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The Influence of siRNA on the Expression of Smo and Gli1 in Human Esophageal Carcinoma EC9706 Cell Line

Author: SongLing
Tutor: ChenKuiSheng;ZhaoZhiHua
School: Zhengzhou University
Course: Pathology and Pathophysiology
Keywords: RNA interference Esophageal cancer Smo Gli1 Boyden ehamber
CLC: R735.1
Type: Master's thesis
Year: 2011
Downloads: 33
Quote: 0
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Abstract


Esophageal cancer is one of the most common malignant tumor with a high incidence, incidence of occult, easy to transfer and high mortality, Henan Province is the high incidence of provinces. Treatment methods include surgery, chemotherapy and radiotherapy in advanced esophageal cancer have certain limitations and the effects can not be satisfactory. Therefore, seek a new target for biological treatment of esophageal cancer and new ways has become in recent years one of the focus. In recent years, the study found that many in the developing embryo, play a regulatory role in the process of differentiation signaling pathways play an important role also in the process of tumorigenesis, including Sonic hedgehog signaling pathway, EGFR signaling pathway, TGF-beta signaling pathway, Wnt signaling pathway. Smoothened (Smo) protein Sonic hedgehog signaling pathway converter to convert extracellular Shh signal cells Glil signal, which initiates gene transcription in the nucleus, the Sonic hedgehog signaling pathway activation. Normal tissues and cells, SMO is not expressed or low expression to maintain normal metabolic needs of the cell. Smo overexpression, abnormal activation of the Hedgehog pathway, transformed and malignant cells produce large amounts of cancer-promoting factor. The study showed that SMO in some human tumors, such as malignant glioma, gastric cancer, hepatocellular carcinoma, pancreatic cancer, colon cancer, breast cancer, prostate cancer, malignant lymphoma, such as basal cell carcinoma tissues with high expression. Some scholars believe that SMO is expected to become a new target for early diagnosis of cancer gene therapy, new indicators to assess the prognosis. Gli gene family was initially amplified in human malignant glioma Glil been confirmed. Human kind the Gli homologue gene: Gli, Gli2, Gli3. The Gli1 gene encodes a protein of 1106 amino acids, located on chromosome 12q13, as vertebrate transcriptional effector shuttle between the cytoplasm and nucleus, the transcription signal passed from the cytoplasm into the nucleus. The Glil protein can not be the protease hydrolysis, can only exist in the cytoplasm to the total length of the state, through its own length from the nucleus downstream gene transcription or inhibition, so, Glil protein transcriptional activation. Another study showed that the use of RNA interference suppression of Smo expression in gastric cancer cell lines can be reduced at the same time amount of Glil expression. Inhibition of SMO in esophageal cancer using RNAi technology, study of Glil expression has not been reported in the literature. RNA interference (RNA interference, RNAi) is a ubiquitous in plants and animals at the mRNA level induced sequence specific gene silencing by double-stranded RNA (double Strand RNA, dsRNA) process. dsRNA into the cell, the role of dsRNA nuclease (Dicer), is cleaved into 21bp-23bp small interfering RNA (small interfering RNA, siRNA), siRNA enzyme complex with a polynucleotide binding to form an RNA-induced silencing complex body (RNA-induced silence complex, RISC), RISC with siRNA complementary mRNA binding, the role of RNA enzyme hydrolysis mRNA belongs to the post-transcriptional gene silencing (post-transcriptional gene silencing, PTGS) a. Application of RNAi technology in cell signaling pathway, anti-viral, anti-tumor and other areas of extensive experimental study in depth, marks that RNAi will become the indispensable tool of the study of gene function. In this study, RNAi technology specific inhibition of Smo expression in EC9706 cells before and after transfection using immunocytochemistry, in situ hybridization to detect EC9706 cells in SMO GT; Gli1 protein and mRNA expression, application Boyden chamber in vitro invasion assay transfection was observed the cell invasion force before and after changes to provide a theoretical basis for the clinical application of RNAi technology targeted treatment of esophageal cancer. Materials and Methods 1. Esophageal cancer EC9706 cells were kindly provided by the Cancer Research Institute of the Chinese Academy of Medical Sciences, Molecular Oncology, State Key Laboratory. 2 in EC9706 cells in culture: in EC9706 cells in RPMI-1640 medium (containing 10% fetal bovine serum, penicillin 100U/ml, streptomycin 100U/ml), 37 ° C, 5% CO2 incubator paste adherent culture. 3 siRNA in vitro synthesis, identification and selection: First design synthetic siRNA oligonucleotide templates, specific binding to the 5 'end of the promoter sequence for T7 polymerase in vitro and T7RNA transcription purpose siRNA. Were synthesized two-stage-specific siRNA and a period of no meaning for the control siRNA, the application of a 4% agarose gel electrophoresis, to the quantitative template DNA as a reference, the length and concentration of siRNA was measured; filter out some interference better specificity by the pre-experiment siRNA. 4 transfected: application LipofusinX liposomal siRNA transfected EC9706 cells. Immunocytochemistry SP detected in each experimental group before and after transfection, the expression control group of Smo and Glil protein. 6 application cell in situ hybridization, transfected cells were detected in each experimental group, the control group of Smo and Glil mRNA expression. Application Boyden chamber invasion in vitro experiments to observe changes in cell invasion force before and after transfection. 8 Statistical analysis: application SPSS 13.0 statistical software for statistical analysis, measurement data using mean ± standard deviation (X ± S); two groups were compared with more than two groups of the t-test (t-test); compared by analysis of variance (ANVOA); a = 0.05 significantly standards. Results successful in vitro synthesis of two siRNA length of 21bp. After 2.EC9706 morphological changes: SMO-specific siRNA transfection in EC9706 cells, showing that cells gradually round, refractive enhancement, and then began to shrink, floating. 3 immunocytochemistry results: 150ng/μl, 200ng/μl 250ng/μl three different concentrations of specific SMO siRNA transfected esophageal cancer EC9706 cells 24h, 48h, 72h after the SMO Glil protein expression were decreased, which to 250ng/μl the inhibitory effect of transfected 72h most obvious, compared to the experimental group and the control cells (transfection) and blank control group (empty liposomes), nonsense control group (nonsense siRNA transfection) differences were statistically significant (P lt; 0.05); each concentration group with the transfection increased the inhibitory effect of enhanced pair-wise comparison of the three time periods was no significant difference (P gt; 0.05); period of time with the transfection dose increase inhibitory effect gradually increased three concentrations compared the difference was statistically significant (P lt; 0.05); blank control group, the nonsense control group cells compared to the control group were not showing Smo, Glil protein expression inhibitory effect, compared to the three control two groups was no significant difference (P gt; 0.05) Situ hybridization: 150ng/μl, the 200ngng/μl, 250ng/μl three different concentrations of specific SMO siRNA transfected esophageal cancer EC9706 cells 24h, 48h, 72h after, Smo, Glil mRNA expression were decreased , which to 250ng/μl the transfection 72h inhibitory effect of the most obvious, the experimental group and the cell control group and blank control group, no justice compared to the control group, the differences were statistically significant (P lt; 0.05); each concentration group transfected with time to increase the inhibitory effect of enhancement, the three time periods compared the difference was not statistically significant (P gt; 0.05); each time period with the transfection dose gradually increased inhibitory effect three concentrations of two two the difference was statistically significant (P lt; 0.05); cells in the control group and blank control group and nonsense compared to the control group were not showing on SMO, Glil mRNA expression of the inhibitory effect, three control groups, two-phase The difference was not statistically significant (P gt; 0.05). Boyden chamber in vitro invasion assay results: SMO siRNA were transfected into EC9706 cells, significantly less than the number of cells through the Matrigel cells, blank and unrelated control group, the difference was statistically significant (P lt; 0.05). Conclusion Smo siRNA specific inhibition of Smo protein and mRNA expression in EC9706 cells. Smo protein and mRNA expression of Smo siRNA down in EC9706 cells, Glil protein and mRNA expression also come down, suggesting that Smo through the Sonic Hedgehog pathway affect Glil expression. Specific SMO siRNA Smo down in EC9706 cells, expression of Glil the same time, the slow cell growth, some cell death, cell invasion force was significantly reduced, indicating that Smo siRNA can inhibit the invasion and metastasis of esophageal cancer EC9706 cells.

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