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Application of miR-26a in preparation of medicine for treating endometritis

A mir-26a, 1.mir-26a technology, applied in the field of biomedicine, can solve the problem of unclear endometrium inflammation damage relief and other problems

Pending Publication Date: 2022-03-11
TARIM UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, it remains unclear whether the alleviating effect of IFN-τ on endometrial inflammatory injury could also be mediated by these differentially expressed miRNAs

Method used

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  • Application of miR-26a in preparation of medicine for treating endometritis
  • Application of miR-26a in preparation of medicine for treating endometritis
  • Application of miR-26a in preparation of medicine for treating endometritis

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] Example 1: Determining the effect of miR-26a overexpression on cellular inflammatory response

[0046] The miR-26a mimics (mimics) were transfected into bovine endometrial epithelial cells to overexpress miR-26a, and the construction of bEECs inflammatory injury model was detailed in figure 2 . bEECs were transfected with miR-26a mimic (50nM) or inhibitor (100nM) liposomes, and 24 hours after transfection, the relative expression level of miR-26a was detected by qPCR (n=6) using U6 as an internal control. Cells were transfected with miR-26a mimics or inhibitors for 24 hours and then stimulated with 2 μg / mL LPS for 24 hours. Using GAPDH as an internal reference, the mRNA levels of inflammatory cytokines IL-1β, IL-6 and TNF-α were detected by qPCR. The secretion levels of cytokines IL-1β, IL-6 and TNF-α were detected by ELISA. In addition, to verify the expression of miR-26a in bEEC and the total expression in mouse endometrial tissue, it can be obtained by high-throu...

Embodiment 2

[0048] Example 2: Effect of miR-26a on PI3K / AKT pathway

[0049] miR-26a mimics and NC mimics were transfected into bEECs for 24 h. The phosphorylation level of NF-κB p65 and the expression level of TLR4 were determined by western blotting. Image-ProPlus (IPP) 6.0 software calculates the gray scale of protein. GAPDH was used as an internal reference.

[0050] The effects of miR-26a mock transfection on PI3K / AKT pathway-related proteins were studied. Western blot results showed that LPS stimulation significantly inhibited the protein levels of p-PI3K and p-AKT, but overexpression of miR-26a restored the LPS-induced suppression of p-PI3K and p-AKT levels, see Figure 5 (A and B) in . In addition, immunofluorescence staining results also showed that overexpression of miR-26a blocked LPS-induced downregulation of p-PI3K and p-AKT, see Figure 5 (C and D) in. also, Figure 6 It was verified that the overexpression of miR-26a can activate the PI3K / AKT signaling pathway.

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Embodiment 3

[0052] Example 3: Validation of PTEN as a target of miR-26a

[0053] A dual-luciferase reporter assay was used to confirm whether PTEN is a molecular target of miR-26a. PTEN was selected as a potential target of miR-26a by predictive analysis of biological databases (Fig. 7A). in Figure 7 Western blot experiments in (A) showed that overexpression of miR-26a reversed the LPS-mediated downregulation of p-PI3K and p-AKT expression.

[0054] The results showed that overexpression of miR-26a could significantly reduce the luciferase activity of the wild-type PTEN mRNA 3'-UTR vector without affecting the luciferase activity of the mutant vector, see Figure 7 (B); this result indicates that there is indeed a binding site for miR-26a in the 3'-UTR of PTEN mRNA. Further studies found that miR-26a mimics significantly inhibited LPS-induced PTEN protein expression, see Figure 7 (D-E) in. However, the mRNA level of PTEN did not change significantly, see (C) in Figure 7. In summar...

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Abstract

The invention discloses application of miR-26a in preparation of a medicine for treating endometritis, and belongs to the field of biological medicine. The invention verifies that the overexpression of the miR-26a obviously inhibits the secretion of the proinflammatory cytokines IL-1beta, IL-6 and TNF-alpha (Tumor Necrosis Factor-alpha). The miR-26a inhibits the translation of the miR-26a by targeting PTEN3 '-UTR, and further activates a PI3K / AKT pathway, so that NF-kappa B signal transduction is inhibited. According to the present invention, the overexpression of the miR-26a can target the PTEN gene so as to inhibit the inflammatory injury of the bEECs; the invention provides an important therapeutic target for accurate treatment of endometritis, verifies the action mechanism of miR-26a, provides a potential miRNA molecule with a function of inhibiting inflammatory injury of bEECs for clinical use, and provides a theoretical basis for research of related drug targets.

Description

technical field [0001] The invention relates to a new application of endogenous non-coding small RNAs, in particular to the application of miR-26a in the preparation of endometritis medicine, and belongs to the field of biomedicine. Background technique [0002] Endometritis is one of the main causes of endometrial injury, a common disease in reproductive system infection, and one of the main causes of abortion in dairy cows. Its molecular mechanisms and pathways of injury are elusive, and the incidence of endometritis is high. Endometrial epithelial cells are important effector cells for the host to resist external stimuli, and play a crucial role in the pathogenesis of endometrial inflammation involved in endometrial injury. [0003] MicroRNA is a kind of evolutionarily conserved non-coding small molecule RNA, which guides the silencing complex (RISC) to degrade mRNA or hinder its translation by complementary bases with target gene mRNA. MicroRNAs can regulate gene expre...

Claims

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Application Information

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IPC IPC(8): A61K31/7105A61P15/00C12Q1/6883
CPCA61K31/7105A61P15/00C12Q1/6883C12Q2600/158C12Q2600/178
Inventor 刘俊峰郭雪峰
Owner TARIM UNIV
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