Preliminary study of microRNA-126 as a novel therapeutic target for primary hypertension

Abstract

The present study aimed to explore microRNA-126 (miR-126) as a novel therapeutic target for primary hypertension. The lentiviral vector containing human immunodeficiency virus 1 (HIV‑1), the miR‑126 gene knockdown viral vector (lenti-miR-126-KD), and control lentiviral vector (lenti‑scramble‑miR) were constructed. Spontaneously hypertensive rats were randomly divided into 4 groups, which received a high dose of lenti‑miR‑126‑KD (1x108, n=5), low dose of lenti‑miR‑126‑KD (1x107, n=6), scramble‑miR (5x107, n=6), and PBS (n=6). Lentiviral vectors were injected into the tail vein. Data on the systolic blood pressure, diastolic pressure, mean arterial pressure, and heart rate were collected weekly. After 8 weeks of virus administration, the distribution of lentiviral vectors in different tissues was observed by fluorescence microscopy. Picric acid Sirius red and H&E staining were used to observe the target organ damage, and the ELISA kit was used to determine the serum nitric oxide (NO) content. The lentiviral vector was found to be constructed successfully. Eight weeks after the lentiviral vector injection, green fluorescent protein was observed in different tissues in each group. The blood pressure and heart rate were not significantly altered after lentiviral vector injection (P>0.05). No significant differences in the heart‑to‑body weight ratio among the four groups were observed (P=0.23). Picric acid Sirius red and H&E staining revealed that there was no significant difference in morphology among the four groups. No significant difference in the serum NO level among the four groups was noted (P=0.23). The miR‑126 gene knockdown lentiviral vector was constructed successfully. No significant antihypertensive effect was observed by the knockdown of miR‑126 for the treatment of primary hypertension. The target organs were not protected significantly after the treatment. The increased level of miR‑126 expression in hypertensive patients may be due to a compensatory mechanism.

Figure 1

(A) The microRNA-126 expression level in hypertension. The hypertensive patients demonstrated significantly higher 3′-UTR of miR-126 (1.410±0.369 vs. 0.838±0.274, P=0.03) and 5′-UTR of miR-126 (1.799±0.490 vs. 0.997±0.437, P=0.03) expression levels compared with the healthy controls. *P<0.05. (B) MicroRNAs in primary hypertensive patients and healthy controls were detected by gene chips. HTN-1-6, primary hypertensive patients; control 1–4, healthy controls. Red color indicates high expression; green color indicates low expression. UTR, untranslated region.

Figure 1

(A) The microRNA-126 expression level in hypertension. The hypertensive patients demonstrated significantly higher 3′-UTR of miR-126 (1.410±0.369 vs. 0.838±0.274, P=0.03) and 5′-UTR of miR-126 (1.799±0.490 vs. 0.997±0.437, P=0.03) expression levels compared with the healthy controls. *P<0.05. (B) MicroRNAs in primary hypertensive patients and healthy controls were detected by gene chips. HTN-1-6, primary hypertensive patients; control 1–4, healthy controls. Red color indicates high expression; green color indicates low expression. UTR, untranslated region.

Figure 2

DNA sequencing results of the lentiviral vector for miR-126 gene knockdown. The highlighted areas indicate the antisense sequence completely complementary to the mature miR-126. Sequencing results with primer (A) MS2117_1 and (B) MS2117_2.

Figure 3

Comparison of (A) systolic pressure (SP), (B) mean blood pressure (MAP), (C) diastolic pressure (BP), and (D) heart rate (HR) among the groups.

Figure 4

Comparison of body weights among the groups before and after experiments.

Figure 5

Green fluorescence expression in each organ under fluorescence microscope. 1st column, HD; 2nd column, LD; 3rd column, scramble-miR. (A) Brain, (B) liver, (C) lung, (D1) longitudinal section of cardiac muscle, (D2) cross section of cardiac muscle, (E1) kidney under low power, (E2) kidney under high power. HD, high dose; LD, low dose.

Figure 6

H&E and Sirius red F2B in carbazotic acid staining of heart and kidney. 1st column, HD; 2nd column, LD; 3rd column, scramble-miR; 4th column, control. (A) H&E staining of cardiac muscle, (B) H&E staining of kidney, (C) Sirius red F2B in carbazotic acid staining of cardiac muscle, (D) Sirius red F2B in carbazotic acid staining of kidney. HD, high dose; LD, low dose.