| Part one Effects of different concentrations of hydrogen intervention on non-small cell lung cancer cellsObjectives:To investigate the effects of different hydrogen concentrations on proliferation,migration,invasion,colony formation and apoptosis of non-small cell lung cancer A549 and H1975 cell lines.Methods:1.The IC50 value of BEAS-2B in human normal lung epithelial cells with different concentrations of hydrogen were evaluated by CCK-8 assay.2.Cell proliferation of different groups treated with hydrogen were confirmed by plate colony formation and CCK-8 assay.3.Cell migration and invasion of different groups treated with hydrogen were investigated by wound-healing experiments and transwell invasion assay.4.Cell cycle and apoptosis of different groups treated with hydrogen were detected by flow cytometry.Results:1.The IC50 of BEAS-2B treated with hydrogen was 77+1.36%.2.CCK-8 results showed that 60%and 80%hydrogen treated groups had significant inhibitory effects on cell activity at 72h compared with the control group(P<0.01),and 80%hydrogen group has the most significant inhibitory effects(P<0.001).The experimental results showed that the number in 60%and 80%hydrogen treated groups for 14 days were lower than that of the control group(P<0.05),and the effect of 80%hydrogen group was the most obvious(P<0.001).Hydrogen inhibits the proliferation of lung cancer cells in a concentration-dependent manner.3.The results of flow cytomeytry showed that after treated with hydrogen,the G2/M phase cells in the 40%,60%and 80%hydrogen groups were significantly increased compared with the control group(P<0.05).Apoptosis was promoted in 40%,60%and 80%hydrogen treated groups compared with the control groups(P<0.05),and the 80%hydrogen group has the most significant effect(P<0.001).Hydrogen promoted apoptosis of lung cancer cells in a concentration-dependent manner.4.The results of wound healing assay were shown compared with the control group,the migration activity of 60%and 80%hydrogen treated groups were significantly inhibited(P<0.01),and its inhibition effect in a concentration-dependent manner.5.The results of Transwell assay were shown after treated with hydrogen,40%,60%and 80%hydrogen group cells were significantly less than that the control group cells that passed through the cell matrix adhesive(P<0.05),and the effect of 80%hydrogen group was the most obvious(P<0.001).Summary:1.The IC50 of BEAS-2B treated with hydrogen was 77+1.36%.2.Treated with 60%,80%hydrogen groups significantly inhibited the proliferation and colony formation capacity of A549 and H1975 cell lines compared with the control group and led to significantly cell accumulation in the G2/M phase.Hydrogen exerted its effcet in a concentration-dependent manner.3.Treated with 60%,80%hydrogen groups significantly suppressed A549 and H1975 cell migration and invasion,which showed in a concentration-dependent manner.4.Treated with 40%,60%and 80%hydrogen groups could promote apoptosis of A549 and H1975 cells in a concentration-dependent manner.Part two Transcriptome analysis of non-small cell lung cancer cells treated with hydrogenObjectives:To study the mechanism and signaling pathway of the effect of hydrogen on lung cancer cellsin the first part,transcriptome sequencing was performed on A549 cells treated with hydrogen.Target signaling pathways were selected and differentially expressed genes were screened for validation experiment.Methods:RNA was extracted from cells and sequenced by transcriptome.Differential gene signaling pathways were analyzed by bioinformatics analysis.Results:1.Compared with the control group,823 differentially expressed genes were screened out in the hydrogen group,of which 168 genes were up-regulated and 655 genes were down-regulated.2.The enrichment of GO Terms results indicated that the down regulated differential gene function involves biological processes related to cell cycle,such as chromosome binding,DNA replication and repair.3.KEGG signal pathway analysis showed that the differentially expressed genes in A549 cells treated with hydrogen were mainly related to cell cycle,DNA replication and mitosis.4.The SMC3,SMC5,SMC6 and NIPBL genes related to cell cycle and chromosome condensation were screened out.Summary:1.The results of transcriptome sequencing showed that the cell cycle and chromosome replication-related signaling pathways of lung cancer were significantly affected after treated with hydrogen.It was revealed that 60%hydrogen group inhibited the growth of A549 cells mainly by regulating cell cycle.2.The SMC2,SMC3,SMC4,SMC5,SMC6 and NIPBL genes associated with chromosome condensation,which were significantly down-regulated by hydrogen,were screened out for further study.Part three Hydrogen inhibits lung cancer cell proliferation by inhibiting chromosome condensationObjectives:To analyze the differentially expressed genes screened in the second part,detect the effect of hydrogen on the stability of SMC3 protein,and investigate the effects of SMC3 over-expression on migration,invasion and apoptosis of A549 and H1975 cells.Methods:1.Expression levels of SMC2,SMC3,SMC4,SMC5,SMC6 and NIPBL in A549 and H1975 cells were determined by qPCR and Western Bolt.2.Detection of SMC3 protein expression in A549 and H1975 cells by immunofluorescence.3.Treated with cycloheximide in the control group and 60%hydrogen group,SMC3 protein stability were determined by Western Bolt.Immunoprecipitation assay to detect the effect of hydrogen on ubiquitination of SMC3 protein.4.Lung cancer cell lines with SMC3 over-expressed were established.Cell migration,invasion and apoptosis were investigated in the control group,hydrogen treated group and SMC3 over-expressed hydrogen treated group.Results:1.The results of qPCR and Western Blot showed that the expression level of SMC3,SMC5,SMC6 and NIPBL were significantly lower than that in the control group,and SMC3 protein showed the most obvious downward trend in hydrogen group.2.The results of immunofluorescence assay showed that SMC3 protein expression in the hydrogen group was lower than that in the control group during cell division.3.Compared with the control group,the expression levels of SMC3 was significantly reduced,while over-expression of SMC3 could partly reverse the effects of hydrogen on migration,invasion inhibition and apoptosis of A549and H1975 cells.Summary:1.The results of qPCR and Western Blot showed that the expression level of SMC3 was significantly lower than that in the control group.It was revealed that 60%hydrogen inhibited the progression of lung cancer by regulate chromosome condensation.2.Lung cancer cell lines transfected with over-expressed SMC3 vector were established.3.Overexpression of SMC3 in lung cancer cells partially reversed the inhibitory effect of hydrogen on lung cancer progression.It is revealed that60%hydrogen inhibited the progression of lung cancer by regulating the expression of SMC3 protein.Part four Effects of hydrogen on the growth of non-small cell lung cancer in vivoObjectives:To study the effect of hydrogen on the proliferation of subcutaneous transplanted tumor in nude mice.To investigate the expression of SMC3 in lung cancer tissues and adjacent tissues and the relationship between SMC3 expression and survival time in patients with lung cancer.Methods:1.Nude mice were inoculated subcutaneously in the right flank with A549 cells,tumor size was measured by a slide caliper and tumor volume was calculated as(length×width~2)/2.2.Immunohistochemistry was used to detect the expression of Ki67,VEGF,SMC3 and COX-2 in the implanted tumor and normal lung tissues of nude mice.3.The expression of SMC3 and the relationship between SMC3 and survival time of lung cancer patients was analyzed in selected specimens using the network public specimens database.Results:Hydrogen significantly inhibits the proliferation of subcutaneous transplanted tumor in nude mice.1.The transplanted tumors weights of hydrogen treated group and cisplatin group were significantly lower than that in control group(P<0.05),and cisplatin group has greater effect on tumor growth.2.Hydrogen did not cause morphologic and pathological changes in normal lung tissue of nude mice.3.Immunohistochemistry results showed that the expression of Ki67,VEGF,SMC3 and COX-2 in the subcutaneous transplanted tumor cells of the hydrogen group was significantly reduced.4.The results of network analysis tools showed that the expression level of SMC3 in lung cancer tissues was higher than that in the normal tissues and the survival time of lung cancer patients with low expression of SMC3 was significantly longer than that of patients with high expression of SMC3(P(27)0.05).Summary:1.Hydrogen did not cause morphologic and pathological changes in normal lung tissue of nude mice.2.Subcutaneous nude mouse xenograft model has been successfully constructed.60%hydrogen group could significantly inhibit the growth of subcutaneous transplanted tumor and reduce the weight of the tumor,and its effect was no more than that of cisplatin group.3.Decline of Ki67,VEGF,SMC3 and COX-2 in hydrogen treated xenografts tissue indicates the proliferation activity of lung cancer cells is inhibited.4.SMC3 expression was significantly up-regulated in cancer tissue compared to normal lung tissue.The survival time of lung cancer patients with low expression of SMC3 was significantly longer than that of patients with high expression of SMC3,suggesting that SMC3 may be one of the tumor markers of lung cancer.Conclusions:1.60%,80%hydrogen could inhibite the proliferation,migration and invasion activity of A549 and H1975 cell lines,and promoted cell apoptosis,which in a concentration-dependent manner.2.Hydrogen inhibited the proliferation of A549 and H1975 cell lines and led to cell accumulation in G2/M phase,possibly through inhibiting the expression of SMC3.3.60%hydrogen did not cause morphologic and pathological changes in normal lung tissue of nude mice.4.60%hydrogen could significantly inhibit the proliferation of subcutaneous transplanted tumor in nude mice.The expression of VEGF,Ki67,SMC3 and COX-2 proteins in the tumor were down-regulated in hydrogen treated group.5.Increased expression of SMC3 was a disadvantageous factor for the total survival time of lung cancer patients,and SMC3 may become a new target for the treatment of lung cancer. |
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