PiRNA-30473 Contributes To Tumorigenesis And Poor Prognosis By Regulating RNA M6A-methylation In DLBCL

Objective:N6-methyladenosine(m6A),the most abundant modification on eukaryote messenger RNA(mRNA),functions in various fundamental bioprocesses.However,the role of m6A in diffuse large B-cell lymphoma(DLBCL)remains poorly understood.Our study highlights the functional importance of the m6A modification machinery in DLBCL,and provides profound insights into the molecular mechanisms underlying tumorigenesis by revealing a previously unrecognized mechanism of gene regulation in DLBCL.Methods:1.Microarray analysis was performed to screen differentially expressed piRNAs in patients with favorable prognosis and patients with poor prognosis.The expression levels of differentially expressed piRNAs in DLBCL were further confirmed by using qPCR.The univariate and multivariate Cox survival regressions were carried out to validate whether piRNA-30473 was related to prognostic.ALJC analysis with cross-validation was performed to assess NCCN-IPI and NCCN-IPI+piRNA models.2.In vitro,antagomir-30473 was transiently transfected into SU-DHL-8 and Farage cells to inhibit piRNA-30473 expression.Cell proliferation,cell cycle distribution and apoptosis were detected.In vivo,xenograft DLBCL model was established to evaluate the effect of piRNA-30473 on DLBCL progression.3.Antagomir-30473 was transfected into SU-DHL-8 and Farage cells to inhibit piRNA-30473 expression.The global m6A-methylation was detected by dot blot and M6A RNA Methylation Quantification Kit.The expression of m6A methyltransferase(METTL3,METTL14 and WTAP)and m6A demethylase(FTO and ALKBH5)were detected by qPCR and western blot.To confirm the relationship between piRNA-30473 and WTAP,the expression of WTAP was analyzed in DLBCL xenografts by immunohistochemistry assay.co-IP was performed to verify whether piRNA-30473 influenced the binding of WTAP to m6A methyltransferase complex.4.Immunohistochemistry was performed to observe the expression of WTAP in patients with DLBCL.The univariate and multivariate Cox survival regressions were carried out to validate whether WTAP was related to prognostic.5.The cell proliferation,cell cycle distribution and apoptosis were detected after using specific siRNA against WTAP in DLBCL cells.6.The cell proliferation and cell cycle distribution were measured after overexpression of WTAP to abolish the decreased level of WTAP by antagomir-30473.7.RNA FISH and qPCR of nuclear and cytoplasmic fractions were performed to examine the cellular distribution of piRNA-30473 and WTAP mRNA.To identify the putative binding site of piRNA-30473 at WTAP 3' untranslated region,bioinformatics analysis was performed using miRanda algorithm.To further verify WTAP was a directed target gene of piRNA-30473,dual luciferase reporter assay was performed with WTAP 3'untranslated region wild-type or mutant binding sites.The stability of HK2 was measured by mRNA stability experiment.8.m6A-seq and RNA-seq were performed to screen potential target genes of WTAP.qPCR and MeRIP-qPCR were used to further confirm target genes of WTAP.Pearson correlation analysis was employed to determine the correlation between the expressions of WTAP and target gene in GEO database.9.The univariate and multivariate Cox survival regressions were carried out to validate whether HK2 was related to the prognostic of patients.10.siRNA-IGF2BP 1,siRNA-IGF2BP2 and siRNA-IGF2BP3 were transfected into SU-DHL-8 and Farage cells to inhibit corresponding genes expression.Then the expression of HK2 was determined by qPCR and the stability of HK2 was measured by mRNA stability experiment.Pearson correlation analysis was employed to determine the correlation between the expressions of IGF2BP2 and HK2 in GEO database.To further verify HK2 was a directed target gene of IGF2BP2,dual luciferase reporter assay was performed with HK2 5' untranslated region wild-type or mutant binding sites.Results:1.The expression level of piRNA-30473 significantly increased in patients with poor prognosis compared with patients with favorable prognosis.piRNA-30473 could be used as a biomarker of prognosis and improve on the prognostic stratification of patients with DLBCL.2.Depletion of piRNA-30473 in DLBCL cells resulted in less proliferation as compared to controls,and no evidence of apoptosis.Moreover,cell-cycle analysis showed that inhibition of piRNA-30473 in DLBCL cells increased the proportions of cells in G1 phase whereas it decreased the proportions of cells in S phase and G2 phase.Injection of antagomir-30473 into B-NSG mice led to a significant reduction in tumor volume compared with control.3.The global m6A level on mRNAs was decreased in antagomir-30473-transfected cells relative to controls.Silencing piRNA-30473 showed inhibition of WTAP in mRNA and protein levels.However,the expression of METTL3,METTL14,FTO,ALKBH5 had no significant difference between two groups.Immunohistochemical showed that treatment with antagomir-30473 partly reduced WTAP expression in xenograft DLBCL models.Co-immunoprecipitation indicated that antagomir-30743 treatment decreased WTAP association with m6A methyltransferase complex.4.Iinmunohistochemical analysis showed that WTAP was highly expressed in patients with poor prognosis.The analysis of GEO database(GSE10846)indicated that the elevated expression of WTAP predicted poor prognosis.5.Depletion of WTAP in DLBCL cells resulted in less proliferation as compared to controls,without evidence of apoptosis.Moreover,cell-cycle analysis showed that inhibition of WTAP in DLBCL cells increased the proportions of cells in G1 phase and G2 phase whereas it decreased the proportions of cells in S phase.6.Reconstituting the expression of WTAP in piRNA-30473-downregulated cells could reverse the biological activity.7.RNA FISH and qPCR of nuclear and cytoplasmic fractions suggested that piRNA-30473 and WTAP mRNA were located both in the nucleus and the cytoplasm.Results of prediction by miRanda suggested that piRNA-30473 had one binding site within WTAP 3' untranslated region.Silencing of piRNA-30473 expression,significantly decreased luciferase activity of the reporter construct carrying wild-type WTAP 3' untranslated region,relative to the control.And this decrease was abrogated when the putative binding site was mutated.mRNA stability experiment revealed that WTAP tended to have shorter half-lives in piRNA-30473-deficient cells.8.m6A-seq and RNA-seq data indicated that WTAP targeted the 5' untranslated region of HK2 transcripts,WTAP knockdown caused a significant decrease in the m6A level of the 5' untranslated region.Gene-specific m6A qPCR and qPCR assays for HK2 confirmed that m6A-level and mRNA-level of HK2 were significantly downregulated in WTAP-deficient cells.The expression of HK2 was correlated with WTAP.9.The expression level of HK2 significantly increased in patients with poor prognosis compared with patients with favorable prognosis.HK2 signature could be used as a biomarker of prognosis and improve on the prognostic stratification of patients with DLBCL.10.qPCR demonstrated that the transcript levels of HK2 were reduced significantly in IGF2BP2-deficient cells.The expression of HK2 was correlated with IGF2BP2.MRNA stability experiment revealed that HK2 tended to have shorter half-lives in IGF2BP2-deficient cells.Luciferase activity of pGL3-HK2-wild type vector was reduced through inhibition of IGF2BP2,whereas this effect was lost in the pGL3-HK2-mutant vector.Conclusion:1.piRNA-30473 regulated global m6A RNA modification in DLBCL cells via targeting WTAP.2.The imbalance of m6A methylation could promote the expression of HK2,contributing to DLBCL development.3.piRNA-30473 was not only used as a biomarker for the prognosis of DLBDL,but also as a target for the treatment of DLBCL.