| Lung cancer,as one of malignant tumors,is also the first cause of death in cancer-related deaths,seriously affecting human health and quality of life,and still lack of effective prevention and treatment.Smoking is the leading risk factor for the development of human lung cancer.Many epidemiological studies have confirmed that the prevalence of lung cancer among smokers is significantly higher than that of non-smokers.Although cigarette smoke can increase the risk of lung disease,but the induction of malignant transformation of cells caused by the development of lung cancer,the specific molecular mechanism is complex and difficult.Many recent studies have shown that the development of many diseases is not only related to the encoding protein RNA,but also related to the expression of ncRNA.The ncRNA includes the most well-known microRNA(miRNA)and the recently studied long non-coding RNA(lncRNA)and circRNA.Among them,lncRNA is a new type of ncRNA,which is more than 200 nt in length and can be used as a new marker for environmental exposure.It is very likely to play an important role in driving the exposed disease.Studies have shown that lncRNA is involved in the development of tumors by interacting with miRNAs,but little is known about the interaction between lncRNA and miRNAs in the pathogenesis of environmental hazards,especially smoking-induced diseases.ObjectiveThis study investigates the effect of long non-coding RNA CCAT1(CCAT1)on the malignant transformation of HBE cells induced by chronic treatment of cigarette smoke extract(CSE)through miR-218 and let-7c,thus revealing the part of molecular mechanism of malignant transformation of HBE cells induced by CSE.To provide scientific clues to finding early biomarkers of lung injury and lung cancer caused by smoking.Methods1.The effects of CSE on the expression of lncRNAs and miiRNAs in HBE cellsHBE cells were treated with 20 ?g/mL CSE for 0,6,12 and 24 h,or 0,20,30 and 40 passages the levels of CCAT1?MALAT1?HOTAIR and H19 were detected by RT-PCR.The expression of CCAT1?miR-21?miR-125a?miR-218 and let-7c was detected by qRT-PCR.We are going to observe the effect of CSE on the expression of lncRNAs and miRNAs in HBE cells.2.The effects of CSE on the expression of BMI1 in HBE cellsHBE cells were treated with 0 or 20 ?g/mL CSE for 0,6,12 and 24 h,or 0,20,30 and 40 passages.The expression of BMI1 was detected by Western blot.We are going to observe the effects of CSE on the expression of BMI1 in HBE cells.3.The roles of CCAT1 in regulation of miR-218 in the increased expression of BMI1 in HBE cells induced by CSEThe cells were treated with 0 or 20 ?g/mL CSE after co-transfected with pGL3-CCAT1-WT/pGL3-Ctrl and miR-218 mimic/Con mimic,or treated with 100 ppm CCAT1 siRNA or Con siRNA before treated with 0 or 20 ?g/mL CSE,or transfected with 50 nM let-7c mimic or Con mimic and then treated with 0 or 20?g/mL CSE for 24 h.The luciferase activities were detected by luciferase reporter gene assay.The the CCAT1 knockdown efficiency was detected by qRT-PCR.The expression of miR-218 was detected by qRT-PCR.The expression of miR-218 in HBE cells was detected by qRT-PCR and the expression of BMI1 in HBE cells was detected by Western blot.100ppm CCAT1 siRNA/Con siRNA and miR-218 inhibitor/Con inhibitor was co-transfected in HBE cells for 24 h and then treated with 0 or 20 ?g/mL CSE for 24 h respectively.The expression of BMI1 in HBE cells was detected by Western blot.We are going to observe the Effects of CCAT1 in regulation of miR-218 on the increased expression of BMI1 in HBE cells induced by cigarette smoke extract.4.The effects of CCAT1 in regulation of BMI1 via miR-218 on the maglinant transformation of HBE cells induced by CSE100 ppm CCAT1 siRNA/Con siRNA and miR-218 inhibitor/Con inhibitor was co-transfected in CSE-induced malignant transformed HBE(T-HBE)cells.Or treated with 50 nM BMI1 siRNA or Con siRNA.The expression of miR-218 was detected by qRT-PCR and the expression of BMI1 was detected by Western blot.The malignancy of malignant transformed HBE cells was observed by colony formation assay.The invasion and migration ability of malignant transformed HBE cells were observed by Transwell assay.We are going to observe the effects of CCAT1 in regulation of BMI1 via miR-218 on the maglinant transformation of HBE cells induced by cigarette smoke extract.5.The effects of CSE on the expression of c-Myc in HBE cellsHBE cells were treated with 0 or 20 pg/mL CSE for 0,6,12 and 24 h,or 0,20,30 and 40 passages.The expression of c-Myc was detected by Western blot.We are going to observe the effect of CSE on the expression of c-Myc in HBE cells.6.The roles of c-Myc in the increased expression of CCAT1 in HBE cells induced by CSEAfter HBE cells treated with 50 nM c-Myc siRNA or Con siRNA for 24 h respectively,HBE cells were treated with 0 or 20 ?g/mL CSE for 24 h respectively.Or HBE cells were treated with 0 or 20 ?g/mL CSE for 24 h respectively.The levels of c-Myc in HBE cells were detected by Western blot,and the expression of CCAT1 in HBE cells was detected by qRT-PCR.The binding of c-Myc to CCAT1 promoter region was detected by ChIP assay.We are going to observe the roles of c-Myc in the increased expression of CCAT1 in HBE cells induced by CSE.7.The roles of let-7c in the increased expression of c-Myc in HBE cells induced by CSEHBE cells were transfected with pmirGLO-c-Myc-3-UTR-WT/pmirGLO--Ctrl and let-7c mimic/Con mimic,respectively.Or treated with 50 nM let-7c mimic or Con mimic for 24 h.The luciferase activities were detected by luciferase reporter gene assay.The let-7c levels were detected by qRT-PCR.Western blot was used to detect the expression of c-Myc in HBE cells.We are going to observe the roles of let-7c in the increased expression of c-Myc in HBE cells induced by CSE.8.The roles of CCAT1 in the increased expression of c-Myc inHBE cells induced by CSEHBE cells were treated with 100 ppm CCAT1 siRNA or Con siRNA for 24 h,and then treated with 0 or 20 ?g/mL CSE for 24 h.Or transfected with pGL3-CCAT1-WT/pGL3-Ctrl and let-7c mimic/Con mimic for 24 h.Or treated with 100 ppm CCAT1 siRNA/Con siRNA and let-7c inhibitor/Con inhibitor for 24 h respectively.The expression level of c-Myc in HBE cells was detected by Western blot.The luciferase activities were detected by luciferase reporter gene assay.The expression of let-7c was detected by qRT-PCR.We are going to observe the roles of CCAT1 in the increased expression of c-Myc in HBE cells induced by CSE.9.The roles of feedback loop between CCAT1 and c-Myc via let-7c in the CSE-induced malignant transformation of HBE cells.The T-HBE cells were cotransfected with 100ppm CCAT1 siRNA/Con siRNA and let-7c inhibitor/Con inhibitor respectively,or treated with 50 nM c-Myc siRNA or Con siRNA.The expression of c-Myc was detected by Western blot.The malignancy of T-HBE cells was observed by colony formation assay.The invasion and migration ability of T-HBE cells were observed by Transwell assay.We are going to observe the roles of this feedback loop between CCAT1 and c-Myc via let-7c in the CSE-induced malignant transformation of HBE cells.Results1.The effects of CSE on the expression of lncRNAs and miRNAs in HBE cellsThe results showed that after treatment with CSE,the expression of CCAT1?MALAT1?HOTAIR and miR-21 were increased,the expression of miR-218 and let-7c was decreased.The results indicate that CSE increases the expression of CCAT1 and decreases the expression of miR-218 and let-7c in HBE cells.2.The effects of CSE on the expression of BMI1 in HBE cellsThe results showed that after treatment with 20 ?g/mL CSE for 6 h or after 20 passages of chronic treatment with 20 ?g/mL CSE,the expression of BMI1 in HBE cells was significantly higher than that in control group.The results indicate that CSE increases the expression of BMI1 in HBE cells.3.The effects of CCAT1 in regulation of miR-218 on the increased expression of BMI1 in HBE cells induced by CSEThe luciferase activity of miR-218 mimic and pGL3-CCAT1-WT cotransfected in CSE treated group was significantly lower than that of CSE treated group.In CCAT1 siRNA combined with CSE treatment group,the levels of CCAT1 significantly decreased and the levels of miR-218 significanly increased.After treated with miR-218 mimic in the CSE-treated group,the expression of miR-218 was significantly increased,and the expression of BMI1 was significantly decreased.The expression of BMI1 in CCAT1 siRNA and miR-218 inhibitor cotransfected in CSE-treated HBE cells was significantly higher than that of CCAT1 siRNA treated cells.These results indicate that CCAT1 regulated the expression of BMI1 through miR-218 in HBE cells induced by cigarette smoke extract.4.The effects of CCAT1 in regulation of BMI1 via miR-218 on the maglinant transformation of HBE cells induced by CSEThe results showed that CCAT1 siRNA treated T-HBE cells could significantly increased the expression of miR-218 and decreased the expression of BMI1,and the number of colonies and invasion/migration cells were significantly lower than those of T-HBE cells.The expression of BMI1 in CCAT1 siRNA and miR-218 inhibitor cotransfected in T-HBE cells was significantly higher than that of CCAT1 siRNA treated T-HBE cells,and the number of colonies and invasion/migration cells were significantly higher than those of CCAT1 siRNA treated T-HBE cells.The number of cell colonies formed on the soft agar and the number of cells invading/migrating in HBE cells transfected with BMI1 siRNA was significantly lower than that of T-HBE cells(P<0.05).These results indicate that CCAT1 in regulation of BMI1 may be influence the malignancy of T-HBE cells through miR-218.5.The effects of CSE on the expression of c-Myc in HBE cellsThe results showed that after treatment with 20 ?g/mL CSE for 6 h or 20 passages of chronic treatment,the expression of c-Myc in HBE cells was significantly higher than that in control group.The results indicate that CSE increases the expression of c-Myc in HBE cells.6.The roles of c-Myc in the increased expression of CCAT1 in HBE cells induced by CSEThe results suggested that the expression of c-Myc and CCAT1 in 20 ?g/mL CSE treated group were significantly higher than those in control HBE cells,and the levels of c-Myc and CCAT1 in c-Myc siRNA combined with 20 ?g/mL CSE treated group were significantly lower than those in CSE treated group.CSE increased c-Myc binding to CCAT1 promoter region.These results indicate that c-Myc plays an important role in the elevation of CCAT1 expression in HBE cells induced by CSE.7.The roles of let-7c in the increased expression of c-Myc in HBE cells induced by CSEThe results suggested that after treated with let-7c mimic,the expression of let-7c in the CSE-treated group was significantly increased.After cotransfected with pmirGLO-c-Myc-3'UTR-WT and let-7c mimic,the luciferase activities were decreased in CSE treated HBE cells,compared with that in CSE treated group.These results indicate that let-7c inhibits the expression of c-Myc in CSE-treated HBE cells via binding to the 3'-UTR of c-Myc.8.The roles of CCAT1 in the increased expression of c-Myc in HBE cells induced by CSEThe results suggested that the expression of CCAT1 and c-Myc in CCAT1 siRNA combined with 20 ?g/mL CSE treatment group were significantly lower than those in CSE treatment group,while the expression of let-7c did not change significantly.The luciferase activity of let-7c mimic and pGL3-COAT1-WT cotransfected in CSE treated group was significantly lower than that of CSE treated group.The expression of c-Myc in CCAT1 siRNA and let-7c inhibitor cotransfected in CSE treatment group was significantly higher than that of in CCAT1 siRNA combined with CSE treatment group,and the expression of let-7c was significantly decreased.These results indicate that CCAT1 may be influence the expression of c-Myc via let-7c.9.The roles of feedback loop between CCAT1 and c-Myc via let-7c in the CSE-induced malignant transformation of HBE cells.The results showed that CCAT1 siRNA treated T-HBE cells could significantly decreased the expression of c-Myc,and the number of colonies and invasion/migration cells were significantly lower than those of T-HBE cells.The expression of c-Myc in CCAT1 siRNA and let-7c inhibitor cotransfected in T-HBE cells was significantly higher than that of COAT1 siRNA treatedT-HBE cells,and the number of colonies and invasion/migration cells were significantly higher than those of CCAT1 siRNA treated T-HBE cells.The number of cell colonies formed on the soft agar and the number of cells invading/migrating in T-HBE cells transfected with c-Myc siRNA was significantly lower than that of T-HBE cells.These results indicate that the feedback loop between CCAT1 and c-Myc via let-7c may be influence the malignancy of CSE transformed HBE cells.Conclusions1.CSE induces the increases of CCAT1 levels and the decreases of miR-218 and let-7c levels in HBE cells.2.CCAT1 via miR-218 regulating of BMI1 plays an important role in CSE-induced malignant transformation of HBE cells.3.The feedback loop between CCAT1 and c-Myc is involved in CSE-induced malignant transformation of HBE cells... |
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