端锚酶和端粒酶反义寡核苷酸联合作用对人肺腺癌细胞A549端粒的影响

Effect on telomere of antisense tankyrase and telomerase oligonucleotide in human lung adenocarcinoma A549 cell line

目的探讨端锚酶及端粒酶反义寡核苷酸联合作用对A549细胞端粒相关蛋白表达和翻译及对端粒缩短和细胞周期的作用。方法将A549细胞随机分组为空白对照、sTANKS、shTERT、asTANKS、ashTERT和asTANKS+ashTERT组,经不同处理,检测hTERTmRNA、端粒酶及端锚酶活性、端粒平均长度及A549细胞寿命。结果ashTERT能抑制hTERTmRNA表达及蛋白质活性;asTANKS不影响hTERTmRNA表达,却能抑制端锚酶活性。asTANKS或ashTERT均可致A549端粒长度缩短,asTANKS+ashTERT则缩短更为明显,其减少A549细胞平均传代数的作用也明显大于asTANKS或ashTERT。结论asTANKS对A549端粒长度的抑制有别于端粒酶途径,不仅能通过影响端锚酶活性缩短肿瘤细胞A549端粒的平均长度,而且可与端粒酶抑制剂协同作用明显缩短肿瘤细胞的生存周期,这可能成为抗癌作用的新靶点。

Objective To determine the effect of transcription and translation in telomeric related proteins, and synergism of progressive telomere shortening and cell cycle alteration in human lung adenocarcinoma A549 cell line, which is induced by antisense tankyrase oligonucleotide（asTANKS） combinated with antisense human telom- erase reverse transcriptase （ashTERT） oligonucleotide. Methods A549 cells were randomly assigned as 3 test groups： ashTERT, ashTERT ＋ asTANKS and asTANKS, three control groups （shTERT, sTANKS and blank）. With individual intervention for different hours, the effect of transcription in hTERT mRNA was evaluated by RT-PCR, and telomerase activity was tested by ELISA-PCR, tankyrase activity was tested by Western blot as well. Moreover, telomere average length was analyzed by Q-FISH, and duration of proliferation was observed by population double test. Results Transcription in hTERT mRNA and telomerase activity for 48 hrs was inhibited obviously by ashTERT, but not by asTANKS. Progressive telomere shortening in A549 ceils for 48 hrs was induced by either asTANKS or ashTERT （vs control, P 〈 0. 01 ）, but it was more significant when induced by combination of them （vs ashTERT / asTANKS, P 〈 0. 01 ）. Furthermore, continuous treatment of ashTERT was similar to asTANKS in duration of proliferation, which was observed 53 ~ 57 PD and 56 ~ 58 PD respectively （vs control, P 〈 0. 01 ）, and combinative effect of them conduced to a shorter survival （22 - 26 PD） and earlier cell crisis onset （vs ashTERT / asTANKS, P 〈 0.01 ）. Conclusion asTANKS leads to progressive telomere shortening, and works in coordination with ashTERT in A549 cell line, which enhances the efficacy of telomere shortening and hastened earlier cellular crisis. This study supports the strategy for telomere-based molecular cancer therapy.