ZMIZ2 Promotes Lung Adenocarcinoma EMT,Invasion And Metastasis By Binding PIN1

Background and Objectives:Lung cancer has long been the cancer with the highest incidence rate and mortality in China.Non-small cell lung cancer(NSCLC)accounts for approximately 85%of lung cancer cases,and lung adenocarcinoma(LUAD)is one of its main histological subtypes.The high mortality rate of lung cancer is mainly related to uncontrolled tumor metastasis.Metastasis is the process by which tumor cells escape from their originating site and migrate to lymph nodes and distal organs.To initiate metastasis,it is common for cancer cells to undergo an epithelial-mesenchymal transition(EMT)process,exhibiting the expression changes of various epithelial and mesenchymal factors that endow plastic changes in cancer cells.The occurrence of EMT is regulated by multiple signaling pathways,among which AKT signaling pathways can directly or indirectly regulate EMT in various ways and play an important role in metastasis.Recent studies have shown that zinc finger MIZ-type containing 2(ZMIZ2)is abnormally overexpressed in a variety of malignant tumors and affects biological processes such as cancer cell invasion and metastasis.However,whether ZMIZ2 affects NSCLC invasion and metastasis remains unclear.Peptide-proline cis/trans isomerase(PIN1)mainly mediates the isomerization of phosphorylated Ser/Thr-Pro motifs,thereby regulating the function,stability,or subcellular distribution of its target protein.Pathological correlations between the expression levels of PIN1 and AKT-pSer473 in hman cancers.In addition,PIN1 overexpression has been shown to be associated with lymph node metastasis in NSCLC.This study aims to clarify the molecular mechanism of ZMIZ2 regulating LUAD cell invasion and metastasis,which will provide a new experimental basis for fully understanding the mechanism of LUAD metastasis,in order to provide a certain theoretical basis for the molecular diagnosis and clinical treatment of LUAD.Methods:(1)The Cancer Genome Atlas(TCGA)database was used to analyze the expression of ZMIZ2 in NSCLC tissues,and the relationship between ZMIZ2 and clinical stage,prognosis were further analyzed.(2)A549 and H1299 cells were infected with lentivirus to construct stable ZMIZ2 overexpression and knockdown cell lines.The mRNA and protein levels of ZMIZ2 were detected by qRT-PCR and Western blot,respectively,to analyze the overexpression and knockdown effects.(3)Western blot was used to detect the expression of EMT-related markers in A549 and H1299 cells with stable overexpression or knockdown of ZMIZ2.The effects of ZMIZ2 on EMT,migration,invasion and metastasis of LUAD cells were investigated by cell scratch healing assay,Transwell assay and nude mouse metastasis model.(4)The effect of ZMIZ2 on the downstream pathways was analyzed by high-throughput transcriptome sequencing and KEGG enrichment,and further verified by Western blot.(5)The protein interacting with ZMIZ2 was identified by IntAct molecular interaction database,and the interaction between ZMIZ2 and PIN1 and their localization in cells were verified using protein immunoprecipitation(IP)experiments and immunofluorescence techniques,respectively,and quantitative analysis experiment and half-life experiment were used to explore whether ZMIZ2 affected the expression of PIN1 and its possible regulatory mechanism.(6)SiRNAs interference technology were used to transiently knock down PIN1 expression in A549 and H1299 cells,qRT-PCR and Western blot were used to detect the knockdown effects of PIN1 mRNA and protein,and Western blot was used to detect the expression of EMT markers and phosphorylated AKT,Transwell assay was used to detect the migration and invasion ability of LUAD cells.(7)The recovery experiment was carried out to transiently knock down PIN1 in A549 and H1299 cells with stable overexpression of ZMIZ2.Western blot was used to detect the expression of EMT markers and phosphorylated AKT,and cell scratch and Transwell experiments were used to detect the migration and invasion ability of LUAD cells.Results:(1)Analysis of TCGA database showed that ZMIZ2 was highly expressed in LUAD tissues compared with adjacent tissues,and the survival time of LUAD patients with high expression of ZMIZ2 was significantly reduced.(2)Overexpression of ZMIZ2 could significantly promote EMT,migration and invasion,and metastasis of LUAD cells,and vice versa,while knockdown of ZMIZ2 could significantly inhibit EMT,migration and invasion of LUAD cells.(3)Through high-throughput transcriptome sequencing and KEGG enrichment analysis,the AKT Signaling pathway was identified,and it was found that ZMIZ2 could activate AKT Signaling pathway to regulate EMT and metastasis.(4)ZMIZ2 and PIN1 colocalize and interact with each other in the nucleus.ZMIZ2 can increase PIN1 protein levels.(5)Transient knockdown of PIN1 inhibited EMT,migration and invasion as well as AKT signaling activation in LUAD cells.(6)Knockdown of PIN1 attenuated ZMIZ2-promoted EMT,migration and invasion,as well as AKT signaling activation in LUAD cells.Conclusion:This study revealed that ZMIZ2 is highly expressed in LUAD tissues,and LUAD patients with high ZMIZ2 expression have a significantly reduced survival.ZMIZ2 promotes EMT,migration,invasion and metastasis in vivo and activates AKT signaling pathway in LUAD cells.Furthermore,it was found that the above promotion effects were dependent on PIN1.Mechanistically,We demonstrated that ZMIZ2 interacts with PIN1 and increases PIN1 protein levels,to activates AKT signaling pathway,which in turn promotes EMT,migration,invasion and metastasis of LUAD cells.These results provide a new insight into the mechanism by which ZMIZ2 promotes LUAD invasion and metastasis.