Fig. 5

MALAT1 directly interacts with c-MYC for binding to the promoter region of KTN1 gene to mediate the transcriptional regulation of KTN1 expression. a Chromatin isolation by RNA purification (ChIRP) with both even and odd antisense oligos tiling MALAT1 showing degree and specificity of MALAT1 retrieval. LacZ tiling oligo retrieval of MALAT1 RNA was used as a negative control. b Relative retrieval of genomic DNAs corresponding to the KTN1 promoter or GAPDH locus following MALAT1 or LacZ ChIRP. c Identification of the E-box element for c-MYC binding at the KTN1 promoter region. TSS, transcription start site. d Electrophoretic mobility shift assay (EMSA) was used to detect the direct association between the E-box element and purified c-MYC protein. Black arrow indicates the binding complex between c-MYC protein and probe containing E-box motif while the white arrow indicates the supershift generated by the association of anti-c-MYC antibody with the above complex. e c-MYC siRNA and NC oligos were transfected into A431 cells. The enrichment of c-MYC at the KTN1 promoter was measured by ChIP-PCR. f MALAT1 and NC ASOs were transfected into A431 cells. The enrichment of c-MYC at the KTN1 promoter was measured by ChIP-qPCR. g Biotin-labeled fragment 1, fragment 2, and fragment 3 domains of MALAT1 RNA transcripts were used to retrieve c-MYC protein by RNA pull-down assay. Random selected protein, SIRT6, was used as a negative control. h RNA immunoprecipitation (RIP) assay was performed using antibodies against c-MYC. The retrieved MALAT1 transcripts were detected by RT-PCR. EGFR transcripts were used as a negative control. i RNA interference (RNAi) with siRNAs targeting c-MYC was applied to A431 cells and the protein expression of c-MYC, KTN1, and EGFR was detected using western blot. All statistical data represent the average of three independent experiments ± s.d. *P < 0.05, **P < 0.01, ***P < 0.001