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 study was also approved by the ethics committee of the First People’s Hospital of Jiangxia District (No.20201029). The data of 163 patients with primary osteosarcoma admitted to Department of Orthopaedics between July 1, 2014 and July 1, 2018 were retrospectively collected. Exclusion was conducted based on following criteria: preoperative comorbidity, relapse and metastasis, incomplete clinical and histopathological data and life expectancy less than 4 months. All enrolled patients received the standard preoperative neoadjuvant chemotherapy, then resection and postoperative chemotherapy according to the 2018 European Sarcoma Network Working Group Clinical Practice Guidelines for osteosarcoma25. 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 confirmed. 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 defined as from the date of enrollment to the date of death or endpoint.
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).
Quantification 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). Amplifications 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 confirmed as an undetectable level. The relative expression level of serum miR-217 was quantitatively evaluated by using the 2–ΔΔCT method.
All statistical analyses were conducted by using the SPSS 20.0 (IBM, USA). The statistical results were considered as significant 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.