NUDIX hydrolase 4 promotes tumor development by modulating the cell cycle in lung cancer
Objective The role of NUDIX hydrolase 4 in tumor progression is unclear.This study aims to explore its mechanisms in lung cancer development to provide new therapeutic directions or theoreti-cal bases.Methods Targeting the 4th and 5th exons of NUDIX hydrolase 4 DNA,short hairpin RNA(shRNA)fragments against its mRNA were designed and corresponding plasmids were synthesized.Then lentivirus was constructed to infect tumor cell lines,which were from Cell Bank in Chinese Academy of Sci-ences.Real-time quantitative reverse transcriptase-polymerase chain reaction(RT-qPCR)was used to con-firm the knockdown efficiency of NUDIX hydrolase 4.The effect on cell proliferation was assessed using the cell counting kit-8(CCK-8)assay,while cell cycle was confirmed by DNA analysis.The impact on cell migration was evaluated using Transwell chambers.The cell line with NUDIX hydrolase 4 knockdown was set as the experimental group,and the cell line containing the empty plasmid was set as the control group.The two independent sample t-test was used to compare the statistical differences between the experimental group and the control group.Results The expression level of NUDIX hydrolase 4 in A549 shRNA1 exper-imental group was lower than that in the control group[(21.57±3.14)％vs.(101±11.85)％,t=11.15,P＜0.01].The expression level in A549 shRNA2 experimental group was lower than that in the control group[(45.76±4.91)％vs.(101±11.85)％,t=7.39,P＜0.01].The expression level in A549 shRNA3 experimental group was lower than that in the control group[(11.69±0.25)％vs.(101±11.85)％,t=12.98,P＜0.01].The expression level of NUDIX hydrolase 4 in H1299 shRNA1 experi-mental group was lower than that in the control group[(12.05±0.98)％vs.(100.00±2.47)％,t=57.43,P＜0.01],the expression level in H1299 shRNA2 experimental group was lower than that in the control group[(30.24±2.16)％vs.(100.00±2.47)％,t=36.88,P＜0.01],the expression level in H1299 shRNA3 experimental group was lower than that in the control group[(15.21±1.77)％vs.(100.00±2.47)％,t=48.36,P＜0.01].CCK-8 assay results showed that tumor cell proliferation capa-bility of A549 shRNA1 experimental group was lower than control group(0.46±0.01 vs.3.52±0.20,t=27.02,P＜0.01).A549 shRNA2 experimental group was lower than control group(0.61±0.01 vs.3.52±0.20,t=25.62,P＜0.01).A549 shRNA3 experimental group was lower than control group(0.56±0.01 vs.3.52±0.20,t=26.13,P＜0.01).H1299 shRNA1 experimental group was lower than control group(1.12±0.05 vs.2.66±0.06,t=35.15,P＜0.01).H1299 shRNA2 experimental group was lower than control group(0.81±0.04 vs.2.66±0.06,t=46.94,P＜0.01),H1299 shRN A3 ex-perimental group was lower than control group(0.93±0.01 vs.2.66±0.06,t=52.28,P＜0.01).Fur-ther DNA analysis of H1299 revealed that the S phase proportion in H1299 shRNA1 experimental group was higher than control group[(19.83±0.99)％vs.(14.08±1.54)％,t=5.43,P＜0.01].The S phase proportion in H1299 shRNA2 experimental group was higher than control group[(24.35±1.34)％vs.(14.08±1.54)％,t=8.72,P＜0.01].The S phase proportion in H1299 shRNA3 experimental group was higher than control group[(32.07±0.29)％vs.(14.08±1.54)％,t=19.84,P＜0.01].The Tr-answell assay found no statistical difference in cell migration between the experimental and control groups,such as cell migration number in A549 shRNA1 experimental group compared with control group[(131.30±11.59)counts vs.(127.30±17.39)counts,t=0.33,P＞0.05],and cell migration number in H1299 shRNA1 experimental group compared with control group[(104.30±8.15)counts vs.(16.00±11.27)counts,t=1.45,P＞0.05].Conclusion Interfering with NUDIX hydrolase 4 expres-sion induces cell cycle arrest at the S phase,which inhibits tumor cell proliferation,but does not affect mi-gration ability in lung adenocarcinoma cell lines.
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