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posted on 30.07.2021, 08:43 by Zewu Zhu, Xiaoqiong Zhang, Yingcheng Jiang, Shuhao Ruan, Fang Huang, Huimin Zeng, Minghui Liu, Weiping Xia, Feng Zeng, Jinbo Chen, Yu Cui, Hequn Chen
(A) Human renal interstitial fibroblasts (hRIFs) were transfected with lentivirus carrying the full length of NEAT1 (Len-NEAT1) or lentivirus carrying different shRNAs targeting NEAT1 (Len-sh1-NEAT1; Len-sh2-NEAT1; Len-sh3-NEAT1), followed by osteogenic induction for 7 days, and qRT-PCR determined the NEAT1 expression to assess the efficiency. (B) The relative EGR1 and BMP2 protein level detected by WB in hRIFs transfected with EGR1 plasmids (p-EGR1) or control plasmids (p-ctrl). (C) QRT-PCR determined the relative miR-129-5p expression in hRIFs transfected with mimic-miR-129-5p or inhibitor-miR-129-5p. (D-E) The relative BMP2 protein level detected by WB in hRIFs transfected with mimic-miR-129-5p or inhibitor-miR-129-5p. (F) The relative alkaline phosphatase (ALP) activity in lysis from hRIFs co-transfected with either Len-NEAT1 or Len-ctrl and either mimic-miR-129-5p (mimic-129-5p) or mimic-ctrl 7 days after osteogenic induction. (G) The relative ALP activity in lysis from hRIFs co-transfected with either Len-sh-NEAT1 or Len-ctrl-sh and either inhibitor-miR-129-5p (inhibitor-129-5p) or inhibitor-ctrl 7 days after osteogenic induction. (H) QRT-PCR determined the enrichment of miR-129-5p expression in EGR1-coprecipitation to that of negative control IgG. (I) HRIFs were transfected with lentivirus carrying different shRNA targeting BMP2 (Len-sh1-BMP2; Len-sh2-BMP2; Len-sh3-BMP2), followed by osteogenic induction for 7 days, and qRT-PCR determined the BMP2 expression to assess the efficiency. (J) QRT-PCR determined the relative NEAT1 expression in hRIFs co-transfected with Len-ctrl or Len-NEAT1 and Len-ctrl-sh or Len-sh-BMP2 7 days after osteogenic induction. (K) Correlation analysis between the relative NEAT1 expression and the relative mRNA expression of BMP2 in NRP (n=19).

Funding

This work was supported by the National Natural Science Foundation of China (81770705 to Hequn Chen; 82000761 to Yu Cui) and Natural Science Foundation of Hunan Province (2017JJ3482 to Xiaoqiong Zhang).

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