| Deregulation of cell proliferation and cell apoptosis underlies neoplastic initiation and development, while inhibition of tumor cell apoptosis indicates reduction of curative effect. It not only involve multiple regulators and signal transduction pathways, but also are regulated by complicated mechanism. Thus, the inhibitor of apoptosis protein (IAP) family is a sort of important factors of inhibiting apoptosis, which is characterized by the presence of one or more baculoviral IAP repeat (BIR) domains at the amino terminal, possessing or not a carboxyl terminal RING zinc-finger domain. Survivn is a novel member of IAP family, which has potential antiapoptosis activity and is generally overexpressed in tumor tissues. Recently, it has indicated by many investigation that survivin has a close connection with human common neoplastic initiation, development and prognosis embracing cervical neoplasm,and its overexpression in the majority of human tumors may lead to radiotherapy resist and chemotherapy endure.RNA interference (RNAi) represents a common biological phenomenon of double-stranded RNA (dsRNA) mediated post-transcriptional gene silencing (PTGS), which lies in altitude plants and animals. The mechanism of this process is described as that: the dsRNA in cells are cut into double-stranded small interfering RNA (siRNA). By joining with an effector complex termed RISC (RNA-induced silencing complex), siRNA binds to the cellular RNA with homologous sequences, and contributes to the degradation of the corresponding mRNA, the inhibition of specific gene expression. Since discovered by researchers in 1998, RNAi has been widely applied in the research of gene function and gene therapy as an efficient tool for specific gene silencing dueing to its convenience and efficiency.Cervical carcinoma is one of the most frequent female malignancy. Although we have made big progress in its prevention and cure, the incidence of cervical carcinoma still rises steadily because of high HPV infection and changes in society life model.The proportion of young patients is on the rise,and the cervical adenocarcinoma is more and more familiar in recent 20 years.Because the adenocarcinoma is easy to occur lymphous nodes transfer early, the radiosensitivity of adenocarcinoma is low, and the chemotherapy is easy to be endured, it is difficult to treat cervical adenocarcinoma and the prognosis is not ideal. So, it has become a hotspot in the field of cervical cancer therapy to seek a scientific and applicable way to enhance the curative effect and improve the prognosis of cervical carcinoma . AIMTo observe the influence of survivin gene expression reduction on the radiosensitivity and chemosensitivity in cervical carcinoma, and by seeking the possible mechanism to investigate the feasibility of RNAi targeting survivin gene in increasing radiosensitivity and chemosensitivity of cervical carcinoma.Thus,lay a solid foundation for survivin gene therapy for cervical carcinoma。METHODSThis research is composed of the following 3 parts:①Construction of short hairpin RNA (shRNA) eukaryotic expression vector targeting survivin gene and observation of its influence on survivin expression in human cervical carcinoma cell HeLa.Designed and synthesized 2 couples of survivin specific siRNA primer (S1-1,2,S2-1,2) based on survivin gene sequence and abide by those design rules. Cloned them into eukaryotic expression vector pSilencer 2.1-U6 neo respectively by DNA recombined techniques after annealing connection reaction. Confirmed by restrict endonuclease digestion and DNA sequencing before transfecting them also respectively into human cervical carcinoma cell HeLa using LipofectAMINE2000, then selected the positive clones by G418, detected survivin mRNA expression by semi-quantitative RT-PCR, detected its protein steady expression by Western blot.Finally, selected out the higher inhibitive efficiency recombined shRNA eukaryotic expression vector and the stable tranfected cells to the following reseach work. ②Influence of survivin gene RNAi on cell proliferation, apoptosis, radiosensitivity and chemosensitivity in human cervical cancer cell HeLa.Protracted cell growth curve by methyl thiazolyl tetrazolium (MTT) assay, examined the changes of cell cycle distribution by flow cytometry, observed cell apoptosis by flow cytometry and Hoechst staining, assessed the changes of caspase-3 activity by kinase activity test, examined the expression of Ku70 protein by immunofluorescence. Observed the changes of cell radiosensitivity by plate clone formation assay and survival curve; measured cell apoptosis and proliferation inhibition at 24h, 48h and 72h after irradiated with 2Gy X ray by flow cytometry and MTT assay. Examined cell viability by MTT assay and counted out 50% inhibitive concentration (IC50) of cisplatin at 72h after delt with it, measured cell apoptosis and growth inhibition at 24h, 48h and 72h after delt with 5ug/ml cisplatin by flow cytometry and MTT assay.③Impact of RNAi targeting survivin gene on tumorigenesis, tumor growth, radiosensitivity and chemosensitivity in nude mice.Inoculated HeLa-S2,HeLa-NC,HeLa-U6 neo and HeLa cells respectively in flank subcutaneous tissue of nude mice to establish xenograft models of human cervical carcinoma, observed the tumor growth status and compared the tumorigenesis ability of the adove 4 cells.To observe the impact of RNAi targeting survivin gene on tumor growth, measured the tumor volume termly, drew the tumor growth curve and investigated the tumor weight after they were killed. Examined the expression of survivin protein in tumor tissues by immunohistochemistry SP method. Examined the expression of FⅧRAg in tumor tissues also by immunohistochemistry SP method, and cunted out MVD.Observed cell apoptosis in tumor tissues by HE staining, TUNEL method and counted out AI.To observe the impact of RNAi targeting survivin gene on tumor radiosensitivity after irradiated by X ray and chemosensitivity after delt with cisplatin, measured the tumor volume termly and drew the tumor growth curve, investigated the tumor weight after they were killed and examined cell apoptosis by TUNEL method.RESULTS①Successfully constructed survivin shRNA eukaryotic expression vector : pSilencer2.1-S1 and pSilencer2.1-S2. Established stable transfected cell line: HeLa-S1, HeLa-S2, HeLa-NC and HeLa-U6 neo.②Detected different decreases of survivin expression in HeLa cells after transfected with pSilencer2.1-S1 and pSilencer2.1-S2, the interference efficiency of the latter was preferred to the former, and the expression inhibitive rates of survivin gene mRNA and protein in HeLa-S2 were 62.8% and 60.1% respectively.③Cell proliferation HeLa-S2 was inhibited, and the A at every checkpoint decreased obviously compared with HeLa(P<0.05); the changes of cell cycle distribution in HeLa-S2 were significant, many cells were blocked in G0/G1 phase (72.7±3.1)%, G2/M phase (5.1±2.9)% reduced sharply; the apoptotic rate of HeLa-S2 was (19.2±1.4)%, rising up obviously. ④Caspase-3 activity of HeLa-S2 had been enhanced and A405 was 1.26±0.04.The expression of Ku70 protein in HeLa-S2 decreased sharply.⑤With the rise of radiation dose, cell clone formation rate declined in all groups of cells and at the same dose of radiation, clone formation rate of HeLa-S2 declined notably; the cell survival curve also showed a significant decrease of HeLa-S2 D0 and Dq, were 3.15 and 1.21 respectively and the radiation enhancement ratios of HeLa-S2 were 2.01(a ratio of D0)and 1.77(a ratio of Dq). With time went by, cell apoptotic rate and proliferation inhibitive rate rose up significantly after irradiated with 2Gy X ray, and the cell apoptotic rates and proliferation inhibitive rates of HeLa-S2 at every checkpoint were obviously lower than those of untransfected HeLa cells.⑥With the rise of cisplatin concentration cell viability declined in all groups of cells and at the same concentration of cisplatin, cell viability HeLa-S2 declined sharply , the IC50 at 72h after delt with cisplatin was ( 0.74±0.02)μg/ml. With time went by, cell apoptotic rate and proliferation inhibitive rate rose up significantly after delt with 5ug/ml cisplatin, and cell apoptotic rates and proliferation inhibitive rates of HeLa-S2 at every checkpoint were obviously lower than those of untransfected HeLa cells.⑦Establish 4 groups of xenograft models of human cervical carcinoma.Compared with HeLa group,the tumorigenesis time of HeLa-S2 group delayed, tumor grew slow, the mean tumorigenesis time of HeLa-S2 group was (21.33±0.51)d,while which of HeLa group was(7.22±0.37)d(P<0.05).⑧The tumor volume of HeLa-S2 group was obviously less than that of HeLa group at every checkpoint.At the terminal of observation,the tumor weight of HeLa-S2 group was obviously lower than that of HeLa group,and they were:(0.369±0.043)g and (1.150±0.136)g respectively(P<0.05).The tumor growth inhibitive rate of HeLa-S2 group was 67.9%.⑨The expression of survivin protein examined by immunohistochemistry in tumor tissues of HeLa-S2 group decreased sharply. The expression of FⅧRAg examined by immunohistochemistry in tumor tissues of HeLa-S2 group also decreased, and MVD went down to 23.38±3.14.Apoptotic cells increased in in tumor tissues of HeLa-S2 group examined by HE staining and TUNEL method, AI was (22.73±1.37)%.⑩The tumor volume of HeLa-S2 group was obviously less than that of HeLa group at every checkpoint after irradiated with X ray and the tumor gowth was inhibited. At the terminal of observation,the tumor weight of HeLa-S2 group was obviously lower than that of HeLa group,they were:(0.407±0.056)g and(1.381±0.289)g respectively(P<0.05).Cell apoptosis increased in tumor tissues of HeLa-S2 group,and compared with HeLa group, AI of HeLa-S2 group rose up notably,they were:(30.06±0.98)% and (4.17±0.64)%(P<0.05). (11)The tumor volume of HeLa-S2 group was obviously less than that of HeLa group at every checkpoint after delt with cisplatin and the tumor gowth was inhibited. At the terminal of observation,the tumor weight of HeLa-S2 group was obviously lower than that of HeLa group,they were: (0.323±0.058)g and(1.347±0.173)g respectively(P<0.05);cell apoptosis increased in tumor tissues of HeLa-S2 group,and compared with HeLa group, AI of HeLa-S2 group rose up notably,they were:(37.38±1.01)% and(5.19±0.61)%(P<0.05).CONCLUSION①Survivin gene RNAi can inhibite the expression of survivin mRNA and protein in human cervical caecinima cell HeLa.②Survivin gene RNAi can inhibit cell proliferation and induce cell apoptosis by reducing survivin expression in HeLa cell.③Survivin gene RNAi can make cell cycle distribution be blocked in G0/G1 phase, reduce the expression of Ku70 protein, weaken the ability of DNA break repair by inhibiting the expression of survivin in HeLa cell and finally significantly increase cell radiosensitivity to X ray.④Survivin gene RNAi can increase cell apoptosis induced by X ray irradiation and enhance its inhibitive efficiency.⑤Survivin gene RNAi can enhance caspase-3 activity, induce cell apoptosis by inhibiting the expression of survivin in HeLa cell and finally significantly increase cell chemosensitivity to cisplatin .⑥Survivin gene RNAi can increase cell apoptosis induced by cisplatin and enhance its inhibitive efficiency.⑦Survivin gene RNAi can weaken tumorigenesis ability of HeLa cell by inhibite its proliferation.⑧Survivin gene RNAi can reduce MVD in xenograft by inhibiting survivin protein expression and finally inhibite tumor growth and accelerate cell apoptosis.⑨Survivin gene RNAi can increase cell apoptosis induced by X ray irradiation, enhance its inhibitive efficiency, consequently improve tumor radiosensitivity.⑩Survivin gene RNAi can increase cell apoptosis induced by cisplatin, enhance its inhibitive efficiency, consequently improve tumor chemosensitivity.
|
PDF Full Text Request