A Novel Insight From The Relationship Between The Activity Of Telomerase And The Expression Of HPV E6, P53 And PinX1 In The Patients With Cervical Squamous Carcinoma

Background and ObjectiveCervical cancer is the third most common cancer in women worldwide with a serious threat to women’s reproductive health and an increasing incidence in young women. The pathogenesis is poorly understood, therefore, it is greatly difficult to develop the management of cervical cancer.Continuous high-risk human papillomaviruses(HR-HPV) is an important factor for cervical cancer.99% of the cervical cancer women are attacked by HPV. The activation of oncogene initiates through the mechanism of HPV to cause the transformation of normal cells. It has been reported that oncoprotein E6 can promote the activity of telomerase. The normal regulation of cells can be attacked by HPV E6, including the regulation of acute injuries, stabilization of the initial gene and the maintenance of cancer expression.Recent studies have found, telomere is an important component to protect the terminal region of the chromosome. The over activation of the telomerase is closely related to the development of tumors. Meanwhile, it leads to the inaccurate repair of the seriously shortened telomere, so that cells could not come to a normal apoptosis, but tend to cancer cell instead.Wild type p53 is an vital signal regulatory factor and anti-oncogene. p53 gene mutates in half of human’s tumors, which makes the damaged DNA cells undergo malignant copy and turns to be a oncogene. However, in human cervical cancer, the p53 is mainly under inactivation instead of mutation. Thus, the inactivation of the wild type p53 may play an important role in the development of cervical cancer.In addition, PinX1, the strong inhibitor of the telomerase, also a suppressor of cancer, is almost expressed in all of normal tissues, but the absence of PinXl can be observed in most tumors. PinXl not only strongly inhibits the activity of telomerase, but also plays a crucial role in the maintenance of telomere length. A new study has reported that PinXl is a new target gene of p53 with "p53-PinXl" pathway, which can be silenced by HPV E6 in cervical cancer cell lines, however, this has not been confirmed in the tissue samples.Cancer samples, adjacent tissues of squamous cervical carcinoma and normal tissues were obtained to explore the expressions of HPV E6, p53 and PinX1 and the activities of telomerase activities, the telomere length, as well as the relationships among them. Therefore, the evidence of "p53-PinXl" pathway and a new target can be provided for the pathogenesis and cervical cancer.Part I The expression of HPV E6, p53 and PinXl in cervical squamous carcinoma1. To detect the expressions of the mRNA level of HPV E6, p53 and PinXl in three groups.2. To detect the expressions of HPV E6, wild type p53 and PinX1 protein level in three groups.3. Immunohistochemistry was also applied to detect the protein level of HPV E6, p53 and PinXl in three groups.Part II The change of the telomerase activities and the telomere length in cervical squamous carcinoma1. To test the telomerase activities and the telomere length in cervical cancer;2. To determine the correlation among HPV E6, p53, PinX1, telomerase activities and telomere length.Method:1. HPV gene sequencing was applied by BGI Shenzhen to pick the squamous carcinoma with high-risk HPV infection and the normal cervical samples without HPV infection.17 cases of squamous carcinoma tissues without chemoradiotherapy were collected from March 2012 to February 2013, and 10 cases of normal samples were collected as control group.2. qPCR and Western blot were applied to detect all these elements in the mRNA and protein level.3. Immunohistochemistry was applied to detect HPV E6, p53, PinXl in these three groups;4. Telomerase activities(by TRAP method) was proved in three groups.5. Gene DNA as template, according to the particular region, primer was designed, thus the telomere length was measured.6. Spearman correlation analysis was made between the HPV E6, p53, PinX1, telomerase activities and telomere length.Results:1. qPCR showed that the HPV E6 had a higher expression than that in the adjacent samples, and, as a result of no expression in the normal tissues, there was no normal control group. qPCR also showed that the p53 and PinX1 had a lower expression than those in adjacent tissues and the normal.2. Western blot showed the same results of HPV E6, p53 and PinXl in squamous carcinoma.3. Immunohistochemistry also showed the similar results of HPV E6, p53 and PinXl in squamous carcinoma.4. Compared with those in the adjacent tissues and normal tissues, the telomerase activities length increased in squamous carcinoma.5. Spearman correlation analysis showed that the HPV E6 was negatively related with p53 and PinX1, positively related with the activities of telomerase; the p53 was positively related with PinX1.Conclusion:The high levels of HPV E6, the low levels of p53 and PinXl, the increased activities and length of telomerase in squamous carcinoma not only provide a strong evidence for "p53-PinX1", but also suggest that the mechanism may play a vital role in the pathogenesis of cervical cancer.