Screening Of The Target MicroRNA In Non-Small Cell Lung Cancer 95D Cells And Observation Of The Invasion Ability Of 95D Cells Regulated By Let-7a In Vitro

PartⅠScreening of the Target microRNA in 95D cells and the determination of let-7a expression level in NSCLC tissue samples[Objective] To screen the target microRNA in 95D cell line.[Methods] The target microRNA of 95D cell lines was determined by miRNA microarray treated with CpG ODN. The determined target microRNA expression was detected with Real-time PCR and compared between lung cancer and corresponding normal lung tissues in 20 NSCLC patients.[Results] Most microRNAs were downregulated after the treatment of CpG ODN and let-7a was the one with the greatest extent(decreased by 6.9 times). The low level of let-7a expression was found in 70% NSCLC tissue samples and its expression in lung cancer tissues was significantly lower than that in normal lung tissues (about 60%, p<0.05).[Conclusions] let-7a as the target microRNA in 95D cell lines was determined. Part II The construction and identification of let-7a lentivirus vectors[Objective] To construct and identify the two lentivirus vectors of let-7a mimics and let-7a inhibitor.[Methods] The lentivirus vectors of let-7a mimics and let-7a inhibitor were constructed and packaged respectively. The titers of the two lentivirus vectors were determined and then the two lentivirus vectors were transfected into 95D cells respectively.[Results] The sequence of the two lentivirus vectors were both correct. The efficiency of the transfected test could be reached at 95% when the titer of each lentivirus vector was 1×108TU/ml. The results of real-time PCR showed that the expression level of let-7a was significantly higher in 95D cells transfected with lentivirus vector of let-7a mimics than that of 95D cells transfeccted with lentivirus vector of let-7a inhibitor (P<0.05).[Conclusions] The lentivirus vectors of let-7a mimics and let-7a inhibitor were constructed successfully.PartⅢThe possible mechanism of reduced proliferation and invasion ability in 95D cells induced by let-7a[Objective] To observe the effect of let-7a expression on proli feration and invasion ability in 95D cells and discuss the possible mechanism.[Methods] The 95D cells were divided into lot-7a over-expressed group or let-7a inhibited group according to the two different lentivirus vectors with which were transfected. The proliferation and invasion ability in 95D cells of the two groups were tested and compared by CCK8 kits and Transwell test respectively. The mRNA and protein expressions of KRAS and HMGA2 in the two groups were detected and compared by real-time PCR and Western blot test respectively. [Results] The proliferation and invasion ability were significantly lower in let-7a over-expressed group than those in let-7a inhibited group (P<0.05). The mRNA expressions of KRAS and HMGA2 were significantly higher in let-7a over-expressed group than those in let-7a inhibited group (P<0.05) while the protein expressions of KRAS and HMGA2 were significantly lower in let-7a over-expressed group than those in let-7a inhibited group (P<0.05).[Conclusions] High level of let-7a could inhibit the proliferation and invasion ability of 95D cell lines. The inhibited effect on HMGA2 and KRAS proteins expression induced by let-7a resulted from the post-transcriptional regulation on HMGA2 and KRAS mRNAs may partly be the important mechanism.