微小RNA-22对造血干细胞TF-1糖代谢的调控及其分子机制

Regulation of microRNA-22 on glycometabolism of hematopoietic stem cell TF-1 and its molecular mechanism

目的研究微小RNA－22（microRNA－22，miR－22）对造血干细胞TF－1糖代谢的调控作用及其分子机制。方法低氧条件培养TF－1细胞2 h，通过实时定量PCR（quantitative real－time PCR，qRT－PCR）检测miR－22、葡萄糖转运因子4（glucose transporter 4，G1ut4）和过氧化物酶体增殖剂激活受体γ（peroxisome proliferators－activated receptor－γ，PPAR－γ）表达水平。构建miR－22基因的小向导RNA（small guide RNA，sgRNA）载体，利用成簇规律间隔的短回文重复序列（clustered regularly interspaced short palindromic repeats，CRISPR）/CRISPR结合核酸内切酶（CRISPR－associated endonumclease，Cas9）CRISPR/Cas9技术对TF－1细胞进行基因敲除。构建过表达载体，导入miR－22基因敲除的细胞后，诱导miR－22过表达，qRT－PCR检测miR－22、Glut4和PPAR－γ的mRNA水平，蛋白质免疫印迹（Western blot）检测Glut4和PPAR－γ蛋白表达水平，以鉴定miR－22表达水平对糖代谢的影响，及其作用机制。结果与对照组比较，低氧培养2 h后TF－1细胞miR－22的表达量降低（0.015±0.000比0.056±0.001），Glut4（0.351±0.038比0.152±0.005）和PPAR－γ（0.421±0.017比0.248±0.008）表达升高，差异均有统计学意义（均P〈0.05）。与对照组比较，miR－22基因敲除后Glut4（0.019±0.000比0.008±0.000）和PPAR－γ（0.038±0.001比0.019±0.000）的表达水平均升高，差异均有统计学意义（均P〈0.05），过表达miR－22后Glut4（0.005±0.000比0.008±0.000）和PPAR－γ（0.137±0.000比0.019±0.000）的表达水平，差异均有统计学意义降低（均P〈0.05）。结论miR－22可通过下调PPAR－γ的表达对造血细胞TF－1的糖代谢发挥负调控作用。

ObjectiveTo study the regulation of microRNA-22（miR-22） on glycometabolism of hematopoietic stem cell TF-1 and its molecular mechanism.MethodsTF-1 cells were cultured for 2 h under hypoxic conditions.The expression levels of Glut4 and miR-22 was detected by quantitative real-time PCR（qRT-PCR）. The sgRNA vector of the miR-22 gene was constructed and miR-22 gene of TK-1 cells was knocked out by the CRISPR/Cas9 technique.Overexpression vectors were constructed, and miR-22 knocked-out cells were introduced to overexpress miR-22, the expression of miR-22 was detected by qRT-PCR and the expression levels of Glut4 and PPAR-γ were detected by qRT-PCR and Western blot.ResultsCompared with the control group, the expression of miR-22 in TF-1 cells decreased （0.015±0.000 vs. 0.056±0.001） and the expression of Glut4 （0.351±0.038 vs. 0.152±0.005） and PPAR-γ （0.421±0.017 vs. 0.248±0.008） increased, when TF-1 cells were cultured for 2 h under hypoxic conditions, and the differences were statistically significant （all P〈0.05）. Compared with the control group, the expression levels of Glut4 （0.019±0.00 vs. 0.008±0.000） and PPAR-γ （0.038±0.001 vs. 0.019±0.000） were significantly increased after miR-22 gene silencing, and they were significantly decreased （Glut4： 0.005±0.000 vs. 0.008±0.000; PPAR-γ： 0.137±0.000 vs. 0.019±0.000） after overexpression of miR-22, and the differences were statistically significant （all P〈0.05）.ConclusionsIt suggests that miR-22 exerts a negative regulation on glycometabolism of hematopoietic stem cell TF-1 by downregulating the expression of PPAR-γ.A new regulatory factor of TF-1 glycometabolism and the mechanisms are identified, which has provided new ideas for the targeted medication of diseases induced by hematopoietic stem cell dysfunction.