| Background：Endometrial cancer is a malignant tumor which seriously threatens women’s health.The morbidity and mortality of endometrial cancer has risen in recent years and it hasbecome an important public health problem which drawn extensive attention all over theworld. In America, there are about47130women who was diagnosed with the endometrialcarcinoma and eventually8010patients died from it. China health statistics yearbook of2012shows that from2004to2005, the ministry of health of malignant tumor in thereview, Endometrial cancer mortality ranked10th among the malignant tumor mortality inwomen with about2.71/100000. Although most of the endometrial cancers are early casesand have a good prognosis, the advanced, poorly differentiated ones still have a very badprognosis and unsatisfactory5-year survival rate. So, it is important to give endometrialcancer an early diagnosis and timely treatment.Heat shock factor1(HSF1) is the major transcription factor in regulating theexpression of HSPs. HSF1can change the HSP gene locus quickly and enhance theexpression of them when the heat-shock reaction was activated. HSPs can help refold themisfolding polypeptide and inhabit protein polymerization which has a benefit effect oncells to restrain protein stress toxicity and keep protein balance in vivo.Some studies in recent years have revealed that HSF1plays an important role in thedevelopment of several kinds of carcinomas. As the main transcriptional activators of heatshock reaction, the function of HSF1has been fully described by the cell test, organ modeltest and biological analysis including nematodes and rodents. Although the role of HSF1 in protecting cells and organisms from serious harmful stimulus have been widely studiedand some positive outcomes have been achieved, there are still large amount of questionsto be solved. Whether the regulation effect of HSF1on downstream factors involved in thedevelopment of tumor? What is the role of HSF1in the development of endometrialcancer and some key cell reactions such as oxidative stress or cell cycle control? What isthe effect of these cells to respond to the intervention of HSF1?To test the expression and the role of HSF1in endometrial cancer, differentconcentrations of hydrogen peroxide were given to the endometrial carcinoma cell line.The survival of cells, changes of HSF1expression, injury of cells, function changes ofantioxidant systems and mitochondrial were observed. The mechanism of anti-apoptoticrole of HSF1in the endometrial carcinoma cells was studied. Our study put forward a newtarget for endometrial cancer treatment, which may improve the prognosis of endometrialcancer.Aims：1.To explore the expression and the role of HSF1in endometrial cancer,2.To explore the effect and mechanism of anti-apoptotic role of HSF1on theendometrial carcinoma cell line.Methods：RT-PCR and Western blot were implemented to assess the expression of HSF1inendometrial carcinoma.RT-PCR was performed to evaluate the expression changes of HSF1after stimulatingwith different concentrations of hydrogen peroxide to the endometrial carcinoma.MTT assays were implemented to assess the inhabitation of cells to differentconcentrations of hydrogen peroxide.Using interference RNA to down-regulating the expression of HSF1in theendometrial carcinoma and using RT-PCR and Western blot to test the expression of HSF1at the down-regulating situation.Annexin V-PI double staining was performed to detect cell apoptosis in each group before and after down-regulating of HSF1.The change of cell cycle in each group before and after down-regulating of HSF1wasobserved by FCM.Using ELIASA to detect MDA, GSH concentration, Catalase enzyme activity, thetotal antioxidant ability and the ATP generation of cells in each group.The concentration of ROS in each group before and after down-regulating of HSF1was observed by FCM.Results：1. The expression level of HSF1mRNA was evaluated; it was highest in Ishikawa cell,then in HEC-1-B and lowest in RL95-2.2. The expression level of HSF1was highest in Ishikawa cell, then in HEC-1-B andlowest in RL95-2.3. The expression of HSF1in Ishikawa cell was obviously increased as stimulating with300μmol/L hydrogen peroxide and the expression was continuously increased as theconcentration of hydrogen peroxide achieved500μmol/L. While the expression wasslightly reduced as the concentration700μmol/L compared with500μmol/L, thedifferences among groups have statistically significance. The expression of HSF1inHEC-1B cell was obviously increased as stimulating with300μmol/L hydrogenperoxide, but the expression was decreased as the concentration achieved500μmol/Land the expression was lower than the base level when the concentration achieved700μmol/L, the differences among groups have statistically significance. Theexpression of HSF1in RL95-2cell was obviously increased as stimulating with50μmol/L hydrogen peroxide. As the concentration achieved300μmol/L, theexpression of HSF1was lower than the expression in50μmol/L group and with aconcentration of500μmol/L, the expression of HSF1was below the base level, thedifferences among groups have statistically significance.4. The cell proliferation inhibition rate was obviously increased in Ishikawa cell andHEC-1-B cell at a concentration of700μmol/L and500μmol/L H2O2stimulus respectively，the differences have statistically significance. While the cell proliferationinhibition rate was obviously increased in RL95-2cell at a concentration of just300μmol/L H2O2stimulus.5. After transfer with siRNA to Ishikawa cell, the expression of HSF1mRNA wasreduced and there are significant differences compared with blank group and thenegative control group(P<0.05)，while there are not significant differences betweenblank group and the negative control group(P<0.05).6. After transfer with siRNA to Ishikawa cell, the expression of HSF1was obviouslyreduced and there are significant differences in transfer group compared with blankgroup and the negative control group（P<0.05），while there are not significantdifferences between blank group and the negative control group（P<0.05）.7. The MTT outcome displayed that the cell proliferation inhibition rate was obviouslyincreased in transfer group when Ishikawa cell was treated with H2O2and thedifferences have statistically significance.8. The percentage of viable apoptotic cell (right lower quadrant) increased moreobviously in transfer group than in control group as the Ishikawa cell was treated withH2O2and the differences have statistically significance.9. The proportion of cells in G0/G1phase was increased in transfer group comparedwith control group as the Ishikawa cell was treated with H2O2,while the proportionof cells in G2+S phase was in transfer group compared with control group and thedifferences have statistically significance（P<0.05）.10. The MDA content was increased more evidently in transfer group than in controlgroup as the Ishikawa cell was treated with H2O2and the differences have statisticallysignificance（P<0.05）.11. The activity of H2O2was reduced in normal and stressed Ishikawa cells comparedwith control group and the differences have statistically significance（P<0.05）.12. The GSH content was reduced in Ishikawa cells compared with control group and thedifferences have statistically significance（P<0.05）. 13. The total ability of anti-oxidant was reduced in transfer group compared with controlgroup as the Ishikawa cells was treated with H2O2and the differences havestatistically significance（P<0.05）14. The ATP level was reduced in transfer group compared with control group as theIshikawa cells was treated with H2O2and the differences have statisticallysignificance（P<0.05）.15. The ROS content was increased obviously in transfer group compared with controlgroup as the Ishikawa cells was treated with H2O2and the differences havestatistically significance（P<0.05）Conclusion：1.The MTT outcome was related to the measuring results of the basal level of HSF1in the three cell lines. Ishikawa cells, which have the highest basal level of HSF1, had thehighest stimulating H2O2concentration of700μmol/L when the cell inhibition rate wasover50%.；RL95-2cells, which have the lowest basal level of HSF1, had the loweststimulating H2O2concentration of300μmol/L when the cell inhibition rate was over50%.；While HEC-1-B cells, which have a basal level between Ishikawa and RL95-2, hadan middle stimulating H2O2concentration of500μmol/L when the cell inhibition rate wasover50%. These suggested that HSF1expression may be associated with tumor stressability and eventually promote the occurrence of endometrial cancer.2.A series of tests revealed that once the expression of HSF1was down regulated, itwas associated with the apoptotic resistance of endometrial cancer cells. HSF1may be anendometrial carcinoma promoter and can enhance the resistance of the cancer, thuscontributed to the occurrence and development of endometrial carcinoma. In conclusion,HSF1may be a good candidate marker and a potential target for endometrial carcinomaprogression intervention... |
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