| The tumor ,the malignant tumor is endangering human health. At present , to study the effective therapy of tumor is becomeing a hot and difficult topic. With the increasing of people's living standards, Incidence of colorectal cancer is rising. The main clinical therapy is the integrated therapy of chemo and radiotherapy to assist the surgery, but the effect is not satisfactory. Therefore, The more effective therapy need to be explored, and one of the most perspective and challenging research is gene therapy.It has been shown that the occurrence , development and prognosis of colon carcinoma are closely concerned with the abnormal expression of telomerase. Telomerase is composed of six components, among which the major components are: human telomerase RNA(hTR), human telomerase reverse transcriptase (hTERT), telomerase related protein(TP1). hTERT is not expressed in normal tissues and cells, whlie which is expressed higher in more than 90% tumor tissues. hTERT is closely related to the telomerase activity, which is the rate-limiting factor of telomerase. Therefore , in recent years , for the target of gene therapy of malignant tumor, domestic and international researchers have interfered the transcription or expression of hTERT, which have tried to apply hTERT gene and its promoter to become the key of gene therapy.RNA-induced interference (RNAi) was induced by small interfering ribonucleic acid (siRNA),and then silenced the target gene expression at the post-transcription mRNA level.At present, RNAi has applied in the research of the correlation between up and down stream of the gene function and signal transduction system, which may provide the new strategy for the gene therapy. The tumor is the disease of polygenes and multiple factors. The inhibition of a single oncogene has no effect often. However, RNAi can inhibit many different genes at the same time,and inhibition effects have no interference. In addition, the identification of RNAi can be accurate to a ribonucleotide.at present, RNAi showed great potential future in the gene functional research and gene therapy.Obiective:To design, constuct and screen the siRNA of targeting hTERT gene.To observe the effects of RNAi on telomerase in HT-29 cells and the alteration of malignant proliferation and phenotye, and to explore the feasibility of RNAi strategy on colon carcinoma therapy. At the same time, the specificity of siRNA knocking out or inhibiting target gene can also be studied.Methods: Through gene blast, three siRNA sequences of targeting hTERT genes were chosen:â… (563-581): TTGCAA AGCATTGGAATCA,â…¡(740-757):AGAACGTTCCGCAGAGAA,â…¢(1657-1676):GTACAGGTTTCACGCATGT.The interfe- ring vectorâ… ,â…¡,â…¢and the control vector were constructed respectively . Green fluorescent cells were observed through fluorescent microscope. The expression of hTERT mRNA was determined by RT-PCR and the telomerase activity was determined by TRAP-ELISA after the vectors were transfected into the HT-29 cells for 48 hours, while the growth rate among different groups were detected by MTT assay.apoptotic cells were detected FCM assay.Results:1 Identification of the recombinant plasmid vector by PCR using bacteria and by sequencing: the Control vector and Interferingâ… ,Interferingâ…¡,Interferingâ…¢vectors are match to the design.2 A great number of green fluorescent cells could be seen in each transfected group through fluorescent microscope after transfected for 48 hours.3 The absorbency of telomerase activation of the HT-29 after transfected by different vectors for 48 hours,which had shown: there was no significant difference between the Control vector group and the Normal group(P>0.05), interfering vector groupsâ… ,â…¡,â…¢had difference compared with the Control vector group and the Normal group(P<0.05), interfering vector groupâ… had difference compared with interfering vector groupsâ…¡,â…¢(P<0.05), the inhibition rate of interfering vectorâ… ,â…¡,â…¢on the telomerase activition were 69%,39%,36%,the maximum inhibition rate was interfering vectorâ… .4 The RT-PCR results showed that there was no obvious alteration of hTERT expression between the Control vector group and the Normal group(P>0.05),while the expression of hTERT was significantly decreased in cells treated with interfering vectorâ… ,and hTERT mRNA expression was declined to 52.5%of the Control vector group.5 MTT results: there was no obvious alteration of OD value in the Control vector group and the Normal group(P>0.05), Interfering vector groupâ… had difference compared with the Control vector group and the Normal group(P<0.05), the inhibition rate of cell growth was 24.36%.6 The alteration of cell cycle was determined by flow cytometry and cell cycle which has shown that there was no obvious alteration of the cell number in G2/M,G0/G1 phrase in the Control vector group and the Normal group(P>0.05), and both were main in phrase S.The cell number in phrase S was obviously declined,while the number in G0/G1 phrase was significantly increased in Interfering vector groupâ… (P<0.05), and the cell apoptotic rate had difference compared with the Control vector group and the Normal group(P<0.05), which obviously increased.Conclusions:â‘ The inhibition effect is different with different sequence of the hTERT ;â‘¡The method of RNAi-DNA vector to inhibit the hTERT genes is feasible;â‘¢The treatment for malignant tumors by knocking out or down regulating hTERT gene with RNAi technology is feasible;â‘£The mechanism of RNAi inhibition the colon carcinoma cells HT-29 proliferation invovles down- regulating the telomerase activition at mRNA post-transcription level of hTERT, inhibiting the cells proliferation, and accele- rating colon carcinoma cells apoptosis.
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