Survivin Expression in Human Colorectal Carcinoma and Specific Inhibition of Survivin Gene Expression by RNA Interference
|School||Tianjin Medical University|
|Keywords||colorectal carcinoma Survivin MVD Angiogensis survivin SW620 real time PCR western-blot FCM apoptosis xeno graft|
The newly discovered survivin, encodes 142 amino acids and belongs to inhibitor of apoptosis proteins(IAPs) . Its chromosomal location is 17q25.survivin is bifuctional member of the inhibitor of apoptosis gene family that counteract apoptotic cell death and regulate mitosis . The keynote of this research is to detect the expression of survivin and its correlation with angiogenesis in colorectal carcinoma . the specific RNA interference technology was used to construct plasmid expressing siRNA for survivin gene. SW620 cells were tansfected with the siRNA expressing plasmid .Survivin mRNA and protein levels were detected with real-time PCR and western blot. In vitro cell apoptosis was using flow cytometry with Annexin V staining. The in vivo inhibitory magnitude tumor, gravimetric variation and toxic effect of interference plasmid were determined using human colorectal carcinoma nude mice model.Part one : The Expression of Survivin and its Correlation withAngiogenesis in colorectal carcinoma Objective: To evaluate the expression of survivin and its correlation withMVD(microvessel density) in colorectal carcinoma. Methods : immunohistochemistry (S-P method) were used to detect survivin and CD34 incolorectal carcinoma (n=30). MVD was counted under microscope. Results : theexpression of survivin protein normal tissue distal to the tumor is low . Positive rate of Survivin protein was 67% ,significant relationship was found between survivin expression and histological type,lymph node metastasis(p<0.05), but not with the invasive depth(P>0.05);CD-34 protein was highly expressed in carcinoma , has significant relationship with node metastasis, invasive depth (P<0.01),but not with the histological type (P>0.05);There was appositive correlation betweenSurvivin and MVD. Conclusions: Survivin increases in colorectalcarcinoma ,plays an important role in occurrence and progression ,these results suggest that surviving play an important role in carcinogenesis and related to histological type, lymph node metastasis and angiogenesis.SECOND PART: In vitro and in vivo study of siRNA interference on survivin in colorectal carcinomaExperiment one :the expression of siRNA interference on survivin in colorectal carcinoma cell line SW620OBJECTIVE : To investigate the effect and mechanism of RNA interference on surviving expression of survivin in transfected colorectal SW620 cells. METHOD: the RNA interference plasmid for survivin gene was constructed, SW620 cells tansfected with the plasmid, survivin mRNA and protein levels were determined with real-time PCR, and western blotting . Immmunofluorescence microscopy used to observe the cellular morphology following hoechest staining.RESULTS: DNA sequencing confirmed the plasmid for surviving gene was successfully constructed .Apoptotic body were observed in the nuclei of cells that transfected with survivin shRNA plasmid under immuno-microscope . Real time PCR showed that mRNA suppress rate by survivin 1 on colorectal SW620 cells line was 29.3%,survivin2 was 37.6% and survivinl+2 was 60.8%,lower than the normal control(p<0.05);western blot assay indicated that the expression of survivin protein in SW620 cells interfered by survivinI,survivin2 and survivinl+2 was significantly lower than those of normal control;FCM analysis demonstrated that the apoptosis rate of transfected cells induced by survivin 1 was 7.51% ,survivin2 was 10.03%,survivinl+2 was 15.49%, higher than the normal control(p<0.05);CONCLUSIONS : RNA interference results in decreased survivin mRNA and protein expression in colorectal carcinoma SW620 cells and induce tumor cell apoptosis .Experiment Two: Survivin gene interference therapy of colorectal carcinoma in nude mice SW620 xeno graft modelOBJECTIVE : to Construct the xeno graft model of colorectal carcinoma SW620 in nude mice, observe the tumor rate and investigate the therapeutic and toxtic effect of the interference plasmid for survivin gene on grafted colorectal carcinoma in this nude mice model. METHODS: The SW620 cell was injected into the nude mice subcutaneoulyj two weeks later, the control ,HK,survivinl+2 plasmid were injected into the graft tumor respectively , by measuring the tumor size and calculating the tumor weight ,the therapeutic effect of interference plasmid were evaluated , toxic effect of the plasmid were observed through calculating theweight of nude mice . RESULTS: There is no difference for tumor volume before the fourth day(p>0.05) ,the treatment administration was observed on day 8,12,16 after plasmid administration, significant difference between survivin and other groups(p<0.01). there was no difference between control and HK group, comparing the tumor volume of day 19, the suppress rate of survivin was 73.4%, HK was 4.45%, there are significant difference between the two groups . the weight of nude mice increase gradually but there is no difference between different groups.( p>0.05). CONCLUSIONS: the interference plasmid for survivin gene suppress the growth of colorectal carcinoma xeno graft in nude mice,but without obvious toxic effect.