The Establishment And Mechanism Study Of Xuanwei Area C1 Coal Combustion-induced F344 Rat Lung Cancer Model

[Background and Objective]Xuanwei area (including Xuanwei, Fuyuan, Qilin,Yunnan Province and Zhanyi, Pan County, shuicheng, liupanshui, Guizhou Province), located in southwest China, the region has a population of about 3.1 million, is a high incidence of lung cancer in China and the world, some scholars have called the local lung cancer in Xuanwei lung cancer or zhujiangyuan lung cancer. Previous studies showed that the mortality rate of lung cancer in Xuanwei geographical distribution of the Late Permian bituminous coal output C1 geographical distribution in space highly consistent, regional lung cancer incidence C1 bituminous coal output. Wherein Xuanwei area laibin town is the highest incidence of lung cancer local towns, reaching 128.31/100,000, is 2-3 times the national incidence of lung cancer; and the same text in Xuanwei wenxing town, lung cancer incidence rate of only 5.83/100,000, well below the national lung cancer incidence level, inhabitants of the laibin town is mainly used as a fuel for life C1 bituminous coal, while the text promotes a town inhabited mainly by using M30 coal as fuel for life. Our previous study on Xuanwei laibin town C1 bituminous showed, C1 bituminous coal in a large number of nano-silica (Silicon dioxide, SiO2) is one of the important factors leading to the occurrence of local high incidence of lung cancer. The study of the high incidence of lung cancer in Xuanwei HE Xing Zhou, also suggest that the high incidence of lung cancer in Xuanwei indoor air benzopyrene (Benzoapyrene, BaP) pollution-related. High incidence of lung cancer in Xuanwei reason may be due to prolonged physical and chemical stimuli and genetic factors common cause. Early in vitro experiments also show that coal dust exposure small non-coding RNA (MicroRNA, miRNA) expression Bease-2B abnormal cells appear. miRNA is considered in the regulation of developmental process of tumorigenesis have played an important role. Part miRNA genes may play a role in cancer and tumor suppressor genes, in part by regulating miRNA proto-oncogene or tumor suppressor gene or genes involved in the formation of cancer form tumors. But the study of the material composition of coal-fired combustion products and the impact on the human body, etc. toxicology studies are currently limited to a single substance, a true reflection of lack of indoor air pollution Xuanwei local state apparatus to establish appropriate animal disease models; and the establishment of an animal model of lung cancer can be dynamically study the pathogenesis, the study Xuanwei C1 bituminous mechanisms leading to high incidence of lung cancer has important significance, provide a theoretical basis for looking for targets for prevention and treatment.In this study, PM2.5 concentration control based on the experimental apparatus constructed in Xuanwei open "fireplace"; the different coal-fired (C1 bituminous coal, M30 coal) combustion products induce F344 rat models constructed with primary lung cancer in Xuanwei, Discussion Effect of bituminous coal combustion products of the ingredients to build an animal model of lung cancer; the use of miRNAs miRNAs abnormal expression microarray, the process of finding a target gene in lung cancer. Reveal the molecular mechanism of the high incidence of lung cancer in Xuanwei, provide experimental evidence for the prevention and treatment of local lung cancer.[method]The study is divided into three parts:1, coal characteristics research of different Xuanwei regions and experimental established:(1) by thermal cracking apparatus for C1 bituminous coal and M30 coal at different temperatures and pyrolysis, using pyrolysis GC-MS to release organic analysis. (2) by simulating combustion using air sampler for C1 and M30 bituminous coal combustion products within the experimental device PM2.5 collected on filters, using ICP-MS for inorganic constituents on the filter is analyzed; (3) adopted jointly controlled fan and SCM, simulation equipment based Xuanwei open "fireplace" state of indoor air pollution, to build a relatively constant PM2.5 closed environment, in experimental animals exposed to the air for exposure.2, building C1 bituminous coal combustion products exposed F344 rats with lung cancer:using equipment F344 rats were exposed to C1 and M30 bituminous coal combustion products of exposure, to establish lung cancer model F344 rats; (1) 90 female F344 rats were randomly divided into C1 bituminous coal high-dose group, C1 bituminous low-dose group, M30 seam high dose group, M30 seam low-dose group and normal control group,18 in each group, were exposed to coal dust; daily in the low dose group exposed for 1 hour high-dose group was exposed 2 hours a day; (2) the total exposure time was 6 months, each month, rats were sacrificed 3, the lung tissue was observed in rats Macroscopic appearance changes, it has been in the right lung after the trachea and fixed in formalin changes embedded, sliced, HE staining of lung tissue and tumor cases, glutaraldehyde-fixed portion is electron microscopy with the left lobe were detected -80℃ Freezing prepare gene chips and western blot spare; (3) using a transmission electron microscope to observe ultrastructure of lung tissue changes.3, miRNA C1 bituminous coal combustion products in infected lung F344 role modeling process (1), using the seventh generation of rats miRCURY LNATM microRNA microarray expression C1 bituminous modeling F344 rat lung tissue were miRNA testing, screening in the modeling process abnormal expression of Mi-RNA spectrum; (2) using STEM method miRNA abnormal expression of trend analysis, screening sustained abnormal expression miRNA; (3), using mirbase, miranda database and mirdb sustained abnormal expression of Mi-RNA target gene prediction information, select a more credible target gene; (4) using qPCR method for miRNA abnormal expression verified; (5), using western blot for F344 target protein within lung tissue were detected; (6), dual luciferase reporter system to validate target genes.[result]1, (1), coal pyrolysis product analysis: ①300℃ when C1 fired pyrolysis products 23 substances, including 6 kinds of PAHs categorys, accounting for 6.08% relative content; C1 coal-fired thermal decomposition products at 500℃ 72 species, including 20 kinds of PAHs category, accounting for 33.70% of the relative content; 700℃ when C1 fired pyrolysis products 150 species, including 37 kinds of PAHs category, accounting for 37.81% of the relative content; ②M30 fired pyrolysis products 300℃ when there are 12 substances, no PAHs class product release; 500℃ when M30 fired pyrolysis products 63 species, including 17 kinds of PAHs category, accounting for 19.32% of the relative content; 700℃ when M30 fired pyrolysis products have 126 kinds in which 38 kinds of PAHs class,34.38% relative content; ③C1 different bituminous coal pyrolysis product with M30 pyrolysis products of organic ingredients, compared with the M30 coal, C1 bituminous coal there PAHs released at low temperature, and the same at a temperature higher PAHs content C1 bituminous release, the structure is more complex; (2), PM2.5 component analysis:①chromium content C1 of PM2.5 in the bituminous coal (4.59±0.8ug/ m3) ratio M30 coal production chromium content (0.57±0.02ug/m3)-between the high and the difference was statistically significant (p<0.05); ② C1 coal PM2.5 in iron content (54.4 ± 2.3ug/m3) is higher than the iron content M30 coal production (23.1 ± 0.2ug/m3), there was significant difference between them (p <0.05); the content of silicon ③C1 coal PM2.5 in (3.74 ± 0.21μg/m3) than coal production of silicon content M30(1.18 ± 0.04μg/m3), there was significant difference between them (p<0.05) (3), controlled by PM2.5 successfully constructed of indoor air pollution experimental device.2, (1) bituminous coal high dose group successfully induced lung cancer in rats 6, carcinogenesis success rate 33.3%; M30 seam high dose group 1 induced lung tumor induction success rate of 5.6%, more than in each group showed no lung cancer formation, C1-dose group compared with bituminous coal story among the group, the difference was statistically significant (p<0.05).(2) C1 F344 rats exposed to coal-fired combustion products of alveolar type Ⅱ cells lamellar bodies significant reduction in lamellar structure disappeared, emptying abnormalities, vacuolization; ultrastructure mainly in alveolar type Ⅱ cell injury based.3, (1) compare with normal control group, there are 30 days of exposure down 2 miRNA differential expression was statistically significant (P<0.05); exposure of 60 days three months there have been regulated miRNA expression difference was statistically significant (P<0.05); there were 90 days of exposure 3 regulated miRNA and miRNA expression difference down three statistically significant (P<0.05); exposure of 120 days,9 regulated miRNA and miRNA expression difference down two statistically significant (P<0.05); exposure of 150 days, respectively,10 up-regulated the expression of miRNA and miRNA down two differences were statistically significant (P<0.05); exposure of 180 days, respectively,17 up-regulated miRNA and miRNA expression difference was statistically lower 13 significance (P <0.05); (2) STEM analysis found that miR-101B-3P, sustained expression upward miR-433-5P, miR-872-3P, miR-465-3P, miR-331-5P, the difference statistically significant (p<0.05); (3), mirbase predict miR-872-3P 436 target genes, miR-101B-3P 794 target genes; miranda predict miR-872-3P 726 target genes, miR-101B-3P has a 1083 target genes, miR-465-3p 33 target genes, miR-331-5p 82 target genes; mirdbl predict miR-872-3P 51 target genes, miR-lOlB-3P 296 target genes; a single database predicted target genes 2359; 2197 databases simultaneously predicted target genes; three databases simultaneously predicted 18 target genes; 3 each database are predicted miRNA target genes only sustained expression of miR-101B-3P and miR-872-3P (4) with the extension of modeling time, C1 bituminous coal combustion products in high dose group F344 rat lung tissue; miR-101B-3P and miR-872-3P increased; (5) Rat lung tissue expression Has2, Ing3, ogt, Plcgl, Serpini in C1 bituminous group exposed rats F344 model group; Protein expression Ing3 and Serpini 1 was gradually decreased. (6) Ing3 is a target gene of miR-101b-3p; Serpini1 is a target gene of miR-872-3p; Has2 not miR-101b-3p target gene; Ogt not miR-101b-3p target gene; Plcgl not miR-101b-3p target genes.[conclusion]1, PM2.5 concentration control can be constructed in Xuanwei open "fireplace" experimental device caused by indoor air pollution;2, the C1 bituminous coal combustion chromium, iron, silicon and PAHs content may be one of the important factors in the high incidence of lung cancer in Xuanwei;3, C1 bituminous coal combustion products exposed to air exposure can be constructed with primary lung cancer in animal models;4, the combustion products build bituminous C1 lung model, miR-101b-3p sustained expression and inhibiting the function of the target gene Ing3 inhibit tumor suppressor gene function, Ing3 maybe a C1 bituminous important mechanism to induce lung cancer; miR-872-3p Length expression and inhibiting the function of the target gene Serpinil, Serpinil gene function may C1 bituminous important mechanism to induce lung cancer; Ing3 and Serpini1 may be a potential target for prevention and treatment.