Prognostic value of Serum miR-217 Level in Osteosarcoma Patients

Background: MicroRNA (miR)-217 is a tumor suppressor signicantly associated with osteosarcoma. We try to evaluate serum levels miR-217 in osteosarcoma patients and evaluate its prognostic signicance. Methods: A total of 163 consecutive osteosarcoma patients and 96 healthy participates were enrolled. Serum miR-217 levels were evaluated by using real-time quantitative reverse transcription polymerase chain reactions(RT-PCR). The association between serum miR-217 level and survival outcomes was evaluated by univariate and multivariate analysis. Results: Serum miR-217 levels in osteosarcoma patients was signicantly lower than healthy volunteers (P<0.05). Low serum miR-217 was signicantly related to advanced cancer and metastasis (both P<0.05). Moreover, patients with a low serum miR-217 had a poorer overall survival than those with a high serum miR-217 levels (P<0.05). Serum miR-217 level also been showed as independent risk factor for osteosarcoma in multivariate analysis (HR, 0.42; 95%CI: 0.12–0.98; p<0.01). Conclusions: Serum miR-217 levels was signicantly downregulated in osteosarcoma patients and remarkably associated with poor prognosis, indicating that serum miR-217 might serve as a useful diagnostic and prognostic indicator for osteosarcoma. osteosarcoma patients from healthy subjects. Furthermore, we evaluated the signicance of serum miR-217 in predicting prognosis of osteosarcoma patients, and found that a low serum miR-217 level was signicantly associated with shorter survival in osteosarcoma patients. We also observed a signicant association between serum miR-217 level and classical unfavorable clinical characteristics for osteosarcoma patients. Based on such results, we suggested that miR-217 can be used as a diagnostic biomarker for osteosarcoma patients, which also can serve as a promising prognostic indictor.


Introduction
Osteosarcoma is one of the most common and lethal primary sarcoma of the bone in adolescents 1,2 .
Despite considerable advancements of therapeutic and diagnostic strategies over the previous decades, the long-term prognosis of osteosarcoma remains unsatisfactory, with 5-year survival rates less than 60%, even with the use of combined chemotherapy 3,4 . Recurrent and metastasis were considered the main contributors to the low 5-year survival rates in patients with osteosarcoma 5,6 . Lung metastasis is frequently observed in newly diagnosed osteosarcoma, approximately 10-20% of patients are diagnosed with metastatic OS upon identi cation of the disease 7 . Moreover, the potential molecular mechanisms underlying the histological heterogeneity, response to treatments and recurrent are still unclear. Therefore, furtherly understanding of the complex and de nite molecular mechanisms concerning the progression and aggressiveness of osteosarcoma is important for risk strati cation and individual treatment.
MicroRNAs (miRNAs) is a subset of endogenous small non-coding RNAs, and is released though passive leakage from cells in setting of chronic in ammation or injury, active secretion, complex formation with lipoproteins or RNA binding proteins, which can speci cally regulate gene expression by inhibiting the translation and/or decreasing of the stability of speci c protein-coding gene 8,9 . Previous studies showed that dysregulation of miRNAs was signi cantly associated with development of various malignancies, including osteosarcoma [10][11][12][13] . It has been revealed that miRNAs can maintain the stable state in serum samples with appropriate and measurable concentration [14][15][16] . In details, serum miRNAs can be resistant to endogenous ribonuclease activity by being packaged into apoptotic bodies or exosomes, or association with other molecules (e.g., in a RNA-protein complex) or modi cations of the miRNAs that make them resistant to RNase activity 17,18 . In addition, the inherent regulatory function of miRNAs makes it likely that many miRNAs expressed in tumor tissue in uence the biological behavior and clinical phenotype of the tumor. Thus, various serum miRNAs expression have been con rmed as valuable indictors for diagnosis or prognosis of cancer 19,20 .
MiRNA-217 is a tumor suppressor miRNA targeting several oncogenes in different cell type 21,22 . MiR-217 could serve as a tumor suppressor which can inhibit tumor growth 23 . Moreover, miR-217 could also function as a oncogene 24 . Therefore, additional studies will be needed to explore the role of miR-217 in cancers. However, no previous study has been showed to evaluate the value of miR-217 in osteosarcoma, especially for serum miR-217. Therefore, in current study, we try to evaluate serum level of miRNA-217 in osteosarcoma patients and analyze its prognostic value.

Patients
This study protocol was planned by basing on the relevant guidelines or regulations, and conformed to the Declaration of Helsinki. All subjects have provided signed informed consent prior to enrollment. This  25 . The tumor specimens of all patients were pathologically diagnosed as osteosarcoma. A control group enrolled 96 age and sex-matched healthy participates.
All relevant clinical and pathological data of each patient were collected and con rmed. The clinical stages were evaluated basing on the Enneking staging system (ESS). All Patients were regularly followed up though clinical visiting or telephone. Follow-up was lasted from the enrollment to death or June 2019. the primary outcome of interest was survival status. Overall survival (OS) was de ned as from the date of enrollment to the date of death or endpoint.

RNA isolation
Peripheral venous blood (5mL) was collected from each subject prior to treatments for osteosarcoma.
Serum was extracted and transferred to RNase/DNase-free tubes and immediately stored at −80 °C for further process. Total RNA was extracted from each serum sample by using of a miRNA easy Serum/Plasma Kit (Qiagen, Valencia, CA, USA). The RNA concentration and integrity were evauated by using a NanoDrop ND-1000 spectrophotometer (Nanodrop technologie, USA).

Quanti cation of miRNA by qRT-PCR
Total RNA from each subjects was used to reversely transcribe miRNAs to a strand cDNA by using a miScript Reverse Transcription Kit (Qiagen, Valencia, CA, USA). Ampli cations were conducted by using a miScript SYBR Green PCR kit (Qiagen, Valencia, CA, USA). The RT-PCR was performed on Applied Biosystems 7500 Real-Time PCR System (Applied Biosystems, Foster City, CA, USA). Sequence of primer for miR-217 was GTC GTA TCC AGT GCA GGG TCC GAG GTA TTC GCA CTG GAT ACG ACG AAA CCC A. The miR-217 expression levels in each sample were normalized against the miR-16 expression. And the threshold cycle (Ct) values ≥40 was con rmed as an undetectable level. The relative expression level of serum miR-217 was quantitatively evaluated by using the 2-ΔΔCT method.

Statistical Analysis
All statistical analyses were conducted by using the SPSS 20.0 (IBM, USA). The statistical results were considered as signi cant while P <0.05 (two sided). Continuous variables that expressed as mean ± SD were compared by using of analysis of variance (ANOVA), whereas comparisons of categorical variables were conducted by using chi-square or Fisher's exact test, which were presented as frequencies (%). Receiver operating characteristic curve (ROC) analysis was performed to evluate the prognosis value of serum miRNA-217 levels in predicting survival. Kaplan-Meier survival curves for serum miRNA-217 levels predicting survival were analyzed by log-rank test. Univariate and multivariate Cox hazard regression model was employed to evaluate the prognostic factors for osteosarcoma.

Results
Serum miR-217 expression in patients with osteosarcoma Serum miR-217 level was signi cantly downregulated in osteosarcoma patients compared with the healthy subjects (P<0.05, see Figure 1). Furthermore, ROC curve analysis revealed that serum miR-217 level could be used to distinguish osteosarcoma patients from healthy subjects, with a sensitivity of 68.6% and a speci city of 73.2%. The area under the curve (AUC) was 0.75 (95%CI:0.66-0.96, P<0.05, Figure 2).

Relationship between Serum 217 and clinical characteristics
The median value of serum miR-217 level in all 163 osteosarcoma patients was considered as the cut-off point to divide patients into the high miR-217 group (n=77) or low miR-217 group(n=86). The association between serum miR-217 levels and clinical characteristics were evaluated by using Chi-squared test. A statistically signi cant difference was observed between a high serum miR-217 levels and distance metastasis and clinical stage (both P<0.01, Table1). However, other clinical variables, including age, sex, tumor site and size, and pathology were not closely associated with serum miR-217 expression (all p> 0.05). (Table 1). Prognostic signi cance of serum miR-217 level in osteosarcoma patients The results of Kaplan-Meier method and log-rank test showed that patients with a low expression of serum miR-217 had a signi cantly poorer OS than those with a high expression of miR-217 (p=0.03, The univariate and multivariate analysis enrolled age and gender of patients, tumor size, distant metastasis, clinical stage, histological type and serum miR-217 level to determine independent prognostic indictor for osteosarcoma patients. After adjustment for potential confounders, a low serum miR-217 level (hazard ratio, 0.42; 95% con dence interval [CI]: 0.12-0.98, P<0.01) was also an independent predictive factor for survival outcome of osteosarcoma patients (Table 2.)

Discussion
In this study, the results showed that the serum level of miR-217 was signi cantly decreased in osteosarcoma patients comparing with healthy participates. Moreover, serum miR-217 level can be used as a useful marker to discriminate osteosarcoma patients from healthy subjects. Furthermore, we evaluated the signi cance of serum miR-217 in predicting prognosis of osteosarcoma patients, and found that a low serum miR-217 level was signi cantly associated with shorter survival in osteosarcoma patients. We also observed a signi cant association between serum miR-217 level and classical unfavorable clinical characteristics for osteosarcoma patients. Based on such results, we suggested that miR-217 can be used as a diagnostic biomarker for osteosarcoma patients, which also can serve as a promising prognostic indictor.
MiR-217, as a novel tumor biomarker, play critical roles in biological process of cancer development 26 .
The miR-217 has been con rmed as a potential tumor suppressor in many malignancies including osteosarcoma 27,28 . Shen et al. reported that miR-217 was decreased both in cancer cell lines and tissues, which was signi cantly correlated with distant metastasis, and functioned as a tumor suppressive miRNA and inhibits the osteosarcoma tumorigenesis through targeting WASF3 29 . Moreover, miR-217 may be involved in inhibiting of tumor cells proliferation and metastasis through targeting KRAS oncogene 30 . In our study, miR-217 was remarkably downregulated in osteosarcoma patients and signi cantly correlated with poor prognosis, which is consistent with the previous studies mentioned above.
However, the underlying function and origin of serum miR-217 in malignancy have not yet been fully understood. Several potential mechanisms for circulating miRNAs releasing have been reported, including passive leakage from cells in setting of chronic in ammation or injury, active secretion, complex formation with lipoproteins or RNA binding proteins [31][32][33] . Yan et al. reported that serum miR-217 expression was signi cantly decreased in acute myeloid leukemia (AML) patients compared to controls, which was identi ed as an independent marker for the diagnosis and prognosis of AML 34 . It has been reported that low expressions of certain miRNAs were remarkably associated with advanced cancer stage 35 . In this study, we found that low serum level of miR-217 was signi cantly associated with clinical stage of osteosarcoma patients, which is consistent with previous study 36 .
Our study rstly reported that the downregulation of serum miR-217 level in a considerable scale osteosarcoma patients group, which can serve as a serum diagnostic and prognostic biomarker, as well as a novel therapeutic target for osteosarcoma. However, there were several limitations in this study. One limitation was a single center, small sample size and retrospective design of study. A large-scale, prospective and multicenter study is required to furtherly reevaluate such results. Furthermore, the underlying roles and mechanisms of miR-217 in development of osteosarcoma have not yet been fully evaluated. Future experiments are need to be performed to elucidate the mechanisms of serum miR-217 in carcinogenesis.

Conclusions
In this study, we found that serum miR-217 levels were downregulated in osteosarcoma patients. Moreover, low serum miR-217 level was signi cantly associated with poor survival of osteosarcoma patients, indicating that miR-217 acting as a tumor suppressor might not only serve as a diagnostic and prognostic indictor for osteosarcoma, but also a potential novel treatment target.

List Of Abbreviations
MiR, microRNA; RT-PCR, real-time quantitative reverse transcription polymerase chain reactions; ESS, Enneking staging system; OS, overall survival; ROC, receiver operating characteristic curve; AUC, area under the curve.

Declarations
Ethics approval and consent to participate: This study was approved by the ethics committee of the First People's Hospital of Jiangxia District(No.20201029).

Consent for publication
All subjects have provided signed informed consent prior to enrollment.

Availability of data and materials
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Competing interests
The authors declare that they have no competing interests Funding There was no funding for this study.