| [Background]Cervical cancer is the second more frequent type of cancer in women worldwide,preceded only by breast cancer, and a considerable cause of morbidity and mortalityamong them. its incidence is rising year by year and the incidence was younger trend[1]. WTO reported that each year about530,000new cases of cervical cancer in2008.The preferred method of early cervical cancer is mainly based on surgery, but foradvanced, recurrent and metastatic patients, surgical treatment less effective. Sochemotherapy more and more attention, radiosensitization and treatment of advancedand recurrent cervical cancer, chemotherapy as conventional secondary treatment, andachieved a certain degree of efficacy. The same time, in patients with advanced cervicalcancer resistant to chemotherapy, and poor physical and other reasons, the curativeeffect is not very ideal, which to some extent limit the application of chemotherapydrugs. Therefore, the search can reduce the dose of chemotherapy drug application,while the improvement of the drug effect in the chemotherapy of chemosensitizer hasimportant clinical significance.Piwil2an AGO/PIWI family, mainly expressed in embryonic stem cells(embryonic stem cells, ESC) and germline stem cells (germ stem cell GSC), displayedin different body gametogenesis in a very important role. Normal individuals after birth,Piwil2only expressed in testicular germ cells and other tissue showed no expression. Inrecent years, researchers have in the different tumor tissues and tumor cell lines detectedthe expression piwil2. Promoter gene which can selectively be activated, resulting in a large number have tumorigenic effects of the variant protein is activated. Even, we alsofound that is closely related to the development of piwil2expression and tumor stemcells. However, the conversion of the PIWIL2mediated cell and tumor formationmechanism is still unclear. AGO/PIWI PIWI family protein contains two regions withdifferent biological functions and PAZ, although the contact of PAZ area and siRNA isnot clear, but piwil2displayed a very important role a different in organismgametogenesis [2].Cisplatin as commonly and effective used in the treatment of cervical cancer is oneof the chemotherapy drugs, and showed better efficacy in the treatment of advancedrecurrent cancer of cervical cancer radiotherapy sensitizers. However, more and morepatients with resistance to cisplatin dose caused toxicity makes it become hot researchfield of Cancer Prevention and in order to increase the cancer sensitivity to cisplatinprograms are problems.Histone deacetylase inhibitors (histone deacetylase inhibitors, HDACis) werefound in recent years, a new class of targeted anticancer drugs, antitumor mechanism onperformance in the re-balancing of cancer cells in disorders of histone acetylation status,suppressiononcogene expression, promote the expression of tumor suppressor genes,block tumor cell growth, induction of tumor cell apoptosis and promoting differentiation.HDACI research the hot concentrated in the combination of HDACIs with a variety ofdrugs (including traditional cytotoxic drugs paclitaxel, cisplatin, carboplatin, Dorsey heraces), and biological agents aspirin efficacy studies, the purpose is looking to make fulluse of the HDACI anticancer mechanism different from traditional chemotherapy drugs,thus making it a good synergistic anticancer drugs auxiliary agent [6]. Therefore areHDACs be considered a target for the treatment of cervical cancer.[Objective]To observed silence PIWIL2expression can enhance the study of cervical cancercell sensitivity to cisplatin and its mechanism. [Method]1. in vitro:(1) shGFP-PIWIL2plasmid transfected cervical carcinoma cell line Hela, Siha andC33A silence Piwil2expression;(2) Filter stable cell lines, using quantitative PCR and Western-blot method to furtherverify the effect of stable transfection;(3) Colony forming assay: crystal violet staining transfected cell colony formationability;(4) Using CellTiter96AQueous cell proliferation assay concentration DDP beforeand after transfection of C33A and SiHa cell growth inhibition;(5) The use of flow cytometry (FCM) to detect changes in apoptosis;(6) Western-blot method to detect p-STAT3, P53, P21, caspase-3, BCL-2expressiondifferences;(7)3mmol/LVPA treated as a controlgroup, using CellTiter96Aqueous detect cellgrowth inhibition rate and Western-blot method to detect cell histone H3acetylationlevels analysis Piwil2regulation of gene expressi2. In Vivo:(1) Nude mice were randomly divided into four groups: group transfected with emptyplasmid (vector group); transfected shRNA (sh-piwil2group); transfected with shRNAtransfected with empty plasmid+DDP group (vector+DDP group);+DDP group(sh-piwil2+DDP group), groups of six mice. Were taken Siha and stable transfection ofcell lines,5×106cells were grown in CD1nu/nu nude armpit subcutaneous beformed palpable tumors DDP treatment group intraperitoneal injection given the DDP2mg/kg/d, non-treatment group was given the saline treatment time for four weeks.(2) every four days with a vernier caliper measurement of tumor size, in accordancewith the formula: tumor volume=(length×width2)/2tumor volume and tumorgrowth curve.[Results]1．in vitro: (1) The quantitative PCR and Western-blot results showed that the the after transfectionshPiwil2plasmid expression of Hela, Siha C33A cells piwil2significantly lowered andthe differences were statistically significant (P <0.05) compared with beforetransfection.(2) Colony results show that the C33A and Siha cells after transfection shPiwil2plasmid colony-forming ability decreased significantly, and the difference wasstatistically significant (P <0.05).(3) CellTiter96AQueous cell proliferation experiment results show after the C33Aand Siha cells transfected shPiwil2plasmid, can significantly enhance their sensitivityto DDP transfected cells growth inhibition rate increased significantly, and thedifference was statistically significant (P <0.05).(4) Flow cytometry (FCM) to detect apoptosis results show that transfection shPiwil2plasmid transfected with empty plasmid silence Piwil2expression Siha cellssignificantly increased apoptosis rate, and the difference was significant (P <0.05). Twocells were treated with cisplatin treatment, the former rate of apoptosis was significantlyhigher than the latter, and the difference was statistically significant (P <0.05).(5) Western-blot method test results show that, to reduce the levels of p-STAT3proteinin the the silence Piwil2expression. P21, P53, upregulation of caspase-3protein, thedifference was statistically significant (P <0.05).(6) VPA treated control, silence Piwil2expression can also significantly improve thelevel of acetylation of histone H3, compared with the control group, the difference wasstatistically significant (P <0.05). CellTiter96AQueous experimental results showthat the silence Piwil2expression VPA treatment, may increase the sensitivity of C33Aand Siha cells of the DDP, the inhibition of cell proliferation rate was significantlyhigher than the untreated group, the difference was statistically significant (P <0.05).2. In Vivo:Transfection to sh Piwil2plasmid transfected with empty plasmid compared cells innude mice tumor volume decreases, the difference was statistically significant (P <0.05); in the DDP treated with, silent Piwil2expression the DDP suppression can significantlyimprove tumor effects, tumor volume was significantly smaller than the vector-DDPgroup, the difference was statistically significant (P <0.05).[Conclusion]Silence Piwil2expression can reduce the level of p-STAT3, raised P21, P53, andcaspase-3expression in cervical cancer cells for sensitivity to DDP destruction. Andincrease histone H3acetylation levels thus enhancing its regulatory gene expressionpathways. |
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