The novel long noncoding RNA lncRNA-Adi regulates adipogenesis

FIGURE 1. Identification of adipose tissue‐derived stromal cell (ADSC) pluripotency and homogeneity, and adipogenic induction. A, ADSCs displayed typical fibroblast‐like morphology. Primary cell culturing for 1 day (a), 4 days (b), 7 days (c), and 10 days (d). B, ADSC surface markers were analyzed by flow cytometry. CD29(+) 99.9%, CD90(+) 99.9%, CD31(−) 3.41%, and CD45(−) 1.73%. C, The identification of ADSC multiple differentiation potential. (a) Oil red O staining showing ADSCs differentiating into adipocytes in which the lipid droplets were stained red. (b) Positive alizarin red staining showing calcifying nodules formed after ADSC induction into osteoblasts. (c) Toluidine blue staining showing ADSCs differentiating into chondrocytes. (d) Immunofluorescent staining with NF antibody after ADSC with neurogenic induction for 1 day. (e) DAPI staining of ADSC with neurogenic induction for 1 day. (f) Merged image of (d) and (e)

FIGURE 2. Overview of transcriptome microarrays. A, Cell morphology changing during adipogenic induction. (a) Uninduced adipose tissue‐derived stromal cells (ADSCs). (b) ADSCs with adipogenic induction for 3 days. (c) ADSCs with adipogenic induction for 5 days. (d–f) ADSCs with adipogenic induction for 7 days under magnifications of 40, 100, and 200. (g–i) Oil red O staining of ADSC with adipogenic induction for 7 days under magnifications of 40, 100, and 200. B, Cluster of coding transcripts (left) and adipocyte gene transcripts (right). Adipocyte differentiation‐related mRNAs such as Slc2a4, Fabp4, PPARγ, and CEBPα were unsurprisingly significantly upregulated in adipogenic‐induced ADSCs (right). C, Cluster of noncoding transcripts (left) and long noncoding (lnc)RNAs (right)

FIGURE 3. Long noncoding (lnc)RNA‐Adi is specifically and highly expressed in mitotic clonal expansion of adipose tissue‐derived stromal cell (ADSC) adipogenic differentiation. A, The expression level of lncRNA‐Adi was significantly upregulated in ADSCs with adipogenic induction for 7 days compared to uninduced ADSCs detected by qRT‐PCR; n = 3. B, Expression pattern of lncRNA‐Adi during adipogenic induction detected by qRT‐PCR in which the expression of lncRNA‐Adi was strikingly upregulated in days 1 and 3 and then was slowly downregulated in days 5 and 7 while the expression level of lncRNA‐Adi was still higher than that of uninduced ADSCs (day 0); n = 3. C, Relative expression of lncRNA‐Adi in ADSCs with adipogenic induction for 3 days when the induction began and 7 days when the mature adipocytes formed; n = 3. D, Relative expression of lncRNA‐Adi in ADSCs with osteogenic induction for 3 days when the induction began and 21 days when the calcifying nodules formed; n = 3. E, Relative expression of lncRNA‐Adi in ADSCs with chondrogenic induction for 3 days when the induction began and 21 days when the chondrogenic induction completed; n = 3. F, Relative expression of lncRNA‐Adi in ADSCs…

FIGURE 4. Long noncoding (lnc)RNA‐Adi is indispensable for adipogenic differentiation of adipose tissue‐derived stromal cells (ADSCs). A, GFP positive cytoplasm image (right) showed that packaged lentivirus with lncRNA‐Adi shRNA sequence successfully transfected into ADSCs. B, ADSCs transfected with lentivirus with lncRNA‐Adi shRNA sequence (lnc‐Adi‐KD) expressed little lncRNA‐Adi during adipogenic induction compared to ADSCs transfected with nothing (ADSCs(BC)) and with lentivirus with nontargeting control sequence (lnc‐Adi‐NC), which verified that the effects of interfering lncRNA‐Adi expression in lnc‐Adi‐KD group ADSCs; n = 3. C, Oil red O staining of ADSCs in BC, NC, and KD groups at day 7. D, Microscopic observation of cell morphology changes in BC, NC, and KD groups at days 0, 3, 5, and 7 after adipogenic induction. E, Microscopic observation of oil red O staining of ADSCs in BC, NC, and KD groups during adipogenic induction at days 0, 3, 5, and 7 under magnifications of 100, 200, and 400. F, Expression level of key adipocytes genes including C/EBPα, PPARγ, aP2, LPL, GLUT4, and leptin in BC, NC, and KD groups during adipogenic induction at days 0, 3, 5, and 7; n = 3

FIGURE 5. Long noncoding (lnc)RNA‐Adi locates in the cytoplasm and is a decoy of miR‐449a. A, RNA fluorescence in situ hybridization (FISH). (a) lncRNA‐Adi probe detection. (b) DAPI staining. (c) Merged image of (a) and (b). B, Relative lncRNA‐Adi RNA levels in cytoplasm and nuclear fractions in uninduced adipose tissue‐derived stromal cells (ADSCs; day 0) and adipogenic‐induced ADSCs (days 3 and 7). C, RNA binding protein immunoprecipitation (RIP) result showed that the binding ability with lncRNA‐Adi had no difference between IgG and PPARγ; n = 3. D, Predicted target site of lncRNA‐Adi binding with miR‐449a by Targetscan. E, Dual luciferase reporter assay to verify that lncRNA‐Adi could bind with miR‐449a; n = 3

FIGURE 6. Long noncoding (lnc)RNA‐Adi controls miR‐449a to regulate the CDK6‐pRb‐E2F1 axis in the control of adipogenesis. A, Adipose tissue‐derived stromal cell (ADSC) proliferation rate in KD, NC, and KD groups during adipogenic induction; n = 3. B, ADSC cell cycle analysis on day 1 after adipogenic induction in BC (upper panel) and KD (lower panel) groups. C, Percentage of cells in G1, S, and G2 phases in BC and KD groups; n = 3. D, Expression pattern of miR‐449a in ADSCs during adipogenic induction; n = 3. E, Dual luciferase reporter assay verification of the impact of lncRNA‐Adi and miR‐449a interaction on CDK6 and CDC25A expression during adipogenic induction of ADSCs; n = 3. F, Relative expression of CDK6 and CDC25A in ADSCs with adipogenic induction for 0 and 1 days in BC and KD groups; n = 3. G, Western blot analysis of CDC25A, CDK6, pRb, and E2F1 in BC and KD groups. H, Integrated model explains the mechanism of lncRNA‐Adi in regulating adipogenesis