ABSTRACT
BACKGROUND: Osteosarcoma is a primary cause of cancer-associated death in children and adolescents worldwide. Long non-coding RNAs SNHG16 (lncRNA SNHG16) and integrin subunit-a 6 (ITGA6) are recently reported to be involved in the tumorigenesis of osteosarcoma by multiple mechanisms. However, the correlation between SNHG16 and ITGA6 in osteosarcoma remains undetermined. METHODS: Expression of miR-488, SNHG16 and ITGA6, as well as epithelial-mesenchymal transition (EMT) associated markers in osteosarcoma tissues and cell lines were examined by qRT-PCR or Western blotting. Effects of miR-488, SNHG16 and ITGA6 on cell migration, invasion were evaluated by wound-healing assay and transwell assay. Bioinformatics analysis and dual-luciferase reported assays were applied to assess the interaction among miR-488, SNHG16 and ITGA6. RNA immunoprecipitation (RIP) was also used to verify SNHG16 and miR-488 interaction. Finally, animal study was used to detect the effect of SNHG16 on osteosarcoma in vivo. RESULTS: SNHG16 and ITGA6 were significantly increased while miR-488 was decreased in osteosarcoma. ITGA6 was screened as a target gene of miR-488, and SNHG16 was sponged by miR-488 in osteosarcoma cells. MiR-488 overexpression and SNHG16 knockdown suppressed migration, invasion and EMT of osteosarcoma cells. Moreover, rescue assays proved that the influences of SNHG16 on osteosarcoma cells migration, invasion and EMT were dependent on miR-488 and ITGA6. In addition, the promotive effects of SNHG16 on osteosarcoma tumor growth and metastasis were further supported by xenograft tumor growth assay. CONCLUSION: SNHG16 promoted migration, invasion and EMT of osteosarcoma by sponging miR-488 to release ITGA6.
ABSTRACT
This study designs to investigate the role and potential mechanism of lncNRA HOTTIP in OS progression in vitro and in vivo. HOTTIP, PTBP1, and KHSRP expression levels were tested through qRT-PCR and western blot in OS tissues or cell lines. Cell proliferation was examined via CCK-8 and colony formation. Cell cycle and apoptosis were analyzed via flow cytometry analysis. The invasive and migratory abilities of OS cells were evaluated by transwell and wound-healing assays. The localization of HOTTIP in OS cells was determined by subcellular fractionation assay. RNA pull down and RNA immunoprecipitation were allowed to assess the interaction between HOTTIP and PTBP1. Xenograft tumor growth assay was employed to test the role of HOTTIP and KHSRP in OS progression. Our data demonstrated HOTTIP was upregulated in OS tissues. HOTTIP knockdown resulted in a suppression of OS cell proliferation, invasion and migration, as well as a promotion of OS cell apoptosis, while HOTTIP overexpression exhibited opposite effects. In mechanism, PTBP1 and KHSRP highly expressed in OS and HOTTIP was identified to interact with PTBP1 to promote KHSRP expression. Meanwhile, we found that overexpression of KHSRP or PTBP1, individually, can partially remove the repression of HOTTIP suppression for OS cell progression. Moreover, xenograft tumor growth assay revealed that HOTTIP knockdown significantly inhibited tumor growth, and this inhibitory effect was abolished by KHSRP overexpression. Collectively, these findings confirmed that HOTTIP facilitates OS cell proliferation, invasion and migration by binding to PTBP1 to promote KHSRP level. Abbreviation: LncRNA: long noncoding RNA; HOTTIP: HOXA distal transcript antisense RNA; KHSRP: KH-Type Splicing Regulatory Protein; qRT-PCR: quantitative real-time PCR; OS: osteosarcoma; OST: osteosarcoma tissues; ANT: adjacent normal tissue.
Subject(s)
Bone Neoplasms/metabolism , Cell Proliferation/physiology , Heterogeneous-Nuclear Ribonucleoproteins/metabolism , Osteosarcoma/metabolism , Polypyrimidine Tract-Binding Protein/metabolism , RNA, Long Noncoding/metabolism , RNA-Binding Proteins/metabolism , Trans-Activators/metabolism , Adolescent , Animals , Bone Neoplasms/genetics , Cell Movement/physiology , Child , Female , Heterogeneous-Nuclear Ribonucleoproteins/genetics , Humans , Male , Mice , Mice, Nude , Neoplasm Invasiveness/genetics , Neoplasm Invasiveness/pathology , Osteosarcoma/genetics , Polypyrimidine Tract-Binding Protein/genetics , Protein Binding/physiology , RNA, Long Noncoding/genetics , RNA-Binding Proteins/genetics , Trans-Activators/genetics , Tumor Cells, Cultured , Xenograft Model Antitumor Assays , Young AdultABSTRACT
BACKGROUND: Invasion and metastasis of synovial sarcoma is the leading cause of death in patients. Epithelial mesenchymal transition (EMT) accelerates tumor cell invasion and metastasis. MiR-9 promotes tumor metastasis by inducing EMT. However, the role of miR-9 in synovial sarcoma is still not clear. METHODS: Overexpression or knockdown of miR-9 in human synovial sarcoma (HSS) cell lines was carried out by miR-9 mimics or miR-9 inhibitors transfection. Cell proliferation, apoptosis, migration and invasion were detected using MTS and colony formation assays, flow cytometry, wound healing and transwell assays, respectively. Luciferase reporter assay was applied to study the interaction between miR-9 and CDH1. Nude mice xenograft model was established, and immunohistochemistry staining assessed Ki-67 level. The related mRNA and protein expression levels were evaluated by qRT-PCR and Western blotting. RESULTS: The bioinformatics analyses and luciferase reporter assay showed that miR-9 can target CDH1 3'-UTR. Moreover, miR-9 could induce EMT of HSS cells via targeting CDH1. The negative regulation of miR-9 on CDH1 expression was also confirmed in a mouse xenograft model of synovial sarcoma. Furthermore, miR-9 was observed to induce HSS cell proliferation, migration and invasion and inhibit apoptosis. MAPK/ERK and Wnt/ß-catenin signal pathways were activated by the miR-9 overexpression in HSS cells, and then further enhancing tumorigenesis of HSS, which was further confirmed in the mouse model. CONCLUSION: MiR-9 induces EMT by targeting CDH1, and activates MAPK/ERK and Wnt/ß-catenin signal pathways, thus promoting HSS tumorigenesis.
Subject(s)
Antigens, CD/metabolism , Cadherins/metabolism , Cell Movement , MicroRNAs/metabolism , Neoplasm Proteins/metabolism , RNA, Neoplasm/metabolism , Sarcoma, Synovial/metabolism , Animals , Antigens, CD/genetics , Cadherins/genetics , Carcinogenesis/genetics , Carcinogenesis/metabolism , Carcinogenesis/pathology , Cell Line, Tumor , Epithelial-Mesenchymal Transition/genetics , Humans , Male , Mice , Mice, Inbred BALB C , Mice, Nude , MicroRNAs/genetics , Neoplasm Invasiveness/genetics , Neoplasm Proteins/genetics , RNA, Neoplasm/genetics , Sarcoma, Synovial/genetics , Sarcoma, Synovial/pathologyABSTRACT
We report our experience with video-assisted thoracoscopy in the surgical closure of heart septal defects. Nine patients, aged 10 to 26 years, underwent operation for closure of an atrial septal defect; and 3, aged 10 to 22 years, for closure of a ventricular septal defect. Three minithoracotomies with a diameter of 2 to 3 cm were made in the fourth intercostal space of the right parasternum and the fourth and seventh intercostal spaces of the right middle axillary line, respectively. Through the openings and guided by a thoracoscope, a catheter was inserted into the superior vena cava, femorofemoral extracorporeal circulation was built, the aorta was crossclamped, and the myocardium was protected by cold cardioplegia. The right atrium was opened, and the defect was exposed with a traction suture. Primary closure of defects was performed successfully in all patients. The duration of aortic crossclamping and extracorporeal circulation ranged from 11 to 56 minutes and from 50 to 168 minutes, respectively. Postoperatively, cardiac murmur disappeared and echocardiograms showed no residual shunt. Repair of heart septal defects can be completely done with the assistance of video-assisted thoracoscopy, offering a new option with minimal incision.
