Figure 3.
Lnc-17-92 forms a transcriptional axis with ACACA to promote proliferation and survival of MM cells. (A) Transcriptomic analysis after lnc-17-92 depletion in MM cell lines that have either DROSHA WT (AMO1, H929) or KO (AMO1DR-KO). Venn diagram of commonly downregulated genes (adjusted P < .05; log2FC < −1). Cells were exposed to ASO1 for 24 hours. (B) qRT-PCR analysis of lnc-17-92 targets in CD138+ cells from 3 MM patients exposed to ASO1 for 24 hours. The results shown are average mRNA expression levels after normalization with GAPDH and ΔΔCt calculations. RNA level in cells exposed to NC (vehicle) were set as an internal reference. (C) Correlation analysis between lnc-17-92 targets (mRNA) and lnc-17-92 in CD138+ MM patient cells from 2 large RNA-seq cohorts (DFCI/IFM, n = 360; MMRF/CoMMpass, n = 720). Spearman r obtained in DFCI/IFM (x-axis) and MMRF/CoMMpass (y-axis) data sets. Dotted red lines indicate r = 0.3. Individual correlation plots (below). (D) GLuc/SEAP dual reporter assay showing reduced activity of ACACA, ANO6, CCDC91, EPT1, EXT1, FER, and KIAA1109 promoter activity after lnc-17-92 knockdown using ASO1. The reporter vectors were cotransfected into 293T cells with either ASO1 or control ASO. Cells were harvested for the luciferase activity assay 48 hour after transfection. Results are shown as % of normalized GLuc activity in ASO1-transfected cells compared with control. (E) ChIRP-qPCR analysis showing effective amplification of ACACA promoter in chromatin purified using 2 lnc-17-92 antisense probe sets (ps1 and ps2), compared with chromatin purified using LacZ antisense probes (negative control). (F) (left) Snapshot obtained by dual RNA-FISH analysis of ACACA pre-mRNA (green) and lnc-17-92 (purple) in a representative AMO1 cell; (right) box plot showing the distance (nm) of ACACA pre-RNA spots to the nearest lnc-17-92 spots (n = 57) or to the nearest random spots (160); 300 nm was used as a cut-off determining proximity. (G) CCK-8 proliferation assay in 5 MM cells lines after transfection with siRNAs against lnc-17-92 targets. Two siRNAs were used for each target, plus a scramble siRNA (NC) as a control. Cell viability was measured at the indicated time point, represented as % of NC-transfected cells. ∗P < .05 after Student t test in panels B, D, and G or after Fisher exact test in panels E and F. Pt, patient.

Lnc-17-92 forms a transcriptional axis with ACACA to promote proliferation and survival of MM cells. (A) Transcriptomic analysis after lnc-17-92 depletion in MM cell lines that have either DROSHA WT (AMO1, H929) or KO (AMO1DR-KO). Venn diagram of commonly downregulated genes (adjusted P < .05; log2FC < −1). Cells were exposed to ASO1 for 24 hours. (B) qRT-PCR analysis of lnc-17-92 targets in CD138+ cells from 3 MM patients exposed to ASO1 for 24 hours. The results shown are average mRNA expression levels after normalization with GAPDH and ΔΔCt calculations. RNA level in cells exposed to NC (vehicle) were set as an internal reference. (C) Correlation analysis between lnc-17-92 targets (mRNA) and lnc-17-92 in CD138+ MM patient cells from 2 large RNA-seq cohorts (DFCI/IFM, n = 360; MMRF/CoMMpass, n = 720). Spearman r obtained in DFCI/IFM (x-axis) and MMRF/CoMMpass (y-axis) data sets. Dotted red lines indicate r = 0.3. Individual correlation plots (below). (D) GLuc/SEAP dual reporter assay showing reduced activity of ACACA, ANO6, CCDC91, EPT1, EXT1, FER, and KIAA1109 promoter activity after lnc-17-92 knockdown using ASO1. The reporter vectors were cotransfected into 293T cells with either ASO1 or control ASO. Cells were harvested for the luciferase activity assay 48 hour after transfection. Results are shown as % of normalized GLuc activity in ASO1-transfected cells compared with control. (E) ChIRP-qPCR analysis showing effective amplification of ACACA promoter in chromatin purified using 2 lnc-17-92 antisense probe sets (ps1 and ps2), compared with chromatin purified using LacZ antisense probes (negative control). (F) (left) Snapshot obtained by dual RNA-FISH analysis of ACACA pre-mRNA (green) and lnc-17-92 (purple) in a representative AMO1 cell; (right) box plot showing the distance (nm) of ACACA pre-RNA spots to the nearest lnc-17-92 spots (n = 57) or to the nearest random spots (160); 300 nm was used as a cut-off determining proximity. (G) CCK-8 proliferation assay in 5 MM cells lines after transfection with siRNAs against lnc-17-92 targets. Two siRNAs were used for each target, plus a scramble siRNA (NC) as a control. Cell viability was measured at the indicated time point, represented as % of NC-transfected cells. ∗P < .05 after Student t test in panels B, D, and G or after Fisher exact test in panels E and F. Pt, patient.

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