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The Effect of RNai-mediated Gene Silencing of Livin on the Growth and Apoptosis of SW480Cells

Author: HongZuo
Tutor: CaoWei
School: Suzhou University
Course: General Surgery
Keywords: Colorectal Cancer LIVIN RNAi SW480 Apoptosis
CLC: R735.35
Type: Master's thesis
Year: 2012
Downloads: 19
Quote: 0
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Abstract


Objiective LIVIN is a novel member of the inhibitor of apoptosis proteinfamily,which plays an important role in gene transcription. By reducing or eliminating theoverexpression of Livin gene in tumor cells, we can remove the inhibition of apoptosis,premote the apoptosis of tumor cells and enhance the sensibility to drug threapy. RNAinterference (RNAi) could specifically reduce gene expression efficiency, has been widelyapplied in cancer therapy. The aim of present study was to employ the sequence-sepcificsiRNA silencing the Livin gene and investigate its effects on human colorectal cancerline SW480.Methods①Structure of Livin mRNA was analysed by software to selecttargeted interference sites. The Livin-siRNA was transfected into colon cancer line SW480by positive ion liposome Lipofectamine2000.Transfect efficiency was assessed by flowcytometry.②After human colon cancer cell line SW480was transfected by Livin smallinterfering RNA (siRNA),the mRNA and protein of Livin and Caspase-3were determinedby RT-PCR and Western blot assay respectively and cell proliferation capacity wassetablished by MTT.③The apoptosis and cell cycle were measured by flow cytometry(FCM).Results①Negative control FAM-siRNA was successfully transfected intohuman colon cancer cell line SW480.The transfection efficiency was about93.9%detectedby FCM with cell density at1.2*105/well and siRNA concentration with80pmol in6wellplay.②After Livin-siRNA were successfully transfected into SW480cell48h, detectedby RT-PCR,Livin mRNA levels in cells was significantly inhibited. Contrasted withnegative control and blank control(p<0.05), the expression of Livin protein was obviously decreased72h after Livin-siRNA transfection(p<0.05). On contrast, the level of Caspase-3was enhanced(p<0.05).③Livin-siRNA could effectively inhibit the growth of SW480cells and accelerate apoptosis in vitro(p<0.05), but it seems that there is no relationshipswith its cell cycle(p>0.05).Conclution Livin-siRNA is successfully constructed and efficiently transfectedinto SW480cells. Livin-siRNA can effectively silence the overexpression of Livin in coloncancer cell lines SW480. Livin-siRNA can efficiently inhibit the expression of LivinmRNA levels and on the contrary the expression of Caspase-3mRNA and proteinobviously increased. Using Livin-siRNA transfection could inhibit the growth of SW480cells and accelerate its apoptosis, but it seems that there is no relationships with its cellcycle.All these suggested that Livin gene could be a potential target for gene therapy ofcolorectal carcinoma, RNAi is exected to be an effective measure for tumor gene therapy.

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CLC: > Medicine, health > Oncology > Gastrointestinal Cancer > Intestinal neoplasms > Colon tumor
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