Inhibition Effects Of RNA Interference On Survivin Gene And The Apoptosis In EC-109 Cells

PARTâ… Expression and Clinic Significance of Survivin Gene in Esophageal CancerObjective: Esophageal squamous cell carcinoma (SCC) is one of the most common human malignancies. Surgical resections play a major role in managing patients within stageâ…¢. In addition, combined therapies including chemotherapy and radiation therapy, have been assessed for improving the outcome of advanced esophageal SCC patients. However, the 3-year survival rate was only 35-55% in stageâ…¡esophageal SCC patients, and 28% in stageâ…¢esophageal SCC patients. Therefore, clarifying the mechanism of tumor biology is considered important to improve the clinical outcome of advanced esophageal SCC patients.Survivin is a member of the Inhibitior Apoptosis Protein (IAP) family, which has been causally related to human tumorigenesis, tumor growth and metastasis. However, until now, the complete identity of view about Survivin expression in esophageal carcinoma and its clinical significance has not been reached. The present study was to explore the Survivin expression level in human esophageal carcinoma and its clinical significance.Methods: Surgically resected specimens were collected from 38 patients with esophageal SCC between September 2005 and December 2005 at the Tumor Hospital of Shoutou University Medical College. 31 males and 7 females were between 41 to 78 years old, the median age was 58. None of the patients had received preoperative radiotherapy and/or chemotherapy. Dewaxed paraffin sections were immunostained by the Envision method. A modified protocol based on the manufacturer's instructions was used. Bright field images of immunostained tissues under 400-fold magnification were captured using a Nikon microscope.Results: Survivin expression was predominantly observed in the cytoplasm and nuclear of the tumor cells. The positive rate was 63.16% (24/38). Survivin expression level in the patients with different ages and histopathological grades showed obvious difference. But there were no obvious difference with different sexes and gradings.Conclusions: These results suggested that Survivin expression might be one of the important prognostic indicators for esophageal carcinoma patients, and Survivin might be identified as a potential target in esophageal carcinoma.PARTâ…¡Inhibition Effects of RNA Interference Expressing Vector on Survivin GeneObjective: To inhibit the expression of endogenous survivin gene and induce the apoptosis in human esophageal cancer cell line EC-109, small interfering RNA (siRNA) targeting survivin gene was applied.Methods: The recombinant plasmid pSRIEN/S was constructed and transfected into EC-109 cells. The transfection efficiency was determined using fluorescence microscopy. The mRNA silence of survivin gene was detected by semi-quantitative RT-PCR and the expression level of survivin protein was determined by Western blot and Immunofluorescence staining. The apoptotic rate was observed by the flow cytometry and Genomic DNA Extract Kit.Results: Fluorescence microscopy showed that the transfection efficiency was about 46.7%. Compared with survivin mRNA expression in the control siRNA treated EC-109 cells, the transcription after treatment with survivin-specific siRNA was silenced by 74.04%, and the expression of survivin protein was reduced to 41.64%. The results of DNA ladder and the flow cytometry demonstrated that the apoptotic rate of EC-109 cells was significantly increased to 60.78%.Conclusions: The siRNA targeting survivin gene shows a dramatic inhibitory effect on RNA transcription and protein expression of survivin genes. The transfected groups showed a promoting effect on the apoptosis in EC-109 cells, which offers a reliable base for the further in vivo experiment.