Expression Of P16, P53 And HTERC In HPV Tested Negative Cervical Cancer

Cervical cancer is the second most common malignancy among women worldwide,accounting for about 9.8% of all cancers.There are about 500000 new cases of cervical cancer in the world each year,half of which dies of cervical cancer,and about 80% of deaths occur in developing countries.HPV infection has been considered as the main pathogenic factor of cervical cancer,and both E6 and E7 are the key roles of the development of cervical cancer.However,many studies have shown that there are about 10% of cervical adenocarcinoma and a certain proportion of HPV negative cervical squamous cell carcinoma.As a tumor suppressor,p53 is one of the most important factors to control cell proliferation.HPV E6 oncogenic proteins bind to p53 by E6 AP to form a complex,which leads to down-regulation of p53;In normal cells,p16 is a negative regulator of proliferation,which results in a down-regulation of CDK4 by a negative feedback loop once the Rb gene is inactivated.The interaction between E7 and p Rb protein can lead to the inactivation of the negative feedback loop,which leads to the up-regulation of p16 so that the cell cycle from G1 to S is promoted.The role of telomerase is considered to be a major factor in the formation and development of cervical cancer.Studies have shown that telomerase is activated in human malignant tumors and transduced cell lines,and it is concluded that telomerase activation is a key event in the formation of human malignant tumors.Telomerase consists of two subunits of hTERC which are used as templates in the course of telomere extension and h TERT which is catalytically active and has reverse transcriptase activity.The amplification of hTERC gene can prevent cell apoptosis and eventually lead to the occurrence of tumor.HPV E6 activates h TERT to maintain the length of telomere,leading to cell immortalization and tumor formation.However,we found that both HPV DNA and E6/E7 mRNA were all negative in patients with cervical cancer in clinical practice.In this study,HPV DNA,E6/E7 mRNA and p16,p53,hTERC were detected,the purpose of which was to investigate the presence of HPV tested negative SCC,and to analyze the expression and significance of three indexes in HPV tested negative SCC.Objective Through the detection of HPV DNA and E6/E7 mRNA,we can conclude that HPV negative cervical squamous cell carcinoma is existent.Detecting the expression of p16,p53,hTERC in HPV tested negative cervical squamous cell carcinoma,and exploring the relationship with the occurrence and the development of HPV tested negative cervical squamous cell carcinoma,we can get a new direction for the pathogenesis and treatment of HPV tested negative SCC.Materials and Methods 1.The object of study:This study collected cervical tissues which were fixed using formalin and embedded by paraffin from October 2013 to May 2015 from the record room of pathology department of Henan Province People’s Hospital(Zhengzhou,Henan).The age range of the selected cases was 18-65 years old,with a median age of 42 years old.None of the female patients had a history of cervical lesions.There are 875 cases of cervical tissue which were collected in the study.The study has been approved by the ethics committee of Henan Province People’s Hospital and obtained the consent and signature of the patients or the patients’ s families.2.Research methods:Hematoxylin-eosin(HE)staining method was used to diagnose the pathological changes of 875 cases of cervical tissue,and the pathological examination of HE stained sections was confirmed by 3 senior medical pathologists based on the international standard.The diagnosises were divided into four groups:225 normal or inflammatory(NNL)cases,210 low squamous intraepithelial lesion(LSIL)cases,200 high squamous intraepithelial lesion(HSIL)cases,240 squamous cervical cancer(SCC)cases.The study ruled out 225 NNL cases last.PCRreverse hybridization and b DNA technique were used to detect HPV type and E6/E7 mRNA in 210 cases of LSIL,200 cases of HSIL and 240 cases of SCC,respectively.Then FISH of hTERC gene and the immunohistochemical staining of p16,p53 were used in 15 cases of HPV(-)(DNA-/mRNA-)and 25 cases of HPV(+)(5 cases of HPV DNA-/mRNA+,20 cases of DNA+/mRNA+).3.Statistical methods:This study uses SPSS17.0 statistical software(SPSS company)for data analysis.The χ2 test,Kruskal-Wallis rank sum test method were used to analyse data,and P<0.05 means that the difference was statistically significant.Results 1.The expression of HPV DNA:In this study,HPV DNA of 650 cases of cervical tissue samples were detected.The total infection rate of HPV was 94.62%.In SCC,the infection rate of HPV was 89.58%,which was lower than that of LSIL(95.24%)and HSIL(100%),and the difference between the three groups was statistically significant(P=0.000).There was significant difference each other(P<0.05).2.The expression of HPV E6/E7 mRNA:E6 and E7 are the key genes of HPV carcinogenic.When HPV DNA is integrated into the host cell gene,E6 and E7 can be over expressed,so the detection of E6/E7 mRNA can indicate the state of HPV infection.The data of this study showed that there was a significant difference in the percentage of E6/E7 mRNA between different pathological grades(r=0.387,P=0.000).Comparing the percentage of mRNA each other among all pathological grades,the results showed that there was no significant difference in mRNA expression between LSIL and HSIL group(P=0.054),and there was significant difference in the percentage of mRNA between LSIL and SCC,HSIL and SCC(P<0.001).3.The correlation analysis between HPV DNA and E6/E7 mRNA:In this study,HPV DNA and E6/E7 mRNA in the case of SCC were analyzed and found that the percentages of HPV DNA+/mRNA+,DNA-/mRNA-,DNA+/mRNA-,DNA-/mRNA+ were 87.5%,6.25%,2.08%,respectively.Compared with HSIL,the percentage of DNA+/mRNA+ samples in SCC was relatively higher,while the percentage of DNA-/mRNA-samples was lower,and the differences were statistically significant(P<0.001).4.The analysis of the difference between HPV DNA and E6/E7 mRNA:In this study,the positive rate of HPV DNA and the positive rate of E6/E7 mRNA in cervical lesions were analyzed.In all cervical tissue samples,the positive rate of HPV DNA was 94.62%(615/650),and the positive rate of E6/E7 mRNA was 71.54%(465/650),and the difference was statistically significant(P=0.000).In LSIL and HSIL,the positive rate of HPV DNA was higher than that of E6/E7 mRNA,and the difference was statistically significant(P<0.001).In SCC,the positive rate of HPV DNA was less than E6/E7 mRNA,and the difference was statistically significant(P=0.008).5.The expression of p53,p16 and hTERC genes:In the study,the expression of p16,p53 of HPV(-)SCC and HPV(+)SCC were detected using the immunohistochemical staining,and hTERC gene of HPV(-)SCC and HPV(+)SCC was detected using FISH,the results of which showed that the positive percentage of p53 in HPV(-)group and HPV(+)group was 66.67%,40% respectively.There was no significant difference in p53 positive rate between the two groups(χ2=2.667,P=0.102).The positive rate of p16 in the two groups was respectively 66.67%,100%,and the difference was statistically significant(correction χ2=6.72,P=0.01).The results of hTERC detection showed that the positive expression rate of hTERC gene was 66.67% HPV(-),80% HPV(+)and the difference was not statistically significant between the two groups(correction χ2=0.32,P=0.572).Conclusions 1.There are some cases in cervical squamous cell carcinoma that both HPV DNA and E6/E7 mRNA are negative,which is HPV tested negative SCC.2.Compared with HPV tested positive SCC,the expressions of p53 and hTERC are no significant difference,while p16 expression is weaker in HPV tested negative SCC.And the clinical significance and formation mechanism of HPV tested negative SCC need to be further studied.