| Objective: Non-small cell lung cancer(NSCLC)has become one of the malignant tumors with the highest morbidity and mortality in the world.Recent studies have shown that aminoacyl-t RNA synthetases(AARSs)are involved in malignant tumors,inflammation,immune system diseases,infections,cardiovascular and neurological diseases in addition to their classical catalysis.Isoleucine-trna synthetase 2(IARS2)is a member of AARSs.Recent studies have shown a correlation with tumorigenesis,but its relationship with lung cancer is not known.To investigate the role and mechanism of IARS2 in non-small cell lung cancer,we evaluated 56 lung cancer tissues and adjacent normal tissues in patients with non-small cell lung cancer.Its expression level is related to the clinicopathological features of lung cancer patients.Stable interfering with IARS2 expression by lentiviral sh RNA technology,and using CCK-8,colony formation assay,flow cytometry(Annexin V/7-AAD staining,PI/RNase staining)to evaluate IARS2 proliferation and colony formation in non-small cell lung cancer cells Effects of apoptosis and cycle distribution on ability;evaluation of the effect of IARS2 on tumorigenicity in non-small cell lung cancer using subcutaneous xenografts in nude mice;evaluation of IARS2 using Gene Chip primeview human human expression profiling chip and IPA(Ingenuity? Pathway Analysis)The expression profile and functional analysis of gene silencing;the molecular mechanisms were discussed by Western Blot,RT-PCR and recovery experiments.This study helps to elucidate the pathogenesis of IARS2 in lung cancer and lay the foundation for exploring new driving genes and molecular targeted therapy for lung cancer treatment.Methods: 1.Western Blot analysis was used to detect the abundance of IARS2 protein in human non-small cell lung cancer tissues and lung cancer cell lines(A549,H1299)and bronchial epithelial cell line(BEAS-2B),and statistical analysis of IARS2 protein and clinicopathological features(including tissue type)Correlation of gender,age,smoking history,and TNM staging.2.Lentiviral sh RNA technology was used to establish a stable interfering IARS2 lung cancer cell line,and CCK-8,plate clone formation assay,flow cytometry(Annexin V/7-AAD staining,PI/RNase staining)were used to detect knockdown of IARS2 on lung cancer cell lines.Effects of proliferation,colony formation,apoptosis,and cell cycle distribution.3.Western Blot was used to detect the expression levels of cleaved-caspase3,cleaved-PARP,caspase9,Bax and Bcl-2,and the effect of IARS2 on apoptosis protein in non-small cell lung cancer cells was evaluated.4.Gene Chip primeview human human expression microarray was used to detect differential gene expression before and after IARS2 knockdown and Ingenuity Pathway Analysis software was used to further analyze the relationship between differentially expressed genes and classical pathways,upstream regulatory factors,disease and function.5.The negative control group and the gene silencing group were inoculated subcutaneously in nude mice to establish an xenograft model in vivo,and the effect of IARS2 on the proliferation of lung cancer cells was observed under the condition of in vivo.6.Western Blot was used to detect the expression levels of related proteins(p-AKT Ser473,p-AKT Thr308,AKT,p-MTOR,MTOR)in AKT/MTOR signal transduction pathway,and A549 cells were treated with novel AKT activator SC79.CCK-8 and Western Blot were used to detect proliferation and expression of pathway proteins,and to evaluate the effect of IARS2 on AKT/MTOR signaling pathway.Result: 1.Compared with adjacent tissues,IARS2 protein is significantly increased in non-small cell lung cancer tissues.The high expression rate of IARS2 in adenocarcinoma accounts for 72.73%,and the high expression rate of IARS2 in squamous cell carcinoma is 64.71%.Adenocarcinoma is slightly higher.Squamous cell carcinoma.The expression of IARS2 protein level may be related to the T phase(P=0.036)and N phase(P=0.037)of the patient's tumor,but not related to the pathological type,age,gender,and smoking history.Compared with the bronchial epithelial BEAS-2B cell line,the expression of IARS2 protein in lung adenocarcinoma cell lines(A549,H1299)was significantly increased.2.The A549 and H1299 cell lines stably knocking down IARS2 were successfully established.Compared with the Control group,the cell proliferation activity of shIARS2-1 group and shIARS2-2 group was significantly decreased,and the difference in proliferation activity at 72 h,96h and 120 h was statistically significant(P<0.05);the cell clone formation ability of shIARS2-1 group and shIARS2-2 group was significantly decreased;knockdown of IARS2 gene could inhibit cell cycle arrest in S phase of A549 cell cycle;compared with A549 control group,shIARS2-1 group The apoptosis rate of shIARS2-2 group was significantly increased(P<0.05).3.The results of apoptosis-related protein showed that compared with the Control group,the relative expression of cleaved-caspase3,cleaved-PARP,caspase9 and Bax in shIARS2-1 and shIARS2-2 groups was significantly increased,while the relative expression of Bcl-2 was significantly higher.Decreased,indicating that knockdown of IARS2 gene can promote the expression of apoptotic protein,IARS2 at least to some extent through the regulation of mitochondrial apoptosis pathway to play its growth inhibition.4.Human gene expression profiling chip and IPA analysis showed that IARS2 expression regulated the expression of 742 genes in A549 cells,of which 507 genes were up-regulated after IARS2 silencing,235 genes were down-regulated;differentially expressed genes and classical pathways,upstream regulatory factors,the relationship between disease and function;differentially regulated genes are highly overlapping with 353 typical pathways involved in apoptosis,cancer,cell cycle regulation,cellular immune response,cell growth,proliferation and development;predicting 259 IARS2 silences will be Activated disease or function predicts 132 suppressed diseases or functions;after IARS2 is silenced,951 molecules such as transcription factors,cytokines,small RNAs,receptors,kinases,chemical molecules,and drugs are activated,and 483 molecules are inhibition.5.The results of AKT/MTOR pathway protein assay showed that the relative expression of p-AKT Ser473,p-AKT Thr308,p-MTOR and MTOR in shIARS2-1 and shIARS2-2 groups was significantly lower than that in Control group.However,there was no significant difference in the relative content of AKT protein,indicating that knockdown of IARS2 inhibits the activation of AKT/MTOR protein by inhibiting AKT/MTOR protein phosphorylation in the AKT/MTOR signaling pathway.6.The relative expression levels of p-AKT Ser473,p-AKT Thr308 and p-MTOR protein in shIARS2-1 group and shIARS2-2 group A549 cells after activator SC79 were higher than those without activator group shIARS2-1 group and shIARS2 group.-2 groups,but lower than the Control group;compared with the non-activator group,the cell proliferation rate of the shIARS2-1 group and the shIARS2-2 group was significantly increased after the addition of the activator SC79(P<0.05),but still lower than the Control The group(P<0.05)indicated that the activator SC79 partially restored the inhibition of proliferation of lung cancer cells induced by IARS2 silencing.7.The tumor volume and tumor weight of shIARS2-1 group and shIARS2-2 group were significantly lower than those of the control group,and the tumor growth rate was significantly decreased,indicating that knocking down the IARS2 gene in mice can inhibit tumor proliferation.Conclusion: 1.IARS2 regulates the expression of multiple genes to exert its biological functions and participate in the occurrence of various diseases and functions.2.The expression of IARS2 is up-regulated in human non-small cell lung cancer tissues,which is positively correlated with T and N phases of tumors.3.Knockdown of IARS2 inhibits tumor cell proliferation,colony formation,cell cycle arrest,promotes tumor cell apoptosis,and regulates mitochondrial apoptosis pathway to some extent.Play its growth inhibition.4.IARS2 may regulate the proliferation of lung cancer cells through the AKT/MTOR signaling pathway. |
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