摘要
目的:通过调控miR-205在肾上腺皮质癌(ACC)细胞株SW-13中的表达,探讨miR-205对SW-13细胞生物学行为的影响。方法:通过基因重组技术构建pcDNA3.1(+)-miR-205重组质粒并合成miR-205的反义寡核苷酸作为反向对照,调控miR-205在肾上腺皮质癌SW-13细胞中表达,实时荧光定量PCR法验证miR-205表达情况;分别采用MTS、EdU、TUNNEL及Transwell小室法,检测过表达及抑制miR-205对增殖、凋亡及侵袭的影响。结果:在转染pcDNA3.1(+)-miR-205及miR-205反义寡核苷酸后,SW-13细胞中miR-205分别上调了63.2倍(P=0.001)及下调了86.3%(P=0.002)。MTS结果显示,在24、48、72和96h时,miR-205上调后SW-13细胞增殖率下降了(10.50±1.80)%、(23.67±4.80)%、(30.9±5.40)%和(38.34±6.20)%,P值分别为0.041、0.014、0.016和0.032;miR-205下调后增殖率提高了(25.31±3.20)%、(32.51±4.60)%、(40.15±4.10)%和(38.34±6.20)%,P值分别为0.032、0.026、0.022和0.013。TUNNEL结果显示,pcDNA3.1(+)-miR-205组SW-13细胞凋亡数为(46±4)个,pcDNA3.1(+)组为(25±3)个,P=0.001 9;anti-miR-205转染组为(11±2)个,空白组为(25±3)个,P=0.002 5。Transwell侵袭实验显示,pcDNA3.1(+)-miR-205组穿过小室膜细胞数为(43±8)个,pcDNA3.1(+)组为(120±15)个,P=0.001 4;anti-miR-205转染组为(118±14)个,空白组为(220±20)个,P=0.001 9。结论:miR-205通过抑制细胞增殖、侵袭并促进凋亡在ACC SW-13细胞中起抑癌作用;成功构建pcD-NA3.1(+)-miR-205真核表达质粒,为进一步研究miR-205在SW-13细胞中的基因调控机制奠定基础。
OBJECTIVE：To investigate the effect of miR-205 on SW-13 cell proliferation,apoptosis and invasive abilities by regulating the expression of miR-205 in adrenocortical carcinoma SW-13 cell line. METHODS： To regulate the expression of miR-205 in adrenocortical carcinoma SW-13 cell line by recombinant DNA technology to construct the pcD-NA3. 1（-4-）-miR-205 recombinant plasmid and synthesizing antisense oligonucleotides of miR-205 as the reverse control, The expression of miR-205 mRNA was detected by real-time fluorogenic quantitative-PCR（qRT-PCR）. The effects of pcDNA3.1 （＋）-miR-205 on SW-13 cell proliferation,apoptosis and invasive abilities were detected by MTS, EdU, TUN- NEL and Transwell chamber methods,respectively. RESULTS： qRT-PCR results revealed that miR-205 was increased by 63.2 fold （P=0. 001） and reduced by 86.3% （P=0. 002） in SW-13 cells after pcDNA3.1 （＋）-miR-205 and miR-205 antisense oligonucleotides transfected respectively. The MTS results suggested that the rate of cell proliferation of SW-13 was declined （10.50土1.80）%, （23.67土4.80）%, （30.79土5.40）% and （38. 34土6.20）% after the upreguhition of miR- 205 in 24,48,72 and 96 h （P values were 0. 041,0. 014,0. 016 and 0. 032）, while the rate was increased （25. 31土3.20） %, （32.51土4.60）%, （40.15土4.10）% and （38.34土6.20）% after the downregulation of miR-205 （P values were 0. 032,0. 026,0. 022 and 0. 013）. The TUNNEL results indicated that the number of SW-13 apoptotic cells were （46土4） and （25＋3）per vision in pcDNA3.1 （＋）-miR-205 group and pcDNA3.1 （＋） group （P=0. 001 9） ,While （11土2） and（25土3） per vision in anti-miR-205 group and blank group （P=0. 002 5）. Transwell invasion assay found that the number of SW-13 cells through the small room film were （43土8） and （120土15） per vision in pcDNA3.1 （＋）-miR-205 group and pcD- NA3.1 （＋） group （P=0. 001 4）, while （118土14） and （220土20） per vision in anti-miR-205 group and blank group （P=0. 001 9）. CONCLUNSIONS： miR-205 may play a tumor suppressor role in adrenocortical carcinoma SW-13 cells through inhibiting cell proliferation,invasion and promoting apoptosis. The eukaryotic expression vector of miR 205 is constructed successfully, which provide miR 205 an experimental basis for further study of gene regulation mechanisms in tumors.
出处
《中华肿瘤防治杂志》
CAS
北大核心
2013年第8期566-571,共6页
Chinese Journal of Cancer Prevention and Treatment
基金
国家自然科学基金(81172421)
国家自然科学基金青年科学基金(81001142)
