| Background The modern medicine has gained great achievement, but the morbidity and mortality of the ovarian carcinoma is still very high. The accomplishment of the human genome map and the deep investigation of the human geonme provide a new way to prevent and cure the carcinoma.Lots of recent research has demonstrated that the activation of the oncogene and the inactivation of the anti-oncogene correlated with the cell proliferation and differentiation gene in the genome, cause the occurrence and development of the carcinoma.Based on this theory, some theoretical and clinical researches of the gene therapy have been implemented to the carcinoma. The prerequisite of the gene therapy of carcinoma is to search and definite the target gene. RNA interfering (RNAi) is a sequence-specific post-transcriptional gene silencing mechanism, which is triggered by double-stranded 21- to 23-nucleotide RNA and causes degradation of mRNA homologous in sequence to the dsRNA. RNAi is an effective mode existing universally in the creature to adjust the gene expression and an important way to evaluate the gene function. RNAi can block the gene expression specifically. This new approach has been widely utilized in the areas of gene function, signal transduction and gene therapy.In these years, great progress has been made in the area of gene therapy of carcinoma relating to siRNA. Vector-based systems such as plasmid or retroviral vector for the introduction and stable expression of siRNA in tumor cells have been used for study the function of the oncogene and the anti-oncogene. The Her-2 oncogene (also called erbB-2) and its encoded gene product p185Her-2 belong to the epidermal growth factor receptor tyrosine kinases. Her-2 signaling pathways play cell growth, differentiation, malignants transformation, and resistance to chemotherapeutic agents. The Her-2 oncogene is overexpressed in ~30% of breast and ovarian cancer case and often indicates a poor prognosis. Therapeutic agents against Her-2 have been intensively sought over the past decade. Here wo show that small interfering RNA( siRNA) can silence the expression of Her-2 in models of human ovarian cancer throught retrovirus- mediated thranfer of an siRNA against Her-2. We planed to adjust the expression of Her-2 by using RNAi at transcription level and observe the effect of this method to the biological characteristics of ovarian carcinoma cell, so we can get some theoretical and experimental data of this method, which is very important to the futher clinical using.Objective To explore whether retroviral vector-driven siRNA can cause RNAi in carcinoma cell , and to observe the effects of small interfering RNA(siRNA)specific for Her-2 gene on biological behavior of ovarian carcinma cell.Methods①Design, construct and identify Her-2/siRNA,Her-2/siRNA-negative recombinant retrovirus as the carrier.②Her-2/siRNA,Her-2/siRNA-negative recombinant plasmid were transfected into packing cell line PT67 by liposome, and PT67 was selected by puromycin later. SKOV3 was infected by the virus supernatant of stable transfected PT67 cell lines, and the stable transfected SKOV3 cell lines ( SKOV3/siRNA,SKOV3/siRNA-negative) established by selecting with puromycin were investigated the reduction levels of Her-2 mRNA and protein by RT-PCR and immumohistochemistry.③Cell proliferation and sensitivity to cisplatin was assayed with MTT, and cell cycle distribution, cell apoptosis with flow cytometry.④The tumor growth of the null mice was analyzed after injection transfected SKOV3 cell(SKOV3/siRNA and SKOV3/siRNA-negative )into the skin, and the effectiveness of NK-92 cells on ovarian cancer was compared with the other groups.Results①Construct Her-2/siRNA,Her-2/siRNA-negative recombinant plasmid successfully.②The stable SKOV3 cell lines with a persistent silence of Her-2 were established. The Her-2 gene expression was inhibited obviously in the SKOV-3/siRNA group by RT-PCR and immunohistochemistry.③The percents of SKOV3/siRNA cell in G0/G1 phase,S phase and G2/M were (68.6±10.3)%, (15.1±1.2)% and (11.4±1.4)% respectively; while the percents of SKOV3/siRNA-negative cell in G0/G1 phase , S phase and G2/M were (55.8±9.1)%, (23.3±1.5)% and (18.1±1.7)%, respectively.④The percent of SKOV3/siRNA cell in early apoptosis was(10.500±0.250)%, while the percent of SKOV3/siRNA-negative cell was(0.340±0.010)%(p<0.01).⑤Comparison with SKOV3/siRNA-negative, the proliferation of SKOV3/siRNA cell was delayed clearly(p<0.05), and the tumor growth of the null mice slowed down significantly (p<0.01).⑥Consequently the survival rate of siRNA group affected by DDP at 4th day was (67.7±4.4)%, indicated significant difference between empty vector group (81.7±1.4)% (p<0.05 ). FCM results showed the apoptosis rates of the SKOV3/siRNA groups induced by DDP were increased to ( 38.6±1.1)%. There was significant difference between siRNA groups and the other groups (p<0.05).⑦Ctotoxicity of NK-92 against SKOV3 and SKOV3/siRNA was (21.1±6.8)% and (45.5±8.9)% respectively (p<0.05), Compared with SKOV3, the tumor growth of SKOV-3/ siRNA decreased significantly (p<0.05), especial with injection of NK-92 cells(p<0.05).Conclusions①Retroviral vector-driven siRNA can block the expression of Her-2 effectively.②siRNA can inhibit the expression of Her-2 gene effectively, which restrains the malignant biological behavior of ovarian carcinoma cell.③The sensitivity to DDP can be increased by inhibition the expression of Her-2 gene.④Silencing of Her-2 by RNAi combined with NK-92 can inhibit the cell proliferation in vivo, so this new therapeutic alliance may become a new approach of ovarian cancer therapy. |
PDF Full Text Request