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目的 :体外实验观察乳酸脱氢酶-A(lactate dehydrogenase-A,LDHA)短发夹RNA对胆管癌细胞系Hucct1 LDHA表达的抑制效果,筛选出针对LDHA基因有效的干扰靶点,并检测LDHA表达下调后,细胞增殖活力的改变情况。方法:设计合成干扰LDHA表达的寡核苷酸片段,经退火、连接等步骤克隆到线性化pGPU6/GFP/Neo真核表达载体,转化入DH-5α大肠杆菌中扩增,经质粒抽提纯化后测序鉴定。重组质粒用脂质体法转染胆管癌细胞系,荧光显微镜观察转染效率;并分别利用RT-PCR及Western blot技术检测LDHA mRNA及蛋白表达,验证短发夹RNA的干扰效果。将干扰效率最高的质粒转染Hucct-1,MTT法检测干扰组较对照组细胞增殖的变化。结果:成功构建了靶向LDHA基因的3个shRNA质粒表达载体,脂质体法平均转染效率为(80±5)%。荧光定量RT-PCR和Western blot显示瞬时转染si-LDHA-1、si-LDHA-3的Hucct1细胞LDHA mRNA和蛋白表达均较阴性对照组有不同程度的下降,其中si-LDHA-3干扰效果尤为明显。MTT法检测显示转染组Hucct1细胞较阴性对照组增殖减慢,转染后96 h两组差异具有统计学差异(P<0.05)。结论:重组质粒能有效干扰胆管癌细胞Hucct1中LDHA基因的表达,经瞬时转染筛选出了有效干扰靶位,下调LDHA的表达,明显抑制胆管癌细胞系Hucct-1的增殖活力。
OBJECTIVE: To observe the inhibitory effect of lactate dehydrogenase-A (LDHA) short hairpin RNA on the expression of HuHCct1 LDHA in cholangiocarcinoma cell line in vitro, and to screen the effective interference target of LDHA gene and to detect the expression of LDHA After down-regulation, cell proliferation activity changes. Methods: The oligonucleotide fragments that interfere with the expression of LDHA were designed and synthesized, then cloned into the linearized pGPU6 / GFP / Neo eukaryotic expression vector by annealing and ligation, then transformed into DH-5α E.coli for amplification, and purified by plasmid extraction After sequencing identification. The recombinant plasmids were transfected into cholangiocarcinoma cell lines by lipofectamine. The transfection efficiency was observed by fluorescence microscopy. The mRNA and protein expression of LDHA were detected by RT-PCR and Western blot respectively, and the interference effect of short hairpin RNA was verified. Hucct-1 was transfected into the plasmid with the highest interference efficiency, and MTT assay was used to detect the changes of cell proliferation in the interference group compared with the control group. RESULTS: Three shRNA plasmid expression vectors targeting LDHA gene were successfully constructed. The average transfection efficiency was (80 ± 5)% by liposome method. The mRNA and protein expression of LDHA in Hucct1 cells transiently transfected with si-LDHA-1 and si-LDHA-3 were all lower than those in the negative control group by fluorescence quantitative RT-PCR and Western blot. Among them, the effect of si-LDHA-3 Especially obvious. The results of MTT assay showed that the proliferation of Hucct1 cells in the transfected group was slower than that in the negative control group, and the difference was statistically significant at 96 h after transfection (P <0.05). CONCLUSION: Recombinant plasmids can effectively interfere with the expression of LDHA gene in Hucct1 cholangiocarcinoma cells. After transient transfection, we can effectively knock down the target and down-regulate the expression of LDHA and significantly inhibit the proliferation of Hucct-1 cells.