| Objective (1) Culture several different kinds of lung cancer cell lines. Detection of hTERT mRNA expression in those cell lines using SYBR fluorescence quantitative PCR,and screen out the maximum Htert mRNA expression from those lung cancer cell lines.(2) According to the hTERT gene sequence and principle of siRNA designing, three pairs of siRNA strands were synthesized and transfected into95D 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.(3) In this study, it was constructed that plasmid vector-mediated siRNA interference targeted hTERT gene, and stably transfected into lung cancer cell line H1299. Explore the giant cell lung cancer95D extract of siRNA sequence in lung adenocarcinoma whether a moderating effect H1299cells siRNA interference, whether in different pathological type of cancer have differences.Methods (1) Culture several different kinds of lung cancer cell lines and primary cell line which obtained from lung cancer patients' tissue, total RNA was isolated from those cell lines using TRIZOL method. Detection of hTERT mRNA expression in difference lung cancer cell lines using SYBR fluorescence quantitative PCR, the PCR amplified products were detected by Agarose Gel Electrophoresis.(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 cell95D, the differences of expression of hTERT mRNA between various treated groups were compared by Real Time RT-PCR and western blot. Cells apoptosis induced by siRNA-mediated RNA interference was observed with Flow Cytometry. The inhibitory effect of cell growth and proliferation were analyzed by MTT method.(3) Construction of interference vectors, according to the experimental group study, Reorganization interference vectors and empty vector transfected by cationic liposomes to lung cancer cell H1299. After selection and monoclonal culture, stable transfected cell of G418was obtained. Protein expression level of hTERT was detected by real Time RT-PCR technology and western blot. Cell growth and proliferation capacity were measured by MTT and colony formation count. Cell cycle of lung cancer cells was measured by Flow cytometry. Further verification by the efficiency of siRNA.Results (1) Differential Expression of hTERT mRNA in different lung cancer cell lines, the expression of which human lung giant cell carcinoma cell line(95D) was the highest. For lung cancer cells of primary culture, expression of hTERT mRNA gradually decreased as the passage number increased.(2)48hours after transfection (transfected concentration was100nmol/L), both hTERT siRNA-1and hTERT siRNA-2could significantly inhibit the expression of hTERT mRNA in95D cell(P<0.01).Their interfering efficiency were77.33±5.13%and50.67±8.02%respectively, meanwhile, the interfering efficiency of hTERT siRNA-3was low, merely27.67±10.26%.(3).50nmol/L,80nmol/L and100nmol/L hTERT siRNA-1were transfected into95D cells respectively.48hours after transfection,50nmol/L transfection group had no statistically significant difference with negative control group(P>0.05). Both of80nmol/L and100nmol/L transfection groups had statistically significant difference with negative control group(P<0.01),whereas the difference between80nmol/L transfection group and100nmol/L transfection group was not statistically significant(P>0.05).(4).The apoptosis percentages of50nmol/L80nmol/L and100nmol/L hTERT siRNA transfection groups were12.40±1.511%,18.40±1.35%and22.87±3.37%respectively. They all had statistically significant difference with liposome control group(P<0.01). The difference between50nmol/L transfection group and negative control group was not statistically significant(P>0.05). Meanwhile,80nmol/L and100nmol/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 at12hours after transfection, and then it became significant,reached peak and turned weak at24hours, The most significant at48hours and72hours inhibition effected weaker. The effect was time dependent.(6). PGenesil.1-hTERT transfected cells H1299were obtained after stable transfection of monoclonal cell line H1299-pGenesil.1-hTERT and H1299-pGenesil.1. Compared with stable blank plasmid transfected cells group, expression of hTERT mRNA cells decreased significantly in stable transfection of siRNA plasmid, inhibition rate was93.97±0.83%, P <0.01.(7). The expression of hTERT protein was suppressed Lung cancer H1299-pGenesil.1-h.TERT cells, compared with H1299-pGenesil.1cells.(8). Compared with H1299-pGenesil.1, cell proliferation ability H1299-pGenesil.1-hTERT cells was decreased.(9).Compared with the control group, H1299-pGenesil.1-hTERT, G1phase cells increased and S/G2phase cells were decreased in H1299-pGenesil.l group, Respectively18.3%(P<0.05),10.4%(P<0.05),7.9%(P<0.05).)Conclusion (1)In different lung cancer cell lines, hTERT mRNA has expressed. In lung cancer cells of primary culture, expression of hTERT mRNA gradually decreased as the passage number increased.(2) The expressions of hTERT is detected in lung cancer cell lines of L78,NCI-H520,A549,LTEP-α-2,NCI-H460and95D. The level of hTERT mRNA is highest in the pulmonary giant cell carcinoma cell line 95D among various lung cancer cell lines.(3). 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.(4). The vector of pGenesil.1-hTERT has been constructed successfully. The expression of hTERT mRNA and protein was significantly suppressed by transfection siRNA interference plasmid. It could regulate the activity of telomerase, proliferation and cell cylce of lung cancer cells. |
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