| Background:As a group of endometrial epithelial malignancies, endometrial carcinoma seriously threatens women’s health, which is noted for the endometrial glands of adenocarcinoma with origins. It is the most common malignancy of the female reproductive system, statistics show that 20% to 30% in the incidence of its annual accounts for the female reproductive system cancer, and nearly 200,000 new cases each year, but second only to the incidence of cervical cancer in our country. In some western countries, there are large sample statistics show that the incidence of endometrial carcinoma is much higher than cervical cancer, it is the first rank in the female reproductive system cancer. Recently, although the specific pathogenesis of EC is still not entirely clear, with the recent significant increase in morbidity and mortality, which has become the focus of basic research and clinical research.Heat-shock response is a defense and stress response,which occurs in the effect of heat stress,and its main features are the changes of gene expression and regulation. HSF1 is the primary regulator of heat shock response. HSF1 can be directly or indirectly mediated in vivo biological activity in many cellular activity factor, and involved in the regulation of various physiological functions of the cells. It makes the body in adapting to the adverse environment as closely as possible to extend lifetime, and involved in malignant transformation of cells at the same time. Thus, it has a certain promoting role in tumorigenesis and development.XAF1 is an important negative regulatory factor which antagonist in XIAP, can be directly combined with the XIAP and quickly blocked the inhibitory effects on apoptosis, So as to reverse the XIAP protective effects on cells, including tumor cells. In addition, in view of many qualities on XAF1 structure and function, it has already become an important breakthroughs in Reducing tumor cell resistance, improving tumor cells to chemotherapeutic drugs, cytokine therapy and radiation therapy sensitivity.The blocking of apoptosis, the occurrence of malignancies and cell resistance has close relations, many chemotherapy drugs clear the tumor cell by inducing apoptosis. So when XAF1 showed high expression, it can lead to a large number of tumor cell apoptosis, and significantly enhance the effect of chemotherapy, and effectively inhibit the transformation and formation of tumor cell in vivo. Then, whether the regulation effect of HSF1 on XAF1 in endometrial carcinoma? How did both of them induce apoptosis of tumor cells changed? What kind of impact would be happen to this whole process if intervention of one of these factors?With these questions, we designed this experiment. In the experiment, we test the expression of HSF1 and XAF1 in endometrial carcinoma tissues and cell lines. We found the mutual relationship between them. We observed the changes of proliferation, cell cycle and apoptosis in the lentivirus infection endometrial carcinoma cells. Examined the role of two factors in the occurrence and development of endometrial carcinoma, with a view to provide a reference in the timely diagnosis, treatment and prognosis in endometrial carcinoma.Aims:1. To explore the expression and the mutual role of HSF1 and XAF1 in endometrial carcinoma tissues and cell lines.2. To explore the impact and the role of HSF1 and XAF1 on proliferation, cell cycle and apoptosis in endometrial carcinoma cell.Methods:Immunohistochemical stain pain(SP method) was applied to detect the expreession of HSF1 and XAF1 in normal endometrial tissue and endometrial carcinoma tissues, and the expreession of HSF1 and XAF1 in different histological grade of endometrial carcinoma. Statistical methods were applied to study the relationship between HSF1, XAF1 expreession and clinicopathological features of endometrial carcinoma, to analyze the correlation between HSF1 and XAF1. Through the endometrial carcinoma tissues and normal endometrial tissue detect the expression of HSF1 and XAF1 gene in protein and m RNA levels by western blot and q PCR experiments.By western blot study on HSF1 and XAF1 gene expression levels of three common of endometrial carcinoma cell lines(Ish-ikawa, HEC-1A, RL95-2). We selected the relative stability of expression of endometrial cancer cell RL95-2 as a lentivirus infection model, and succeeded in building a stable transfection of interference HSF1 cell lines and a stable transfection of overexpression XAF1 cell lines.The experiments of western blot and q PCR were applied to detect the changes of XAF1 expression in HSF1 interference experimental group(Lv-sh-HSF1-RL95-2),while to detect the changes of HSF1 expression in XAF1 overexpression experimental group(Lv-XAF1-RL95-2).Through MTT, FCM and cell apoptosis level detection tested the changes of endometrial cancer cells RL95-2 on proliferation, cell cycle and cell apoptosis, in HSF1 been gene interference and XAF1 gene been overexpressed. Experiment was divided into two parts, the first part of HSF1 gene interference group and the specific grouping was as follows: the experimental group Lv-sh-HSF1-RL95-2, group control Lv-scrambleRL95-2, blank control group control. The second part of XAF1 gene overexpression, the specific grouping was as follows: the experimental group Lv-XAF1-RL95-2, group control Lv-cherry-RL95-2, blank control group control.Results:Immunohistochemical staining showed: compared with the control group(normal endometrial ltissue), HSF1 gene expression levels in cancer tissue were higher than normal endometrial tissue. HSF1 is high expression in endometrial carcinoma tissue, the positive expression rate was associated with histological grade(P<0.001), myometrial invasion and lymph node metastasis(P<0.05), and pathologic stage irrelevant. XAF1 is low expression in endometrial carcinoma, the positive expression rate was associated with histological grade, myometrial invasion, clinical stage and lymph node metastasis(P<0.05). In the preliminary validation of both expression in cancerous and non-cancerous tissue, we have also been related experiments, in varying degrees of differentiation of cancer tissues. Immunohistochemistry staining showed: With lower levels of cancer tissue differentiation, HSF1 gene expression showed a gradual increasing trend, while the expression of XAF1 gene is showing a decreasing trend. This result we assume that they are likely to occur in endometrial carcinoma were involved in the development process and played a certain role. In order to further explored the role of them in the occurrence and development in cancer, through the complete data collection for the relevant statistical analysis, the results showed that negative correlation between the two factors(r=-0.582,P<0.001).In vitro environment, HSF1 and XAF1 expression levels in three kinds of endometrial carcinoma cell lines by using western blot indicated: HSF1 expression in three kinds of endometrial carcinoma cell lines, among which Ish-ikawa was of the highest expression, RL95-2 followed, HEC-1A minimum, while the expression level of the lowest XAF1 in Ish-ikawa, RL95-2 followed, HEC-1A maximum. So we selected RL95-2 cells as a lentivirus infection, in order to ensure the stability and authenticity of the experimental results, get the most accurate results in experiment.The expression of XAF1 was significantly higher than its all of group control in HSF1 interference experimental group(Lv-sh-HSF1-RL95-2).At the same time, the expression of HSF1 was no significant change than its all of group control in XAF1 overexpression experimental group(Lv-XAF1-RL95-2).Preliminary test showed HSF1 and XAF1 were negative correlation, and stable transfection of HSF1 and XAF1 cell lines have been built successfully. Our experimental group drawed the cell growth curve by MTT and found that experimental groups Lv-sh-HSF1-RL95-2 cell proliferation capacity was significantly lower in the group of HSF1 gene interference, but in group Lv-scramble-RL95-2 and group control, cell proliferation capacity were significantly higher than experimental groups(P<0.05). The experimental groups Lv-XAF1-RL95-2 cell proliferation capacity was significantly lower than its all of group control in the group of XAF1 gene overexpression(P<0.05).Cell cycle analysis results showed that: the experimental group as above, in the HSF1 gene interference group, the experimental group and two control groups(Lv-scramble-RL95-2, control) compared, the experimental group(Lv-sh-HSF1-RL95-2) appeared clear G1 phase arrest, reducing the number of cells into S phase, after interference HSF1 gene appeared endometrial carcinoma RL95-2 cell cycle arrest. In the XAF1 gene overexpression group, the XAF1 stable overexpression in Lv-XAF1-RL95-2 cells compared with Lv-cherry-RL95-2 group and control group, the experimental group Lv-XAF1-RL95-2 appeared a clear G1 phase arrest, reducing the number of cells into S phase, XAF1 gene stable overexpression can lead to endometrial carcinoma RL95-2 cell also appear cell cycle arrest.Apoptosis detection experiments showed that: in the XAF1 gene overexpression group, the XAF1 stable overexpression in Lv-XAF1-RL95-2 cells compared with Lv-cherry-RL95-2 group and control group, the experimental group Lv-XAF1-RL95-2 appeared a clear trend of early apoptosis(P<0.001), indicating that the stable overexpression XAF1 genes can lead to endometrial carcinoma RL95-2 appeared in a large number of early apoptotic. in the HSF1 gene interference group, the experimental group and two control groups(Lv-scramble-RL95-2, control) compared, the experimental group(Lv-sh-HSF1-RL95-2) appeared consistent with the results of overexpression group(P<0.001), early apoptotic cells was significantly increased in the experimental group. The experimental description of HSF1 gene after gene interference endometrial carcinoma RL95-2 appeared to increase of the rate of early apoptotic cells.Conclusion:HSF1 and XAF1 genes involved in the progression of a variety of human-ralated diseases, including cancer, and in a variety of tumor cell proliferation, cycle and apoptosis play a role in the research. At present, the correlation of them in endometrial carcinoma and the role of the correlation in the occurrence and development in diseases have not been specifically reported. Our group have analyzed the expression of them and association of the expression between the two, and verified the role and mechanism of two factors in the occurrence and development of endometrial carcinoma in vitro. All results prompted that HSF1 and XAF1 gene in endometria carcinoma-targeted studies are likely to provide a new target for gene therapy for the diseases, as well as to provides new directions so as to break through the bottleneck of current treatment of endometrial cancer. |
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