| Background and Objective:Lung cancer is one of the most commonly malignant tumors worldwide. Its incidence and mortality always remain the forefront of all cancer cases. Of the malignant tumor's incidence in our country, lung cancer is the first place for men and the second place for women. Meanwhile, the mortality of lung cancer stays the first place for both men and women respectively. Lung cancer is becoming a severe threat to people's health. Treatment of lung cancer is drawing more and more attention.At present, as the focus of tumor research, gene therapy is developing continuously. RNA interference(RNAi) is a highly defensive guard mechanism generally existing in lower plants and high mammal. It is a post-transcription gene silencing mechanism which is triggered by homologous double-strand RNA(dsRNA), and then causes the degradation of targeting mRNAs. By strengthening RNAi mechanism against aimed gene, pathogenesis nucleic acid expression and abnormal protein synthesizing could be controlled in disease. In recent researches, with its highperformance and specificity, RNAi showed the superiority comparing with other gene therapy technologies. It will induce a new gene therapy trend which possesses potential clinical research and application perspective.Further screening of effective action sites of tumor, gene therapy is also continuously developing. Telomerase activity has been detected in majority of human malignant tumors, and showed negative results in adult somatic cell additionally. Therefore malignant tumors has very high telomerase specific. Meanwhile, high telomerase activity plays an important role in the occurrence and development of lung cancer. As the catalytic subunit of telomerase, human telomerase reverse transcriptase (hTERT) is the key factor of telomerase activity and tumor progression .So that hTERT is possible to become the ideal gene therapeutic target.In this study, the expression of hTERT mRNA in various lung cancer cell lines was determined by Real Time RT-PCR. It demonstrated that the human pulmonary giant cell carcinoma cell line 95D was higher than the other cell lines. According to the hTERT gene sequence and principle of siRNA designing, three pairs of siRNA strands were synthesized and transfected into 95D cells by cationic liposome. RNAi technology was applied aiming to investigate the inhibitory effect of specific siRNA on hTERT expression and the supression of cells proliferation.Methods:1. After optimizing PCR conditions, the relative expression of hTERT mRNA was determined by Real Time RT-PCR in lung cancer cell lines of L78, NCI-H520,A549, LTEP-α-2, NCI-H460 and 95D.2. According to the hTERT gene sequence and principle of siRNA designing, three pairs of siRNA strands were designed and synthesized chemically. After transfecting hTERT siRNA into the human pulmonary giant cell carcinoma cell 95D, the differences of expression of hTERT mRNA between various treated groups were compared by Real Time RT-PCR.3. Cells apoptosis induced by siRNA-mediated RNA interference was observed with Flow Cytometry.4. The inhibitory effect of cell growth and proliferation were analyzed by MTT method.Results:1. The data of experiment and analysis of images showed that differential expression of hTERT mRNA in various lung cancer cell lines. The PCR product of hTERT andβ-actin all showed obvious amplified strips in corresponding position as expected. It indicated that the process of RNA extraction and RT-PCR were favorable simultaneously. The result of Real Time RT-PCR demonstrated that the level of hTERT mRNA was highest in the human pulmonary giant cell carcinoma cell line 95D among various lung cancer cell lines.2. 48 hours after transfection ( transfected concentration was 100nmol/L ), both hTERT siRNA-1 and hTERT siRNA-2 could significantly inhibit the expression of hTERT mRNA in 95D cell .Their interfering efficiency were 77.33±5.13% and 50.67±8.02% respectively, meanwhile, the interfering efficiency of hTERT siRNA-3 was low, merely 27.67±10.26%. siRNA-1 transfection group had statistically significant difference with negative control group(P<0.01) and liposome control group (P<0.01). The differences between siRNA-2 group and the two control groups were also statistically significant(P<0.01). Therefore, hTERT siRNA-1 was chosen for RNAi experiment consequently.3. 50nmol/L ,80nmol/L and 100nmol/L hTERT siRNA-1 were transfected into 95D cells respectively. 48 hours after transfection , 50 nmol/L transfection group had no statistically significant difference with negative control group(P>0.05). Both of 80nmol/L and 100nmol/L transfection groups had statistically significant difference with negative control group(P<0.01),whereas the difference between 80 nmol/L transfection group and 100 nmol/L transfection group was not statistically significant(P>0.05).4. The apoptosis percentages of 50nmol/L ,80nmol/L and 100nmol/L hTERT siRNA transfection groups were 12.40±1.51%,18.40±1.35% and 22.87±3.37% respectively. They all had statistically significant difference with liposome control group(P<0.01). The difference between 50nmol/L transfection group and negative control group was not statistically significant(P>0.05). Meanwhile, 80nmol/L and 100 nmol/L hTERT siRNA transfection groups all had statistically significant difference with negative control group(P<0.01). The difference between liposome control group and negative control siRNA transfection group was also statistically significant (P<0.05). 5. The MTT result showed that hTERT siRNA was able to cause the repression of lung cancer cell proliferation. The inhibitory effect appeared at 12 hours after transfection , and then it became significant ,reached peak and turned weak at 24 hours, 48hours and 72 hours respectively. The effect was time dependent.Conclusions:1. The high expressions of hTERT is detected in lung cancer cell lines of L78,NCI-H520,A549,LTEP-α-2,NCI-H460 and 95D. The level of hTERT mRNA is highest in the pulmonary giant cell carcinoma cell line 95D among various lung cancer cell lines. It lay the foundation for further study on lung cancer gene therapy for inhibiting the expression of lung cancer cell telomerase by using siRNA mediated RNAi technology.2. Effective siRNA were designed and synthesized according to the gene sequence and principle of siRNA designing, which can down-regulate the expression of hTERT so as to inhibit telomerase activity, induce cell apoptosis and suppress cell proliferation. |
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