【作者】 段慧菡；

【导师】 蒋义国；

【作者基本信息】 广州医学院 ， 卫生毒理学， 2009， 硕士

【摘要】 背景与目的miRNA是一类内源性非编码的单链小分子RNA。成熟的miRNA大小在19~23个寡核苷酸(nucleotide ,nt)左右,其与靶基因mRNA 3’UTR上靶序列碱基配对,在转录后水平抑制蛋白质翻译或降解mRNA,是发挥功能的基础。miRNA对基因表达的调控现象在真核生物中广泛存在,不同的组织器官、不同类型的肿瘤、肿瘤的不同发展阶段都存在不同的miRNA的表达,使miRNA表达谱具有组织细胞特异性。苯并[a]芘(B[a]P)是常见的环境污染物,在生产和生活中有许多的暴露机会,也是吸烟相关肺癌的发展中起关键作用的成因之一。肿瘤是一类细胞周期性疾病,致癌作用被看作是细胞周期内在机制紊乱的结果。细胞周期存在多个检查点(checkpoint)调节各期间的转换及细胞周期进程。在课题组前期工作中,已发现miR-320和miR-494在anti-BPDE转化的人类支气管上皮细胞中表达最高。本实验选择miR-320和miR-494 ,以B[a]P染毒小鼠原代支气管上皮细胞,首次探索了miR-320和miR-494在B[a]P染毒细胞的细胞周期中的作用。方法小鼠原代支气管上皮细胞的培养在无菌条件下处死小鼠,取出支气管,立即置于预冷的Collection培养基中;移入PBS液中去除周围结缔组织及血管并纵向切开。用Dissociation培养基4°C消化24h加入Culture培养基轻柔地上下倒置管子12次,重复操作一次。离心,用Culture培养基重悬细胞。将此细胞悬液接种于60mm的培养皿中,2h后收集未贴壁细胞。将细胞接种于已用胎盘胶原包被的24孔板内,用Culture培养基在正常培养条件下,37°C、5% CO2下培养24h。次日弃去Culture培养基,添加Differentiation培养基,37°C、5% CO2下培养。原代细胞的染毒B[a]P粉末溶于DMSO中,细胞培养液中DMSO的终浓度不超过0.1%。用经多氯联苯诱导的大鼠肝匀浆微粒体酶系(Co-factor supplemented post-mitochondrial fraction, S9)并加相应的辅助因子(6-磷酸葡萄糖、辅酶II)配制成体外代谢活化系统。S9混合液随配随用。原代细胞在无血清培养的第二天,分别用0.01μM, 0.10μM和1.00μM并加S9活化的B[a]P无血清培养基染毒细胞,分别作用12h、24h、48h后弃去染毒液,PBS洗涤,继续在正常培养条件下无血清培养。原代细胞的转染转染实验步骤按照Hiperfect转染试剂24孔板转染操作指南进行。miR-320和miR-494抑制物(anti-miR-320,anti-miR-494)在反应体系的终浓度为30nmol/L,转染培养24h后可弃去培养基。用荧光素FAM标记的阴性对照检测抑制物的转染效率可达90%~95%。QRT-PCR检测miR-320和miR-494的表达用Trizol抽提各组样本的总RNA。按下列成分分别组成20μl miR-320、miR-494及18s rRNA的定量PCR反应体系: 10μl 2×Real-time PCR Master Mix, 1.6μl miRNA specific primer set (20μM), 2μl cDNA template, and 0.2μl Taq DNA聚合酶(5U/μl),加d3H2O至20μl。执行如下程序:95℃3min,95℃30s,62℃40s,循环40次。以18s rRNA作为内参照,采用2-△△Ct法对基因表达进行分析。流式细胞仪检测细胞周期的分布将细胞悬液离心,加入缓冲液重悬细胞,参照Cycle TEST Plus DNA reagent试剂盒说明书进行染色。FACSArray流式细胞仪分析细胞DNA含量,并应用Modfit LT 3.0软件分析细胞周期。流式细胞仪检测CDK6的表达水平采用间接免疫荧光标记法进行免疫荧光染色。一抗37°C孵育30min,用缓冲液洗涤2次后加二抗37°C避光孵育30min;缓冲液洗涤、重悬细胞。用FACSArray流式细胞仪进行CDK6含量的检测。以荧光指数(fluorescence index ,FI)表示CDK6的相对含量。7.统计学方法实验数据均以均数±标准差表示,采用SPSS 15.0统计软件处理,采用独立样本t检验进行数据分析,P < 0.05为差异有统计学意义。结果1. miR-320和miR-494在B[a]P暴露的小鼠原代支气管上皮细胞的表达分别以0.01μM、0.10μM和1.00μM的B[a]P并加S9活化染毒细胞。各剂量组B[a]P分别作用12h, 24h及48h后,miR-320和miR-494表达水平均有不同程度的上调。其中,1.00μM组的miR-320和miR-494表达均显示出明显的时间依赖关系。选取1.00μM B[a]P作用24h的条件进行后续的实验。2. miR-320和miR-494表达水平的有效抑制miR-320和miR-494的抑制物能分别特异有效地抑制miR-320和miR-494的表达。与B[a]P组相比,miR-320的表达水平在anti-miR-320组降低96.7%,差异显著(P <0.01);而在anti-miR-494组却无明显的变化,差异无统计学意义(P >0.05);同样地,miR-494的表达水平在anti-miR-494组明显地下调98%,差异显著(P <0.01);而在anti-miR-320组变化不明显,差异无统计学意义(P >0.05)。3. miR-320和miR-494对染毒细胞细胞周期的影响小鼠原代支气管上皮细胞经过1.00μM B[a]P作用24h后,出现了G1期阻滞的现象。与溶剂对照组的62.70%±1.54%的G1期细胞比例相比,B[a]P组细胞周期的进程被阻滞,84.28%±0.36%的细胞停留在此间期,差异具有统计学意义(P <0.05)。在分别转染了miR-320和miR-494的抑制物的anti-miR-320组和anti-miR-494组中,G1期细胞比例分别降低至60.57%±3.41%和57.52%±2.43%,与B[a]P组相比差异均有统计学意义(P <0.05)。而在anti-miR-nc组里,G1期细胞比例无明显的改变。4. miR-320和miR-494对染毒细胞CDK6表达水平的调控细胞经过1.00μM B[a]P染毒处理24h后,与溶剂对照组相比,CDK6的表达值由0.86±0.06降低至0.41±0.03,下降幅度达52.3%,差异显著(P <0.01)。而在分别转染了miR-320和miR-494的抑制物作用24h后,anti-miR-320组和anti-miR-494组均出现CDK6表达水平的恢复,与B[a]P组相比,CDK6的表达量分别有2.0±0.3与2.1±0.6倍的升高,差异具有统计学意义(P <0.01)。结论1.用冷消化和无血清培养的方法成功培养了小鼠原代支气管上皮细胞。2. miR-320和miR-494在B[a]P暴露的小鼠原代支气管上皮细胞中表达增高。3. miR-320和miR-494的高表达促使细胞G1期阻滞。4. miR-320和miR-494在一定程度上通过对CDK6表达的调控影响B[a]P暴露的细胞的细胞周期。本项目特色与创新:在国际毒理学研究领域,较早开展了miRNA研究;首次在原代培养细胞中研究miRNA,并发现miR-320和miR-494对细胞周期的调节作用。更多还原

【Abstract】 Backgroud and objectiveMicroRNAs (miRNAs) are are a novel class of 19-23 nucleotide (nt) noncoding RNA molecules that regulate gene expression by targeting the 3′-untranslated region (3′UTR) of mRNAs with consequent inhibition of protein translation or degradation of target mRNA。MiRNA-mediated regulation of gene expression is a wide-spread phenomena in eukaryotic organisms. MiRNA expression profiles are very informative, and differ between normal tissues and derived tumors and between tumor types. Benzo[a]pyrene (B[a]P is a well-known environmental pollutant. There is potential possibility for either environmental or occupational exposure to B[a]P. It is known to be present in cigarette smoke and implicated as a causative substance in the development of smoking-related lung cancer. Carcinogenesis is considered to be the result of dysregulation of the cell cycle machinery. It is now known that progression through different phases of cell cycle is controlled by several cell cycle checkpoints, including G1/S, G2/M and the spindle checkpoint. In our previous study, miR-320 and miR-494 were the most highly expressed in the human bronchial epithelial cells (16HBE-T) transformed by anti-BPDE, compared with that in the vehicle-treated control cells. In the present work, for the first time, we investigated the roles of miR-320 and miR-494 in cell cycle of primary murine bronchial epithelial cells exposed to B[a]P.Methods1. Isolation and culture of murine bronchial epithelial cells With the use of a sterile technique, mice were sacrificed, tracheas were resected and collected in Collection medium. Tracheas were washed with media to remove blood and debris, opened longitudinally, and then incubated in Dissociation medium, kept for 24 h at 4°C. The tracheas were inverted 12 times, transferred to another tube of Culture medium, inverted again to further release cells. The cells were collected by centrifugation and incubated in Culture medium at 37°C for 2h in 60mm culture dish to remove the non-epithelial cells. Non-adherent cells were collected, washed, and seeded in the 24-well plate, which precoated with Human Placental Collagen. The cells were incubated at 37°C in 5% CO2 in a humidified incubator for 24h. On the second day, the cells was replaced with Differentiation medium.2. Cell treatment The fractions of rat liver homogenates (S9) was added with 5mmol/L glucose-6-phosphate and 2.5mmol/L nicotinamide adenine dinucleotide phosphate. Benzo[a]pyrene (B[a]P) dissolved in dimethylsulphoxide). The final concentration of DMSO did not exceed 0.1% of total incubation volume. On the third day, the cells were treated with 0.01μM, 0.10μM or 1.00μM B[a]P (with S9 mixture) in DMEM/F-12 medium contains the growth factors for 12h, 24h or 48h, then the medium was discarded. After washed with PBS for three times, the cells were cultured in the normal conditions for further studies.3. Cell transfection Cells transfection experiments were performed using Hiperfect Transfection Reagent in 24-well plates according to the manufacturer’s instructions. The inhibitors of miRNA were diluted at a final concentration of 30 nmol/L. Incubated cells with the transfection complexes under their normal growth conditions for 24h before replacing the medium. Transfection efficiencies in range of 90%~95% were confirmed by microscopic analysis.4. RNA extraction and QRT–PCR Total RNA was extracted from the cultured cells using Trizol Reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s protocol. The 20μl miRNA Real-time PCR Reaction System included 10μl 2×Real-time PCR Master Mix, 0.16μl miRNA specific primer set (20μM), 2μl cDNA template, and 0.2μl Taq DNA polymerase (5U/μl). PCR was carried out at 95oC for 3 min denaturing, followed by 40 cycles of 95oC for 30s and 62oC for 40s. The 18S rRNA was used as an internal control. The 2-ΔΔCt method for relative quantitation of gene expression was used to determine miRNA expression levels. 5. Cell cycle analysis Cells were collected by centrifugation, washed and resuspended in Buffer Solution (BD Biosciences, San Jose, CA, USA).The cells were stained with Propidium iodide (PI) using a CycleTEST Plus DNA reagent kit (BD Biosciences, San Jose, CA, USA) according to the manufacturer’s instructions. For each sample, 20, 000 events were acquired using FACSArray Flow Cytometer (Becton Dickerson, San Jose, CA, USA). Modfit LT v3.0 software was used for the acquisition and analysis.6. Detection of expressions of CDK6 The treated cells were quantitatively analysed by flow cytometry (FCM) with indirect immunofluorescence technique.Cells were incubated in water-bath for 30min at 37°C with 100μl CDK6 antibody. The samples were then washed twice with Staining Buffer and incubated in water-bath for 30 min at 37°C with 100μl of the second antibody of phycoerythrobilin (PE)-conjugated goat anti-rabbit IgG. Fluorescence index (FI) was used to quantified CDK6 expression.7. Statistical analysis All data are presented as the mean±SD. The data were evaluated by indepengdent sample t-test between groups with the SPSS 15.0 program (SPSS Inc. Chicago, IL, USA). P value < 0.05 was considered statistically significant.Results1. Expressions of miR-320 and miR-494 in primary murine bronchial epithelial cells exposed to B[a]P The cells were treated with B[a]P activated by the S9 mixture, at concentrations of 0.01μM, 0.10μM and 1.00μM for 12h, 24h and 48h. 0.01μM, 0.10μM and 1.00μM B[a]P treatments all increased the expression levels of miR-320 and miR-494 in different degrees. Typical time-dependent increase were evident in miR-320 and miR-494 expressions at the concentration of 1.00μM. We concentrated on 1.00μM B[a]P for 24h as the treatment condition of cells for further study.2. Inhibition of the expressions of miR-320 and miR-494 with miRNA inhibitors the specific miRNA inhibitors was able to suppress the expression levels of miR-320 and miR-494 effectively. the expression level of miR-320 in anti-miR-494 group were not significantly different (P>0.05), while the expression levels significantly reduced by 96.7% in anti-miR-320 group, compared with that in B[a]P group (P < 0.01). Similarly, the expression levels of miR-494 sharply reduced by 98.0% in anti-miR-494 group (P < 0.01) but showed no significantly difference in anti-miR-320 group (P>0.05), compared with the B[a]P group.3. MiR-320 and miR-494 regulate cell cycle Inspection of the cell cycle progression from B[a]P-treated cells revealed that cells underwent G1 arrest were response of B[a]P exposure. As seen in Figure 4, 1.00μM B[a]P treatment significantly accumulated the number of cells in the G1 phase (84.28%±0.36% of the B[a]P group compared with 62.70%±1.54% of the control group P< 0.05). Anti- miR-320 group and anti- miR-494 group produced a low percentage of G1 cells after transfection with miR-320 inhibitors or miR-494 inhibitors for 24h (60.57%±3.41% and 57.52%±2.43%, respectively, P<0.05). No apparent alterations of G1 cells shown in the anti-miR-nc group.4. The effects of miR-320 and miR-494 on CDK6 the cells treated with B[a]P for 24h resulted in reduced expression levels of CDK6 (0.41±0.03 of the B[a]P group), which decreased by 52.3% compared with 0.86±0.06 of the control group. Howerver, after transfection with miR-320 inhibitors or miR-494 inhibitors for 24h, the cells were led to recovery in CDK6 levels. The expression levels of CDK6 in anti-miR-320 and anti-miR-494 groups were 1.23±0.02 and 1.24±0.02 , which had 2.0±0.3, 2.1±0.6-fold respectively higher than those in B[a]P group.Conclusions1. Primary murine bronchial epithelial cells were cultured successfully with serum-free method.2. The expression levels of miR-320 and miR-494 increase in primary murine bronchial epithelial cells exposed to B[a]P.3. Overexpressions of miR-320 and miR-494 induce G1 arrest in B[a]P-exposed cells.4. MiR-320 and miR-494 should effect G1 arrest partially by regulating CDK6 in B[a]P-exposed cells.更多还原