| [Background]Bladder cancer（BC）is a complex biological procession involving multiple steps,stages and factors.BC is the ninth most common cancer in the world,with an estimated 430,000 new cases a year,according to a 2012 multicenter study of global cancer publication rates.About 165,000 people die of bladder cancer every year worldwide,of which 75%are men.BC is characterized by high recurrence,easy infiltration and metastasis,and high mortality,which has been the main bottleneck in the clinical treatment of bladder cancer and improvement of survival rate.Many studies have proved that the occurrence and development of solid tumors benefit from the tumor microenvironment,which contains not only tumor cells,but also immune cells infiltrating tumors,among which macrophages are one of the important immune cells in the tumor microenvironment.Macrophages in tumor microenvironment are mainly tumorassociated macrophages（TAM）,which promote tumor growth by secreting related factors.IL-6 is a cytokine secreted by both tumor cells and macrophages,it can not only promote the growth of tumor cells,but also induce the polarity transformation of macrophages from M1 to M2.MicroRNA（miRNA）is a kind of single-stranded small molecule non-coding RNA composed of 21-25 oligonucleotide.By binding to the 3’UTR segment of the target gene,microRNAs inhibit the translation of the target gene or directly promotes its degradation,regulate genes expression at the post-transcriptional level,and thus widely participates in tumor growth and regulates tumor immunity.MiR-26b-5p is low expressed in many tumor cells,including bladder cancer,and acts as a tumor suppressor gene.But the specific mechanism is still unclear.Recently,epigenetic regulation of gene expression has attracted more and more attention.Among them,EZH2-catalyzed trimethylation of lysine 27 on histone 3（H3K27me3）regulates chromatin structure density,thereby silencing the expression of tumor suppressor genes or miRNAs,which reveal the cause of low expression of some tumor suppressor miRNAs in tumors,but their specific regulatory mechanisms still need to be further clarified.[Purposes]（1）To investigate the expression of miR-26b-5p in bladder cancer tissues and cells,and identify the effect of miR-26b-5p on malignant biological behavior of bladder cancer cells and macrophage immunity.（2）To analyse the target genes that may be regulated by miR-26b-5p.Experiments were also used to prove that miR-26b-5p participated in bladder cancer cell progression and macrophage immunity by regulating the expression of its target gene.（3）To further investigate the effect of M2 TAM on bladder cancer cells and the possible regulatory mechanism.[Methods]（1）Effects of miR-26b-5p of T24 on cells proliferation,invasion and the macrophages function① RT-qPCR was used to evaluate miR-26b-5p expression in bladder cancer samples and different cell lines.The expressions of miR-26b-5p,IL-6 and EZH2 genes in bladder cancer tissue samples were verified by in situ hybridization and immunohistochemistry.②The effects of miR-26b-5p expression on the proliferation,migration and invasion of bladder cancer were investigated by CCK-8,clonal formation assay and Transwell assay.③ELISA was used to evaluate the effect of the expression of miR-26b-5p in T24 cells on the secretion of IL-6 and IL-10 in macrophages.④PCR was used to detect the effects of miR-26b-5p in T24 cells on macrophage M1-type and M2-type genes expression.⑤ Fluorescence microsphere phagocytosis assay was performed to detect the effect of miR-26b-5p in T24 cells on the phagocytosis of THP-1-derived macrophages.（2）MiR-26b-5p inhibited the proliferation,migration and invasion of T24 cells by targetting IL-6 and EZH2① Bioinformatics predicted the target genes of miR-26b-5p.② The mRNA and protein expressions of IL-6 and EZH2 in different transfection groups were detected by RT-qPCR and Western Blot.③ Luciferase assay was performed to verify whether miR-26b-5p can bind to the 3’UTR of IL-6 and EZH2 mRNA.④ CCK-8 proliferation and Transwell assay were used to detect the effect of IL-6 on the proliferation,migration and invasion of T24 cells.⑤Functional rescue experiments were used to verify that miR-26b-5p regulates bladder cancer progression by targetting IL-6.（3）The effect of IL-6 secreted by T24 cells on macrophages function①CCK8 assay was used to detect the effect of IL-6 in T24 cell on macrophage proliferation.② The effects of the expression of IL-6 in T24 cells on the expression of macrophage polarization markers CD 163 and CD206 were detected by PCR.③The fluorescence microsphere phagocytosis experiment was conducted to detect the expression of IL-6 in T24 cells and whether it affected the phagocytosis function of macrophages in the coculture system.④WB was used to detect the effect of IL-6 secretion by T24 cells on the IL6/STAT3 signaling pathway of macrophages.（4）IL-6 secreted by M2 macrophage feedback regulated the expression of miR-26b-5p in bladder cancer① CCK8 assay and Transwell assay demonstrated the effect of IL-6 secreted by M2 macrophages on the proliferation,invasion and metastasis of T24 cells.② RT-qPCR and WB were used to detect the expression of related pathway genes（IL6,STAT3,pSTAT3,EZH2）and miR-26b-5p in bladder cancer cells after being co-cultured with macrophages.③Histone methylation of H3K27me3 up-stream of miR-26b promoter was analyzed on UCSC website.④PCR and WB experiments verified that EZH2 catalyzed the H3K27me3 of miR26b promoter and inhibited the expression of miR-26b-5p.⑤ 5’RACE method was used to determine the transcriptional initiation site（TSS）of miR-26b.ChiP-PCR assay confirmed that EZH2 catalyzed the epigenetic silencing of H3K27me3 in the specific range of possible promoters of miR-26b.[Results]（1）① miR-26b-5p was poorly expressed in bladder cancer cell lines and tissues,it is negatively correlated with the expression of IL-6 and EZH2,and these three genes may be further correlated with the grade of bladder cancer.② Overexpression of miR-26b-5p can inhibit the proliferation,migration and invasion of bladder cancer T24 cells.③ The expression of miR-26b-5p in T24 cells reduced the secretion of IL-6 and IL-10 in THP-1derived macrophages.④ The expression of miR-26b-5p in T24 cells up-regulated the expression of IL-12,IL-lb and TNF-α genes and down-regulated the expression of IL-1ra,IL-6,IL-10,IL-4 and CCL17 genes in macrophages.⑤ The expression of miR-26b-5p in T24 cells inhibited the M2 polarity transformation and enhanced the phagocytic function of THP-1-derived macrophages.（2）① TargetScan,PicTa and miRanda predicted that EZH2 and IL-6 were both miR26b-5p target genes.② The expression of miR-26b-5p can down-regulate the mRNA and protein expression of IL-6 and EZH2 in bladder cancer.③ miR-26b-5p can bind to the 3’UTR of IL-6 and EZH2 mRNA.④miR-26b-5p inhibited the proliferation,migration and invasion of tumor cells by targeting IL-6.（3）① The expression of IL-6 in T24 cells could enhance the proliferation of macrophages,promote the M2-type polarization of macrophages and inhibit macrophage phagocytosis on fluorescent microspheres.② T24 cells secreted IL-6 could induce M2 polarization of macrophages by activating IL-6/STAT3 pathway in macrophages.（4）① In co-culture system,IL-6 secreted by M2 TAM promoted the migration and invasion of bladder cancer cells.② IL-6 secreted by M2 TAM down-regulated the expression of miR-26b-5p through the IL-6/STAT3/EZH2 pathway.③ Bioinformatics predicted extensive methylation and enrichment of H3K27me3 in the up-stream of miR-26b promoter sequence.④ EZH2 interference reduced the expression of H3K27me3 and IL-6 in bladder cancer cells.⑤ EZH2-catalyzed H3K27me3 silenced the transcription of premiR-26b and down-regulated the expression of miR-26b-5p.[Conclusions]（1）The expression of miR-26b-5p in bladder cancer was down-regulated,which enabled the expression of IL-6 gene that had been targeted by miR-26b-5p to be recovered.IL-6 was highly expressed in bladder cancer cells and can contribute to macrophages in a paracrine way in tumor microenvironment,by activating the IL-6/STST3 signaling pathways in macrophage which induced macrophage differentiate into tumor growth promoted macrophage（the M2 macrophages）.（2）By the way,M2 TAM can feedback produce IL-6 contributing to the bladder cancer cells,by activating the IL-6/STST3/EZH2 signal pathway,raised the transcription of EZH2,silencing the transcription of miR-26b epigenetically,and ultimately suppressing the expression of miR-26b-5p.（3）In conclusion,multiple feedback loops between miR-26b-5p-IL-6-macrophages contributed to the depressed expression of miR-26b-5p,thus promoting the malignant progression of bladder cancer. |
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