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 3

Regulation of apoA1 by miR-541-3p.

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Regulation of apoA1 by miR-541-3p. (A and B) Huh-7 cells were forward tr...
(A and B) Huh-7 cells were forward transfected (n = 3) with miR-541-3p mimics or antimiR-541-3p. After 48 hours, mRNA levels of APOA1 and CASZ1 were quantified in triplicate. (C) SiCasz1-transfected cells (n = 3) were collected to measure mRNA (left) and media to quantify apoB and apoA1 protein levels (right) in triplicate. (D) Cells were transfected (n = 3) with 10 nM siCtrl or siCasz1 or 21 nM miR-541-3p mimics, individually or in combination. Cells were used to measure different mRNAs (left) and media to measure apoB and apoA1 protein (right) in triplicate. (E) Plasmids for the expression of luciferase with or without the 3′-UTR of Casz1 were transfected (n = 3). After 24 hours, cells were reversed transfected (n = 3) with different amounts of miR-541-3p mimics or antimiR-541-3p. After 24 hours, luciferase activity was measured in triplicate in media. (F) Cells were transfected (n = 3) with increasing amounts of miR-541-3p mimics or antimiR-541-3p. After 48 hours, Ago2 immunoprecipitates were used to quantify miR-541-3p and CASZ1 mRNA levels in triplicate and expressed as a percentage of control miR–transfected cells. (G) Transfected cells were treated with actinomycin D (10 μg/mL) in triplicate and collected at different intervals to measure CASZ1 and APOA1 mRNA levels. (H) Cells were transfected (n = 3) with apoA1 promoter–luciferase constructs. The next day, they were reverse transfected (n = 3) with increasing concentrations of miR-541-3p mimics or antimiR-541-3p. After 48 hours, luciferase activity was measured in triplicate. (I) Cells were transfected in triplicate with plasmids expressing luciferase under the control of the wild-type or mutated apoA1 promoter. The next day, cells were equally distributed and transfected (n = 3) with different amounts of siCasz1. After 48 hours, luciferase activity was measured (n = 3) in conditioned media. *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.