MicroRNAs (miRNAs) are a group of endogenous noncoding RNAs that play important roles in many biological processes. This study aimed to check if miRNAs were involved in the response to acupuncture in rats. Microarray analysis was performed to compare the miRNA expression profiles of medulla in spontaneously hypertensive rats (SHRs) treated with or without acupuncture. Our microarray analysis identified 222 differentially expressed miRNAs in the medulla of SHRs treated with acupuncture at taichong acupoint. Among these miRNAs, 23 miRNAs with a significant difference were found in acupuncture-treated SHRs compared to untreated rats. These 23 miRNAs could regulate 2963 target genes which were enriched in at least 14 pathways based on our bioinformatic analysis. miRNA-339, miR-223, and miR-145 were downregulated in the medulla of SHRs compared to normotensive rats. Notably, these miRNAs were upregulated to basal levels in the medulla of SHRs treated with acupuncture at taichong in comparison with SHRs receiving acupuncture at nonacupoint group or SHRs without any treatment. Our findings have revealed significant changes of a panel of selective miRNAs in hypertensive rats treated at taichong acupoint. These data provide insights into how acupuncture elicits beneficial effects on hypertension.
Hypertension, a major risk factor for cardiovascular disease, affects approximately one billion individuals worldwide [
Acupuncture originated in ancient China at least 2,500 years ago. Although there is some controversy in mainstream Western medicine, it has become one of the most widely practiced forms of alternative medicine in the world [
Since the 1970s, a number of animal and clinical studies have demonstrated the effectiveness of acupuncture at specific acupoints to lower blood pressure in essential hypertension [
The small noncoding microRNAs (miRNAs) have critical functions in the regulation of various critical biological processes such as cell metabolism, proliferation, death, and development [
All animal experiments were performed at the Laboratory Animal Center of Guangzhou University of Chinese Medicine, Guangzhou, China. The procedure was approved by the Ethics Committee of Guangzhou University of Chinese Medicine, Guangzhou, China [permit number: SYXK (Yue) 2008-0085].
The 6th generation of miRCURYTM LNA Array (v.16.0) was obtained from KangChen Bio-tech (Shanghai, China), which contains more than 1891 capture probes, covering all human, mouse, and rat microRNAs annotated in miRBase 16.0, as well as all viral microRNAs related to these species. In addition, this array contains capture probes for 66 new miRPlus human microRNAs. All chemicals and reagents used were of analytical grade.
The experiments were performed with SHRs and Sprague-Dawley (SD) rats provided by Beijing Vital River Laboratory Animals Co., Ltd (Beijing, China). The rats were housed in cages maintained in a temperature- and humidity-controlled room at the Laboratory Animal Center of Guangzhou University of Chinese Medicine, Guangzhou, China. The animals were given a standard diet. The SHRs with confirmed blood pressure
A number of clinical and animal studies have reported the efficacy of acupuncture at taichong point in reducing hypertension [
The acupuncture procedure at taichong point and nonacupoint in SHRs. In taichong group of the SHRs, acupuncture was performed at bilateral LR3 located between the 1st and the 2nd metatarsal of dorsal foot, while, in nonacupoint group, acupuncture was done at bilateral nonacupoint located at fossa between the 3rd and 4th metatarsal of dorsal foot.
Total RNA was isolated using TRIzol (Invitrogen, Grand Island, NY, USA) and miRNeasy minikit (QIAGEN) according to manufacturer’s instruction. After RNA isolation from the samples, the miRCURY Hy3/Hy5 Power labeling kit (Exiqon, Vedbaek, Denmark) was used according to the manufacturer’s guideline for miRNA labelling. One microgram of each sample was 3′-end-labeled with Hy3 fluorescent label, using T4 RNA ligase. After stopping the labeling procedure, the Hy3-labeled samples were hybridized on the miRCURY LNA Array (v.16.0) (Exiqon) according to the manufacturer’s manual. The slides were scanned using the Axon GenePix 4000B microarray scanner (Axon Instruments, Foster City, CA, USA). Scanned images were then imported into GenePix Pro 6.0 software (Axon) for grid alignment and data extraction. Replicated miRNAs were averaged and miRNAs in which intensities >50 in all samples were chosen for calculating the normalization factor. The data were normalized using the Median normalization method and after normalization differentially expressed miRNAs were identified through Volcano Plot filtering. In addition, hierarchical clustering was performed using MEV software (v4.6, TIGR).
Computational target prediction of miRNAs was conducted by miRDB online searching program (
The rat medulla was homogenized in 400
Data are presented as the mean ± SD. Multiple comparisons were evaluated by one-way analysis of variance (ANOVA) followed by Tukey’s multiple comparison procedure, with
To reveal miRNAs profiling response to acupuncture therapy in SHRs, microarrays containing 1891 human, rat, and mouse miRNAs were used to examine the expression of miRNAs in medullas after acupuncture treatment at taichong point in SHRs. The analyses were performed using total RNA collected from three rat medullas. Statistical analysis of the miRNA expression data identified 222 miRNAs with significant changes in the expression, with 23 miRNAs having expression levels greater or equal to a 1.5-fold change in SHRs with or without acupuncture treatment (Tables
miRNAs upregulated by acupuncture in the medulla of SHRs.
Mature miRNAs |
Fold-change |
|
miRBase accession number |
---|---|---|---|
339 | 2.13 | 0.0214 | MIMAT0000583 |
223 | 3.34 | 0.0011 | MI0000963 |
145 | 3.02 | 0.0059 | MI0000918 |
451 | 3.32 | 0.0097 | MI0001731 |
193 | 2.88 | 0.0300 | MI0000936 |
378 | 2.30 | 0.0135 | MI0003719 |
423 | 1.97 | 0.0277 | MI0006145 |
let-7b* | 1.70 | 0.0443 | MIMAT0004705 |
Each experiment was performed in triplicate. The fold change is presented as the mean ± SEM.*Indicates antisense mature miRNA.
miRNAs downregulated by acupuncture in the medulla of SHRs.
Mature miRNAs |
Fold-change |
|
miRBase accession number |
---|---|---|---|
7a | 0.52 | 0.0254 | MI0000641 |
9 | 0.48 | 0.0491 | MI0000838 |
128 | 0.45 | 0.0229 | MI0000900 |
132 | 0.42 | 0.031 | MI0000905 |
134 | 0.59 | 0.0037 | MI0000907 |
182 | 0.24 | 0.0093 | MI0006133 |
335 | 0.6 | 0.0475 | MI0000612 |
382 | 0.44 | 0.0381 | MI0003548 |
383 | 0.51 | 0.0324 | MI0003478 |
434 | 0.44 | 0.0155 | MI0006147 |
496 | 0.59 | 0.011 | MI0012622 |
135a | 0.45 | 0.0414 | MI0000908 |
136* | 0.58 | 0.0131 | MIMAT0004733 |
376b-5p | 0.54 | 0.0052 | MIMAT0003195 |
384-3p | 0.57 | 0.0225 | MIMAT0005310 |
Each experiment was performed in triplicate. The fold change is presented as the mean ± SEM. *Indicates antisense mature miRNA.
To validate the microarray profiling data, qRT-PCR was used to confirm the upregulated miRNAs including miRNA-339, miR-223, miR-145, and miR-451. The data showed that miR-339, miR-223, and miR-145 were significantly upregulated in medullas of SHRs treated with acupuncture at taichong point in contrast to the model group untreated with acupuncture (Figure
Confirmation of miR-339 (a), miR-223 (b), and miR-145 (c) expression changes in the medulla of SHRs treated with acupuncture at taichong point (model + acu.). The expression level of miR-339, miR-223, and miR-145 in rats was detected using qRT-PCR. The relative expression of these miRNAs was calculated in relation to levels of U6 RNA using the
Next, we compared the effect of acupuncture at taichong acupoint or at nonacupoint on miRNA expression changes in SHR medulla. The data showed that acupuncture at taichong point significantly increased miRNA-339, miR-223, and miR-145 levels in medullas of SHRs in contrast to the model group, while acupuncture at nonacupoint did not significantly alter miR-339, miR-223, and miR-145 levels compared to the model group. Moreover, compared to acupuncture at nonacupoint, acupuncture at taichong point significantly upregulated the expression of miRNA-339, miR-223, and miR-145 in medullas of SHRs (Figure
Acupuncture treatment at taichong acupoint elicits specific miRNA expression changes in SHRs. Acupuncture at taichong point (Taichong) significantly increased miR-339 (a), miR-223 (b), and miR-145 (c) expression levels in the medullas of SHRs compared to SHRs with nonacupoint treatment (non-acu.).
To explore possible targets and signaling pathways involved in response to acupuncture, we used miRDB to predict the targets of the differentially expressed microRNAs responsive to acupuncture and used DAVID Bioinformatics Resources 6.7 (
The enriched KEGG pathways of acupuncture-responsive miRNAs in SHRs.
Category | Term | Count | % |
|
Benjamini |
---|---|---|---|---|---|
KEGG_PATHWAY | Neurotrophin signaling pathway | 21 | 0.1 |
|
|
KEGG_PATHWAY | Pathways in cancer | 51 | 0.2 |
|
|
KEGG_PATHWAY | MAPK signaling pathway | 42 | 0.2 |
|
|
KEGG_PATHWAY | Wnt signaling pathway | 38 | 0.2 |
|
|
KEGG_PATHWAY | Ubiquitin mediated proteolysis | 31 | 0.1 |
|
|
KEGG_PATHWAY | Cell cycle | 29 | 0.1 |
|
|
KEGG_PATHWAY | Chemokine signaling pathway | 29 | 0.1 |
|
|
KEGG_PATHWAY | Calcium signaling pathway | 28 | 0.1 |
|
|
KEGG_PATHWAY | Oocyte meiosis | 27 | 0.1 |
|
|
KEGG_PATHWAY | Tight junction | 22 | 0.1 |
|
|
KEGG_PATHWAY | T cell receptor signaling pathway | 21 | 0.1 |
|
|
KEGG_PATHWAY | Endocytosis | 42 | 0.2 |
|
|
KEGG_PATHWAY | Axon guidance | 21 | 0.1 |
|
|
KEGG_PATHWAY | TGF-beta signaling pathway | 20 | 0.1 |
|
|
Thresholds: count cut-off ≥20; EASE <0.1.
In this study, we first found that miRNAs responded to acupuncture treatment in SHRs. Previous studies have demonstrated that physical therapy elicited remarkable miRNA profiling changes in humans [
Our finding further showed that acupuncture treatment at specific acupoint elicits selective miRNA expression changes in SHRs (Figure
Our bioinformatics analysis showed that 23 miRNAs responded to acupuncture and these miRNAs might regulate 2963 targets. These miRNAs have many important physiological and pathological functions. miRNA-339, miR-223, and miR-145 are highly conserved and have multiple targets predicted for them in both humans and rats [
Our bioinformatic data also showed that there were 14 pathways regulated by the miRNAs responsive to acupuncture therapy (Table
Proposed role of miRNAs in the antihypertensive effects of acupuncture.
In addition, our bioinformatics data predicted that mitogen-activated protein kinase (MAPK) signaling pathway was possibly involved in the antihypertensive activity of acupuncture in rats. The MAPK signaling molecules have been recognized as important mediators in directing cellular responses to a diverse array of stimuli, such as proinflammatory cytokines and exposure to environmental compounds. They regulate gene expression, differentiation, apoptosis and many other cellular processes [
In summary, our microarray study for the first time identified 222 differentially expressed miRNAs in the medulla of SHRs treated with acupuncture at the taichong acupoint. Among these miRNAs, 23 miRNAs were found to be differentially expressed in acupuncture-treated SHRs compared to untreated control rats. These 23 miRNAs could regulate 2963 target genes based on our bioinformatic analysis. Importantly, our RT-PCR assay has confirmed that miRNA-339, miR-223, and miR-145 were upregulated in SHRs treated with acupuncture at taichong acupoint in comparison with the nonacupoint group. Our findings have demonstrated significant changes of specific and selective miRNAs in rats when taichong acupoint was stimulated. Our data have revealed the specific miRNA profile changes in response to acupuncture treatment and strongly suggest that a selective panel of miRNAs play an important role in the antihypertensive activity of acupuncture therapy.
The authors declare that there is no conflict of interests regarding the publication of this paper.
Jia-You Wang and Hui Li equally contributed to this work as joint first authors.
The microarray experiments were performed by KangChen Bio-tech Inc., Shanghai, China. This project were supported by National Key Basic Research and Development Program (Grant no. 2012CB518504), Natural Science Foundation of Guangdong Province (Grant no. S2013010011547), National Natural Science Foundation of China (Grant no. 81173349), and University of South Florida College of Pharmacy Startup Fund.