Research On The Expression Of New Gene F10 In The Tissue Of Gynecological Malignant Tumors And The Role In Regulation Of Proliferation And Cell Cycle Of Endometrial Cancer Cell

Gynecological malignant tumors are serious threat to women's physical health.Cervical cancer is the most common one,accounting for more than half of the total,followed by ovarian cancer,endometrial cancer.The three kinds of female genital malignant tumor is known as "the three gynecological cancer".The research team found a novel gene F10(GenBank:AB196290)through a series of related previous studies.F10 gene was selected from differential cDNA Library of hydatidiform mole and normal villi and is a new gene associated with invasive behavior of choriocarcinoma.We found that the expression of F10 gene in the hydatidiform mole and choriocarcinoma are expressed and enhanced.F10 gene may be related to malignancy associated changes of hydatidiform mole and invasion of tumor.At the same time,the experimental data show that F10 gene mRNA are positive expression in ovarian cancer,endometrial cancer tissues.This finding suggests that F10 gene is not only closely related to the occurrence and development of trophoblastic tumor,and may regulate the cell cycle,cell proliferation of a variety of tumor cells and plays an important role of tumor development.However,the mode and method of the study need to be further studied.PART ONE EXPRESSION OF F10 GENE IN CERVICAL CANCER CANCER,ENDOMETRIAL CANCER AND OVARIAN CANCEROBJECTIVEIn order to further explore the F10 gene's distribution in different gynecological tumor and compare the expression of F10 between tumor tissues and normal tissues.METHODUsing immunohistochemical method and reverse transcription polymerase chain reaction(RT-PCR)to detect F10 gene expression in cervical cancer,endometrial cancer,ovarian cancer and corresponding normal tissues.RESULTS1.Reverse transcription polymerase chain reaction(RT-PCR)is used to detect the expression of F10 gene mRNA in cervical cancer,endometrial cancer and ovarian cancer.1.1 Through the analysis of paired sample t test,the average expression of FO gene showed in 30 paired cervical cancer is 2.26±0.52,the average expression level is significantly higher than that of adjacent tissues of cervical carcinoma tissues and normal tissues which is 0.81 ± 0.15,with statistical significance(p<0.05).The average expression results show that in 30 paired endometrial carcinoma F10's expression is 8.88±1.60,the average expression level was significantly higher than that of adjacent tissues and normal tissues of F10 4.96±0.68,(p<0.05).The expression of average of F10 gene showed in 20 paired ovarian carcinoma is 1.59±0.26,the average expression level is significantly higher than that of adjacent tissues and normal tissues in F10 0.65±0.07,(p<0.05).1.2 Using two sample t test to compare the RT-PCR results of 10 cervical cancer patients without the preoperative use of chemotherapy in the treatment and 20 cervical cancer patients with preoperative neoadjuvant chemotherapy,the results show that there were statistically significant differences between the two groups of data(p=0.04).Immunohistochemistry showed no statistically significant difference between the two groups(p=0.99).2.Immunohistochemistry is used to detect the expression of F10 gene encoding protein in cervical cancer,endometrial carcinoma and ovarian cancer.Immunohistochemical results show that the average expression of F10 encoding protein in cervical cancer tissues is 0.13±0.01,significantly higher than that 0.04±0.01 in normal tissues and adjacent tissues.The average expression of F10 in endometrial carcinoma tissue is 0.06±0.01,which was significantly higher than that 0.02±0.03 in normal tissues and adjacent tissues.The average expression of F10 in ovarian cancer tissues was 0.04±0.00,which was significantly higher than that 0.02±0.00 in normal tissues and paracancerous tissues.PART TWO THE RULE OF F10 IN REGULATION OF PROLIFERATION AND CELL CYCLE OF ENDOMETRIAL CANCER CELLSOBJECTIVEConstruction of F10 gene over-expression and silencing cells of Ishikawa and HEC-1A endometrial carcinoma cell lines,contrastive study on effect of F10 gene of the proliferation and some known regulator proteins of the cell cycle on the of human endometrial carcinoma cell lines.METHODRespectively to construct the eukaryotic plasmid expression vector,lentiviral vector RNAi,recombinant F10 gene and the empty vector.Through screening and packaging,the recombinant plasmid and empty vector are transfected into Ishikawa and HEC-1A cell lines,to obtain stable F10 gene over-expression and silencing cells and the corresponding empty vector transfected cell lines.RT-PCR method is used to detect the expression of F10 gene in Ishikawa cell lines,HEC-1A cell lines,the untreated groups and the blank vector groups.CCK8 method is used to draw the cell growth curve observed in the F10 silencing and over-expression endometrial cancer cells.Cell immunofluorescence is applied to quantitative analysis of endometrial carcinoma associated cycle regulatory factors:cyclinD1,cyclinE,CDK2,CDK4,CDK6,P16,P21 and related cell cycle regulatory factor:serine/threonine protein kinase 4(PAK4)and proliferating cell nuclear antigen(PCNA)and focal adhesion kinase(FAK)expression.RESULTS(1)Among Ishikawa and HEC-1A cell lines,the expression of F10 gene in the F10 over-expression group is much higher than that in the control group.The expression of F10 gene in F10 gene silencing group is significantly lower than that in control group.There is no significant difference in the expression of F10 gene between the control group and the over-expression and silencing F10 gene group.(2)The results of CCK-8 method shows that Ishikawa cell line and HEC-1 A cell line in the F10 silence group are slower than the untreated group,and the growth rate of F10 over expression group is faster than that of the untreated group.There is no significant difference between F10 gene over-expression empty vector group and F10 gene silencing empty vector group.(2)CCK-8 method shows that the growth rate of F10 silencing group of Ishikawa cell line and HEC-1 A cell line is slower than that of control group,and the growth rate of F10 over-expression group is faster than that of control group.There is no significant difference between F10 gene over-expression group empty vecctor,F10 gene silencing group empty group and control group.(3)Cell immunofluorescence results show:1)Positive cell cycle regulatory factors:compared with control group,Ishikawa and HEC-1 A cell lines of F10 gene silencing group CyclinDl,CyclinE,CDK2,CDK4,CDK6 protein expression levels are significantly lower than the control group(P<0.05),in F10 over-expression group,the expression level of CyclinD1,CyclinE,CDK2,CDK4,CDK6 is significantly higher than that in control group(P<0.05).2)Negative cell cycle regulator:Ishikawa and HEC-1A cell lines of F10 gene silencing group,control group and over-expression group express P16 protein in a rising trend,compared with control group and over-expression group,the F10 expression in silencing group is statistically significant low(P<0.05).Ishikawa cell line of F10 gene silencing group and over-expression group express higher P21 protein level compared with control group.The F10 expression in silencing group is statistically significant higher than in over-expression group(P<0.05).HEC-1A cell line of F10 gene silencing group,control group and over-expression group express P21 protein in a declining trend compared with control group.And the F10 expression in over-expression group is statistically significant low(P<0.05)3)Serine/threonine protein kinase 4(PAK4):Ishikawa and HEC-1A cell lines of F10 gene silencing group's PAK4 protein expression level is significantly lower than the control group(P<0.05),F10 over-expression group express PAK4 significantly higher than control group(P<0.05).4)Proliferating cell nuclear antigen(PCNA):Ishikawa cell line of F10 gene silencing group,control group and over-expression group express PCNA protein in a rising trend,compared with control group and over-expression group,the F10 expression in silencing group is statistically significant low(P<0.05).HEC-1 A cell line of F10 gene silencing group and over-expression group express higher PCNA protein levels than control group,and the F10 expression in silencing group is statistically lower than in over-expression group(P<0.05).5)Focal adhesion kinase(FAK):Ishikawa cell line of F10 gene silencing group,control group and over-expression group express FAK protein in a declining trend.Compared with control group and over-expression group,the F10 expression in silencing group is statistically significant high(P<0.05).HEC-1A cell line of F10 gene silencing group and over-expression group express higher FAK protein level than control group,and the F10 expression in silencing group is statistically significant higher than in over-expression group(P<0.05).6)Ishikawa and HEC-1 A cell lines show no significant difference in the expression of all cell cycle related proteins between F10 over-expression group empty vector,F10 silencing empty vector and control group(P>0.05).CONCLUSIONThe expression of F10 gene in cervical cancer,endometrial cancer and ovarian cancer is significantly higher than that in adjacent tissues.The Ishikawa and HEC-1A cell lines with stable over-expression and down regulated F10 gene are successfully established and tested.It is proved that F10 gene could promote the proliferation of endometrial cancer cells.Cell immunofluorescence technology confirm that F10 gene can promote the positive cell cycle regulatory factors in endometrial cancer cells and decrease the cell cycle negative regulatory factor.It may accelerate the process of cell cycle and promote the proliferation of endometrial cancer by regulating the expression of cyclins.