| ObjectiveTelomerase plays a important role in the carcinogenesis and the development oflung cancer. In recent years, reseachers pay close attention to the study of hTERT as agene therapy target which is the determinant factor of the regulation of telomeraseactivity. We aimed to study the effect of hTERT siRNA silencing the hTERTexpressions,inhibiting the telomerase activity and inducing the apoptosis of lungcancer cells, further to study the effect of hTERT siRNA on the growth of xenograftsof lung cancer.Methods1. After transferring in vitro transcription hTERT siRNA into lung cancer cellsA549 by LipofectaminTM 2000, We compared the differences of the expression ofhTERT mRNA, the expression of hTERT protein, the activity of telomerase and theapoptosis between two groups of lung cancer cells treated with LipofectaminTM 2000and with hTERT siRNA combined with LipofectaminTM 2000 by RT-PCR,immunocytochemistry, TRAP-ELISA and flow cytometry respectively.2. After the lung cancer xenografts of athymic mouse were established, theseathymic mouse were divided into control group and hTERT siRNA group, and wereinjected with LipofectaminTM 2000 or LipofectaminTM 2000 combined with hTERTsiRNA into tumors. Then we observed the differences of tumor volume change,tumorweight and tissue structure of tumor and compared the differences of the expression ofhTERT mRNA,the expression of hTERT protein,the activity of telomerase betweentwo groups of lung cancer xenografts by RT-PCR,immunohistochemistry andTRAP-ELISA respectively.Results1. hTERT siRNA inhibited the expression of hTERTand the telomerase activity,induced the apoptosis of lung cancer cells A549.①The expression of hTERT mRNA: The relative amount of hTERT mRNAexpression of lung cancer cells treated with hTERT siRNA were 44.28±3.02％,35.55±2.08％and 51.50±1.84％at 24 hours, 48 hours and 72 hours respectively.The control groups were 96.20±4.11％,99.24±2.55％and 100.16±1.96％respectively.The differences between the treated group and the control group weresignificant statistically at 24 hours (t=17.636, P＜0.05),48 hours (t=33.619, P＜0.05) and 72 hours (t=31.216, P＜0.05). The percentages of inhibited hTERTmRNA were 53.98％, 64.48％and 48.59％respectively.②The expression of hTERT protein: The percentages of hTERT proteinexpression of lung cancer cells treated with hTERT siRNA were 44.0±5.2％,21.0±5.9％and 47.9±4.7％at 24 hours, 48 hours and 72 hours respectively. Thecontrol groups were 76.5±2.2％, 77.4±2.1％and 72.9±4.6％respectively. Thedifferences between the treated group and the control group were significantstatistically at 24 hours (t=9.923,P＜0.05) , 48 hours (t=15.708,P＜0.05) and 72hours (t=6.548,P＜0.05). The percentages of inhibited hTERT protein were 42.5％,72.9％and 34.3％respectively. The average photodensities of hTERT proteinexpression of lung cancer cells treated with hTERT siRNA were 0.1500±0.0200,0.1533±0.0153 and 0.1700±0.0100 at 24 hours, 48 hours and 72 hours respectively.The control groups were 0.2400±0.0360, 0.2667±0.0055 and 0.2400±0.0265respectively. The differences between the treated group and the control group weresignificant statistically at 24 hours (t=3.781,P＜0.05), 48 hours (t=3.435,P＜0.05)and 72 hours (t=4.287,P＜0.05).③The telomerase activity: The telomerase activities of lung cancer cellstreated with hTERT siRNA were 103.60±10.05, 63.00±6.85 and 110.70±10.10 at 24hours, 48 hours and 72 hours respectively. The control groups were 224.23±9.20,220.30±25.18 and 200.70±6.07 respectively. The differences between the treatedgroup and the control group were significant statistically at 24 hours (t=15.335, P＜0.05),48 hours (t=10.440, P＜0.05) and 72 hours (t=12.226, P＜0.05). The percentages of inhibited telomerase activity were 54.8％,71.4％and 41.6％respectively.④The apoptosis of lung cancer cells: The apoptosis percentages of lung cancercells treated with hTERT siRNA were 9.33±0.30％,15.67±0.81％and 19.30±0.95％at 3 days,9 days and 15 days respectively. The control groups were 3.20±0.36％,5.97±0.35％and 6.70±0.45％respectively. The differences between the treated groupand the control group were significant statistically at 3 days (t=-22.479,P＜0.05),9 days (t=-19.064, P＜0.05) and 15 days (t=-20.622, P＜0.05)⑤The analysis of correlation: the expressions of lung cancer cells hTERTmRNA are positively correlated to the expressions of hTERT protein. The level ofhTERT mRNA expressions is more high, the level of hTERT protein expressions isalso more high (r=0.942,P＜0.05); The telomerase activities of lung cancer cells arenegatively correlated to its apoptosis. The telomerase activity is more high, theapoptosis is more low (r=-0.954,P＜0.05).2. hTERT siRNA inhibited the growth of lung cancer xenografts of nudemouse.①The weight of tumor: the average tumor weight of the control group was344±143mg which was higher significantly than the treated group which was158±97mg at 28th days when hTERT siRNA was injected into tumors. The differencebetween the treated group and the control group was significant statistically (t=2.397, P＜0.05).②The change of tumor volume and the tumor-inhibited percentage: theaverage tumor volume of the control group was 766.7±339.2mm3 which was highersignificantly than the treated group which was 330.0±97.1mm3 at 28th days whenhTERT siRNA was injected into tumors. The difference between the treated groupand the control group was significant statistically (t=2.364, P＜0.05). Thetumor-inhibited percentage reached to 64.28％.③The tumor histology: The control groups showed: the heteromorphisms ofthe tissue and cells is manifested, the cells distribute into the form of nest and trab, the interstitial fibrous tissue is little, the size of cells vary obviously, the nucleus are largeand anachromasis, the nucleolus are obvious, there are much pathological nucleardivisions and polykaryocytes. The treated group showed: the cells also distribute intothe form of nest and trab, there are some structures similar to gland alveolus in someregions, the interstitial fibrous tissue is more than the control group, the size of cellsand nucleolus are smaller than the cells of the control group, the pathological nucleardivisions and polykaryocytes are less than the control group.④The expression of hTERT mRNA: the relative amount of hTERT mRNAexpression of the control group tumor was 99.06±1.84％which was significantlyhigher than the treated group which was 54.47±4.33％at 28th days when hTERTsiRNA was injected into tumors. The difference between the treated group and thecontrol group was significant statistically (t=21.179, P＜0.05), The percentage ofinhibited hTERT mRNA was 45.01％.⑤The expression of hTERT protein: The pergentage of hTERT proteinexpression of the control group tumor was 77.4±4.7％which was higher significantlythan the treated group which was 38.0±4.9％at 28th days when hTERT siRNA wasinjected into tumors. The difference between the treated group and the control groupwas significant statistically (t=12.901, P＜0.05 ), The percentage of inhibited hTERTprotein was 52.2％. The average photodensity of hTERT protein expression of thecontrol group tumor was 0.2780±0.0545 which was higher significantly than thetreated group which was 0.1600±0.0158 at 28th days when hTERT siRNA wasinjected into tumors. The difference between the treated group and the control groupwas significant statistically (t=4.650, P＜0.05).⑥The telomerase activity: The average telomerase activity of the control grouptumor was 210.06±30.06 which was higher significantly than the treated group whichwas 102.38±8.27 at 28th days when hTERT siRNA was injected into tumors. Thedifference between the treated group and the control group was significant statistically(t=7.727, P＜0.05), The percentage of inhibited telomerase activity was 51.26％.⑦The analysis of correlation: RTA of the xenografts are Positively correlatedto the change of tumor volume. RTA of the xenografls is more low, its growth is more slow(r=0.932,P＜0.05).Conclusion1. hTERT siRNA can downregulates the expression of hTERT mRNA andprotein of lung adenocarcinoma cells, consequently inhibitesthe telomerase activity.2. The major mechanism which hTERT siRNA can induces the apoptosis of lungadenocarcinoma cells is that hTERT siRNA can inhibites the telomerase activity.3. hTERT siRNA can inhibits the growth of lung adenocarcinoma xenografts ofnude mouse.It is implied that hTERT may become a good target of gene therapy oflung cancer and hTERT siRNA is active and stable in vivo.Therefore our studiesprovide some data of animal studies to the clinical reseach of gene therapy in thefuture. |
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