[目的]探讨微小RNA(miR)-125b对肺癌细胞95D生物学功能的影响. [方法]以95D细胞为实验对象,分别转染miR-125b模拟物和模拟物阴性对照,未经特殊处理为空白组.应用MTT法、流式细胞仪和Transwell小室法分别检测miR-125b表达改变对95D细胞增殖、凋亡、周期及侵袭的影响. [结果]95D细胞转染48h后,转染组miR-125b的水平提高,表达量为阴性对照组的22.32倍.与阴性对照组相比,miR-125b模拟物转染组细胞凋亡率明显降低[(13.77±0.52)％,(9.90±1.33)％,P＜0.05],细胞增殖、周期、侵袭无显著改变(P＞0.05). [结论]miR-125b基因过表达能够抑制肺癌95D细胞凋亡.

[目的]探讨微小RNA（miR）125b-和miR-27a对肺癌细胞95D靶基因的调控作用,探索肺癌的发病机制。[方法]通过生物信息学对miR-125b和miR-27a进行靶基因预测和GO功能分析,并通过转染模拟物和抑制物的分别上调和下调细胞中miR-125b和miR-27a的表达水平,应用Western blot检测预测靶基因的蛋白表达水平。[结果]靶基因预测和GO基因功能注释结果显示:miR-125b与凋亡的调节功能有关,MAP3K11是其重要靶基因之一;miR-27a与转录和细胞分化功能有关,PLK2是其潜在靶基因之一。Western blot结果显示:与相应阴性对照组相比,miR-125b抑制物转染组MAP3K11蛋白的表达明显增高1.795倍（P＜0.05）,模拟物转染组MAP3K11蛋白的表达未发生明显改变（P＞0.05）;miR-27a抑制物和模拟物转染组PLK2蛋白的表达均未发生明显改变（P＞0.05）。[结论]miR-125b可能通过对MAP3K11的调节参与MAP3K介导的凋亡,影响肺癌的发生发展。

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The present study aimed to investigate the biofunctions of microRNA (miR)‑125b on lung cancer cells. A miR genechip array was used to examine the differential expression of miRs between 95D lung cancer cells and 16 human bronchial epithelial (HBE) cells. Overexpression of miR‑125b was observed in the cell lines and in the lung carcinoma tissues compared with the adjacent tissues, confirmed using reverse transcription quantitative polymerase chain reaction. Bioinformatic analysis of miR‑125b was also performed, including target prediction, gene ontology and pathway analysis. MTT, flow cytometry and Transwell assays were also used to examine the effect of downregulated miR‑125b on the proliferation, apoptosis, invasive ability and cell cycle of 95D cells. Significant differences were observed in the expression of 45 miRs in the 95D cells compared with those in 16HBE cells and the expression of miR‑125b was significantly higher in 95D cells compared with that in 16HBE cells as well as in lung tumor tissues compared with that in adjacent tissues. In addition, inhibition of the expression of miR‑125b in 95D cells induced apoptosis, G1/S phase arrest and reduction of their invasive ability. In addition, bioinformatics software predicted that miR‑125b was involved in the regulation of several pathways associated with cancer, including the transforming growth factor‑β, Wnt and mitogen‑activated protein kinase signaling pathways. These data indicated for the first time, to the best of our knowledge, that miR‑125b may function as an oncogene in lung cancer.

We investigated the effect of low-dose, long-term benzo(a)pyrene exposure on the miRNA expression profile in lung tissue of mice, and the potential mechanism of miRNAs in the benzo(a)pyreneinduced damage to health. Subject mice were treated with 5 μg/kg benzo(a)pyrene twice a week for 8 weeks by intragastrical administration, while control mice were treated with the same volume of olive oil solvent. All mice were then fed for another 8 weeks without exposure to benzo(a)pyrene, after which total RNA was isolated from lung tissue. miRNA expression profiles were generated by SOLiD™3 high-throughput sequencing and the signaling pathways represented were analyzed by DIANA-mirPath. A total of 74 miRNAs were dysregulated in mice lung tissues exposed to benzo(a)pyrene. Signaling pathways regulated by benzo(a)pyrene exposure included those involved in the environmental information process and human tumorigenesis. We conclude that low-dose, long-term benzo(a)pyrene exposure alters specific miRNA expression profiles.

To evaluate the combined cytotoxicity effects between functionalized multi-walled carbon nanotubes (MWCNTs-PC) and cigarette smoke solution (CSS), 16-HBE cells was used as the target cells and exposed to various concentrations of MWCNTs-PC and CSS combined together. Cell proliferation, cell apoptosis, DNA damage were detected by Methyl thiazolyl tetrazolium (MTT) assay, flow cytometry, single cell gel electrophoresis assay (SCGE) and micronuclear assay, respectively. The dose-dependent cytotoxic and genetic effects of CSS were found in our study. However, compared to the control group, the MWCNTs-PC exposed groups showed no significant difference in all concentration, with or without CSS exposure. It suggests that the MWCNTs-PC did not influence cellular toxicity or DNA damage of CSS on 16-HBE cells. No combined cytotoxic effects between NWCNTs-PC and CSS were found in this study.

OBJECTIVE:To study the effect of high-dose long-term benzo(a)pyrene[B(a)P]exposure on miRNAs expression profile in lung tissues of mice and analyze the potential mechanism of miRNAs on the B(a)P-induced health damage.METHODS:40 ICR mice were divided into control group and exposure groups randomly.Each group included 10 male and 10 female mice.Exposure group mice were treated with 50 mg/kg B(a)P(twice a week) for 8 weeks by intragastric administration.Control mice were treated with same volume of olive oil solvent.Then,all mice were fed for another 8 weeks without exposure.The total RNA was isolated from mice lung tissues.The miRNAs expression profiles were evaluated by SOliD high-throughput sequencing technique and the difference expression of miRNAs were analyzed. Real time-PCR was used to confirm the miRNAs expression.The potential targets of miRNAs were calculated by TargetScan,miRanda and picTar software.RESULTS:Most of miRNAs were found at low expression levels in lung tissue.Compared with control mice,109 miRNAs expression changed significantly in lung tissue of B(a)P exposure mice. Of these 109 miRNAs,50 were up-regulated and 59 were down-regulated,the expression changes were 7.43-fold and 40.63-fold,respectively.miR-20b was analyzed by real time-PCR to verify the sequencing.The result of real time-PCR was consistent with sequencing.The potential targeted proteins of miR-20b were involved in cell proliferation,cell cycle,apoptosis and oncogenesis.CONCLUSION:High-dose long-term B(a)P exposure could specifically alter miRNAs expression profiles.The differential expression of miRNAs may play an important role for the B(a)P-induced health damage.

Objective To observe the toxic effect of chlorpyrifos on the ovary of mice and the expression level of apoptotic genes in ovary of exposed mice.Methods Fifty ICR mice were randomly divided into five groups,10 in each group,including a negative control group(corn oil),a positive control group(estradiol) and three experimental groups,7.5,15.0 and 30.0 mg/kg.Female mice were treated by gavage for two weeks.Follicle constituent ratio and apoptosis of ovary granulose cells were observed,and the expression levels of mch3 and bcl-2 gene were checked by RT-qPCR.Results The ratios of primordial follicle in 15.0 and 30.0 mg/kg groups and the ratio of atresic follicle in 30.0 mg/kg group were significantly higher compared with the control(P<0.05).The ratios of growing follicle in 15.0 and 30.0 mg/kg groups and the ratio of mature follicle in 30.0 mg/kg group were significantly lower compared with the control(P<0.05).With the increase in the dose of chlorpyrifos,the ratio of primordialo follicle showed increasing tendency,the ratio of mature follicle suggested decreased,the ratio of growing follicle increased at first then decreased,and the ratio of atresic follicle decreased at first then increased.The apoptosis of ovary granulose cells were significantly increased at the dose of 30.0 mg/kg(P<0.05),and it showed increasing tendency with the increase of the dose of chlorpyrifos.The expression level of mch3 were significantly increased at the dose of 15.0 and 30.0 mg/kg(P<0.05),and bcl-2 gene were significantly decreased at the dose of 15.0 and 30.0 mg/kg in a dose-dependent manner(P<0.05).Conclusion Chlorpyrifos may have female reproductive toxicity,through increasing the expression level of mch3 as well as decreasing the expression level of bcl-2 gene,inducing the apoptosis of ovary granulose cells and inhibiting the development of follicle.

The widespread explored application of water soluble carbon nanotubes makes it important to understand their potential toxic effects on health. This study investigates the effects of phosphoryl choline grafted water soluble multi-walled carbon nanotubes (MWCNTs-PC) on human bronchial epithelial (16-HBE) cells by different cytotoxicity methods in vitro. Various concentrations of MWCNTs-PC were incubated with 16-HBE cells, the effects of cell proliferation, cell apoptosis, cell cycle and DNA damage were detected by methyl thiazolyl tetrazolium (MTT) assay, flow cytometry, single cell gel electrophoresis assay (SCGE) and micronuclear assay, respectively. Compared with the control group, there were no significant differences in the changes of cell proliferation, cell apoptosis, cell cycle and DNA damage. Within the experimental concentrations of MWCNTs-PC, no obviously cytotoxicity and DNA damage was observed on 16-HBE cells in this study.

Objective To evaluate the acute toxicity,the bio-distribution and the other biological effects of three types of water-soluble carbon nanotube derivates.Methods One hundred and forty-four Sprague-Dawley(SD) rats were randomly divided into seven dose groups and a control group and treated with CNTs throughh caudal vein injection at the dosages of 12.5,25,50,70,100,140 and 200 mg/kg,6 rats each group.The injection volume was 10 ml/mg.One week later,rats were sacrificed by exsanguinations' via the femoral aorta.Whole blood as well as the liver,spleen,lung tissues were collected for further assay.Results At extremely high doses,CNT-PEG was easier to cause the death of rats than CNT-PC and CNT-PEG-PC.Compared with CNT-PEG and CNT-PEG-PC,CNT-PC was obviously easier to penetrate into lung tissues and ingested by liver and spleen tissues.The biochemical indices such as ALB,ALT and Cr did not have significant changes compared with the control groups(P0.05).CNT-PC could induce the proliferation and activation of lymphocytes(P0.05).There was no obvious clinical manifestation of liver,spleen or lung injury in rats after 7-day exposure of all of three types of CNT derivates.Conclusion Three kinds of modified CNTs did not show obvious acute toxicity to the rats,but the CNTs with different chemical constitution may induce distinct behaviors of CNTs in vivo such as bio-distribution and other biological effects.

Multi-walled carbon nanotubes (MWCNTs) were modified with three types of hydrophilic moieties: phosphatidylcholine (PC), polyethylene glycol (PEG), and PC-terminated polyethylene glycol (PEG-PC). Based on the high water dispersibility of modified carbon nanotubes (CNTs), the extremely high doses of these CNT derivatives were intravenously injected in rats from 12.5 to 200mg kg(-1). Based upon pathologic results, most of the injected CNT derivatives accumulated in the lung, whereas CNT-PC dispersed throughout the organism compared to CNT-PEG and CNT-PEG-PC, thereby resulting in a broader distribution in liver and spleen which may be biologically relevant. The modified CNT induced only low acute toxicity, which may contribute to biocompatibility of mammals to these grafted functional groups.

MicroRNAs (miRNAs) were recently identified to play crucial roles in tumorigenesis processes. In present study, we explored the potential influence of miRNA-143 on several cellular biological processes related with lung cancer development in human bronchial epithelial cells 16-HBE. Chemosynthesis anti-miR-143 was used to down-regulate expression of miR-143. Cell proliferation was detected by methyl thiazolyl tetrazolium (MTT) assay after 24, 48 and 72 h transfection of anti-miR-143. Apoptosis and cell cycle were detected by flow cytometry after 48 h transfection of anti-miR-143. The results revealed that there were no significant differences in cell activity and apoptosis between transfection group and control group (P>0.05). Compared with control group, DNA content of G1 period of anti-miRNA-143 group had a mild decrease (P<;0.05), DNA content of S period of anti-miRNA-143 group had a mild increase (P<;0.05). The results suggested that down-regulated miR-143 induce S arrest of cell cycle. MiR-143 may associate with lung cancer development through influence cell cycle progression. The cooperation between more miRNAs and environmental carcinogen on the tumorigenesis of lung cancer needs to further investigate.

MicroRNAs (miRNAs) were recently identified to play crucial roles in tumorigenesis. In present study, we investigated the miRNAs expression profile in human bronchial epithelial cell exposed to B(a)P by high-throughput microarray analysis, and screened the different expressed miRNAs in cells exposed to B(a)P compared with control cells. 16-HBE cells of exposure group were treated 3 times with B(a)P at a concentration of 2.0 mu mol/l and had 20 passages. Cells of control group were treated with DMSO solvent by the same way. The total RNA of cells was isolated and the quality of RNA was monitored. Then, RNA was labeled and hybridization was carried out on the Affymetrix miRNA Array. The hybridization signals were detected and scanner images were quantified. The results showed that miR-21, miR-27a, miR130b, miR-345, miR-720 and miR-768 were more than 2 times up-regulated, and miR149, miR-638, miR-663 and miR-1228 were more than 2 times down-regulated in 16HBE cells exposed to B(a)P. The results suggested that altered expression of specific miRNAs may be involved in tumorigenesis induced by B(a)P and their function need to confirm in the future study.

[Objective] To evaluate the potential cytotoxicity of titanium dioxide (TiO2) on mouse embryonic fibroblast (NIH 3T3) cells, either alone or in combination with UV radiation. [Methods] 10nm- anatase, 25nm-anatase, 25nm-rutile and 200nm -anatase TiO2 were dispersed in methylene blue aqueous solution with different concentration, followed by UV radiation, to qualitative analyze the free radical generation. MTT assay was employed to detected 3T3 cell viability after treated with different concentrations of TiO2 (15.6, 31.2, 62.5, 125.0 and 250.0 mu g/ml, respectively), with or without UV exposure and lipid peroxidation products (MDA) was observed as an indicator of oxidative stress damage. [Results] The result of photocatalytic degradation of methylene blue showed that free radical generation is associated with particle size, crystalline form and concentration of TiO2. The photocatalytic degradation of methylene blue decreased when the TiO2 concentration increasing. The anatase TiO2 showed a significant size-dependent decrease of photocatalysis capacity. The photocatalysis of rutile TiO2 was weaker than anatase TiO2 with the same size (P<0.05). The results of cell viability and the level of lipid peroxidation products (MDA) showed that the cytotoxicity of TiO2 increased in a dose-dependent manner, with or without UV radiation. Further, UV exposure may enhance the toxic effect of TiO2 to 3T3 cells. For the same concentration of TiO2, cytotoxicity decreased when the particle size increased, and effect of anatase form was stronger than that of rutile form (P<0.05). [Conclusion] TiO2 showed potential cytotoxicity to 3T3 cells in a dose- and size-dependent manner. Furthermore, UV radiation caused decreases of cell viability, which were most likely due to hydroxyl radicals induced oxidative stress damage.

MicroRNAs(miRNAs) are small non-coding RNAs, which could have associations with the occurrence or development of cancers. In this study, we investigated an effect of the down-regulated miR-143 on human bronchial epithelial cells (16-HBE) exposed to Benzo[a]pyrene [B(a)P]. Anti-miR-143 was transfected into cells to down-regulate the expression of miR-143. Cell proliferation, apoptosis and cycle of transfected cells were detected by methyl thiazolyl tetrazolium (MTT) assay and flow cytometry after 24 h exposed to B(a)P, respectively. Our results confirmed that B(a)P has significant toxic effect on cell proliferation, raised the apoptosis rate and blocked more cells at the S period in cell cycle (P<0.05). However, there is no significant difference between transfected group and non-transfected group after cells exposed to B(a)P (P>0.05). The results suggested that down-regulated miR-143 has no significant influence on cellar bio-effects induced by B(a)P under the presented experimental conditions.

[目的] 运用微阵列芯片技术研究长期低剂量暴露于苯并(a)芘[B(a)P]后小鼠肝组织和肺组织的miRNAs表达谱差异，并对差异表达miRNAs进行靶基因预测，探讨miRNAs与外源性致癌物健康效应的关系。 [方法] 18-22g 小鼠随机分为对照组和染毒组，每组20只，雌雄各半。经口给予小鼠5μg/kg B(a)P，每周2次，持续8周，对照组同时给予同等剂量的橄榄油溶剂。染毒结束后继续饲养8周，取肝脏组织和肺脏组织，Trizol法分别提取组织总RNA，miRNAs芯片杂交，检测小鼠肝脏组织和肺脏组织的miRNAs表达谱，并对暴露组和对照组的miRNAs进行差异表达分析。运用miRanda和TargetScan软件预测miRNAs可能调控的靶基因。 [结果] 对照组的肝、肺组织的信号强度分析结果显示，绝大部分的miRNAs表达丰度较低，少数miRNAs表达丰度较高较高，其中miR-122, miR-762, miR-22, miR-194, miR-494, miR-192只在肝组织中具有较高表达丰度，miR-23a, miR-145, miR-126-3p, miR-143, miR-16, miR-125b-5p, miR-103只在肺组织中具有较高表达丰度。与对照组相比，暴露组的miRNAs表达谱发生明显改变。肝组织中共有21个miRNAs表达异常，其中10个miRNAs上调，11个miRNAs下调，最大差异表达为5.65倍。肺组织中共有24个miRNAs表达异常，其中19个miRNAs上调，5个miRNAs下调，而最大差异表达8.76倍。B(a)P暴露引起的差异表达在肝组织和肺组织中的表现方式具有很大的不同，只有miR-720和miR-699同时在肝组织和肺组织明显下调，绝大部分miRNAs只在肝组织中或者肺组织中有明显改变，有4个miRNAs甚至在肺组织中明显上调而在肝组织中明显下调。对在肝、肺组织中均显著异常表达的miR-720进行信息学分析，筛选出的靶基因中涉及信号转导因子、免疫调节因子、癌症相关基因等。[结论]长期低剂量B(a)P暴露可以引起小鼠肝、肺组织的miRNAs表达产生特异性改变，可能在B(a)P致机体健康效应过程中起着重要作用。