| Renal cell carcinoma(RCC),which is the most common malignancy in kidney,has a high mortality parallel to the incidence.Looking for efficient tumor biomarkers is significantly important for RCC diagnosis and treatment.Human SLC4 7A2(Solute carrier family 47,member 2)gene,which encodes multidrug and toxin extrusion protein 2,is predominantly expressed in kidney.In this study,we investigated the expression of SLC47A2 mRNA and protein in RCC and paired adjacent normal tissues.A dramatical decrease had been observed in tumor tissues from RCC patients at low TNM stage,which made SLC47A2 a potential diagnosis biomarker of RCC.Epigenetics plays an important role in the regulation of gene transcription,it's involved in the tumorigenesis and biomarker development.DNA methylation and histone modification are the most important epigenetic pathways.DNA methylation profiles at the promoter region of SLC47A2 gene in RCC tumor and paired normal tissues were mapped in this study.There was no difference between the tissues in promoter CpG island methylation rate,which meant promoter CpG island methylation had no correlation with SLC47A2 tanscription repression in RCC.Histone modification profiles in RCC tumor and paired normal tissues were detected by Chromatin Immunoprecipitation(ChIP).It was found that SLC47A2 was a bivalent gene whose promoter enriched with both H3K4me3 and H3K27me3.Decreased H3K4me3 enrichment in tumor tissues suggested an involvement of bivalent mechanism in SLC47A2 tanscription repression.In addition,histone acetylations H3K27AC and H4AC were also reduced in RCC tumor tissues compared to paired normal tissues,and it was consistent to SLC47A2 tanscription repression in RCC.The relationship between SLC47A2 expression and histone modificaitons was investigated in RCC cell lines.Histone methyltransferase MLL1 increased the transcriptional activity of SLC47A2,MLLl diminishment induced a decreased H3K4me3 enrichment around transcription start site(TSS)at the gene promoter and a lower mRNA expression of SLC47A2.A much less MLL1 occupancy around TSS at the promoter of SLC47A2 was also found in RCC tumor tissues compared to paired normal tissues.All the results indicated that MLL1 was responsible for H3K4me3 related SLC47A2 transcription repression in RCC.Enhancer of zeste homolog 2(EZH2),a subunit of Polycomb repressive complex 2(PRC2),catalyzes the trimethylation of histone H3 lysine 27(H3K27me3).Knockdown of EZH2 expression reduced H3K27me3 enrichment around TSS at the promoter of SLC47A2,and then induced its mRNA expression.It suggested that H3K27me3 was also involved in SLC47A2 transcription repression in RCC.The combined H3K4me3 and H3K27me3 regulations of SLC47A2 expression in both RCC tissues and cell lines revealed a control of bivalent gene expression by H3K4me3/H3K27me3 ratio.To investigate whether histone acetylations play roles in SLC47A2 expression,a trichostatin A(TSA)treatment was caried out in RCC cell lines.TSA improved both histone acetylation H3K18AC,H3K27AC,H4AC and histone methylation H3K4me2,H3K4me3 enrichment at SLC47A2 promoter,a induction of gene expression was also observed after TSA treatment.Further studies showed histone deacetylase(HDAC)was closely related to SLC47A2 repression in RCC.HDAC10 was responsible for histone deacetylation of H3K18AC,H3K27AC,and also suppressed H3K4me2,H3K4me3 enrichment at SLC47A2 promoter,finally,the transcription of SLC47A2 was affected by these histone acetylation and methylation changes together.The results of inhibiting the expression of a transcription factor E2F1 were similar to HD AC 10 suppression,and a interaction between the two proteins was also found.Therefore,a hypothesis will be raised up:E2F1 can recruit HD AC 10 to SLC47A2 promoter,that it will result in decrease of histone acetylation and methylation enrichment followed by gene transcription repression.It should be considered carefully when using HD AC inhibitors because of the induction of SLC47A2 expression.In summary,it has been clarified that SLC47A2 transcription repression in RCC was regulated by histone modificaitons.These findings will help us understand RCC formation,and may also provide a novel potential target for RCC early diagosis and therapy. |
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