| Retinoblastoma(RB) is an intraocular malignancy arising in the retina and mainly occurred in infant. RB is a rare condition with an incidence of 1 case in 15,000 worldwide and an increasing morbidity in recent years. If without timely treatment, RB grows and spreads easily in the eye, and eventually leads to retinal detachment and necrosis, intracranial invasion along the optic nerve, threatening the child’s eyesight and life. RB treatment consists of photocoagulation, surgical removal, cryotherapy and chemoradiotherapy, but most of the therapies have side effects. Further study on the pathomechanism of RB and seeking more effective therapy is of great significance for clinical treatment of RB.SHH is one of the three homologous genes of hedgehog(HH), which regulates the process of embryo formation, cell growth and differentiation. Abnormal expression of SHH was found in medulloblastoma, basal cell carcinoma, colorectal cancer and lung cancer. SHH was considered to be a marker of tumor with high malignant degree. Previous studies showed that activation of hedgehog pathway was involved in the development of tumor through regulating cyclin D, cyclin E and N-myc, etc. Increased expression of SHH was found in clinical tissue of RB, and the expression level of SHH was closely related to tumor stage, local invasion and metastasis. However, the regulation mechanism of SHH in RB and the effects on biological function of RB cell are not clear, which needs further research.PI3K/Akt signal pathway was continuously activated in a wide variety of tumor. Researches showed that activation of PI3K/Akt signal pathway promoted tumor cell growth, invasion and migration, inhibited apoptosis through inducing the expression of cell cycle related genes and disproportionated of pro-apoptotic proteins and antiapoptotic proteins. Deactivation of hedgehog inhibited tumor cell growth and promoted apoptosis through inhibiting PI3K/Akt signal pathway in pancreatic cancer. Exogenous SHH enhanced activity of hedgehog pathway and the phosphorylation level of Akt in glioblastoma, and the expression of p-Akt was reduced by blocking the hedgehog pathway. LY294002, an inhibitor of PI3K/Akt signal pathway, reduced the expression of MMP2 and MMP9 induced by SHH, inhibited the ability of cell invasion and migration, which indicated that hedgehog pathway regulated tumor cell proliferation and growth through PI3K/Akt signal pathway, but the mechanism is still not clear in retinoblastoma.In this study, the expression of SHH was silenced in WERI-Rb-1 by RNA interference to explore the effects of SHH on cell proliferation and apoptosis of RB cell, and the regulation of PI3K/Akt signal pathway was discussed. The results will provide theoretical basis for the molecular targeted therapy of RB and give the target for developing new drugs. Part 1. Construction and screening of SHH-sh RNA recombinant plasmidObjective: Constructing RNA interference plasmids targeting SHH gene and testifying the inhibitory effect on m RNA and protein expression, to provide effective means for researching the function SHH gene on biological function of retinoblastoma cell.Method: Three RNA-interference sequences and one nonsense sequence targeting SHH gene was designed, synthesized, and connected to the the sh RNA expression plasmid p RNAH1.1. WERI-Rb-1 cells were transfected with recombinant plasmid and the stable transfection cell colony was screened in medium with presence of G418. Real-time PCR was employed to detect the inhibition effect of SHH-sh RNA, and the best sequence was selected, and was further validated by western blot on the protein expression level of SHH in WERI-Rb-1.Result: The RNA interference plasmids targeting SHH, SHH-sh RNA-1, SHH-sh RNA-2, SHH-sh RNA-3 and negative control were constructed successfully. The stable transfection cell was screened by G418. The results of Real-time PCR showed that the m RNA expression of SHH was reduced significantly in human retinoblastoma cell line WERI-Rb-1 transfected with SHH-sh RNA compared with the control group(P<0.01). The inhibition efficiency of SHH-sh RNA-1, SHH-sh RNA-2, SHH-sh RNA-3 reached 82 %, 35 %, 68 %, separately, and SHH-sh RNA-1, with highest inhibition efficiency, was used for subsequent experiment. Western blot results showed the protein expression level of SHH was also inhibited significantly compared with the negative control group, The inhibition efficiency reached 75 %(P<0.01).Conclusion: Constructed RNA-interference plasmids targeting SHH gene can effectively and specifically inhibit the expression of SHH m RNA and protein in WERI-Rb-1 cells, which will provide a useful tool to investigate the role of SHH gene on cell proliferation and apoptosis in human retinoblastoma. Part 2. Effect of silencing SHH gene on cell proliferation and apoptosis in human retinoblastomaObjective: To explore the effect of silencing SHH gene on cell proliferation and apoptosis in human retinoblastoma.Method: The experiment was divided into three groups: WERI-Rb-1, negative control(NC) and SHH-sh RNA. The effect of SHH gene silencing on cell proliferation was detected by MTT assay. The effect of silencing SHH gene on cell cycle was examined by flow cytometry. The cell cloning efficiency in vitro was measured by colony formation assay. The cell apoptosis rate was determined by hoechst staining and flow cytometry following Annexin V/PI staining. The protein expression of Cleaved caspase-3, Cleaved-PARP, Bcl-2 and Bax were determined by Western blot analysis.Result: The si RNA-mediated SHH gene silencing resulted in significant cell growth inhibition in SHH-sh RNA group(P<0.01). There was cell accumulation in the G1/G0 phase and reduction in S and G2/M in SHH-sh RNA group compared with the negative control group(P<0.01). The cell cloning efficiency in vitro was inhibited after SHH silencing. The cell apoptosis rate was increased, which was proved by hoechst staining and flow cytometry. Increased protein expression of Cleaved caspase-3, Cleaved-PARP and Bax, decreased protein expression of Bcl-2 was showed in SHH-sh RNA group.Conclusion: SHH gene silencing inhibits cell proliferation, increases cell apoptosis rete and induces cell cycle arrest in G1/G0 phase in human retinoblastoma WERI-Rb-1 cell by unbalancing the ratio of Bcl-2/Bax and promoting the expression of Cleaved caspase-3 and Cleaved-PARP. Part 3. The molecular mechanism of SHH gene silencing on cell proliferation and apoptosis in human retinoblastomaObjective: To clarify the effect of SHH gene silencing on PI3K/Akt signal pathway in human retinoblastoma WERI-Rb-1 cells, and the mechanism of PI3K/Akt on cell proliferation and apoptosis in human retinoblastoma.Method: The effect of SHH gene silencing on PI3K/Akt signal pathway was testified by Western blot. WERI-Rb-1 cells transfected with SHH-sh RNA or NC were treated with IGF-1, an activator of PI3K/Akt signal pathway. The experiment was divided into four groups: NC, SHH-sh RNA, NC+IGF-1 and SHH-sh RNA+IGF-1. The protein expression of p-PI3 K, PI3 K, p-Akt, Akt and apoptosis related proteins, Cleaved caspase-3, Cleaved-PARP, Bcl-2 and Bax were determined by Western blot analysis. The cell proliferation was detected by MTT assay. The cell apoptosis was examined by flow cytometry.Result: The protein expression of p-PI3 K and p-Akt were decreased in WERI-Rb-1 cells with SHH gene silencing, which was reversed after treatment with IGF-1. IGF-1 also promoted cell proliferation, inhibited cell apoptosis of SHH-sh RNA transfected WERI-Rb-1 cells. Increasing protein expression of Cleaved caspase-3, Cleaved-PARP, Bax and decreasing expression of Bcl-2 were found in SHH-sh RNA+IGF-1 group compared with the negative control group(P<0.01).Conclusion;Silencing SHH gene inhibits the activation of PI3K/Akt signal pathway in human retinoblastoma WERI-Rb-1 cells. SHH may promote WERI-Rb-1 cell proliferation, inhibit cell apoptosis through activating PI3K/Akt signal pathway. |
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