| BackgroundLung cancer is one of the most diagnosed malignant tumors,and has been the most frequent cause of cancer-related mortality worldwide.Despite treatment advances,it is still a highly aggressive and fatal disease.Lung cancer is progressed to metastasis,and is not diagnosed until it has progressed to an advanced stage.So,more studies are needed to throw light upon the inherent molecular mechanism and eventually ideal prognostic markers and therapeutic method need to be developed.Zinc-fingers and homeoboxes(ZHX)is a family of vertebrate transcription factors that contains three members,ZHX1,ZHX2 and ZHX3.These three genes are expressed in many tissues and localized in the nucleus,and have been reported as tumor suppressor genes in many studies.ZHX2 is an important transcriptional repressor,which contains two zinc-finger motifs and five homeodomains.ZHX2 appears to promote cell-cycle repression and involved in tumorigenesis,and the ZHX2 level is associated with the progression and poor outcome of tumor.Furthermore,ZHX2 may be influence drug resistance in cancer cells.Prior study has reported that deficiency of ZHX2 was found in human hepatcellular carcinoma,and ZHX2 deficiency was an early event in the progression of hepatocellular carcinoma.Compared to adjacent none-tumor tissues,the expression of ZHX2 always reduced in human hepatocellular carcinoma tissues.Moreover,decreased ZHX2 expression was associated with hepatocyte proliferation and reduced survival times of the patients.Meanwhile,both in vivo and in vitro experiments,ZHX2 overexpression inhibited the proliferation of hepatocellular carcinoma cells and the growth of tumor xenografts.ZHX2 is an important regulator of hepatic gene regulation in the development of liver cancer,and ZHX2 could suppress the secretion of alpha-fetoprotein(AFP),which suggested that ZHX2 deficiency was associated with AFP reactivation.To the patients with multiple myeloma,the expression of ZHX2 mRNA is low in peripheral blood lymphocytes compared to healthy individuals.Dysregulated ZHX2 levels will increased the risk of multiple myeloma events.The loss of ZHX2 is associated with the poor outcome of this disease,while high ZHX2 expression is associated with a better response and longer survival.9 After high-dose chemotherapy,high ZHX2 expression heralded a better response and longer survival.As a tumor suppressor,the effect of ZHX2 in the development of tumor has been paid more and more attention.However,there is little research on ZHX2 in lung cancer progression.In the present study,the association between ZHX2 and lung cancer was investigated.We designed a series of experiments to test our hypothesis that ZHX2 may suppress the progress of lung cancer and explore the inherent molecule mechanism.ObjectiveThe study aimed to analyze the expression of ZHX2 in lung cancer and its relationship with the prognosis of patients,to explore the influence of ZHX2 on lung cancer and the molecular mechanism involved,and to provide a reliable theoretical basis and a new direction for the clinical diagnosis,treatment and prognosis assessment of lung cancer.MethodsThis study mainly discussed the function and specific mechanism of ZHX2 in lung cancer.This study is mainly divided into two parts.The first part mainly explores the expression and significance of ZHX2 in lung cancer.First,ma-seq analysis of gene expression differences in lung cancer tissues and paracancer tissues showed that ZHX2 expression was down-regulated in lung cancer,but p38MAPK signaling pathway related gene expression was up-regulated.QPCR further confirmed the sequencing results and proved the reliability of the sequencing results.Secondly,TCGA database was used to analyze the negative correlation between the expression of ZHX2 and mmp-9 in lung cancer.Moreover,the KEGG signal pathway integration analysis found that the p38MAPK signaling pathway was abnormal in lung cancer,so it was speculated that ZHX2 might inhibit the occurrence and development of lung cancer by inhibiting the p38MAPK signaling pathway.Subsequently,the phosphorylation expressions of ZHX2,mmp-9 and p38MAPK(qPCR,WB,IHC,etc.)were analyzed in lung cancer samples to further prove the relationship between ZHX2 and p38MAPK signaling pathways in lung cancer.Finally,the relationship between the expression of these genes and the survival time of patients was analyzed by TCGA database.Through the analysis of patient samples,it can be preliminarily determined that ZHX2 inhibits the occurrence of lung cancer by targeting the p38MAPK signaling pathway.In the second part,the expression of ZHX2 on the biological function of lung cancer cell lines was studied.The second part mainly explores the inhibition of ZHX2 on the proliferation of lung cancer cells by targeting the p3 8 MAPK signaling pathway.First,we detected the expression of ZHX2 in several lung cell lines and found a lung cancer cell line with low expression of ZHX2 as the research object.Subsequently,we overexpressed ZHX2 in the low-expressed ZHX2 cell lines to detect the effects of ZHX2 overexpression on cell proliferation,apoptosis,invasion and metastasis,and tumor formation.ResultsIn the first part of the study,in order to analyze gene expression profiles in lung cancer,we selected three pairs of lung cancer tissues and their corresponding paracancer tissues for rna-seq,and detected gene expression differences in lung cancer tissues and their corresponding paracancer tissues through second-generation sequencing and bioinformatics analysis.Through analysis,it was found that ZHX2 was poorly expressed in lung cancer tissues,while c-myc,ETS1,mmp-9,MEF2 and Statl,the related target genes of p38MAPK signaling pathway,were highly expressed in lung cancer tissues.In order to further prove the reliability of rna-seq sequencing results,we further verified genes with different gene expression in rna-seq by qPCR.The results showed that,consistent with ma-seq sequencing results,the expression of ZHX2 was down-regulated in lung cancer tissues,while the expression of related target genes of p38MAPK signaling pathway c-myc,ETS1,mmp-9,MEF2 and Stat1 were up-regulated in lung cancer tissues,with statistically significant differences(p<0.05).Subsequently,we used KEGG signaling pathway analysis to predict the enrichment of different genes in cancer-related signaling pathways.The results showed that p38MAPK signaling pathway is one of the most obvious signaling pathways activated in lung cancer tissues,and also includes TGF-signaling pathway,EGFR signaling pathway,etc.Since rna-seq sequencing found that the expression of ZHX2 was down-regulated in lung cancer,and the expression of p38MAPK signaling pathway related target genes was up-regulated in lung cancer,we speculated that the down-regulated expression of ZHX2 might lead to the activation of p38MAPK signaling pathway in lung cancer,thus promoting the occurrence of lung cancer.Previous sequencing studies found that ZHX2 was low expressed in lung cancer,and the related target genes of p38MAPK signaling pathway c-myc,ETS1,mmp-9,MEF2 and Statl were highly expressed in lung cancer,which was also found by qPCR detection.In order to prove the synergy between the two,we firstly analyzed the mRNA expression of ZHX2 and p38MAPK signaling pathway related target gene mmp-9 in lung cancer through TCGA database.The results showed that compared with the corresponding paracancer tissues,ZHX2 was indeed low expressed in lung cancer,while mmp-9 was high expressed in lung cancer,and the difference was statistically significant(p<0.05).Through further analysis,we found that the expression of ZHX2 and mmp-9 does have negative synergy.Later,we analyzed the relationship between ZHX2 and mmp-9 expression and the overall survival time of lung cancer patients using the TCGA database.The results showed that the overall survival time of patients with high expression of ZHX2 was significantly higher than that of lung cancer patients with low expression of ZHX2,while the overall survival time of patients with high expression of mmp-9 was significantly lower than that of patients with low expression of mmp-9,and the difference was statistically significant(p<0.05).This suggests that in breast cancer,the down-regulation of ZHX2 expression may promote the occurrence of lung cancer by activating the p38MAPK signaling pathway,and affect the survival and prognosis of patients.Through previous sequencing and database analysis,we preliminarily demonstrated the expression of ZHX2 and p38MAPK signaling pathway target genes in lung cancer.In order to further verify the sequencing results,we analyzed the lung cancer tissues and the corresponding paracancer tissues collected clinically by means of Western blot and immunohistochemistry.The results showed that compared with the corresponding paracancer tissues,ZHX2 showed low expression in the lung cancer tissues,while the phosphorylation level of p38MAPK in the lung cancer tissues was significantly increased,and mmp-9 showed high expression in the lung cancer tissues,indicating that abnormal activation of the p38MAPK signaling pathway did exist in the lung cancer tissuesIn the second part of the study,in order to explore the function and mechanism of ZHX2 in lung cancer,we first detected the expression of ZHX2 in human lung cells.We measured the mRNA expression levels of ZHX2 in five cell lines:human normal lung epithelial beas-2b cells,human non-small cell lung cancer A549,H1975,and HCC827.The results showed that the expression of ZHX2 in A549,H1975 and HCC827 of non-small cell lung cancer was significantly lower than that of beas-2b cells in normal lung epithelial cells(p<0.05)(.Among them,the expression of ZHX2 was the lowest in A549.Therefore,in the follow-up experiment,we chose A549 as the research object for the follow-up study.Subsequently,we detected the effect of ZHX2 expression on cell proliferation,invasion and metastasis.We used cck-8 test to detect the effect of ZHX2 expression on cell proliferation.The results showed that,compared with the control group and the NC group,the ZHX2 mRNA expression was significantly increased in the ZHX2 group and ZHX2+Ani group,and the difference was statistically significant(p<0.05).Compared with the control group and the NC group,the proliferation,migration and invasion abilities of the ZHX2 group and the SB group were significantly reduced,and the difference was statistically significant(p<0.05).In addition,compared with the ZHX2 group,there was no significant change in the proliferation,migration and invasion ability of cells in the SB group,and the difference was not statistically significant(p>0.05).The proliferation,migration and invasion of cells in ZHX2+Ani group were significantly increased,and the difference was statistically significant(p<0.05).This suggests that the expression of ZHX2 can inhibit cell proliferation,invasion and metastasis,while the activation of p38MAPK can reverse the inhibition of ZHX2.We examined the effect of ZHX2 expression on apoptosis.Annexin V assay was used to detect apoptosis.The results showed that compared with the control group and the NC group,the apoptosis rate of cells in the ZHX2 group and the SB group was significantly increased,and the expression of apoptosis-related proteins was significantly increased,and the difference was statistically significant(p<0.05).Compared with the ZHX2 group,there was no significant change in cell apoptosis rate and expression of apoptosis-related proteins Bax,Cleaved caspase-3 and Cleaved caspase-9 in the SB group(p>0.05).However,the apoptosis rate and expression of apoptosis-related proteins were significantly reduced in the ZHX2+Ani group,and the difference was statistically significant(p<0.05).These results suggest that the expression of ZHX2 can promote the apoptosis of human lung cancer cells,while the activation of p38MAPK can inhibit the pro-apoptotic effect of ZHX2.We examined the effect of ZHX2 expression on intracellular p38MAPK signaling pathway related protein expression.Compared with the control group and NC group,the expression of mmp-9 protein in ZHX2 group and SB group was significantly down-regulated,and the expression of p-p38 MAPK/P38 MAPK was significantly decreased,and the difference was statistically significant(p<0.05).Compared with the ZHX2 group,there was no significant change in mmp-9 protein and p-p38 MAPK/P38 MAPK expression in the SB group,and the difference was not statistically significant(p>0.05).In the ZHX2+Ani group,mmp-9 protein expression was significantly up-regulated,and p-p38 MAPK/P38 MAPK expression was significantly up-regulated,and the difference was statistically significant(p<0.05).The results showed that the expression of ZHX2 could inhibit the p38MAPK signaling pathway,and the p38MAPK agonist could reverse the inhibition of ZHX2.We examined the effect of ZHX2 expression on tumor formation in subcutaneous tumorigenesis model of nude mice,and the apoptosis of cells in tumor was detected by Tunel staining.The results showed that compared with the control group and the NC group,the tumor growth rate of the ZHX2 group and the SB group was significantly slower,the tumor volume and weight were significantly reduced,the proportion of PCNA positive cells in the tumor tissue was significantly reduced,and the apoptosis index was significantly increased,with statistically significant differences(p<0.05).Compared with the ZHX2 group,there was no significant change in tumor growth and apoptosis in the SB group,and the difference was not statistically significant(p>0.05).In the ZHX2+Ani group,the tumor growth rate was significantly accelerated,the tumor volume and weight were significantly increased,the proportion of PCNA positive cells in the tumor tissue was significantly increased,and the apoptosis index was significantly reduced,with statistically significant differences(p<0.05).These results suggest that ZHX2 expression inhibits tumor growth and promotes apoptosis,while p38MAPK agonists can reverse this process.We detected the expression of ZHX2 and p38MAPK signaling pathway related proteins in mouse tumor tissues.The results showed that compared with the control group and the NC group,the expression of ZHX2 protein was significantly increased in the ZHX2 group and ZHX2+Ani group,and the difference was statistically significant(p<0.05).Compared with the control group and NC group,the expression of mmp-9 protein in ZHX2 group and SB group was significantly down-regulated,and the expression of p-p38 MAPK/P38 MAPK was significantly decreased,and the difference was statistically significant(p<0.05).Compared with the ZHX2 group,there was no significant change in mmp-9 protein and p-p38 MAPK/P38 MAPK expression in the SB group,and the difference was not statistically significant(p>0.05).The expression of mmp-9 protein and p-p38 MAPK/P38 MAPK in ZHX2+Ani group increased significantly,and the difference was statistically significant(p<0.05).These results suggest that ZHX2 inhibits lung cancer cell proliferation and promotes lung cancer cell apoptosis by inhibiting the p38MAPK signaling pathway.ConclusionsHX2 is abnormally low expressed in lung cancer and is associated with the prognosis of patients.In lung cancer,ZHX2 promotes cell apoptosis by inhibiting the p38MAPK signaling pathway,inhibiting cell proliferation,invasion and metastasis,and tumor formation.Therefore,ZHX2 can be used as a potential target for the treatment of lung cancer. |
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