Effect And Mechanism Of MiR-146a Targeted Genes IRAK1,TRAF6 And Its Relevant IncRNA HOXA11-as On Lung Cancer

Lung cancer is the leading cause of cancer-related death worldwide.The morbidity and mortality still rank the first place among all malignancies.Lung cancer is often diagnosed at an advanced stage when effective treatment is no longer possible.Even in early-stage patients treated by surgery,the recurrence rate remains high.The tradition treatment strategies of lung cancer include surgery,radiotherapy,chemotherapy and comprehensive treatment.Simultaneously,the emerging molecular targeted therapy provides new choice of lung cancer treatment strategies.The typical target is epidermal growth factor receptor(EGFR).Since EGFR inhibitors have been approved for treatment in lung cancer,the therapeutic effect has been greatly improved.However,the significant improvement in the five-year mortality rate is far from satisfaction.Thus,there is an urgent need to discover new molecular diagnostic and therapeutic targets for lung cancer patients.Our previous research showed that the expression of miR-146a in lung cancer tissue and its cell lines were both lower than the corresponding control groups.MiR-146a may function as a tumor suppressor microRNA in lung cancer,via targeting EGFR.Besides EGFR,whether there are other target genes for miR-146a?Neither,to date,the molecular mechanism of miR-146a in lung cancer remains enigmatic.Long non coding RNA(lncRNA,>200nt)is a class of non coding protein RNA.LncRNAs are emerging key factors in the regulation of various cellular processes,including X-chromosome silencing,genomic imprinting,chromatin modification,transcriptional activation,transcriptional interference and intranuclear transportation.In recent years,expression of a large number of lncRNAs have been reported to dysregulate in human cancer samples,which regulate the biology function of cancer as oncogenic or tumor suppressor.Our previous work has confirmed that there were binding sites between IncRNA HOXA11-AS and miR-146a,as predicted by bioinformatics.However,to date,there has been no report on the relationship of HOXA11-AS and tumor in literature.Combined with our previous study and bio-information,we speculated that HOXA11-AS may downregulate the expression of miR-146a,hence to regulate the downstream genes IRAK1,TRAF6,EGFR and NF-?B signal path,and to be involved in the molecular mechanism of the carcinogenesis,progression and metastasis of lung cancer.The current thesis focused on the effect of miR-146a potential targeted genes IRAK1 and TRAF6 in lung cancer,as well as the influence of a miR-146a relevant IncRNA HOXA11-AS on the malignant phenotypes of lung cancer,and further to investigate the prospective possibility for HOXA11-AS to act as a molecular target for future lung cancer treatment strategies.Materials and Methods1.The expression and biological effect of miR-146a targeted genes IRAK1 and TRAF6 in lung cancer1.1 The level of miR-146a was detected by using real time RT-qPCR in 65 cases of lung cancer patients.Meanwhile,the protein expression of miR-146a targeted genes IRAK1 and TRAF6 was assessed by using immunohistochemistry.The expression level of miR-146a was compared to that of IRAK1 and TRAF6 protein.1.2 MiR-146a mimic,miR-146a inhibitor,IRAK1 siRNA,TRAF6 siRNA,EGFR siRNA and their controls were transfected into lung cancer A549 and H460 cells by using LipofectamineTM2000.The biological function of miR-146a and its target genes IRAK1 and TRAF6 in lung cancer was then explored with a series of in vitro functional experiments.1.3 IRAK1 and TRAF6 protein expression and their clinical significance were determined in another independent cohort with lung cancer tissues(n=365)and normal lung tissues(n=30)by using immunohistochemistry,and its correlation with clinical pathological parameters was analyzed as well.2.The influence of miR-146a relevant IncRNA HOXA11-AS on the malignant phenotypes of lung cancer2.1.The expression levels of HOXA11-AS in lung cancer tissues(n=6)and lung cancer cell lines(A549,H460,H1299 and PC9)were detected by using real-time qRT-PCR.2.2.Lentivirus-mediated HOXA11-AS RNAi was constructed.Lung cancer cell lines,including A549,H460,H1299 and PC9,were infected with lenti-HOXA11-AS RNAi or lenti-control virus to obtain the stable low HOXA11-AS-expressed cell lines.2.3 To determine the effect of HOXA11-AS knockdown in lung cancer cell lines,we performed the manual cell counting,CCK-8 assay,flow cytometry,wound healing assay,migration and matrigel invasion assays.2.4 In vivo tumorigenicity and angiogenesis was explored by using a chick chorioallantoic membrane(CAM)model.3.The relationship of sequence complementation between HOXA11-AS and miR-146a was verified by using dual luciferase report gene experiment.Results1.Expression of miR-146a targeted protein IRAK1 and TRAF6 were upregulated and could enhance the tumor growth1.1 The expression of miR-146a was inversely correlated IRAK1 and TRAF6 in 65 cases of lung cancer tissues.1.2 In vitro experiments showed that downregulation of miR-146a expression in A549 and H460 lung cancer cells could promote the cell growth and inhibit apoptosis.On the contrary,over-expression of miR-146a markedly reduced the malignant behavior of lung cancer cells.Knockdown of IRAK1 and TRAF6 by using siRNA interference in lung cancer cells could significantly inhibit cell proliferation and induce cell apoptosis,which was comparable to the effect of miR-146a mimic.1.3 The expression of IRAK1 in lung cancer was significantly higher than that in normal lung tissues(p=0.002).Its expression was related to clinical TNM stage(r=0.241,p<0.001),lymph node metastasis(r=0.279,p<0.001)and tumor size(r=0.299,p<0.001)in lung cancer.Meanwhile,the expression of TRAF6 in lung cancer was significantly higher compared with that in normal lung tissues(p=0.001).Further analysis by spearman correlation indicated that the protein level of TRAF6 expression was significantly associated with TNM stages(r=0.230,p<0.001),tumor size(r=0.272,p<0.001)and lymph node metastasis(r=0.225,p<0.001).2.HOXA11-AS could enhance the cell proliferation and suppress apoptosis in lung cancer cells2.1 We primarily demonstrated that HOXA11-AS was upregulated in lung cancer tissues.The expression levels of HOXA11-AS in lung cancer cell lines increased as compared to that in normal bronchial epithelial cell lines.However,this finding needs to be further verified with a larger size cohort.2.2 The number of cells was counted under microscope manually for 7 days and cell growth curve was drawn.The cell growth curve showed that cell proliferation rate was significantly lower in the group of HOXA11-AS knockdown than that in the corresponding control groups(p<0.001).2.3 In vitro experiments showed that cell migration and invasion were significantly inhibited in lung cancer cells with HOXA11-AS being knocked down as compared to control group(p<0.01).2.4 The flow cytometry indicated that the apoptosis rate after knockdown of HOXA11-AS significantly increased,compared with corresponding control groups(p<0.01).2.5 After the downregulation of HOXA11-AS,the cell cycle was retarded in G0/G1 or G2/M phases,whereas significantly less cells were observed in S phases compared to the control groups(p<0.01).2.6 Downregulation of HOXA11-AS expression could obviously inhibit the size of tumor formation(p<0.05)and its angiogenesis in CAM(p<0.01).3.Dual luciferase assay demostrated that miR-146a could specifically bind to the 3'UTR of HOXA11-AS,hence cause a significant reduction of the luciferase activity.This result demonstrated that targeting relationship may exist between HOXA11-AS and miR-146a.Conclusion1.Together with our previous observation,IRAK1 and TRAF6 are target genes of miR-146a in lung cancer.MiR-146a inhibits cell growth and induces apoptosis in lung cancer cells via targeting IRAK1 and TRAF6.2.HOXA11-AS is overexpressed in lung cancer tissues and cultured cell lines.HOXA11-AS could play a vital role as oncogene in cell proliferation,migration and invasion.3.MiR-146a could act as "sponge" of HOXA11-AS.The incorporation of HOXA11-AS and miR-146a may thus reduce the expression level of miR-146a in lung cancer.4.HOXA11-AS/miR-146a/IRAK1,TRAF6,EGFR interaction may be important in the carcinogenesis,progression and metastasis in lung cancer.However,this hypothesis needs further verification.This potential HOXA11-AS/miR-146a/IRAK1,TRAF6,EGFR axis has the hope to benefit the future diagnosis,prediction and treatment of lung cancer.