Using small molecules (natural products) discovery web server for targeting miRNA-mRNA interaction to rescue inhibition of mRNA translation
Refereed conference paper presented and published in conference proceedings


全文

其它資訊
摘要Background and Aims: MicroRNAs (miRNAs) play key roles in a series of physiological and pathological processes [1]. MiRNAs and their specific target mRNAs could form unique loops, which make them promising targets for developing therapeutics in specific diseases. However, there is still lack of selective small molecule discovery strategy aiming at targeting the interaction between miRNAs and their target mRNAs. To address the issue, we constructed a small molecule discovery web server for targeting miRNA-mRNA interaction (SMD-RRI). Recently, we have found that miR-214 could target ATF4 mRNA to inhibit bone formation. It is known that ATF4 is a transcription factor responsible for promoting osteogenic differentiation and osteoblastic function [2]. Thus, we hypothesize that the potential candidate small molecules, screened from a natural product database by SMD-RRI, could specifically target the loop between miR-214 and ATF4 mRNA to rescue inhibition of ATF4 mRNA translation to promote osteoblast activity in vitro.
Methods: The determination of the best small molecule in promoting osteogenic differentiation was detected using QPCR. The effect of this small molecule on ATF4 protein expression and osteogenic potential within osteoblast-like cells was investigated using WB and QPCR. The cell viability of this small molecule was determined using MTT assay.
Results: QPCR data revealed that small molecule #4 demonstrated the highest mRNA expression levels of alkaline phosphatase (ALP) and osteocalcin (BGLAP) among all the candidate molecules, indicating its best ability in promoting osteogenic differentiation. Consistently, compared to the corresponding control groups, small molecule #4 enhanced Alp+ staining and the amount of BGLAP protein in the supernatant of MC3T3-E1 cells in a dose-dependent manner. After treatment with small molecule #4, no change was found in the ATF4 mRNA level, but the amount of ATF4 protein was upregulated. The MTT assay revealed that no obvious cytotoxicity was observed for molecule #4 in the tested concentrations. The MC3T3-E1 cells were transfected with biotin-label miR-214-ATF4 loop synthesized in vitro, and incubated with the small molecule #4 for 12 h. After that, streptavidin beads were added to the lysates of the cells. The liquid chromatography-mass spectrometry (LC-MS) was used to determine the amount of the small molecule #4. The MC3T3-E1 cells were transfected with biotinlabel miR-214-ATF4 loop synthesized in vitro, and incubated with the small molecule #X for 12 h. After that, streptavidin beads were added to the lysates of the cells. The liquid chromatography-mass spectrometry (LC-MS) was used to determine the amount of the small molecule #X.
Conclusions: Further experiments are needed to elucidate whether the effect of the small molecule #4 on ATF4 protein expression and osteogenic potential within osteoblast-like cells after the transfection of the loop formed by miR-214 and ATF4 mRNA.
著者Zhen Jian Zhuo, Zong Kang Zhang, Bao Ting Zhang
會議名稱International Conference on Brain Research in Chinese Medicine: Degeneration and Repair
會議開始日26.07.2018
會議完結日28.07.2018
會議地點Hong Kong
會議國家/地區香港
會議論文集題名International Conference on Brain Research in Chinese Medicine: Degeneration and Repair
出版年份2018
月份7
語言英式英語

上次更新時間 2020-21-01 於 14:59