Study On The Mechanism Of PM2.5 Promoting The Development Of Non-small Cell Lung Cancer And The Effect Of Brucea Javanica Oil Emulsion On The Respiratory Tract Microecology In The Treatment Of Lung Cancer

Purpose: To investigate the mechanism of the progressive development of non-small cell lung cancer in cells and lung cancer-bearing mice after PM2.5 exposure.To explore the mechanism of the change of substance in lung cancer after PM2.5 exposed in vitro and vivo assays.To research the expression of differentially expressed genes in vitro assays,perform the lung cancer-bearing mice to compare the numbers of tumor nodule,analyze the expression of the twenty-four angiogenesis,confirm the levels of the expression with key differentially expressed genes on the tumor tissue,and carry out the respiratory tract microbial floras research in lung cancer after PM2.5 exposure.Finally to further clarify the new direction of the development of lung cancer after PM2.5 regulation,and lay a foundation by forming the new effective methods for prevention and treatment of diseases.Materials and methods: 1.To acquire the concentration of the PM2.5 exposure in cell viability assay.Human NSCLC cell lines A549 was seeded at a density of 1.0 × 104 per well in triplicate in 96-well plates and kept at 37 in 5% CO℃ 2,and grown to 95% confluence and tackled with PM2.5 at the five different concentration for 72 h,and the same amount of PBS(Phosphate Buffered Saline)was added in the control group.Absorbance was taken at a wavelength of 490 nm to confirm the cell viability.2.Cell proliferation assays with the conditioned medium from PM2.5 exposure.We collected the cultured medium from the A549 and H1299 cell lines after PM2.5 exposure.Alternatively,in PM2.5 exposure group,cultured condition is composed of PM2.5-exposed supernatants and RPMI1640 absolute medium at a ratio of 1:3;in control group,cultured condition is composed of cultured medium and RPMI1640 absolute medium at a ratio of 1:3.Cell viability was assessed to confirm the effects on the two cell lines.3.Perform the Monolayer wound healing assay to validate the function.We collected the A549 and H1299 cell lines after PM2.5 exposure,and then PM2.5-exposed cells were plated at a density of 2×106 per well in six-well culture dish,and cultured in a CO2 incubator for 12 h.The PM2.5-unexposed cells were seeded as control groups.Cells were then allowed to scratch after 12 h with the 10μl pipet tip,at the end of which,The media and displaced cells were then removed,and cultured with 2% low serum culture medium.Photos were taken of the scratches,including the reference points,at 0,24,and 48 h to monitor closure of the scratch.4.Perform the invasion assay to validate the function.We collected the A549 and H1299 cell lines after PM2.5 exposure.The upper side,namely Transwell,was coated with 50μl/cm2 reconstituted basement membrane substance.The coated filters were air-dried for l hrs prior to the addition of the cells,including PM2.5-exposed cells and PM2.5-unexposed cells.The lower compartments were filled with RPMI containing 10% FBS,and 1×104 cells were resuspended in 100μl RPMI and placed in the upper part of a transwell plate.The percentage of cell migration was calculated after 20 hrs in a humidified atmosphere of 5% CO2 at 37℃.5.Perform the RNA-Seq to acquire the differentially expressed genes in NSCLC after PM2.5 exposure.We collected the A549 cells after PM2.5 exposure,and carried out the RNA-Seq array to analyze the expression of the differentially expressed genes after PM2.5 exposure.The Gene Ontology(GO)project aims to describe gene and gene product attributes,which covers three domains: biological process,cellular component and molecular function.Pathway analysis is a functional analysis that maps genes to Kyoto Encyclopedia of Genes and Genomes(KEGG)pathways.6.Perform the quantitative real-time PCR to validate the differentially expressed genes.We collected the A549 and H1299 cells after PM2.5 exposure,and control group.Total RNA extracted from experimental samples was performed reverse transcription assay.And we performed the QRT-PCR to validate the expression of the differentially expressed genes on the level of transcription.GAPDH was used as an internal control and the expression of the target RNA was normalized to GAPDH.Thresholds for statistical significance are noted in Results.Relative expression levels were calculated using the 2-ΔΔCt method.7.Perform the Enzyme-linked immunosorbent assay to validate the expression levels of MMP1 and IL1β in culture supernatants.We collected exposed culture supernatants after PM2.5 exposure,and control group.The PM2.5-unexposed culture supernatants were collected as control group.The production of IL1β and MMP1 in PM2.5 exposed group and control group was measured by enzyme-linked immunosorbent assay.8.Perform the Western blot assay to validate the expression levels of MMP1 and IL1β in A549 and H1299 cell lines. We collected the A549 and H1299 cell lines after PM2.5 exposure and control group.The total protein of each group was extracted,and the total protein concentration of each sample was detected by BCA assay.We perform the Western blot assay to validate the expression levels of MMP1 and IL1β.We detected the optical signal intensity of 700 nm and 800 nm using Odyssey two-color infrared fluorescence system,and analyzed the figure with Image J software.9.Established 32 lung cancer-bearing mice with A549 cells in 5×105 each mouse,randomly divided into 2 groups(n=16)using a random number table method.That is to say,PM2.5 exposed group and its control group.We performed the following assays after 6 weeks.We performed the endotracheal intubation and instillation in PM2.5,20 μl one time,and twice one week,totally eight times.In the control group,the same operation on endotracheal intubation and instillation was carried out.10.Measurement of tumor markers by morphometry in vitro.The SCID lung cancer-bearing mice were killed.The lung tissue was taken to describe the tumor.At the same time,biopsy assay was performed to measure the development of tumor in the tissue,and to evaluate the situation of the progressive development in NSCLC.11.Detection of MMP1,IL1β and VEGF expression in tumor tissues.The lung tissues of lung cancer-bearing mice in the experimental group and the control group were treated with formalin and paraffin embedded.We performed the IHC method to detect the expression of MMP1,IL1 and VEGF protein in tumor tissues.By comparing the positive rate of unit area,the the progressive development of transplanted tumor was evaluated in NSCLC.12.Detection of the angiogenesis from blood.Using the ophthalmic forceps to dig out the eyeball,and collect peripheral blood in 1.5 ml centrifuge tube.After room temperature for one hour,centrifuge 4℃,2000 rpm,total 5min.And transfer the centrifugal separation of serum to a new 1.5 ml centrifuge tube at-80℃.We performed the angiogenesis assay to detect the levels of angiogenesis factors.13.Established 32 lung cancer-bearing mice with A549 cells in 5×105 each mouse,randomly divided into 6 groups(n=8)using a random number table method.That is to say,Brucea oil emulsion treatment group and its control group,and combined PM2.5 exposed and emulsion treatment group and its control group.We performed the following assays after 6 weeks.We performed the endotracheal intubation and instillation in PM2.5,20 μl one time,and twice one week,totally eight times.And we performed the lavage with Brucea oil emulsion 100 μl one time,and total 28 times.In the control group,the same operation on endotracheal intubation and instillation was carried out.14.Collected the throat swab samples.lung cancer-bearing mice were anesthetized,and then kept oral opening,pulled out and fixed the tongue with blunt forceps with sterile cotton swab and pharyngeal samples,stored at-80℃.15.The research of Brucea oil emulsion-driven changes in upper airway microbiota in lung cancer-bearing mice after PM2.5 exposure.We performed the Mi Seq high-throughput sequencing platform to complete the sequencing analysis of sample 16 S r RNA.The data were analyzed by cluster analysis of OTU,and the species composition and the difference between samples were analyzed.Results: 1.To confirm the concentration of the PM2.5 exposure was 50 μg/cm2.The results showed that the PM2.5 samples final concentration of 50 μg/cm2 was used as the experimental condition for the treatment of non-small cell lung cancer cell line.2.The cell viability assay showed that the conditioned medium promoted the proliferation.To investigate differences in the proliferation of NSCLC lines A549 and H1299 induced by PM2.5-exposed and unexposed culture supernatants,we performed cell proliferation assays in 96 well plates.Compared with the unexposed culture supernatant group,the PM2.5-exposed culture supernatant group showed higher viability and proliferation.Our results suggested that A549 and H1299 cells had larger populations after PM2.5 exposure.Compared with the control group,the exposure group was statistically significant.3.The results of the monolayer scratch assays showed that PM2.5 exposure enhanced the migration ability of non-small cell lung cancer cell lines.The results from scratch test showed that the A549 and H1299 cell lines had strong migration ability after PM2.5 exposure.Compared with the control group,the PM2.5 exposed cells had stable migration cell lines,which laid the foundation for the further study in the mechanism.4.Invasion experiments showed that PM2.5 exposure enhanced the invasion of non-small cell lung cancer cell lines.We investigated the effects of PM2.5 exposure on the invasion of lung cancer cell lines.The results showed that compared with the control group,the A549 and H1299 cell lines of non small cell lung cancer(PM2.5)had stronger invasion ability than the control group.5.RNA-Seq array.By comparing m RNA expression profiles in the whole genome,we identified 143differentially expressed genes(DEGs)in the exposure group relative to the control group(P < 0.05).22,389 m RNAs were identified in A549 cells.Among them,143 genes with differential expression showed more than 2-fold changes,of which 66 genes were up-regulated and 77 were down-regulated.And we constructed the protein interaction network in partial differentially expressed genes.6.Validation of differentially expressed genes in real-time PCR.Next,to analyze the specific molecular mechanisms induced by PM2.5 exposure,16 DEGs were subjected to further functional analyses.These genes included 12 up-regulated genes and 4 down-regulated genes.We found expression of these genes in the exposure group to significantly differ from their expression in the control group,similarly to the trends in the microarray data.7.Detection of MMP1 and IL1β in supernatant of PM2.5 exposure.A total of 4 groups of A549 and H1299 cell cultured medium were collected from the experimental group and the control group.The levels of IL1β and MMP1 in each group were detected by ELISA kit.Compared with the control group,the expression levels of IL1β and MMP1 in the PM2.5 exposed group were significantly higher,and the results were statistically significant.8.Detection of MMP1 and IL1β in NSCLC cell lines.A total of 4 groups of A549 and H1299 cell lines were collected from the experimental group and the control group.After extracting the total protein,the results of BCA detection,the protein concentration was 181 μg/ml in the H1299 control group;the experimental group was 168 μg/ml;and the protein concentration was 305 μg/ml in the A549 control group;the experimental group was 289μg/ml.We performed the western blot assay,and the results showed that the expression of IL1β and MMP1 in the PM2.5 exposed group was significantly higher than that in the control group.9.Relying on the endoscope of cold light and gel loading tips,a new method was set up for intubated operations.In order to carry out the PM2.5 exposure treatment,all mice were given the operation of tracheal instillation.We performed tracheal instillation 20 μl 20 mg/ml with PM2.5 suspension in experimental group,and performed tracheal instillation 20 μl with normal saline in the control group.After the mice were sacrificed,the location of the lower end of the trachea and the location of the lung were scattered,and the experimental technique was proved to be effective.10.Measurement of tumor tissue in vitro.We performed the morphometry of the lung tissue near the lung membrane in vitro.2.833±0.6009 tumor nodules were observed in the control group,and 6.167±0.7032 tumor nodules were observed in the experimental group.The count of tumor tissue was 10.670±0.7149 in the experimental group and the control group was 7.833±0.7032.Those results showed that PM2.5 exposure promote tumor progressive development from gross tissue observations and sections.11.Detection of expression MMP1,IL1β and VEGF in tumor tissues.We detected the expression of MMP1,IL1β and VEGF in vivo tumor samples by immunohistochemical experiments,and performed semi quantitative analysis by Image J software in the immunohistochemical results of MMP1,IL1β and VEGF.In exposed group,the expression of the three of protein was higher than the control group.12.Detection of expression 24 angiogenesis in lung cancer-bearing mice blood.DNR MF chemi imager was used to analyze the results of PM2.5 exposure group and control group,and the gray value was analyzed by using Image J software.The results showed that the expression of IL1β,IL13,IL6,TIMP-1,TIMP-2,TNF-α,THPO and VEGF was increased in the tumor bearing mice after PM2.5 exposure.13.OTU cluster analysis and taxonomic analysis.The 16 S r RNA sequence with similarity above 97% is usually defined as a OTU,that is,each OTU corresponds to a different microorganism.In this study,OTU cluster analysis found 1836 OTU,namely,the 1836 microorganisms.The choice of species classification information for each OTU corresponding to the 16 S bacterial ribosomal database,and the result shows that the community composition of Brucea javanica oil emulsion in treatment of lung cancer in mice,can improve the colonization in the respiratory tract,among of them,Firmicutes,Actinobacteria,Bacteroidetes,Proteobacteria,fusiformis bacteria accounted for upper respiratory tract bacteria in the phylum the proportion of respiratory tract flora balance can be adjusted.14.Community composition map.Compared with the control group,there are obvious differences in upper respiratory tract flora from Brucea javanica oil emulsion treatment group.As the results showed that in the Brucea javanica oil emulsion treatment group the structure of bacteria is more reasonable,the traditional Chinese medicine Brucea javanica oil emulsion treatment can significantly improve the tumor bearing mice flora diversity characteristics.And brucea javanica oil emulsion which treated on lung cancer-bearing mice can improve respiratory tract flora diversity.And PM2.5 exposure can reduce the numbers of the community in respiratory tract.It indicated that PM2.5 can disturb the balance which established by Brucea javanica oil emulsion treatment.15.Comparative analysis of samples.In the study,we totally performed the two comparative analysis methods within samples including: PCA analysis and Uni Frac hierarchical clustering tree.From the PCA sample analysis and Uni Frac tree hierarchical clustering results can be showed that in Brucea javanica oil emulsion treatment group showed extensive difference with othe groups.And in PM2.5 exposed group and combined PM2.5 exposed and emulsion treatment group have mimimal differences.Conclusion: 1.The whole-genome of m RNA expression profiling was performed on PM2.5 exposed group alignment with control group.A total of 22,389 m RNAs were identified in A549 cells through microarray analysis.We found that 143 genes with differential expression showed more than 2-fold changes,among them 66 genes were upregulated and 77 were downregulated.We performed the real-time PCR assay and found significant differential genes in exposure group,compared with control group,which showed the change trends similar to those revealed by microarray data.Those results indicated that these differentially expressed genes involved in the progressive development in NSCLC after PM2.5 exposure.2.In this study,we investigated the mechanism of PM2.5 exposure on non-small cell lung cancer in two aspects: cell model and animal model.We validated the effect of high expression of MMP1 and IL1β on the proliferation and migration mechanism after PM2.5 exposure in vitro experiments;in animal models,we established in non-small cell lung cancer animal model and confirmed that PM2.5 exposure affects the development and metastasis of lung cancer,also confirmed the high expression of MMP1,IL1β and VEGF genes in tumor tissues,and evaluated the effect of 24 kinds of angiogenesis factors on tumor development in vivo experiments.3.We performed the animal model of PM2.5 exposure,and in this animal model we also treated the lung cancer tumor bearing mice with Brucea javanica oil emulsion.And then we analyzed the effects of PM2.5 exposure and the Brucea javanica oil emulsion treatment on respiratory tract microecological effects.From the results of the community composition,it can be shown that Brucea javanica oil emulsion can the results confirm the community composition of Brucea javanica oil emulsion modulate the respiratory tract flora balance after treatment with lung cancer-bearing mice.And PM2.5 exposure can reduce the diversity of local flora in upper respiratory tract indicating that PM2.5 exposure can interference with a relatively balanced microenvironment.4.In this study,using the endoscope of cold light and gel loading tips,a new method for tracheal instillation is set up in mice,which is rapid,simple and has good repeatability.It could be widely applied for the endotracheal intubation and instillation in mice.