The Tumor-promoting Role And Mechanism Of TG2 In HBV-positive Hepatocellular Arcinoma

Background There are around 2 billion people infected with the hepatitis B virus(HBV)all over the world,of which about 240 million suffer from the chronic hepatitis B.The latest research results show that the HBV infection is the main risk factor for the development of the hepatocellular carcinoma(HCC),and the relative risk degree of suffering from the HCC in those HBV carriers is more than 100 times higher than that in normal persons,and about 80%of HCC is deteriorated from the chronic liver injury,hepatitis,liver fibrosis and liver cirrhosis.The survival rate of HCC patients is around 10%over 5 years,and its morbidity ranks is the 5th while the mortality is the third.Nowadays,the.serum alpha-fetoprotein(AFP)is the only molecular marker applied to clinical diagnosis for HCC,which,however,only reports rise only in form 60%to 70%of the HCC,while noabnormal can be viewed in about one third of the HCC patients.Therefore,it is urgently to screen the tumor molecular markers that can be combined with AFP to improve the positive rate of serological diagnosis of HCC.The tissue transglutaminase(TG2),a inducible transaminyltransferase which i s calcium ion-dependent and protein modified by transamide-catalyzed,has b een found to play a significant role in cell apoptosis,adhesion,proliferation and signal transduction,which is attributed to its non-enzymatic function,an d is usually positive to the metastasis of certain solid tumors.Today's resear ches on TG2 mainly focus on some malignant tumors,such as breast cancer,pancreatic cancer,renal cancer,lung cancer,colon cancer and melanoma,but there is few reports positive to the role,mechanism and positive signal pathway of AFP involved in HBV-positive HCC cells.Objective To explore the role,mechanism and possible signal pathway of TG2 in HBV-positive HCC,the paper intends to discuss from four aspects:tissue,cell,animal and signal pathway.It would be the experimental basis for screening molecular markers for diagnosis and accurate treatment of HCC.MethodChapter One Expression and Role of TG2 in HBV-positive HCC Tissues1.Immunohistochemical(IHC)analysis and RT-PCR determination was performed on 150 paraffin-embedded HBV-positive HCC tissues and paracancerous tissues,to observe the expression of TG2 in HBV-positive tissues and five HCC cell lines.2.To observe the phenotypic changes of the proliferation,migration,and invasion tables of HBV-positive HCC,as well as investigate the roles of TG2 in proliferation,migration and invasion HBV-positive HCC,shRNA was adopted to targeting TG2 gene knock-down for HBV-positive HCC cell lines,and subsequently MTT experiment,scratch assay,and Transwell experiment were carried out on the cells.Chapter Two Expression and Role of TG2 in AFP negative HBV-positive HCC Tissues1.Fresh frozen HCC tissues and matched paracancerous tissues were paraffin-embedded among 58 cases including positive HBsAg,negative HBV DNA and negative AFP,followed by slices immunohistochemical(IHC)analysis,to investigate the expression of TG2 protein in AFP negative HCC tissues.Meanwhile,total RNA was extracted and used to detect the expression of TG2 in the HCC tissues and matched paracancerous tissues by RT-PCR and Western blot.2.The miR-TG2 lentiviral vector was constructed and packaged,and transinf ected into HepG 2.2.15 and Hep3B cells,and then resuscitated and cultivate HBV-positive HCC cell lines(HepG 2.2.15 and Hep 3B),and RT-PCR,CCK8,transwell and flow cytometry double staining detection were applied for detecting the expression of TG2 in the cell lines mentioned above(befor e and after the infection by miR-TG2 lentiviral vector),and also evaluated the cell viability,proliferation,invasion,migration and apoptosis.Chapter Three The effects of HepG2.2.15 on the Neoplasia Inside the Nu de Mice Before and After TG2 shRNA1 Transfection1.HepG2.2.15 and Hep3B were handled with shRNA,and followed by cell experimental result to screen stable lines(HepG2.2.15),and 4 to 6-weeks old BALB/c nude mice were selected to conduct the tumorigenicity assay.3.The size of the tumor was measured per 3 days,and the data of mice4.and tumor growth were recorded and used for drawing the curves.Afterwards,the tumor were removed and photographed and weighed,and subsequently used for the HE staining test,TUNEL labeling test,western-blot test and RT-PCR test.Chapter Four The Mechanism and Pathway of Tumorigenic of TG2 in H BV-positive HCCAfter consulting articles on breast cancer,renal cancer lung cancer and colon cancer,and referring from literature and database,five pathway proteins possibly positive to the tumorigenic mechanism of TG2,including caspase3,caspase8,caspase9,TGF-? and NF?B,were preliminarily selected,and the tumorigenic mechanism was discussed from two aspects,respectively apoptosis and proliferation.Additionally,the tumorigenic mechanism and positive signal pathway of TG2 in HBV-positive HCC were preliminarily explored and validated by the methods of Western blot and RT-PCR in HBV-positive HCC cells(HepG2.2.155 and Hep3B)and the tumorigenic tissues of nude mice.ResultChapter One:The IHC results showed that almost 70%of the tumor specimens were TG2 positive,and which significantly higher than that of the nor mal group(22.86%),and indicated that the expression level of TG2 in paracancerous tissues was significantly lower than that in HCC tissues,while the expression level of TG2 mRNA in the fresh frozen HBV-positive HCC tissues was higher than that in matched paracancerous tissues(P<0.05),and meanwhile the expression level of TG2 in HBV-positive HCC cell lines was significantly higher than that in non-HBV-positive HCC cell lines(P<0.05);an d it was the first revelation that,compared with the paracancerous tissues,th e expression of TG2 mRNA and protein in HBV-positive HCC tissues increased significantly,and both the transcription and protein levels were up-regulated;and it was also firstly found that the targeting knock-down of TG2 in HBV-positive HCC cell lines(HepG 2.2.15,Hep3B)could inhibit the external proliferation,migration and invasion effect of HCC cells;besides,it was also found that the expression of TG2 in 3 non-HBV-positive HCC cell lines was significantly lower than that in HBV-positive HCC cell lines.Chapter Two:Both the IHC and RT PCR results indicated that TG2 was hi ghly expressed in AFP negative HBV-positive HCC tissues,and was significa ntly higher than that in matched paracancerous tissues(P<0.05);furthermore T G2 mRNA was expressed higher AFP negative HBV-positive HCC tissues thanthat in matched paracancerous tissues(P<0.05);and then the stable interference sequences was screened and packaging with lentivirus to LV5 carrier,subsequently used to infect HBV-positive HCC cells and non-HBV-positive HCC cells,then it was found that the expression of TG2 in HBV-positive HCC tissues was higher than that in non-HBV-positive HCC cells(P<0.05);after targeted TG2 knock-down,the CCK8 assay revealed that the proliferation ability of HCC cells decreased;and the flow cytometry double staining detection indicated that the apoptotic ability of HCC cells increased.Chapter Three:The weight loss of nude mice have been viewed significantlyin TG2-NC group(P<0.05)and the tumor size was obviously larger than that of the knock-down group(P<0.05).And HE staining explored that the tumors in the TG2 knock-down group appeared relatively normal and less necrotic tissue,while in the normal group and NC group,both of which were defined as control groups,there were more necrotic tissues and disordered structure with shrinking cell nucleus and cytolysis.TUN EL labeling method was applied to detect the apoptosis of tumorigenic tissues,the positive cell rate in NC group and TG2 knock-down group was 19.34±2.43 and 6,56±2.13(P<0.05)respectively,suggesting that the knock-down of TG2 could increase the apoptosis of HCC cells and then affect the tumor growth.Western blot was applied to detect the expression of protein,revealed that TG2 protein expressed higher in NC group than that in TG2 knock-down group,suggesting that the targeting knock-down of TG2 could inhibit the tumor growth inside BALB/c nude mice,therefore,it can be speculated that TG2 has played a significant role in promoting the proliferation and anti-apoptosis of HCC cells.Chapter Four:The expression of 5 pathway proteins in Hep3B cells and HepG2.2.15 cell lines(normal group,TG2 shRNAl empty vector transfection group and TG2 shRNAl transfection group)indicated that the expression of Caspase3 in the TG2 targeted knockdown group of Hep3B cells decreased,while that of the other 4 groups increased significantly(P<0.05),under the premise that the expression of the total amount of both internal reference and target protein was basically the similar;in the TG 2 knock-down group of HepG2.2.15 cells,the expression of Caspase3 increased while that of Caspase 8,Caspase 9,NF?B and TGF? dropped significantly(P<0.05).In the tumorigenic tissues of TG2 knock-down group,the expression of the apoptosis-positive proteins,including Caspase 3,Caspase8,Caspase 9,TGF?and proliferation-promoted protein NK?B down-regulated uniformly.Western blot was applied to verify the expression of ERK1/2 antibody in the tumor tissues of the nude mice,and revealed that the expression of ERK1/2 in TG2 knock-down group decreased significantly(P<0.05),indicated that the tumor promoting effect of TG2 can also be achieved through ERK1/2-NF?B pathway by adjusting proliferation of tumor cells.There are two kinds of effects in HBV-positive HCC,namely apoptosis and proliferation,it means that it cannot only promote the apoptosis of HCC by up-regulating the expression of apoptosis positive proteins,but also promote the proliferation of HCC by up-regulating the expression of proliferation positive proteins,but the latter has always been dominant.In this research,it was the first report that TG2 in HCC activated and regulated the proliferation of HCC by ERK1/2-NF?B pathway.ConclusionTG2 in HBV-positive HCC was highly expressed in matched paracancerous tissues,especially in AFP negative HBV-positive HCC,the expression was significantly up-regulated;and TG2 in HBV-positive HCC expressed significantly higher than that in non-HBV-positive HCC cell lines,indicated that TG2 combined with AFP might be served as a new tumor marker for diagnosing HCC.It inhibited the proliferation,migration and invasion of HCC cells in vitro,and the tumor growth of nude mice,while expedited apoptosis,when TG2 was knocked down,indicated that TG2 in HBV-positive HCC cells had effect on promoting the development of malignant phenotype.In addition,as it tol d above,TG2 in HCC regulated the proliferation of HCC by ERK1/2-NF?B pathway,might serve as an important molecular target for the treatment of HCC in the future.TG2 involved in the migration,invasion,proliferation and apoptosis of HCC through multiple pathway,which provided laboratory basis to for screening novel molecular markers and precise molecular targeted therapy of HCC.