Fibronectin-1 modulated by the long noncoding RNA OIP5-AS1/miR-200b-3p axis contributes to doxorubicin resistance of osteosarcoma cells.

Chemoresistance has been an obstacle in the further improvement of 5-year survival rates of osteosarcoma (OS) patients, but the underlying mechanism of chemo-resistance remains unclear. A comprehensive analysis of mRNAs and noncoding RNAs related to OS chemo-resistance could help solve this problem. In the current study, we first identified that fibronectin-1 (FN1), screened by microarray analysis in three paired chemo-resistant and chemo-sensitive OS cell lines, was significantly upregulated in the chemo-resistant OS cell lines and tissues and was related to unfavourable prognosis. Further functional assays revealed that FN1 inhibition greatly increased the sensitivity of OS cells to doxorubicin in vitro and in vivo, whereas FN1 overexpression had the opposite effect. Moreover, mechanistic investigation demonstrated, by a series of assays that included luciferase reporter gene, RNA immunoprecipitation, RNA pull-down and rescue assays, that FN1 expression was regulated by the oncogenic long noncoding RNA (lncRNA) OIP5-AS1 through sponging miR-200b-3p. Thus, these results indicated the role and potential application of the lncRNA OIP5-AS1/miR-200b-3p/FN1 regulatory pathway as a promising target in treatment of OS chemo-resistance.

Overexpressed circPVT1, a potential new circular RNA biomarker, contributes to doxorubicin and cisplatin resistance of osteosarcoma cells by regulating ABCB1.

Circular RNAs (circRNAs) represent a widespread class of non-coding RNAs generated from back-splicing, with a circular loop structure. Many circRNAs have been reported to play essential roles in cancer development and have the potential to serve as a novel class of biomarkers for clinical diagnosis. However, the role of circRNA in osteosarcoma (OS) remains largely unknown. In the current study, we examined the expression level of circular RNA PVT1 (circPVT1), previously screened and identified the oncogenic role in gastric cancer, in OS and found that circPVT1 was significantly up-regulated in the OS tissues, serums and chemoresistant cell lines, correlated with poor prognosis of OS patients. Besides, ROC curve demonstrated that circPVT1 may be a better diagnostic biomarker than alkaline phosphatase (ALP) in OS with more sensitivity and specificity. In addition, functional assays revealed that circPVT1 knockdown by siRNA could weaken the resistance to doxorubicin and cisplatin of OS cells through decreasing the expression of classical drug resistance-related gene ABCB1. These findings may provide a new insight into the role of circPVT1 as a biomarker for the diagnosis and treatment target of OS.

Circular RNA circ_HIPK3 is down-regulated and suppresses cell proliferation, migration and invasion in osteosarcoma.

Circular RNA (circRNA) is associated with human cancers, however, few studies have reported its value in the diagnosis and prognosis prediction of osteosarcoma (OS). In this study, we investigated the expression level of eight selected cancer-related circRNAs including circ-Cdr1as, circ_HIPK3 and circ-ITCH in OS cell lines, tissues and plasmas by quantitative real-time polymerase chain reaction (qRT-PCR) and found that only circ_HIPK3 could stably down-regulate in the OS cell lines, tissues and plasmas than the corresponding controlled. One-way analysis of variance was further conducted to analyze the relationship between circ_HIPK3 expression level and clinic pathological factors of OS patients. Receiver operating characteristic (ROC) curve was built to evaluate the diagnostic values of circ_HIPK3. Circ_HIPK3 expression was significantly correlated with Enneking stage (P=0.042) and lung metastasis (P=0.036). The area under the ROC curve was 0.783 and the sensitivity and specificity were 0.56 and 0.84, respectively. Kaplan-Maier analysis also showed that lower expression of circ_HIPK3 correlated with shorter overall survival time and poor prognosis of OS patients. Besides, function analysis demonstrated that circHIPK3 overexpression significantly suppressed OS cell proliferation, migration and invasion in vitro. Overall, our data suggest that circ_HIPK3 may become a novel potential biomarker for diagnosis and treatment target of OS.

A novel circulating hsa_circ_0081001 act as a potential biomarker for diagnosis and prognosis of osteosarcoma.

Chemo-resistance and lung metastasis have been the two obstacles in the osteosarcoma (OS) treatment, which is still lack of effective biomarkers for prediction, diagnosis and treatment. Circular RNA (circRNA) is a new type of endogenous noncoding RNA that could serve as ideal biomarkers in cancer because of its stable loop structure. However, little is known about the diagnostic value of circRNAs in OS as well as their associations with clinicopathologic characteristics of OS patients. In the current study, we identified a novel circRNA, hsa_circ_0081001, screened by the RNA sequencing in the three paired chemo-resistant and chemo-sensitive OS cell lines (MG63/DXR vs MG63, KHOS/DXR vs KHOS, U2OS/DXR vs U2OS), and found that hsa_circ_0081001 was significantly up-regulated in the OS cell lines, tissues and serums, associated with poor overall survival and cox multivariate analysis showed that hsa_circ_0081001 was a novel independent prognostic factor for OS patients. Then, receiver operating characteristic (ROC) curve analysis revealed that hsa_circ_0081001 could act as a biomarker for the OS diagnosis and prognosis prediction, better than alkaline phosphatase (ALP) and lactate dehydrogenase (LDH). In addition, we preliminarily found that hsa_circ_0081001 expression level may dynamically monitor and reflect the condition changes of OS patients in a small-scale prospective clinical pretest. In conclusion, our study suggested that circulating hsa_circ_0081001 could serve as a potential biomarker and therapeutic target for OS patients.

Screening circular RNA related to chemotherapeutic resistance in osteosarcoma by RNA sequencing.

AIM: To identify circular RNAs (circRNAs) related to osteosarcoma (OS) chemoresistance. MATERIALS & METHODS: CircRNA expression profile was performed in three paired human chemoresistant and chemosensitive OS cell lines by next-generation sequencing. Quantitative real-time-PCR (qRT-PCR) was used to confirm next-generation sequencing data. Bioinformatics analysis was conducted to predict their functions. RESULTS: Eighty circRNAs were dysregulated in the chemoresistant OS cells compared with the control, after validated by qRT-PCR. Bioinformatics analysis showed that some pathways related to drug metabolism were significantly enriched. Additionally, hsa_circ_0004674 was distinctly increased in OS chemoresistant cells and tissues, related to poor prognosis. CircRNA-miRNA-mRNA pathways related to hsa_circ_0004674 were constructed by TargetScan and miRanda. CONCLUSION: CircRNAs may play a role in OS chemoresistance and hsa_circ_0004674 might be a candidate target.

A New Surgical Method of U-Shaped Myometrial Excavation and Modified Suture Approach with Uterus Preservation for Diffuse Adenomyosis.

OBJECTIVE: To evaluate the feasibility, safety, and efficacy of a new surgical method of U-shaped myometrial excavation and modified suture approach with uterus preservation for diffuse adenomyosis. METHODS: From January 2012 to December 2014, 198 patients with diffuse adenomyosis were surgically treated using this novel procedure in Zhengzhou Hua-Shan Hospital. Degree of dysmenorrhea, menstrual blood volume, serum CA 125, and uterine size before and at 1 month, 3 months, 6 months, 12 months, and 24 months after surgery were compared. RESULTS: Postoperatively, VAS score of dysmenorrhea, menstrual blood volume, serum CA 125 level, and uterine size significantly decreased at 1 month, 3 months, 6 months, 12 months, and 24 months from presurgical levels (all p < .001), but there were no differences at the follow-up time points. Two patients recurred at 18 months and 23 months after surgery, but both recovered after repeat surgery. Interestingly, 2 other patients recrudesced at 10 months and 12 months after surgery. In addition, only one patient was found to have a postoperative anaemia with fever, conservatively managed without surgery. CONCLUSION: U-shaped myometrial excavation and modified suture approach with uterus preservation is a safe and feasible surgical approach to treat diffuse adenomyosis, with favourable outcomes.

LncRNA FENDRR sensitizes doxorubicin-resistance of osteosarcoma cells through down-regulating ABCB1 and ABCC1.

Long noncoding RNAs (LncRNAs) act as crucial regulators in various cancers including osteosarcoma (OS), yet their potential roles and molecular mechanisms in OS chemoresistance remain unclear. In the present study, we investigated the role and potential regulatory mechanism of the most down-regulated expressed lncRNA, FENDRR screened by our previous lncRNA microarray analysis between the paired doxorubicin-resistant and sensitive human osteosarcoma cell lines (MG63/DXR vs MG63). FENDRR expression was down-regulated in the doxorubicin-resistant OS cell lines and tissues and negatively correlated to the poor prognosis of OS patients. Overexpression of FENDRR suppressed doxorubicin-resistance, G2/M phase of cell cycle, and promoted cell apoptosis of osteosarcoma cells in vitro and tumor growth in vivo whereas FENDRR knockdown had the opposite effects. In addition, we found that FENDRR was mainly located in the cytoplasm and could regulate the drug resistance of osteosarcoma cells by negatively affecting posttranscriptional expression of ABCB1 and ABCC1. Together, our study demonstrated that lncRNA FENDRR may act as an inhibitory molecule of doxorubicin-resistance through down-regulating the expression of ABCB1 and ABCC1 genes in osteosarcoma cells. These findings may extend the function of FENDRR in tumor progression and provide a novel target for reversing OS chemoresistance.

Antisense lncRNA FOXF1-AS1 Promotes Migration and Invasion of Osteosarcoma Cells Through the FOXF1/MMP-2/-9 Pathway.

Osteosarcoma (OS) is the most common primary malignant bone cancer in children and adolescents. Long non-coding RNAs (lncRNAs) have been shown to play significant role in various cancers, including OS. In a previous study, we have reported that a novel antisense lncRNA FOXF1-AS1, also known as FENDRR, could sensitize doxorubicin-resistance of OS cells through down-regulating ABCB1 and ABCC1. Here in, the critical role of FOXF1-AS1 in regulating OS progression was further investigated. Firstly, we found that FOXF1-AS1 and its antisense transcript FOXF1 expression were positively up-regulated in OS tissues and cell lines and correlated with poor prognosis of OS patients. Besides, FOXF1-AS1 as well as FOXF1 silencing significantly inhibited cell proliferation, migration, invasion of OS cells and tumor growth both in vitro and vivo through decreasing the expression of MMP2 and MMP9, whereas enhanced expression of FOXF1-AS1 had the opposite effects. In addition, mechanistically, both of FOXF1-AS1 and FOXF1 could regulate the expression of MMP2 and MMP9 at mRNA and protein levels, whereas FOXF1-AS1 could influence the FOXF1expression but FOXF1 did not have the same effect on FOXF1-AS1. Rescue assay further showed that FOXF1-AS1 overexpression efficiently reversed the knockdown of MMP2 and MMP9 expression induced by si-FOXF1. Thus, we concluded that FOXF1-AS1 may promote migration and invasion of OS cells through the FOXF1/MMP-2/-9 pathway. Taken together, these findings demonstrated the underlying mechanism of FOXF1-AS1 in the regulation of OS progression and provide a novel potential target in the OS therapy.