Overexpressing miR-122-5p Inhibits the Relaxation of Vaginal Smooth Muscle in Female Sexual Arousal Disorder by Targeting Vasoactive Intestinal Peptide Receptor 1

Abstract

Introduction: Female sexual arousal disorder (FSAD) is a common issue causing physical and psychological pain, but it has no standard diagnostic criteria or treatment. So its pathogenesis desiderates to be explored.

Aim: To investigate the specific function of miR-122-5p in FSAD.

Methods: 18 subjects were grouped into FSAD and normal control groups according to the Chinese version of the Female Sexual Function Index, and the expression levels of miR-122-5p and vasoactive intestinal peptide receptor 1 (VIPR1) protein in their tissue were verified through real-time quantitative polymerase chain reaction (qRT-PCR) and western blot (WB) analysis. Then in vitro experiment, miR-122-5p was overexpressed or inhibited in rat vaginal smooth muscle cells (SMCs). The relaxation of rat vaginal SMCs was reflected by the cell morphology, intracellular free cytosolic calcium ion (Ca²⁺) levels, cell proliferation and apoptosis, together with the cyclic adenosine monophosphate (cAMP) concentration and protein kinase A (PKA) activities. Additionally, the expression levels of relaxation-related proteins, including VIPR1, stimulatory G protein (Gs), adenylate cyclase (AC), and PKA, were detected based on WB analysis. Furthermore, a rescue experiment that simultaneously overexpressed or silenced miR-122-5p and VIPR1 was conducted, and all the indicators were evaluated.

Main outcomes measure: The expression level of VIPR1 and downstream proteins, cell morphology, cell proliferation and apoptosis, and intracellular free Ca²⁺ levels were examined.

Results: We verified that women with FSAD had higher miR-122-5p and lower VIPR1 protein. Then overexpressing miR-122-5p decreased relaxation of rat vaginal SMCs, which was manifested as a contractile morphology of cells, an increased intracellular free Ca²⁺ concentration, and lower cAMP concentration and PKA activity. Moreover, by rescue experiments, we inferred that VIPR1 was the target of miR-122-5p and affected the relaxation function of vaginal SMCs.

Conclusion: miR-122-5p regulates the relaxation of vaginal SMCs in FSAD by targeting VIPR1, ulteriorly providing an underlying diagnostic and therapeutic target for FSAD. Cong S, Gui T, Shi Q, et al. Overexpressing miR-122-5p Inhibits the Relaxation of Vaginal Smooth Muscle in Female Sexual Arousal Disorder by Targeting Vasoactive Intestinal Peptide Receptor 1. Sex Med 2021;9:100390.

Keywords: Female sexual arousal disorder; Vaginal smooth muscle cell; Vasoactive intestinal peptide receptor 1; miR-122-5p.

Figure 1

Expression levels of miR-122-5p and vasoactive intestinal peptide receptor 1 (VIPR1) protein in vaginal tissue of FSAD patients. (A) Conservation of the binding region of VIPR1 3′UTR with miR-122-5p. (B) Prediction of miR-122-5p and VIPR1 targeting points. (C) The relative miR-122-5p expression level was analyzed in 6 pairs of vaginal tissues from FSAD and the normal control group by quantitative real-time PCR (qRT-PCR). (D) The expression level of VIPR1 protein was evaluated by western blot (WB) analysis with another 3 pairs of tissues in 2 groups. All indicators after being extracted from 18 samples between 2 groups were detected 3 times. The results are the mean ± standard error of the mean (SEM) and the differences were analyzed by t-tests. *: P-value<0.05; **: P value<0.01.

Figure 2

MiR-122-5p overexpression attenuates the relaxation function of vaginal smooth muscle cells (SMCs). (A) Morphology of rat vaginal SMCs under an inverted microscope. (B) Length of rat SMCs. (C) Free Ca²⁺ content in rat vaginal SMCs. (D) The proliferation rate of rat vaginal SMCs measured with a Cell counting kit-8 (CCK8) assay. (E) The apoptosis rate of rat vaginal SMCs with flow cytometry and terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick end labeling (TUNEL) assay. (F) Cyclic adenosine monophosphate (cAMP) concentration and protein kinase A (PKA) activity. (G) mRNA expression levels of vasoactive intestinal peptide receptor 1 (VIPR1), stimulatory G protein (Gs), adenylate cyclase (AC), and PKA. (H) VIPR1, Gs, AC, and PKA protein expression determined by western blotting. Vaginal SMCs were isolated from one rat. All indicators extracted from SMCs were detected three times except for cellular morphology. The results are the mean ± standard error of the mean (SEM) and were analyzed by t-tests. *: P-value<0.05; **: P-value<0.01.

Figure 3

MiR-122-5p silencing results in increased relaxation function of rat vaginal smooth muscle cells (SMCs). (A) Morphology of rat vaginal SMCs under an inverted microscope. (B) Length of rat SMCs. (C) Free Ca²⁺ content in rat vaginal SMCs. (D) The proliferation rate of rat vaginal SMCs detected with a Cell counting kit-8 (CCK8) assay. (E) The apoptosis rate of rat vaginal SMCs with flow cytometry and terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick end labeling (TUNEL) assay. (F) Cyclic adenosine monophosphate (cAMP) concentration and protein kinase A (PKA) activity. (G) mRNA expression levels of vasoactive intestinal peptide receptor 1 (VIPR1), stimulatory G protein (Gs), adenylate cyclase (AC), and PKA. (H) VIPR1, Gs, AC, and PKA protein expression determined by western blotting. Vaginal SMCs were isolated from one rat. All indicators extracted from SMCs were detected three times except for cellular morphology. The results are the mean ± standard error of the mean (SEM) and were analyzed by t-tests. *: P-value<0.05; **: P-value<0.01.

Figure 4

Overexpression of vasoactive intestinal peptide receptor 1 (VIPR1) redressed the inhibitory effect of miR-122-5p overexpression on rat vaginal smooth muscle cells (SMCs) relaxation. (A) Morphology of rat vaginal SMCs under an inverted microscope. (B) Length of rat SMCs. (C) Free Ca²⁺ content in rat vaginal SMCs. (D) The apoptosis rate of rat vaginal SMCs with flow cytometry and terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick end labeling (TUNEL) assay. (E) Cyclic adenosine monophosphate (cAMP) concentration and protein kinase A (PKA) activity. (F) VIPR1, stimulatory G protein (Gs), adenylate cyclase (AC), and PKA protein expression determined by western blotting. Vaginal SMCs were isolated from one rat. All indicators extracted from SMCs were detected three times except for cellular morphology. The results are the mean ± standard error of the mean (SEM) and were analyzed by One-factor ANOVA and the least significant difference (LSD) test. *: P-value<0.05; **: P-value<0.01; maternal group: the blank control group; NC group: the unloaded group.

Figure 5

Simultaneous silencing of miR-122-5p and vasoactive intestinal peptide receptor 1 (VIPR1) leads to a decrease in the relaxation function of rat vaginal smooth muscle cells (SMCs). (A) Morphology of rat vaginal SMCs under an inverted microscope. (B) Length of rat SMCs. (C) Free Ca²⁺ content in rat vaginal SMCs. (D) The apoptosis rate of rat vaginal SMCs with flow cytometry and terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick end labeling (TUNEL) assay. (E) Cyclic adenosine monophosphate (cAMP) concentration and protein kinase A (PKA) activity. (F) VIPR1, stimulatory G protein (Gs), adenylate cyclase (AC), and PKA protein expression determined by western blotting. Vaginal SMCs were isolated from one rat. All indicators extracted from SMCs were detected 3 times except for cellular morphology. The results are the mean ± standard error of the mean (SEM) and were analyzed by One-factor ANOVA and the least significant difference (LSD) test. *: P-value<0.05; **: P-value<0.01; maternal group: the blank control group; NC group: the unloaded group.