decreased microrna-182-5p helps alendronate promote ... · decreased microrna-182-5p helps...

Bioscience Reports (2018) 38 BSR20180696https://doi.org/10.1042/BSR20180696

* These authors are regardedas co-first authors.

Received: 04 May 2018Revised: 16 October 2018Accepted: 06 November 2018

Accepted Manuscript Online:09 November 2018Version of Record published:21 December 2018

Research Article

Decreased microRNA-182-5p helps alendronatepromote osteoblast proliferation and differentiationin osteoporosis via the Rap1/MAPK pathwayBao-Long Pan1,*, Zong-Wu Tong2,*, Shu-De Li3, Ling Wu4, Jun-Long Liao5, Yu-Xi Yang1, Hu-Huan Li1,Yan-Juan Dai1, Jun-E Li1 and Li Pan1

1Department of Laboratory, People’s Hospital of Yuxi City, Yuxi 653100, P.R. China; 2Department of Nephrology, People’s Hospital of Yuxi City, Yuxi 653100, P.R. China; 3Departmentof Biochemistry and Molecular Biology, School of Basic Medical Science, Kunming Medical University, Kunming 650500, P.R. China; 4Department of Quality Management, CentralBlood Station of Yuxi City, Yuxi 653100, P.R. China; 5Department of Rehabilitation Medicine, People’s Hospital of Yuxi City, Yuxi 653100, P.R. China

Correspondence: Ling Wu ([email protected]) or Li Pan ([email protected])

Osteoporosis (OP) is a serious health problem that contributes to osteoporotic structuraldamage and bone fragility. MicroRNAs (miRNAs) can exert important functions over boneendocrinology. Therefore, it is of substantial significance to clarify the expression andfunction of miRNAs in bone endocrine physiology and pathology to improve the poten-tial therapeutic value for metabolism-related bone diseases. We explored the effect ofmicroRNA-182-5p (miR-182-5p) on osteoblast proliferation and differentiation in OP ratsafter alendronate (ALN) treatment by targeting adenylyl cyclase isoform 6 (ADCY6) throughthe Rap1/mitogen-activated protein kinase (MAPK) signaling pathway. Rat models of OPwere established to observe the effect of ALN on OP, and the expression of miR-182-5p,ADCY6 and the Rap1/MAPK signaling pathway-related genes was determined. To deter-mine the roles of miR-182-5p and ADCY6 in OP after ALN treatment, the relationship be-tween miR-182 and ADCY6 was initially verified. Osteoblasts were subsequently extractedand transfected with a miR-182-5p inhibitor, miR-182-5p mimic, si-ADCY6 and the MAPKsignaling pathway inhibitor U0126. Cell proliferation, apoptosis and differentiation werealso determined. ALN treatment was able to ease the symptoms of OP. miR-182-5p nega-tively targeted ADCY6 to inhibit the Rap1/MAPK signaling pathway. Cells transfected withmiR-182 inhibitor decreased the expression of ALP, BGP and COL I, which indicated that thedown-regulation of miR-182-5p promoted cell differentiation and cell proliferation and inhib-ited cell apoptosis. In conclusion, the present study shows that down-regulated miR-182-5ppromotes the proliferation and differentiation of osteoblasts in OP rats through Rap1/MAPKsignaling pathway activation by up-regulating ADCY6, which may represent a novel targetfor OP treatment.

IntroductionAs a chronic systemic bone disease, osteoporosis (OP) manifests as less bone mass and a disorder of bonestructure, which eventually contributes to the risk of fracture [1,2]. Changes in osteoblast or osteoclastproduction or the life span contribute to an imbalance between bone formation and resorption, whicheventually results in OP [3]. According to statistics, fractures caused by OP will occur in individuals over50 years old, among whom 50% are female and 20% are male; moreover, hip fracture is the most devastat-ing because of the high rates of disability and mortality and the expensive costs, both to individuals andsociety as a whole [4]. By inhibiting phosphatase, alendronate (ALN) treatment is an effective anti-boneabsorbent, which can promote the apoptosis of osteoclasts under the condition of extremely high doses

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[5]. China has approved a special formulation of ALN 70 mg/vitamin D3 5600 IU (ALN/D5600) considering thatpatients with OP can cure their disease and absorb sufficient vitamin D at the same time [6]. Although ALN is highlyrecommended in international treatment, the accompanying side effects cannot be ignored, for example, weight loss,sciatica, rash and asthma [7]. Moreover, in the differentiation of bone cells, microRNAs (miRNAs) serve irreplaceablefunctions, regulating the lineage commitment and cell progression of mesenchymal stem cells [8].

As a member of the miR-183–182 cluster that encodes miR-96, miR-182 and miR-183, MiR-182 plays an impor-tant role in regulating the apoptosis, growth and differentiation of cells [9,10]. Adenylyl cyclase isoform 6 (ADCY6)is one of the colon membrane-bound isoforms of adenylyl cyclase, and it has been shown to participate in elevatedblood pressure and the cardiovascular response [11]. Rap1, a small GTP-ase, dynamically controls the coordinationof cell spreading and endothelial barrier function by regulating cell–cell adhesion, cell–matrix adhesion and actinrearrangement [12]. Based on the activation of Rap1, the excitability of neuronal and cocaine-induced behavioral re-ward responses can be induced through the mitogen-activated protein kinase (MAPK)/extracellular signal-regulatedkinase (ERK) pathway [13]. The MAPK/ERK signaling code also functions as a more common original cancer signalwith the exception of a tumor suppressor according to recent evidence [14]. However, research regarding the connec-tion between miR-182-5p and the Rap1/MAPK signal pathway and its controlling function in OP remains limited.In the present study, we will explore the mechanism of the miR-182-5p, ADCY6 and Rap1/MAPK signaling pathwayin osteoblast proliferation and differentiation in OP rats after ALN treatment. We aim to provide a new basis for thedevelopment of targeted therapy for OP.

MethodsEthics statementsThe present study was carried out in strict accordance with the recommendations in the Guide for the Care and Useof Laboratory Animals of the National Institutes of Health. The protocol was approved by the Institutional AnimalCare and Use Committee of People’s Hospital of Yuxi City.

Model establishment and specimen collectionRats were fed adaptively for 1 week during which there was no death. Thirty rats were randomly divided into sham(with no treatment) group, operation group (OP group) and ovariectomy + ALN sodium treatment (ALN group) with10 rats per group (ALN sodium was purchased from Yangtze River Pharmaceutical Group, Taizhou, Jiangsu, China).The OP model was established in the OP group and the ALN group via bilateral ovariectomies [15]. Fasting for 12 hbefore surgery, 3% pentobarbital sodium (1 ml/kg) was injected into the abdominal cavity of the rats for anesthesia.The abdominal region was fixed to a surgical board, and a dorsal approach was used under sterile conditions. Alongitudinal incision was subsequently made (1 cm) in the central back to separate the skin from both sides. Thedorsal muscles were incised on either side of the spine (incision should be as small as possible) with the aim to separatethe cellulite. The stump was ligated by a surgical line after the exposed pink ovaries were removed. After releasing thehemostatic forceps and gently placing the uterus into the abdominal cavity, the muscle layer and skin were sutured,respectively. The incision was subsequently cleaned using an alcohol cotton ball, the other ovary was removed andthe wound was sutured as previously described. The ovaries of the rats in the sham group were only exposed and werenot removed. Only a small amount of surrounding adipose tissues was removed and sutured. A gentamycin sulfateinjection was continuously administered into the muscle to resist infection 3 d after the operation. One week after themodel was successfully established, the ALN sodium was crushed at a dosage of 0.1 mg kg−1 d−1 [16] and preparedin a suspension with distilled water. The rats in the ALN group were subsequently fed the suspension. The rats in thesham group and the OP group were administered normal saline for 4 weeks continuously.

After 4 weeks of drug treatment, the rats in each group were fasted for 12 h with water deprivation for 6 h. Theabdominal cavities were injected with 3% pentobarbital sodium (1 ml/kg). Blood was collected from the femoralartery and centrifuged at 2863 × g for 10 min to separate the serum. The femurs were separated, and the left femurswere rapidly removed. After being washed with normal saline, the left femurs were stored in liquid nitrogen, and theright femurs were fixed with 10% formalin.

Determination of blood phosphorus, serum calcium and bone mineraldensityThe fresh left femur after isolation was arranged on the test board and scanned by a dual energy X-ray bone densit-ometer (HOLIGIC QDR-4500w, Waltham, MA, USA). Software was employed to analyze the bone mineral density(BMD) of the whole femur using the small animal model during the scanning. Rat serum was extracted, and the

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calcium and phosphorus levels were determined by an automatic biochemical analyzer (TBA-30, Toshiba, Otawara,Japan), in which the serum calcium was tested by the alkaline picrate method, and the blood phosphorus were verifiedvia direct ultraviolet spectrophotometry.

Hematoxylin–eosin (HE) staining and immunohistochemistryFemur tissues fixed with 10% formaldehyde were decalcified in 10% nitric acid solution and then washed overnight,embedded in paraffin wax and cut into 4 μm sections. The sections were subsequently placed in the oven at 65 ◦Cfor 6 h. The sections were conventional dewaxed, washed for 1–2 min, stained with hematoxylin for 3–6 min andthen washed under running water for 1–2 min. The sections were combined with 1% hydrochloric acid ethanol for1–3 s, slightly washed for 1–2 s, returned to blue with ammonia for 5–10 s, washed for 15–30 s and stained by 0.5%eosin solution for 2–3 min. The sections were washed with distilled water again for 1–2 s, dehydrated sequentially in80%, 95% and 100% ethanol for 15–30 s, 15–30 s and 1–2 s, respectively, cleared with xylenes (xylene I/II) for 2–3 seach and then sealed by neutral gum. The HE staining of the femoral tissues in each group was observed under anoptical microscope (B561T-PH2-J11, Olympus Optical Co., Ltd., Tokyo, Japan), and the microphotograph (×400)was performed. The HE staining results were quantified with ImageJ Software. The paraffin sections of the femurtissues of the rats were prepared (steps were performed as previously described), blocked with 5–10% normal goatserum [diluted with phosphate buffered saline (PBS)] and incubated at room temperature for 10 min. The serumwas discarded without washing. The sections were combined with a proper proportion of diluted primary, rabbitantibody to CD34 antibody (1:200, ab81289) and rabbit antibody to receptor activator of nuclear factor-kappa B(RANK, 1:200, ab216484) and incubated at 37◦C for 12 h or 4◦C overnight. The antibodies were purchased fromAbcam Inc. (Cambridge, MA, USA). Following three washes in PBS for 5 min each, the sections were stained withdiaminobenzidine (DAB). The sections were then fully washed with tap water, counterstained and sealed.

TdT-mediated dUTP-biotin nick end-labeling assayThe apoptosis of osteoblasts was observed by staining with the TdT-mediated dUTP-biotin nick end-labeling(TUNEL) kit (C1086, Beyotime Biotechnology Co., Ltd., Shanghai, China). Ten percent formaldehyde fixed femoraltissues were embedded in paraffin, cut into sections and dewaxed with conventional paraffin. The sections weresubsequently rehydrated with gradient alcohol and soaked in 3% H2O2 solution at room temperature for 10 min.After being washed with PBS for 5 min, the sections were combined with 50 μl of 20 μg/ml proteinase K (P6556,Sigma–Aldrich Chemical Company, St Louis, MO, USA) solution and hydrolyzed at room temperature for 20 minto remove tissue protein. The sections were washed three times with PBS (5 min each time) and combined with cit-rate buffer for antigen retrieval for 30 min. The sections were subsequently washed two times with PBS (5 min eachtime). TdT enzyme reaction solution (50 μl) was added to the sections and placed in a wet box at 37◦C for 1 h underconditions void of light. The TdT enzyme-free reaction solution was used as the negative staining control. After threewashes with PBS (5 min each time), the sections were combined with 50 μl of peroxidase-labeled anti-digoxigeninand placed in a wet box at 37◦C for 30 min under conditions void of light. After three washes with PBS (5 min eachtime), the sections were combined with 4′,6-diamidino-2-phenylindole (DAPI) solution (C1002, Beyotime Biotech-nology Co., Shanghai, China) to develop at room temperature for 10 min. After two washes with PBS (5 min eachtime), the sections were mounted with neutral gum, and a fluorescence microscope (BX53, Olympus Optical Co.,Ltd., Tokyo, Japan) was used for observation and photograph. The nuclei of positive apoptotic cells were stained withgreen, and those of the normal cells were stained with blue. Ten views were randomly selected to count the ratio ofthe positive cells to the total number of cells as the apoptosis index (AI).

Reverse transcription quantitative polymerase chain reaction (RT-qPCR)The left femurs were grinded into homogenate. Total RNA from the femur tissues was extracted according to theinstructions of the Trizol kit (15596-026, Invitrogen, Gaithersburg, MD, USA). A nucleic acid and protein analyzer(BioPhotometer D30, Eppendorf, Hamburg, Germany) was used to measure the RNA purity. RNA was reverselytranscripted into cDNA using the reverse transcription kit (K1621, Fermentas, Maryland, N.Y., USA). The primersequences of miR-182-5p, ADCY6, extracellular signal-regulated kinase 1/2 (ERK1/2), P38 MAPK, proliferating cellnuclear antigen (PCNA), Ras-related protein 1 (Rap1), bone morphogenetic protein-2 (BMP2), bone gla protein(BGP), alkaline phosphatase (ALP), type I collagen (COL IA1), U6 andβ-actin (Table 1) were synthesized by ShanghaiGenechem Co., Ltd. (Shanghai, China). A fluorescent quantitative PCR Kit (Takara, Dalian, Liaoning, China) wasused to determine the mRNA levels of the genes, and the reaction system was: 5.3 μl of 2 × Taq Master Mix, 1μl of forward primer (5 μM), 1 μl of reverse primer (5 μM), 1 μl of cDNA and 11.7 μl of RNase Free H2O. The


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Table 1 The primer sequences for RT-qPCR

Gene Sequence

miR-182-5p F: 5′-GGGTCTAGCTGCCGGAGG-3′

R: 5′-CGGTGTGAGTTCTACCATTGC-3′

U6 F: 5′-GCTCGCTTCGGCAGCACA-3′

R: 5′-GAGGTATTCGCACCAGAGGA-3′

ADCY6 F: 5′-CAGCAGGGTAGTGTGTGCAG-3′

R: 5′-TCTGCATTTGATTTTGGCCT-3′

ERK1/2 F: 5′-CTCAAGCCTTCCAACCTC-3′

R: 5′-TTCCACGGCACCTTATTT-3′

P38 MAPK F: 5′-GCATAAAGCCCAAGAGAACCAAC-3′

R: 5′-GACAGGACAAGCTCCAGAGCACA-3′

Rap1 F: 5′-GCGTCTGGTCA GAGAGCTG-3′

R: 5′- TCAATCCTCCGAGCTACATTCT-3′

β-actin F: 5′-GGCACCACACTT TCTACAAT-3′

R: 5′-AGGTCTCAAACATGATCTGG-3′

PCNA F: 5′-CGGCGTGAACCTACAGA-3′

R: 5′-TCGCAGCGGTATGTGTCGAA-3′

BMP2 F: 5′-ACGATGCCGCCATTTGTG-3′

R: 5′-CGCCTCGCCTTCTTCAGT-3′

BGP F: 5′-AGCGACTCTGAGTCTGACAAA-3′

R: 5′-AACGGTGGTGCCATAGATGCG-3′

ALP F: 5′-CAGTGGTATTGTAGGTGCTGTG-3′

R: 5′-TTTCTGCTTGAGGTTGAGGTTAC-3′

COL1A1 F: 5′-GTCCTATGGCTATGATGAGAAATC-3′

R: 5′-CACCATCCAAACCACTGAAAC-3′

Abbreviations: ADCY6, adenylate cyclase 6; ALP, alkaline phosphatase; BGP, bone gla protein; BMP2, bone morphogenetic protein-2; COL I, typeI collagen; ERK1/2, extracellular signal-regulated kinase 1/2; F, forward; miR-182-5p, microRNA-182-5p; PCNA, proliferating cell nuclear antigen; R,reverse; Rap1, Ras-related protein 1; RT-qPCR, quantitative real-time polymerase chain reaction.

reaction conditions were as follows: predenaturation at 95◦C for 5 min, a total of 40 cycles for the denaturation at94◦C for 45 s, anneal at 56◦C for 45 s and extension at 72◦C for 45 s. A real-time fluorescence quantitative PCRinstrument (ABI 7500, ABI, Foster City, CA, USA) was applied for measurement. U6 served as the internal controlof miR-182-5p, and β-actin was the internal control of the other related genes. The relative expression of each targetgene was calculated based on the 2−��Ct method, and each experiment was repeated three times. The expression ofmRNA: ��Ct [Ct (target gene of the experimental group) − Ct (internal control of the experimental group)] − [Ct (target gene of the NC group) −Ct (internal control of the NC group)], the relative transcription level of mRNA = 2−�Ct.

Western blot analysisLeft femur tissue samples (50 mg) were cut into pieces, combined with protein lysate (R0010, Beijing Solabio LifeSciences Co., Ltd., Beijing, China), lysed and centrifuged, and the supernatant was collected to determine the pro-tein concentration. Total protein was separated via 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis(P1200, Beijing Solabio Life Sciences Co., Ltd., Beijing, China), transferred to a polyvinylidene fluoride (PVDF) mem-brane (HVLP04700, Millipore, Bedford, MA, USA) using the semidry transfer method, and stained with Ponceau S(P0012, Beijing Solabio Life Sciences Co., Ltd., Beijing, China) to observe protein transfer. The membrane was sub-sequently washed two times with Tris-buffered saline with Tween 20 (TBST), blocked at room temperature for 2 h in5% skimmed milk powder and washed three times with TBST. The membrane was then incubated with the follow-ing primary antibodies overnight at 4◦C: anti-rabbit ADCY6 (1:500, ab14718), anti-rabbit ERK1/2 (1:1000, ab17942),anti-rabbit P38 MAPK (1:1000, ab197348), anti-rabbit Rap1 (1:1000, ab92836), anti-rabbit PCNA (1μg/ml, ab18197),anti-rabbit BMP2 (1:500, ab14933), anti-rabbit BGP (1:1000, ab93876), anti-rabbit ALP (1:500, ab83259), anti-rabbitCOL I (1:1000, ab34710) and anti-rabbit β-actin (1:1000, ab8227) (Abcam, Cambridge, MA, UK), as well as theanti-rabbit p-ERK and anti-rabbit p-P38 MAPK (1:2000, 4370; 1:1000, 4511, Cell Signaling Technology, Beverly, MA,USA). After being rinsed with TBST three times (10 min per time), the membrane was incubated with horseradishperoxidase (HRP)-labeled secondary goat anti-rabbit IgG (1:2000, ab6721, Abcam, Cambridge, MA, UK) at roomtemperature for 2 h and then washed three times (10 min each time) with TBST. DAB reaction solution was used to


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develop the color. A gel imager (Gel Doc XR, Bio-Rad, Hercules, CA, USA) was used for photograph, and the ratiosof the gray values of the target bands to the internal reference bands serve as the relative protein expression levels.This method was also suitable for the determination of the protein levels in the cell experiment.

Osteoblast isolation, culture and identificationLeft femur tissues of the OP group were obtained. The blood vessels and connective tissues were cleaned, placed in 1ml of PBS, cut into 1 mm3 tissue blocks, washed two times with PBS and centrifuged. With the PBS aspirated, cellswere combined with 5 ml of 0.25% trypsin solution for 20 min in a 37◦C water bath, followed by the addition of5 ml of 0.1% collagen II. Cells were then detached in a 37◦C water bath for 60 min. M199 culture medium (3 ml,31100035, Gibco, Carlsbad, CA, USA) that contained 10% fetal bovine serum (FBS, 16000-044, Gibco, Carlsbad, CA,USA) was added to the cells. Following centrifugation for 10 min at 179 × g , the supernatant was removed. Thecells were subsequently combined with 3 ml of M199 culture medium that contained 10% FBS for cell resuspensionand incubated in a 5% CO2 incubator (ZXKR-1150, Shanghai Zhicheng Analytical Instrument Manufacturing Co.,Ltd., Shanghai, China) at 37◦C. After 24 h, the medium was changed for the first time; the medium was subsequentlychanged every 2–3 d. The third generation of cells in the logarithmic growth phase was used in this experiment, andthe cell morphology was observed under an inverted microscope (IX53, Olympus Optical Co., Ltd., Tokyo, Japan).

ALP stainingAfter the third generation of cell detachment and culture, 5-bromo-4-chloro-indolyl-phosphatase (BCIP) and the

ALP assay kit (Nanjing Jiancheng Biology Company, Nanjing, Jiangsu, China) were used for staining. After being fixedwith formalin, the cell samples were washed three times with PBS solution (3 min each time). The proper amountsof BCIP and BI staining solution (which contained 3 ml of ALP staining buffer, 10 ml of BCIP solution and 20 mlof nitroblue tetrazolium) were added to fully cover the sample. After incubating in conditions void of light at roomtemperature for 2 h, BCIP was removed and BI staining solution was added. Next, 1–2 times distilled water washeswere conducted to terminate the color development, and the sample was observed under a fluorescence microscope(BX53, Olympus Optical Co., Ltd., Tokyo, Japan).

Calcium nodules staining (alizarin red staining)The third generation of cells were cultured for two weeks and then mineralized to form opaque calcified nodules. Thecell samples were washed 1–2 times with PBS, fixed with 95% ethanol for 10 min, washed 1–2 times with PBS again,covered and stained with 0.1% alizarin red solution for 10 min, rinsed with PBS and observed under an invertedmicroscope.

Cell transfection and groupingThe third generation of femoral osteoblasts extracted from the OP group were allocated into eight groups: blank(no transfected plasmid), negative control (NC, transfected with NC plasmid), miR-182-5p mimic (transfected withmiR-182-5p mimic plasmid), miR-182-5p inhibitor (transfected with miR-182-5p inhibitor plasmid), U0126 group(treated with U0126), miR-182-5p inhibitor + U0126 group (transfected with miR-182-5p inhibitor plasmid andtreated with U0126), siRNA-ADCY6 (transfected with ADCY6 interference plasmid) and miR-182-5p inhibitor +si-ADCY6 (cotransfected with miR-182-5p and ADCY6 interference plasmid). After being detached with 0.25%trypsin, cells were resuspended with M199 culture medium that contained 10% FBS to adjust the density to 1 × 105

cells/ml and seeded in a 6-well plate. When the confluence of the cells reached 70%, the culture medium was replacedwith serum-free M199 culture medium, and cells were transfected after 24 h of culture. Lipofectamine 2000 (6 μl,11668-019, Invitrogen Inc., Carlsbad, CA, USA) was diluted in 200 μl of serum-free Opti-MEM medium (31985070,Gibco, Carlsbad, CA, USA), and 2 μg of target plasmid were diluted in 100 μl of serum-free Opti-MEM medium,mixed respectively and cultured at room temperature for 10 min. These two solutions were mixed together and in-cubated for 20 min at room temperature. The culture medium in the six-well plate was aspirated, and 1.7 ml ofserum-free M199 culture medium was added to each well; the transfected compound was added to the correspond-ing wells. The final concentration of U0126 treated cells was 10 μM [17]. After being incubated for 18 h in a 5% CO2incubator at 37◦C, fresh complete medium was replaced, and cells were collected after transfection for 48 h.

Dual-luciferase reporter gene assayAccording to the online prediction website microRNA.org, the target gene of miR-182-5p was predicted. WhetherADCY6 was a direct target of miR-182-5p was identified via a dual-luciferase reporter gene assay. Endonuclease


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sites (SpeI and HindIII) were introduced into the pMIR-reporter to design complementary mutation sites of seedsequences on the wild type (WT) ADCY6. The target fragment was inserted into the pMIR-reporter plasmid using T4DNA ligase after restriction endonuclease digestion. Correctly sequenced luciferase-reporter plasmid WT or mutated(MUT) were cotransfected into HEK-293T cells (CRL-1415, Shanghai Xin Yu Biotech Co., Ltd, Shanghai, China) withmiR-182-5p, respectively. After transfection for 48 h, the culture medium was discarded and washed two times withPBS; the cells were then collected and lysed. The activity of luciferase was tested using the luciferase reporter genedetection system (Dual-Luciferase R© Reporter Assay System, E1910, Promega Corporation, Madison, WI, USA). Theactivity of firefly luciferase was measured by the addition of 50 μl of firefly luciferase solution into every 10 μl ofsample. Then, 50 μl of Renilla luciferase solution were added to evaluate the activity of Renilla luciferase; the ratio ofthe firefly luciferase activity to the Renilla luciferase activity was the relative luciferase activity. The experiment wasrepeated three times.

3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assayEach transfected group of osteoblasts in the logarithmic growth period was inoculated into 96-well plates at a densityof 1 × 104 cells/well, with eight replicates in each group. There was a blank well that contained only medium withoutcells. When the cell confluence reached 70%, 10 μl of 5 mg/ml 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT) reagent (ST316, Shanghai Beyotime Biotechnology Co., Ltd., Shanghai, China) were added to eachwell, and the cells were cultured in a 5% CO2 incubator at 37◦C for 4 h. With the supernatant aspirated, the 96-wellplate was washed one time with PBS, and 100 μl of dimethyl sulfoxide (DMSO, D5879, Sigma–Aldrich ChemicalCompany, St Louis, MO, USA) were added to each well. The 96-well plate was subsequently vibrated at a low speedon the shaking table for 10 min. The optical density (OD) at 490 nm was measured using a microplate reader (MK3,Thermo, Pittsburgh, PA, USA) for each well. Cell survival rate = (OD value of the experiment well − OD value ofthe blank well)/OD value of blank well. The experiment was conducted three times to obtain the mean value.

Detection of ALP and osteocalcin secretionThe P-nitrophenyl phosphate (PNPP) method was used to determine the ALP content: third generation osteoblastsin the ALN group were seeded in a 12-well plate with a density of 1 × 105 cells/ml and combined with 250 μl of 0.05%TritnoX-100 for cell lysis. Cell lysates (50 μl) were transferred to a 96-well plate, combined with 50 μl of 4.5 mmol/lPNPP (Am- resco, Solon, OH, USA) and water-bathed at 37◦C for approximately 30 min. Finally, 50 μl of 0.1 mol/lsodium hydroxide were added to terminate the reaction. The absorbance (A) value at 405 nm was measured using amicroplate reader (Bio-Rad, Inc., Hercules, CA, USA). Cell lysates (20 μl) were extracted in each well and combinedwith 180 μl of Coomassie staining solution (Sigma–Aldrich Chemical Company, St Louis, MO, USA). The value at595 nm was measured using a microplate reader. Total protein (mg/l) was obtained according to the standard curveof protein.

Osteocalcin secretion measurement: third generation osteoblasts from the ALN group were seeded in a 48-wellplate with a density of 2 × 104 cells/ml (0.5 ml each well). After 24 h, the cells were cultured in Dulbecco’s Mod-ified Eagle’s Medium (DMEM, Gibco, Carlsbad, CA, USA) that contained 10% calf serum (0.45 ml each well) andincubated in a 5% CO2 incubator (ZXKR-1150, Shanghai Zhicheng Analytical Instrument Manufacturing Co., Ltd.,Shanghai, China) at 37◦C. When the cell confluence reached 80%, the serum-containing medium was replaced withserum-free bovine serum albumin (BSA) culture medium (Gibco, Carlsbad, CA, USA). After 3 d of culture, the cellculture medium was collected. The osteocalcin secretion was obtained according to the instructions of the osteocalcinradioimmunoassay kit (Beijing East Asia Immunological Technology Institute, Beijing, China).

Flow cytometryWith transfection for 48 h, the culture medium was discarded, and the cells were washed one time with PBS. The cellswere treated with 0.25% trypsin. Cells were collected and centrifuged at 179 × g for 5 min at 4◦C. The supernatantwas removed, and the cells were washed two times using PBS and centrifuged at 179 × g for 5 min. The supernatantwas discarded, and the cells were fixed with precooled 70% ethanol at 4◦C overnight. The cells were rinsed with PBS,centrifuged again at 179 × g for 5 min and incubated with 10 μl of RNase enzyme at 37◦C for 5 min. Followingthe addition of 1% propidium iodide (PI) staining buffer (40710ES03, Shanghai QCbio Science & Technologies Co.,Ltd., Shanghai, China) under conditions void of light for 30 min, the samples were analyzed using a flow cytometer(Becton, Dickinson and Company, Franklin Lakes, NJ, USA) to detect the cell cycle by red fluorescence at an excitationwavelength of 488 nm. The experiment was repeated three times.


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Table 2 ALN significantly increased serum calcium, decreased serum phosphorus and enhanced BMD in OP

Group Serum calcium (mmol/l) Serum phosphate (mmol/l) Bone mineral density (g/cm3)

Sham 2.47 +− 0.15 2.75 +− 0.26 1.53 +− 0.07

OP 2.54 +− 0.18 2.36 +− 0.25* 1.03 +− 0.06*

ALN 2.52 +− 0.20 2.44 +− 0.31* 1.31 +− 0.05*#

Abbreviations: ALN, alendronate; OP, osteoporosis. *, P < 0.05 vs. the sham group; #, P < 0.05 vs. the OP group.

After transfection for 48 h, cells were detached with ethylenediaminetetraacetic acid (EDTA)-free trypsin and col-lected in a flow tube, followed by centrifugation at 4◦C, 179 × g for 5 min. The supernatant was discarded, and the cellswere washed with precooled PBS and centrifuged again at the speed of 179 × g for 5 min. An Annexin-V-fluoresceinisothiocyanate (FITC)/PI apoptosis detection kit (CA1020, Beijing Solabio Life Sciences Co., Ltd, Beijing, China) wasused to detect the apoptosis of cells after the supernatant was discarded. Binding buffer was used to wash cells. Thecells were resuspended in a mixed solution (Annexin-V-FITC and binding buffer at a ratio of 1:40), incubated at roomtemperature for 30 min, and combined with mixed solution (PI and Binding buffer at a ratio of 1:40). The cells wereincubated at room temperature for 15 min. The flow cytometer was used to detect the cell apoptosis. The lower leftquadrant showed living cells as Annexin V−/PI−, and the right upper quadrant was the advanced apoptotic cell orthe secondary necrosis cell as Annexin V+/PI+, while the right lower quadrant was the early apoptotic cell as AnnexinV+/PI−. The experiment was repeated three times.

Statistical analysisStatistical analyses were conducted using SPSS 21.0 statistical software (IBM Corp., Armonk, N.Y., USA). Measure-ment data are expressed as the mean +− standard deviation. Multiple groups were compared by one-way analysis ofvariance. P < 0.05 indicated that the difference was statistically significant.

ResultsALN significantly enhances BMD and serum calcium while decreasingserum phosphorus in OPAfter 4 weeks of the drug intervention, there was no death of rats. The content of the serum calcium, blood phosphorusand BMD were determined. There were significant differences in the blood phosphorus and BMD between the OPgroup and the sham group. Compared with the OP group, the ALN group had a significantly increased BMD, showingits therapeutic effect. Compared with the sham group, the OP and ALN groups had increased serum calcium (P >

0.05) and significantly decreased blood phosphorus content and BMD (P < 0.05). Compared with the OP group, theALN group had increased serum calcium and decreased serum phosphorus with no significant difference (P > 0.05),as well as a significantly increased BMD (P < 0.05). ALN treatment could elevate the BMD (Table 2).

ALN treatment alleviates the pathological changes of femoral tissue in OPHE staining was used to observe the pathological changes of femoral tissue in OP. The trabecular trabeculae werethick and well-arranged into mesh, with a small gap, deep coloring, complete shape and structure, density of bonetrabeculae, normal area and a relatively small medullary cavity in the sham group. In the OP group, the trabecu-lar bone was thinner, cortical thinning was observed, and the morphological structure was poor with a break. Thegap between the trabecular increased, the bone density and area were reduced, the number of hematopoietic cellsdecreased, the fat vacuole was elevated, the osteoblasts decreased and the osteoclasts increased, which are typicalosteoporotic signs, indicating that the model was successfully established. In the ALN group, there was no obviousthinning of the cortical bone and no obvious thinning of the trabecular bone with normal trabecular connectivity.The gap between the trabecular enlarged slightly, the area of the trabecular space did not significantly decrease, thenumber of hematopoietic cells in the medullary cavity was elevated and the osteoclasts decreased (Figure 1A). Theexpression of CD34 and RANK was determined by immunohistochemistry, and positive expression was identified asyellow or brown granules on the cell membrane (Figure 1B). The statistical results showed that the positive expressionof the hematopoietic marker CD34 in the OP group was lower than that in the sham group, while the positive expres-sion of the osteoclast marker RANK was increased. Compared with the OP group, the ALN group showed increasedexpression of the hematopoietic marker CD34 and decreased expression of the osteoclast marker RANK (Figure 1C).


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Figure 1. ALN treatment significantly improves OP symptoms

(A) HE staining (40×) shows the effect of ALN treatment on the histopathological changes of femoral tissues in rats; (B) immuno-

histochemical staining of CD34 and RANK in the sham, OP and ALN groups (400×); ALN, alendronate; HE, hematoxylin–eosin; OP,

osteoporosis; RANK, receptor activator of NF-KappaB.

Apo

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Figure 2. ALN treatment decreases cell apoptosis in OP

(A) TUNEL staining reflects apoptotic cells (400 ×); (B) AI of femoral tissues; *, P < 0.05 vs. the sham group, #, P < 0.05 vs. the OP

group; ALN, alendronate; OP, osteoporosis; TUNEL, TdT-mediated dUTP-biotin nick end-labeling assay; AI, apoptosis index.

ALN treatment inhibits the apoptosis of cells in femoral tissue in OPTUNEL staining was applied for cell apoptosis determination. The positive cells of apoptosis were stained with darkbrown. The AI of the femur tissues in the sham group was (24.35 +− 2.89)%. The AI of the femur tissues in the ALNgroup was significantly lower (25.8 +− 2.97)% than that of the OP group (44.9 +− 4.53)% (P < 0.05). ALN treatmentcould inhibit the apoptosis of cells in rats with OP (Figure 2).


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Figure 3. MiR-182-5p is up-regulated, whereas ADCY6 and the Rap1/MAPK signaling pathway are inhibited after treatment

with ALN

(A) miR-182-5p expression and mRNA levels of ADCY6, ERK1, ERK2, Rap1 and P38 MAPK in response to ALN treatment; (B) the

gray value of ADCY6, ERK1/2, p-ERK1/2, P38 MAPK, p-P38 MAPK and Rap1 protein bands in response to ALN treatment; (C)

protein levels of ADCY6, ERK1/2, Rap1 and P38 MAPK and the extent of ERK1/2 and P38 MAPK phosphorylation in response to

ALN treatment; *, P < 0.05 vs. the sham group; #, P < 0.05 vs. the OP group. MiR-182-5p, microRNA-182-5p; OP, osteoporosis;

ADCY6, adenylate cyclase 6.

MiR-182-5p is down-regulated, whereas ADCY6 is up-regulated and theRap1/MAPK signaling pathway is activated after treatment with ALNThe miR-182-5p expression and the mRNA and protein levels of ADCY6 and the Rap1/MAPK signalingpathway-related factors in femoral tissues were determined by RT-qPCR (Figure 3A) and Western blot analysis (Figure3B,C). Compared with the sham group, the OP group had a significantly higher expression of miR-182-5p, whereas ithad significantly down-regulated mRNA and protein levels of ADCY6, ERK1, ERK2, Rap1 and P38 MAPK, as well asthe extent of ERK1/2 and P38 MAPK phosphorylation (all P < 0.05). Compared with the OP group, the ALN grouphad a decreased expression of miR-182-5p and increased mRNA and protein levels of ADCY6, ERK1, ERK2, Rap1and P38 MAPK, as well as the extent of ERK1/2 and P38 MAPK phosphorylation. It was determined that miR-182-5pwas highly expressed in the rats with OP, while ADCY6, ERK1, ERK2 and Rap1, as well as the extent of ERK1/2 andP38 MAPK phosphorylation were lowly expressed. The ALN group showed the opposite trend, indicating that it hasan inhibitory effect on rats with OP.

Osteoblasts are successfully culturedMicroscopic observation showed that the osteoblasts were triangular or multispindle and the cells were morphologi-cally diverse after several days. The osteoblasts were spindle shaped, triangular or polygonal, with protuberance. Thenuclei were round or oval. In the growth stage, cell division was more common, and the cells were multiple and con-nected. At the time of confluence, the cells are stony and could be overlapped. The overlapping cells gradually formedcell nodules, followed by collagenous accumulation and calcium salt deposition, and finally formed an opaque min-eralized nodule (Figure 4A). Alkaline phosphatase staining showed that most cells were colored (black or gray). Thecytoplasm showed a positive reaction of gray-black granule or block deposit (Figure 4B). Calcium nodule staining(alizarin red staining): typical orange calcified nodules were shaped (Figure 4C). These results were consistent withthe characteristics of osteoblasts, which can be used to verify the success of the osteoblast culture.

ADCY6 is a direct target gene of miR-182-5pAccording to the online prediction website analysis, there is a binding site between miR-182-5p and ADCY6 3′UTR,and ADCY6 is the target gene of miR-182-5p (Figure 5A). Dual-luciferase reporter gene assays were used to verifythat ADCY6 is the target of miR-182-5p (Figure 5B). Compared with the NC group, the miR-182-5p mimic groupsignificantly inhibited the activity of luciferase in the ADCY6-WT 3′UTR (P < 0.05), while miR-182-5p had nosignificant effect on the activity of luciferase in the MUT 3′UTR (P > 0.05). MiR-182-5p could specifically bind toADCY6 3′-UTR, and ADCY6 is the target gene of miR-182-5p.


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(A) (B) (C)

50 µm 50 µm 50 µm

Figure 4. Osteoblasts are cultured successfully

(A) The morphology of osteoblasts (the original magnification is 100×); (B) ALP images of osteoblasts (the original magnification is

200×); (C) alizarin red staining images of osteoblasts (original magnification is 100×). ALP, alkaline phosphatase staining.

3' gccaCACUCAAGAUGGU-AACGGUUu 5' rno-miR-182

544:5' ucagGGGAAUGUUUCCAUUUGCCAAa

mirSVR score: -0.8389PhastCons score: 0.6429

rno-miR-182/ADCY6 Alignment

(A)

(B)

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C

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Figure 5. ADCY6 is the target gene of miR-182-5p

(A) Binding regions between ADCY6 3′UTR and miR-182-5p sequence according to the online prediction website analysis; (B)

luciferase activity of the ADCY6 Wt and ADCY6 Mut after transfection; *, P < 0.05 vs. the NC group. ADCY6, adenylate cyclase 6;

NC, negative control; miR-182-5p, microRNA-182-5p.

MiR-182-5p inhibits the expression of ADCY6 and the Rap1/MAPKsignaling pathway activationRT-qPCR (Figure 6A) and Western blot analysis (Figure 6B,C) were conducted to measure the expression ofmiR-182-5p, ADCY6 and the Rap1/MAPK signaling pathway-related genes after transfection. There was no signifi-cant difference in the gene expression in the NC group compared with the blank group (P > 0.05). Compared with theblank and NC groups, the miR-182-5p mimic group showed promoted miR-182-5p expression, and the miR-182-5pmimic and si-ADCY6 groups had down-regulated mRNA and protein levels of ADCY6, ERK1, ERK2, P38 MAPK andRap1, as well as the extent of ERK1/2 and P38 MAPK phosphorylation (all P < 0.05). The expression of miR-182-5pin the miR-182-5p inhibitor group was down-regulated, and the mRNA and protein levels of ADCY6, ERK1, ERK2,P38 MAPK and Rap1, as well as the extent of ERK1/2 and P38 MAPK phosphorylation were up-regulated in themiR-182-5p inhibitor group (all P < 0.05). The expression of miR-182-5p was down-regulated in the miR-182-5pinhibitor + si-ADCY6 group with no other significant difference (P > 0.05). After the addition of the signal trans-duction pathway inhibitor U0126, the expression trend of the related signal transduction protein was similar to thatof the miR-182-5p mimic group and the si-ADCY6 group. The ADCY6 expression was enhanced; however, there wasno difference in the expression of the related signal transduction protein between the miR-182-5p inhibitor + U0126group and the blank group. MiR-182-5p could reduce the expression of ADCY6, thus suppressing the activation ofthe Rap1/MAPK signaling pathway.

Down-regulation of miR-182 promotes osteoblast proliferationMTT assay and Western blot analysis were employed to evaluate the osteoblast proliferation (Figure 7A) and PCNAprotein level, respectively (Figure 7B,C). The OD values of the cells in each group were all increased with time. Therewas no difference in the PCNA protein level and OD values between the blank and NC groups (P > 0.05). Compared


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Figure 6. MiR-182-5p inhibits the expression of ADCY6 and suppressed the Rap1/MAPK signaling pathway

(A) The expression of miR-182-5p and mRNA levels of ADCY6, ERK1, ERK2, P38 MAPK and Rap1 in response to the treatment

of miR-182-5p mimic, miR-182-5p inhibitor, si-ADCY6, miR-182-5p inhibitor + si-ADCY6, U0126 or miR-182-5p inhibitor + U0126;

(B) the gray value of ADCY6, ERK1/2, p-ERK1/2, P38 MAPK, p-P38 MAPK and Rap1 protein bands in response to the treatment of

miR-182-5p mimic, miR-182-5p inhibitor, si-ADCY6, miR-182-5p inhibitor + si-ADCY6, U0126 or miR-182-5p inhibitor + U0126; (C)

protein levels of ADCY6, ERK1,/2, Rap1 and P38 MAPK and the extent of ERK1/2 and P38 MAPK phosphorylation in response to the

treatment of miR-182-5p mimic, miR-182-5p inhibitor, si-ADCY6, miR-182-5p inhibitor + si-ADCY6, U0126 or miR-182-5p inhibitor

+ U0126; *, P < 0.05 vs. the blank and NC groups; #, P < 0.05 vs. the miR-182-5p inhibitor group; OP, osteoporosis; ADCY6,

adenylate cyclase 6; Rap1, Ras-related protein 1; MAPK, mitogen-activated protein kinase; ERK, extracellular signal-regulated

kinase; miR-182-5p, microRNA-182-5p.

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Figure 7. The down-regulation of miR-182-5p promotes osteoblast proliferation

(A) OD value in response to the treatment of miR-182-5p mimic, miR-182-5p inhibitor, si-ADCY6, miR-182-5p inhibitor + si-ADCY6,

U0126 or miR-182-5p inhibitor + U0126 measured by MTT assay; (B) the gray value of PCNA protein band in response to the

treatment of miR-182-5p mimic, miR-182-5p inhibitor, si-ADCY6, miR-182-5p inhibitor + si-ADCY6, U0126 or miR-182-5p inhibitor

+ U0126; (C) PCNA protein level in response to the treatment of miR-182-5p mimic, miR-182-5p inhibitor, si-ADCY6, miR-182-5p

inhibitor + si-ADCY6, U0126 or miR-182-5p inhibitor + U0126; *, P < 0.05 vs. the blank group and the NC group; #, P < 0.05 vs.

the miR-182-5p inhibitor group. NC, negative control; PCNA, proliferating cell nuclear antigen; miR-182-5p, microRNA-182-5p.


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with the blank and NC groups, the miR-182-5p mimic, U0126 and si-ADCY6 groups had a decreased PCNA pro-tein level and proliferation decreased (P < 0.05); the protein level of PCNA in the miR-182-5p inhibitor group wasincreased and proliferation accelerated (P < 0.05). There was no significant difference in the cell proliferation in themiR-182-5p inhibitor + si-ADCY6 group and miR-182-5p inhibitor + U0126 group (P > 0.05). Compared with themiR-182-5p inhibitor group, the PCNA protein level and the cell proliferation ability were significantly decreased inthe miR-182-5p inhibitor + si-ADCY6 and miR-182-5p inhibitor + U0126 groups (all P < 0.05). MiR-182-5p couldinhibit the proliferation of osteoblasts by inhibiting the Rap1/MAPK signaling pathway, and ADCY6 can promotethe proliferation of osteoblasts.

Down-regulated miR-182-5p enhances osteoblast differentiationThe activities of ALP (Figure 8A) and BGP (Figure 8B), as well as the mRNA and protein levels of ALP, BMP2,BGP and COL I (Figure 8C–K) were determined to investigate the effect of miR-182-5p on osteoblast differentiation.There was no significant difference in the levels of ALP and BGP between the blank and NC groups (P > 0.05).Compared with the blank and NC groups, the miR-182-5p mimic, siRNA-ADCY6 and U0126 groups had significantlydecreased ALP, BMP2 and BGP levels and down-regulated COL I expression (all P < 0.05), whereas the miR-182-5pinhibitor group had elevated ALP, BMP2 and BGP levels and up-regulated COL I expression (all P < 0.05). Therewas no significant change in the levels of ALP, BMP2, BGP and COL I in the miR-182-5p inhibitor + si-ADCY6 andmiR-182-5p inhibitor + U0126 groups (P > 0.05). MiR-182-5p could inhibit the expression of differentiation factors,and ADCY6 could promote the expression of differentiation factors. Down-regulation of miR-182-5p can activatethe Rap1/MAPK signaling pathway by targeting ADCY6, thus promoting the differentiation of osteoblasts.

Down-regulated miR-182-5p inhibits cell cycle progression and cellapoptosis of osteoblastsFlow cytometry was employed to determine the cell cycle distribution (Figure 9A,B) and cell apoptosis (Figure 9C,D).There was no significant difference in the cell cycle distribution and apoptosis rate in the blank and NC groups (P> 0.05). Compared with the blank and NC groups, the miR-182-5p mimic, siRNA-ADCY6 and U0126 groups hadmore cells in the G1 phase and less cells in S phase, as well as significantly increased cell apoptosis (P < 0.05). Inthe miR-182-5p inhibitor group, there were less cells in the G1 phase and increased cells in the S phase, and the cellapoptosis was significantly lower (P < 0.05). MiR-182-5p could promote osteoblast apoptosis, while ADCY6 couldinhibit osteoblast apoptosis. The down-regulation of miR-182-5p can reverse the apoptosis induced by a low expres-sion of ADCY6, and the Rap1/MAPK signaling pathway inhibitor U0126 can reverse the down-regulated phenotypeof miR-182-5p.

DiscussionOP, a frequently occurring disease, is a metabolic bone disorder that affects millions of individuals worldwide whosehigh morbidity leads to a high rate of disability, extreme pain and a reduced quality of life [18,19]. MiRNAs havevarious functions, and it has been reported that they could play a role in bone formation, such as the differentiationof osteoblasts [20]. The therapeutic potentials of miRNAs, including miR-138, miR-338-3p and miR-188, have beenproven in OP [21]. Given that each miRNA has many potential functions, it is beneficial for disease treatment toresearch the mechanism of miRNAs. In the present study, we explored the mechanism of miR-182-5p, ADCY6 andthe Rap1/MAPK signaling pathway. Consequently, the present study demonstrates that miR-182-5p regulates theapoptosis and proliferation of osteoblasts through the Rap1/MAPK signaling pathway by negatively targeting theADCY6 gene.

Initially, we found that in OP, the trabecular bone was thinner, cortical thinning occurred, and the morphologicalstructure was poor with a break. The bone density and area were reduced, the osteoblasts decreased and the osteoclastsincreased. Consistent with our study, a study observed through HE staining that there were osteoporotic changes inOP rats, including an increase in the intertrabecular space, thinning of the bone trabeculae and microfractures ofthe bone trabeculae [22]. Moreover, we found ALN had increased serum calcium, decreased serum phosphorus, anincreased BMD and a lower AI of the femur tissues in rats with OP, which indicates that ALN has a therapeuticeffect on OP. ALP and COL I expression followed by the maturation of the extracellular matrix in the early stage andALP, COL and BGP are the main regulatory genes in bone formulation [23]. In many therapies, ALN is currentlythe most commonly used treatment for OP in patients, with an increasing use of the 70 mg weekly dosages, andthe latest formulations of this drug are expected to reduce side effects and increase adherence to the treatment ofanti-fractures, aiming at better clinic effects [24]. ALN at a low concentration is beneficial to the formation of bone


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Figure 8. Down-regulated miR-182-5p enhances osteoblast differentiation

(A and B) ALP activity and BGP activity in response to the treatment of miR-182-5p mimic, miR-182-5p inhibitor, si-ADCY6,

miR-182-5p inhibitor + si-ADCY6, U0126 or miR-182-5p inhibitor + U0126; (C) the gray value of ALP, BGP, COL I and BMP2 protein

bands in response to the treatment of miR-182-5p mimic, miR-182-5p inhibitor, si-ADCY6, miR-182-5p inhibitor + si-ADCY6, U0126

or miR-182-5p inhibitor + U0126; (D and E) mRNA and protein levels of COL I in response to the treatment of miR-182-5p mimic,

miR-182-5p inhibitor, si-ADCY6, miR-182-5p inhibitor + si-ADCY6, U0126 or miR-182-5p inhibitor + U0126; (F and G) mRNA and

protein levels of ALP in response to the treatment of miR-182-5p mimic, miR-182-5p inhibitor, si-ADCY6, miR-182-5p inhibitor +

si-ADCY6, U0126 or miR-182-5p inhibitor + U0126; (H and I) mRNA and protein levels of BMP2 in response to the treatment of

miR-182-5p mimic, miR-182-5p inhibitor, si-ADCY6, miR-182-5p inhibitor + si-ADCY6, U0126 or miR-182-5p inhibitor + U0126. *,

P < 0.05 vs. the blank group and the NC group; #, P < 0.05 vs. the miR-182-5p inhibitor group. ALP, alkaline phosphatase; BGP,

Bone gla protein; COL I, type I collagen; NC, negative control; miR-182-5p, microRNA-182-5p.


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(C)

(A) (B)

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Figure 9. Down-regulated miR-182-5p inhibits cell cycle progression and cell apoptosis of osteoblasts

(A) Cell cycle diagram depicting cell progression; (B) quantitative analysis for cell cycle progression in response to the treatment of

miR-182-5p mimic, miR-182-5p inhibitor, si-ADCY6, miR-182-5p inhibitor + si-ADCY6, U0126 or miR-182-5p inhibitor + U0126; (C)

flow cytometry map demonstrating cell apoptosis conditions; (D) apoptosis rate analysis for cells in response to the treatment of

miR-182-5p mimic, miR-182-5p inhibitor, si-ADCY6, miR-182-5p inhibitor + si-ADCY6, U0126 or miR-182-5p inhibitor + U0126; *,

P < 0.05 vs. the blank group and the NC group; #, P < 0.05 vs. the miR-182-5p inhibitor group; NC, negative control; miR-182-5p,

microRNA-182-5p.

through promoting the mineralization ability of osteoblasts to a certain extent [25]. According to one experiment, thecell viability was decreased in high concentrations of ALN, indicating that a high dose was cytotoxic, with damage toits therapeutic effect, ultimately resulting in osteonecrosis [26].

According to our research, miR-182-5p directly targets ADCY6 as verified by the dual luciferase reporter gene assay,and following the transfection of miR-182-5p inhibitor, the expressions of ADCY6, ERK1, ERK2, P38 MAPK andRap1, and the extent of ERK1/2 and P38 MAPK phosphorylation were up-regulated, indicating that miR-182-5p couldreduce the expression of ADCY6, thus inhibiting the activation of the Rap1/MAPK signaling pathway. Consistentwith our study, there are six predicted target sites in both ADCY6 and miR-182 transcripts, which demonstrates thatADCY6 is a target gene of miR-182 and miR-182 down-regulates the expression of ADCY6 in circadian variation[27]. Interestingly, recent research suggests that the expression of ADCY6 may be directly modulated by miR-182,which is the way ADCY6 participated in circadian rhythm regulation [28]. The MAPK signaling pathway plays acrucial role in regulating osteogenesis, which is a disorder that affects the bones; for example, in ovariectomy-inducedbone loss, MAPK was up-regulated [29]. In addition, hsa-miR-205-5p negatively affects osteoblast differentiation viathe P38 MAPK signaling pathway [30]. It has been reported that the constitutive activation of the MAPK/ERK andAKT/PKB signaling pathway, as well as the up-regulation of microRNAs, including miR-96, miR-182 and miR-183,were factors that contributed to the inactivation of FOXO1 [31], which indicates that there may be a correlationbetween miR-182-5p and the Rap1/MAPK pathway.

Furthermore, inhibition of miR-182-5p could promote the ALP and BGP levels and the COL I expression, aswell as cell proliferation and differentiation through the Rap1/MAPK signaling pathway via negative regulation ofADCY6. LGR4, a novel RANKL receptor, has been found to suppress osteoclast differentiation and survival induced


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by RANKL in vivo and in vitro [32]. A recent publication noted that miR-182 negatively regulates osteoblast differ-entiation, in which its down-regulation increased osteoblast differentiation [33]. Research has shown that with thedown-regulation of p38MAPK and the ERK1/2 signal pathway, the ALP, COL I and BGP expression levels were in-hibited [23]. A positive regulation exists between BMP2 induced osteogenic differentiation and p38 MAPK, and theinhibition of MAPK will reduce the differentiation of osteoblasts [34].

In this research, we demonstrate the regulation mechanism of miR-182-5p on osteoblast proliferation and differen-tiation. The mechanism of miR-182-5p is mediated by the Rap1/MAPK signaling pathway via targeting the ADCY6gene. We speculate that the miR-182-5p regulated mechanism may be a promising new direction in the developmentof therapeutic treatments for OP and aims to provide a new basis for the development of targeted therapy. However,we intend to further explore the effects of miR-182-5p on the differentiation of osteoblasts in animals.

AcknowledgmentWe thank the individual who provided assistance and helpful discussions on our manuscript.

Author contributionB.L.P. and S.D.L. conceived and designed the study. L.P., J.L.L., Y.X.Y., H.H.L., Y.J.D. and J.E.L. performed all experiments andanalyses. L.W. and Z.W.T. wrote the manuscript. All authors participated in revising the manuscript, and have read and approvedthe manuscript.

Competing interestsThe authors declare that there are no competing interests associated with the manuscript.

FundingThe authors declare that there are no sources of funding to be acknowledged.

AbbreviationsADCY6, adenylyl cyclase isoform 6; ALN, alendronate; BCIP, 5-bromo-4-chloro-indolyl-phosphatase; BSA, bovine serum albu-min; DAPI, 4′,6-diamidino-2-phenylindole; DMEM, Dulbecco’s Modified Eagle’s Medium; EDTA, ethylenediaminetetraacetic acid;ERK, extracellular signal-regulated kinase; FBS, fetal bovine serum; FITC, fluorescein isothiocyanate; MAPK, mitogen-activatedprotein kinase; miRNA, microRNA; MUT, mutated; NC, negative control; OD, optical density; OP, osteoporosis; PI, propidiumiodide; PNPP, P-nitrophenyl phosphate; WT, wild type.

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