| Signal transducer and activator of transcription (STAT3) is known to be oncogene which is connected with many tumors closely.The content of STAT3 mRNA (5kb) is high in the brain, heart, liver, testes and thoracic gland thymus, it also can be detected in spleen. STAT3 is expressed in different types of cells and tissues widely, and its signal transduction pathway is connected with cell proliferation, differentiation and apoptosis closely. It has been found that STAT3 has a high expression in leukemia, multiple myeloma, squamous cell carcinoma on head and neck, melanoma, breast cancer, prostate cancer and lung cancer. Study shows that STAT3 expression has positive correlation with the high degree of tumor malignancy and poor prognosis. Therefore, a variety of strategies including antisense oligonucleotides, ribozymes was taken to reduce the expression of STAT3 so as to weaken the founction of promting desintegration and transfer to tumor cells of JAK-STATs.It is a promising strategy to inhibit tumor cell proliferation and induce tumor cell apoptosis by inhibiting the expression of STAT3.There are many ways to inhibiting the ptoteinum repression of STAT3 ,but they generally have some disadvantages of low transduction efficiency, short fonuction time,etc.The discovery and application of RNA interference (RNA interference, RNAi) phenomenon provide us a new thinking way to inhibit the expression of genes. RNAi is a RNA-induced post- transcriptional regulation and control method with a high degree of sequence-specific which can make specific gene silencing and functional incapacitation .RNAi has become an effective way to study gene function and cure tumor gene .In this experiment,we select the adenovirus vector as the tool to transport siRNA.We look torward to inhibit the proliferation of glioma cell and induce apoptosis of cell by inhibiting the expression of STAT3 by viral vector-mediated RNAi technology, make theoretical foundation for gene therapy for tumor.First of all, in accordance with the principle of RNA interference, we design the targeting point of RNA interference, make synthetic primers containing shSTAT3, gain Human U6 promoter and reverse complementary target sequence fragments HU6shSTAT3 with STAT3 by PCR method, construct recombinant adenovirus vector (rAdUSTAT3) containing the short hairpin RNA of STAT3 under the supervision of fluorescent protein adenovirus vector(Ad eGFP) through Microbix adenovirus AdMaxTM the company's packaging systems and transfer it to the C6 cells. Thenchoose the cells of non transduction virus as basic reference, monitor efficiency of transduction by GFP, choose the cells transducted by the virus vector with STAT3 irrelevant shRNA as non-specific inhibiting reference ,choose P-actin as a inner reference, detect the levels of STAT3 mRNA by RT - PCR, detect the levels of protein of STAT3 by Western Blot. Finally, assay the proliferation capacity of STAT3 cells by MTT. Detect transfected cell morphology changes and apoptosis by acridine orange staining.Detect the levels of PCNA expression of transfected cell by immunohistochemical.The results showed that: the relative expression of mRNA of C6 cells transducted by rAdUshSTAT3(l) decreased by 41.36% (p <0.01) at 48h compared to rAdUshSTAT3 (4),the relative expression of mRNA of C6 transducted by rAdUshSTAT3(2) decreased by 20.51% (p <0.01), the relative expression of mRNA of C6 transducted by rAdUshSTAT3(3) decreased by 15.6% (p <0.01); Compared to the cells of untransduced by virus, the relative expression of mRNA of C6 cells transducted by rAdUshSTAT3 (1) decreased by 46.77% (p <0.01), the relative expression of mRNA of C6 cells transducted by rAdUshSTAT3 (2) decreased by 27.85% (p <0.01), the relative expression of mRNA of C6 cells transducted by rAdUshSTAT3 (3) decreased by 33.38% (p <0.01), the relative expression of mRNA of C6 cells transducted by rAdUshSTAT3 (4) decreased by 3.6% (p> 0.05). Compared to rAdUshSTAT3 (4) at 72h, the relative expression of mRNA of C6 cells transducted by rAdUshSTAT3 (1) decreased by 65.24% (p <0.01), the relative expression of mRNA of C6 cells transducted by rAdUshSTAT3 (2) decreased by 51.98% (p <0.01), the relative expression of mRNA of C6 cells transducted by rAdUshSTAT3 (1) decreased by 50.95% (p <0.01); Compared to the cells of untransducted by virus, the relative expression of mRNA of C6 cells transducted by rAdUshSTAT3 (1) decreased by 68.82% (p <0.01), the relative expression of mRNA of C6 cells transducted by rAdUshSTAT3 (2) decreased by 56.93% (p <0.01), the relative expression of mRNA of C6 cells transducted by rAdUshSTAT3 (3) decreased by 56.95% (p <0.01); the relative expression of mRNA of C6 cells transducted by rAdUshSTAT3 (4) decreased by 10.29% (p <0.05).The relative expression of STAT3 protein did not change (p> 0.05) 48h after recombinant adenovirusvector transducting C6 cells ; after 72h the expression of STAT3 of the cells untransducted the virus has no significant difference (p> 0.05) compared to the negative control group .Compared to the negative control group rAdUshSTAT3 (4) , the relative expression of mRNA of C6 cells transducted by rAdUshSTAT3 (3) decreased by 31% (p <0.05), the relative expression of mRNA of C6 cells transducted by rAdUshSTAT3 (2) decreased by 37% (p <0.05), the relative expression of mRNA of C6 cells transducted by rAdUshSTAT3 (1) decreased by 56.25% (p <0.01). Compared to the cells of untransduced by virus, the relative expression of mRNA of C6 cells transducted by rAdUshSTAT3 (1) decreased by 68% (p <0.01), the relative expression of mRNA of C6 cells transducted by rAdUshSTAT3 (2) decreased by 44% (p <0.01). the relative expression of mRNA of C6 cells transducted by rAdUshSTAT3 (3) decreased by 38% (p <0.01).Compared to the cells of untransduced by virus, the inhibiting rate of AdUshSTAT3 (1-3)to the proliferation of C6 cells at 24h, 48h, 72h were 20.16%, 56.25%, 73.18%; 19.76%, 52.06%, 70.2%; 12.93% , 49.59%, 60.39%; compared to rAdUSTAT3 (4). The results show that, the inhibiting rate of rAdUSTAT3 (4)to the proliferation of C6 cells at 24h, 48h, 72h were 18.76%, 41.22% and 62.35%; 18.34%, 35.58% and 58.16%; 11.4%, 31.7% and 48.33%.The results of acridine orange staining showed: the quantity of transfected cells by rAdUshSTAT3 (1-3) decieased significantly, cytoplasmic shrinkage, cell nucleus enlarged and apoptotic bodies appeared. Especially the cell transfected by rAdUshSTAT3 (1) is the most obviously.The results of immunohistochemistry showed: the level of PCNA expression of transfected cells by rAdUshSTAT3 (1) was significantly lower than the level of PCNA expression of transfected cells by rAdUshSTAT3(4)(P <0.05).We can draw the following conclusions from this experiment: adenovirus vector transduction can efficiently transduct C6 cells, is an effective carrier for gene therapy to glioma; the short hairpin RNA to STAT3 by adenovirus vector can effectively inhibit the expression of STAT3 of neural glioma cells, block JAK-STAT3 signaling pathway, thus inhibiting the proliferation of glioma cells. The secondary structure of mRNA should be fully taken into account in choosing target of the RNAi-specific interference, try to avoid too much stem-loop structure. At the same time, consider the domain of structure of STAT3 protein in choosing the target sequence ,we found the effect of interference in SH2 is superior than the other domains.the effect of inhibition to C6 cell by SiRNA of SH2 domain was the most obviously. The effect of inhibition to C6 cell by SiRNA of the CCD and TAD domains was obviously, but weaker than the effect of inhibition to C6 cell by SiRNA of SH2 domain.These estabilish solid foundation for the further resarch of RNAi against STAT3-depth studyTo sum up, the next step is to make effective siRNA by the selection of specific and effective target to SH2 domain of STAT3,and enhance the role of aging by transforming the adenovirus vector; stuff on JAK-STAT3 signaling pathway and other tumor-related genes by the strategy of multi-genes combined application of RNAi ,eventually develop a specific and efficient drug of gene therapy to inhibiting the cell proliferation of glioma.
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