Relationship Between High Incidence Of Lung Cancer Among Non-smoking Women And Naturally Occurring Silica In Bitu Minous Coal And The Possible Mechanism Of Carcinogenesis In Xuanwei, China

[Background and Objective]:China’s Xuanwei County in Yunnan Province have the worid’s highest incidence of lung cancer in non-smoking women-20times higher than the rest of China. Previous studies showed, this high lung cancer incidence may be associated with the silica particles embedded in the production combustion from C1coal, and no significant associated with smoking and industrial pollution. But, previous studies are not statistically quantified nor separated and characterization of silica particles in the production combustion from C1coal, these silica particles’size distribution, morphology, containing trace elements are important factors for the lung cancer epidemic. The International Agency for Research on Cancer (IARC) has classified crystalline silica was stated to be a class I carcinogen, because it can cause pulmonary fibrosis. But, we can’t know may be carcinogen mechanism of naturally occurring silica.We simulated the natural condition of women in Xuanwei County exposure to silica particles, for long time and low concentration transtracheal drip of silica nanoparticles in rat colon cancer, and found that the inflammation is the main form of lung injury. However, the molecular mechanism of from the inflammatory response to cancer is unclear, in recently studies, finding a no-code small RNA (microRNA, miRNA) may be serve as a bridge between inflammation and cancer. Also is the inflammatory cytokines produced from the inflammatory response can stimulate the miRNA expression up-regulated, and the miRNA expression up-regulated can expanding the inflammatory response and can promote the tumorigenesis. We study based on the following objects:(1) separateding the silica particles from the production combustion from C1bituminous, how unusual is the naturally occurring silica of morphology, size distribution, surface characteristics compared to the industrially occurring silica and crystalline silica by physical method.(2) Simulation of combustion test to calculation of daily total exposure of women living in the Xuanwei area to silica nanoparticles, which can provide a proof for cell experiment in vitro and animal experiment in vivo.(3) Gene chip technique screening miRNAs expression which associated with Xuanwei non-smoking female lung cancer.(4) Study the effect of naturally occurring silica caused miRNA expression and inflammatory cytokines excretion to bronchial epithelial cells, and preliminary exploration that the may be carcinogenic mechanism of naturally occurrlly occurring silica to human.[Methods]:(1)The20samples C1bituminous coal widely used in the high-incidence area of lung cancer in Xuanwei was selected as experiment group, while the C2+1, K7and M30bituminous coal that was mined and used in the low-incidence area of lung cancer in Xuanwei were selected as control group. Twenty paraffin-embedded cancer tissue samples from the non-smoking women with no small cell lung cancer who were born in Xuanwei and were at least the3rd generation of the family living there were collected from the department of pathology, the third affiliated hospital of Kunming medical university (tumor hospital of Yunnan province). Titrimetric potassium silicofluoride method was used to measure the content of silica in raw coal and its bottom ashes in20samples from the experimental group and control group. Scanning electron microscope (SEM) was used to observe the morphology of silica particles in C1bituminous coal and its bottom ashes, and scanning electron microscopy coupled with energy dispersive X-ray analyzer used to observe the morphology of silica particles in the bottom ashes and coal soot of C1bituminous coal as well as the lung cancer tissue from the non-smoking women in Xuanwei,and transmission electron microscope coupled with energy dispersive X-ray analyzer (TEM-EDX) was used to analyze the microscopic composition. The combustion experiments to simulate the consumption and cooking practice, soot was collected using air pollution monitoring equipment, separateding silica particles from soot by physical methods, particle size distribution was measured by a laser particle size analyzer, the surface areas of the particles were determined by BET nitrogen adsorption analysis, the women living in the Xuanwei area to silica nanoparticles every woman’s daily total exposure amount from indoor air was calculated by an indirect method.(2) Selecting2pairs cancer tissue and corresponding non-malignant lung tissue samples with no small cell and non-smoking female lung cancer from the department of pathology of the third affiliated hospital of Kunming medical university (tumor hospital of Yunnan province). Detecting miRNAs and mRNAs differentially expression between lung cancer tissues and their corresponding normal lung tissue, testing the miRNAs relative expression levels in20cases came from Xuanwei County by real time PCR technology which significant upregulate and downregulate in lung cancer tissue, biological information genetics predict the target genes controlled by significant upregulated miRNAs, gene ontology (GO) analysis that was used to categorize changes in mRNA expression in lung cancer tissues,(3) Respectively make the mass and surface area as a metric, assesssment the bronchial epithelial cells viability change exposed to silica after48-h at differential concentration, observing the subcellular distribution of silica nanoparticles in human brochial epithelial cells after24-h at100μg/mL by TEM, Enzyme Linked Immunosorbent Assay (ELISA) detecting Interleukin8(IL-8), y-interferon (y-IFN), Basic Fibroblast Growth Factor/Fibroblast Growth Factor2(bFGF/FGF-2) and Vascular Endothelial Growth Factor (VEGF) secretion changes of bronchial epithelial cells exposed to silica particles at100μg/mL after24-h and48-h, detecting the miRNAs expression levels by real-time PCR at0-h,24-h and48-h, Pearson correlation analysis the relationship between inflammatory cytokines secretion and the expression of miRNAs.[Results]:(1)The silica content in C1bituminous coal and its bottom ashes in the high-incidence area of lung cancer in Xuanwei was significently higher than that in C2+1, K7, and M30bituminous coal in the low-incidence area of lung cancer in Xuanwei(P<0.05). We can’t found the free silica particles in raw coal and can found free silica particles in combustion products (coal ashes and soot), and can observed the free silica particles in lung cancer tissue which come from high-incidence area of lung cancer in Xuanwei. We can separated the silica particles from combustion products (coal ashes and soot) by physical methods, these naturally occurring silica particles were mostly nanoscale, have various shape, large surface area, and contained such as iron,aluminium, calcium, and potassium. We calculated the daily silica nanoparticle exposure of women living in the Xuanwei area as2.25±0.35μg by indirectly method.(2) Genes chip analysis shows that have495miRNAs expression levels in non-smoking female lung cancer tissues differently from the corresponding non-malignant lung cancer tissues, which have251miRNAs significant up-regulared (≥2folds) and have208miRNAs significant down-regulated (<2folds), miR-508-5p, miR-1270, and miR501-5p significant up-regulated and miR-24-3p and miR-103a-3p significant down-regulated in non-smoking female lung cancer tissue of Xuanwei. Founding the miR-508-5p, miR-1270, and miR501-5p have a stablely up-regulate expression levels and the miR-24-3p and miR-103a-3p have a stablely down-regulate in20cases came from Xuanwei County by real time PCR technology, mostly target genes which controlled by significantly up-regulated miRNAs are contribute to inflammatory response through biological information genetics predict. Genes chip analysis27958mRNAs, finding4263genes significant up-regulated and1922genes significant down-regulated in non-smoking female lung cancer tissue of Xuanwei. Gene ontology (GO) analysis all differentially expression (DE) genes, finding have79.4%genes are contribute to inflammatory response.(3) The silica particles which separated from C1coal combustion production (soot) have a large affect to cell viability in human bronchial epithelial cells (BEAS-2B) compared to the50-nm silica and crystalline silica, TEM revealed that the naturally occurring silica particles more easily enter human bronchial epithelial cells, and all silica nanoparticles can’t enter cell nucleus. IL-8and y-IFN were released from BEAS-2B cells exposed to the naturally occurring silica particles and50-nm silica particles, bFGF/FGF-2and VEGF were released from BEAS-2B cells exposed to the crystalline silica. The miR-508-5p, miR1270and miR501-5p expression levels in human bronchial epithelial cells increased5,7and6times compared to blank control group exposed to naturally occurring silica particles at100μg/mL after24h, The miR-508-5p, miR1270and miR501-5p expression levels in human bronchial epithelial cells increased7.5,10and10times compared to blank control group exposed to naturally occurring silica particles at100μg/mL after48h, significant higher than the50-nm silica particles group and crystalline silica group (p<0.01). Detecting the target genes by three miRNAs control of Phosphoinositide3Kinase Adapter Protein (PIK3AP) expression levels by fluorescent probe, finding the expression level in naturallying occurring silica group significant lower than other group (p<0.01). Results of the correlation analyses indicate that the correlation between IL-8and开，啊-INF contains and three miRNAs expression level respectively are0.925and0.948, and the bFGF/FGF2and VEGF contain and three miRNAs expression level respectively are-0.947and-0.964after24-h, the correlation between IL-8and γ-INF contains and three miRNAs expression level respectively are0.978and0.933, and the bFGF/FGF2and VEGF contain and three miRNAs expression level respectively are-0.630and-0.732after48-h.[Conclutions]:(1) In Xuanwei, the incidence of lung cancer among non-smoking women is high in the area where silica-rich C1bituminous coal is produced. There are silica particles enriched in both the combustion products of Cl bituminous coal and the cancer tissue from the non-smoking women with non-small cell lung cancer, with similar morphology and microscopic composition. We hypothesize that the silica particles from combusted C1bituminous coal in Xuanwei are mixed with indoor air and inhaled along with other suspended particles.(2) The high lung cancer incidence in the Xuanwei area may thus result from low-dose, chronic exposure to naturally occurring silica nanoparticles over a longer time.(3) The high lung cancer incidence in the Xuanwei area may have pathogenic pathways: low-dose, chronic exposure to naturally occurring silica nanoparticles over a longer time, chronic nonspecific inflammation response can cause inflammatory cytokines released and stimulate the miRNAs expression up-regulate, and finally give rise to the lung cancer.