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Objective: The aim of this study was to investigate sclerostin (SOST) expression in a rat model of experimental tympanosclerosis (TS) and its possible role in the formation of TS. Materials and Methods: Thirty-four SD rats were randomly divided into 2 groups: experimental group (n = 17) and normal group (n = 17). The left tympanic cavities in the experimental group were inoculated with methicillin-resistant Staphylococcus aureus. The changes of tympanic membranes were examined and recorded under otoendoscope. Haematoxylin-eosin staining was adopted to detect the morphological changes in the tympanic membrane and middle ear mucosa. Immunohistochemistry and Western blot analysis were used to observe the expression of SOST, Wnt3a, β-catenin, and P-ERK1/2. Results: In the experimental group, sclerotic lesions were observed in 54.5% ears in the end of 6 weeks. Morphological changes such as mucosa incrassation, inflammatory cells infiltration, fibrous tissue proliferation, and interstitial tissue incrassation prominently appeared in the tympanic membrane and middle ear mucosa. SOST protein was mainly distributed in the cytoplasm of epithelial cells and gland cells, the expression of which increased significantly in the calcified experimental ears. In addition, expression levels of Wnt3a, β-catenin, and P-ERK1/2 increased significantly in the calcified group too. Conclusion: The upregulated expression level of SOST may be involved in the formation of TS, first, through the pro-phosphorylation of ERK1/2 in the inflammatory stage, and then through the enhancement of Wnt3a in the osteogenic stage.

Purpose: Some epidemiological studies and animal studies have reported a relationship between leukocyte telomere length (LTL) and bone mineral density (BMD). However, the causality underlying the purported relationship has not been determined. Here we performed a two-sample MR analysis to test the causal link between telomere length and BMD. Results: Our research suggested no causal link of LTL and BMD using IVW method. The weighted median, MR-Egger regression and MR.RAPS method yielded a similar pattern of effects. MR-Egger intercept test demonstrated our results were not influenced by pleiotropy. Heterogeneities among the genetic variants on heel estimated BMD and TB-BMD vanished after excluding rs6028466. “Leave-one-out” sensitivity analysis confirmed the stability of our results. Conclusion: Our MR analysis did not support causal effect of telomere length on BMD. Methods: We utilized 5 independent SNPs robustly associated with LTL as instrument variables. The outcome results were obtained from GWAS summary data of BMD. The two-sample MR analysis was conducted using IVW, weighted median, MR-Egger regression and MR.RAPS method. MR-Egger intercept test, Cochran’s Q test and I2 statistics and “leave-one-out” sensitivity analysis were performed to evaluate the horizontal pleiotropy, heterogeneities and stability of these genetic variants on BMD.

STUDY DESIGN Meta-analysis to collect relevant studies to assess the association between COL11A1 and GDF5 genetic variants and susceptibility to IDD. OBJECTIVE The aim of this study was to assess whether or not COL11A1 and GDF5 genetic variants were associated with susceptibility to IDD. SUMMARY OF BACKGROUND DATA IDD or LDH is a major public health problem. There have been several studies evaluating the relationship between COL11A1 and GDF5 genetic variants with risk of intervertebral disc degeneration (IDD). However, the studies were limited in discrete outcome and sample size, and some of the results were contradictory. METHODS We systematically searched the relevant publications in electronic databases. Eligible studies were included based on the defined criteria. The pooled odds ratios (ORs) with its 95% confidence intervals (CIs) were received using STATA 15. Subgroup analysis, sensitivity analysis, publication bias, and the "Trim and fill" method were performed in the meta-analysis. RESULTS A total of 3287 IDD cases and 5115 controls were incorporated into the meta-analysis. Our results demonstrated that COL11A1 rs1676486 was significantly associated with increased IDD susceptibility under all genetic models (allele model T vs. C: OR = 1.40, 95% CI 1.23-1.59, P = 0.000; homozygote model TT vs. CC: OR = 1.89, 95%CI 1.40-2.56, P = 0.000; dominant model TT+TC vs. CC: OR = 1.52, 95% CI 1.29-1.80, P = 0.000; recessive model TT vs. TC + CC: OR = 1.58, 95% CI 1.18-2.12, P = 0.002). However, GDF5 rs143383 was not (allele model T vs. C: OR = 1.15, 95% CI 0.91-1.44, P = 0.244; homozygote model TT vs. CC: OR = 1.22, 95% CI 0.75-2.00, P = 0.429; dominant model TT vs. CC+CT: OR = 1.22, 95% CI 0.95-1.57, P = 0.112; recessive model TC + TT vs. CC: OR = 1.12, 95% CI 0.73-1.73, P = 0.594). Subgroup analysis indicated ethnicity was not the source of heterogeneity. Sensitivity analysis, publication bias, and the "Trim and fill" method demonstrated the meta-analysis was of reliability. CONCLUSION Our results suggested that COL11A1 rs1676486 was significantly associated with IDD and the T allele was a risky factor. However, GDF5 rs143383 was not. LEVEL OF EVIDENCE 1.

Background: Osteoporosis is a complex disorder, with multiple biopsychosocial contributors. As a principal influencing factor of socioeconomic status, education is commonly regarded associated with osteoporosis. However, the associations between education and osteoporosis are inconsistent in different researches, and we aim to investigate it under two-sample Mendelian randomization analysis framework. Methods: A series of quality control steps were taken in our analysis to select eligible instrumental SNPs which were strongly associated with exposure. To make the results more robust and reliable, we utilized several robust analytical methods (inverse-variance weighting, weighted median, MR-Egger regression and MR.RAPS) that based on different assumptions of two-sample Mendelian randomization analysis. MR-Egger intercept test, Cochran’s Q test and “leave-one-out” sensitivity analysis were performed to remove outliers and evaluate the horizontal pleiotropy, heterogeneities and stability of these genetic variants on outcomes. Outlier variants identified by MR-PRESSO outlier test were removed to reduce heterogeneity and the effect of horizontal pleiotropy. Two-sample Mendelian randomization approach was also employed to determine the mechanism by which education affects osteoporosis. Results: Our two-sample Mendelian randomization analyses suggested a causal effect of longer educational attainment on lower risk of osteoporosis and higher heel-BMD, FN-BMD and LS-BMD, but lack of evidence on lower risk of osteoporostic fracture or higher FA-BMD. Our MR analyses also determined the probability of serum lipid level, smoking, magnesium supplements, selenium supplements and physical activity working as mediators in the causal effect of educational attainment on osteoporosis. Interpretation: Our results can be helpful for education policy making to increase BMD, or reminding clinicians of taking integrated biopsychosocial measures and efforts to prevent or intervene with bone loss when patients are high risk population of osteoporosis. Funding Statement: Not applicable Declaration of Interests: The authors declare that they have no competing interests. Ethics Approval Statement: Our analysis used published study or publicly available GWAS summary data. No original data was collected for this manuscript, and thus, no ethical committee approval was required. Each study included was approved by their institutional ethics review committees, all participants provided written informed consent.

Background An increasing number of studies have demonstrated that long non-coding RNAs (lncRNAs) play pivotal roles in cancer onset and development. LncRNA AFAP1-AS1 has been validated to be abnormally upregulated and play oncogenic roles in various malignant tumors. The biological role and mechanism of AFAP1-AS1 in OS (osteosarcoma) remains unclear. Methods Quantitative reverse transcription PCR (qRT-PCR) is applied to examine AFAP1-AS1 expression in OS tissues and OS cell lines. The function of AFAP1-AS1 in OS cells is investigated via in-vitro and in-vivo assays. Western bolt and rescue experiments are applied to detect the expression changes of key molecules including epithelial-mesenchymal transition markers and identify the underlying molecular mechanism. RNA immunoprecipitation is performed to reveal the interaction between AFAP1-AS1 and RhoC. Results AFAP1-AS1 expression is upregulated in human OS tissues and cell lines. AFAP1-AS1 knockdown induces OS cell apoptosis and cell cycle G0/G1 arrest, suppresses OS cells growth, migration, invasion, vasculogenic mimicry formation and epithelial-mesenchymal transition (EMT), and affects actin filament integrity. AFAP1-AS1 knockdown suppresses OS tumor formation and growth in nude mice. AFAP1-AS1 knockdown elicits a signaling inhibition including decreased levels of RhoC, ROCK1, p38MAPK and Twist1. Moreover, AFAP1-AS1 interacts with RhoC. Overexpression of RhoC can partly reverse AFAP1-AS1 downregulation-induced cell EMT inhibition. Conclusions AFAP1-AS1 is overexpressed in osteosarcoma and plays an oncogenic role in osteosarcoma through RhoC/ROCK1/p38MAPK/Twist1 signaling pathway, in which RhoC acts as the interaction target of AFAP1-AS1. Our findings indicated a novel molecular mechanism underlying the tumorigenesis and progression of osteosarcoma. AFAP1-AS1 could serve as a promising therapeutic target in OS treatment. Electronic supplementary material The online version of this article (10.1186/s13046-019-1363-0) contains supplementary material, which is available to authorized users.

Binwu Hu,1 Deyao Shi,1 Xiao Lv,1 Fashuai Wu,1 Songfeng Chen,2 Zengwu Shao1 1Department of Orthopaedics, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China; 2Department of Orthopaedic Surgery, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, China Background: DEP domain containing mammalian target of rapamycin (mTOR)-interacting protein (DEPTOR), a recently discovered endogenous inhibitor of mTOR, has been found to be abnormally expressed in various tumors. Recent studies have demonstrated that DEPTOR could serve as a potential prognostic biomarker in several kinds of cancer. However, the prognostic value of DEPTOR is still controversial so far. Patients and methods: PubMed, Embase and Web of Science were systematically searched to obtain all relevant articles about the prognostic value of DEPTOR in cancer patients. ORs or HRs with corresponding 95% CIs were pooled to estimate the association between DEPTOR expression and the clinicopathological characteristics or survival of cancer patients. Results: A total of nine eligible studies with 974 cancer patients were included in our meta-analysis. Our results demonstrated that the expression of DEPTOR was not associated with the overall survival (OS) (pooled HR=0.795, 95% CI=0.252–2.509) and event-free survival (EFS) (pooled HR=1.244, 95% CI=0.543–2.848) in cancer patients. Furthermore, subgroup analysis divided by sample size, type of cancer, Newcastle–Ottawa Scale (NOS) score and evaluation of DEPTOR expression showed identical prognostic value. In addition, our analysis also revealed that there was no significant association between expression level of DEPTOR and clinicopathological characteristics, such as tumor stage, lymph node metastasis, differentiation grade and gender. Conclusion: Our meta-analysis suggested that despite the fact that DEPTOR could be overexpressed or downregulated in cancer patients, it might not be a potential marker to predict the prognosis of cancer patients. Keywords: DEPTOR, cancer, overall survival, event-free survival, meta-analysis

Purpose: Programmed cell death 1 ligand-1 (PD-L1) and PD-1 as prognostic biomarkers have spurred considerable interest in several types of malignant tumors. In the present meta-analysis, we aimed to elucidate the clinicopathological and prognostic values of PD-L1/PD-1 in osteosarcoma. Methods: We systematically searched PubMed, Web of Science, EMBASE, Scopus, CBM and the Cochrane Library databases up to March 3, 2018. Eligible studies assessing the relationship between PD-L1 or PD-1 expression and clinicopathological and prognostic outcomes in osteosarcoma were incorporated. Pooled relative risks (RRs) and 95% confidence intervals (CIs) were used to estimate the outcomes. Results: Eight studies involving 413 patients were incorporated into our meta-analysis. Pooled results showed that PD-L1/PD-1 overexpression was significantly associated with metastasis (RR = 1.54, 95% CI: 1.12-2.11, p = 0.008) in osteosarcoma. Furthermore, osteosarcoma patients exhibited a remarkably higher total mortality risk (RR = 1.86, 95% CI: 1.09-3.17, p = 0.021) with PD-L1/PD-1 overexpression. However, no significant reduced overall survival rate (RR = 0.70, 95% CI: 0.46-1.07, p = 0.103) was detected in the study. Conclusion: Our meta-analysis indicates that PD-L1/PD-1 may serve as an important biomarker for adverse clinicopathologic features and poor prognosis in patients with osteosarcoma.

// Binlong Zhong 1, * , Donghua Huang 1, * , Kaige Ma 1 , Xiangyu Deng 1 , Deyao Shi 1 , Fashuai Wu 1 and Zengwu Shao 1 1 Department of Orthopaedic Surgery, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022, People’s Republic of China * These authors contributed equally to this study and share first authorship Correspondence to: Zengwu Shao, email: szwpro@163.com Keywords: COL1A1 polymorphism, rs1800012, musculoskeletal degenerative diseases, osteoarthritis, intervertebral disc degeneration Received: June 27, 2017 Accepted: August 23, 2017 Published: September 08, 2017 ABSTRACT It has been reported that the single nucleotide polymorphism (SNP) rs1800012 in COL1A1 gene might be linked to the susceptibility of musculoskeletal degenerative diseases, such as osteoarthritis (OA) and intervertebral disc degeneration (IVDD). However, the data from different studies is contradictory. Here we aimed to comprehensively summarize and clarify the relationship between the SNP and musculoskeletal degenerative diseases. Seven eligible studies including 1339 cases and 5406 controls were screened out from PubMed, Web Of Science and Cochrane library databases. Significant association was identified in sub group analysis of IVDD in homozygote model (GG versus TT: OR = 0.33, 95% CI 0.14–0.78, P = 0.012), heterozygote model (GT versus TT: OR = 0.29, 95% CI 0.11–0.72, P = 0.008) and dominant model (GG/GT versus TT: OR = 0.31, 95% CI 0.13–0.74, P = 0.008). Additionally, significant relationship was also found in sub group analysis of severe degree of IVDD in homozygote model (GG versus TT: OR = 0.37, 95% CI 0.15–0.91, P = 0.031), heterozygote model (GT versus TT: OR = 0.33, 95% CI 0.13–0.87, P = 0.024) and dominant model (GG/GT versus TT: OR = 0.36, 95% CI 0.14–0.88, P = 0.025). Although no significance was observed, there is a trend that the more G allele at COL1A1 rs1800012 site, the less possibility of IVDD and severe IVDD would happen. Our results indicate that COL1A1 rs1800012 polymorphism associates with the susceptibility of IVDD. However, this polymorphism may not be associated with OA risk.

Osteosarcoma (OS) is the most common malignant bone tumor. The prognosis of metastatic and recurrent OS patients still remains unsatisfactory. Cisplatin reveals undeniable anti-tumor effect while induces severe side effects that threatening patients’ health. Dynasore, a cell-permeable small molecule that inhibits dynamin activity, has been widely studied in endocytosis and phagocytosis. However, the anti-tumor effect of dynasore on OS has not yet been ascertained. In the present study, we suggested that dynasore inhibited cell proliferation, migration, invasion, and induced G0/G1 arrest of OS cells. Besides, dynasore repressed tumorigenesis of OS in xenograft mouse model. In addition, we demonstrated that dynasore improved the anti-tumor effect of cisplatin in vitro and in vivo without inducing nephrotoxicity and hepatotoxicity. Mechanistically, dynasore repressed the expression of CCND1, CDK4, p-Rb, and MMP-2. Furthermore, we found that dynasore exerts anti-tumor effects in OS partially via inhibiting STAT3 signaling pathway but not ERK-MAPK, PI3K-Akt or SAPK/JNK pathways. P38 MAPK pathway served as a negative regulatory mechanism in dynasore induced anti-OS effects. Taken together, our study indicated that dynasore does suppress cell proliferation, migration, and invasion via STAT3 signaling pathway, and enhances the antitumor capacity of cisplatin in OS. Our results suggest that dynasore is a novel candidate drug to inhibit the tumor growth of OS and enhance the anti-tumor effects of cisplatin.

TP53 is a tumor suppressor gene which is essential for regulating cell division and preventing tumor formation. Several studies have assessed the associations of TP53 single-nucleotide polymorphisms (SNP) with susceptibility of malignant bone tumors, including osteosarcoma and Ewing sarcoma, but the results are inconsistent. In the present meta-analysis, we aimed to elucidate the associations of TP53 rs1042522 genetic polymorphism with the risk of osteosarcoma or Ewing sarcoma. We systematically searched Medline, PubMed, Web of Science, Embase, and the Cochrane Library databases. Eligible studies assessing the polymorphisms in the TP53 rs1042522 gene and risk of malignant bone tumors were incorporated. The pooled odds ratio (OR) with its 95% confidence intervals (95% CIs) were used to assess these possible associations. Five studies with a total of 567 cases and 935 controls were finally included the meta-analysis. Meta-analysis of TP53 rs1042522 polymorphism was significantly associated with an increased risk of malignant bone tumors (G versus C: OR = 1.27, 95% CI 1.08–1.50, P=0.005; GG versus GC/CC: OR = 1.55, 95% CI 1.21–2.00, P=0.001). Moreover, in a stratified analysis, a statistically significant correlation between this SNP and osteosarcoma risk was also observed. Our results suggest that there are significant associations of TP53 rs1042522 polymorphism with malignant bone tumors risk. More studies based on larger sample sizes and homogeneous samples are warranted to confirm these findings.

The primer sequences for PCR and the sequences of siRNAs used in this study. (DOCX 15 kb)

Clinicopathological characteristics of the patients enrolled in this study. (DOCX 16 kb)

// Fashuai Wu 1, * , Deyao Shi 1, * , Shidai Mu 2 , Yu Huang 3 , Feng Gao 1 , Xiangcheng Qing 1 and Zengwu Shao 1 1 Department of Orthopaedics, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022, China 2 Institute of Hematology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022, China 3 Department of Otorhinolaryngology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022, China * These authors contributed equally to this work Correspondence to: Zengwu Shao, email: profszw1962@163.com Keywords: lncRNA; osteosarcoma; prognosis; biomarker; meta-analysis Received: May 23, 2017 Accepted: December 26, 2017 Published: January 12, 2018 ABSTRACT Long non-coding RNAs (lncRNAs) contribute to progression of various cancers including osteosarcoma through abnormal regulation of cancer-related cellular processes. Recent researches have shown that various lncRNAs are abnormally expressed in osteosarcoma and associated with the prognosis. With the aim of gaining a better insight into the association between expression level of lncRNAs and prognosis of osteosarcoma, multiple databases including PubMed, Embase, Cochrane Library and Web of Science were carefully searched for available studies up to March 14, 2017. Finally, 19 publications with 1298 patients matched our inclusion criteria and were evaluated in our meta-analysis. The quality of each study was scored using the Newcastle-Ottawa Scale and studies not reaching a minimum threshold were excluded. Results of the meta-analysis demonstrated that abnormal expression level of lncRNAs predicted poor overall survival (pooled HR 3.064, 95% CI: 2.487–3.775) and event-free survival (pooled HR 2.642, 95% CI 1.759–3.970) in osteosarcoma and subgroup analysis showed consistent prognostic value. Furthermore, combining data of Cox multivariable analysis indicated that abnormal expression level of lncRNAs was an independent prognostic marker for overall survival (pooled HR 2.864, 95% CI: 2.246–3.651) in osteosarcoma patients. The clinicopathological parameters analysis further showed that abnormal expression level of lncRNAs was correlated with tumor size, tumor stage, metastasis and differentiation grade of osteosarcoma. Limitations of the meta-analysis included variation of cut-off value definition, Chinese provenance of most studies, publication bias and so on. In conclusion, this meta-analysis suggested that abnormal expression level of lncRNAs has a promising future for predicting the prognosis of patients with osteosarcoma.

Table S3. Summary of meta-analysis for the association of FasL rs5030772 and rs763110 polymorphisms with musculoskeletal degenerative diseases leveled by diagnosis. (DOCX 24 kb)

Table S1. Summary of meta-analysis for the association of Fas rs1800682 and rs2234767 polymorphisms with musculoskeletal degenerative diseases leveled by diagnosis. (DOCX 25 kb)

Table S2. Summary of meta-analysis for the association of FAS rs1800682 and rs2234767 polymorphisms with musculoskeletal degenerative diseases leveled by race groups. (DOCX 23 kb)

Table S4. Summary of meta-analysis for the association of FASL rs5030772 and rs763110 polymorphisms with musculoskeletal degenerative diseases leveled by race groups. (DOCX 22 kb)

Background LncRNA CCAT1 is significantly overexpressed in various types of cancers, suggesting that it might be associated with prognosis and clinicopathological features in patients with cancer. Methods A comprehensive search was performed in Pubmed, Web of Science, OVID and CNKI databases. We also retrieved articles from other sources, such as retrieving from the reference lists of relevant articles. Eligible studies were included based on defined exclusion and inclusion criteria to perform a meta-analysis. STATA 14.0 was used to estimate pooled hazard ratios (HRs) with 95% confidence interval (95% CI), the heterogeneity among studies and publication bias to judge the prognostic value. Results A total of 1587 patients from 11 eligible studies were included in the meta-analysis. The results showed that high expression level of CCAT1 was significantly associated with shorter overall survival in cancer patients (HR 2.335, 95% CI:1.551–3.517); in the subgroup analysis, region (China or UK), sample size (more or less than 100), type of cancer (digestive or non-digestive disease) and paper quality (score more or less than 7) did not alter the association between CCAT1 expression and cancer prognosis but preoperative treatment did. And CCAT1 expression was an independent prognostic marker for overall survival in patients with cancer (pooled HR 2.195, 95%CI:1.316–3.664) using Cox multivariate analyses. The clinicopathological parameters analysis further showed that increased expression level of CCAT1 was correlated with tumor size, lymph node metastasis, TNM stage, distant metastasis, microvascular invasion and capsular formation in relevant cancers. Conclusions The meta-analysis results from present study suggested that increased expression level of CCAT1 was associated with poor prognosis and can serve as an independent biomarker. And the expression level of CCAT1 was associated with clinicopathological features in relevant cancers.

Objective. Excessive apoptosis of nucleus pulposus cells (NPCs) induced by various stresses, including compression, contributes to the development of intervertebral disc degeneration (IVDD). Mesenchymal stem cells (MSCs) can benefit the regeneration of NPCs and delay IVDD, but the underlying molecular mechanism is poorly understood. This study aimed to evaluate the antiapoptosis effects of bone marrow-derived MSC (BMSC) on rat NPCs exposed to compression and investigate whether the mitochondrial pathway was involved. Methods. BMSCs and NPCs were cocultured in the compression apparatus at 1.0 MPa for 36 h. Cell viability, apoptosis, mitochondrial function, and the expression of apoptosis-related proteins were evaluated. Results. The results showed that coculturing with BMSCs increased the cell viability and reduced apoptosis of NPCs exposed to compression. Meanwhile, BMSCs could relieve the compression-induced mitochondrial damage of NPCs by decreasing reactive oxygen species level and maintaining mitochondrial membrane potential as well as mitochondrial integrity. Furthermore, coculturing with BMSCs suppressed the activated caspase-3 and activated caspase-9, decreased the expressions of cytosolic cytochrome c and Bax, and increased the expression of Bcl-2. Conclusions. Our results suggest that BMSCs can protect against compression-induced apoptosis of NPCs by inhibiting the mitochondrial pathway and thus enhance our understanding on the MSC-based therapy for IVDD.