The Study Of RNA Interference-mediated Suppression Of CAR In Human Lung Squamous Carcinoma NCI-H520Cell Line In Vitro

Objective:To investigate the effects of CAR gene on proliferation and invasion of human lung cancer cells, we established a lung squamous carcinoma cell line in which CAR gene expression was stably suppressed by RNA interference, colony forming experiments and Transwell chamber were employed to detect the correlation of CAR and tumor formation, invasion and metastasis.Method:1Three small interfering RNAs (siRNA) targeting CAR gene were transfected into a lung squamous carcinoma NCI-H520cell line. G418was used to select a cell line in which CAR gene expression was stably suppressed by RNA interference.2The expression levels of CAR mRNA and protein were detected by RT-PCR and Western blot in three NCI-H520cell lines. One of the three cell lines in which CAR gene expression was most suppressed was selected.3colony forming experiments and Transwell chamber were employed to detect the effects of CAR gene on proliferation, invasion and migration of NCI-H520cells after CAR expression was reduced.Results:1Three NCI-H520cell lines in which CAR gene expression was stably suppressed was obtained.2RT-PCR and Western blot results showed CAR expression was successfully reduced on mRNA and protein levels. Among the three NCI-H520cell lines, shRNA-2showed the strongest inhibition effect, with inhibition rates of82.5%and79.8%for the expression of CAR mRNA and protein respectively.4A significant correlation between CAR and tumour invasion, migration was observed. Colony forming assays suggested reduced CAR levels are associated with weakened proliferation ability, however, these data did not reach statistical significance.Conclusion:1A cell line NCI-H520in which CAR gene expression was stably and efficiently suppressed was successfully constructed. 2Low expression level of CAR gene in NCI-H520cell inhibited cell invasion and migration. These data predicted that CAR expression might be necessary for invasion and migration of lung cancer cells.3Low expression level of CAR gene in NCI-H520cells did not influence tumor cell growth, this observation need further study toward delineating the mechanisms underlying the role of CAR in carcinogenesis.