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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 the biological behavior of SW-13 cells by regulating the expression of miR-205 in the adrenocortical carcinoma (ACC) cell line SW-13. METHODS: Antisense oligonucleotides of recombinant plasmid pcDNA3.1 (+) - miR-205 and miR-205 were constructed by reverse transcription polymerase chain reaction to regulate the expression of miR-205 in adrenocortical carcinoma SW-13 cells The expression of miR-205 was verified by real-time fluorescence quantitative PCR. The effects of miR-205 on proliferation, apoptosis and invasion were detected by MTS, EdU, TUNNEL and Transwell chamber method respectively. Results: After transfected with pcDNA3.1 (+) - miR-205 and miR-205 antisense oligonucleotides, miR-205 was up-regulated 63.2-fold (P = 0.001) and down-regulated by 86.3% (P = 0.002). The results of MTS showed that the proliferation rate of SW-13 cells decreased by (10.50 ± 1.80)%, (23.67 ± 4.80)%, (30.9 ± 5.40)% and (38.34%) after miR-205 was upregulated at 24,48,72 and 96 h ± 6.20)% and P values were 0.041, 0.014, 0.016 and 0.032, respectively. The proliferation rates of miR-205 decreased by 25.31 ± 3.20%, 32.51 ± 4.60%, 40.15 ± 4.10% and 38.34 ± 6.20)%, P values were 0.032, 0.026, 0.022 and 0.013, respectively. The results of TUNEL showed that the number of apoptotic SW-13 cells in pcDNA3.1 (+) - miR-205 group was (46 ± 4) and that in pcDNA3.1 (+) group was (25 ± 3) The number of anti-miR-205 transfection group was (11 ± 2) and that of the blank group was (25 ± 3), P = 0.002 5. Transwell invasion assay showed that there were 43 ± 8 cells in pcDNA3.1 (+) - miR-205 group and 120 ± 15 in pcDNA3.1 (+) group (P = 0.001 4) The number of anti-miR-205 transfection group was (118 ± 14), (220 ± 20) in blank group, P = 0.001 9. CONCLUSION: miR-205 plays a role of anti-tumor in ACC SW-13 cells by inhibiting cell proliferation, invasion and promoting apoptosis. To construct pcD-NA3.1 (+) - miR-205 eukaryotic expression vector, -205 gene regulation mechanism in SW-13 cells.