Mechanism Of A1CF/FAM224A/miR-590-3p/ZNF143 Positive Feedback Loop Regulatingthe Malignant Biological Behaviors Of Glioma Cells

Objective: Glioma is considered to be the most common and lethal type of malignant brain tumor,accounting for about 40% to 50% of primary intracranial tumors.Despite recentadvances in surgery,chemotherapy and radiotherapy,the prognosis of glioma has not improved significantly.Therefore,precise gene-targeted therapy is expected to become an effective therapeutic strategy for glioma.RNA binding proteins(RBPs)play a pivotal role in the post-transcriptional regulation of gene expression and exert important functions in various biological processes.APOBEC1 complementation factor(A1CF)participates in the post-transcriptional cytidine(C)to uridine(U)RNA editing of apolipoprotein B(APOB)mRNA by cooperating with its partner APOBEC1,further modulating lipid metabolism.Recent studies have indicated that A1CF is closely related to malignant tumor progression.However,the potential role of A1CF in glioma remains unclear.Long non-coding RNAs(lncRNAs)are a type of non-coding RNAs with transcripts longer than 200 nucleotides in length with limited protein-coding ability.However,to date,there have been no reports on the function of the lncRNA family with sequence similarity 224 member A(FAM224A)in human glioma.microRNAs(miRNAs)are a class of endogenous small non-coding RNAs of approximately 20–22 bp in length.They are involved in the regulation of post-transcriptional gene expression mainly through binding to the 3’ UTR of target genes and causing either degradation or repression of mRNAs.In particular,miR-590-3p has also been found to be downregulated in glioma.However,the detailed regulatory mechanism of miR-590-3p in glioma still remains unclarified.Transcription factor zinc finger protein 143(ZNF143)is a human homolog of the Xenopus transcriptional,activator Staf and its expression can be induced by treatment with various DNA-damaging agents.Recent studies have shown that dysregulated expression of ZNF143 is closely related to the malignant progression of tumors.To date,the mechanisms underlying the function of ZNF143 in glioma have not been elucidated.In the present study,the endogenous expression of A1CF,FAM224A,miR-590-3p and ZNF143 were investigated in glioma tissues and cell lines.Their roles in modulating malignant progression of glioma and the cross-talk between A1CF,FAM224A,miR-590-3p and ZNF143 were elucidated.Our findings may provide a novel target for glioma therapy.Methods:qRT-PCR and western blot analysis were employed to detect the endogenous expression levels of A1CF,FAM224A,miR-590-3p,ZNF143 and ArfGAP with SH3 domain,ankyrin repeat and PH domain 3(ASAP3)in glioma tissues and U87 and U251 cell lines.A1CF,FAM224A,miR-590-3p,ZNF143 and ASAP3 were overexpressed or knockdown by cell transfection in U87 and U251 cells.CCK-8,transwell assays and flow cytometry analysis were conducted to evaluate the function of A1CF,FAM224A,miR-590-3p,ZNF143 and ASAP3 in the malignant biological behaviors of glioma cells.RNA immunoprecipitation(RIP),RNA stability measurement and nascent RNA capture assays were used to determine the regulation of A1CF on the RNA stability of FAM224 A.Dual-luciferase reporter and RIP assays were carried out to investigate the targeted binding between miR-590-3p and FAM224 A,and miR-590-3p and ZNF143.Direct effects of ZNF143 on promoter regions of ASAP3 and MYB were detected by chromatin immunoprecipitation(Ch IP).The tumor xenografts assay in nude mice was used to assess the effects of A1CF,FAM224A,and miR-590-3p on tumorigenicity and survival time of tumor-bearing nude mice.Results: A1CF and FAM224 A expression were significantly upregulated in glioma tissues and U87 and U251 cell lines,and their expression levels were positively correlated with the pathological grade of glioma.Moreover,high A1CF and FAM224 A expression levels indicated a poorer prognosis for glioma patients.A1CF exerted carcinogenic functions in glioma cells via stabilizing and upregulating FAM224 A expression,thus promoted the proliferation and migration and invasion abilities while inhibited the apoptosis of glioma cells.Conversely,miR-590-3p was significantly downregulated in glioma tissues and in U87 and U251 cell lines,and the expression level of miR-590-3p was negatively correlated with the progression of glioma pathological grade.Overexpression of miR-590-3p significantly decreased the proliferation and migration and invasion abilities while facilitated apoptosis of U87 and U251 glioma cells.FAM224 A was a putative target of miR-590-3p,and FAM224 A knockdown,which was associated with the overexpression of miR-590-3p,remarkably inhibited the biological behaviors of U87 and U251 glioma cells.ZNF143 was upregulated in glioma tissues and in U87 and U251 cell lines,and the expression level of ZNF143 was positively correlated with the pathological grade of glioma.Inhibition of ZNF143 significantly restrained cell proliferation,migration and invasion,and promoted apoptosis of U87 and U251 glioma cells.miR-590-3p could negatively modulate the expression of ZNF143 via binding to the ZNF143 3′ UTR and inhibited the biological behaviors of glioma cells.ASAP3 was upregulated in glioma tissues and in U87 and U251 cell lines,and the expression level of ASAP3 was positively correlated with the pathological grade of glioma.ASAP3 knockdown significantly inhibited the malignant biological behaviors of glioma cells.ZNF143 could bind to the promoter regions of ASAP3 and MYB and transcriptionally activate their expressions.ZNF143 knockdown downregulated the expression of ASAP3 and MYB and inhibited the biological behaviors of glioma cells.In addition,ZNF143 could directly target the promoter of FAM224 A and transcriptionally stimulate its expression,collectively forming a positive feedback loop.Finally,knockdown of A1CF and FAM224 A combined with overexpression of miR-590-3p inhibited tumor xenograft growth and prolonged the survival of nude mice.Conclusion: 1.A1CF,FAM224A,ZNF143 and ASAP3 were significantly upregulated whilemiR-590-3p was downregulated in glioma tissues and U87 and U251 cell lines.Downregulated expression of A1CF,FAM224A,ZNF143 and ASAP3 andoverexpression of miR-590-3p significantly decreased the proliferation and migrationand invasion abilities while facilitated apoptosis of U87 and U251 glioma cells.2.A1CF upregulated the expression of FAM224 A by enhancing its stability andmiR-590-3p specifically bound to FAM224 A.3.Transcriptional regulatory factor ZNF143 was a downstream target of miR-590-3p.ZNF143 could bind to the promoter regions of MYB,ASAP3 and FAM224 A andpositively regulated their transcription.4.Silencing A1CF downregulated the expression of FAM224 A by redusing its stability;enhanced the negative regulatory effect of miR-590-3p on ZNF143,downregulatedthe expression of ZNF143,and restrained the proliferation and migration andinvasion abilities while promoted apoptosis of glioma cells by inhibiting thetranscription of MYB,ASAP3,and FAM224 A.5.A1CF-FAM224A-miR-590-3p-ZNF143 positive feedback loop conducts critical regulatory effects on the malignant biological behaviors of glioma cells.