THE RELATIONSHIP BETWEEN SERUM RHOA AND MIR-133B IN ACUTE ISCHEMIC STROKE

Yong-Peng Yu, Ya-Li Zheng, Ting-Hong Yu, Jia-He Bai and Ting-Ting Jiang Yongpeng Yu Innovation Studio and Department of Neurology, the Affiliated Weihai Central Hospital of Qingdao University and Weifang Medical College, Weihai, Shandong, 264400. ...................................................................................................................... Manuscript Info Abstract ......................... ........................................................................ Manuscript History Received: 07 January 2020 Final Accepted: 10 February 2020 Published: March 2020

The proportion of history of hypertension, diabetes, hyperlipidemia, and stroke was significantly higher in the patient group than in the control group (p< 0.05) ( Table 1). There were no significant differences in age, sex, and smoking rates between the two groups (p>0.05).

ELISA:
RhoA was detected by ELISA performed as previously described [13]. Elbow venous blood was collected in the morning and stored in a procoagulant tube. After 2 hours, cubital venous blood was centrifuged at 3,000g for 15 min at 4ºC at room temperature and ELISA assay (Nanjing dongji biology co. LTD) follows the manufacturer's agreement. Samples were read at 450 nm using a microplate reader.

RNA extraction:
Total RNA was isolated from whole blood samples (5ml, collected in test tubes containing EDTA) according to the manufacturer's protocol. The concentration of the RNA samples were determined by measuring optical density (OD) 260/OD280.

Real-time quantitative PCR:
A total of 1μg of RNA was reverse transcribed using Moloney murine leukemia virus (MMLV) reverse transcriptase (Nanjing dongji biology co. LTD) and specific primers. PCR amplification was performed in a 10μl reaction system, which contained 5μl SYBR Green Supermix, 0.4μL forward primer, 0.4μl reverse primer, 2.2μl double-distilled H2O, and 2μl template cDNA. The temperature protocols used for RT are as follows: 72ºC for 10min; 42ºC for 60min, 72ºC for 5min and 95ºC for 2min thermocycling conditions were set as follows: 95ºC for 10min followed by 40 cycles of 95ºC for 15 seconds and 60ºC for one min. DNA fragments were collected to be tested by PCR with primer located in miR-133b promoter: forward 5'-TGCAAA-CACTTGCAGCTGAG-3'and reverse 5'-TCTACTCTGCCTGCTTGT TG-3' (198bp).

Statistical Analysis:
Statistical analysis was performed using SPSS l7.0 software. The measurement data are expressed as mean ± standard deviation. A one-way ANOVA multiple comparison test followed by a Turkish post hoc test was used to compare two or more groups. Student's test, Pearson's correlation coefficient, and Spearman's rank test were used to evaluate the normality of the distributions of RhoA levels, NIHSS scores, and miR-133b values. The difference was statistically significant p <0.05.

Clinical analysis of AIS patients and control samples:
One hundred and twenty individuals were enrolled into the present study including 60 patients with AIS and 60 healthy controls. In total, 53% were male, and 47% were female. Age of patients ranged from 45 to 78 years. The 360 NIHSS assigns scores to stroke patients based on seriousness: 0 indicates stroke symptoms, 1-4 indicates minor stroke, 5-15 indicates moderate stroke, 16-30 indicates severe stroke. The clinical parameters were tabulated and are presented in Table 1.

Serum RhoA level and miR-133b in AIS patients and controls:
The relationship between RhoA and miR-133b has benn clarified in the previous study.It indicated that miR-133b+MSC treatment further significantly decreased RhoA at day 14 after MCAO compared with that after naive MSC treatment, and the RhoA expression after miR-133b-MSC treatment was sustained at a similar elevated level at day 14 after MCAO compared with naive MSC treatment [14]. These finding suggests that miR-133b plays a crucial role in regulating RhoA expression.
In the present study, the serum miR-133b level was lower in AIS patients than that in healthy controls (Fig. 1).While the miR-133b-related RhoA level was higher in AIS patients than that in healthy controls (Fig. 2). The correlation between RhoA and miR-133b was analyzed by Spearman's coefficient. The data revealed a negative correlation between serum RhoA level and miR-133b (Spearman's = −0.632; p= 0.009) in AIS patients and healthy controls.These results confirm that miR-133b is the putative targe that regulates the expression of RhoA, which may be causative of AIS.

Levels of miR-133b and RhoA related with NIHSS scores:
The expression levels of RhoA in the patients with severe neurological deficits were significantly higher than those of patients with moderate and mild neurological deficits (p<0.05). At the same time, the levels of RhoA in the patients with moderate neurological impairment were significantly higher than those of patients with mild neurological impairment. (p<0.05) (Fig.3) The level of miR-133b in the above patients with different degrees of neurological impairment showed the opposite change with RhoA (Fig.4).This finding suggests that the serum miR-133b level is associated with the NIHSS scores, which may contribute to AIS diagnosis.

Discussion:-
RhoA, a kind of bird nucleoside triphosphate binding protein, is a member of the Ras superfamily,whose downstream effecter Rho kinase (ROCK) play a vital role in various physiological and pathological processes, such as contraction of vascular smooth muscle cell, actin cytoskeleton organization, cell adhesion, et al. Increasing evidences show that RhoA/ROCK has played a key role in increasing the permeability of blood-brain barrier (BBB) [15].Under the activation of cerebral ischemia reperfusion injury, ROCK increased phosphorylation of myosin regulatory light chain 2 (p-MLC2) expression and enhanced the contraction force of actin and myosin, by increasing BBB leakage at the same time, the increase of p-MLC2 may also contribute to the destruction of ZO-1. Consequently the expression of MMP-9 increasing causes hydrolysis of the extracellular matrix, which results in BBB damage and brain edema. Inhibition of RhoA/ROCK pathway can downregulate expression of MMP-2/9, which can reduce the degradation of tight connections and BBB damage [16].
In the present study, the serum RhoA level was significantly increased in patients with AIS. MiRNAs are a class of endogenous non-coding small RNAs consisting of 18-24 nucleotides. They bind to the 3'-UTR of the target gene messenger RNA to regulate degradation of the messenger RNA or inhibit protein translation to regulate the target gene expression [17]. Previous studies have shown that microRNAs are widely distributed in various body fluids, such as serum, plasma, saliva, tears, and amniotic fluid, and that the types and amounts of microRNAs in body fluids vary with physiological and disease conditions [18]. It is involved in growth and development of many diseases [19]. Compared with healthy controls, the levels of serum miR-133b in patient with AIS reduced. Exosomes from MSCs mediate the miR-133b transfer to astrocytes and neurons, which regulate gene expression, subsequently benefit neurite remodeling and functional recovery after stroke [14]. Exosome-mediated transfer of miR-133b overexpression contributes to neurite outgrowth [20].At the same time, MSCs were used to deliver miR-133b to increase the expression level of miR-133b in ischemic lesion and further improve therapeutic effects [21]. Here, we evaluated for the potential of miR-133b as a biomarker for AIS in clinical practice.
In addition to confirming the downregulation of serum miR-133b expression in the patients with AIS, we also found that there was a significant negative correlation between the expression of miR-133b and RhoA, suggesting that the expression of miR-133b and RhoA can be used as a biomarker for AIS. There is a good correlation between miR-361 133b and RhoA in patients with moderate neurological deficits, which indicates that miR-133b and RhoA have a high degree of value in estimating to moderate neurological dysfunction in AIS. Therefore, in patients with severe neurological deficits, further exploration of protein markers that are better related to miR-133b will become the focus of future research.
This study suggests that serum miR-133b may play a protective role in the pathogenesis, development, and prognosis of ischemic stroke. Serum miR-133b is a useful biomarker for AIS as well as its degrees of neurological impairment.However, the specific mechanism still remains elusive, further research is needed to determine whether there is a relationship between miR-133b and BBB damage in AIS. Large scale studies are still needed to confirm these findings.