| 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. GATA-3 is in fact a well-known, crucial transcription factor in T-cell development and differentiation. It plays a significant role in the early development of thymocytes and the differentiation of adult T-cells in a subtype of T helper cells(Th2), pruducing a specific set of cytokines and promoting humoral immunity. Recent reports have also illustrated altered expression patterns of Gata-3 in other human cancers, such as in breast, pancreatic, cervical and esophageal carcinoma. Our group first found both abroad and domestic the expression of GATA-3 in the HO-8910, an ovarian carcinoma cell line. How the expression of GATA-3 affect the biological characteristics of the carcinoma and the immune escape is not fully understood now.We planed to adjust the expression of GATA-3 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 GATA-3 gene on biological behavior of ovarian carcinma cell.Methods①Design, construct and identify GATA-3/siRNA-1,GATA-3/siRNA-2 and GATA-3/siRNA-negative recombinant retrovirus as the carrier.②GATA-3/siRNA-1,GATA-3/siRNA-2 and GATA-3/siRNA-negative recombinant plasmid were transfected into packing cell line PT67 by liposome, and PT67 was selected by puromycin later. HO-8910 was infected by the virus supernatant of stable transfected PT67 cell lines, and the stable transfected HO-8910 cell lines (HO-8910/siRNA-1,HO-8910/siRNA-2 and HO-8910/siRNA-negative) established by selecting with puromycin were investigated the reduction levels of GATA-3 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 HO-8910 cell(HO-8910/siRNA-1,HO-8910/siRNA-2 and HO-8910/siRNA-negative )into the skin.Results①Construct GATA-3/siRNA-1,GATA-3/siRNA-2 and GATA-3/ siRNA-negative recombinant plasmid successfully.②The stable HO-8910 cell lines with a persistent silence of GATA-3 were established.③The percents of HO-8910/siRNA-1 cell in G0/G1 phase,S phase and G2/M were (66.3±10.3)%, (8.6±1.2)% and (27.1±3.4)% respectively; the percents of HO-8910/siRNA-2 cell in G0/G1 phase , S phase and G2/M were (64.3±14.6)%, (7.9±0.9)% and (26.2±2.9)%, respectively; while the percents of HO-8910/siRNA- negative cell in G0/G1 phase , S phase and G2/M were (50.3±9.1)%, (9.2±1.5)% and (33.6±4.7)% ; the percents of HO-8910 cell in G0/G1 phase, S phase and G2/M were (46.3±5.2)%, (8.9±0.7)% and (35.5±6.1)% ,respectively④The percent of HO-8910/siRNA-1 cell in early apoptosis wa(s0.05±0.01)%, the percent of HO-8910/siRNA-2 cell in early apoptosis was(0.04±0.01)%, while the percent of HO-8910/siRNA- negative cell was(0.03±0.01)%(P﹥0.05), while the percent of HO-8910 cell was(0.03±0.01)%(P﹥0.05).⑤Comparison with HO-8910/siRNA-negative, the proliferation of HO-8910/siRNA cell was delayed clearly(P<0.05), and was more sensitive to cisplatin . The growth of the corresponding implanted tumor slowed down significantly(P<0.01).Conclusions①Retroviral vector-driven siRNA can block the expression of GATA-3 effectively.②The blocking of GATA-3 gene increase the percentage of G0/G1 stage cell, which means the lower growth speed of the ovarian carcinoma cell.③The sensitivity of HO-8910 cell to cisplatin became higher after the blocking of GATA-3 gene. So our conclusion can be drawn that down-regulation of GATA-3 by RNAi may affect the malignant biological behavior of ovarian carcinoma cell. |
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