Effect Of LKB1 Overexpression On Proliferation And Migration Of Human Thyroid Papillary Carcinoma Cells

Objectives To investigate the effect of LKB1 overexpression on the proliferation and migration of human thyroid papillary carcinoma cells.Methods Firstly,Western Blot was used to detect the expression of LKB1 protein in normal Thyroid Nthy-ori3-1 cells and papillary thyroid carcinoma K1 cells.Then the well-growing K1 papillary thyroid carcinoma cells were divided randomly into Control group,NC group and Lenti-h-LKB1 group.The Control group was treated without any treatment,while the NC group was transferred with empty vector and the Lenti-h-LKB1 group was transferred with LKB1 recombinant eukaryotic carrying green fluorescent protein(GFP).The expression of GFP was observed under inverted fluorescence microscope to determine the transfection efficiency after 72 hours,Take the transfection efficiency of more than 80% of the cells spare.The expression of LKB1 mRNA in each group was detected by Realtime PCR,and the expression of LKB1 protein was detected by Western blot to verify whether the LKB1 overexpression cell model was constructed successfully.Each group of cells were cultivated continuously for 24,48,72,96 hours,the proliferation of cells in each group(OD)was detected by MTT assay.The cells were cultured for 24 hours after successful transfection,Transwell invasion assay was used to detect cell migration and Flow cytometry was used to detect cell cycle changes.Results Compared with normal thyroid Nthy-ori3-1 cells,the expression of LKB1 protein in K1 cells decreased,and the difference was statistically significant(t=40.376,P<0.01).72 hours after transfection,the transfection efficiency of the virus was more than 80% when the complex number of infection(MOI)was about 60.Realtime PCR results showed that the relative expression level of LKB1 mRNA in Lenti-h-LKB1 group was(5.992 ± 0.610),higher than that in Control group(1.000 ± 0.000)and NC group(1.065 ± 0.198),the difference among them was statistically significant(F=444.557,P<0.01),while there was no significant difference between the negative control group and the blank group(P> 0.05).Western blot results showed that the relative expression levels of LKB1 protein in Lenti-h-LKB1 group was(1.152±0.042),higher than that in Control group(0.339±0.021)and NC group(0.343± 0.049),the difference was statistically significant(F=428.530,P<0.01),indicating that LKB1 overexpression cell model was constructed successfully.The results of MTT assay showed that the OD value of the Lenti-h-LKB1 group at different time points was lower than that of the Control group and the NC group(P<0.05),but there was no significant difference between the Control group and the NC group(P>0.05).Transwell experimental results showed that the number of transmembrane cells in the Lenti-h-LKB1 group was(118.000±7.714)/field,lower than that in Control group(334.800±16.829)/field and NC group(332.400±18.528)/field,the difference among them was statistically significant(F=338.831,P<0.01),while there was no significant difference between the negative control group and the blank group(P> 0.05).The results of flow cytometry showed that,in Control group,NC group and Lenti-h-LKB1 group,the percentages of cells in G0/G1 phase were(44.065 ± 0.021)%,(43.740 ± 0.226)% and(58.260±1.044)%,the percentage of cells in S phase were(43.755±0.205)%,(43.425±0.304)%and(34.890±0.506)%respectively.Compared with the Control group and the NC group,the number of cells in the Lenti-h-LKB1 group increased at G0/ G1 phase,decreased in the S phase(F=310.187,P<0.01;F=3352.726,P<0.01),but there was no significant difference between the Control group and the NC group(P> 0.05).Conclusions 1 The expression of LKB1 protein in thyroid papillary carcinoma K1 Cells was reducted.2 the overexpression of LKB1 can inhibit the proliferation and migration of K1 cells in human papillary thyroid carcinoma.