| Esophageal carcinoma is a kind of the most common malignancies in our country, which is characterized by high incidence, nonspecific appearance, easy metastasis and high death rate. The incidence of esophageal carcinoma in Henan is higher than many other provinces. The treatments include surgery, chemotherapy, radiotherapy and so on, but the efficacy of existing treatment is limited and not satisfactory for advanced esophageal carcinoma, so searching for new targets or new ways for biological treatment of esophageal carcinoma has become a research hotspot in recent years. In recent years, the research shows that many signaling pathways which contuol the embryonic development and tissue differentiation also play an important role in the process of tumor formation, the signal transduction pathway includes Sonic hedgehog pathway, EGFR pathway, TGF-βpathway, Wnt pathway and so on. Smo protein is the information converter in the Sonic hedgehog pathway, which can change the SHh signal extracellular into the Glil signal intracellular, then start the gene transcription in the nucleus, so the Smo protein also plays a role to active the Sonic hedgehog pathway. In normal tissue and cells, there is no or low expression of Smo protein that maintain the requirement of normal cell metabolism. However, the overexpression of Smo protein can active the Sonic hedgehog pathway abnormally and also produce a large number of cancer-promoting factor which lead to the cell malignant transformation. The existing research shows that Smo present high expression in some human tumors such as malignant glioma, gastric cancer, hepatocellular carcinoma, pancreatic cancer, colon cancer, breast cancer, prostate cancer, malignant lymphoma, basal cell carcinoma and so on. Some scholars believe that the Smo is going to become the new target of gene therapy against cancer, and also the indicators for early diagnosis and assessment of prognosis.Gli gene family is initially confirmed in the expansion of Gli1 in human malignant glioma. Humans have three Gli homologous genes:Gli1, Gli2, Gli3. Gli1 gene positioning 12q13 in chromosomes that encoding a protein contains 1106 amino acids, shuttling between cell plasma and the nucleus, transferring the transcription signal from cell plasma into the nucleus. Gli1 protein cannot be hydrolyzed by protease but only exists in the cytoplasm by full-length state, and transcribe or inhibit the downstream genes through its own different lengths. So the Gli 1 protein only has the transcription-actived function. Another study shows that the use of RNA interference inhibiting the expression of Smo in gastric carcinoma cell lines can also reduce the expression of Gli1 simultaneously. The studies that using RNAi technology to inhibit the expression of Smo and Gli1 in esophageal carcinoma have not been reported in the literature.RNA interference (RNAi) is a widespread sequence-specific gene silencing process in the mRNA levels induced by the adoption of double-stranded RNA (dsRNA) in animals and plants. The dsRNA introduced or generated in cells is subjected to digestion with a dsRNA-specific endonuclease called Dicer and cleaved into 21bp -23bp small interfering RNAs(siRNAs), and the resultant duplexes just as siRNAs combine with a kind of poly-nuclease complex called RNA-induced silence complex(RISC). The RISC combines with mRNA which is complementary to siRNA and plays a role in digestion of mRNA as RNase. The effects of the implementation of RNAi belongs to the technology of post-transcriptional gene silencing (PTGS). At present the application of RNAi technology in cell signaling pathways, anti-virus, anti-tumor and other areas of experimental study indicates that RNAi will become an indispensable tool to study gene functions. In this study, the application of RNAi is used to inhibit the expression of Smo specifically in esophageal carcinoma EC9706 cells. The expression of Smo, Glil protein and mRNA were detected using immunocytochemistry and in situ hybridization before and after transfection; Boyden chamber experiment in vitro was used to detect the changes of invasion ability of EC9706 cells before and after the transfection, and provide a theoretical basis for the clinical application of RNAi technology targeting against malignancies such as esophageal carcinoma.Materials and methods1. Human esophageal carcinoma strain EC9706 is presented by the Chinese Academy of Medical Sciences Cancer Institute, State Key Laboratory of Molecular Oncology.2. Culture of the esophageal carcinoma EC9796 cells:The EC9706 cells were cultured adherently in RPMI-1640 culture medium (containing 10%fetal bovine serum, penicillin 100u/ml, streptomycin 100u/ml) under the condition of 37℃and 5%CO2.3. Synthesis and identification of siRNA in vitro:The oligonucleotide templates of siRNA were designed and synthesized firstly which contained T7 RNA polymerase promoter sequence at 5'terminal that could bind to the T7 RNA polymerase to transcript the target siRNA in vitro. Two pieces of specific siRNA and a piece of nonsense siRNA were synthesized respectively, the application of 4% agarose gel electrophoresis was used to detect the length and concentration of siRNA, screening out one with better interference effect by advance experiment.4. Transfection:LipofusinX siRNA Transfection Reagent was used to transfect siRNA into EC9706 cells.5. The expression of Smo and Glil protein in every experimental group and control group was detected by immunocytochemistry.6. The expression of Smo and Glil mRNA in every experimental group and control group was detected by in situ hybridization.7. Boyden chamber experiment in vitro was used to detect the changes of invasion ability of EC9706 cells before and after the transfection. 8. Statistical analysis:The application of SPSS 13.0 statistical software was used for statistical treatment, measurement data were expressed by mean±standard deviation(X±S); data between two groups were analyzed by t test(t-test); data more than two groups were analyzed by the ANVOA; the level of significant difference is a=0.05.Results1. Two pieces of siRNAs were synthesized successfully in vitro and both of their length were 21bp.2. Morphological changes of EC9706 cell:After the transfection of Smo-specific siRNA, it was observed that the EC9706 cells became round gradually, the refractive index increased and then began to shrink.3. Immunocytochemical results:The expression of Smo and Glil protein in esophageal carcinoma EC9706 cells was lower in experimental groups which were transfected with specific Smo siRNA(150ng/μl,200ng/μl and 250ng/μl) for 24h,48h and 72h compared with cell control group(no transfection), blank control group (empty liposomes) and non-sense control group(transfected with non-sense siRNA), especially the group at the concentration of 250ng/μl for 72h showed the most obvious inhibitory effect. There was a significant difference between experimental group and control group(P<0.05). The inhibitory effect on the expression of Smo and Glil protein was in time-dependent manner at every dosage, but there was no significant difference in three kinds of time(P>0.05). The inhibitory effect on the expression of Smo and Glil protein was in dosage-ependent manner at every acting time and there was a significant difference in three kinds of dosage(P<0.05). Compared with the cell control group, no inhibitory effect on the expression of Smo or Glil protein was shown in both blank control group and non-sense control group, there was no significant difference among the three control groups(P>0.05).4. In situ hybridization results:The expression of Smo and Glil mRNA in esophageal carcinoma EC9706 cells was lower in experimental groups which were transfected with specific Smo siRNA(150ng/μl,200ng/μl and 250ng/μl) for 24h,48h and 72h compared with cell control group, blank control group and non-sense control group, especially the group at the concentration of 250ng/μl for 72h showed the most obvious inhibitory effect. There was a significant difference between experimental groups and control groups(P<0.05). The inhibitory effect on the expression of Smo and Glil mRNA was in time-dependent manner at every dosage, but there was no significant difference in three kinds of time(P>0.05). The inhibitory effect on the expression of Smo and Gli 1 mRNA was in dosage-dependent manner at every acting time and there was a significant difference in three kinds of dosage(P<0.05). Compared with the cell control group, no inhibitory effect on the expression of Smo or Gli 1 mRNA was shown in both blank control group and non-sense control group, there was no significant difference among three control groups(P>0.05).5. Boyden chamber results:Compared with all the control groups, the number of cells traversed Matrigel is decreased obviously in experiment group, and there is a significant difference(P<0.05).Conclusions1. Smo siRNA can specifically inhibit the expression of Smo protein and mRNA in esophageal carcinoma EC9706 cells.2. When the expression of Smo protein and mRNA in esophageal carcinoma EC9706 cells was depressed by specific Smo siRNA, the expression of Gli 1 protein and mRNA was also reduced at the same time, which suggested that Smo may affect the expression of Glil through the Sonic Hedgehog pathway.3. At the time when specific Smo siRNA depressed the expression of Smo and Glil in esophageal carcinoma EC9796 cells, the cells grew slowly and died partly, the cellular invasive potential was suppressed significantly after transfection, which indicated that Smo siRNA inhibited the proliferation of esophageal carcinoma EC9706 cells. |
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