MicroRNA‐497 elevation or LRG1 knockdown promotes osteoblast proliferation and collagen synthesis in osteoporosis via TGF‐β1/Smads signalling pathway

Abstract MicroRNAs (miRNAs) have been corroborated to engage in the process of cellular activities in osteoporosis. However, few researches have been conducted to expose the integrated role of miR‐497, leucine‐rich alpha‐2‐glycoprotein‐1 (LRG1) and transforming growth factor beta 1 (TGF‐β1)/Smads signalling pathway in osteoporosis. Thereafter, the study is set out to delve into miR‐497/LRG1/TGF‐β1/Smads signalling pathway axis in osteoporosis. Osteoporosis bone tissues and normal bone tissues were collected. Rat osteoporosis models were constructed via ovariectomy. Model rats were injected with restored miR‐497 or depleted LRG1 to explore their roles in osteoporosis. Rat osteoblasts were extracted from osteoporosis rats and transfected with restored miR‐497 or depleted LRG1 for further verification. MiR‐497 and LRG1 expression in femoral head tissues and osteoblasts of osteoporosis rats were detected. TGF‐β1/Smads signalling pathway‐related factors were detected. MiR‐497 was poorly expressed while LRG1 was highly expressed and TGF‐β1/Smads signalling pathway activation was inhibited in osteoporosis. MiR‐497 up‐regulation or LRG1 down‐regulation activated TGF‐β1/Smads signalling pathway, promoted collagen type 1 synthesis and suppressed oxidative stress in femoral head tissues in osteoporosis. MiR‐497 restoration or LRG1 knockdown activated TGF‐β1/Smads signalling pathway, promoted viability and suppressed apoptosis of osteoblasts in osteoporosis. Our study suggests that miR‐497 up‐regulation or LRG1 down‐regulation promotes osteoblast viability and collagen synthesis via activating TGF‐β1/Smads signalling pathway, which may provide a novel reference for osteoporosis treatment.


| INTRODUC TI ON
Osteoporosis is widely recognized as a bone disease which is manifested with low bone mass and microarchitectural deterioration of bone tissues, resulting in bone fragility and thus posing a great threat on bone fracture. 1 An enormous number of people of both genders and all races are affected by osteoporosis, and its prevalence will increase in the process of ageing. 2 There are risk factors contributing to the incidence of osteoporosis, including age, smoking, low weight, alcohol intake, oestrogen status, physical inactivity, low calcium intake and low vitamin D status. 3 Thus, antiresorptive therapy, tobacco avoidance, adequate combined calcium and vitamin D intake, moderate alcohol intake and weight-bearing exercise are introduced for patients with osteoporosis. 4 More importantly, it is essential to recognize the optimal treatment of osteoporosis.
MicroRNAs (miRNAs) are small non-coding endogenous RNAs regulating gene expression at post-transcriptional level which interfere with translation of specific target mRNAs and are thought to regulate many cellular processes. 5 Deregulated miRNA-mediated mechanisms are found to become the crucial pathological factor in bone deterioration and other bone-related diseases. 6 Specifically, circulating miR-497-5p is regarded as a promising biomarker for osteoporosis diagnosis and prognosis due to its participation in bone metabolism and bone loss. 7 A study has elucidated that miR-497 ~ 195 cluster plays an unique but irreplaceable role in regulating angiogenesis combined with osteoporosis and its function may widen our vision to treat with age-related osteoporosis. 8 It has been reported that transforming growth factor β1 (TGF-β1)/Smads pathway is involved in dexamethasone-induced osteoporosis progression. 9,10 Moreover, regulation of TGF-β1/Smads pathway has been documented to promote bone formation and preclude bone resorption. 11 Widely, TGF-β could improve the repair capacity following bone injury through promoting cell division and osteoblast generation, so that the proliferation effect of TGF-β notably increases the number of osteoblasts. 12 In addition, a conclusion drawn from a previous investigation points out that leucine-rich alpha-2-glycoprotein-1 (LRG1) promotes angiogenesis by modulating endothelial TGF-β signalling pathway. 13 Wang et al have stated that LRG1 induces endothelial cell angiogenesis, mesenchymal stem cell (MSC) migration and bone formation. 14 Taking the above-mentioned findings into consideration, the present study was conducted to identify the molecular mechanism of miR-497/LRG1/TGF-β1/Smads axis in osteoporosis and their potentials in the progression of osteoporosis.

| Ethics statement
The study was approved by the Ethics Committee of the Third Affiliated Hospital, Southern Medical University, Academy of Orthopedics of Guangdong Province, and informed written consent was acquired from all patients. All animal experiments went along with the Guide for the Care and Use of Laboratory Animal by International Committees.

| Study subjects
Bone mineral density of patients, who were admitted from January 2017 to September 2018, was measured by a dual-energy X-ray bone density meter (DEXA, Norland, USA). According to the diagnostic criteria issued by World Health Organization, osteoporosis is diagnosed if the bone mass density T value is greater than −2.5. The inclusive standard was as follows: osteoporosis patients confirmed by DEXA had complete clinical data and imaging data; specimen collection and use were agreed with the informed consent of the patients and the matching control individuals; the patients were free of severe combinations such as malignant tumour, diabetes, hypertension, or heart disease.
Twenty clinically diagnosed osteoporosis patients who had received hip replacement caused by femur neck fracture were collected as an osteoporosis group, of which 17 cases of female and 3 cases of male whereas 20 patients who had received hip replacement caused by bilateral hip dysplasia were collected as a control group, of which 14 cases of female and 6 cases of male. The cancellous bone located at femoral neck was removed by an aseptic rongeur and preserved at −80℃.

| Experimental animals
Healthy female Sprague Dawley (SD) rats (n = 70) of specificpathogen-free grade, ageing 3-4 months and weighting 180-250 g, were purchased from the Experimental Animal Center of Guangddong (Guangzhou, China). Adaptively raised for 7 d (free food and drinking water, 24 ± 2℃, 12-h day/night cycle), rats were applied for experiments. promotes osteoblast viability and collagen synthesis via activating TGF-β1/Smads signalling pathway, which may provide a novel reference for osteoporosis treatment.

| Osteoporosis rat modelling and grouping
Fasted for 8-12 h, 70 female SD rats were assigned into the control group (n = 8) and osteoporosis model group (n = 62) by random number table. Anaesthetized with 3% pentobarbital sodium at 0.1 mL/100 g, rats were fixed in a prone position, cut off back hair and disinfected on the back skin with iodine wine and alcohol. On both sides of the spine, vertical incisions were made to separate muscles to open the abdominal cavity to perform ovariectomy (ovary, a fleshred particles visible on the adipose tissue, pathologically confirmed as ovarian tissues). After that, if there was no active bleeding in abdominal cavity, muscles and skin were stitched and disinfected with 75% alcohol, and 100,000 units of penicillin were injected to prevent infection. Normally raised for 2 months, the rats were administrated.
Company (Shanghai, China). Rats were euthanized 2 weeks after injection. Metabolic cages were used to retain urine within 24 hours to detect biochemical indicators. With rats anaesthetized with 3% pentobarbital sodium at 0.1 mL/100 g and fixed on the operating table, the abdominal cavity was opened to obtain abdominal aorta blood (5-8 mL). The blood samples were centrifuged at 3000 rpm/ min to take serum and stored at −70°C for detection of bone formation-related indicators. Part of femoral head tissues was applied for histological observation, and the other part was preserved in liquid nitrogen for reverse transcription quantitative polymerase chain reaction (RT-qPCR) and Western blot analysis, etc.

| Detection of blood and urine biochemical indices
Urinary calcium and blood calcium were determined by methyl thymol blue colorimetric method, urinary phosphorus and blood phosphorus by reduction by ferrous ammonium sulphate hexahydrate and hydroxyproline (Hyp) by dimethylaminobenzaldehyde method. These kits were provided by Beijing Leagene Biotech. Co., Ltd, (Beijing, China). Alkaline phosphatase (ALP) was detected by aminoantipyrine colorimetric method, and the detection kit was provided by NanJing JianCheng Bioengineering Institute (Nanjing, China).

| Detection of bone formation-related indices
Stood at room temperature for 30 minutes, the blood samples were centrifuged at 3000 r/min to separate serum, in which osteocalcin (OC), bone alkaline phosphatase (BALP), aminoterminal (PINP) and carboxyterminal (PICP) contents were determined by enzymelinked immunosorbent assay (ELISA) kits (Shanghai Enzyme-linked Biotechnology Co., Ltd., Shanghai, China). Optical density (OD) values were measured at 450 nm (within 10 minutes after termination) on a microplate reader (infinite M200, Tecan, Austria). A standard curve was drawn using a computer CurveExpert 1.3 analysis software to calculate the sample content.
Then, the samples were sequentially dehydrated in ethanol of differ-

| Detection of oxidative stress-related indices
The preserved femoral head tissues in liquid nitrogen were diluted with normal saline, homogenized and centrifuged at 3000 r/min to obtain the supernatant. Superoxide dismutase (SOD) activity was determined by xanthine oxidase method, malondialdehyde (MDA) content by thiobarbituric acid method and glutathione peroxidase (GSH-Px) activity by colorimetric method. The SOD, MDA and GSH-Px kits were purchased from Nanjing Jiancheng Bioengineering Institute. Followed by that, the osteoblasts were covered by the freshly prepared substrate application solution and with a hydrophobic membrane.

| Osteoblast isolation, identification and treatment
Incubated in a wet box at 37°C in the dark, the osteoblasts were counterstained with haematoxylin and observed by a microscope.

| Western blot analysis
Total proteins were extracted from bone tissues and cells, followed by protein concentration determination and deionized water adjustment. The 10% sodium dodecyl sulphate separation gel and concentrated gel were prepared. Mixed with the loading buffer, the protein samples were boiled at 100°C which was followed by ice bath and centrifugation. Then, the protein samples were processed with electrophoresis separation and electroblotting onto a nitrocellulose membrane. Next, the membrane was blocked with 5% skim milk powder overnight and probed with the primary antibodies

| MiR-497 expression reduces and LRG1 expression increases in femoral head tissues of patients with osteoporosis
RT-qPCR results ( Figure 1A) showed that miR-497 and miR-195 expression in femoral head tissues of the osteoporosis patients were reduced (all P < 0.05) in contrast to controls. As miR-497 showed a more obvious difference, miR-497 was selected for the studied miRNA.
RT-qPCR and Western blot analysis demonstrated that LRG1 expression was increased in femoral head tissues of the osteoporosis patients by comparison with controls ( Figure 1B-D).
A negative relation was observed between miR-497 and LRG1  Figure 1G).

| Up-regulation of miR-497 or downregulation of LRG1 activates TGF-β1/Smads signalling pathway in rats with osteoporosis
RT-qPCR and Western blot analysis (

F I G U R E 2
Up-regulation of miR-497 or down-regulation of LRG1 activates TGF-β1/Smads signalling pathway in rats with osteoporosis. A, MiR-497 expression in femoral head tissues of rats; B, LRG1 mRNA expression in femoral head tissues of rats; C, LRG1 protein bands in femoral head tissues of rats; D, LRG1 protein expression in femoral head tissues of rats; E, TGF-β1 protein bands in femoral head tissues of rats; F, TGF-β1 protein expression in femoral head tissues of rats; G, Smad3, Smad4 and Smad7 protein bands in femoral head tissues of rats; H, Smad3, Smad4 and Smad7 protein expression in femoral head tissues of rats; I, p-Smad2/3 protein bands in femoral head tissues of rats; J, p-Smad2/3 protein expression in femoral head tissues of rats; * represented P < 0.05; **represented P < 0.01; *** represented P < 0.001; n = 8. The data were expressed as the mean ± standard deviation. Comparisons among multiple groups were analysed using ANOVA, followed by Tukey's post hoc test for pairwise comparisons

F I G U R E 4
Up-regulation of miR-497 or down-regulation of LRG1 attenuates pathological femoral tissue damage, raises Col-1 expression and inhibits oxidative stress in femoral head tissues of rats with osteoporosis. A, HE staining of femoral head tissues of rats; B, Col-1 mRNA expression in femoral head tissues of rats; C, Col-1 protein bands in femoral head tissues of rats; D, Col-1 protein expression in femoral head tissues of rats; E, SOD activities in rat femoral head tissues; F, MDA contents in rat femoral head tissues; G, GSH-Px activities in rat femoral head tissues; * represented P < 0.05; **represented P < 0.01; *** represented P < 0.001; n = 8. The data were expressed as the mean ± standard deviation. Comparisons among multiple groups were analysed using ANOVA, followed by Tukey's post hoc test for pairwise comparisons

| MiR-497 is down-regulated while LRG1 is upregulated in osteoblasts of rats with osteoporosis, and miR-497 elevation or LRG1 depletion activates TGF-β1/Smads signalling pathway
ALP staining ( Figure 5A and B): After ALP staining, it could be seen that the cell membrane and intracytoplasmic particles were stained red-brown, that was, the cell staining was positive, so the successful culture of osteoblasts was identified.

F I G U R E 5
MiR-497 is down-regulated while LRG1 is up-regulated in osteoblasts of rats with osteoporosis, and miR-497 elevation or LRG1 depletion activates TGF-β1/Smads signalling pathway. A, Osteoblast morphology at different time-points; B, ALP staining of osteoblasts; C, MiR-497 expression in rat osteoblasts; D, LRG1 mRNA expression in rat osteoblasts; E, LRG1 protein bands in rat osteoblasts; F, LRG1 protein expression in rat osteoblasts; G, TGF-β1 protein band in rat osteoblasts; H, TGF-β1 protein expression in rat osteoblasts; I, Smad3, Smad4 and Smad7 protein bands in rat osteoblasts; J, Smad3, Smad4 and Smad7 protein expression in rat osteoblasts; K, p-Smad2/3 protein bands in rat osteoblasts; L, p-Smad2/3 protein expression in rat osteoblasts; * represented P < 0.05; **represented P < 0.01; *** represented P < 0.001; N = 3. The data were expressed as the mean ± standard deviation. Comparisons among multiple groups were analysed using ANOVA, followed by Tukey's post hoc test for pairwise comparisons

| Up-regulation of miR-497 or downregulation of LRG1 inhibits osteoblast apoptosis and promotes Col-1 synthesis of rats with osteoporosis
Detected by RT-qPCR and Western blot analysis ( Figure 6A-G   Another study has revealed that female with osteoporosis presented with lower GSH-Px and SOD enzyme activity and higher level of MDA. 32 Concerning to the study performed previously, miR-497 with up-regulated expression is documented to possess target genes to promote proliferative activity, which is indicative of the promoting function of up-regulated miR-497 in proliferative activity. 33 In addition, another research outcomes state that overexpressed miR-497 can promote the proliferation capability of cardiomyocyte and inhibit apoptosis of cardiomyocyte in myocardial ischaemia-reperfusion injury. 34 Moreover, it is reported that miR-497 restoration impaired lung cancer cell viability and colony-forming ability by activating TGF-β signalling. 35 Commonly discussed in cancers, LRG1

| D ISCUSS I ON
reduction is surveyed to inhibit cell viability and promote apoptosis of leukaemia cells. 36 Mechanistically, silencing of LRG1 inhibits oesophageal squamous cell carcinoma cell proliferation and facilitates apoptosis. 37 Moreover, our study has revealed that miR-497/LRG1 axis mediated TGF-β1/Smads pathway to affect osteoporosis. Currently, the activation of TGF-β/Smad signalling pathway prevented osteoporosis via enhanced bone formation and disrupted bone resorption. 9 Besides, activating TGF-β1/Smads pathway was also functional for attenuating glucocorticoid-induced osteoporosis. 11 Functionally, the activated TGF-β1/Smads signalling pathway induced by miR-202-3p up-regulation was indicated to ameliorate oxidative stress and cell apoptosis in myocardial ischaemic-reperfusion injury. 38 In papillary thyroid carcinoma, promoting the activation of TGF-β1/Smads signalling pathway enhanced malignant cell apoptosis and impaired cell proliferation. 39 Anyway, the activated TGF-β1/Smads signalling pathway has shown its conducive effects on diseases.
In conclusion, our study reveals that miR-497 is down-regulated and LRG1 is upregulated in osteoporosis, and the increased miR-497 or decreased LRG1 acts to alleviate the progression of osteoporosis via activating TGF-β1/Smads pathway ( Figure 8). Therefore, more potential therapeutic strategies pivoting on miR-497 and LRG1 are emerged.
Further investigations of the mechanism of miR-497 and LRG1 should be explored in detail and performed with a larger cohort, so as to support a promising clinical application for patients with osteoporosis.

ACK N OWLED G EM ENT
We would like to acknowledge the reviewers for their helpful comments on this paper.

CO N FLI C T O F I NTE R E S T
The authors declare that they have no conflicts of interest.

E TH I C A L S TATEM ENT
This study was approved and supervised by the animal ethics committee of the Third Affiliated Hospital, Southern Medical University,

F I G U R E 8
The mechanistic diagrams indicate that in osteoporosis, miR-497 could promote osteoblast proliferation and collagen synthesis, and inhibit osteoblast apoptosis through inhibiting LRG1 and activating TGF-β1/Smads signalling pathway. The miR-497 mimics inhibited the expression of the LRG1 gene and activated TGF-β1/Smads signalling pathway, including elevated expression of TGF-β1, Smad3, Smad4 and p-Smad2/3, and decreased expression of Smad7, thereby promoting osteoblast proliferation and collagen synthesis, and suppressing osteoblast apoptosis Academy of Orthopedics of Guangdong Province. The treatment of animals in all experiments conforms to the ethical standards of experimental animals.

CO N S E NT FO R PU B LI C ATI O N
Not applicable.

DATA AVA I L A B I L I T Y S TAT E M E N T
Not applicable.