Casz1 and Znf101/Zfp961 differentially regulate apolipoproteins A1 and B, alter plasma lipoproteins, and reduce atherosclerosis
Abulaish Ansari, Pradeep Kumar Yadav, Liye Zhou, Binu Prakash, Laraib Ijaz, Amanda Christiano, Sameer Ahmad, Antoine Rimbert, M. Mahmood Hussain
Abulaish Ansari, Pradeep Kumar Yadav, Liye Zhou, Binu Prakash, Laraib Ijaz, Amanda Christiano, Sameer Ahmad, Antoine Rimbert, M. Mahmood Hussain
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Research Article Vascular biology

Casz1 and Znf101/Zfp961 differentially regulate apolipoproteins A1 and B, alter plasma lipoproteins, and reduce atherosclerosis

Abstract

High apolipoprotein B–containing (apoB-containing) low-density lipoproteins (LDLs) and low apoA1–containing high-density lipoproteins (HDLs) are associated with atherosclerotic cardiovascular diseases. In search of a molecular regulator that could simultaneously and reciprocally control both LDL and HDL levels, we screened a microRNA (miR) library using human hepatoma Huh-7 cells. We identified miR-541-3p that both significantly decreases apoB and increases apoA1 expression by inducing mRNA degradation of 2 different transcription factors, Znf101 and Casz1. We found that Znf101 enhances apoB expression, while Casz1 represses apoA1 expression. The hepatic knockdown of Casz1 in mice increased plasma apoA1, HDL, and cholesterol efflux capacity. The hepatic knockdown of Zfp961, an ortholog of Znf101, reduced lipogenesis and production of triglyceride-rich lipoproteins and atherosclerosis, without causing hepatic lipid accumulation. This study identifies hepatic Znf101/Zfp961 and Casz1 as potential therapeutic targets to alter plasma lipoproteins and reduce atherosclerosis without causing liver steatosis.

Authors

Abulaish Ansari, Pradeep Kumar Yadav, Liye Zhou, Binu Prakash, Laraib Ijaz, Amanda Christiano, Sameer Ahmad, Antoine Rimbert, M. Mahmood Hussain

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Figure 2

Regulation of apoB by miR-541-3p.

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Regulation of apoB by miR-541-3p. (A and B) Huh-7 cells were transfected...
(A and B) Huh-7 cells were transfected (n = 3) with different amounts of miR-541-3p or antimiR-541-3p, and different mRNA levels were quantified in triplicate. (C) Cells were transfected (n = 3) with different concentrations of siZnf101. After 48 hours, mRNA levels were quantified (left). Media were used to quantify apoB/apoA1 protein levels (right). (D) Huh-7 cells were transfected (n = 3) with siZnf101 (21 nM) or miR-541-3p mimics (10 nM), alone or in combination. Changes in mRNA were quantified in cell lysates (left), and proteins in media (right). (E) Cells were forward transfected (n = 3) with plasmids (5 μg) expressing the dual reporter Gaussia luciferase/secreted alkaline phosphatase under control of the Znf101 3′-UTR or a control plasmid without the Znf101 3′-UTR. The next day, cells were reverse transfected (n = 3) with miR-541-3p mimics or antimiR-541-3p. After 48 hours, media were assayed for luciferase and alkaline phosphatase activities. (F) Cells transfected (n = 3) with miR-541-3p mimics (left) or antimiR-541-3p (right) were used for Ago2 immunoprecipitation and measurements of miR-541-3p and Znf101 mRNA. (G) Cells were transfected (n = 3) with 20 nM miR-541-3p mimics or a control miR. After 18 hours, cells were treated with actinomycin D (10 μg/mL). At indicated times, Znf101 and apoB mRNA levels were quantified. (H) Cells were transfected (n = 3) with plasmids expressing luciferase under the control of the apoB or cytomegalovirus promoter. The next day, cells were distributed and transfected (n = 3) with different amounts of miR-541-3p mimics or antimiR-541-3p. After 48 hours, luciferase activity was measured in conditioned media. (I) Cells were transfected (n = 3) with plasmids expressing luciferase under control of the wild-type or mutated apoB promoter. The next day, cells were transfected with different amounts of siZnf101 (n = 3). After 48 hours, conditioned media were used to measure luciferase activity. *P < 0.05; **P < 0.01, ***P < 0.001; ****P < 0.0001 by ordinary 1-way ANOVA pairwise multiple comparisons, where different groups were compared with control.