| [Objective]1. To explore the expressions differences of the Bmi-1, WWOX and PUMA gene in gallbladder carcinoma, chronic cholecystitis tissue with mild-moderate atypical hyperplasia and normal gallbladder tissues, and to analyze the relationship between Bmi-1, WWOX and PUMA gene expressions and clinicopathological features of gallbladder cancer, and to reveal the role and Clinical significance of their expression differences in the occurrence and development process of gallbladder cancer, so as to lay the foundation for the follow-up study on the occurrence mechanism of gallbladder cancer.2. To successfully construct targeting shRNA-Bmi-1recombinant vector to inhibit proto-oncogene Bmi-1expression by RNAi interference techniques in vitro experiment; to observe its effects on gallbladder cancer cell proliferation, apoptosis and cell cycle; To explore the molecular mechanisms of Bmi-1regulating gallbladder cancer cell growth through regulating WWOX/P73/PUMA/Bax/Bcl-2/Caspase-3signaling pathways.3. By successfully constructed pcDNA3.0-WWOX eukaryotic expression vector in vitro experiment, To explored the effects of WWOX gene overexpression on gallbladder cell proliferation, apoptosis and cell cycle, and to further study molecular mechanisms that WWOX/P73/PUMA/Bax/Bcl-2pathway regulates the cell growth of gallbladder cancers, and to lay a foundation for studying whether there is a feedback loop between Bmi-1/WWOX pathway.4. To successfully establish subcutaneously transplantation tumor Model of human gallbladder carcinoma in BALB/c Nude Mice. To assess the therapeutic efficacy of targeting inhibition Bmi-1on transplanted tumor through in vivo experiment.to further investigate its impact on gallbladder cancer cell growth and molecular mechanism of regulating Bmi-1/WWOX/P73/PUMA/Bax/Bcl-2pathway, so that results in vitro can be verified.5. Through clinical correlational research, experimental studies in vitro and in vivo on nude mice, to systematically describe effects and molecular mechanisms that Bmi-1regulates cell proliferation, apoptosis and cell cycle of human gallbladder cancer, and its WWOX signaling pathway, which will help screening markers of gallbladder cancer, assessment of prognosis, provide a theoretical basis and experimental data for the early diagnosis of gallbladder cancer and its molecule targeted therapy.[Methods]1. Clinical correlative research55cases of patients with primary gallbladder adenocarcinoma of complete the clinicopathological data and its surgical specimens to keep in the archives with paraffin blocks were collected.30cases with chronic cholecystitis tissue with mild-moderate atypical hyperplasia from the same time period and22cases with normal gallbladder tissues from patients of the right hemihepatectomy with the calculus of intrahepatic duct or with liver tumor, which without stones and tumors invasion in the gallbladder were collected as controls.Five cases with fresh tissues were included In each group. Bmi-1/WWOX/PUMA mRNA and protein expression difference in the above specimens were determined by IHC-MaxVision, RT-PCR and Real-time PCR. The relationship between the protein and mRNA expression differences in three groups and the clinicopathological factors was analyzed.2. In vitro studies, with proto-oncogene Bmi-1mRNA as a targetTo successfully construct shRNA-Bmi-1recombinant vector and transfect it into gallbladder cancer GBC-SD cells by RNAi interference technology in vitro experiment. The transfection efficiencies were evaluated using an inverted fluorescence microscope and flow cytometry. The change levels of transcription and translation of gallbladder cancer cells after transfection were determined by RT-PCR and Westernblot assay. The best recombinant plasmid interfering Bmi-1target sequence was selected for subsequent experimental groups. Using an inverted microscope, Brdu essay, AnnexinV/7-AAD single and double-stained essay, transmission electron microscope and cell cycle analysis techniques, the influencing mechanisms of targeting shRNA inhibition of Bmi-1on gallbladder cancer cell proliferative activity, apoptosis progress and cell cycle and their relationship of biological effects and time gradient were investigated, to further explored the molecular mechanisms of regulation influence of targeting shRNA interfering with Bmi-1expression on WWOX/P73/PUMA/Bax/Bcl-2/Caspase-3pathways through Using Real-time PCR, Western Blot, immunofluorescence stain and flow cytometry techniques.3. In vitro studies, with anti-oncogene WWOXmRNA as a targetThe total RNA of GBC-SD cells was extracted. The complete WWOX sequence was amplified by RT-PCR and cloned into the eukaryotic expression vector pcDNA3.0. Through using restriction enzyme digesting and DNA sequencing to identify, the recombinant pcDNA3.0-WWOX eukaryotic expression vector was successfully constructed. GBC-SD cells were transfected by liposome-mediation, at same time, the empty vector and Liposomes was respectively transfected as a negative control and GBC-SD cells as a blank. The changes of transcription and translation levels of gallbladder cancer cells after transfection was determined by RT-PCR, Western blot and immunofluorescence techniques. Using MTT assay, Annexin V/7-AAD double staining, TUNNEL assay, cell cycle detection technology, transmission electron microscopy, mitochondrial transmembrane potential detection technique, the influencing mechanisms of over-expressed WWOX on proliferative activity, apoptosis and cell cycle of gallbladder cancer cell and their relationship with biological effects and time gradient were investigated. To further explored the molecular mechanisms of eukaryotic expression vector pCDNA3.0on regulating WWOX/P73/PUMA/Bax/Bcl-2pathways through Using Real-time PCR,Western Blot, immunofluorescence, and flow cytometry techniques. 4. In vivo studiesBased on results from in vitro studies, establishing subcutaneously transplantation tumor Model of human gallbladder carcinoma in BALB/c nude mice.By using shRNA-Bmi-1recombinant plasmid for multi-point injection within and around the transplantation tumor, the growth curve of the transplantation tumor was drawed, and the inhibition rate of the transplantation tumor volume was calculated at the end of the experiment.Through specimens anatomy and histopathology detection measure, anti-tumor effect of targeting shRNA interfering with Bmi-1expression on subcutaneously transplantation tumor in nude mice was explored.Through using Max Vision, TUNEL essay, transmission electron microscopy, RT-PCR and Western blot techniques, the molecular mechanisms were further investigated on the effect that Bmi-1regulated the gallbladder cancer cell growth and key gene expression of WWOX pathway.[Results]1. Results of clinical correlative studies(1) IHC-MaxVision test results showed that the positive expressions of Bmi-1, WWOX and PUMA protein in gallbladder carcinoma tissue were respectively83.6%(46/55),38.2%(21/55) and34.5%(19/55). The positive expression of Bmi-1protein in gallbladder carcinoma tissue was significantly higher than chronic cholecystitis with atypical hyperplasia and normal gallbladder tissue, while the positive expression of WWOX and PUMA protein in gallbladder carcinoma tissue was significantly lower than chronic cholecystitis with atypical hyperplasia and normal gallbladder tissue (P <0.05).(2) Relationship between differential expression of Bmi-1, WWOX and PUMA and clinicopathological factors:Expression levels of Bmi-1and WWOX protein were correlated with the clinicopathological stage, tissue differentiation and lymph node metastasis of gallbladder cancer(P<0.05), but uncorrelated with gender, age, tumor size, tumor location, and existence of cirrhosis and gallstones(p>0.05). The expression level of PUMA protein was correlated with tissue differentiation and lymph node metastasis of gallbladder cancer (P<0.05), but uncorrelated with gender, age, tumor size, the clinicopathological stage, tumor location, and existence of cirrhosis and gallstones (p>0.05).(3) Real-time PCR and RT-PCR tests showed Bmi-lmRNA expression level is that gallbladder carcinoma>chronic cholecystitis with atypical hyperplasia> normal gallbladder tissue;while WWOX and PUMAmRNA expression levels are that gallbladder carcinoma<chronic cholecystitis with atypical hyperplasia<normal gallbladder tissue (P<0.05). Experiments show that Bmi-1, WWOX and PUMA genes might be involved in the occurrence and development of gallbladder cancer. Gallbladder carcinoma may occur if the persistent high expression of Bmi-1or the persistent lower expression of WWOX or PUMA in chronic cholecystitis with atypical hyperplasia tissue.2. Results of in vitro study on the proto-oncogene Bmi-1mRNA as a target(1) Constructing and screening results of shRNA-Bmi-lrecombinant vector:the amplified sequence of four groups of Bmi-1genes was proved to be correct by gene sequencing after recombinant vector shRNA-Bmi-1was constructed.The cell transfection efficiency was about70%using inverted fluorescence microscope and flow cytometry; After four groups plasmids were transfected into gallbladder GBC-SD cells for48h, the fourth group, in which the sequence had significant inhibition with the transcription and translation levels by RT-PCR and Westernblot essay, was selected as the subsequent experimental group (i.e. shRNA-Bmi-1group).(2) The effect of targeting shRNA to interfere Bmi-1expression on gallbladder cancer cell proliferation, apoptosis and cell cycle:By Targeted inhibition of Bmi-1, the levels of Bmi-1mRNA and protein expression were significantly lower than the control groups determined by RT-PCR and Western blot (p<0.05). And there was no significant difference in the expression quantity between the control groups (p>0.05).The cell proliferation inhibition rate of shRNA-Bmi-1was very obvious over time than the control groups. And the strongest inhibitory effect and most death of cells were observed72h after transfection using inverted fluorescence microscope. Brdu tests show proliferation activity of gallbladder cancer cells in shRNA-Bmi-1group was lower than the control groups, and cell proliferation was time-dependent. The proliferation activity was significantly decreased with over time. The strongest inhibition of cell proliferation was observed72h after transfection (p<0.05). Annexin V/7-AAD single and double-stained show that cell apoptosis rate was significantly increased over time (24h<48h<72h) after transfected with shRNA-Bmi-1recombinant plasmid for24h,48h and72h. And late apoptotic rate increased mainly(p<0.05).The transmission electron microscope detections show concentration, pycnosis and became apoptotic body of gallbladder cells in shRNA-Bmi-1transfected group. Cell cycle detection show G0/G1phase of the cells were increased, while G2/M and S phase cells were decreased. The cells were blocked at the G0/G1phase and cell apoptosis rates were significantly increased and proliferation index were decreased(p<0.05).(3)Molecular mechanisms on targeting shRNA to interfere the expression of Bmi-1to regulate the WWOX pathway:Bmi-1mRNA and protein expression quantity and protein fluorescence intensity were decreased compared with the control groups, while expression quantities of WWOX, P73, PUMAmRNA and protein were significantly increased compared with the control groups and the expression intensity of the protein fluorescence was significantly enhanced in shRNA-Bmi-1transfection group determined by Real-time PCR, Western Blot essay, and immunofluorescence staining techniques (p<0.05); Flow cytometry technique analysis showed that protein expressions of Bax and Caspase-3of gallbladder cancer cells were increased compared with the control groups, while the protein expression of Bcl-2was decrease compared with the control groups (p<0.05).3. The results of in vitro experiments with anti-oncogene WWOX mRNA as a target(1) The construction of the WWOX gene eukaryotic expression vector:the amplified WWOX gene sequence was proved to be correct by double restriction enzyme digestion and gene sequencing of pcDNA3.0-WWOX recombinant plasmid.(2) The effect of pcDNA3.0-WWOX recombinant plasmid on gallbladder cancer cell proliferation, apoptosis and cell cycle:RT-PCR and Westernblot analysis showed WWOXmRNA and protein expression were significantly higher than the control groups48hours after transfection with pcDNA3.0-WWOX (p<0.05); There was no significant difference in WWOXmRNA and protein expression levels between the control groups (p>0.05). Immunofluorescence detection showed WWOX protein fluorescence intensity around the nucleus of gallbladder carcinoma cell in pcDNA3.0-WWOX group was stronger than the control groups, while the change in WWOX protein fluorescence intensity between the control groups was not significant. Inverted microscope observation found that the number of adherent GBC-SD cells in pcDNA3.0-WWOX group significantly reduced and suspended cells and cell debris increased, while it showed normal cell proliferation and good adherent growth in the control groups. MTT assay showed that cell proliferation of pcDNA3.0-WWOX group significantly decreased and the cell proliferation inhibition effect was more significant over time, while there was no significant inhibition of cell proliferation in the control groups (P<0.05). BrdU essay showed that the cell proliferation rate of pcDNA3.0-WWOX group was lower than the control groups (P<0.05). Annexin V/7-AAD double staining showed that the early and late apoptotic rates of gallbladder cancer cells in pcDNA3.0-WWOX group are higher than the control groups (p<0.05); while there was no significant difference in early and late apoptotic rates between the control groups (P>0.05). TUNNEL essay showed apoptosis ratio of gallbladder cancer cell increased compared with the control groups24h/48h/72h after transfection with the recombinant plasmid pcDNA3.0-WWOX, with more pronounced48h after transfection (p<0.05). Cell cycle analysis found that the G0/G1phase of gallbladder cancer cells in pcDNA3.0-WWOX group increased, the G2/M and S phase cells decreased, the apoptosis rate increased and proliferation index decreased (p<0.05); while there was no significant difference between the control groups (p>0.05). The transmembrane potential of chondriosome (ΔψFm) determined by JC-1staining showed the green fluorescence signal was significantly higher than the empty vector, liposomes and blank control group by overexpressed WWOX genes in the chondriosome of gallbladder cancer cells, suggesting that WWOX mainly induced early apoptosis of gallbladder cancer cells(p<0.05). The transmission electron microscope observed that typical apoptotic morphology of gallbladder cancer cells changed with pycnosis, nuclear fragmentation, formation of apoptotic bodies in the pcDNA3.0-WWOX group.(3) The effect of pcDNA-WWOX recombinant plasmid on the key gene of WWOX pathway:Real-time PCR, Western Blot and immunofluorescence assay showed that the expression levels of the transcription and translation of P73and PUMA in gallbladder cancer cells were higher than the control groups by WWOX expression upregulated pecifically (p<0.05); Flow cytometry essay showed that expression levels of Bax protein in gallbladder cancer cells were significantly higher than the control groups, while the expression levels of Bcl-2protein decreased compared with the control group by WWOX expression upregulated pecifically(p<0.05).4. Results of in vivo experiments of transplantation tumorsThe formation rate of the transplantation tumor was100%5-7days after nude mice inoculated subcutaneously with GBC-SD. The tumor growth in the shRNA-Bmi-1group was slower than the control groups. The tumor volume increased in each group after treatment for6weeks when the experiments were terminated, but the transplantation tumor volume in the shRNA-Bmi-1group was significantly smaller than the control groups (shRNA-Scramble group, Lipofectamine group, GBC-SD group). And its tumor inhibition rate was60.6%(p<0.05). There was no significant difference in the tumor volume between the control groups (P>0.05). HE staining essay showed obvious cell necrosis, fuzzy morphology, inflammatory cell infiltration, and unclear histological structure in the shRNA-Bmi-1group;while it showed obvious the cell heteromorphism, fewer inflammatory cells, a clear histological structure in the control groups. MaxVision stainings found that Ki67and VEGF protein expression of the transplantation tumor in the shRNA-Bmi-1group decreased compared with the control groups, suggesting reduced tumor angiogenesis and significantly inhibited cell proliferation in the shRNA-Bmi-1group. TUNEL and transmission electron microscope analysis showed that targeting shRNA to inhibit Bmi-1in vivo may induce gallbladder cancer cell apoptosis, which is consistent with the in vitro experiments. Real-time PCR, Western Blot and immunohistochemistry detection showed targeting shRNA interfering the Bmi-1expression can specifically promote Bmi-1mRNA degradation and inhibit the protein expression, and at the same time promote the expression of critical WWOX/P73/PUMA/Bax gene and inhibit the expression of Bcl-2gene in the downstream pathway on Bmi-1.[Conclusions]1. Bmi-1,WWOX and PUMA expression were involved in the occurrence and development process of gallbladder cancer. The increased positive expression rate of Bmi-1or decreased WWOX and PUMA positive expression rate or loss of expression indicate increased tumor malignancy and metastasis. The expression difference of Bmi-1,WWOX and PUMA may be involved in tumor invasion and progression.To combine detecting the expression of Bmi-1,WWOX and PUMA in gallbladder carcinoma tissues using immunohistochemical method was helpful to assist the early diagnosis and to help make a reasonable appraisal of their prognosis.2. The shRNA-Bmi-1recombinant vector was successfully constructed in vitro, targeted silencing Bmi-1expression can effectively promote the degradation of Bmi-1mRNA and inhibit its protein expression in gallbladder cancer cells, specifically inhibit cell proliferation, induce apoptosis of gallbladder cancer cells, and arrest at G0/G1phase of cell cycle, resulting in DNA synthesis blocked in gallbladder cancer cells. Studies suggest that Bmi-1is involved in the regulation of proliferation and apoptosis of gallbladder cancer cells, and maintaining cell cycle operation.Bmi-1is an important factor inhibiting apoptosis and promoting abnormality cell proliferation. Meanwhile, targeting to silent the Bmi-1expression can effectively promote the expression of critical WWOX, P73, PUMA, Bax and Caspase-3gene and inhibit the expression of Bcl-2protein in the WWOX pathway, which may be related to the regulation mechanism of gallbladder cancer cell growth.Bmi-1and its Bmi-1/WWOX pathway was involved in the regulation of gallbladder cancer cell growth through the mitochondrial dependent apoptosis pathway.3. The pcDNA3.0-WWOX eukaryotic expression vector was successfully constructed in vitro.The overexpression of WWOX gene can effectively inhibit degradation of WWOXmRNA, promote its protein expression in gallbladder cancer cells. It can effectively inhibit proliferation activity and induce the gallbladder cancer cell apoptosis. It can also block the cell cycle at the G0/G1phase. At the same time, it can up-regulate and promote the chelating accumulation of73protein in the cytoplasm of gallbladder cancer cells, promote the transcription and translation of PUMA and Bax, and inhibit the transcription and translation of Bcl-2, which may be one of important mechanisms regulating gallbladder cancer cell growth. WWOX/P73/PUMA pathway was involved in the regulation of gallbladder cancer cell proliferation, apoptosis and cell cycle changes through the mitochondrial dependent apoptosis pathway, which further verified the molecular mechanisms of WWOX pathway regulating gallbladder cancer cells.4. The animal model of subcutaneous transplanted tumor of gallbladder cancer in nude mice was successfully constructed. Targeting to silent Bmi-1expression can induce the cell apoptosis of transplanted tumors of gallbladder cancers, significantly inhibit tumor growth and the formation of new vessels, and regulate the expression of key genes in Bmi-1/WWOX/P73/PUMA/Bax/Bcl-2pathway of subcutaneous transplanted tumor in nude mice. The findings are consistent with those by the in vitro experiments. It was further confirmed by in vivo experiments that Bmi-1/WWOX pathway is involved in the cell growth of gallbladder cancer through the mitochondrial dependent apoptosis pathway.5. Bmi-1and WWOX may provide a potential molecular target for the gene targeted therapy of gallbladder cancer. It can be one of the potential therapeutic tools for gallbladder carcinoma combined with gene therapy through targeting to silent Bmi-1expression and specifically up-regulating the WWOX expression. Blocking the Bmi-1/WWOX pathway plays an important role in target therapeutic strategies for gallbladder cancer and also provides a new experimental basis for the diagnosis of early gallbladder cancer and multiple targeted molecular therapies for gallbladder cancer. |
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