| Objective LASS2/TMSG1 is a newly discovered tumor metastasis suppressor gene in recent years.Studies have shown that it contains two conserved domains,TLC and Homeo-domain,which affect tumor cell growth,invasion,metastasis,metabolism,apoptosis,differentiation,etc.To explore the apoptosis-related genes and signal transduction pathways regulated by LASS2/TMSG1 in human lung cancer A549 cells based on genome-wide expression profile microarray detection technique,to explore the relationship between genes,to find the auxiliary venation of differential genes affecting organisms,and to systematically explore the mechanism of LASS2/TMSG1 gene involved in the apoptosis of human lung cancer cells tumor cell apoptosis in complex action chains.Method Lentivirus infection experiment was used to construct LASS2/TMSG1 gene silencing cell line,scratch test was used to detect the effect of LASS2/TMSG1 gene silencing on A549 cell migration,plate clone formation test was used to detect the effect of LASS2/TMSG1 gene silencing on A549 cell proliferation.Tunel/DAPI double staining test and Hoechst/PI double staining test were used to detect the effect of LASS2/TMSG1 gene on A549 cell apoptosis.Human lung cancer A549 cells before and after LASS2/TMSG1 silencing were detected by genome-wide expression microarray,differential genes were screened,significance of differential genes and signal transduction pathways were analyzed,global signal transduction network,signal pathway action network and co-expression regulatory network were obtained,and some of the results of genome-wide expression microarray were verified by fluorescence real-time quantitative PCR and Westernblot.Small interference RNA transient test was used to construct p38MAPK interference cell line to inversely verify the relationship between p38MAPK and LASS2/TMSG1.SPSS22.0 statistical software was used for data analysis and processing,and t-test was used to compare the mean of samples between the two groups,P<0.05,there was significant difference between the two groups.Results The levels of LASS2/TMSG1 mRNA and protein in the LASS2/TMSG1 silence group were significantly lower than those in the blank control group and the negative control group(real-time PCR method:t=3.36,P=0.030;t=5.56,P=0.005 Western blot:t=4.30,P=0.013;t=5.22,P=0.006).Compared with the blank control group and the negative control group,the cell migration rate of the LASS2/TMSG1 silence group was significantly higher than that of the other two groups(t=29.72,P=0.000;t=53.06,P=0.000),and the cell proliferation ability of the LASS2/TMSG1 silence group was significantly higher than that of the blank control group and the negative control group(t=17.20,P=0.000;t=12.27,P=0.000).The results of Tunel/DAPI double staining showed that the apoptosis rate in the LASS2/TMSG1 silence group was significantly lower than that in the blank control group and negative control group(t=3.21,P=0.033;t=4.54,P=0.011).The results of Hoechst/PI double staining showed that the early apoptosis rate in LASS2/TMSG1 silencing group was significantly lower than that in blank control group and negative control group(t=4.61,P=0.010;t=3.31,P=0.030),but there was no significant difference in late apoptosis and necrosis among the three groups(Late apoptosis:t=0.42,P=0.697;t=0.23,P=0.832;t=0.34,P=0.754 Necrosis:t=0.74,P=0.501;t=0.44,P=0.682;t=0.96,P=0.389).2289 differential genes were screened by genome-wide expression profile microarray,including 1237 up-regulated genes and 1051 down-regulated genes.The significance analysis of up-regulated differential genes is mainly distributed in the biological processes such as cell division,mitosis,G2max M conversion of mitotic cell cycle,cell proliferation and so on.The up-regulated signal pathways mainly include cell cycle,DNA replication,MAPK signal pathway,PI3K-Akt signal pathway and so on.The significance analysis of down-regulated differential genes is mainly distributed in biological processes such as cell adhesion,inflammatory response,angiogenesis,regulation of ERK1 and ERK2 cascade,down-regulated signal pathways mainly include complement and coagulation cascade,cytokine-cytokine receptor interaction,tumor necrosis factor signal pathway,autophagy-animal,cell apoptosis and so on.The global signal transduction network showed that the degree values of MAP2K6(MEK6)and MAP3K7(TAK1)were the high and down-regulated;the signal pathway network showed that the degree value of apoptosis signal pathway ranked first and down-regulated,while the degree value of MAPK signal pathway ranked second and up-regulated;the co-expression regulatory network showed that the top 10 degree values were TMX3,KIF23,HMMR,WISP2,PAPPA,UACA,CDC25C,CHP1,DBF4 and TOP2A.UACA is an anti-apoptotic gene related to apoptosis.The mRNA and protein levels of MAP2K6(MEK6),MAP3K7(TAK1)and p38MAPK in LASS2/TMSG1 silent group detected by fluorescence real-time quantitative PCR and Western blot were significantly lower than those in blank control group and negative control group,and the difference was statistically significant(all P<0.01,see the results in detail),which was consistent with the results of gene chip.The levels of p38MAPK mRNA and protein in the p38MAPK interference group were significantly lower than those in the blank control group and negative control group(real-time PCR method:t=23.46,P=0.000;t=26.09,P=0.000;Western blot t=9.24,P=0.000;t=14.81,P=0.000).The levels of LASS2/TMSG1 mRNA and protein white in the p38MAPK interference group were also significantly lower than those in the blank control group and negative control group(real-time fluorescence quantitative PCR:t=4.02,P=0.007;t=10.37,P=0.000;Western blot:t=3.63,P=0.022;t=4.60,P=0.010).Conclusion LASS2/TMSG1 gene silencing promotes the migration and proliferation of human lung cancer A549 cells,inhibites the early apoptosis of A549 cells,and inversely proved that LASS2/TMSG1 gene may be involved in inhibiting the migration and proliferation of human lung cancer cells and inducing tumor cell apoptosis.Combined with the previous research of our group and the results of genome-wide expression microarray analysis,LASS2/TMSG1 may reduce the activation of p38MAPK by down-regulating the signal transduction pathway of MAPK family,that is,down-regulating p38MAPK gene and its upstream factors MAP2K6(MEK6)and MAP3K7(TAK1),thus inhibiting the downstream biological behaviors regulated by p38MAPK(including the process of apoptosis,etc.).In addition,this experiment also screened the anti-apoptosis gene UACA,which is quite different,which provides the direction for the next research of this topic. |
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