Weighted Gene Co-expression Network Analysis Revealed That CircMARK3 Is a Potential CircRNA Affects Fat Deposition in Buffalo

Figures

• 

  Figure 1. Screened-out circRNAs significantly associated to buffalo fat deposition. (A–D) CircRNA co-expression network of adipose tissue in the Xinyang buffalo revealed by WGCNA; (A) Hierarchical cluster tree of co-expression modules, where every leaf on a tree is a circRNA and main branches are made up of 21 color-coded modules; (B) Construction of co-expression modules by WGCNA. Progressively more saturated red colors indicate higher overlap among these functional modules and blocks of lighter color along the diagonal are the gene modules. Module assignment gene and dendrograms are at the top and left, respectively; (C) Association analysis of gene co-expression network modules with trait, where each row corresponds to a module (name displayed on the left) and each column corresponds to a particular trait. Colors of the row/column intersection cells indicate the correlation coefficient between module and trait (positive-green and negative-red); (D) Scatterplot of Gene Significance (GS) For Traits vs. Module Membership (MM) in the turquoise module, with a highly significant correlation between GS and MM (one dot represents one gene in the turquoise module); (E) Overlapping circRNAs…

• 

  Figure 2. Characteristic analysis of circMARK3. (A) Basic information of circMARK3 composition structure (bases C and T labeled red were mutated into T and C in the sequence); (B) Semiquantitative PCR results of circMARK3 and MARK3 before and after RNase R treatment; (C) RT-qPCR results of circMARK3 and MARK3 before and after RNase R treatment; (D) Cell localization of circMARK3 by semiquantitative PCR; (E) Cell localization of circMARK3 by RT-qPCR; (F) Sequence alignment results of circMARK3 in mouse and buffalo; (G) CircMARK3 conservation in adipose tissues of different species by semiquantitative PCR detection; (H) Expression profile of circMARK3 in heart, liver, spleen, lung, kidney, muscle and adipose tissues of mouse. In panels B-E, bovine-β-actin was used to normalize the expression level of the reference gene. In panel H, mouse-β-actin was used to normalize the expression level of the reference gene. Data are presented as the mean ± SD, n = 3, **P < 0.01, ***P < 0.001. (B–D) The samples derive from the same experiment and gels were processed in parallel.

• 

  Figure 3. Expression pattern of circMARK3 in tissues and adipocytes of buffalo. (A) Expression profile of circMARK3 in heart, liver, spleen, lung, kidney, muscle and adipose tissues of buffalo; (B–D) expression pattern during buffalo adipocyte differentiation of PPARG (after 0, 2, 6, and 10 days) (B), C/EBPα (C) and FABP4 (D); (E) Oil Red O staining for detection of buffalo adipocyte lipid deposition (differentiation day 10); (F) Expression pattern of circMARK3 during buffalo adipocyte differentiation. In (A–D) and (F), bovine-GAPDH was used to normalize the expression level of the reference gene. In panel (A), data was normalized to the heart. In panels (B–D) and (F), all data were normalized to day 0. Data are presented as the mean ± SD, n = 3, #:no significance, *P < 0.05, **P < 0.01, ***P < 0.001.

• 

  Figure 4. Overexpression of mouse circMARK3 promotes adipogenic differentiation of 3T3-L1 adipocytes. (A) Strategy for mouse circMARK3 overexpression, adipogenic differentiation, RT-qPCR and Oil Red O staining in 3T3-L1 cells; (B) Oil Red O staining in 3T3-L1 cells transfected with pCD2.1-ciR and mouse pCD2.1-circMARK3 on day 6 after adipogenic differentiation. (C) Histogram corresponding to the quantification of Oil Red O staining by spectrophotometry; (D–G) RNA expression 48 h after transfection of circMARK3 (D), PPARG (E), C/EBPα (F) and FABP4 (G). In panels (D–G), mouse-β-actin was used to normalize the expression level of the reference gene in 3T3-L1 cells. Control means the pCD2.1-ciR group, circMARK3 means the mouse pCD2.1-circMARK3 group. NC means negative control. Data are presented as the mean ± SD, n = 3, **P < 0.01, ***P < 0.001.

• 

  Figure 5. Overexpression of buffalo circMARK3 promotes adipogenic differentiation of buffalo adipocytes. (A) Micrographs of GFP-positive cells in the Ad_GFP and Ad_circMARK3 groups on day 2 after cell transfection; (B) Oil Red O staining of buffalo adipocytes transfected with Ad_GFP and Ad_circMARK3 group on day 6 after adipogenic differentiation. (C) Histogram corresponding to the quantification of Oil Red O staining by spectrophotometry; (D–G) RNA expression two days after transfection of circMARK3 (D), PPARG (E), C/EBPα (F), and FABP4 (G). Bovine-GAPDH was used to normalize the expression level of the reference gene in buffalo adipocytes. NC means negative control. Data are presented as the mean ± SD, n = 3, **P < 0.01, ***P < 0.001.

• 

  Figure 6. Schematic illustration of the experimental procedure and main results. RNA-seq analysis is from a previous study (31).