The Function And Mechanism Of LncRNA ZFPM2-AS1 In Gastric Cancer And Tumor Microenvironment

Objective: With the advancement of bioinformatics and RNA-sequencing techniques,the essential role of long non-coding RNAs(lncRNAs)in gastric cancer(GC)has attracted more and more attention.LncRNAs could not only regulate the proliferation and invasion of GC cells but also modulate the immune escape,thus affecting the occurrence and development of GC.In this study,we aimed to determine an immune-related lncRNA(IRlncRNA)and explore regulatory mechanisms in GC and tumor microenvironment.Methods: The lncRNA data of GC and normal samples were extracted from the TCGA,and the list of immune-related genes was downloaded from the Imm Port database.The correlation and differential expression analyses were utilized to identify the differentially expressed IRlncRNAs(DE-IRlncRNAs).Then,the core IRlncRNA(lncRNA ZFPM2-AS1)was determined via WGCNA and survival analysis,and q RT-PCR assay was performed to detect its expression levels.Meanwhile,CCK-8,colony formation,wound-healing,Transwell migration,and invasion assays were carried out to explore the effect of lncRNA ZFPM2-AS1 on GC cells.Bioinformatics analysis was carried out to predict potential miRNAs interacting with lncRNA ZFPM2-AS1 and the potential target genes of miRNAs.Then,q RT-PCR,Western Blot(WB),RNA Binding Protein Immunoprecipitation(RIP),and dual luciferase experiments were conducted to verify the above results.Cell function experiments were utilized to explore whether lncRNA ZFPEM2-AS1 could modulate the malignant biological behavior of GC cells by miR-139-5p/ABL2/NF-κB signaling pathway.Furthermore,different subsets of macrophages were constructed,and lncRNA ZFPM2-AS1 expression levels were detected via q RT-PCR.In vitro experiments were conducted to determine whether lncRNA ZFPM2-AS1 could affect the migration,invasion,and epithelial-mesenchymal transition(EMT)of GC cells via modulating the M2 polarization of tumor-associated macrophages(TAMs)and its specific regulatory mechanisms.Results: The results of bioinformatics analysis revealed that the core IRlncRNA(lncRNA ZFPM2-AS1)was significantly overexpressed in GC samples and cells and associated with the poor prognosis of patients.The results of CCK8 and colony formation assays indicated that lncRNA ZFPM2-AS1 enhanced the proliferation of GC cells.The data from flow cytometry analysis indicated that lncRNA ZFPM2-AS1 promoted the cycle process of GC cells.The results of Transwell migration and invasion assays exhibited that lncRNA ZFPM2-AS1 enhanced the migration and invasion of GC cells.The results of WB assay showed that lncRNA ZFPM2-AS1 promoted the EMT of GC cells.The results of mechanism study showed that lncRNA ZFPM2-AS1 could upregulate the expression of ABL2 through competitive binding to miR-139-5p and further activated the NF-κB signaling pathway,thus promoting the proliferation,migration,invasion,and EMT of GC cells.In addition,lncRNA ZFPM2-AS1 was highly expressed in M2 and TAMs,suggesting that it might promote the M2 polarization of TAMs.Further studies indicated that lncRNA ZFPM2-AS1 could induce the M2 polarization of TAMs via activating JAK2/STAT3 pathway,thereby promoting the invasion and metastasis of GC.Conclusions: LncRNA ZFPM2-AS1,as the core IRlncRNA in GC,could promote the progression of GC through the miR-139-5p/ABL2/NF-κB pathway.Furthermore,lncRNA ZFPM2-AS1 could indirectly promote the invasion and metastasis of GC by inducing the M2 polarization of TAMs.Part Ⅰ: The Expression and Biological Function of LncRNA ZFPM2-AS1 in GCObjective: GC is one of the most common digestive system malignancies characterized by low early diagnosis rates,high recurrence and metastasis rates,and poor prognosis.Therefore,human health is seriously threatened.In recent years,the crucial role of lncRNAs in tumors has attracted increasing attention.In this study,we aimed to determine a novel lncRNA involved in the initiation and progression of GC and explore the effect on the biological behavior of GC cells.Methods: Bioinformatics analysis was performed to identify DE-IRlncRNAs.Based on the expression of DE-IRlncRNAs,a novel molecular subtype of GC was identified by the non-negative matrix factorization(NMF)algorithm.The Kaplan–Meier analysis and ESTIMATE algorithm were applied to analyze the overall survival(OS)and tumor microenvironment(TME)differences in different molecular subtypes.Subsequently,the core immune-related lncRNA(lncRNA ZFPM2-AS1)was identified using WGCNA.To investigate the role of lncRNA ZFPM2-AS1 in the development of GC,q RT-PCR was utilized to detect its expression levels.Meanwhile,GC cell lines with stable knockdown or overexpression of lncRNA ZFPM2-AS1 were established,and cell function experiments were carried out to explore the effect of lncRNA ZFPM2-AS1 on GC cells.WB was further performed to explore the effect of lncRNA ZFPM2-AS1 on the EMT of GC cells.Results: Based on the expression of these 406 DE-IRlncRNAs,all GC patients were divided into two molecular subtypes,including Cluster 1 and Cluster 2.The Kaplan–Meier analysis showed that Cluster 1 patients had a worse OS rate than Cluster 2.Furthermore,Immune Score,Stromal Score,and ESTIMATEScore were significantly elevated in Cluster 1 compared with Cluster 2.Then,the core DE-IRlncRNA(lncRNA ZFPM2-AS1)was identified by WGCNA analysis.Based on the TCGA database,lncRNA ZFPM2-AS1 was found to be significantly overexpressed in GC samples and associated with the survival of patients.The result of q RT-PCR also showed that lncRNA ZFPM2-AS1 was highly expressed in GC cell lines.The results of cell function experiments indicated that the knockdown of lncRNA ZFPM2-AS1 inhibited the proliferation,migration,and invasion of GC cells.In contrast,overexpression of lncRNA ZFPM2-AS1 promoted the proliferation,migration,and invasion of GC cells.Furthermore,the results of WB assay showed that after lncRNA ZFPEM2-AS1 knockdown,the expression of E-cadherin was significantly upregulated,and the expression of N-cadherin,Vimentin,p-p65,and p-IKBα was significantly downregulated.In contrast,after lncRNA ZFPM2-AS1 overexpression,the levels of E-cadherin were remarkably reduced,and the levels of N-cadherin,Vimentin,p-p65,and p-IKBα were remarkably increased.Conclusions: LncRNA ZFPM2-AS1 is significantly overexpressed in GC and associated with the prognosis of patients.Furthermore,lncRNA ZFPM2-AS1 plays an oncogenic role by activating the NF-κB signaling pathway and could be a promising diagnostic biomarker and therapeutic target in GC.Part Ⅱ: LncRNA ZFPM2-AS1 Regulates the Proliferation,Migration,Invasion,and EMT of GC Cells via miR-139-5p/ABL2/NF-κB Signaling PathwayObjective: LncRNAs could competitively bind miRNAs and reduce the inhibition of miRNAs on target genes,therefore promoting the expression of target genes and making them play pro-tumorigenic or anti-tumorigenic functions.The above regulatory mechanism,also known as the competing endogenous RNA(ceRNA)mechanism,provides a crucial direction for tumor research.Therefore,we further investigated whether lncRNA ZFPM2-AS1 could modulate GC cell malignant biological behaviors through the ceRNA mechanism.Methods: Bioinformatics analysis was utilized to predict potential miRNAs interacting with lncRNA ZFPM2-AS1 and the potential target genes of miRNAs.Then,the lncRNA ZFPM2-AS1-miR-139-5p-ABL2 regulatory network was constructed,and q RT-PCR,RIP,and dual luciferase experiments were carried out to verify the predicted results.q RT-PCR and WB assays were utilized to detect the levels of miR-139-5p after lncRNA ZFPM2-AS1 knockdown and overexpression in GC cells,as well as the levels of ABL2 after silencing and upregulating miR-139-5p.RIP and dual luciferase assays were further applied to demonstrate the interaction between lncRNA ZFPM2-AS1 and miR-139-5p,as well as miR-139-5p and ABL2.To reveal the molecular mechanisms of lncRNA ZFPM2-AS1 in GC,cell function experiments were performed to determine whether lncRNA ZFPEM2-AS1 could regulate the proliferation,cell cycle,migration,invasion,and EMT of GC cells through miR-139-5p/ABL2/NF-κB signaling pathway.Results: According to bioinformatics analysis,we constructed the lncRNA ZFPM2-AS1-miR-139-5p-ABL2 regulatory network.The q RT-PCR and WB assay results showed that the knockdown of lncRNA ZFPM2-AS1 remarkably increased miR-139-5p expression in GC cell lines.In contrast,the overexpression of lncRNA ZFPM2-AS1 significantly reduced miR-139-5p expression.Furthermore,the knockdown of miR-139-5p remarkably upregulated the expression of ABL2,while the overexpression of miR-139-5p significantly downregulated the expression of ABL2.RIP and dual luciferase experiments revealed that miR-139-5p could directly bind to lncRNA ZFPM2-AS1,and ABL2 could directly bind to miR-139-5p.Then,LV-ZFPM2-AS1 and miR-139-5p mimics were co-transfected into GC cells,and rescue experiments were applied to demonstrate.The rescue assays indicated that upregulating miR-139-5p could remarkably suppress the promotive effects of lncRNA ZFPM2-AS1 on the proliferation,migration,invasion,and EMT of GC cells.The results of WB revealed that miR-139-5p could also significantly inhibit the activation of lncRNA ZFPM2-AS1 on ABL2 and the NF-κB signaling pathway.Furthermore,in vitro experiments indicated that highly expressed ABL2 could partially rescue the inhibitory effects of miR-139-5p on the proliferation,migration,invasion,and EMT of GC cells.The results of WB also showed that upregulated ABL2 could partially rescue the suppression of miR-139-5p on the NF-κB signaling pathway.Conclusions: LncRNA ZFPEM2-AS1 could upregulate the expression of ABL2 by competitively binding to miR-139-5p,and further activates the NF-κB signaling pathway,thereby promoting the initiation and progression of GC.The regulatory mechanism of lncRNA ZFPM2-AS1 enriches the molecular network of lncRNA biological functions in GC and provides a novel theoretical basis for the prevention,early diagnosis,and treatment of GC.Part Ⅲ: LncRNA ZFPM2-AS1 Regulates the Migration,Invasion,and EMT of GC Cells via Inducing the M2 Polarization of Tumor-Associated MacrophagesObjective: TAMs are the most abundant immune cells in the tumor microenvironment(TME),which are characterized by high heterogeneity and plasticity.The conversion between M1 and M2 polarization status is an essential factor that affects the function of TAMs in anti-tumorigenesis and pro-tumorigenesis.As the core immune-related lncRNA in GC,the role of lncRNA ZFPM2-AS1 in the TME remains unknown.Therefore,we further explored whether lncRNA ZFPM2-AS1 could promote the progression of GC by regulating the M2 polarization of TAMs.Methods: THP-1 monocytes were utilized to induce differentiation into M0,M1,and M2 macrophages and TAMs.q RT-PCR was carried out to detect lncRNA ZFPM2-AS1 expression levels in different subsets of macrophages.Then,TAMs with stable knockdown or overexpression of lncRNA ZFPM2-AS1 were constructed.The expression levels of M1 and M2 macrophage markers in TAMs and the secretion levels of IL-10 in the supernatant of TAMs were detected by q RT-PCR,WB and ELISA assays.Finally,WB and cell function assays were performed to explore whether lncRNA ZFPM2-AS1 affects the migration,invasion,and EMT of GC cells by regulating the polarization of macrophages and investigate the mechanisms of lncRNA ZFPM2-AS1-induced polarization of M2 macrophages.Results: LncRNA ZFPM2-AS1 was significantly highly expressed in M2 and TAMs,suggesting that lncRNA ZFPM2-AS1 might promote the M2 polarization of TAMs.Then,the results of q RT-PCR and WB assays showed that the levels of M2 macrophage markers were significantly decreased after the knockdown of lncRNA ZFPM2-AS1 in TAMs.ELISA assay demonstrated that the levels of IL-10 were remarkably reduced or increased in the supernatant of TAMs after silencing or upregulating lncRNA ZFPM2-AS1 in TAMs.The above experiments indicated that lncRNA ZFPM2-AS1 could enhance M2 TAMs polarization and IL-10 secretion.The results of Transwell assays showed that knockdown of lncRNA-ZFPM2-AS1 in TAMs significantly suppressed GC cell migration and invasion,while overexpression of lncRNA ZFPM2-AS1 remarkably enhanced GC cell migration and invasion.The results of WB also showed that the knockdown of lncRNA-ZFPM2-AS1 in TAMs inhibited the EMT of GC cells,while the overexpression of lncRNA ZFPM2-AS1 in TAMs promoted the EMT of GC cells.To further reveal the mechanisms of lncRNA ZFPM2-AS1-induced M2 macrophage polarization,we applied WB assays to estimate JAK2/STAT3 signaling pathway-related proteins.The results indicated that IL-10,p-JAK2,and p-STAT3 expression were remarkably downregulated after silencing lncRNA ZFPM2-AS1 in TAMs.In contrast,IL-10,p-JAK2,and p-STAT3 expression were remarkably upregulated in TAMs after overexpressing lncRNA ZFPM2-AS1 in TAMs.Conclusions: LncRNA ZFPM2-AS1 induces the M2 polarization of TAMs via activating the JAK2/STAT3 pathway,thereby promoting the development and metastasis of GC.The regulatory mechanism of lncRNA ZFPM2-AS1 in the TME provides new ideas and directions for exploring GC pathogenesis,and diagnosis and treatment strategies.