The Effect Of Silent GATA6 Expression On Oral Squamous Cell Carcinoma SCC-4 Cell Growth And NF-κB Signaling Pathway

Objective:To clarify the role of GATA6 in the proliferation of oral squamous cell carcinoma cells,preliminarily discuss the effects of silencing GATA6 on NF-κB signaling pathway in oral squamous cell cancer cells,and explore the possible mechanism.Methods:1.The cell culture,serial subcultivation:Take out oral squamous cell carcinoma cell lines CAL-27,SCC-4,normal oral epithelial cell lines HOK inliquid nitrogen storage.The cell suspension was inhaled into a cell dish containing appropriate medium for culture and serial subcultivation after recovery.2.Reverse transcription polymerase chain reaction RT-PCR was used to detect the m RNA expression of GATA6 in oral squamous cell carcinoma cell and normal oral epithelial cell.3.Western blot was used to detect the protein expression of GATA6 in oral squamous cell carcinoma cell and normal oral epithelial cell.4.Three interfering fragments of GATA6(siRNA for GATA6)were designed and synthesized and transfected into the oral squamous cell carcinoma cell SCC-4 with the highest up-regulated GATA6 expression and the control cell line.The GATA6 gene silencing efficiency was verified by RT-PCR and Western blot.5.CCK8 method was used to observe the proliferation of cells in each group after transfection.6.Cell cycle was detected by Flow cytometry.7.Western blot was used to detect the expressions of key proteins(IκB、p-IκB-α、IKKα、IKKβ、p-IKKα/β、NF-κB p65、p-NF-κB p65)involved in the NF-κB signaling pathway in the transfected cells.Results:1.Changes in m RNA and protein expressions of GATA6 in oral squamous cell carcinoma cells and normal oral epithelial cells:compared with HOK cells,the m RNA expression levels of GATA6 in CAL-27 and SCC-4 cells were significantly increased,with statistically significant difference.Compared with HOK cells,the protein expression level of GATA6 in SCC-4 cells was significantly increased,with statistically significant difference.The protein expression level of GATA6 in CAL-27 cells had no significant difference compared with HOK cells.2.The GATA6 siRNA was transfected into SCC-4 cells with the highest up-regulated expression,and the down-regulated effect was determined by RT-PCR and Western blot:the GATA6 siRNA-2 and GATA6 siRNA-3 groups in the three transfected interference fragments were obviously lower than the control group,the difference was significant.Compared with the blank control group,the GATA6 siRNA-1 group had no significant difference in the expression of GATA6 m RNA,and the interference efficiency was not up to 50%,suggesting that the interference was not successful.β-actin was used as internal reference antibody.Protein expression levels of GATA6 in transfected SCC-4 cells were measured.Moreover,the protein expression level of GATA6 in SCC-4 cells in GATA6 siRNA-2 and GATA6 siRNA-3 successfully interfered group was significantly lower than that in the pure cell blank control group,the difference was statistically significant.3.Cell proliferation in each group was detected by CCK8 method(continuous detection for 7 days):in each period,Compared with the blank control group and the ineffective interference group GATA6 siRNA-1,the absorbance value in the effective interference group was decreased.Cell proliferation was slow and growth was significantly inhibited.On the 7th day after transfection,the inhibition of cell growth was the greatest,the difference was significant.It is suggest that targeted inhibition of the expression of GATA6 may potentially inhibit the proliferation of oral squamous cell carcinoma.4.The cell cycle was analyzed by flow cytometry in each group showed that:the effective interference groups GATA6 siRNA-2 and GATA6 siRNA-3 had significantly higher G0/G1 cell ratio than the blank control group and the ineffective interference group GATA6 siRNA-1,with statistically significant difference.The cell ratios in S and G2/M phase were downregulated.It is suggested that the groups that interferes with the expression of GATA6 may inhibit the cell proliferation ability through G0/G1 phase cell cycle arrest.5.Western blot was used to detect the key proteins of NF-κB signaling pathway(IκB、p-IκB-α、IKKα、IKKβ、p-IKKα/β、NF-κB p65、p-NF-κB p65)in the transfected cells:the expression of p-IκB-α、IKKα and IKKβ was significantly decreased in GATA6 siRNA-2 and GATA6 siRNA-3 groups compared with the blank control group and GATA6 siRNA-1 group.Furthermore,the activation of NF-κB signaling pathway was suppressed to some extent.Conclusion:GATA6 is highly expressed in SCC-4 cells of oral squamous cell carcinoma cell line.The GATA6 in SCC-4 was silenced by siRNA interference,which can significantly inhibit the proliferation of oral squamous cell carcinoma cells,block its cell cycle,and inhibit the activation of NF-κB signaling pathway.