The medical application of mir-574-5p in diabetes and related diseases
A technology for type 2 diabetes and its application, which is applied in the field of biomedicine and can solve problems such as unclear effects and mechanisms
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Embodiment 1
[0057] Example 1 The expression of miR-574-5p is reduced in the serum of obese people with insulin resistance and mice.
[0058] (1) Extraction of total RNA from each mouse tissue.
[0059] Normal C57BL / 6 mice were sacrificed by neck dislocation under isoflurane anesthesia, 100 mg of each tissue was extracted, and total RNA was extracted using TRIzol reagent (Invitrogen, Carlsbad, CA). Using PrimeScript with gDNA Eraser TM RT kit (TakaRa) and specific RT primers (Guangzhou Ruibo Biological Co., Ltd.), reverse transcribed 2 μg of total RNA into cDNA. In CFX96Touch TM On the real-time PCR detection system (Hercules), use Fast qPCR mixture (TakaRa) and specific primers (Guangzhou Ribo Biotech Co., Ltd.) were used for real-time quantitative PCR. U6 small nuclear RNA was used as an internal standard for miRNA. Each reaction was repeated three times and analyzed by the 2-ΔΔCt method.
[0060] (2) It was found that miR-574-5p had specific high expression in adipose tissue of ...
Embodiment 2
[0065] Example 2 Fat damage causes miR-574-5p expression to decrease.
[0066] (1) Differentiated 3T3L1 cells were treated with palmitic acid (PA).
[0067] After PA (400 mM) was added to the supernatant of 3T3L1 cells differentiated in vitro for 7 days and cultured for 24 hours, the cells were collected, RNA was extracted, and the expression of miR-574-5p was detected. It was found that PA treatment significantly reduced miR-574-5p expression, as figure 2 As shown in A.
[0068] (2) Obesity model of C57 mice treated with HFD.
[0069] Male C57BL / 6 mice (8-10 weeks old) were purchased from Nanjing Model Animal Center (Nanjing, China). This study was approved by the Northern Theater Command General Hospital Institutional Animal Care and Use Committee. All animal experimental protocols were in accordance with the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health. Mice were fed a high-fat diet (HFD) for 12 weeks and then divided...
Embodiment 3
[0072] Example 3 miR-574-5p initiates the differentiation of preadipocytes into adipocytes.
[0073] To clarify whether miR-574-5p is involved in adipocyte differentiation. Transfect overexpression of miR-574-5p vector (miR-574-5p mimic) or miR-574-5p inhibitor (inhibitor) in differentiated 3T3L1 cells, and detect its effect on 3T3L1 cell differentiation.
[0074] (1) Cell transfection.
[0075]X-tremeGENE HP DNA transfection reagent (invitrogen) was used to transfect miRNA mimic (100nM), miRNA inhibitor (100nM) and its control (Guangzhou Ruibo Biotech Co., Ltd.) into 3T3-L1 cells. After culturing for 48 hours, the cells were collected, proteins were extracted, and Western blot was performed to detect adipocyte differentiation marker proteins.
[0076] (2) Oil red staining of cells.
[0077] Oil red O solution was purchased from Sigma. Mix 4ml of Oil Red O solution with 6ml of deionized water to get 10ml of Oil Red O working solution. The working solution of Oil Red O mus...
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