Study On Expression, Function And Mechanism Of TNKS1Gene In Human Brain Astrocytoma

Part One:Expression and significance of TNKS1in human brain astrocytomaObjective:To investigate the expression of TNKS1and its potential clinical significance in human brain astrocytoma.Methods:Immunohistochemistry SP, RT-PCR, Western blot were used to detect the expression of TNKS1in51cases of fresh human brain astrocytoma and6cases of normal brain tissue. Statistical analysis was performed to investigate the relationship between the mRNA and protein expression levels of TNKS1and the clinicopathologic features.Results:The mRNA and protein expression levels of TNKS1in tumor tissues were significantly higher than those in the normal brain tissues(P<0.01), and the TNKS1mRNA and protein were positively associated in tumor tissues(r=0.958, P<0.01). With the increased grade of astrocytomas, the mRNA and protein expression level of TNSK1was gradually increased, which had a significant difference compared to each grade (P<0.01). But no correlation was found between TNKS1gene expression and patient age or sex (P>0.05)Conclusion:The TNKS1gene was upregulated in both mRNA and protein levels in astrocytomas compared to normal brain tissues. The enhanced expression of the TNKS1gene was clearly correlated with the pathological grades of the tumor, suggestting that TNKS1has an oncogenic role in astrocytoma carcinogenesis and might be associated with tumor progression. Part Two:siRNA targeting TNKS1gene inhibits the expression of TNKS1in U251cellsObjective:To design, synthesize and filter out the effective siRNA sequence silencing human TNKS1gene in U251cells.Methods:The human brain glioblastoma cell line U251was choosed as the research object, RT-PCR was used to detect the expression level of TNKS1in U251cells first. Then three small interfering RNA (siRNA) fragments targeted for TNSK1gene were designed and transfected into U251cells via lipofectamine. And inverted fluorescence microscope was used to observe the effect of transfecion. Real time PCR and Western blot were employed to detect the mRNA and protein expression of TNKS1gene to select the siRNA interference fragment (siRNA-TNKS1) that could most efficiently inhibit the expression of TNKS1gene. Then the protein expression of TNKS1was detected by Western blot after the transfection of the selected siRNA-TNKS1in U251cell.Results:TNKS1mRNA expressed in U251cells. The transfecion efficiency reached (84.53±4.39)%observed by inverted fluorescence microscope. Real time PCR and Western blot which were employed to detect that TNKS1-siRNA-2239sequence had the best silencing effect in inhibiting TNKS1expression in U251cells. The protein expression of TNKS1of siRNA transfection group was0.175±0.020, lower than that of the blank control group (0.667±0.015) and negative control group (0.630±0.024)(P<0.01)Conclusion:The siRNA targeting TNKS1gene can inhibit the expression of TNKS1in U251cells. Part Three:The influence on biological effects of U251cells by the TNKS1gene silencingObjective:This study was carried out to investigate the influence on biological effects of U251cells by silence of TNKS1. Then we could figure out the possible function of TNKS1in the the genesis and progression of astrocytoma.Methods:Designed three different intervention U251cells:blank control group (used the normal U251), negative control group (the inclusion of non-specific siRNA/Lipofectamine complexes in U251cell), siRNA transfection group (TNKS1-siRNA-2239-Liposome-transfected U251cell). U251cell lines were cultured in vitro, then MTT method, Transwell assay, flow cytometer (FCM) analysis and Annexin V/PI method were applied to measure cell growth, cell invasiveness, cell cycle and apoptosis of U251cells respectively.Results:1. Detected at the same time by the MTT, the OD of the siRNA transfection group was lower than that of blank control group and negative control group (P<0.01).2. The result of Transwell assay showed that cell number through the artificial polycarbonate basal membrane of the siRNA transfection group became decreased as compared with the blank control group and negative control group (P<0.01).3. The result of the flow cytometry showed that cells at Anterior G0/G1phases were obviously increased and those at S phase were significantly decreased in the siRNA transfection group as compared with those in the blank control group and negative control group (P<0.05).4. Annexin V/PI method detected that the early-stage apoptosis rate and the late-stage apoptosis rate of the siRNA transfection group were much higher than those of the blank control group and negative control group (P<0.01)Conclusion:The siRNA silencing expression of TNKS1gene can inhibit the proliferation and invasion of U251cells, also it can induce the apoptosis of U251cells. Part Four:The initial study on the mechanism of TNKS1in the human brain astrocytomaObjective:This study was to explore the possible ways how TNKS1involved in the genesis and progression of human astrocytoma.Methods:1. hTERT and TRF1were choosed as the research objects, then the protein expression of hTERT and TRF1was detected by Western blot after the transfection of the selected siRNA-TNKS1in U251cells.2. β-catenin, the core component of Wnt/β-catenin pathway, was choosed as the research object, immunohistochemistry was used to detect the expression of β-catenin in51cases of fresh human brain astrocytoma and6cases of normal brain tissue. Statistical analysis was performed to investigate the relationship between TNKS1and β-catenin.Results:1. The expression of hTERT protein of siRNA-TNKS1transfection group (0.182±0.031) was significantly lower than that of the blank group (0.352±0.026) and negative control group (0.347±0.046)(P<0.01). The expression of TRF1protein of siRNA-TNKS1transfection group(0.271±0.056) was significantly higher than that of the blank group (0.094±0.023) and negative control group (0.133±0.037)(P<0.01).2. Immunostaining analysis showed that β-catenin positive cells were abundant in astrocytomas of various grades. The β-catenin IRS was significantly positively correlated with increasing WHO grades (P<0.01). TNKS1IRS was positively correlated with β-catenin IRS (r=0.848, P<0.01).Conclusion:1. The siRNA silencing expression of TNKS1gene can decrease the expression of hTERT, and increase the expression of TRF1, implies that TNKS1plays an important role in the maintenance of telomere length in U251cells;2. The positive correlation between TNKS1and P-catenin expression implies that the TNKS1might exert its function through the Wnt/β-catenin pathway in astrocytomas.