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Application of small-molecule RNA and Rho kinase inhibitor

A technology of kinase inhibitors and small molecules, which is applied in the field of biomedicine and can solve problems such as different physiological effects

Inactive Publication Date: 2015-12-16
SHENZHEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, only Fasudil hydrochloride is marketed as a Rho kinase inhibitor for clinical use, but there are many potential adverse reactions in the clinical application of Rho kinase inhibitors. The main reasons are: Rho kinase is widely distributed in tissue cells and inhibits the activity of different tissue cells. Rho kinases have different physiological effects; existing Rho kinase inhibitors also inhibit other protein kinases while inhibiting Rho kinases

Method used

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  • Application of small-molecule RNA and Rho kinase inhibitor
  • Application of small-molecule RNA and Rho kinase inhibitor
  • Application of small-molecule RNA and Rho kinase inhibitor

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0052] Example 1 Preparation of small molecule RNAmiR-223

[0053] The mature sequence of miR-223 is 5'-UGUCAGUUUGUCAAAUACCCCA-3' (22bp, SEQ ID NO: 1), which can be prepared in the following manner.

[0054] 1. Chemical synthesis

[0055] The mimic of miR-223 (miRNAmimic or agomir) was synthesized by Guangzhou Ruibo Biotechnology Co., Ltd., which is a double-stranded RNA that can mimic the high-level expression of endogenous mature miR-223; standard purification, stored at -20°C. Dissolve in RNase-free water to prepare a 25 μM stock solution, store at -80°C for later use, and dilute to the required concentration when used.

[0056] 2. Viral vector mediates the expression of miR-223

[0057] (1) Preparation of expression pri-miR-223 vector

[0058] The mature sequence and precursor sequence (pre-miRNA) information of human miR-223 was obtained in miRBase (http: / / www.mirbase.org / ) database. Find flanking sequences of about 200-300 bp on both sides of the mature sequence in t...

Embodiment 2

[0064] Example 2 miR-223 targets to inhibit the expression of RhoB and MLC2

[0065] Cell culture: 293A cells (purchased from ATCC, Manassas, VA) were cultured in 10% FBS-DMEM medium (FBS was purchased from Gibco, DMEM was purchased from Hyclone). Cells in good growth state were collected, counted by centrifugation, and counted at 6×10 4 Inoculate / well in a 24-well plate at 37°C, 5% CO 2 Cultivate for 24h.

[0066] Transfection: When the cell density reaches 60-70%, cells are transfected with PEI, co-transfected with 3'-UTR reporter vector or mutated 3'-UTR reporter vector (50ng), miRNA overexpression vector pLVX-CMV-miR -223 or pLVX-CMV-control (500ng) and the internal reference carrier phRL-TK (10ng), each group of experiments was done in 3 replicate wells. Two days after transfection, discard the culture medium, wash twice with PBS, and then lyse the cells with 50 μL of 1×PassiveLysisBuffer.

[0067] Luciferase detection: Use the Dual-Luciferase Reporter Assay System (E...

Embodiment 3

[0070] Example 3 miR-223 inhibits the Rho kinase signaling pathway by targeting RhoB and MLC2

[0071] Cell culture: Human PASMC (primary pulmonary artery smooth muscle cells) were purchased from Lonza (Walkersville, MD) and cultured in SmGM-2 complete medium (Lonza) containing 5% FBS. Cells in good growth state were collected and counted by centrifugation. 6×10 5 Inoculate each well in a 60mm dish at 37°C, 5% CO 2 Cultivate for 24h.

[0072] Transfection: chemically synthesized miRNA mimics (mimic) and inhibitors (inhibitor) were used to overexpress or inhibit the expression of miR-223-3p, and cel-miR-67-3p was used as a negative control (mimiccontrol and inhibitorcontrol), All were purchased from Ribo (Guangzhou Ruibo Biological). The cells were planted in culture dishes, and when the cell density reached 70% overnight, the negative control, miR-223mimic (50nM) or miR-223inhibitors (100nM) were transfected by Lipofectamine2000 (Invitrogen). After 4-6 hours of transfectio...

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Abstract

The invention discloses application of small-molecule RNA and a Rho kinase inhibitor, wherein the small-molecule RNA is miR-223 which serves as a Rho kinase inhibitor and is used for preparing medicines for treating diseases caused by aberrant activation of a Rho kinase signaling pathway. The small-molecule RNA, taking key factors RhoB and MLC2 in the Rho kinase signaling pathway as targets, is capable of significantly inhibiting the Rho kinase signaling pathway. The small-molecule RNA, as a novel Rho kinase inhibitor, can be used for preparing medicines for treating cardiovascular disease, bronchial asthma, glaucoma, erectile dysfunction, nervous system diseases, tumors and the like.

Description

technical field [0001] The present invention relates to the field of biomedicine, in particular to the application of a small molecule RNA and a Rho kinase inhibitor, in particular to the use of hsa-miR-223-3p in the preparation of medicines for diseases caused by abnormal activation of the RhoB / Rho signaling pathway application. Background technique [0002] The key signaling molecules of Rho / Rho kinase signaling pathway include Rho protein, Rho kinase, myosin light chain phosphatase (MLCP), myosin light chain (MLC) and so on. The Rho protein family is a class of small molecule GTP-binding proteins, including RhoA, RhoB, RhoC, Rac1, Rac2, and Cdc42. Under the stimulation of external signals, the Rho protein changes from the GDP-bound inactive state to the GTP-bound active state, and the activated Rho protein then transmits the signal to the downstream effector molecule Rho kinase, which causes Rho kinase to be phosphorylated and activated; activation The Rho kinase then p...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K31/7088A61P9/00A61P11/06A61P27/06A61P15/10A61P25/00A61P9/12A61P35/00C12N15/867C12N15/113
Inventor 苟德明康康张小英曾艳陈继栋
Owner SHENZHEN UNIV
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