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Study on Specific siRNA Inhibiting Expression of hTERT and Cell Growth in Lung Cancer Cell Line 95D

Author: CuiDong
Tutor: GeLinHu
School: Guangzhou Medical College
Course: Surgery
Keywords: RNA interference Small interfering RNA Telomerase reverse transcriptase Lung tumors Apoptosis
CLC: R734.2
Type: Master's thesis
Year: 2009
Downloads: 83
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Abstract


Background and Purpose: Lung cancer is one of the most common malignancy in the world today, the morbidity and mortality of all cancers, lung cancer is always at the forefront. Men and women, the incidence of malignant tumors of lung cancer were ranked first and second, and the men and women are lung cancer mortality. Lung pose a serious threat to human health has been. Therefore, people attach importance to the traditional treatments of surgery, radiotherapy, chemotherapy, are also actively explore new therapies. Tumor gene therapy in recent years as a new cancer treatment research hotspot continuous development. RNA interference (RNA interference, RNAi) is a biological phenomenon, from lower plants to higher mammals widespread by small interfering RNA (small interference RNA, siRNA) guide degradation of target mRNA post-transcriptional gene silencing. By RNA interference gene silencing effect of the key, can inhibit the pathogenic nucleic acid expression or abnormal protein synthesis. Compared with other means of gene therapy and RNA interference technology shows the efficiency and specificity, ease of strengths, is leading the new trend of gene therapy, has considerable application prospects. Gene therapy requires a valid target locus, so screening effective gene therapy targets important. Known that telomerase activity is almost can be detected in the vast majority of human malignancies are to negative, but in almost all into human cells, and therefore have a high correlation with malignancy between. High activity of telomerase in the incidence of lung cancer development also plays an important role in the catalytic subunit of human telomerase reverse transcriptase (human telomerase reverse transcriptase, hTERT) is the decisive factor in the telomerase activity and tumor development. which is expected to become an ideal target for gene therapy of lung cancer. In this study, the use of real-time quantitative PCR technique for quantitative analysis of hTERT mRNA expression in lung cancer cells, screening out the relative expression level of the highest lung cancer cell line 95D. Chemically synthesized siRNA transfected into 95D cells, to explore for hTERT-specific siRNA-mediated RNA interference silencing the expression of hTERT gene in lung cancer cells and inhibition of proliferation of lung cancer cells. Research content and methods: 1. Optimize PCR conditions, L78 Real Time RT-PCR technology to detect lung cancer cell lines, NCI-H520, A549, LTEP-α-2, NCI-H460 and 95D hTERT mRNA relative expression levels. HTERT gene sequence and siRNA design principles, design and chemical synthesis of the three pairs siRNA, siRNA transfection into high hTERT mRNA expression in lung giant cell carcinoma 95D Application Real Time RT-PCR technology to detect the transfection hTERT mRNA changes in expression levels. 3. Flow cytometry of hTERT siRNA transfection mediated RNA interference level of apoptosis of lung cancer induction and impact. 4. MTT method to detect the hTERT siRNA for the growth of lung cancer cells, the inhibition of the proliferative capacity. Results: 1 target gene and internal reference gene PCR product fragment size and expected values ??were similar, indicating each cell RNA extraction and RT-PCR process. Real Time RT-PCR results showed that hTERT mRNA are highly expressed in multiple lung cancer cell lines, in which the giant-cell lung cancer 95D highest relative expression level. RNA interference research subjects to 95D. 2. HTERT siRNA transfected 95D cells after 48 hours (concentration of transfection 100nmol / L), the comparison with the control group and negative control group of blank liposomes, siRNA-1, siRNA-2 were significantly inhibited the expression of hTERT mRNA (P <0.01), the inhibition rates were 77.33 ± 5.13% and 50.67 ± 8.02%, lower siRNA-3 inhibition rate was only 27.67 ± 10.26%. 3.Select inhibition of lung cancer cells of the most significant effect of the hTERT siRNA-1 95D from low to the transfection of the three high concentration, the concentration of the transfection were 50nmol / L, 80 nmol / L, 100nmol / l. 50nmol / L concentration turn hTERT mRNA expression level differences between the transfection group and negative control group without statistical significance (P gt; 0.05). 80nmol / L group 100nmol / L concentration transfected group, respectively, compared with the negative control group of hTERT mRNA expression level differences were statistically significant (P less than 0.01), while no statistically significant difference between the two groups ( P gt; 0.05). 48h after transfection, compared with the blank liposome control group 50nmol / L, 80mol / L, 100nmol / L concentration hTERT siRNA-1 transfection group apoptosis rates were significantly increased (P values ??less than 0.01 ). Compared with the negative control group, 50nmol / L concentration apoptosis rate of transfected group no significant increase (P gt; 0.05), while 80 nmol / L, 100nmol / L rate of apoptosis significantly increase (P values ??were less than 0.01 ). The blank liposome control group and negative control group showed significant differences (P lt; 0.05). The 5. MTT test results showed that lung cancer cells 12 hours after transfection proliferative effect began to emerge, but the effect is still weak, the 24 hours have been obvious, 48 ??hours, most significantly, the inhibitory effect of 72 hours weakening. The hTERT siRNA can effectively inhibit the proliferation of lung cancer cells, the inhibitory effect of a time-dependent manner. Conclusion: human lung cancer cell lines L78, NCI-H520, A549, LTEP-α-2, NCI-H460 and 95D hTERT mRNA were highly expressed, including lung giant cell carcinoma 95D highest relative expression level. Cell model selection for the next step of RNA interference experiments laid the foundation. Effective design and synthesis of specifically targeting of hTERT siRNA can effectively cut lung giant cell carcinoma 95D telomerase hTERT mRNA expression, thereby inhibiting telomerase activity, induced apoptosis in lung cancer cells, and inhibit the proliferation of lung cancer cells.

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