Journal: bioRxiv

Article Title: Genome-wide, integrative analysis implicates circular RNA dysregulation in autism and the corresponding circular RNA-microRNA-mRNA regulatory axes

doi: 10.1101/712331

Figure Lengend Snippet: Experimental validation of the backspliced junction of an upregulated circRNA (circARID1A) and the corresponding circRNA-miRNA regulatory axis. (a) Validation of the NCL junction of circARID1A. Top and middle: the schematic diagrams displaying seven predicted target sites of one single downregulated miRNA (miR-204-3p) on the circle of circARID1A (top) and the designed divergent primers around the NCL junction of circARID1A (middle). Bottom: comparisons of two different RTase products (MMLV- and AMV-derived products) of circARID1A in TC/FC samples and four types of neuronal cell lines (hNPC (or ReN), NHA, SH-SY5Y, and U118), followed by Sanger sequencing the RT-PCR amplicons for the NCL event in the TC. ReN, ReNcell VM. (b) Experimental validation of the circRNA (or backspliced) junction of circARID1A. The figure shows the expression fold changes (as determined by qRT-PCR) for circARID1A, ARID1A mRNA, and GAPDH (negative control) in the indicated tissues/cell lines before and after RNase R treatment. (c) Experimental examination of the evolutionary conservation of circARID1A across the brains of vertebrate species from primates to chicken. Comparison of MMLV- and AMV-derived-RTase products (left) and the corresponding sequence chromatograms (right) for the circARID1A event in the brains of the indicated six species are shown. (d) Comparison of the expression profiles of circARID1A and its corresponding co-linear mRNA counterpart in 10 normal human tissues. The expression levels of brain are used to normalize the relative expression values of the other tissues. (e) The relative expression of circARID1A and its corresponding co-linear mRNA counterpart in 10 normal human tissues. (f) qRT-PCR analysis of the cytoplasmic to nuclear expression ratios for circARID1A and ARID1A mRNA. GAPDH and U6 snRNA are examined as controls. (g,h) qRT-PCR analyses of the correlations between the expression of circARID1A and miR-204-3p after circARID1A knockdown (g) or overexpression (h) in various human neuronal cell lines. The top panels of (g) and (h) represent that circARID1A knockdown (g) or overexpression (h) did not significantly affect the ARID1A mRNA expression. CT, control. KD, knockdown. OE, overexpression. (i) Luciferase reporter assay for the luciferase activity of Gaussia luciferase (GLuc)-circARID1A in ReN, NHA, SH-SY5Y, and U118 cells transfected with miR-204-3p mimics to validate the binding between circARID1A and miR-204-3p. The entire circle sequence of circARID1A was cloned into the downstream region of the GLuc gene (i.e., GLuc-circARID1A; top). The luciferase activity of GLuc was normalized with secreted alkaline phosphatase (SEAP). (j) qRT-PCR analysis of the expression level of circARID1A in ReN and NHA cells after transfection with miR-204-3p mimics. All the qRT-PCR data are the means ± SEM of three experiments. P values are determined using two-tailed t test. Significance: * P value < 0.05 and *** P value < 0.001. NS, not significant.

Article Snippet: The cells were grown on 20μg/ml laminin (Merck) coated culture plates containing ReNCell NSC maintenance medium (Merck) supplemented with 20ng/ml of bFGF and EGF (Merck).

Techniques: Derivative Assay, Sequencing, Reverse Transcription Polymerase Chain Reaction, Expressing, Quantitative RT-PCR, Negative Control, Over Expression, Luciferase, Reporter Assay, Activity Assay, Transfection, Binding Assay, Clone Assay, Two Tailed Test

Journal: bioRxiv

Article Title: Genome-wide, integrative analysis implicates circular RNA dysregulation in autism and the corresponding circular RNA-microRNA-mRNA regulatory axes

doi: 10.1101/712331

Figure Lengend Snippet: Experimental validation of the circARID1A regulatory role of miR-204-3p sponges and the corresponding circRNA-miRNA-mRNA regulatory interactions. (a) Schematic diagram representing the circARID1A-miR-204-3p regulatory axis and its regulated miRNA targets (171 genes, including 12 ASD risk genes). (b-d) Microarray analysis of the target mRNA log 2 (fold change) in response to overexpression of circARID1A and miR-204-3p. (b) Distribution of the target mRNA log 2 (fold change) in response to overexpression of circARID1A and miR-204-3p. P value is determined using Kolmogorov-Smirnov test. (c) A negative correlation between mRNA fold changes after circARID1A overexpression and miR-204-3p overexpression. The red dots represent the 12 ASD risk genes. The Pearson’s correlation coefficient ( R ) and P value are shown. (d) Heat map of the 12 ASD risk mRNA log 2 (fold change) in response to overexpression of circARID1A and miR-204-3p. P value is determined using one-tailed paired t test. (e) qRT-PCR analyses of ASD risk gene expression in NHAs (top) and hNPCs (ReNcell VM or ReN) (bottom) cells after circARID1A knockdown, miR-204-3p mimics, and miR-204-3p mimics with circARID1A expression vector (p-circARID1A), respectively. P values are determined using two-tailed t test. Significance: * P value < 0.05, ** P value < 0.01, and *** P value < 0.001. (f) Imaging of immunostained ReN cells after two weeks of differentiation. Immunostaining shows undifferentiated (top) and differentiated (bottom) ReN cells with the neural stem cell marker nestin (left), the neuronal marker βIII-tubulin (middle), and the mature glial cell marker GFAP (right). Nuclei are stained with DAPI (blue). Scale bar = 100 μm. GFAP, glial fibrillary acidic protein. (g) Relative expression of circARID1A and two ASD risk genes ( NLGN1 and STAG1 ) during hNPC differentiation. The Pearson correlation coefficients ( R ) between the expression of circARID1A and the two ASD risk genes and P values are shown in in parentheses. All the qRT-PCR data are the means ± SEM of three experiments.

Article Snippet: The cells were grown on 20μg/ml laminin (Merck) coated culture plates containing ReNCell NSC maintenance medium (Merck) supplemented with 20ng/ml of bFGF and EGF (Merck).

Techniques: Microarray, Over Expression, One-tailed Test, Quantitative RT-PCR, Expressing, Plasmid Preparation, Two Tailed Test, Imaging, Immunostaining, Marker, Staining