Preliminary Study On The Expression And Effect And Regulation Mechanism Of Programmed Cell Death 4 In Cervical Cancer

BackgroundCervical cancer is the second most common malignancy in women worldwide,it is the leading cause of cancer deaths of women.As a result of early cervical cancer patients are lack of early specific clinical symptoms and effective screening,the majority found already belongs to the late cancer.The cure rate of carcinoma in situ is nearly 100%,while that in stage Ⅳ is only about 10%.Indicating prognosis depends on informations of carcinoma obtained from surgery.And there are no valuable molecular markers.According to Shibahara’s study,programmed cell death 4(PDCD4),was frst discovered as a gene associated with apoptosis.Further work showed that the expression of PDCD4 is reduced in in a variety of tumors and the loss of PDCD4 expression is associated with tumor progression and poor prognosis.As a suppressor of gene transcription and protein translation,PDCD4 inhibits the malignant phenotype of cancer cells.Together,these observations have suggested that Pdcd4 is a tumor suppressor gene.However,the expression and function of PDCD4 in cervical cancer has not been reported.The objective of the current study was to provide theoretical basis and experimental basis for the clinical application of PDCD4 according to investigation of the expression level and function and modulation of PDCD4 in cervical cancer cells.Chapter Ⅰ The Expression of PDCD4 and its clinical significance in human cervical cancer tissuesObjectiveClarify the expression and clinical significance of PDCD4 in human cervical cancer.Materials and methods1.We collected tissue specimens from patients who underwent cervical conization,including normal,cervical intraepithelial neoplasia(CIN),carcinoma in situ(CIS)and invasive cervical cancer(ICC).We also obtained fresh cervical cancer tissues and the corresponding adjacent tissues.2.We detected PDCD4 protein expression in cervical tissues by western blot and immunohistochemistry.Results1.According to the immunohistochemical stainings,most of the PDCD4 protein was located in the nucleus of normal cervical epithelial basal and stratum spinosum cells.PDCD4 expressions in both CIN/CIS tissues and invasive cervical cancer were signifcantly lower than that in normal tissues(P=0.009 and P<0.001,respectively).And PDCD4 level of invasive cervical cancer was signifcantly lower than that of CIN/CIS(P=0.003).Western blot showed that PDCD4 was lowly expressed in cervical cancer tissues compared to adjacent tissues.2.The loss of PDCD4 in cervical cancer tissues was found to be signifcantly associated with lymph node metastasis(P=0.046),histological type(P=0.003)and p53(P=0.001).Chapter Ⅱ The effect of PDCD4 in cervical cancer cells ObjectiveObserve the effect of PDCD4 in cervical cancer cells.Materials and methods1.We detected PDCD4 mRNA and protein expression of Hela and Caski and Siha by RT-PCR and western blot.We selected a PDCD4-less-expressing cervical cancer cell line Siha and introduced PDCD4 into these cells using lentivirus containing PDCD4 gene.2.CCK8 assay,plate clone formation assay,cell cycle analysis,cell apoptosis analysis,cell migration and invasion assay were performed to observe the effect of PDCD4 on Siha cells.Results1.The expression level of PDCD4 in cervical cancer cell lines Compared to Hela and Caski,the expression level of PDCD4 in Siha cells was the lowest,and the difference was statistically significant2.PDCD4 induced cell apoptosis,inhibited cell migration and invasion(1)Lv-NC and Lv-PDCD4 were transfected to Siha cells respectively,and we observed cells under the fluorescence microscope.The infection efficiency was above 90%.RT-PCR and Western blot showed that after transfected Lv-PDCD4 the expression level of PDCD4 was significantly increased(2)Overexpression of PDCD4 in Siha cells induced cell apoptosis,inhibited cell viability and cell migration and cell invasion.Overexpression of PDCD4 had no significant effect on plate clone formation capacity and cell cycle transition.Chapter Ⅲ The regulation mechanism of PDCD4 in cervical cancer cellsObjectiveElucidate the regulation mechanism of PDCD4 in cervical cancer cells.Materials and methods1.siRNA-198 was transfected to Siha cells to silence HPV 16 e6/e7,detected the effect of e6/e7 interference on the expression level of PDCD4.2.Lv-NC and Lv-PDCD4 were transfected to Siha cells respectively,and PathScan intracellular signaling array kit was used to detect changes in phosphorylation level of important and well-characterized intracellular signaling molecules.Results1.Knockdown of e6/e7 expression had no significant effect on the expression level of PDCD4.2.PathScan intracellular signaling array kit was used to detect PDCD4-triggered signal transduction.Overexpression of PDCD4 obviously induced the activation of STAT1 and p53.Conclusions1.PDCD4 expression was down-regulated in cervical cancer tissues,and the depression of PDCD4 was signifcantly associated with lymph node metastasis,histological type and p53 status.2.Overexpression of PDCD4 in Siha cells induced cell apoptosis,inhibited cell migration and invasion.3.HPV 16 e6/e7 didn’t regulate PDCD4 expression.PDCD4 affected the biological functions of Siha cell probably via activing p53 and STAT1.