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目的探讨结肠癌LS174T细胞及结直肠腺癌组织中缺氧诱导因子-1α(HIF-1α)的表达及与NDRG1的相关性。方法构建靶向HIF-1α的质粒pSilence-2.1-U6-siRNA并鉴定,采用阳离子脂质体转染法将其转染LS174T细胞,低氧培养24h,运用逆转录-聚合酶链反应(RT-PCR)检测HIF-1α、NDRG1 mRNA表达。Western blots检测NDRG1蛋白表达。采用原位杂交技术检测人结直肠腺瘤和腺癌组织中HIF-1αmRNA,应用免疫组织化学方法检测NDRG1蛋白的表达。结果siRNA从转录水平抑制了HIF-1α基因表达。同时,NDRG1 mRNA、蛋白表达也显著受抑制。结直肠腺癌组织中HIF-1αmRNA阳性表达率为67.7%(42/62),腺瘤为44.4%(8/18)。从Dukes A期到Dukes C+D期演变过程中HIF-1αmRNA表达阳性率不断增加(P<0.05)。腺癌组NDRG1阳性表达率显著高于腺瘤组。结直肠腺癌中NDRG1阳性表达与Dukes分期显著相关。HIF-1α与NDRG1均呈正相关(P<0.05)。结论HIF-1α可能通过上调NDRG1表达参与了结直肠腺的进展。
Objective To investigate the expression of hypoxia-inducible factor-1α (HIF-1α) in colon cancer LS174T cells and colorectal adenocarcinoma and its relationship with NDRG1. Methods Plasmid pSilence-2.1-U6-siRNA targeting HIF-1α was constructed and identified. The recombinant plasmid was transfected into LS174T cells by cationic liposome transfection and then cultured in hypoxia for 24h. The expression of pSilence- PCR) to detect HIF-1α, NDRG1 mRNA expression. Western blots detected NDRG1 protein expression. HIF-1αmRNA was detected by in situ hybridization in human colorectal adenoma and adenocarcinoma. The expression of NDRG1 protein was detected by immunohistochemistry. Results siRNA inhibited HIF-1α gene expression from the transcriptional level. At the same time, NDRG1 mRNA and protein expression were also significantly inhibited. The positive rate of HIF-1αmRNA in colorectal adenocarcinoma was 67.7% (42/62) and that of adenoma was 44.4% (8/18). The positive rate of HIF-1α mRNA expression increased from Dukes A stage to Dukes C + D stage (P <0.05). The positive rate of NDRG1 in adenocarcinoma group was significantly higher than that in adenoma group. The positive expression of NDRG1 in colorectal adenocarcinoma was significantly associated with Dukes stage. HIF-1α and NDRG1 were positively correlated (P <0.05). Conclusion HIF-1α may be involved in the progression of colorectal gland by up-regulating NDRG1 expression.