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Construction of siRNA Lentivector Specific for c-Met and Screening of Stable Cell Line

Author: HuangWanGang
Tutor: LiZhongXin；ZhouDeMin
School: Hebei Medical University
Course: Oncology
Keywords: c-Met siRNA human colon cancer cells SW620 Lentivector stable transfection
CLC: R735.3
Type: Master's thesis
Year: 2011
Downloads: 141
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Abstract


Colorectal cancer (CRC) is the third most common malignancy in China. The main cause of CRC-related death is formation of hematogenous and lymphatic metastases ,30–40% of patients have metastatic disease at the initial diagnosis.The most common sites of metastasis from colon cancer are the regional lymph nodes, the liver, the lung, and the peritoneum, in which the liver being the most frequent site of metastases. Because the majority of deaths with colon cancer are due to metastatic disease, inhibitions of metastasis of colon cancer are expected to become effective treatment. Previous studies have shown that HGF / c-Met signaling pathway is closely related to colorectal cancer liver metastases.Hepatocyte growth factor (HGF), otherwise known as scatter factor, its receptor is c-Met, the combination of HGF and c-Met would activate downstream signaling pathways, strongly promote mitosis and organ formation, induce epithelial cell migration, invasive and angiogenesis. In malignant tissue including malignant colorectal cancer, c-Met is overexpressed or mutated and closely related to cancer occurrence, development, metastasis and poor prognosis. Research has shown that there are such relationships in patients combined with liver metastasis in colorectal cancer: liver metastases > normal liver tissue, liver metastases > primary CRC . Therefore, HGF/c-Met is likely to be new therapeutic targets for colorectal cancer , especially with liver metastasis.Small interfering RNA (siRNA) is one of the important post-genome era research tool.The mechanism of siRNA: siRNA duplexes into the body unwinding, the guide chain (refer to siRNA antisense strand, antisense siRNA) into the RNA-mediated silencing complex (RNA-induced silencing complex, RISC), and cut the target mRNA, the cut off point in the middle of small interfering RNA, and then degradate the target mRNA.Objective: To screen siRNA specific for c - Met in colon cancer cells and construct its Lentivector. To package lentivirus and establish stable cell line.Method:1 Design 3 genes for c-Met siRNA sequence and cloned into the lentiviral vector pSD31 with Lipofectamine?2000 and transfected the lentivirus into human colon cancer cell line SW620, also set up negative control and control.2 The mRNA and protein of c-Met were detected by RT-PCR,Western Blot,after transfection of SW620 cells pSD31-met, the experiment group significantly lower than control group.3 Select stable cell line with puromycin ,siRNA designed for c-Met was detected being inserted into SW620 gensome with PCR; protein detected by Western Blot.4 Inverted microscope observed 48 hours after transfection, transfection pSD31-met group, cell proliferation reduced, cell number decreased and cell aggregation inhibited.Conclusion:1 Different siRNAs are designed to target the relevant c-Met mRNA of human colon cancer cell.2 The Lentivector of siRNA specific for c-Met is of potent interference ability for target tissue.3 We packaged lentivirus and established stable cell line.

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