Journal: Molecular Therapy. Nucleic Acids

Article Title: A Simplified System to Express Circularized Inhibitors of miRNA for Stable and Potent Suppression of miRNA Functions

doi: 10.1016/j.omtn.2018.09.025

Figure Lengend Snippet: Efficient Antagonization of hsa-miR223 with the Circularized ARRB1-Derived RNA Sponge in T-ALL Cell Lines (A) Schematic representation depicting hsa-miR223-binding site (MBS) at the 3′ end UTR (or BUTR) of human β-Arrestin1 (ARBB1). A 402-bp fragment containing MBS was PCR amplified and subcloned into the CimiR system, resulting in cirBUTR. Its linear counterpart linBUTR was also engineered as a control. Upon transfection into HEK293 cells, a specific circular product was readily detected by RT-PCR in the cirBUTR transfection group (indicated by the arrow), but not in the linBUTR group. (B) The circularized RNA sponge is more stable than the linear transcript. HEK293 cells were transfected with cirBUTR or linBUTR plasmid DNA and treated with actinomycin D for the indicated time, and total RNA was isolated and subjected to qRT-PCR analysis of the abundance of linear and circular forms of BUTR transcripts, while RFP mRNA was used as an exogenous transcript control. **p < 0.01. (C and D) Effective reversal of the expression of hsa-miR223-targeted genes by cirBUTR in T-ALL lines. The T-ALL cell lines Jurkat (C) and CCRF-CEM (D) were infected with the retrovirus stably expressing linBUTR, cirBUTR, or the seed sequence scrambled circRNA (SCR). The expression of miR223-targeted genes, such as ARRB1 , FBXW7 , and RHOB , was assessed by qPCR analysis. GAPDH was used as an internal control. *p < 0.05, **p < 0.01.

Article Snippet: To synthesize the cDNA for mRNA or mature miRNA quantification, random hexamer, the miRNAs’ specific stem-loop primers, and the reverse primer of Gapdh were incubated together with total RNA at 70°C for 5 min and then reverse transcribed with M-MuLV Reverse Transcriptase (New England Biolabs).

Techniques: Derivative Assay, Binding Assay, Amplification, Transfection, Reverse Transcription Polymerase Chain Reaction, Plasmid Preparation, Isolation, Quantitative RT-PCR, Expressing, Infection, Stable Transfection, Sequencing

Journal: Molecular Therapy. Nucleic Acids

Article Title: A Simplified System to Express Circularized Inhibitors of miRNA for Stable and Potent Suppression of miRNA Functions

doi: 10.1016/j.omtn.2018.09.025

Figure Lengend Snippet: The Circular Bulged Anti-miR21 Effectively Inhibits hsa-miR21 Functions and Suppresses Cell Proliferation in Human Breast Cancer Cells (A & B) qPCR analysis of the expression of hsa-miR21 target genes in breast cancer cells. The cirBulg21, linBulg21 vectors and miR21 seed sequence scrambled circRNA expression vector were constructed in the same fashion as that for antimiR223. Human breast cancer cell lines MCF7 (A) and SKBR3 (B) were infected with linBulg21, cirBulg21 or scrambled retroviral vector (SCR) for 72h. Total RNA was isolated and subjected to qPCR analysis using the qPCR primers for known miR21 target genes. All assays were done in triplicate. GAPDH was used a reference gene. “ ∗ ” p<0.05; “ ∗∗ ” p<0.01. (C) Western blotting analysis of hsa-miR21-regulated expression of BCL2 and PTEN in breast cancer cells. Human breast cancer cell line SKBR3 was infected with linBulg21, cirBulg21 or scrambled vector (SCR) for 72h. Total cell lysate was subjected to SDS-PAGE and Western blotting with BCL2 or PTEN antibody. GAPDH expression was used as internal/loading control. (D) Colony formation assay. SKBR3 and MCF7 cells were infected with linBulg21, cirBulg21 or scrambled retroviral vector (SCR) for 24h and subsequently seeded in 60mm cell culture dishes at 1X103 or 2X103 cells. Colonies were fixed and stained with crystal violet on day 12. Representative images are shown. (E) Cell cycle analysis. SKBR3 cells were infected with linBulg21, cirBulg21 or scrambled retroviral vector (mock) for 48h. Cells were collected and subjected to cell cycle analysis. Each assay condition was done in triplicate. “ ∗ ” p<0.05, “ ∗∗ ” p<0.01. Scale bar, 1 cm.

Article Snippet: To synthesize the cDNA for mRNA or mature miRNA quantification, random hexamer, the miRNAs’ specific stem-loop primers, and the reverse primer of Gapdh were incubated together with total RNA at 70°C for 5 min and then reverse transcribed with M-MuLV Reverse Transcriptase (New England Biolabs).

Techniques: Expressing, Sequencing, Plasmid Preparation, Construct, Infection, Isolation, Western Blot, SDS Page, Colony Assay, Cell Culture, Staining, Cell Cycle Assay