Role of differentially expressed microRNA-139-5p in the regulation of phenotypic internal anal sphincter smooth muscle tone

Abstract

The present study focused on the role of microRNA-139-5p (miRNA-139-5p) in the regulation of basal tone in internal anal sphincter (IAS). Applying genome-wide miRNA microarrays on the phenotypically distinct smooth muscle cells (SMCs) within the rat anorectrum, we identified miRNA-139-5p as differentially expressed RNA repressor with highest expression in the purely phasic smooth muscle of anococcygeus (ASM) vs. the truly tonic smooth muscle of IAS. This pattern of miRNA-139-5p expression, previously shown to target ROCK2, was validated by target prediction using ingenuity pathway (IPA) and by qPCR analyses. Immunoblotting, immunocytochemistry (ICC), and functional assays using IAS tissues and cells subjected to overexpression/knockdown of miRNA-139-5p confirmed the inverse relationship between miRNA-139-5p and ROCK2 expressions/IAS tone. Overexpression of miRNA-139-5p caused a decrease, while knockdown by anti-miRNA-139-5p caused an increase in the IAS tone; these tissue contractile responses were confirmed by single-cell contraction using magnetic twisting cytometry (MTC). These findings suggest miRNA-139-5p is capable of significantly influencing the phenotypic tonicity in smooth muscle via ROCK2: a lack of tone in ASM may be associated with the suppression of ROCK2 by high expression of miRNA-139-5p, whereas basal IAS tone may be associated with the persistence of ROCK2 due to low expression of miRNA-139-5p.

Figure 1

miRNA microarray data and its validation via qPCR. (A) Microarray analysis heat map of microRNAs isolated form rat IAS, RSM and ASM SMCs. Nine miRNAs shown in the heat map follow the gradient trend of ASM > RSM > IAS. (B) Graph showing that miRNA microarray identified nine differentially expressed miRNAs in an ascending gradient manner from the purely tonic, mixture of tonic and phasic to the purely phasic SMCs as IAS > RSM > ASM. The IAS represents tonic, while RSM and ASM represent semi tonic and purely phasic smooth muscles, respectively. Among nine differentially expressed miRNAs, miRNA-139-5p is the most differentially expressed, with ~4-fold higher expression in the ASM vs. the IAS. (C) qPCR analysis graph showing relative expression calculated by using the equation: Δ Ct = Ct miRNA-Ct (RNU6B) and 2^(−ΔΔCT) = 2^(−ΔCt IAS, RSM, ASM−ΔCt IAS) confirms the highest expression of miRNA-139-5p in the ASM vs. the IAS SMCs with pattern similar to that in the differential miRNA microarray (*p < 0.05; n = 4).

Figure 2

Ingenuity pathway analysis (IPA) showing ROCK2 as one of the direct targets of miRNA-139-5p. miRNA microarray data was subjected to ingenuity pathway analysis (IPA) and various validated targets of miR-139-5p from the literature were plotted as an Interactome. The analysis shows that human ROCK2 is one of the direct targets of miR-139-5p as it has a direct miR-139-5p binding site at its 3′UTR. Some of the other validated targets mentioned in literature, which may also be down-regulated in tonic smooth muscles are given in this interactome.

Figure 3

Effect of miRNA-139-5p overexpression on RhoA/ROCK machinery in IAS SMCs. (A) Western blot data showing that miRNA-139-5p overexpression in the IAS SMCs decreases the expression of RhoA/ROCK signal transduction machinery proteins which is selectively blocked by the anti-miRNA-139-5p. (A) (upper portion). Western blots comparing the expression levels of RhoA, ROCK1, and ROCK2 before and after miRNA-139-5p, anti-miRNA-139-5p alone, and following miRNA-139-5p + anti-miRNA-139-5p. The lower portion of the panel provides the corresponding quantitative data showing that miRNA-139-5p causes significant (*p < 0.05) downregulation of RhoA and ROCK2 without any significant (p > 0.05) effect on the levels of ROCK1. Conversely, anti-miRNA-139-5p by itself causes significant upregulation of RhoA and ROCK2 (*p < 0.05). (As indicated, RhoA, ROCK1, and ROCK2 expressions were compared with GAPDH.) (B) (upper portion).Western blots comparing the expression levels of p-MYPT1and p-MLC₂₀, before and after miRNA-139-5p, anti-miRNA-139-5p alone, and following miRNA-139-5p + anti-miRNA-139-5p. The lower portion of this panel provides the quantitative data showing that miRNA-139-5p significantly (*p < 0.05) downregulates, while anti-miRNA-139-5p causes significant (*p < 0.05) upregulation of p-MYPT1 and p-MLC₂₀. (As shown, p-MYPT1, and p-MLC₂₀ expressions were compared with MYPT1 and MLC₂₀, respectively.) (C) Model representing the effect of miRNA-139-5p on RhoA/ROCK2 signaling cascade. The IAS smooth muscle is characterized by upregulated RhoA/ROCK signaling which may be either constitutively active or GPCR-activated. Data suggest that higher levels of miRNA-139-5p in the IAS attenuate ROCK2 expression, which in turn leads to activation of MLCP (via decrease in p-MYPT1), and decrease in p-MLC₂₀ and basal tone.

Figure 4

Immunocytochemistry data before and after miRNA-139-5p. (A) Immunocytochemical images showing expression of RhoA, ROCK2, p-MYPT1, and p-MLC₂₀ following transfection of IAS SMCs with miRNA-139-5p. (B) Quantitative data showing significant (*p < 0.05) downregulation of immunofluorescence intensity of RhoA, ROCK2, p-MYPT1, and p-MLC₂₀ in the IAS SMCs following transfection with miRNA-139-5p as compared with controls (cells treated with scrambled miRNA). BG = background.

Figure 5

Effect of miRNA- and anti-miRNA-139-5p. (A) miRNA-139-5p (100 nM) produces significant (*p < 0.05; n = 5) decrease in the IAS tone, as compared with scrambled miRNA (control), which is significantly blocked by pre-treatment with 100 nM antagomir (*p < 0.05; n = 5). Conversely, 100 nM antagomir by itself significantly (*p < 0.05; n = 4) increases the IAS tone as compared to control. (B) miRNA-139-5p significantly shifts U46619 CRC causing an increase in the IAS tone towards right (*p < 0.05; n = 6–8), which is attenuated by the antagomir. In contrast, the antagomir causes a significant shift in the control CRC to left (p > 0.05; n = 6–8). (C) Time course data with U46619 (1 μM) before and after miRNA-139-5p show a significant (*p < 0.05) decrease in the maximal increase in the IAS tone and increase in the time taken to achieve it following miRNA-139-5p transfection. The above described effect of miRNA-139-5p is significantly (*p < 0.05) attenuated by the pre-transfection of the smooth muscles with anti-miRNA-139-5p. Conversely, anti-miRNA-139-5p significantly (*p < 0.05) increases the maximal effect and produces left-ward shift in time-course of U46619-induced contraction. Changes in the shifts in the kinetic velocities following U46619 are shown by the regression analyses with dashed lines.

Figure 6

Effect of miRNA- and anti-miRNA-139-5p on IAS kinetics. (A,B) Graphs showing rate of relaxation (A) and of recovery (B) of IAS tone following 0 Ca²⁺ and normal Ca²⁺ replenishment, respectively, following transfection of the smooth muscle strips. miRNA-139-5p significantly (*p < 0.05) decreases the rates of relaxation and of recovery of the IAS tone which are significantly reversed by anti-miRNA-139-5p pre-treatment (*p < 0.05; n = 6–8).

Figure 7

MTC data showing the effect of miRNA- and anti-miRNA-139-5p. (A) Baseline cell stiffness of individual IAS SMCs before and after 72 h transfection with ROCK2 siRNA, miRNA-139-5p, anti-miRNA-139-5p (inhibitor), and miRNA-139-5p plus the inhibitor. SMCs transfected with ROCK2 siRNA and miRNA-139-p showed decreases in baseline cell stiffness (basal tone) while cells transfected with anti-miRNA-139-5p showed increases in basal tone. Increased basal tone in response to anti-miRNA-139-5p was attenuated with co-transfection with miRNA-139-5p. Data are presented as Mean ± s.e.m. (n = 89–149 individual cell measurements). (B) Dynamic changes the stiffness of IAS SMCs in response to 100 nM U46619. Consistent with the rat smooth muscle data, anti-miRNA-139-5p causes faster and greater cell stiffening in response to U46619.