Pathological Observation Of Lung Cancer Induced By Radon Inhalation In BALB/c Mice And The Regulatory Mechanism Madiated By MAP3K1 Gene

Objective:Radioactive radon(Rn)and its daughters are the second major risk factors for lung cancer after smoking,and the biological mechanisms are not fully understood.In this study,a mouse model of lung cancer induced by radon exposure was used to observe the effects of radon inhalation on lung tissue damage and tumorigenesis.To reveal the effects,changes in tumor markers and the expression of relative genes in the lung cancer induced by radon exposure were detected and the MAP3K1 gene was selected as a target to observe the expression changes in its mRNA and protein level,and changes of phosphorylated and non-phosphorylated protein of JNK,ERK and P38 were also determined before and after silencing of the Map3k1 gene.Using in vitro and in vivo tests on human lung adenocarcinoma A549 cells,the molecular mechanism of radon induced lung cancer mediated by MAPK signaling pathway was explored.Method:1.4-5 week old SPF BALB/c mice were randomized grouping into radon exposure group and control group,60 rats,half male and half female in each group.In radon exposure group,the mice were exposed to radon(100000 Bq/m3)in the HD-3 type multi-function ecological radon chamber every day,and the exposure time 6 hours per day,6 days a week.A half of animals were sacrificed at 6th and 9th months after radon exposure respectively.In 6 and 9 months groups,the time of radon exposure were 930 h and 1392 h respectively,and the cumulative dose were148 and 221 working level month(WLM)respectively.In control group,all of animals fed environment with less than 50 Bq/m3 of radon concentration.Respectively,half of the animals were treatment at each time point,the lung coefficient,pathological changes and tumorigenesis of lung tissue were observed.2.The serum was extracted in each group animals at above mentioned.Using enzyme-linked immunosorbent assay(ELISA)detection the expression levels of tumor marker neuron specific enolase(NSE)and cytokeratin 19 fragment(CYFRA21-1),carcinoembryonic antigen(CEA)and gastrin releasing peptide(ProGRP).The expression of p53,BCL2,HSP90 and IL12 genes in lung in each group animals were detected by Real-Time PCR method.3.Respectively using real-time PCR and western blot technology to detect the Map3kl mRNA and MEKK1 protein expression levels in lung tissue in each group,and using western blot technology to detect phosphorylated and non-phosphorylated protein change levels of JNK,ERK and P38 signal downstream pathways.4.RNA interference technology was used to specifically silence the expression of Map3k1 and to detect the effect of A549 on the biological behavior and ability of human lung adenocarcinoma cell line.First,the siRNA sequences were synthesized,the interference efficiency was verified in human lung adenocarcinoma A549 cells,and the sequence with the highest interference efficiency was selected for the following experiments.Second,the migration ability and invasion ability of human lung adenocarcinoma A549 cells before and after Map3k1 interference were compared by scratch healing and transwell invasion assay.Colony forming ability of A549 cells was detected by soft agar colony forming assay after Map3kl down-regulation.The proliferation,cycle and apoptosis of A549 cells before and after Map3kl interference were compared by MTT and flow cytometry.Nude mice model was used to detect the effect of targeted inhibition of Map3k1 on the tumorigenicity of A549 cells in nude mice.Finally,western blot technology was used to detect phosphorylated and non-phosphorylated protein change levels of JNK,ERK and P38 three signal pathways.Result:1.The lung tissue of mice in the radon group was characterized by chronic bronchitis,pneumonia and fibrous tissue hyperplasia,with increased lung coefficients.Pathological observation confirmed lung adenocarcinoma.The incidence rate was 6.7%in in 6 month exposure group and 20%in 9 month exposure group,with a significant higher incidence in female animals than in male animals.2.Radon exposure induced the content of tumor markers NSE,CYFRA21-1,CEA and increased ProGRP in BALB/c mice.The content of NSE in serum in female animals was significantly higher than that in male animals at 6th month.Radon exposure induced the mRNA expressions of cancer relative genes of p53,BCL-2 and HSP90,and the mRNA levels of p53 and HSP90 in female animals were significantly higher than those in male animals at 6th month.The mRNA expression levels of IL-12 were significantly decrease with time.3.The expressions of Map3kl gene were significantly increased in lung of mice after radon exposure,in which the expressions of Map3kl mRNA levels in female mice were higher than those in male mice at 6th month group.At 9th month group,the expressions of Map3kl in mRNA and protein levels were both higher than those in male mice.The levels of JNK,phosphorylation of JNK and ERK were significantly suppressed after the up-regulation of MEKK1,and the levels of ERK,P38 and phosphorylation of p38 were not obvious changes.4.Three chains of Map3kl-siRNA could inhibit the expression of target gene in mRNA and protein levels,in which the inhibitory effect of Map3k1-siRNA 1 was the strongest with 70%interference efficiency.Targeted inhibition of Map3kl gene could significantly inhibit the migration and invasion ability,the colony forming ability and cell proliferation in A549 cells.The cell cycle was blocked in S phase,and cell apoptosis was altered.In vivo tumor formation assay in nude mice showed that the targeted silencing Map3kl gene could significantly inhibit the tumorigenic ability of A549 cell line.The levels of JNK,phosphorylation of JNK and ERK were significantly increased after the down-regulation of MEKK1.Conclusion:1.Long term radon inhalation could induce chronic inflammatory hyperplastic changes and lung adenocarcinoma in BALB/c mice,with the incidence of 6.7%and 20%at 6th month and 9th month respectively.The incidence in female mice were significant higher than that in male mice.2.The expressions of tumor markers in serum of mice increased after radon exposure.The expression of some oncogenes and tumor suppressor genes were up-regulated or down-regulated respectively in lung tissues,which was more obviously in female mice compared with that in male mice.3.The expressions of Map3k1 in the lung tissue of mice were significantly increased after radon exposure,with a significant inhibitory effect for phosphorylation levels of JNK and ERK suggesting an involvement of Map3k1 gene in the regulation of radon induced lung cancer.4.Targeted silencing of Map3kl gene could significantly inhibit the ability of tumor formation of human lung adenocarcinoma A549 cell line in nude mice,indicating the role of Map3kl gene in mediation of radon induced lung adenocarcinoma via the JNK and ERK signaling pathway.