The Expression Of IEX-1 In Cervical Cancer And Its Relationship With HPV Infection

Background and ObjectiveCervical cancer Cervical cancer is one of the most common malignant tumors of the female reproductive system.The morbidity of cervical cancer is the third one in female which is behind lung cancer and stomach cancer, There are about 90% of death cases taking place in developing country.It is seriously threatening woman's life and health. The main two types of cervical cancer are Squamous Cell Carcinoma(SCC) and adenocarcinoma(ADCA),The former one is about 80%.The morbidity of cervical adenocarcinoma is increasing in recent years.Persistent infections of the high risk human papilloma virus(HR-HPV) is the main carcinogenic factor for cervical cancer,and the most common types are HPV-16 and HPV18. Early proteins E6 and E7 are the main cancer proteins of HR-HPV and P53 can be affected by E6 in a way of ubiquitinational Hydrolysis,leading to the aging of p53-dependent apoptosis and/or losses,and the combination of E7 and pRB results in the disturbance of cell cycle.Therefore, HR-HPV infection may lead to malignant transformation of cells and tumor development.In1993,Charles found out immediate early response gene X-1(IEX-1) in mouse fibroblasts as a number of immediate early gene(IEG),it can express shortly and fast in various stimulating conditions.These stimulating factors are growth factors,cytokines, ionizing radiation,viral infections and other types of stress factors.IEX-1 isinvolved in cellmetabolism, increment and apoptosis, differentiation and the cell cycle regulation of many biological effects.IEX-1plays a complex or even the opposite effect in the regulation of cell cycle and apoptosis. Alough studies have shown that IEX-1 expressed in a disordered way in many human malignant tumors,but there are less researches about the function of IEX-1 in cervical cancer.There have no ideas about the expression of IEX-1 in cervical cancer and the association between IEX-1 and HPV infection in cervix.This study will analyse the expression of IEX-1 in cervical cancer ?CIN tissue and normal cervical tissue by Immunohistochemistry method, then we analyse the relationship between IEX-1 expression and clinical cases of cervical cancer,then it can predict the role of IEX-1 in development of cervical cancer.And we can analyse different expression of IEX-1 in different HPV infection status of cervical carcinoma cells through the RT-qPCR method and the Western Blot method.Subsequently,we can design siRNA according to HPV16 E6, then we can use it to specificly knock down and reduce the expression of E6 in SiHa cells.Then we can detect the expression of IEX-1 in SiHa cells before and after E6 silence to confirm the correlation between HPV infection and expression of IEX-1.Materials and Methods1.Cell lines in human cervical cancer are given for free by the Huazhong University of science,such as SiHa, HeLa, C-33 a and favours.We gathered 132 paraffin block samples in the department of pathology of the first affiliated hospital of Zhengzhou University from January 2010 to October 2014.There are 29 cases of cervical benign lesion,aged 32-61,46 cases of CIN(CIN?is 19 cases CIN? is 17 cases CIN? is 10 cases),aged 29-58; 57 cases of cervical cancer(squamous cell carcinoma is 35 cases, adenocarcinoma is 22 cases), aged 27-65. According to FIGO clinical staging of cervical carcinoma:phase I is 23 cases, 26 cases of phase II, phase III in 6 cases, two patients with IV stage. According to differentiation of levels:there are high differentiation in 13 cases, middle differentiation in 18 cases, poorly differentiated in 26 cases.2.We use SP Immunohistochemistry to detect the expression of IEX-1 in cervical carcinoma,CIN tissue and normal cervical tissue, then we can analyse its relationship between clinicopathological features and the expression of IEX-1.3.We can detect the expression of IEX-1 in SiHa(HPV16+) and HeLa(HPV18+), C-33a(HPV-) in cervical cancer cell line in a real-time fluorescent quantitative PCR method, also we can use Western Blot method to detect the expression of IEX-1 protein in cervical cancer cell line about Si Ha(HPV16+) ?HeLa(HPV18+) and C-33a(HPV-).4.We can use RT-qPCR and Western Blot to detect the mRNA level and the expression of protein about E6 ? E7 and IEX-1 in SiHa cells before and after E6-specific siRNA interfered SiHa cells.5.Statistical treatment:we use SPSS17.0 to statistical analysis. Qualitative data were compared with Chi-square test while quantitative data were compared with ANOVA.The difference with P<0.05 is considered to be of significance. Double comparison use the Bonferroni method to correct test coefficient.Results1.The expression of IEX-1 decreased in cervical benign lesion, CIN organization and cervical carcinoma.There was a statistically significant difference between the three(2=75.905,P=0.000<0.017). The expression of IEX-1 in cervical cancer has nothing to do with the patients ' age, histological type and FIGO stage(P>0.05). The expression of IEX-1 are closely related with differentiation of cervical cancer? invasion depth?lymph node metastases and muscle layers.The lower degree of differentiation it is, the lower the IEX-1 protein expresses(P<0.05).and lymph node metastasis positive rates of IEX-1 significantly lower than no lymph node metastasis(P<0.05); IEX-1 protein positive rates of the deep muscle invasion protein expression was significantly lower than only superficial myometrium invasion(P<0.05).2.IEX-1mRNA expression in C-33 a cells(21.652 ± 9.020) is higher than SiHa(1) and HeLa(1.742±0.854) cells(P<0.017). There are no significant difference of IEX-1mRNA expression in HeLa cells(P>0.017)and SiHa.IEX-1 protein expressionin C-33 a cells(18.017 1.351) is higher than it is in SiHa(1.028 ± 0.039) cells and HeLa(2.004 ± 0.363) cells(P<0.017).IEX-1 protein expression in HeLa cells and SiHa had no significant difference(P>0.017).3.mRNA and protein levels of E6 in interference group were significantly lower than the negative control group and the control group(P<0.05), E6 mRNA and protein levels in the negative control group and the control group difference was not statistically significant(P>0.05).mRNA and protein levels of IEX-1 in Interference group were significantly higher than the negative control group and the control group(P<0.05), and mRNA and protein levels of IEX-1 in the negative control group and the control group was not statistically significant(P>0.05).Conclusion1.IEX-1 expression in cervical cancer to reduce the expression level and cervical malignancy was negatively correlated with cervical cells IEX-1 expression is reduced or absent may be involved in the development of cervical cancer.2.IEX-1 expression levels in cancer cells or absence of infection with HPV negative correlation with infection HPV type unrelated, IEX-1 and HPV in cervical lesions may play during the opposite effect.3.E6 SiRNA can silence the expression of specific cervical cancer cell line E6 protein. Cervical cancer cell lines HPV E6 silence, IEX-1 upregulation, HPV E6 oncoprotein possible by reduced expression of IEX-1.