Study Of Survivin Expression In Colorectal Carcinoma And Gene Therapy On Colorectal Carcinoma By Antisense Oligodeoxynucleotides Targeting The Messenger RNA Encoding Survivin

Background : Colorectal cancer is one of the most common malignant tumor in the world. It carries a poor prognosis and the improvement in survival rates over the past 30 years has been disappointing. To search a new method for prognosis and treatment colorectal carcinoma is an important task for medical workers.Regulation of apoptosis ,or programmed cell death ,is crucial to the preservation of homeostasis and morphogenesis of human tissue. Disturbance of this process by aberrantly extending cell viability or favouring accumulation of transforming mutation is thought to contribute to carcinogenesis . Survivin is a new member of the inhibitors of apoptosis proteins family, it has been described to be selectively expressed in the most common human neoplasms and to be associated with clinical tumour progression. Moreover, the protein appears to be involved in tumour cell resistance to some anticancer agents and ionizing radiation. On the basis of these findings survivin has been proposed as a promising target for new anticancer interventions.Antisense technique, one of the gene therapy, is based on the priciple of base pair when antisense oligodeoxynucleotides (ASODNs) incubated with tumor cells,sequence specific complementary to the target sequence would block transcription and expression of the relevant gene. Because of its effectiveness,safety, simplicity and no vector, antisense technique is a promising approach in the treatment of colorectal carcinoma.In this study , we select survivin as target gene to analyse the relationship between the expression levels of survivin mRNA and the colorectal carcinoma patient's characteristics. Then we design antisense oligodeoxynucleotides complementary to the messenger RNA of survivin, which would inhibit the growth , proliferation of colorectal cells and induce their apoptosis. Our research display that survivin antisense oligodeoxynucleotides may be a valuable gene therapy for the colorectal carcinoma. This study is composed of three parts.Part OneExpress of Survivin in human colorectal adenocarcinoma and Its cilinal implicationObjective: To explore the correlation between expression of survivin gene in human colorectal adenocarcinoma and its clinical effects. Methods: By using semi-quantitative reverse transcription polymerse chain reaction (RT-PCR) technique, survivin gene expression in 29 previously untreated colorectal adenocarcinoma patients was studyed. We analyzed the relationship between the expression levels of survivin mRNA and the patient's characteristics, including tumor size,patients age.lymph node or liver metastisis, presence of invasion to > 1/2 myometrium,clinical stage, and histological grade. Results: Survivin gene expression levels in tissues colorectal adenocarcinoma patients at diagnosis were significantly higher than that in normal colorectal mosuca (0.614 ±0.39 vsO.16± 0.059 P< 0.01). Further analysis showed that the expression level of survivin mRNA was significantly associated with clinical stage, lymph and liver metestasis (PO.05, respectively). Conclusions: Survivin is expressed in the majority of human colorectal adenocarcinoma and correlates with its clinicopathological factors. These findings suggest that survivin overexpression may play a pivotal role in theprogression of colorectal tumors and may provide an important prognostic implicationfor colorectal carcinomas.We conclude that the survivin mRNA is a definingdiagnostic marker for colorectal carcinomas that may also yield prognosticinformation.Part TwoEffects of survivin antisense oligodeoxynucleotides on HT-29 colorectal carcinoma cellsObjective: To investigate the effect of survivin gene on the growth of human colorectal cancer HT-29 cells and to explore the feasibility of gene therapy in human colorectal carcinoma. Methods: The antisense oligodeoxynucleotides (ASODNs) were complementary to survivin sequences.The human colorectal cancer HT-29 cell line was treated with ASODNs .The effects of the survivin-ASODN on the growth of HT-29 and apoptosis were studied by cell colony assay,flow cytometry, reverse transcription polymerse chain Reaction . Low concentration 5-Fu was added to study the sensitivity of colorectal cancer cell induced by ASODNs to chemotherapy. Results :The number of HT-29 cells colony was inhibited by 61% after the cells were treated for 48 hours. The cell proliferation was inhibited effectively by antisense oligodeoxynucleotides to survivin gene, and the inhibitory effect depended on the concentration of ASODN and the time of incubation ,but they could not last very long. The expression of survivin mRNA was decreased sharply in HT-29 cells treated with ASODNs. The characteristics of apoptosis was revealed in HT-29 cells treated with ASODNs,i.e. sub-Gl peak and morphological changes . Compared with the control group ,the percentage of the survival cells significantly decreased in the presence of both 5-Fu and survivin ASODNs. Conclusions : The antisense oligodeoxynucleotides were capable of inhibiting the proliferation of human colorectal carcinoma HT-29 cells and inducing the apoptosis.Combination of survivin ASODN and 5-Fu might improve the treatment outcome of colorectal carcinoma.