Experimental And Clinic Research On The Growth Of Human Hepatocellular Carcinoma Cells Via Regulation Of FHIT Gene And The Methylation Of Its Promotor Region

[Objective]1. To explore the expressions differences of the FHIT gene in human hepatocellular carcinoma tissues and para-carcinoma tissues, and to analyze the relationship between FHIT gene expressions and clinicopathological features of liver cancer, and to reveal the role and clinical significance of their expression differences in the occurrence and development process of liver cancer, so as to lay the foundation for the follow-up study on the occurrence mechanism of liver cancer.2. To detect the methylation status of FHIT in HepG2,Hep3B and Huh7 cells with the different expression of FHIT; To study the relationship of the methylation status of FHIT and its protein expression level; To explore the partial molecular mechanisms of the methylation of FHIT gene and the occurrence and development process of liver cance.3. To successfully construct targeting pLVX-IRES-FHIT slow virus carrier recombinant vector; To observe its effects on proliferation, apoptosis, invasion and cell cycle of human hepatocellular carcinoma cells:HepG2,Hep3B and Huh7; To explore the molecular mechanisms of FHIT regulating human hepatocellular carcinoma cell growth through inhibiting of cyclin D1 via β-catenin/TCF4 signaling pathways.4. By successfully constructed pcDNA3.0/pHA-FHIT recombinant vector in vitro experiment, and co-transfection with β-catenin into HepG2 cells;To observe transcription activity and expression level of protein of cyclin D1;To further study molecular mechanisms that FHIT regulates the cell growth of liver cancers via classic Wnt-β-catenin/TCF-4 pathway.5. Through clinical correlational research, experimental studies in vitro, to systematically describe effects and molecular mechanisms that FHIT regulates cell proliferation, apoptosis, invasion and cell cycle of human hepatocellular carcinoma cells, and its Wnt-β-catenin/TCF-4 pathway signaling pathway, which will help screening markers of liver cancer, assessment of prognosis, provide a theoretical basis and experimental data for the early diagnosis of liver cancer and its molecule targeted therapy.[Methods]1. Clinical correlative research90 cases of patients with primary liver cancer of complete the clinic pathological data and its surgical specimens to keep in the archives with paraffin blocks were collected. FHIT gene expressional differences in the above specimens were determined by IHC-tissue chip. The relationship between the FHIT gene and the clinic pathological factors and postoperative survival of liver ancer was analyzed.2. The human hepatocellular carcinoma cells HepG2, Hep3B and Huh7 were cultured. Then the total proteins were colleted to explore expression level of FHIT of these cells by Western blot. The DNA of HepG2,Hep3B and Huh7 were collected, then the DNA was treated with bisulfite. The methylation level of promoter region of FHIT was deteced by PCR(Methylation-special PCR, MSP).With 5-azadc treated, the total protein of FHIT of HepG2,Hep3B and Huh7 was collected to explore the expression of FHIT of these cells.3. In vitro studies, to successfully construct pLVX-IRES-FHIT slow virus recombinant vector and transfect it into human hepatocellular carcinoma cells HepG2, Hep3B and Huh7. To explore cell proliferation, cell invasion and cell cycle of these cells. The total RNA of Huh7 cells were extracted. The full sequence of FHIT was amplification by RT-PCR. According DNA sequencing, the pLVX-IRES-FHIT slow virus recombinant vector was constructed successfully by double enzyme digestion with T-FHIT vector and pLVX-IRES-mCherry virus vector.The pLVX-IRES-FHIT slow virus recombinant vector was transfected into HepG2, Hep3B and Huh7 cells by Lipofectamine 2000, while the control vector was transfected into the same cells. To explore the changes of transcription and translation level of these three cells by RT-PCR and Westernblot. To detect the infection of cell proliferation, apotosis, invasion and cell cycle of these cells with overexpression of FHIT and the relationship of biological effects and time gradient by MTT, Annexin V/PI, cell cycle detection technology, Transwell experiment.4. Through using restriction enzyme digesting and DNA sequencing to identify, the recombinant pHA-FHIT recombinant fusion gene expression vector was successfully constructed. The pHA-FHIT vector and the control pcDNA3.0 vector were transfected into HepG2 cells respectively, so FHIT-HA and pcDNA3.0 group (control) were produced. After cultured 48 hours, the protein of cells were collected to explore the expression of FHIT and using coloning formation assay to detect the growth of HepG2 cells. Using Westernblot to explore expression of cyclinDl. With co-transfection of pHA-FHIT and CD1 luciferase reporter into HepG2 cells (the control group is co-transfection of P-catenin and CD1 luciferase reporter), the luciferase activity of cyclinD1 was detected.[Results]1. Results of clinical correlative studies(1) IHC test results showed that there is significant difference in expressions of FHIT protein between hepatocellular carcinoma tissue and para-carcinoma tissue. The positive expression of FHIT protein in para-carcinoma tissue was significantly higher than hepatocellular carcinoma tissue (P<0.05).(2) Different expression levels of FHIT protein were correlated with tissue differentiation of liver cancer(P<0.05,CC=0.589) and lymph node metastasis (P<0.05,CC=0.326).(3) The overall survival of patient with postoperation was correlated with low expression level of FHIT protein with poor expression, tissue differentiation of liver cancer, clinic pathological T stage, TNM stage and tumor size (P<0.05), but uncorrelated with gender, age, existence of capsule, portal vein-emboli, AFP content, existence of cirrhosis and lymph nodemetastasis (p>0.05).2. Research on the methylation level of promoter region of FHIT in HepG2, Hep3B and Huh7.(1) Westernblot showed that low expression of FHIT was in HepG2, Hep3B cells, but higher expression of FHIT was in Huh7 cells.(2) MSP showed that the partial methylation of promoter region of FHIT was in HepG2, Hep3B cells.(3) With 5-azadc treated, the expression of FHIT of HepG2, Hep3B was higer after treating with 5-Azadc.(4)In HepG2, the methylation of promoter region of FHIT was highest3. Results of cell proliferation, apoptosis, invasion and cell cycle of human hepatocellular carcinoma cells HepG2, Hep3B and Huh7 with transfection of pLVX-IRES-FHIT slow virus recombinant vector.(1) According DNA sequencing, the pLVX-IRES-FHIT slow virus recombinant vector was constructed successfully by double enzyme digestion with T-FHIT vector and pLVX-IRES-mCherry virus vector.(2)In vitro research to study cell proliferation and apotosis of human hepatocellular carcinoma cells with pLVX-IRES-FHIT slow virus recombinant vector. MTT tests showed that proliferation activity of HepG2,Hep3B and Huh7 with pLVX-IRES-FHIT transfection were reduced specially after 48 hours, but in 72 hours the inhibition was not obviously while the control group was not significant inhibition (P<0.05). AnnexinV/PI double staining method showed that there wasn’t significant difference after 48h apoptosis level among three hepatoma cells (P>0.05) and the order of inhibitional proliferation activity from high to low is HepG2,Hep3B and Huh7;(3) In vitro research to study cell invasion of human hepatocellular carcinoma cells with pLVX-IRES-FHIT slow virus recombinant vector. Transwell tests showed that invasive activity of HepG2, Hep3B and Huh7 with pLVX-IRES-FHIT transfection were inhibited (P<0.05). The control group was not significant inhibition (P<0.05). All these showed that the order of invasive activity was HepG2>Hep3B>Huh7. This points out that the different expression of FHIT was correlated to degree of cell differentiation;(4) In vitro research to study cell cycle of human hepatocellular carcinoma cells with pLVX-IRES-FHIT slow virus recombinant vector. Cell cycle test showed that there are many cells in G0/G1 stage, little cells in M and S stage. The control group was not significant inhibition (P>0.05) in Hep3B and Huh7 cells.The influence of over expression FHIT to these cells is that cell cycle stop in S stage, and sequence of cell number in G0/G1 is HepG2<Hep3B<Huh7.4. Results of FHIT gene regulate transcriptional activity of cyclinD1 via classic Wnt-β-catenin/TCF-4 pathway.Through using RT-PCR, Westernblot and cyclinDl lucfierase assay, we found that special up-raising FHIT expression significantly inhibited the transcriptional activity of the cyclinD1 promoter and decreased the expression of cyclinD1 in HepG2 cells (P<0.05).[Conclusion]1. FHIT gene is a cancer suppressor gene and differences of FHIT expression are correlated with tissue differentiation of liver cancer and lymph node metastasis. FHIT gene may be involved in tumor invasion and progression as so to correlate with the overall survival of patient with postoperation. Poor expression of FHIT gene on hepatoma cells indicated increased tumor malignancy. To use immunohistochemical method was helpful to assist the early diagnosis and make a reasonable appraisal of their prognosis.2. The methylation level of promoter region of FHIT is observed in HepG2 and Hep3B cells, especially in HepG2. Moreover, the methylation leads to poor expression of FHIT in HepG2 cells and promotes tumor proliferation of HepG2 cells.3. To successfully construct targeting pLVX-IRES-FHIT slow virus carrier recombinant vector in vitro, over expression of FHIT gene effectively inhibited proliferation activity and invasion of HepG2,Hep3B,Huh7 hepatoma cells, In addition, over expression of FHIT gene can stop cell cycle to S stage and increase cells of G0/G1 stage.4. The increased expression of FHIT gene inhibited the growth of HepG2, similiarly inhibited transcripitional activity and expression of cyclinDl gene.5. FHIT gene maybe regulate transcriptional activity of cyclinDl via classic Wnt-β-catenin/TCF-4 pathway.