The Study On Relaitionship Between TIZ Gene And Ovarian Cancer

Ovarian cancer is a serious threat to women's lives and health,its Mortality in the female malignancy tumor is in the top,and there is an upward trend.Due to ovarian physiology locate in the deep of pelvic,it is lack of typical clinical symptoms with early ovarian cancer in patients.The majority of patients with ovarian cancer have been found disease is advanced. Therefore it is important to study the level of molecular biology and malignant biological behavior of the mechanism,In order to find methods for early diagnosis and to explore means of effective treatment for improving the prognosis and improve the survival rate,we should try our best to find typical Serum markers.Our laboratory use SEREX technology screening ovarian cancer-associated antigen TIZ.TIZ protein express higher in serum of patients with malignant ovarian tumors,and combined with other serum markers help to improve the sensitivity and specificity of diagnosis.To learn more about the relationship of TIZ gene with ovarian cancer,the experiment detect ovarian tissue in a different expression of TIZ gene through RT-PCR,and we construct eukaryotic expression vector and RNA interference experiment.Our aim is to know the relationship between TIZ gene and ovarian cancer,and to know the impact of TIZ gene to ovarian cell line.Malignant ovarian tissues of TIZ mRNA Expression and clinical valueObjective:To investigate the TIZ gene in malignant ovarian tissues and its clinical significance. Methods:We use RT-PCR to detected 48 cases of ovarian cancer,22 cases of ovarian benign tumor and 24 cases of normal ovarian tissue of TIZ mRNA expression,and analysis relationship between TIZ gene and ovarian cancer.Results:TIZ mRNA express in normal ovarian tissue,ovarian benign and malignant ovarian tissue.TIZ mRNA express in ovarian malignant tissues lower than normal ovarian tissue (p＜0.05),TIZ mRNA express in ovarian malignant tissues lower than benign ovarian tissue (p<0.05);On the other hand,no significant associations were found between TIZ expression and histological typing,stages,prognosis of ovarian malignant.(p＜0.05).Conclusion:TIZ mRNA in ovarian cancer express lower than other tissue.TIZ gene can inhibit the occurrence of ovarian cancer.Amplification and eukaryotic vector construction of TIZ gene from human ovarian cancer tissueObjective:Through genetic engineering technology,TIZ cDNA was amplified to construct its eukaryotic expression vector.It aims to study the relationship between ovarian cancer and TIZ gene.Methods:We choose a piece of serous ovarian cancer tissue as a template,we extracted total RNA from mucus ovarian cancer tissue and then performed reverse transcription polymerase chain reaction to amplify the full length of heparanase gene.The amplicon was cloned into the vector of pcDNA3.1.The result was confirmed by enzyme digestion and sequencing.Results:We successfully amplified the full length of human TIZ gene and cloned it into the vector of pcDNA3.1.The homology was 99.9%confirmed by sequencing.Sequencing results showed that the T mutation in 1551 as C,but it does not alter the amino acid and it is a meaningless change,can be used for experimental study of the follow-up.Conclusion We successfully amplified the full length of TIZ and cloned it into eukaryotic vector.The study of transfection with eukaryotic by TIZ gene affect the biological behavior of ovarian cancer cell linesObjective:To study TIZ on ovarian function in cancer cells,and analysis its affection on ovarian cancer invasive and metastasis,and pilot its role in early diagnosis,treatment targeting treatment.Methods:TIZ gene which successfully constructed the eukaryotic expression vector will be transfected into the non-TIZ gene expressed ovarian cancer cell line HO8910,G418 selection identified by RT-PCR to confirm the stable transfection HO8910/pcDNA3.1-TIZ cell line. And then proceed to cell growth characteristics,cell cycle,clone formation,cell migration, adhesion,cell invasion experiments.Results:We got stable expression of TIZ protein ovarian cancer cell line after transfection screening.Growth curves showed that the transfection of TIZ gene ovarian cancer cell line growth slowed.Flow cytometry showed that cells in the in HO8910/pcDNA3.1-TIZ group which is in proliferated state of(S+G2+M) is 32.6%,the G1 period is 67.4%,while the corresponding control group was 60.7%and 39.3%,it is in reducing the proliferation cycle of cells.The colony formation rate of the two groups, migration,adhesion ability of invasion was no significant difference(p＜0.05).Conclusion:The results show that the cell functions,TIZ can enhance ovarian cancer HO8910 cell growth capacity,their migration,invasion,adhesion had no different. The study of RNA interference TIZ gene affect the biological behavior of ovarian cancer cell linesObjective:To construct the shRNA gene TIZ expression vector and transfected into ovarian cancer,then detected interference of the efficiency of gene TIZ.Methods:We design three different siRNA fragments and lipofect them into SKOV3,in which TIZ is highly expression,we use real-time PCR to test the interfere rate and choose the best inference siRNA fragrment.Then we construct fragment interference vector and Lipofect into ovarian cancer cell line SKOV3 to obtain stable cell lines.RT-PCR test TIZ gene inhibited situation,and then we carry the experiment of cell function.Results:Real-time PCR showed that the copy of TIZ-573 group is 0.844±0.756,compared with the other two groups were statistically significant differences(p＜0.05).TIZ-573 group has the highest rate of inhibition,the inhibitory rate is 61%.We construct siRNA interference vector pGPU6/GFP/Neo-TIZ-573,and transfected ovarian cancer cell line SKOV3,then obtained the stable interfered cell lines Skov3/pGPU6/GFP/Neo-TIZ-573.And we use RT-PCR to test TIZ mRNA expression.We found that TIZ mRNA expression decreased significantly.Growth curve shows that pGPU6/GFP/Neo-TIZ-573 group of cells grow faster than other two.Flow cytometry showed that cells in the Skov3/pGPU6/GFP/Neo-TIZ-573 group which was in proliferated state of(S+G2+M) was 65.1%,G1 percent was 34.9%, corresponded to the control group were 36.6%and 63.4%,the cells in the cell cycle increased. The colony formation rate of the two groups,migration,adhesion ability of invasion had no significant difference(p＜0.05).Conclusion:The results show that interference with the expression of TIZ can enhance the growth of Skov3 ovarian cancer cells.the ability of their migration,invasion,and adhesion had no effect.