Effects of NFAT5 inhibition on proliferation, invasion, migration and apoptosis of lung adenocarcinoma cells / 国际肿瘤学杂志

ABSTRACT

Objective:To investigate the expressions of nuclear factor of activated T cell 5 (NFAT5) in lung adenocarcinoma tissues and cells, and the effects of NFAT5 on the proliferation, invasion, migration and apoptosis of lung adenocarcinoma cells.Methods:The clinical pathological specimens and paracancerous tissues of 61 patients with lung adenocarcinoma diagnosed and treated in 904th Hospital of Joint Logistic Support Force of People′s Liberation Army from June 2017 to June 2019 were collected. The expression levels of NFAT5 in lung adenocarcinoma tissues and paracancerous tissues were detected by quantitative real-time PCR (qRT-PCR), and the relationships between the expression of NFAT5 and clinicopathological features of patients were analyzed. H1975 cells were divided into control group (no treatment), NC group (transfecting siRNA-NC) and si-NFAT5 group (transfecting siRNA-NFAT5) . qRT-PCR was used to detect the expression level of NFAT5 in cell line. MTT and clone formation assay were used to detect cell proliferation. Transwell and scratch test were used to detect cell invasion and migration ability. Flow cytometry was used to detect cell apoptosis. The expressions of mitogen-activated protein kinase (MAPK) signaling pathway related proteins were detected by Western blotting.Results:The expression level of NFAT5 mRNA in lung adenocarcinoma (3.22±0.20) was significantly higher than that in paracancerous tissues (1.00±0.12), and there was a statistically significant difference ( t=75.662, P<0.001). The expression level of NFAT5 in lung adenocarcinoma tissue was associated with TNM stage ( χ2=10.357, P=0.001) and lymph node metastasis ( χ2=18.268, P<0.001). The expression levels of NFAT5 in the control group, NC group and si-NFAT5 group were 1.00±0.06, 1.01±0.05 and 0.31±0.06, and there was a statistically significant difference ( F=140.498, P<0.001). The absorbance ( A) values in the control group, NC group and si-NFAT5 group were 0.70±0.01, 0.55±0.01 and 0.35±0.01 at 24 h after transfection, 0.92±0.03, 0.87±0.06 and 0.57±0.06 at 48 h after transfection, 1.05±0.01, 0.90±0.01 and 0.66±0.01 at 72 h after transfection, and there were statistically significant differences ( F=9.815, P=0.013; F=45.977, P<0.001; F=129.494, P<0.001). Further pairwise comparison showed that the proliferation abilities of the si-NFAT5 group at 24, 48 and 72 h were significantly lower than those of the control group and NC group (all P<0.001). The cell clone numbers in the three groups were 452.33±31.50, 421.00±17.35 and 129.00±17.35 respectively, with a statistically significant difference ( F=128.200, P<0.001). The cell clone number in the si-NFAT5 group was significantly lower than that in the control group and NC group (both P<0.001). The invasion numbers of cells in the three groups were 262.67±28.02, 278.00±27.50 and 46.00±12.00 respectively, and there was a statistically significant difference ( F=89.896, P<0.001). The cell invasive ability in the si-NFAT5 group was significantly lower than that in the control group and NC group (both P<0.001). The relative scratch widths in the three groups were 0.28±0.04, 0.32±0.04 and 0.54±0.04 respectively, and there was a statistically significant difference ( F=42.889, P<0.001). The relative scratch width in the si-NFAT5 group was significantly increased than that in the control group and NC group (both P<0.001). The apoptosis rates in the three groups were (3.38±0.56)%, (3.14±0.62)% and (13.82±0.75)% respectively, and there was a statistically significant difference ( F=264.705, P<0.001). The apoptosis rate in the si-NFAT5 group was significantly higher than that in the control group and NC group (both P<0.001). The differences of protein expressions of NFAT5, p-P38/P38, p-ERK1/2/ERK1/2, p-JNK/JNK among the three groups were statistically significant ( F=91.245, P<0.001; F=132.896, P<0.001; F=243.332, P<0.001; F=118.358, P<0.001). The protein expressions of NFAT5, p-P38/P38, p-ERK1/2/ERK1/2, p-JNK/JNK in the si-NFAT5 group were all significantly lower than those in the control group and NC group, and there were statistically significant differences (all P<0.001). Conclusion:The expression of NFAT5 is increased in lung adenocarcinoma tissues and cells. Inhibition of NFAT5 can inhibit proliferation, invasion and migration of lung adenocarcinoma H1975 cells, and promote apoptosis of H1975 cells. The mechanism may be related to the inhibition of MAPK signal pathway by NFAT5.