17 b-Estradiol alleviates oxidative damage in osteoblastsby regulating miR-320/RUNX2 signaling pathway

YANYAN XU�, HAO XU

�, XIUPING YIN, XIANLI LIU, ZHONGXI MA andZHIGANG ZHAO*

Department of Orthopaedics, Wuhan Fourth Hospital, Puai Hospital, Tongji Medical College,Huazhong University of Science and Technology, Wuhan 430033, China

*Corresponding author (Email, gtjzwkzzg@163.com)

�Yanyan Xu and Hao Xu have contributed equally to this work.

MS received 26 May 2021; accepted 18 November 2021

The aim of this study is to investigate the effect and mechanism of 17 b-estradiol (E2) on oxidative stress in theosteoblasts. An oxidative stress-induced damage model was established using H2O2 in MC3T3-E1 cells, andH2O2-induced cells were treated with E2. The results indicated that E2 attenuated oxidative stress in H2O2-induced MC3T3-E1 cells. In addition, H2O2 upregulated the expression of miR-320-3p and downregulated thatof RUNX2, but these changes were counteracted by E2. Thereafter, we verified the interactive relationshipbetween miR-320-3p and RUNX2. Then, H2O2-induced MC3T3-E1 cells were transfected with miR-320-3pmimics or inhibitors and treated with E2. The results indicated that miR-320-3p inhibition suppressed H2O2-induced inflammation, apoptosis, and oxidative stress and promoted the osteogenic differentiation of MC3T3-E1 cells by regulating RUNX2, ALP, and OCN, and this effect was further strengthened by E2. In conclusion,the findings suggest that E2 alleviates oxidative damage in osteoblasts by regulating the miR-320/RUNX2signaling.

Keywords. 17 b-Estradiol; miR-320/RUNX2 axis; osteoporosis; oxidative stress

1. Introduction

Osteoporosis is a chronic metabolic bone disordercharacterized by loss of bone mass per unit volume thatoften occurs in postmenopausal women and older men(Black and Rosen 2016b; Eastell and Szulc 2017; Wattsand investigators 2014). It significantly reduces theamount of bone, alters bone microstructure, andincreases bone fragility, leading to an increased risk offracture (Cauley 2017; Farr and Khosla 2015).Approximately 50% of postmenopausal women sufferfrom osteoporosis due to decreased estrogen secretion,which results in an imbalance between osteoclastresorption and formation (Bolognese 2010; Sasser et al.2005; Taguchi et al. 2007). Numerous therapeuticmethods of treating osteoporosis are presently availableincluding the use of parathyroid hormone, bisphos-phonates, and calcitonin and hormone replacementtherapy. However, there are side effect that vary in

severity. Parathyroid hormone is associated withosteosarcoma occurrence (Nikitovic et al. 2016), andbisphosphonates treatment has been shown to beassociated with jaw osteonecrosis jaw, gastrointestinalside effects, and subtrochonteric fractures (Black andRosen 2016a; Rosini et al. 2015). This prompts a highdemand of the development of novel ways of managingosteoporosis.Estrogen deficiency is a primary cause of post-

menopausal osteoporosis and estrogen replacementtherapy has been effective in preventing bone lossand decreasing the frequency of osteoporotic frac-tures (Passos-Soares et al. 2017; Yang et al. 2013;Zhang et al. 2020). Thus, there is considerableinterest in uncovering the mechanisms by whichestrogen exerts its protective effects on bone. Estra-diol, a type of estrogen, is an important substance inthe maintenance of bone health by directly targetingosteoblasts, one of the most essential cell types for

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bone formation and sustaining normal bone density(Harada and Rodan 2003; Khosla et al. 2012; Yanget al. 2013). Yang et al. reported that estradiolinhibited osteoblast apoptosis by promoting ER-ERK-mTOR-mediated autophagy (Yang et al. 2013)and Liu et al. demonstrated that 17 b-estradiol (E2)attenuated bone deterioration via the inhibition of theephA2/ephrinA2 pathway (Liu et al. 2018). Inaddition, E2 was shown to suppress oxidative stressand mitigate oxidative stress-mediated neuroinflam-mation, memory impairment, and neurodegenerationin vivo (Khan et al. 2019). Accumulating evidencesuggests that oxidative stress is associated with thepathophysiology of osteoporosis (Geng et al. 2019),but the effect of E2 on oxidative stress in osteo-porosis remains unclear.MicroRNAs (miRNAs) are small endogenous

RNAs that regulate gene-expression post-transcrip-tionally (Lu and Rothenberg 2018). A number ofmiRNAs have been demonstrated to play vital rolesin regulating bone formation, absorption, andosteoporosis. MiR-133 was significantly upregulatedin estrogen deficiency-induced osteoporosis, regu-lating osteogenic differentiation of bone marrowmesenchymal stem cells and the occurrence ofosteoporosis (Lv et al. 2015). MiR-365a-3p sup-pressed osteogenic differentiation and enhancedosteoporosis development by targeting runt-relatedtranscription factors (RUNX2) (Cheng et al. 2019).Kong et al. revealed that miR-320a, a pro-osteo-porotic miRNA induced oxidative stress response inosteoblasts (De-Ugarte et al. 2018), was overex-pressed in individuals experiencing postmenopausalosteoporosis and promoted MC3T3-E1 cell apoptosis(Kong et al. 2019). In addition, the mutual regulatoryrelationship between estradiol and miR-320 has beendemonstrated (Ernst et al. 2016). However, whetherE2 is involved in mediating oxidative stress inosteoporosis by modulating miR-320 is currentlyunknown.Evidence has shown that RUNX2, a factor that

activates osteoblast differentiation, is targeted by miR-320 (Dong et al. 2020). We hypothesized that E2alleviates oxidative damage in osteoblasts by regulat-ing the miR-320/RUNX2 signaling pathway. Herein,an oxidative stress-induced model of cell damage wasestablished by H2O2 in the osteoblastic cell lineMC3T3-E1. H2O2-induced MC3T3-E1 cells weretreated with E2 and/or transfected with miR-320-3pmimics/inhibitors. Oxidative stress and miR-320-3p/RUNX2 signaling activation were examined inMC3T3-E1 cells.

2. Materials and methods

2.1 Cell culture and treatment

MC3T3-E1 cells were purchased from the ShanghaiInstitutes for Biological Sciences (Chinese Academy ofScience) and maintained in Dulbecco’s modified Eaglemedium (Hyclone) supplemented with 10% fetalbovine serum (Gibco) at 37�C with 5% CO2. When thecell confluence reached 80–90%, the cells were cul-tured with 0.3 mM H2O2 for 6 h to construct anoxidative stress model. The levels of superoxide dis-mutase (SOD) and malondialdehyde (MDA) and theproportion of cells with an increase in reactive oxygenspecies (ROS) production were determined.H2O2-induced MC3T3-E1 cells were subsequently

treated with E2 at different concentrations (10-9, 10-8,10-7, and 10-6 M). The cell viability, levels of SODand MDA, proportion of cells with increased ROSproduction, miR-320-3p (also known as miR-320a-3p)expression, and RUNX2 expression were evaluated.The interactive relationship between miR-320-3p andRUNX2 was evaluated using a dual-luciferase activityassay.Furthermore, H2O2-induced MC3T3-E1 cells were

transfected with miR-320-3p mimics (miR-mimic),miR-320-3p inhibitors (miR-inhibitor), or their corre-sponding negative controls (NC), with or without E2treatment. Cell viability, apoptosis, inflammatoryresponse, oxidative stress, and the expression ofosteogenic differentiation-related factors wereevaluated.

2.2 Biochemical assay

After treatment, the levels of SOD and MDA level inMC3T3-E1 cells were detected using correspondingcommercial assay kits (SOD, A001-1; MDA, A003-1),purchased from Nanjing Jiancheng BioengineeringInstitute, according to the manufacturer’s instruction.

2.3 Flow cytometry

Flow cytometry was performed to detect the proportionof cells with increased ROS production and the pro-portion of apoptotic MC3T3-E1 cells. To measure theproportion of cells with increased ROS production,harvested cells were resuspended in diluted DCFH-DA(Bioswamp, 10 lmol/l) at 1 9 107 cells/ml and main-tained at 37�C for 10 min. After three washes with

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serum-free medium, the cells were subjected to flowcytometry (Novo). To measure the proportion ofapoptotic cells, 1 107 harvested cells were resus-pended in 1 ml of phosphate-buffered saline and cen-trifuged at 1000g at 4�C for 5 min (repeated twice).The cells were then resuspended in 200 ll of bindingbuffer and stained with 10 ll of annexin V-fluoresceinisothiocyanate (FITC, BD) and 10 ll of propidium

iodide (PI, BD) in the dark at 4�C for 30 min. There-after, 300 ll of binding buffer was added and the cellswere subjected to flow cytometry (Novo).

2.4 Cell Counting Kit-8 (CCK-8) assay

MC3T3-E1 cells were seeded into a 96-well plate at5 9 103 cells/well (180 ll per well) and incubatedovernight. After E2 treatment and/or transfection, thecells were cultured with 10 ll of CCK-8 solution at37�C with 5% CO2 for 4 h. The absorbance of the platewas detected using an AMR-100 apparatus (Allsheng)at 450 nm.

2.5 Quantitative reverse transcription polymerasechain reaction (qRT-PCR)

Total RNA was extracted from MC3T3-E1 cells usingTrizol (Ambion), reverse-transcribed into cDNA using acorresponding commercial kit (Takara), and amplified byPCR using the SYBRGreen PCR kit (KAPABiosystems).The sequences are as follows: miR-320-3p forward, 50-GGGTGCTGGATCAGTGG-30, reverse, 50-AACTGGTGTCGTGGAGTCGGC-30; U6 (endogenous control of miR-NAs) forward, 50-CTCGCTTCGGCAGCACATATACT-30, reverse, 50- ACGCTTCACGAATTTGCGTGTC-30;alkaline phosphatase (ALP) forward, 50-GGGGCAACTCCATCTTT-30, reverse, 50-TTTTCCCGTTCACCGTC-30; RUNX2 forward, 50-CAACTTCCTGTGCTCCGT-30, reverse, 50-GAAACTCTTGCCTCGTCC-30;osteocalcin (OCN) forward, 50-TTTCTGCTCACTCTGCTG-30, reverse, 50-ACTACCTTATTGCCCTCC-30;GAPDH (endogenous control of mRNAs) forward, 50-CCTTCCGTGTTCCTAC-30, reverse, 50-GACAACCTGGTCCTCA-30. The data were analyzed using the 2-DDCt

method.

2.6 Western blot

Total proteins were extracted from MC3T3-E1 cells.After protein content was quantified, 20 lg of proteinsfrom each sample were separated and transferred ontopolyvinylidene fluoride membranes (Millipore, MA,USA). After blocking with 5% skim milk, the mem-branes were incubated for 1 h with primary antibodiesagainst RUNX2, ALP, OCN, and GAPDH (all fromBioswamp). Thereafter, the membranes were incubatedwith goat anti-rabbit IgG secondary antibodies (Bios-wamp) for 1 h. GAPDH acted as the internal reference.

Figure 1. H2O2 induces oxidative stress response inMC3T3-E1 cells. (A) Biochemical assay was performed todetect the SOD and MDA level in MC3T3-E1 cells.(B) Flow cytometry was performed to detect the percentagecells with increase in ROS in MC3T3-E1 cells. Datarepresents mean ± SD (n = 3), *p\ 0.05.

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2.7 Enzyme-linked immunosorbent assay (ELISA)

The levels of the inflammation-related factors tumornecrosis factor-a (TNF-a) and interleukin-6 (IL-6)were detected by ELISA using commercial kits (TNF-a, MU30030; IL-6, MU30044), purchased from Bios-wamp, according to the manufacturer’s instruction.

2.8 Dual-luciferase activity assay

Wild type (WT) and mutant (MUT) RUNX2 cDNAcomprising the predictive binding sites of miR-320-3pand point mutations of the miR-320-3p seed regionbinding site, respectively, were inserted intopmirGLOvectors. MC3T3-E1 cells were co-transfectedwith miR-320-3p mimics/NC and RUNX2-WT,RUNX2-MUT, or empty vectors for 4 h using Lipo-fectamine� 2000 (Invitrogen). Luciferase activity wasdetected by the dual luciferase reporter assay kit(Genecopoeia) according to the experimental protocol.

2.9 Statistical analysis

Data are presented as the mean ± standard deviation(SD). Statistical differences among data were analyzedusing one-way analysis of variance followed byTukey’s tests. *p \ 0.05 was considered to be statis-tically significant.

3. Results

3.1 H2O2 induces oxidative stress responsein MC3T3-E1 cells

Figure 1 shows that H2O2 decreased the level ofSOD while increasing that of MDA level in

MC3T3-E1 cells. In addition, H2O2 increased theproportion of MC3T3-E1 cells with enhanced ROSproduction. The results confirmed the successfulconstruction of the oxidation stress model inMC3T3-E1 cells.

3.2 E2 suppresses H2O2-induced oxidative stressand regulates miR-320-3p and RUNX2 expressionin MC3T3-E1 cells

H2O2-induced MC3T3-E1 cells were treated with E2at various concentrations. CCK-8 assay indicatedthat E2 promoted the proliferation of H2O2-inducedMC3T3-E1 cells in a concentration- and time-de-pendent manner (figure 2A). Since the cell viabilityremained static beyond 48 h of treatment, 48 h wasselected as the time point for subsequent experi-ments. Further experiments revealed that E2 sup-pressed oxidative stress in H2O2-induced MC3T3-E1cells in a concentration-dependent manner, asdemonstrated by the increase in SOD level, thedecrease in MDA level, and the decrease in theproportion of cells enhanced ROS production(figure 2B–D). We observed that E2 exerted the besteffect at 10-8 M and thus, this concentration waschosen for subsequent experiments. Next, the effectof E2 on the expression of miR-320-3p (figure 2E)and RUNX2 (figure 2F) was evaluated in MC3T3-E1cells. The resulted indicated that while H2O2

upregulated miR-320-3p and downregulatedRUNX2, these changes were reversed to a certainextent by E2. Furthermore, the binding relationshipbetween miR-320-3p and RUNX2 was verified usinga dual-luciferase activity assay (figure 2G).

3.3 E2 accentuates suppressive effect of miR-320-3p inhibition on H2O2-induced inflammation,apoptosis, and oxidative stress in MC3T3-E1 cells

We then assessed the effect of miR-320-3p and E2 onH2O2-induced MC3T3-E1 cells. The results showedthat miR-320-3p mimics increased miR-320-3pexpression and the levels of IL-6 and TNF-a butdecreased the viability of H2O2-induced MC3T3-E1cells, and these changes were reversed by E2 (fig-ure 3). Flow cytometry indicated that miR-320-3pmimics promoted the apoptosis of H2O2-inducedMC3T3-E1 cells, which was reversed by E2 admin-istration (figure 4A). In addition, miR-320-3p mim-ics enhanced oxidative stress in H2O2-induced

bFigure 2. E2 suppresses H2O2-induced oxidative stress andregulates miR-320-3p and RUNX2 expression in MC3T3-E1cells. (A) CCK-8was performed to detect viability ofMC3T3-E1 cells. (B) Biochemical assay was performed to detect theSOD andMDA level inMC3T3-E1 cells. (C) Flow cytometrywas performed to detect the percentage cells with increase inROS in MC3T3-E1 cells. (D) Quantitative analysis of (C).(E) qRT-PCR was performed to detect the miR-320-3pexpression in MC3T3-E1 cells. (F) Western blot wasperformed to detect the RUNX2 protein expression.(G) Dual-luciferase activity assay was performed to verifythe binding relationship between miR-320-3p and RUNX2.Data represents mean ± SD (n = 3), *p\ 0.05.

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MC3T3-E1 cells, as indicated by the proportion ofcells with increased ROS production, the increase inMDA level, and the decrease in SOD level(figure 4B–C). The positive effect of miR-320-3pmimics on oxidative stress in H2O2-induced MC3T3-E1 cells was suppressed by E2. MiR-320-3p inhibi-tors showed the opposite effect as that of miR-320-3p mimics.

Figure 3. E2 accentuates suppressive effect of miR-320-3p inhibition on H2O2-induced inflammation in MC3T3-E1 cells.(A) qRT-PCR was performed to detect the miR-320-3p expression in MC3T3-E1 cells. (B) CCK-8 was performed to detectviability of MC3T3-E1 cells. (C) ELISA was performed to detect the IL-6 and TNF-a level in MC3T3-E1 cells. Datarepresents mean ± SD (n = 3), *p\ 0.05.

cFigure 4. E2 accentuates suppressive effect of miR-320-3pinhibition on H2O2-induced apoptosis and oxidative stressin MC3T3-E1 cells. (A) Flow cytometry was performed todetect the percentage apoptotic cells in MC3T3-E1 cells.(B) Flow cytometry was performed to detect the percentagecells with increase in ROS in MC3T3-E1 cells. (C) Bio-chemical assay was performed to detect the SOD and MDAlevel in MC3T3-E1 cells. Data represents mean ± SD(n = 3), *p\ 0.05.

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3.4 E2 accentuates the effect of miR-320-3pinhibition in promoting osteogenic differentiationof MC3T3-E1 cells

Western blot and qRT-PCR were performed to detectthe expression of osteogenic differentiation-relatedfactors. The results demonstrated that miR-320-3pinhibition upregulated the mRNA (figure 5A) andprotein (figure 5B) expression of ALP, RUNX2, andOCN, and this effect was accentuated by E2. MiR-320-3p overexpression showed the opposite effect as that ofmiR-320-3p inhibition.

4. Discussion

The current work demonstrates that E2 attenuatesoxidative stress in H2O2-induced MC3T3-E1 cells. Inaddition, H2O2 increased the expression of miR-320-3pand decreased that of RUNX2 in MC3T3-E1 cells, butthese changes were counteracted by E2. MiR-320-3pinhibition suppressed H2O2-induced inflammation,apoptosis, and oxidative stress in MC3T3-E1 cells andpromoted their osteogenic differentiation by upregu-lating RUNX2, and this effect was further strengthenedby E2.

Figure 5. E2 accentuates the effect of miR-320-3p inhibition in promoting osteogenic differentiation of MC3T3-E1 cells.(A) qRT-PCR was performed to detect the ALP, RUNX2, and OCN mRNA expression in MC3T3-E1 cells. (B) Western blotwas performed to detect the ALP, RUNX2, and OCN protein expression in MC3T3-E1 cells. Data represents mean ± SD(n = 3), *p\ 0.05 vs. MOD group, #p\ 0.05 vs. miR-mimic group, %p\ 0.05 vs. miR-inhibitor group.

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Oxidative stress is characterized by excessive ROSproduction or impaired ROS, leading to increased ROSlevels in tissues or cells. It is closely related to bio-logical phenomena such as aging, tumor progression,and diabetes (Luca et al. 2019; Poprac et al. 2017;Zhang et al. 2015a) and could lead to cellular dys-function by activating apoptotic pathways and inflam-matory response (Sinha et al. 2013; Tian et al. 2017).In bone tissues, ROS levels are increased during thepathogenesis of postmenopausal, senile, and gluco-corticoid osteoporosis, suggesting that oxidative stressplays a crucial role in the pathological process of var-ious types of osteoporosis (Cervellati et al. 2013;Manolagas 2010). Oxidative stress-induced osteoblas-tic apoptosis and inflammatory response are maincontributing factors in the pathogenesis of osteoporosis(Li et al. 2017; Zhang et al. 2015b). In turn, these twoprocesses are closely linked by triggering each other toestablish a vicious cycle wherein inflammation isaggravated (Lugrin et al. 2014). A previous study hasshown that E2 suppressed oxidative stress andinflammation in Raw 264.7 cells, consistent with ourfindings demonstrating that E2 attenuated oxidativestress and inflammation in MC3T3-E1 cells. In addi-tion, we suggest that the effect of E2 on oxidative stressand inflammation in MC3T3-E1 cells is mediated bymiR-320, a miRNA associated with oxidative stress(Ke et al. 2019). We thus speculate that the effect of E2on inflammatory response in MC3T3-E1 cells ismediated by its regulatory effect on oxidative stress,which needs to be further investigated.The current study also showed that E2 enhanced the

expression of RUNX2, a target of miR-320, in H2O2-induced MC3T3-E1 cells. RUNX2 activation promotedosteoblastic differentiation, during which ALP and typeI collagen are produced in the extracellular matrix.Phosphorus mineralization and calcium deposition thusoccur in the extracellular matrix to promote the for-mation of the new bone (Arora et al. 2020), therebyalleviating osteoporosis.

5. Conclusion

Overall, our findings confirmed that hypothesis that E2alleviates oxidative damage in osteoblasts throughoxidative stress inhibition by regulating the miR-320/RUNX2 axis. This study provides a theoretical basisfor the use of E2 and reveals a potential target (miR-320) in osteoporosis therapy. In vivo experiments willbe designed in follow-up studies to supplement ourcurrent conclusions.

Acknowledgements

This study was financially supported by ScientificResearch Project of Health Commission of HubeiProvince (No. WJ2019F016).

Funding

Funding was provided by Scientific Research Project ofHealth Commission of Hubei Province (Grant No.WJ2019F016).

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Corresponding editor: RITA MULHERKAR

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