Sp1 Cooperates With Sp3 To Upregulate MALAT1 Expression In Human Hepatocellular Carcinoma

Long noncoding RNAs(LncRNAs)are non-protein coding transcripts containing more than 200 nucleic acids.LncRNA functions to regulate gene transcription via post-transcriptional modification and interacts with microRNA(miRNA).It plays a crucial role in regulation of human carcinogenesis.In hepatocellular carcinoma(HCC),altered LncRNA expression is associated with tumor progression.Therefore,LncRNA could be a potential target for HCC treatment and should be further investigated in HCC development and progression.The metastasis-associated lung adenocarcinoma transcript 1(MALAT1)is highly conserved amongst mammals and is primarily expressed in the nucleus.MALAT1 overexpression is associated with elevated levels of alpha-feto-protein(AFP)and numbers of tumor lesions,and has been identified in host reaction after liver transplantation.MALAT1 could interact with various target genes,such as serine/arginine-rich splicing factor 1(SRSF1)and potential gene signaling pathways associated with human carcinogenesis.Nevertheless,transcriptional regulation of MALAT1,per se,remains unknown.Specificity protein(Sp)family is the famous transcription factor.The members Spl and Sp3 recognize the similar amino acid sequences of DNA binding domains and are widely distributed throughout the tissues.In HCC,Sp1 promotes carcinogenesis by regulating transcription of VEGF and ?-catenin expression.Our previous data also showed the same mechanism in Sp3.The sequence database,ChIpBase,was established using 543 ChIP-Seq's results.Analysis of this database revealed the potential regulation between transcription factors and Noncoding RNA(NcRNA),indicating a possible connection of Sp1 with MALAT1 expression.Moreover,the role and binding sites of Spl/3 in regulation of MALAT1 transcription in HCC remains unknown.Part 1.Expression of Spl/3 and MALAT1 in HCC and their relationshipObjective:To detect the expression of Sp1,Sp3 and MALAT1 in tissues and cell lines of HCC,analyze the correlation of expression level with clinicopathologic features,and preliminarily confirm the relationship between Sp1/3 and MALAT1.Methods:Thirty-two cases of HCC and paratumorous tissues were collected to detect the expression of Sp1,Sp3 and MALAT1 by qRT-PCR.Correlation with clinicopathologic features was analyzed.And then in three HCC cell lines,including Bel-7402,Huh7 and HepG2,the expression of Sp1,Sp3 and MALAT1 was detected by qRT-PCR and Western Blot(WB).Moreover,RNAi was preformed to respectively silence(iSpl or iSp3 group)and co-silence(iSpl/3 group)the gene Spl and Sp3 for investigating the alteration of MALAT1 mRNA level.In addition,HepG2 cells were treated with a certain concentration(IC50 for 72 h confirmed by MTT)of Sp1 inhibitor Mithramycin A to observe its influence on Sp1,Sp3 and MALAT1.Results:We confirmed high expression of Sp1,Sp3 and MALAT1 in HCC vs.paratumorous tissues,which was associated with AFP level(Sp1:r=7.44,P=0.0064;MALAT1:r=12.37,P=0.0004).Co-silencing of Sp1 and Sp3 synergistically repressed MALAT1 expression,whereas slight change of MALAT1 was observed in iSpl or iSp3 group(P<0.05).And Sp1 binding inhibitor,Mithramycin A,also inhibited MALAT1 expression in HCC cells(P<0.05).Conclusion:The high expression of Sp1,Sp3 and MALAT1 promotes the carcinogenesis.Potential relationship was investigated,but binding sites remained unknown for further proof to verify.Mithramycin A effectively down-regulated expression of MALAT1 in HCC cells.Part 2.Transcriptional regulation of Spl/3 on MALAT1Objective:To detect the effect of Spl/3 on transcriptional activity of MALAT1 promoter and identify the potential binding sites,and preliminarily discuss the mechanism of transcriptional regulation.Methods:Chromatin immunoprecipitation assay(ChIP)was performed to observe the direct combination between Sp1/3 and MALAT1.Further,electrophoretic mobility shift assay(EMSA)was employed to prove the combination between Sp1/3 and binding site#4.At last,MALAT1 reporter plasmid expressing region-400/-1bp was constructed,then Spl and Sp3 expression plasmids were transiently transfected into HEK293T cells separately or together.According to different ratios of Spl/3 expression plasmid to MALAT1 reporter plasmid,the transcription activity of MALAT1 in different groups was tested by Dual Luciferase Reporter Gene Assay.And mutation of predicted site#4 in reporter plasmid was designed to verify the effect on transcription activity.Results:Five Sp1/3 binding regions were identified by ChIP,especially the region-85/-4bp which obviously combined with Spl/3.Bio-labeled probe Spl/3 showed powerful combination with the nuclear extract.Besides,we confirmed the up-regulation,which was 5.8 or 4.9 times compared with control group,of transcriptional activity at the ratio of 25:1(Spl/3:MALAT1,P<0.05).After co-upregulating Sp1 and Sp3,the promotion of transcriptional activity was also investigated at the ratio of 75:1(P<0.05).Almost no change was detected in mutation group(P<0.05).Conclusion:In this study,we investigated the role of Sp1/3 in regulation of MALAT1 transcription in HCC cells.Our findings provide mechanistic information for future investigation of regulation of HCC progression mediated by Sp1/3 transcription of MALAT1 expression.The upstream of MALAT1 contains Spl/3 binding sites,especially the region-85/-4bp,which could be responsible for MALAT1 transcription.Inhibitors,like Mithramycin A,may provide a potential therapeutics trategy for HCC patients with MALAT1 overexpression.