The Effects Of HGF By RNAi On The Proliferation And Migration Of SW620

Colorectal carcinoma is the second common malignant tumor in digestive tract. Nowadays, the clinicle treatments such as operation, radiotherapy and therapy have some success on colorectal carcinoma. But, it has the big wound,bad adverse reaction and worse prognosis. Because of the younger of the colorectal carcinoma patients and the patient's demand of high quality of life in post-treatment ,a new mothed which has the micro-wound,fewer adverse reaction even the radical cure of tumer is needed. It has been proved that the development of colorectal carcinoma included lots of steps, stages and facrors. Especially, the variation and the signal transduction of oncogene are closely associated with tumor progression and metastasis.One of the most important signal transductions is protein tyrosine kinase(PTK) ,which is the first link of the signaling of hepatocyte growth factor (HGF) and its acceptor c-Met .HGF, also known as scatter factor (SF), is one of polypeptide growth factor (PGF), with pleiotropic effects clinicle treatments such as operation,radiotherapy and chemo have some success on colorectal carcinoma.But,it has the big wound,bad adverse reaction and worse prognosis.Because of the including mitogen, organogenesis, invasive growth and migration of endothelial cells, angiogenesis and so on[1].Recently, it has been shown that gene mutation and over-expression of HGF/c- Met are occurred in some procedure of pathology especially in some malignancies. Moreover, the mutation and over-expression of HGF/c- Met have a close relationship with tumor genesis, progression, metastasis andprognosis. Udai found that the over-expression of HGF and c-Met was closely associated with tumer metastatic phenotype, metastases and survival. HGF will be worthy of diagnosing and deciding the stage and prognosis about colorectal carcinoma as a sign of serology. HGF was in the hope of finding a new therapeutic target to treat colorectal carcinoma.RNA interference (RNAi) is a highly evolutionally conserved process of post-transcriptional gene silencing (PTGS) by which double stranded RNA (dsRNA), when introduced into a cell, causes sequence-specific degradation of homogolous mRNA sequences . Nowadays, RNAi causes specific gene silencing to take the place of traditional antisense nucleotide which is used in a hot wide variety of fields, including identification of gene function,regulation of post-transcriptional gene expression and so on. Additionally, RNAi provides the new way on gene therapy in kinds of disease especially makes the hot spot of gene therapy in tumor. As the intermediate product of RNAi, the small interfering RNA (siRNA) launched the RNAi in the cells and the result was known as knockout.We had proved that HGF can promote the proliferation and migration of SW620 in a concentration-dependent manner previously . In this experiment, we would make down the gene expression of HGF by RNAi to investigate the effect of HGF on the proliferation and migration of SW620 in order to find a new therapeutic target of colorectal cancer.Objective:To investigate the effects of the inhibition of HGF expression by RNAi on the proliferation and migration of SW620 .Methods:1 In this study, the specific HGFα/βsiRNA (h) designed and synthetized by Santa was transfected by lipidosome LIPOFECTAMINETM 2000 into SW620 . We also made negative control and blank group .The efficiency of cell transfection was examined by the inverted fluorescence microscope.2 The expressions of HGF were analyzed by Western Blot and reverse-transcription PCR .3 The morphology of SW620 was observed under the inverted microscope.4 MTT was used to analyse the effect on the proliferation after RNAi and to draw the growth curve of each group.5 The effect on the cell cycle after RNAi was determined by FCM.6 The effect on the migration of cells after RNAi was studied by restoration of basal membrane.7 The ultrastructure of SW620 after RNAi was observed under SEM and TEM.Results:1 The efficiency of cell transfection examined by the inverted fluorescence microscope was about 70-80%.2 The expression of HGF in SW620 after the transfection of specific HGFα/βsiRNA was down- regulated significantly.3 It can been seen under the inverted microscope that the proliferation and the quantity of cells were decreased apparently after 48h of the transfection of HGFα/βsiRNA.And cell aggregation was suppressed significantly.4 The cell proliferation of HGFα/βsiRNA group was suppressed deeply as shown by MTT, Aditionaly, the most obvious effect happened at 48h.5 The results of FCM showed that the cells of HGFα/βsiRNA group in G1 phase increased, but the cells in S phase decreased compared with the control groups.6 As shown by restoration of basal membrane, the capability of migration of the cells of HGFα/βsiRNA group was degraded 58.2% than the negative control group and 60.3% than the untreated group.7 The results of SEM showed that there were less parapodiums on the cell surface ,and the intensive and visible scabrosities can be seen after treated with HGFα/βsiRNA.However,the result of cells .in both control groups was converse.8 As shown by TEM, the microtubules and microfilaments of SW620 decreased or disappeared emarkably after being treated with HGFα/βsiRNA.Conclusion:The proliferation and migration of SW620 was suppressed obviously by RNAi of HGF .