Identification of Drug Targets and Agents Associated with Ferroptosis-related Osteoporosis through Integrated Network Pharmacology and Molecular Docking Technology.

BACKGROUND: Osteoporosis is a systemic bone disease characterized by progressive reduction of bone mineral density and degradation of trabecular bone microstructure. Iron metabolism plays an important role in bone; its imbalance leads to abnormal lipid oxidation in cells, hence ferroptosis. In osteoporosis, however, the exact mechanism of ferroptosis has not been fully elucidated. OBJECTIVE: The main objective of this project was to identify potential drug target proteins and agents for the treatment of ferroptosis-related osteoporosis. METHODS: In the current study, we investigated the differences in gene expression of bone marrow mesenchymal stem cells between osteoporosis patients and normal individuals using bioinformatics methods to obtain ferroptosis-related genes. We could predict their protein structure based on the artificial intelligence database of AlphaFold, and their target drugs and binding sites with the network pharmacology and molecular docking technology. RESULTS: We identified five genes that were highly associated with osteoporosis, such as TP53, EGFR, TGFB1, SOX2 and MAPK14, which, we believe, can be taken as the potential markers and targets for the diagnosis and treatment of osteoporosis. Furthermore, we observed that these five genes were highly targeted by resveratrol to exert a therapeutic effect on ferroptosis-related osteoporosis. CONCLUSION: We examined the relationship between ferroptosis and osteoporosis based on bioinformatics and network pharmacology, presenting a promising direction to the pursuit of the exact molecular mechanism of osteoporosis so that a new target can be discovered for the treatment of osteoporosis.

A Comparative Analysis of Between Percutaneous Cannulated Screw Fixation and Traditional Plate Internal Fixation in Treatment of Sanders II and III Calcaneal Fractures.

The objective of this study is to compare the clinical efficacy of percutaneous cannulated screw fixation and traditional plate internal fixation in the treatment of Sanders II and III calcaneal fractures. The records of 64 patients were retrospectively analyzed. Thirty-three cases were fixed by percutaneous cannulated screws. Thirty-one cases were fixed with traditional steel plates. The preoperative preparation time of the screw group and plate group was 3 ± 1.7 days and 4.6 ± 2.1 days. The surgery time was 118.9 ± 43.8 minutes and 146.9 ± 47.6 minutes. The length of hospitalization was 8.7 ± 3.9 days and 17.0 ± 7.9 days. Intraoperative blood loss was 38.2 ± 27.7 mL and 67.1 ± 58.8 mL. The postoperative drainage volume of the plate group was 85.1 ± 53.7 mL, and no wound drainage was needed in the screw group after surgery. Postoperative wound complications occurred in 2 cases of the screw group and 8 cases of the plate group. The recovery effects of Gissane angle and Bohler angle are similar in the 2 groups. The excellent and good rate of the American Orthopaedic Foot and Ankle Society ankle-hindfoot Scale in the screw group was 96.8% at 12 months after surgery, whereas the rate was 93.5% in the plate group. Compared with the traditional plate internal fixation, the percutaneous cannulated screw group achieved a similar excellent and good rate of clinical treatment. It has the advantages of less trauma, less bleeding, low incidence of complications, short preoperative preparation, and hospitalization time.

Mesenchymal stem/stromal cells-derived exosomes for osteoporosis treatment.

Osteoporosis is a systemic bone disease, which leads to decreased bone mass and an increased risk of fragility fractures. Currently, there are many anti-resorption drugs and osteosynthesis drugs, which are effective in the treatment of osteoporosis, but their usage is limited due to their contraindications and side effects. In regenerative medicine, the unique repair ability of mesenchymal stem cells (MSCs) has been favored by researchers. The exosomes secreted by MSCs have signal transduction and molecular delivery mechanisms, which may have therapeutic effects. In this review, we describe the regulatory effects of MSCs-derived exosomes on osteoclasts, osteoblasts, and bone immunity. We aim to summarize the preclinical studies of exosome therapy in osteoporosis. Furthermore, we speculate that exosome therapy can be a future direction to improve bone health.

Effect of micro/nano-sheet array structures on the osteo-immunomodulation of macrophages.

The immune response induced by surface topography crucially determines the implant success. However, how the immune response is mediated by the size of surface topography remains unclear. Hence, various biocompatible Mg-Al layered double hydroxides sheet-array films with different sizes (nano, micro and nano/micro mixture) were constructed on the biomedical titanium, and their osteo-immunomodulation effects on the macrophages were explored. The nano-sheet array structures significantly promoted the polarization of M2 macrophages by activating the PI3K-AKT-mTOR signaling pathway with high gene expressions of integrin ß2 and FAK. While the micro-sheet array structures enhanced osteogenic differentiation of mouse bone marrow mesenchymal stem cells (mBMSCs) via ROCK-YAP/TAZ-mediated mechanotransduction. Moreover, the nano-sheet array structures promoted the osteogenic differentiation of mBMSCs with a high proportion of M2 macrophages through a shared medium. This study gave further information concerning integrin-induced focal adhesions in cells of different sheet array structures and their role in macrophage polarization and osteogenic differentiation of mBMSCs, which might help to design biomaterial surfaces with optimal geometry for a desired immunemodulation.

Comprehensive analysis of lncRNA expression profiles in postmenopausal osteoporosis.

To explore the key lncRNAs affecting postmenopausal osteoporosis (PMOP) progression, the transcriptome sequencing of peripheral blood mononuclear cells from fifteen early postmenopausal women, according to bone mineral density, were divided into groups of osteoporosis, osteopenia and normality, in each of which the expression profiles of lncRNAs was investigated. From the results we observed nine candidates of lncRNAs, which were to be compared with miRBase, and found that MIR22HG as one candidate of lncRNA was most likely to be directly used as miRNA precursor. Based on the KEGG annotation and lncRNA-miRNA-mRNA-KEGG network, we analyzed the potential role of candidate lncRNAs. The results showed that the expression profiles of lncRNAs could help identify the novel ones involved in the progression of PMOP, and that MIR22HG could serve as a miRNA precursor to regulate FoxO signaling pathway in bone metabolism. Our findings can be of great help in predicting and diagnosing early PMOP.

miR-27b attenuates dexamethasone-inhibited proliferation and osteoblastic differentiation in MC3T3-E1 cells by targeting PPARγ2.

Osteoporosis is a metabolic bone illness characterized by low bone density and a high risk of fracture. It is estimated that there are >60 million individuals in China suffering from this disease, which highlights an urgent requirement for the development of novel and safe drugs for the long-term treatment of osteoporosis. MicroRNAs (miRNAs/miRs) have previously been identified as critical regulators in the progression of osteoporosis. As an intronic miRNA, miR-27b enhances the osteoblastic differentiation of stem cells from the bone marrow and the maxillary sinus membrane. However, the mechanism underlying miR-27b in osteoporosis remains to be elucidated. In the present study, MC3T3-E1 pre-osteoblasts were treated with dexamethasone (DEX) to establish an in vitro model of osteoporosis. The results of the present study demonstrated that DEX treatment markedly inhibited the viability of MC3T3-E1 cells, and downregulated the expression level of miR-27b. The results of reverse transcription-quantitative PCR, western blotting and dual-luciferase assays revealed that miR-27b directly regulated and suppressed the expression of peroxisome proliferator-activated receptor γ2 (PPARγ2) in MC3T3-E1 cells. Furthermore, overexpression of miR-27b by transfection of cells with miR-27b mimic attenuated DEX-mediated inhibition of cell viability, alkaline phosphatase (ALP) activity and the expression levels of bone morphogenetic protein-2 (BMP2), runt-related protein 2 (Runx2) and osteocalcin (OCN). The results of the present study indicated that miR-27b alleviated DEX-inhibited proliferation and osteoblastic differentiation. Moreover, miR-27b knockdown repressed MC3T3-E1 cell viability, ALP activity and protein levels of BMP2, Runx2 and OCN. However, these effects were abrogated by small interfering RNA-mediated PPARγ2 silencing. In conclusion, the results of the present study demonstrated that miR-27b attenuated DEX-inhibited proliferation and osteoblastic differentiation in MC3T3-E1 pre-osteoblasts by targeting PPARγ2.

The therapeutic potential of mesenchymal stem cells in treating osteoporosis.

Osteoporosis (OP), a common systemic metabolic bone disease, is characterized by low bone mass, increasing bone fragility and a high risk of fracture. At present, the clinical treatment of OP mainly involves anti-bone resorption drugs and anabolic agents for bone, but their long-term use can cause serious side effects. The development of stem cell therapy and regenerative medicine has provided a new approach to the clinical treatment of various diseases, even with a hope for cure. Recently, the therapeutic advantages of the therapy have been shown for a variety of orthopedic diseases. However, these stem cell-based researches are currently limited to animal models; the uncertainty regarding the post-transplantation fate of stem cells and their safety in recipients has largely restricted the development of human clinical trials. Nevertheless, the feasibility of mesenchymal stem cells to treat osteoporotic mice has drawn a growing amount of intriguing attention from clinicians to its potential of applying the stem cell-based therapy as a new therapeutic approach to OP in the future clinic. In the current review, therefore, we explored the potential use of mesenchymal stem cells in human OP treatment.

miR-452-3p inhibited osteoblast differentiation by targeting Smad4.

Osteoblast differentiation is a complex process that is essential for normal bone formation. A growing number of studies have shown that microRNAs (miRNAs) are key regulators in a variety of physiological and pathological processes, including osteogenesis. In this study, BMP2 was used to induce MC3T3-E1 cells to construct osteoblast differentiation cell model. Then, we investigated the effect of miR-452-3p on osteoblast differentiation and the related molecular mechanism by RT-PCR analysis, Western blot analysis, ALP activity, and Alizarin Red Staining. We found that miR-452-3p was significantly downregulated in osteoblast differentiation. Overexpression miR-452-3p (miR-452-3p mimic) significantly inhibited the expression of osteoblast marker genes RUNX2, osteopontin (OPN), and collagen type 1 a1 chain (Col1A1), and decreased the number of calcium nodules and ALP activity. In contrast, knockdown miR-452-3p (miR-452-3p inhibitor) produced the opposite effect. In terms of mechanism, we found that Smad4 may be the target of miR-452-3p, and knockdown Smad4 (si-Smad4) partially inhibited the osteoblast differentiation enhanced by miR-452-3p. Our results suggested that miR-452-3p plays an important role in osteoblast differentiation by targeting Smad4. Therefore, miR-452-3p is expected to be used in the treatment of bone formation and regeneration.

Integrated Phytochemical Analysis Based on UPLC-MS/MS and Network Pharmacology Approaches to Explore the Effect of Odontites vulgaris Moench on Rheumatoid Arthritis.

Odontites vulgaris Moench has the effect of clearing away heat, detoxification, dispelling wind, and clearing dampness. In this study, the potential anti-inflammatory compounds of O. vulgaris were investigated using ultra-high-performance liquid chromatography-mass spectrometry (UPLC-MS/MS) combined with the network pharmacology approach and further confirmed on an LPS-activated RAW 264.7 macrophage model. Monomer compounds were prepared from the active fraction using modern advanced separation and purification methods. UPLC-Q-Exactive HRMS was used to identify the chemical compounds in the active fractions of O. vulgaris. D-mannitol, geniposidic acid, salidroside, shanzhiside methyl ester, eleutheroside B, geniposide, 7,8-dihydroxycoumarin, gardoside methyl ester, arenarioside, vanillic acid, p-hydroxy-cinnamic acid, melampyroside, syringaresinol, tricin, and diosmetin were isolated from O. vulgaris for the first time. A compound database of O. vulgaris was established based on the existing literature to predict the mechanism of O. vulgaris in the treatment of rheumatoid arthritis. The results suggest that the PI3K-Akt pathway mediates O. vulgaris and deserves more attention in the treatment of RA. Finally, the anti-rheumatoid arthritis effects of the four target compounds were validated with the decreased levels of NO, TNF-α, IL-6 and IL-1ß in RAW 264.7 macrophage cells treated with LPS. The present study explored the potential targets and signaling pathways of O. vulgaris in the treatment of RA, which may help to illustrate the mechanisms involved in the action of O. vulgaris and may provide a better understanding of the relationship between O. vulgaris and RA. This study provides novel insights into the development of new drugs and utilization of Mongolian traditional Chinese medicine resources.

Floating elbow combining ipsilateral distal multiple segmental forearm fractures: A case report.

BACKGROUND: Floating elbow along with ipsilateral multiple segmental forearm fracture is a rare and high-energy injury, although elbow dislocation or fracture of the ulna and radius may occur separately. CASE SUMMARY: We report the case of a 37-year-old woman with open (IIIA) fracture of the right distal humerus with multiple shaft fractures of the ipsilateral radius and ulna with a history of falling from a height of almost 20 m from a balcony. After providing advanced trauma life support, damage control surgery was performed to debride the arm wound and temporarily stabilize the right upper limb with external fixators in the emergency operating room. Subsequently, one-stage internal fixation of multiple fractures was performed with normal values of biochemical indicators and reduction in limb swelling. The patient achieved good outcome at the 7 mo follow-up. CONCLUSION: One- or two-stage treatment must be performed according to the type of injury; we efficiently used the "damage control principle."

Comparative profile of exosomal microRNAs in postmenopausal women with various bone mineral densities by small RNA sequencing.

To explore the role of plasma miRNAs in exosomes in early postmenopausal women. Small RNA sequencing was implemented to clarify the expression of miRNA in plasma exosomes obtained from 15 postmenopausal women, divided into groups of osteoporosis, osteopenia, and normal bone mass based on bone mineral density. Differentially expressed miRNAs (DEMs) were identified by comparing miRNA expression profiles. Five putative miRNAs, miR-224-3p, miR-25-5p, miR-302a-3p, miR-642a-3p, and miR-766-5p were confirmed by real-time PCR; miRNA target genes were obtained from 4 databases: miRWalk, miRDB, RNA22, and TargetScan. The miRNA-mRNA- Kyoto Encyclopedia of Genes and Genomes (KEGG) networks were analyzed, and the DEMs' potential role was investigated by gene ontology terms and KEGG pathway annotation. The results suggest that characterizing plasma exosomal miRNA profiles of early postmenopausal women by small RNA sequencing could identify novel exo-miRNAs involved in bone remodeling, and miR-642a-3p maybe contribute to the prediction and diagnosis of early postmenopausal osteoporosis.

Postoperative complications of concomitant fat embolism syndrome, pulmonary embolism and tympanic membrane perforation after tibiofibular fracture: A case report.

BACKGROUND: Fat embolism syndrome (FES) is a rare disease characterized by pulmonary distress, neurologic symptoms, and petechial rash and seriously threatens human life and health. It is still neglected clinically because of the lack of verifiable diagnostic criteria and atypical clinical symptoms. No studies on FES with pulmonary embolism (PE) and tympanic membrane perforation have been reported to date. Here, we report a rare case of concomitant FES, PE and tympanic membrane perforation after surgery in a patient with a tibiofibular fracture. CASE SUMMARY: A 39-year-old man presented with right lower extremity pain due to a car accident while driving a motorbike on the road. X-ray and computed tomography scans revealed a fracture of the right mid-shaft tibia and proximal fibula categorized as a type A2 fracture according to the AO classification. A successful minimally invasive operation was performed 3 d after the injury. Postoperatively, the patient developed sudden symptoms of respiratory distress and hearing loss. Early diagnosis was made, and supportive treatments were used at the early stage of FES. Seven days after surgery, he presented a clear recovery from respiratory symptoms. The outcome of fracture healing was excellent, and his hearing of the left ear was mildly impaired at the last follow-up of 4 mo. CONCLUSION: Concomitant FES, PE and tympanic membrane perforation are very rare but represent potentially fatal complications of trauma or orthopedic surgery and present with predominantly pulmonary symptoms. Early diagnosis and treatment can reduce the mortality of FES, and prevention is better than a cure.

The therapeutic potential of mesenchymal stem cells in treating osteoporosis

Osteoporosis (OP), a common systemic metabolic bone disease, is characterized by low bone mass, increasing bone fragility and a high risk of fracture. At present, the clinical treatment of OP mainly involves anti-bone resorption drugs and anabolic agents for bone, but their long-term use can cause serious side effects. The development of stem cell therapy and regenerative medicine has provided a new approach to the clinical treatment of various diseases, even with a hope for cure. Recently, the therapeutic advantages of the therapy have been shown for a variety of orthopedic diseases. However, these stem cell-based researches are currently limited to animal models; the uncertainty regarding the post-transplantation fate of stem cells and their safety in recipients has largely restricted the development of human clinical trials. Nevertheless, the feasibility of mesenchymal stem cells to treat osteoporotic mice has drawn a growing amount of intriguing attention from clinicians to its potential of applying the stem cell-based therapy as a new therapeutic approach to OP in the future clinic. In the current review, therefore, we explored the potential use of mesenchymal stem cells in human OP treatment.

LKB1 Promotes the Transformation of Bone Marrow Mesenchymal Stem Cells into Adipocytes Under Oxidative Stress via AMPK-mTOR Signaling Pathway.

Bone marrow mesenchymal stem cells (BM-MSCs) are cells with the potential to differentiate into adipocytes in oxidative stress. In this study, tert-butyl hydroperoxide is used as a stimulator that promotes reactive oxygen species in BM-MSCs. The results demonstrate that knockdown of LKB1 inhibits the transformation of BM-MSCs into adipocytes in the presence of oxidative stress. In addition, ß3 adrenergic receptor agonists, a positive stimulatory molecule for the transformation of BM-MSCs into adipocytes, restores the transformation ability of BM-MSCs caused by LKB1-siRNA. As an upstream signal of adenosine monophosphate-activated protein kinase (AMPK), LKB1 activates the AMPK pathway and promotes the expression of PPARγ and CCAAT/enhancer binding proteins (C/EBPα). This indicates that the regulation of LKB1 on BM-MSCs is dependent on the AMPK pathway. Immunofluorescence localization experiments reveal that the LKB1 and AMPK localizations partially overlap, oxidative stress promotes their expression in the cytoplasm. In general, LKB1 promotes the transformation of BM-MSCs to adipocytes by activating AMPK pathway under oxidative stress.

Upregulated lncRNA THRIL/TNF-α Signals Promote Cell Growth and Predict Poor Clinical Outcomes of Osteosarcoma.

BACKGROUND: The immunosuppressive facet and tumorigenic role of TNF-α have been revealed in osteosarcoma (OS). Long noncoding RNA THRIL is identified to regulate TNF-α expression and participates in immune response. Thus, investigations on the clinical expression pattern of THRIL/TNF-α signal in OS would provide a potential target premise for OS patients. METHODS: We collected OS (n=83), nontumor tissues (n=37) and serum samples (n=83 for OS and n=40 for healthy control) to determine the expressions and clinical significance of THRIL/TNF-α signal. Knockdown of THRIL in OS cell lines MG63 and Saos2 in vitro and in vivo was performed to confirm its function in the development of OS. RESULTS: Elevated expression of THRIL was associated with increased TNF-α levels in OS tissues and serum samples. Combination of THRIL and TNF-α in tissues showed a more efficient diagnostic value for OS patients than either of them. Moreover, high-expressed THRIL was associated with larger tumor size, advanced Enneking stage and lung metastasis, whereas high TNF-α expression was found in patients with high histologic grade and patients who simultaneously harbor high THRIL and TNF-α showed the worst overall survival and metastasis-free survival. TNF-α signals increased OS cell vitalities in vitro but knockdown of THRIL inhibited TNF-α expressions, leading to impaired cell vitality, increased apoptosis and also downregulated epithelial to mesenchymal transition (EMT) phenotype and the ability of invasion, but these processes were restored by the treatment of TNF-α. The oncogenic role of THRIL/TNF-α signal was also confirmed in the xenograft model of MG63 cells. CONCLUSION: Overexpressed THRIL and TNF-α promoted OS progression with efficient diagnostic and prognostic value. THRIL/TNF-α signal supported cell growth and EMT phenotype of OS cells in vitro and in vivo.

Relative Incidence of Proximal Fibula Fractures with Tibial Plateau Fractures: An Investigation of 354 Cases.

We aimed to investigate the incidence of proximal fibula fractures in patients with tibial plateau fractures and to identify risk factors for such combined injuries. From January 2011 to December 2015, 354 patients with tibial plateau fractures who had been admitted to a level 1 trauma center were retrospectively evaluated by an orthopaedic trauma surgeon and two skeletal radiologists. Anteroposterior plain radiography and computed tomography (CT) were used to characterize the injuries, and the incidence of associated proximal fibula fractures was determined. The tibial plateau fractures were classified according to the Schatzker's and three-column classifications. Associated proximal fibula fractures were simultaneously classified with a new fibula fracture classification system. Finally, we determined whether there were statistically significant associations between the presence of a proximal fibula fracture and different types of tibial plateau fractures, as well as sex and age. Proximal fibula fractures were detected in 192 (54.24%) patients using plain radiography and in 215 (60.73%) patients using CT. Logistic regression analysis indicated an increasing trend in the incidence of fibula fractures detected by the CT-based three-column classification system as follows: "age × sex > three columns > age > single posterior column > lateral column + posterior column > medial column + posterior column > medial column + lateral column > single lateral column > single medial column." Proximal fibula fracture associated with tibial plateau fracture is a common phenomenon worthy of attention. In women, age increases the likelihood of complex tibial plateau fractures, particularly those involving the posterolateral articular surface and diaphysis, as well as the likelihood of developing proximal fibula fractures. This s Level IV diagnostic study.

MicroRNA-664a-5p promotes osteogenic differentiation of human bone marrow-derived mesenchymal stem cells by directly downregulating HMGA2.

Osteogenic differentiation of human bone marrow-derived mesenchymal stem cells (BMSCs) has been regarded as a central issue in fracture healing. MicroRNAs (miRNAs, miRs) participate in diverse physiological processes such as osteoblastic differentiation of BMSCs. In this study, we found that miR-664a-5p was upregulated during osteogenic differentiation of human BMSCs, and this upregulation positively correlated with the expression of osteogenic genes Runt-related transcription factor 2 (RUNX2), alkaline phosphatase (ALP), and osteocalcin (OCN). Overexpression of miR-664a-5p promoted the osteogenic differentiation of BMSCs, whereas a knockdown of miR-664a-5p suppressed it. Additionally, high-mobility group A2 (HMGA2) mRNA was identified as a direct target of miR-664a-5p that mediates the function of this miRNA. Overexpression of HMGA2 obviously attenuated miR-664a-5p-induced osteogenic differentiation of BMSCs. Thus, the newly identified miR-664a-5p-HMGA2 pathway expands our understanding of the mechanisms underlying the osteogenic differentiation of human BMSCs, may provide deeper insights into the regulation of this differentiation, and can point to new effective methods for treating osteoporosis.

TMEM18 inhibits osteogenic differentiation of rat bone marrow-derived mesenchymal stem cells by inactivating ß-catenin.

Transmembrane protein 18 (Tmem18) is an obesity-associated gene essential for adipogenesis; however, its function in the osteogenic differentiation of bone marrow-derived mesenchymal stem cells (BMSCs) is still unclear. In this study, we found that Tmem18 was significantly downregulated in rat BMSCs after osteogenic induction. TMEM18 overexpression remarkably downregulated osteo-specific genes including alkaline phosphatase (Alp), Runt-related transcription factor 2 (Runx2), osteocalcin (Ocn), and osteopontin (Opn), and reduced the number of mineral deposits and ALP activity in vitro, whereas knockdown of Tmem18 yielded the opposite results. In vivo assays also indicated that TMEM18 knockdown BMSCs have an increased bone formation potential in a rat model of calvarial defects. Analyses of the mechanism suggested that TMEM18 overexpression decreased ß-catenin expression, whereas the TMEM18 knockdown enhanced ß-catenin expression and promoted its nuclear translocation. The positive effects on osteogenic differentiation of rat BMSCs owing to the TMEM18 knockdown were attenuated by ß-catenin downregulation. Taken together, these results indicate that TMEM18 plays an inhibitory role in osteogenic differentiation of BMSCs via inactivation of ß-catenin.

Roles of microRNA-22 in Suppressing Proliferation and Promoting Sensitivity of Osteosarcoma Cells via Metadherin-mediated Autophagy.

OBJECTIVE: To analyze the effect of microRNA-22 on autophagy and proliferation and to investigate the underlying molecular mechanism of osteosarcoma cell chemotherapy sensitivity. METHODS: MG-63 cells were divided into four groups, including a control group, a negative control (NC) group, a cisplatin group, and a cisplatin + miR-22 group. Proliferation of MG-63 cells that had been treated with cisplatin and transfected with miR-22 mimics was determined using MTT assay and colony formation assay. We assessed the degree of autophagy using flow cytometry through cellular staining of the autophagy lysosomal marker monodansylcadaverine (MDC). The effect of microRNA-22 on autophagy was observed along with the expression levels of Beclin1, LC3, metadherin (MTDH) and ATG5 by western blot and quantitative reverse transcription polymerase chain reaction (qRT-PCR). Luciferase reporter assay revealed the targeted binding site between miR-22 and the 3'-untranslated region (3'-UTR) of MTDH mRNA. Western blot and qRT-PCR were used to explore the level of MTDH in the control group, the NC group, the cisplatin group, and the miR-22 group for 6, 12, and 24 h. RESULTS: In the in vitro study, the MTT results indicated that the MG-63 cells with overexpression of miR-22 exhibited a significant decline in the proliferation capacity compared with the control group (0.513 ± 0.001, P < 0.0005). Similar to the MTT results, MG-63 cells that were transfected with miR-22 mimic (101.0 ± 10.58) formed fewer colonies compared with the cisplatin group (129.7 ± 4.163). MDC staining revealed that miR-22-overexpressing osteosarcoma (OS) cells treated with cisplatin showed a significant decrease in the expression of autophagy (7.747 ± 0.117, P < 0.0001). Our data revealed that miR-22 could regulate not only autophagy but also proliferation in the chemosensitivity of osteosarcoma cells. We found that miR-22 sensitized osteosarcoma cells to cisplatin treatment by regulating autophagy-related genes. In addition, Luciferase Reporter Assay revealed that miR-22 negatively regulated autophagy through direct targeting of MTDH. We performed western blot analysis to detect the MTDH expression level. The results revealed that the overexpression of miR-22 obviously decreased the expression of MTDH (1.081 ± 0.023, P < 0.001). CONCLUSION: Inhibition of miR-22 ameliorated the anticancer drug-induced cell proliferation decrease in osteosarcoma cells. MTDH was identified as the miR-22 target in OS cells and MTDH-triggered autophagy played a key function in the miR-22-associated chemotherapy sensitivity.

CircRNA CDR1as/miR-7 signals promote tumor growth of osteosarcoma with a potential therapeutic and diagnostic value.

BACKGROUND: The circular RNA (circRNA) antisense to the cerebellar degeneration-related protein 1 transcript (CDR1as)/micro RNA-7(miR-7) signal axis has been investigated in many diseases via regulation of the target genes of miR-7, which participates in the carcinogenesis and metastasis. However, the clinical role and function of CDR1as/miR-7 pathway in osteosarcoma (OS) remain to be identified. MATERIALS AND METHODS: Noncancerous bone tissues (n=18) and OS tissues (n=38) were used to determine the expressions and roles of CDR1as and miR-7. We knocked down the expression of CDR1as via siRNAs in OS cell lines to analyze its function in vitro and in vivo. RESULTS: CDR1as was upregulated in OS tissues with significant diagnostic value (cutoff value: 1.613). OS patients with high tumor size, Enneking stage, and distant metastasis have high CDR1as levels, but the miR-7 as tumor suppressor negatively correlated with CDR1as. Inhibition of CDR1as in OS cell lines U2OS and MG63 with high CDR1as levels, leading to de-repressed miR-7 levels, impaired cell vitality and increased apoptosis and G1/S arrest in parallel with reduced ability of cell migration, which, however, could be restored by miR-7 inhibitor. Mechanistically, knockdown of CDR1as could restore the availability of miR-7 and inhibit the target genes of miR-7 including EGFR, CCNE1, PI3KCD, and RAF1. Moreover, CDR1as also upregulated N-cadherin and inhibited E-cadherin to promote the epithelial-mesenchymal transition via miR-7 for cell migration. CDR1as inhibition in vivo also induced tumor regression with decreased PCNA levels, and miR-7 inhibitor could reverse these effects via upregulation of EGFR, CCNE1, PI3KCD, and RAF1. The expressions of these genes were confirmed to be higher in CDR1as-high OS samples than in CDR1as-low OS samples. CONCLUSION: These findings suggested that the CDR1as/miR-7 signal axis could be the molecular target for the treatment of OS.