regulators of DBH-AS1,we used siRNA technology to knockdown TP53 gene in HepG2 and LO2 cells.Then used Western blot to detect the p53 protein level between the test groups and negative control?NC?groups.The change of DBH-AS1 was determined by Real-Time PCR.4.Recombinant lentiviruses containing HBx?Lv-HBx?or control?Lv-con?were used to infect HepG2 and LO2 cells to construct stable cell lines.The infection efficiency was confirmed by qRT-PCR and western blot.Real-Time PCR was used to detect the change of DBH-AS1 between the test groups and NC groups.RESULTS1.Real-Time PCR showed that overexpression of DBH-AS1 in HepG2 cells elevated the expression of oncogenic cell-cycle regulators including CDK6,CCND1,CCNE1,but reduced the expression of cyclin-dependent protein kinase inhibitors including p16,p21,p27.While the expression of CDK6,CCND1,CCNE1 were down regulated and p16,p21,p27 were up regulated in Hep3B cells with DBH-AS1 silenced.2.Western blot results indicated that overexpression of DBH-AS 1 in HepG2 cells elevated the protein levels of CDK6,CCND1,CCNE1 and the activation level of p-ERK,p-p38,p-JNK,decreased the protein levels of p16,p21,p27.By contrast,in Hep3B cells with DBH-AS1 silenced,the protein levels of CDK6,CCND1,CCNE1 and the activation level of p-ERK,p-p38,p-JNK were all decreased,while the protein levels of p16,p21,p27 were up regulated.3.TP53 gene was satisfactorily silenced in HepG2 and LO2 cells which was confirmed by Real-Time PCR and Western-blot of its mRNA and protein level.The expression of DBH-AS1 were significantly up regulated in the TP53 silenced groups compared with NC group.4.The up-regulation of HBx in HepG2 and LO2 cells infected by Lv-HBx were confirmed by Real-Time PCR and Western-blot analysis.The expression level of DBH-AS1 was significantly increased in the HBx up-regulated groups compared to NC groups.CONCLUSIONActivation of the ERK/p38/JNK MAPK signaling pathway partially accounts for DBH-AS 1-induced cell growth and proliferation.Furthermore,lncRNA DBH-AS1 is negative regulated by p53,and its expression is induced by HBx protein. |
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