反式-7,8-二羟-9,10-环氧苯并芘诱导恶变细胞中miR-494和miR-22的功能及PTEN的表达  

Function of miR-494,miR-22 and Expression of PTEN in Transformed Cells Induced by Anti-BPDE

作　　者：刘林华[1] 蒋义国[1] 

机构地区：[1]广州医学院化学致癌研究所呼吸疾病国家重点实验室,广东广州510182

出　　处：《环境与健康杂志》2010年第5期379-382,共4页Journal of Environment and Health

基　　金：国家自然科学基金资助项目(30571546;30771780);教育部留学回国人员科研启动基金资助项目(2007-24);广东省自然科学基金资助项目(07117550);广东省高校自然科学重点项目(06Z021);广州市教育局科技项目(08A093)

摘　　要：目的分析反式-7,8-二羟-9,10-环氧苯并芘(anti-BPDE)诱导转化人支气管上皮细胞第30代(16HBE-T30)及其前期第15代细胞(16HBE-T15)miR-494及miR-22的表达水平,探讨其在化学致癌过程中的作用及对靶基因PTEN抑癌基因的调控。方法以16HBE-T30和16HBE-T15细胞为实验组,溶剂处理组分别为其相应的对照组细胞(16HBE-N30和16HBE-N15),用茎环结构逆转录引物定量RT-PCR检测四组细胞miR-494及miR-22成熟体表达水平。运用定量RT-PCR和Western blotting分别检测四组细胞PTEN mRNA和蛋白表达水平。运用miRNA前体及抑制剂对miNRA表达进行调控后,再检测PTEN的表达改变,并进行报告基因信号改变、细胞凋亡、软琼脂集落形成率等分析。结果 16HBE-T30中miR-494和miR-22表达水平分别是16HBE-N30的12.6和2.3倍,而16HBE-T15中miR-494、miR-22表达水平较16HBE-N15下降。16HBE-T30中PTEN蛋白表达量低于16HBE-N30。与前体阴性对照组相比,上调的miR-494和miR-22分别将PTEN蛋白的表达量降低至0.8和0.5倍,而降低的miR-494和miR-22分别将PTEN蛋白的表达量上调至1.3和1.5倍。在16HBE-T30细胞中,miR-494和miR-22表达下调均增强caspase-3/7活力,miR-494和miR-22表达上调则减弱其活力。miR-494和miR-22下调能够降低转化细胞的恶性程度。结论 anti-BPDE恶性转化细胞中miR-494和miR-22在转录后水平反向调控靶基因PTEN的表达,这两种miRNA在anti-BPDE致癌过程中发挥类癌基因作用。Objective To explore the functionality of miR-494 and miR-22 in carcinogenesis and regulatory role on PTEN tumor suppressor gene through detecting the expression of miR-d94 and miR-22 in transformed human bronchial epithelial cells from the 30th passage （16HBE-T30） and its early 15th passage （16HBE-T15） induced by anti-benzo[a]pyrene-7,8-diol-9,10-epoxide （anti-BPDE）. Methods The expression of mature miR-494 and miR-22 in 16HBE-T30 and 16HBE-T15 cells and their respective vehicle control cells （16HBE-N30 and 16HBE-N15） was detected by miRNA specific stem-loop reverse transcription primer real- time RT-PCR. Real-time RT-PCR and Western blot were used to determine the expression of PTEN mRNA and protein. Following enhance or inhibition of mature miRNA expression with precursors or antisense inhibitors, PTEN expression, luciferase activities, cell apoptosis, cell growth in soft agar and cell mobility were analyzed. Results The expression levels of miR-494 and miR-22 in 16HBE-T30 cells were 12.6-fold and 2.3-fold as high as those of 16HBE-N30 cells respectively,while expression of both miR-494 and miR-22 decreased in 16HBE-T15 cells. PTEN protein appeared to be lower in 16HBE-T30 cells compared with those of 16HBE-N30 cells. The expression of PTEN protein regulated by enforced miR-494 and miR-22 decreased, which were 0.8-fold and 0.5-fold as high as that of negative precursor control group respectively. The expression levels of PTEN protein regulated by inhibited miR-494 and miR-22 increased ,which were 1.3-fold and 1.5-fold as high as that of negative inhibitor control group, respectively. MiR-494 and miR-22 with decreasing expression up-regulated the easpase-3/7 activity in 16HBE-T30 and miR-494 and miR-22 with increasing expression down-regulated the caspase-3/7 activity. The miR-494 and miR-22 with decreasing expression down-regulated the malignancy of transformed cells. Conclusion MiR-494 and miR-22 regulate the expression of PTEN post-transcriptionally, and function as micro-oncogenes in carcino

关 键 词：反式-7  8-二羟-9  10-环氧苯并芘 microRNA PTEN基因 人支气管上皮细胞 

分 类 号：R994.6[医药卫生—毒理学]