The Regulation Of Hedgehog Pathway On Invasion Of Non-small Lung Cancer Cells

Objective:To investigate the regulatory effects of two key moleculars in Hedghog pathway,SMO and GLI1,on invasion of non-small cell lung cancer cells,by overexpression or knockdown of these two genes in H1299 and A549 cells,and to explore their molecular mechanisms by analizing the potential regulation of SMO and GLI1 on EMT markers.Methods:(1)Construct human SMO and GLI1 overexpression vectors base on plasmid PC DNA 3.1,transfecting h 1299 and A549 cells by liposome(Lip 2000),and constructing overexpressed non-small cell lung canc cell lines;Small interfering RNA(Si RNA)targeting human SMO or GLI1 was designed and synthesized.The two lung cancer cells were transfected with liposome to construct gene knock-down cell lines.Gene and protein overexpression or knockdown was detected by fluorescent quantitative PCR and western blot.(2)The effects of SMO and GLI1 overexpression / knockdown on cell migration and infiltration in non-small cell lung cancer were examined by transwell chamber test.A1 mg / ml matrigel was laid on the polycarbonate film of the upper chamber,50 ul per well,simulating the extracellular matrix,after the gel had solidified,cultured cells were inoculated for 16 h and fixed and DAPI chromatin was fixed.(3)The effects of SMO and GLI1 overexpression / knockdown on E-cadherin,N-cadhenin and Vimentin were examined by western blot in H1299 cells.(4)Using SPSS 16.0 statistical software for data analysis.Two – sample heteroscedasticity hypothesis was used.Data were analyzed by independent sample t test.P ? 0.05 is the significant difference standard,and p ? 0.01 is the standard of extremely significant difference.All experiments were independently repeated for three times.Results:(1)The results of restriction enzyme digestion and sequencing showed that SMO,GLI1 overexpression vectors p3xha-SMO and P3 x flag-GLI1 were successfully constructed.Fluorescent quantitative PCR and Western blot results showed that transfected expression vector or Si RNA significantly increased / decreased the expression of SMO and GLI1 in H1299 and A549 cells of non-small cell lung cancer.at m RNA level,SMO expression was increased by 2.927 times(P= 0.00041)and2.790 times(P= 0.0045),and GLI1 expression was increased by 3.446 times(P =0.00049)and 2.660 times(P= 0.0036),respectively.SMO expression was reduced by98 %(P = 0.0211)and 53.3 %(P= 0.0098)in H1299 / A549 cells transfected with Si RNA,and GLI1 expression was reduced by 67.9 %(P = 0.0017)and 74.4 %(P =0.0018),respectively.(2)Transwell test showed that in H1299 / A549 cells,the invasiveness of SMO knockdown group was 66.5 %(P < 0.05)and 74.2 %(P < 0.05)lower than that of the control group,respectively.The invasiveness of GLI1 knockout group decreased by 55.2 %(P < 0.05)and 80.1 %(P < 0.05),respectively.(3)Transwell test showed that in H1299 / A549 cells,SMO invasion increased by1.623 times(P < 0.05)and 1.664 times(P < 0.05),respectively.The invasive ability of GLI 1 was 1.680 times(P < 0.05)and 1.588 times(P < 0.05),respectively.(4)Western blot showed that in H1299 cells SMO / GLI 1 overexpression group,the expression of E-cadherin was significantly down-regulated and the expression of EMT marker gene N-cadherin / Vimentin was significantly up-regulated.Conclusion:Hedgehog signaling pathway key molecules SMO and GLI1 can promote the invasion of non-small cell lung cancer cells,which may promote the metastasis of clinical lung cancer.This effect may be achieved by promoting EMT;SMO and GLI 1 gene interference can significantly inhibit the invasion of non-small cell lung cancer cells so it can be used as a potential target for clinical treatment of non-small cell lung cancer.