MiR193a Modulation and Podocyte Phenotype

Abstract

Apolipoprotein L1 (APOL1)-miR193a axis has been reported to play a role in the maintenance of podocyte homeostasis. In the present study, we analyzed transcription factors relevant to miR193a in human podocytes and their effects on podocytes' molecular phenotype. The motif scan of the miR193a gene provided information about transcription factors, including YY1, WT1, Sox2, and VDR-RXR heterodimer, which could potentially bind to the miR193a promoter region to regulate miR193a expression. All structure models of these transcription factors and the tertiary structures of the miR193a promoter region were generated and refined using computational tools. The DNA-protein complexes of the miR193a promoter region and transcription factors were created using a docking approach. To determine the modulatory role of miR193a on APOL1 mRNA, the structural components of APOL1 3' UTR and miR193a-5p were studied. Molecular Dynamic (MD) simulations validated interactions between miR193a and YY1/WT1/Sox2/VDR/APOL1 3' UTR region. Undifferentiated podocytes (UPDs) displayed enhanced miR193a, YY1, and Sox2 but attenuated WT1, VDR, and APOL1 expressions, whereas differentiated podocytes (DPDs) exhibited attenuated miR193a, YY1, and Sox2 but increased WT1, VDR, APOL1 expressions. Inhibition of miR193a in UPDs enhanced the expression of APOL1 as well as of podocyte molecular markers; on the other hand, DPD-transfected with miR193a plasmid showed downing of APOL1 as well as podocyte molecular markers suggesting a causal relationship between miR193a and podocyte molecular markers. Silencing of YY1 and Sox2 in UPDs decreased the expression of miR193a but increased the expression of VDR, and CD2AP (a marker of DPDs); in contrast, silencing of WT1 and VDR in DPDs enhanced the expression of miR193a, YY1, and Sox2. Since miR193a-downing by Vitamin D receptor (VDR) agonist not only enhanced the mRNA expression of APOL1 but also of podocyte differentiating markers, suggest that down-regulation of miR193a could be used to enhance the expression of podocyte differentiating markers as a therapeutic strategy.

Keywords: APOL1; RXR; Sox2; VDR; WT1; YY1; miR193a.

Figure 1

Podocyte molecular profiles in undifferentiated (UPD) and differentiated conditions (DPD). Podocytes were incubated in Petri dishes in media either at 33 °C for 48 h (UPD) or at 37 °C for 10 days (DPDs) (n = 4). (A) RNAs were extracted and assayed for miR193a (n = 4). Cumulative data are displayed in bar graphs (means ± SD). * p < 0.05 compared with UPD. (B) Protein blots from four independent lysates were probed for CD2AP, WT1, VDR, YY1, Sox2, and Glyceraldehyde -3-phosphate dehydrogenase (GAPDH). (C) Protein blots from four different lysates were probed for Nephrin, APOL1, and GAPDH. (D–J) Cumulative densitometric data (protein: GAPDH ratio) are shown in dot plots. * p < 0.05 compared with respective UPD.

Figure 2

Transcription factor binding on miR193a promoter. (A). DR1, DR3 and DR4 elements for nuclear receptor binding. (B). Sox2 motif on the miR193a gene.

Figure 3

Evaluation of the transcription factors enhancing miR193a expression and associated downstream signaling. (A) YY1-miR193a complex. YY1 (Cyan) binds on miR193a promoter; the interacting residues (Magenta) are displayed. (B) Sox2-miR193a complex. Sox2 (Pink) binds on miR193a promoter and interacting residues (Magenta) are shown. (C) UPDs were transfected with siRNA-YY1 or SiRNA-Sox2 (n = 3). After 48 h, RNAs were extracted from control (C) and siRNA-YY-1 and siRNA-Sox2-transfected UPDs. RNAs were assayed for miR193a. Cumulative data are shown in a bar diagram. * p < 0.05 compared with control (C). (D) cDNAs were prepared from the RNAs extracted from the protocol A. cDNAs were amplified with specific primers for YY1, Sox2, VDR, WT1, CD2AP, and Nephrin (n = 3). Cumulative data are shown in a bar diagram. * p < 0.05 compared with respective controls (C). (E) UPDs were transfected with either scrambled (SCR), siRNA-YY1, or siRNA-Sox2 (n = 3). After 48 h, proteins were extracted from control, SCR-, siRNA-YY1- and siRNA-Sox2-transfected podocytes. Protein blots were probed for YY1, Sox2, CD2AP, WT1, VDR, and GAPDH. Representative gels from two different lysates are displayed. (F) Cumulative densitometric data from protein blots of protocol C are displayed in a bar diagram. * p < 0.05 compared with respective control and SCR.

Figure 4

Determination of repressors contributing to the downing of miR193a expression. (A) Vitamin D receptor- Retinoid X receptor (VDR-RXR) heterodimer-miR193a complex. DNA binding domain (DBD) of RXR (Marine blue) and substrate-binding domain (SBD) of RXR (Marine blue) are involved in binding with DNA (miR193a). Some interacting residues of RXR-DBD and RXR-SBD are represented in Magenta color, and VDR-DBD and VDR-SBD are represented in Brown color. VDR substrate-binding domain (SBD) and DNA binding domain (DBD) is represented in Cyan color. (B) DPDs were transfected with either siRNA-VDR or siRNA-RXR (n = 3). After 48 h, RNAs were extracted from control (C) and siRNA-VDR- and siRNA-RXR-transfected DPDs. RNAs were assayed for miR193a. Cumulative data from three independent experiments are shown in bar graphs. * p < 0.05 compared with control (C). (C). WT1 recruits EZH2 and binds at miR193a promoter. WT1 (Marine blue) binds on miR193a promoter with interacting residues (Magenta). (D). DPDs were treated with buffer or transfected with siRNA-WT1 (n = 3). After 48 h, RNAs were extracted from control (C) and siRNA-WT1-transfected DPDs. RNAs were assayed for miR193a. Cumulative data from three independent experiments are shown in a bar diagram. * p < 0.05 compared with control (C).

Figure 5

Analysis of VDR-RXR repressor complex. (A) The structural construct of the VDR-RXR repressor complex. The complex shows VDR (cyan) and RXR (marine blue) heterodimer. VDA (red) bound with VDR and RXR heterodimer interacts with miR193a promoter and recruits SMRT (pink) and HDAC3 (magenta) and forms a repressor complex. (B) A schematic diagram is displaying the formation of the VDR-RXR repressor complex at the miR193a promoter. The VDA bound VDR makes a heterodimer with RXR and recruits SMRT and HDAC3 and forms a repressor complex. (C) Protein blots of three different cellular lysates of UPDs and DPDs were probed for RXR, VDR, SMRT, HDAC3, and GAPDH. Gels of three independent lysates are displayed. (D–G). Cumulative densitometric data (protein GAPDH) are shown as dot plots. * p < 0.05 compared with respective UPD. (H) Cellular lysates from protocol C were immunoprecipitated (IP) with the RXR antibody. Protein blots of RXR-IP fractions were probed for RXR, VDR, SMRT, HDAC3, and IgG. Gels are displayed. (I–L). Cumulative densitometric data (protein/GAPDH) from the protocol H, are shown as dot plots. * p < 0.05 compared with respective UPD.

Figure 6

Analysis of the WT1-EZH2 repressor complex. (A) The structural construct of the WT1-EZH2 repressor complex. (B) A schematic diagram is displaying the formation of the WT1 repressor complex at the miR193a promoter. WT1 recruits EZH2, SMRT, and HDAC1 inducing methylation at lysine 27 residues at Histone (H) 3 tail. (C) Protein blots of three different cellular lysates of UPDs and DPDs were probed for WT1, EZH2, HDAC1, and H3K27me3, and GAPDH. Gels of three independent lysates are shown. (D–G). Cumulative densitometric data (protein/GAPDH) are shown as dot plots. * p < 0.05 compared with respective UPD. (H) Cellular lysates from protocol C were immunoprecipitated (IP) with the WT1 antibody. Protein blots of WT1-IP fractions were probed for WT1, EZH2, HDAC1, and H3K27me3, and IgG. Gels are displayed. (I–L) Cumulative densitometric data (protein/GAPDH) from the protocol H, are shown as dot plots. * p < 0.05 compared with respective UPD.

Figure 7

APOL1 3′ UTR and miR193a Secondary structures. (A) miR193a-5p structure conformation and hybrid structure conformations of APOL1 3′ UTR and miR193a-5p. The APOL1 3’ UTR structure conformation consists of secondary structure elements such as External loop, Helices, bulge loop, Interior loop, and Hairpin loops. i. The hybrid structure is showing miR193a-5p and APOL1 3′ UTR pairing with a 3′ overhang. ii. The hybrid structure conformation shows intensive pairing. (B) Tertiary structure of APOL1 3′ UTR and miR193a-5p (Magenta) complex. The miR193a-5p binding site on APOL1 3′ UTR is represented in Cyan. (C) UPDs were transfected with either control plasmid (empty vector, EV) or a specific inhibitor of miR193a (25 nM) (n = 3). In parallel sets of experiments, DPDs were transfected with either control plasmid (EV) or miR193a plasmid (50 nm) (n = 3). RNAs were assayed for miR193a. Cumulative data are displayed in bar graphs. * p < 0.0 compared to respective EV. (D) cDNAs from EV- and miR193a inhibitor-treated UPDs from 7C were amplified with specific primers for APOL1, VDR, WT1, and Nephrin (n = 3). Cumulative data are shown in a bar diagram. * p < 0.05 compared to respective EV. (E) cDNAs from EV and miR193a-transfected DPDs from 7C were amplified with specific primers for APOL1, VDR, WT1, and Nephrin (n = 3). Cumulative data are shown in bar graphs. * p < 0.05 compared with respective EV.

Figure 7

APOL1 3′ UTR and miR193a Secondary structures. (A) miR193a-5p structure conformation and hybrid structure conformations of APOL1 3′ UTR and miR193a-5p. The APOL1 3’ UTR structure conformation consists of secondary structure elements such as External loop, Helices, bulge loop, Interior loop, and Hairpin loops. i. The hybrid structure is showing miR193a-5p and APOL1 3′ UTR pairing with a 3′ overhang. ii. The hybrid structure conformation shows intensive pairing. (B) Tertiary structure of APOL1 3′ UTR and miR193a-5p (Magenta) complex. The miR193a-5p binding site on APOL1 3′ UTR is represented in Cyan. (C) UPDs were transfected with either control plasmid (empty vector, EV) or a specific inhibitor of miR193a (25 nM) (n = 3). In parallel sets of experiments, DPDs were transfected with either control plasmid (EV) or miR193a plasmid (50 nm) (n = 3). RNAs were assayed for miR193a. Cumulative data are displayed in bar graphs. * p < 0.0 compared to respective EV. (D) cDNAs from EV- and miR193a inhibitor-treated UPDs from 7C were amplified with specific primers for APOL1, VDR, WT1, and Nephrin (n = 3). Cumulative data are shown in a bar diagram. * p < 0.05 compared to respective EV. (E) cDNAs from EV and miR193a-transfected DPDs from 7C were amplified with specific primers for APOL1, VDR, WT1, and Nephrin (n = 3). Cumulative data are shown in bar graphs. * p < 0.05 compared with respective EV.

Figure 8

Evaluation of the effect of VDA on downing of miR193a and associated expression of podocyte molecular markers. (A) UPDs (at 33 °C) and DPDs (at 37 °C) were incubated in media containing either buffer (Control, C) or VDA (EB1089, 100 nM) for 48 h (n = 3). RNAs were assayed for miR193a expression. Cumulative data of three independent experiments are displayed in a bar diagram. * p < 0.05 compared with respective control (C). (B) B. cDNAs were prepared from RNA extracted from the protocol A, amplified with specific primers for WT1, VDR, APOL1, CD2AP, and Nephrin. Cumulative data are shown as bar graphs. * p < 0.05 compared respective controls (C).

Figure 9

Schematic diagram showing modulation of miR193a expression determining the phenotype of podocytes. (A) Increased YY1/Sox2 expression enhances but own regulation of WT1 and VDR de-represses the expression of miR193a in UPDs. (B) Downing of YY1/Sox2 and escalation of WT1/VDR expression downregulates the expression of miR193a and maintains podocytes in a differentiated state.