mTOR磷酸化水平对成骨MC3T3E1细胞增殖、自噬和分化的作用及其机制

doi:10.13481/j.1671-587X.20210306

Abstract

Abstract: Objective

To inhibit and activate the phosphorylation of mammalian target protein of rapamycin （mTOR） by rapamycin （RAPA） and MHY1485，and to discuss the effect of level of phosphorylated mTOR（p-mTOR） on the proliferation， autophagy and differentiation of osteoblasts and its mechanism.

Methods

The mouse MC3T3-E1 osteoblasts were divided into control group， 20 nmol·L^－1 RAPA group， 60 nmol·L^－1 RAPA group， 160 nmol·L^－1 RAPA group and 2.0 μmol·L^－1 MHY1485 group. The percentages MC3T3E1 osteoblasts at different cell cycles in various groups were detected by flow cytometry；the expression levels of mTOR and downstream signals—IF4E-binding protein 1 （4E-BP1） and ribosome protein subunit 6 kinase 1 （S6K1），B cell lymphoma-2（Bcl-2），Bcl-2 related X protein（Bax），and Caspase-3，alkaline phosphatase （ALP）， Runt-related transcription factor 2 （Runx2），and osteoblast-related transcription factor Osterix were detected by Western blotting and Real-time fluorescence quantitative PCR（RT-qPCR） methods；the mineralization ability of MC3T3-E1 osteoblasts in various groups was observed by Alizarin red staining.

Results

Compared with control group， the proliferation activities of MC3T3-E1 osteoblasts in 20 and 60 nmol·L^－1RAPA groups were significantly increased（P<0.05 or P<0.01）， the percentages of MC3T3-E1 osteoblasts at G₁ phase were increased（P<0.05 or P<0.01），the percentages of the MCET3-E1 osteoblasts at S phase were increased（P<0.05 or P<0.01），the expression levels of p-mTOR and its downstream pathway of p-4E-BP1 and p-S6K1， as well as p62，Bax，and Cleaved-Caspase-3 were decreased（P<0.05 or P<0.01），and the expression levels of LC3-Ⅱ， Bcl-2， ALP and Runx2 were increased （P<0.05 or P<0.01）.Compared with control group，the proliferation activity of MC3T3-E1 osteoblasts in 160 nmol·L^－1 RAPA group was significantly decreased（P<0.01），and the expression levels of p-mTOR，p-4E-BP1， and p-S6K1 were decreased （P<0.05 or P<0.01）；the proliferation activity of MC3T3-E1 osteoblasts in 2.0 μmol·L^－1 MHY1485 group was decreased（P<0.01），and the expression levels of p-mTOR，p-4E-BP1， and p-S6K1 were decreased（P<0.01）；however，the expression levels of Bax and Cleaved-Caspase-3 in both 160 nmol·L^－1 RAPA and 2.0 μmol·L^－1 MHY1485 groups were increased（P<0.05 or P<0.01），and the expression levels of Bcl-2， ALP， Runx2， and Osterix were decreased（P<0.05 or P<0.01）.Twelve days after treatment， the mineralized nodules were found in the MC3T3-E1 osteoblasts in 20 and 60 nmol·L^－1RAPA groups， and only a very small amount of calcium salt were found in the MC3T3-E1 osteoblasts in 160 nmol·L^－1 RAPA and 2.0 μmol·L^－1 MHY1485 groups.

Conclusion

Mild to moderate inhibition of mTOR phosphorylation can promote the proliferation， autophagy and differentiation of the MC3T3-E1 osteoblasts，while excessive inhibition and enhancement of mTOR phosphorylation have the opposite effect.

Key words: mammalian target of rapamycin, osteoblasts, cell proliferation, autophagy, cell differentiation

CLC Number:

R329.28

Xining LI, Wei WENG, Zheyuan SHEN, Xiaojie DOU, Yu ZHAO, Jikang MIN. Effect of mTOR phosphorylation level on proliferation,autophagy，and differentiation of MC3T3-E1 osteoblasts and its mechanism[J].Journal of Jilin University(Medicine Edition), 2021, 47(3): 575-586.

Figures/Tables 14

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Primer	Primer sequence	Length of product （bp）
p62	Forward 5'-GTGTGCCCAGACTACGACCT-3'	242
	Reverse 5'-GACTCAGCTGTAGGGCAAGG-3'
LC3	Forward 5'-CCGACCGCTGTAAGGAGG-3'	322
	Reverse 5'-AAGCCGAAGGTTTCTTGGGA-3'
Bcl-2	Forward 5'-CGGGAGAACAGGGTATGA-3'	210
	Reverse 5'-CAGAGACAGCCAGGAGAA-3'
Bax	Forward 5'-AGGGTTTCATTCCAGGATCGAGC-3'	195
	Reverse 5'-AGGCGGTGAGGACTCCAGCC-3'
Caspase-3	Forward 5'-ATGGACAACAACGAAACCTCCGTG-3'	306
	Reverse 5'-CCACTCCCAGTCATTCCTTTAGTG-3'
ALP	Forward 5'-TGCCCTGAAACTCCAAAAGC-3'	250
	Reverse 5'-CTTCACGCCACACAAGTAGG-3'
Osterix	Forward 5'-CATCCCTATGGCTCGTGGTA-3'	225
	Reverse 5'-TGGGTTAAGGGGAGCAAAGT-3'
Runx2	Forward 5'-CCCAGCCACCTTTACCTACA-3'	150
	Reverse 5'-TATGGAGTGCTGCTGGTCTG-3'
GAPDH	Forward 5'-ATGGGTGTGAACCACGAGA-3'	229

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Effect of mTOR phosphorylation level on proliferation,autophagy，and differentiation of MC3T3-E1 osteoblasts and its mechanism

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