Effect Of ADAM17-shRNA On Proliferation, Migration And Invasion Of Thyroid Carcinoma Cell

Objectives To study the effect of ADAM17 silence on proliferation,migration and invasion of human thyroid papillary carcinoma K1 cell.Methods 1 Applying lentivirus transfection technique,small interfering RNA（ADAM17-shRNA）of ADAM17 gene carrying red fluorescent protein（RFP）expression frame transferred into human papillary thyroid carcinoma cell line K1 to silent ADAM17 gene.Setting MOI value gradient:30、40、50、60、70、80、90、100,under inverted fluorescence microscope,the expression of RFP was observed to determine the transfection efficiency.Thus,the optimal MOI value of 80%transfection efficiency was found out.2 K1 cells were divided into control group,nonsense shRNA group and ADAM17-shRNA group.ADAM17-shRNA group cells were added to the lentivirus vector integrated with ADAM17-shRNA according to the groped MOI value.Nonsense shRNA group cells were added to the lentivirus vector integrated with nonsense-shRNA according to the same MOI value.The same amount of PBS was added into control group.3 The expression of ADAM17 mRNA in K1 cells was detected by RT-PCR method and the expression of ADAM17 protein was detected by Western blotting assay,which testing the efficiency of ADAM17 gene silencing.4 MTT assay and flow cytometry were used to assess the ability of K1 cell proliferation.The K1 cell migration ability was detected by Transwell migration assay and the invasion ability was assessed by Transwell invasion assay.Results 1 When the MOI value was 80,the transfection efficiency of the virus was more than 80%,proving that lentivirus vector was successfully transfected into K1 cells and expressed stably.2 Calculating of relative expression of ADAM17 mRNA by 2^-ΔΔCTΔΔCT method,control group（1.000±0.000）,nonsense shRNA group（1.064±0.156）,ADAM17-shRNA group（0.082±0.017）,there was no significant difference between nonsense shRNA group and control group（P>0.05）,and the expression level of ADAM17-shRNA group was significantly decreased（P<0.05）.ADAM17 protein expression,control group（0.868±0.183）,nonsense shRNA group（0.841±0.332）,ADAM17-shRNA group（0.267±0.066）,there was no significant difference between nonsense shRNA group and control group（P>0.05）,and the expression level of protein in ADAM17-shRNA group was significantly decreased（P<0.05）.The expression of ADAM17 gene in ADAM17-shRNA group cell was significantly inhibited,and the ADAM17 gene was successfully silenced.The optical density（OD）of K1 cells in control group,nonsense shRNA group and ADAM17-shRNA group were measured by MTT method at 24 h,48 h and 72 h after transfection:24 h（0.431±0.041、0.444±0.050、0.307±0.030）,48 h（0.745±0.028、0.788±0.019、0.450±0.041）,72 h（1.229±0.059、1.255±0.067、0.681±0.036）.The OD value of ADAM17-shRNA group at different time points was lower than that of control group and nonsense shRNA group（P<0.05）,but there was no significant difference between control group and nonsense shRNA group（P>0.05）.The results of flow cytometry showed the percentages of cells in G0/G1 phase were that,control group（35.16±1.59）%,nonsense shRNA group（32.27±2.42）%,ADAM17-shRNA group（51.17±2.14）%.There was obvious G₀/G₁ phase arrest.Compared with control group and nonsense shRNA group,the number of ADAM17-shRNA group at G0/G1 phase sharply increased（P<0.05）,but there was no significant difference between the control group and nonsense shRNA group（P>0.05）.Under 100 times mirror,the number of cells through the membrane in Transwell migration experiment showed that,control group（422.800±24.108）,nonsense shRNA group（432.600±25.550）,ADAM17-shRNA group（149.200±20.753）.The number of cells inADAM17-shRNA group was less than that of control group and nonsense shRNA group（P<0.05）,while there was no significant difference between control group and nonsense shRNA group（P>0.05）.Under 100 times mirror,the number of cells through the membrane in Transwell invasion experiment showed that,control group（216.400±13.088）,nonsense shRNA group（211.800±18.363）,ADAM17-shRNA group（83.800±11.903）.The number of cells inADAM17-shRNA group was less than that of control group and nonsense shRNA group（P<0.05）,while there was no significant difference between control group and nonsense shRNA group（P>0.05）.Conclusions ADAM17-shRNA could successfully silence the ADAM17 gene in K1 cells of papillary thyroid carcinoma.Deletion of ADAM17 gene can inhibit the proliferation,migration and invasion of K1 cells.