| Renal Cell Carcinoma(RCC)is the seventh most common site for tumors and the most common malignant tumor in urinary system,with an increasing incidence year by year.Long non-coding RNAs(IncRNAs)play important roles in regulating the abnormal expression of cancer genes,and closely related with tumor development.Depending on their functional models,lncRNAs can be divided into two types:they can regulate the adjacent or own genes by cis transcriptional regulation;or regulate distal genes by trans transcriptional regulation.The expression of SLC47A2 is strongly suppressed in RCC,which will significantly change the metabolism paths of kidney cells in excreting the anti-cancer drugs and endogenous substances.According to the annotation information from UCSC Genome Browser database,the SLC47A2 loci exists four potential promoters and three transcription factor-binding regions.It suggests that the SLC47A2 loci might express several SLC47A2 associated non-mRNA Transcripts(SANTs),which can cis regulate the expression level of its coding gene and might also be closely related to the specially low expression level of MATE 2-K in RCC.We determined the expression levels of non-mRNA sequences,which located in the downstream of the regulation regions by qPCR.We found that a sequence regulated by Section 3 shown significantly higher expression level in normal tissue than paired cancer tissue,and we predicted it as a characteristic sequence of SANT1.We succesfully obtained the full-length sequence of SANT1 via 5'/3' RACE,and cloned the SANT1 into the expression vector.We confirmed the cis transcriptional regulation model of SANT 1 by high-express or knockdown the expression level of SANT1 in 786-O,ACHN,769-P and Caki-1 cell lines.Knockdown the expression of SLC47A2 significantly strengthened the inducible expression of GLUT1 by FH or SDH B/SDH D deficiency in 769-P cells.It seemed that the low expression of SANT1 involved in metabolic reprogramming of RCC cells by inhibit the expression of MATE 2-K.What's more,SANT1 might involve in the post-transcriptional regulation of SLC47A2 by competitive binding the miR-17 family members,which might induce the degradation of mRNA.However,the results from gene report assay shown that both miR-20a and miR-93(two members from miR-17 family)had no direct binding activities with SANT1 and mRNA 3' UTR,and the SANT1 exhibited no function in preventing the mRNA from degradation via ceRNA model(competing endogenous RNA).However,when we up-or down-expressed miR-20a in four RCC cell lines,the expression level of SLC47A2 was significantly changed.Interestingly,miR-20a displayed a bi-directional modulation in regulating the expression of SLC47A2:the transcription of mRNA in different cell lines can be promoted or restrained under same treatment(up-or down-express the miR-20a).We conjectured that miR-20a is involved in regulating two kinds of SLC47A2 transcription factors:one of them is an activator,and another one is an inhibitor.The regulatory network reprogramming followed by miR-20a abnormal expression determines the final expression level of SLC47A2.As reported by UCSC Genome Browser database and references,SLC47A2 was mainly regulated by both H3K4me3,H3K27ac and H3K27me3,and the associated histone modifiers and transcription factors are c-Myc/MLLl,E2F1/HDACs and EZH2,respectively.Encouragingly,E2F1,c-Myc and miR-20a were reported to forming a negative feedback loop,which contains a SLC47A2 activator(c-Myc)and an inhibitor(E2F1).We confirmed the existence of the c-Myc/MLL1 and E2F1/HDAC1 complexes in 769-P cells via Co-IP,and then ensured the bi-directional modulation model of c-Myc.We strengthened or suppressed the function of c-Myc in 769-P cells,and then,we tested the protein levels of c-Myc and E2F1,and the epigenetic modification signals in SLC47A2 promoter.As a result,the protein levels of c-Myc and E2F1 were significantly higher after transfecting the c-Myc expression vector.Both of the binding rates of c-Myc/MLL1 and E2F1/HDAC1 in promoter were up-regulated,which resulting in a higher level of H3K4me3 signal and lower level of H3K27ac modification.The binding rate of S-5-P RNAP II was suppressed,indicating the transcriptional inhibition of SLC47A2.However,the protein levels of c-Myc and E2F1 were strongly suppressed after 10058-F4(c-Myc inhibitor)treatment.The deficiency of c-Myc and E2F1 resulted in significant lower binding of c-Myc/MLL1 and E2F1/HDAC1 in promoter,while the restrained H3K4me3 and significantly strengthened H3K27ac modification activating the transcriptional activity of SLC47A2 via enhancing the binding rate of S-5-P RNAP II in promoter region.The RCC tissue exhibited higher protein levels of c-Myc and E2F1 than paired normal tissue.Higher expression level of E2F1 resulted in higher binding rate of E2F1/HDAC1 and lower H3K27ac modification signal in SLC47A2 promoter region.However,the biding activity of c-Myc/MLL1 was inhibited,resulting in lower H3K4me3 modification signal.We found that the abnormal expression of HIF(Hypoxia-inducible factor)suppressed the target binding activity of c-Myc.We successfully showed a picture about a bi-directional modulation model of c-Myc/E2F1/miR-20a loop via regulating the H3K4me3 and H3K27ac modification signals in SLC47A2 promoter.The SANT1 might play an important role in activing the transcription of SLC47A2 by up-regulating the binding rate of S-5-P RNAP II in mRNA promoter.IncRNAs can also act as a gene regulator via translating small peptides or forming RNP complex(RNA-protein complex).We constructed two SANTI mutants:they have same fundamental sequence and translation potencies,but dramatically different secondary structures.The three kinds of SANT1 were transfected into four RCC cell lines,and only the SANT1-S1 shown similar inductive effect with full-length SANT1.It suggested that the perfect secondary structure is the primary functional factor of SANT1.According to the software analysis report,the SFPQ protein was forecasted to be a strong partner with SANT1 and SANT1-S1.We confirmed the direct combination between SANT1/SANT 1-S1 and SFPQ via CLIP,and the binding activity was weakened in SANT1-S2.The binding rate of SFPQ in SLC47A2 promoter was significantly suppressed in SANT1 high-expressing 769-P cells and normal tissue,indicating the inhibiting ability of SFPQ-associated functional complex.SFPQ/HDAC1 is a well-known inhibiting complex in gene regulation via suppressing the H3K27ac modification in gene promoter.Considering the primary function of E2F1/HDAC1 in c-Myc/E2F 1/miR-20a induced bi-directional modulation model,we predicted SFPQ/E2F1/HDAC1 complex is the functional performer in SLC47A2 regulation.The existence of SFPQ/E2F1/HDAC1 complex was confirmed in 769P cells by Co-IP assay.We further ensured that the perfect secondary structure of SANT1 is necessary for the formation of RNP complex,and the high expressing of SANT1 resulted in higher H3K27ac modification and S-5-P RNAPII binding in SLC47A2 promoter via inducing off-target binding of E2F1/HDAC1.As a conclusion,the c-Myc/E2F 1/miR-20a loop displayed a bi-directional modulation of SLC47A2 via regulating the H3K4me3 and H3K27ac modification in promoter.In RCC tissue,the abnormal expression of HIF caused off-target binding of c-Myc,resulting in lower modification level of H3K4me3;the higher expression level of E2F1 caused lower modification level of H3K27ac in promoter.On the contrary,off-target binding of E2F1/HDAC1 induced by abundant expression of SANT1,as well as lower expression level of E2F1,resulted in significantly higher level of H3K27ac modification at promoter region in normal tissue.As a result,the expression level of SLC47A2 was significantly enhenced in normal tissue than cancer tissue. |
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