Inhibition Of HPOT1Enhanced The Radiosensitivity Of Cervical Cancer Cells

Part Ⅰ Expression of hPOT1in cervical cancer and normal tissue adjacent to carcinoma and its clinical significancePurpose:To study the expressions of telomeres protect protein1(hPOT1) in cervical cancer and normal tissue adjacent to carcinoma, and to explore its roles in the development and progression of cervical cancer.Methods:Immunohistochemistry was applied to detect the expressions of hPOT1in94cases of cervical cancer and12cases of normal tissue adjacent to carcinoma. Nuclear immunoreactivity was judged as positive. According to the literature, hPOT1positivity was examined as follows:500nuclei were observed in hPOT1immunostained slides under×200magnification microscopy. The hPOT1index was designated as a percentage.Results:hPOT1was mainly in the nucleus. The hPOT1index was significantly higher in the cervical cancer (31.52±8.95%) than those in normal tissue adjacent to carcinoma (15.50±4.26%), P<0.001. We compared the hPOT1index and clinicopathological parameters. The94cervical cancer cases were divided into two groups by hPOTl index:hPOT1-low (with lower hPOT1index≤30%) and hPOT1-high (with higher hPOT1index>30%). The hPOT1indexes of hPOT1-low and hPOT1-high groups were23.04±6.36%and40.76±6.49%, respectively (P<0.001). There was no difference between the two groups in age, histological types, pathological grade, N-factor (nodal metastasis); however, T-staging was advanced in hPOT1-high. The number of T2-3cases was29out of45in the hPOT1-high group, and,14out of49in hPOT1-low group; in contrast, that number of T1in hPOT1-high group and hPOT1-low group were16/45and35/49, respectively (P<0.001). The results indicated that the expression of hPOT1was related to T-staging, the more advance of T-staging, the higher expression of hPOT1protein.Conclusions:The high level of expression of hPOT1of was closely associated with the development of cervical cancer. hPOT1may become a potential target of anti-tumor treatment. Part II Inhibition of hPOT1enhanced the radiosensitivity of cervical cancer cellsPurpose:To study the influence of inhibition of hPOT1expression on the radiosensitivity of cervial cancer cells.Methods:First, we constructed a lentiviral vector harboring RNAi sequence targeting the hPOT1gene (pGMLV-SC5-hPOTl-shRNA) and negative control sequence (pGMLV-SC5-Neg-shRNA), then transfected them into the C33A cells to establish stable interfering cell line. Then mRNA and protein expression levels of hPOTl were detected by qRT-PCR and Western blot, respectively. Radiosensitivity was tested by clone formation experiment. The cell survival curve was splined with GraphPad Prism5.0software according to multitarget-single hitting model, and SF2, DO, Dq, N, SERsF2and SERDq were obtained.Results:Recombinant lentivirus vector targeting inhibition of hPOT1were successfully built and transfected into C33A cells. Two stable interfering cell models of C33A/hPOT1-shRNA and C33A/Neg-shRNA were successfully established. Inhibition effects of hPOT1-shRNA were confirmed by qRT-PCR and; Western blot. After hPOT1expression was suppressed, the SF2of C33A/hPOT1-shRNA cells dropped to0.380±0.059, compared with C33A/Neg-shRNA (0.563±0.072, P=0.0003), C33A/hPOT1-shRNA cells had lower DO (1.428) and Dq (0.983) than C33A/Neg-shRNA cells (1.658,2.393, respectively). SERSF2and SERDq were1.482and2.432respectively when hPOT1expression was suppressed.Conclusions:Inhibition of hPOT1increased the radiosensitivity of C33A cells. hPOT1may become a target of radiosensitization in the treatment of cervical cancer. Part Ⅲ Mechanism of radiosensitization of inhibiting hPOT1expression on cervical cancer cells Purpose:In our previous study, we found that inhibiting hPOT1expression enhanced the radiosensitivity of C33A cells. Here we conducted preliminary research on the mechanisms.Methods:After targetedly surpressing hPOT1expression, telomerase activity was detected by TRAP-PCR-ELISA, and mean telomere lengths of8-10th generation of subculture was tested by Southern Blot. Apoptosis level and cell cycle distribution were detected using flow cytometry at different times after X-ray irradiation. γ-H2AX foci in the nucleus induced by X-ray radiation, which was considered as one of the standard examination methods of DNA damage, were detected by immunofluorescent assay and obversed with confocal laser scanning microscope.Results:The mean telomere lengths extended significantly after hPOT1was inhibited, while telomerase activity did not change obviously. Spontaneous apoptosis significantly increased with suppression of hPOT1(4.07±0.52%vs.5.16±0.67%, t=2.57, p=0.042). After5Gy X-ray treated, apoptosis induced by irradiation was significantly higher in C33A/hPOT1-shRNA cells than in C33A/Neg-shRNA cells at24hours (34.95%±4.56vs.43.16±3.11%, t=2.97, p=0.025). Inhibition of hPOT1significantly reduced G2/M ratio in C33A/hPOT1-shRNA cells after5Gy X-ray radiation (12h t=5.995; p<0.001;24h t=9.527, p<0.001;48h t=12.81, p<0.001), and decreased the length of time of G2/M arrests. At0.5h After1Gy X-ray treated, y-H2AX foci increased rapidly in both cells, and there were much more y-H2AX foci in C33A/hPOT1-shRNA cells than that in C33A/Neg-shRNA cells (P<0.001). At12h after IR, there were still more residual γ-H2AX foci in C33A/hPOT1-shRNA cells (p<0.05). These results indicated that inhibition of hPOT1increased DNA damage after irradiation, and might impair or delay DNA damage repair.Conclusions:Inhibition of hPOT1increased radiosensitivity of C33A cells. It might due to decrease of G2/M retardation. Inhibiting hPOT1expression extended the length of telomere without increasing telomerase activity, and led to increase of radiosensitivity of C33A cells through inducing apoptosis and inhibiting repair of DNA damage. In conclusion, hPOT1had close relationship with telomere stability and radiosensitivity, and could be recognized as a new target of radiosensitization of cervical cancer.