LDH-A shRNA对人胰腺癌细胞系PANC-1增殖凋亡和LDH-A表达的影响

Effect of LDH-A shRNA on the growth, apoptosis and expression of LDH-A in a human pancreatic cancer cell line PANC-1

目的通过构建乳酸脱氢酶A（lactate dehydrogenase，LDH—A）shRNA并转染PANC-1细胞，分析其对胰腺癌细胞生物学特性的影响。方法构建3条LDH—AshRNA质粒，脂质体转染质粒至PANC-1细胞中，实时荧光定量PCR法检测不同质粒转染后LDH-AmRNA的表达变化。将抑制效率最高的shRNA质粒-3转染PANC-1细胞，MTT法检测转染前后细胞增殖的变化，流式细胞仪检测细胞凋亡的变化。RT-PCR检测LDH—A表达以及酶化学染色检测转染前后LDH活性的变化。结果3种LDH—AshRNA质粒转染胰腺癌细胞后，LDH-AshRNA质粒-3的2-ΔΔCt值为（0．47±0．02），较正常细胞（0．71±0．01）小，存在抑制作用，且抑制效率较高。PANC-1细胞在转染shRNA质粒-3后12h转染组吸光度值显著低于对照组，细胞出现增殖抑制，24、36、48和72h，转染组的吸光度值均显著低于对照组（P〈0．01）。转染质粒组细胞凋亡也明显增高，其凋亡率达到61．74％；转染shRNA质粒-3的胰腺癌细胞株，其LDH—AmRNA的表达明显抑制，酶化学染色显示LDH活性明显减弱。结论LDH—AshRNA通过抑制胰腺癌细胞LDH—AmRNA的表达抑制其增殖及诱导细胞凋亡。

Objective To construct a short hairpin RNA （shRNA） targeting LDH-A, and evaluate the effects on growth, apoptosis and the expression of LDH-A in PANC-1 cells. Method Three shRNAs targeting LDH-A were combined to pGCsilencer vector, and transfected into PANC-1 cells. The expression of LDH-A after transfected by the three shRNAs in pancreatic cancer cell lines PANC-1 was detected by quantitative real time PCR. After tranfected by LDH shRNA-3 with the highest inhibited rate, cell growth was analyzed by MTTassay, apoptosis was detected by flow cytometry. The LDH-A expression was detected by reverse transcription polymerase chain reaction, LDH activity was observed with enzyme cytochemical method. Results The 2-AACt of LDH-A shRNA-3 was （ 0. 47 ± 0. 02 ）, less than the untransfected pancreatic cancer cell（0. 71 ± 0. 01 ）, the LDH-A shRNA-3 could inhibit the expression of the LDH-A most effectively. The growth of the pancreatic cancer cell was inhibited after 12 h trausfected by LDH-A shRNA- 3, all the absorbance value of transfected cell in 24 h ,36 h ,48 h,72 h decreased obviously compared to the untreated pancreatic cancer cell（P 〈0. 01 ）. The apoptosis rate of the transfeeted cell increased to 61.74%. The inhibition of LDH-A expression in PANC-1 cells transfeeted by shRNA-3 was significantly and the activity of LDH reduced. Conclusion LDH-A shRNA inhibits the expression of LDH-A, the proliferation of cancer cells inducing the apoptosis of PANC-1 cells.