Ichiro Itonaga

microRNA-93 promotes cell proliferation via targeting of PTEN in Osteosarcoma cells

BackgroundAberrant microRNA (miRNA) expression plays an essential role in osteosarcoma (OS) pathogenesis. Recent studies have shown that dysregulation of miRNA expression is associated with increased tumorigenesis and poor prognosis in several types of cancers, including OS. The aim of this study was to investigate the relevant microRNAs involved in the development of OS.MethodsTo explore possible oncogenic factors in OS, we used a microarray-based approach to profile changes in the expression of miRNAs and their target mRNAs in five OS cell lines and human mesenchymal stem cells (hMSCs). An miRNA, miR-93, was significantly up-regulated, whereas phosphatase and tensin homologue (PTEN) expression was significantly down-regulated in all tested OS cells, when compared with hMSCs.ResultsWhen anti-miR-93 was transfected into OS cell lines, PTEN expression was greatly increased, suggesting that PTEN might be a target of miR-93 in ES cells. The expression of phosphorylated Akt protein, which is known to be inversely correlated with that of PTEN, was significantly down-regulated in anti-miR-93-transfected cells. Furthermore, transfection of anti-miR-93 inhibited the proliferation and cell cycle progression of ES cells. In addition, the down-regulation of miR-93 in these cells significantly suppressed tumor growth in vivo.ConclusionEctopic expression of miR-93 decreased PTEN protein levels. Furthermore, miR-93 increased proliferation and decreased apoptosis in OS cells, whereas its silencing in these cells inhibited such carcinogenic processes. Taking these observations together, miR-93 can be seen to play a critical role in carcinogenesis through suppression of PTEN, and may serve as a therapeutic target for the treatment of OS.

The effect of selective cyclooxygenase-2 inhibitor on human osteoclast precursors to influence osteoclastogenesis in vitro

The inducible prostaglandin synthesis enzyme, cyclooxygenase-2 (COX-2), is involved in bone resorption and osteoclastogenesis, and acts indirectly through prostaglandin E2 (PG E2) produced by osteoblastic cells. This study was undertaken to investigate whether celecoxib (a selective COX-2 inhibitor) has a direct effect on human osteoclast precursors to influence osteoclastogenesis in vitro. Human peripheral blood mononuclear cells (PBMCs) were cultured on glass coverslips and dentine slices with soluble receptor activator of NF-kB ligand (sRANKL) and macrophage colony stimulating factor (M-CSF). COX inhibitors including celecoxib were added to the cultures. Osteoclast formation was assessed as the number of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells (MNCs), and the functional evidence of lacunar resorption pits on dentine slices was assessed. Celecoxib and indomethacin inhibited osteoclast formation and the extent of lacunar resorption in a dose-dependent manner, but the effect of indomethacin was less than that of celecoxib. Mofezolac affected neither the number of TRAP-positive MNCs nor the extent of lacunar resorption pits. These results indicate that celecoxib influences not only osteoclast formation through osteoblastic cells but also acts directly on circulating osteoclast precursors to influence human osteoclast differentiation. The effect of celecoxib on osteoclast precursors may be related to the COX-2 signal pathway.

Synovectomy, debridement, and continuous irrigation for infected total knee arthroplasty

Since 1990, a total of ten joints in nine patients with infected total knee arthroplasty have been treated in our department within 21 days of the onset of infection. Their radiographs showed no evidence of implant loosening or “moth-eaten” appearance. They underwent synovectomy, debridement, and continuous irrigation without implant removal. Continuous irrigation was maintained for 7–29 days. It was possible to retain implants in eight joints of seven patients. Two joints of two patients were removed. Pain disappeared in all eight joints in which the implants were retained. Four patients could walk with one cane; one patient could walk with one crutch. Range of motion in five joints remained over 100°. We recommend synovectomy, debridement, and continuous irrigation to cure an early stage infection of total knee arthroplasty.RésuméDepuis 1990, dix articulations chez neuf malades avec une arthroplastie totale infectée du genou ont été traité dans notre département dans les 21 jours après le début de l’infection. Les radiographies n’ont pas montré de descellement d’implants ou d’anomalie de la texture osseuse. Ils ont subi synovectomie, débridement et irrigation continue sans ablation de l’implant. L’irrigation continue a été maintenue pour sept à 29 jours. Il a été possible de conserver les implants de huit articulations chez sept malades. Deux articulations de deux malades ont subi l’ablation de l’implant. La douleur a disparu de toutes les articulations ou les implants avaient été conservés. Quatre malades devaient marcher avec une canne et un avec une béquille. L’amplitude de mobilité est restée au-dessus de 100° pour cinq articulations. Nous recommandons la synovectomie, le débridement et l’irrigation continue pour traiter une infection précoce d’arthroplastie totale du genou.

Joint Motion of Bipolar Femoral Prostheses

From 1982 to 1992, 251 bipolar hip arthroplasties were performed on 213 patients. Among them, 117 bipolar femoral prostheses were randomly selected to examine the behavior of abduction motion under weight-bearing loads. Roentgenographic motion study was performed at an average of 46.5 months after surgery (range, 2-110 months). One hundred one prostheses used in dysplastic osteoarthritic, rheumatoid, and revised failed total hip arthroplasty patients moved 18.2% at the outer bearing and 81.8% at the inner bearing, while 16 prostheses used in femoral neck fracture and osteonecrosis of the femoral head patients moved 49.7% at the inner bearing and 50.3% at the outer bearing. There was a statistical difference in the motion pattern between the two groups. The abduction motion behavior of the bipolar femoral prostheses was not affected by the length of the follow-up period, the diameter of the outer heads, or the position of the prostheses on immediate postoperative roentgenograms.

c-Myc Represses Tumor-Suppressive microRNAs, let-7a, miR-16 and miR-29b, and Induces Cyclin D2-Mediated Cell Proliferation in Ewing’s Sarcoma Cell Line

Myc oncogenic transcription factor is known to inhibit tumor suppressive microRNAs (miRNAs), resulting in greater expression of their target protein related to cell cycle, invasion or anti-apoptotic factors in human cancer cells. To explore possible oncogenic factors in Ewing’s sarcoma (ES), we conducted microarray-based approach to profile the changes in the expression of miRNAs and its downstream mRNAs in five ES cell lines and human mesenchymal stem cells (hMSCs). Three miRNAs, let-7a, miR-16 and miR-29b were significantly down-regulated, whereas c-Myc and cyclin D2 (CCND2) were significantly up-regulated in all tested ES cells compared with hMSCs. To verify that let-7a, miR-16 and miR-29b were the targets of c-Myc in ES cell lines, we transfected siRNA against c-Myc and confirmed the coordinate up-regulation of let-7a, miR-16 and miR-29b through the repression of c-Myc. The ES cells transfected with c-Myc-siRNA and let-7a, miR-16 and miR-29b exhibited the inhibition of the cell cycle progression. The increased expression of let-7a, miR-16 and miR-29b resulted in the reduction of CCND2 protein expression. We also demonstrated that c-Myc-siRNA treatment of ES cells was associated with the decreased expression of CCND2 as a down-stream of three miRNAs. Furthermore, the introduction of let-7a, miR-16 and miR-29b in ES cells could inhibit the c-Myc-mediated up-regulation of CCND2 resulted in the prevention of cell cycle progression. In addition, the transfection of let-7a, miR-16 and miR-29b in ES cells suppressed tumor growth ex vivo treatment. These findings suggests that the up-regulation of c-Myc inhibited the expression of let-7a, miR-16 and miR-29b subsequently induced CCND2 expression in ES cells. The present study might identify a novel oncogenic axis that c-Myc regulates the expression of CCND2 via let-7a, miR-16 and miR-29b, leading to the development new therapeutic targets for ES.

Subchondral cyst of the tibia secondary to Wilson disease

We present the case of a 40-year-old male patient who had been suffering from Wilson disease for over 20 years, whose knee was diagnosed as osteoarthritis combined with subchondral cyst of the tibia. Preoperative examinations (X-ray, CT and MRI) confirmed the diagnosis. The microscopic examination detected thickening of the synovial membrane, and histopathological findings revealed that lymphoid cells and plasma cells were infiltrated at the synovial membrane. On copper-specific staining, no copper pigmentation was identified. However, the energy-dispersive X-ray (EDX) microanalysis revealed copper pigmentation in high concentration. These findings may contribute to our better comprehension of the development process of the arthropathy in patients with Wilson disease. The combination of subchondral cyst with Wilson disease is extremely rare, as only about 16 such cases have been reported in the English literature.

Analysis of measured D-dimer levels for detection of deep venous thrombosis and pulmonary embolism after spinal surgery.

Study Design A retrospective clinical study. Objectives To show the prevalence of deep venous thrombosis (DVT) and pulmonary embolism (PE) after spinal surgery using a D-dimer assay followed by screening with computed tomographic (CT) pulmonary angiography and CT venography. Summary of Background Data A few studies on DVT development after spinal surgery have been reported. Methods A complete surveillance examination for DVT and PE was conducted in 88 patients who underwent spinal surgery [male patients, 48; female patients, 40; average age at operation, 62.4 y (range, 17 to 85 y)] through a D-dimer assay combined with CT pulmonary angiography and CT venography. The operation levels were the cervical spine (21 cases), the thoracic spine (16 cases), and the lumbar spine (51 cases). We adopted a D-dimer cut-off point of 10 &mgr;g/mL, and classified the patients into high D-dimer (HD; D-dimer level ≥10 &mgr;g/mL) and low D-dimer (LD; D-dimer level <10 &mgr;g/mL) groups. Results Nine (10.2%) patients showed D-dimer levels of ≥10 &mgr;g/mL (HD group); of these, 5 patients (5.7%) had DVT. Two (2.2%) of the 5 DVT patients had PE. DVT was evident in 1 (6.2%) of the 16 patients who underwent thoracic procedures and 4 (7.8%) of the 51 patients who underwent lumbar procedures. Statistical comparison between the HD (excluding 5 patients with DVT or PE) and LD groups showed a significant difference in intraoperative blood loss between the groups (P=0.02). Conclusions The D-dimer assay was useful in predicting DVT development. A D-dimer level of ≥10 &mgr;g/mL is considered to be a risk factor for thromboembolic disease after spinal surgery. False-positive cases of thromboembolic disease preclude the use of this assay as a stand-alone test for DVT diagnosis. CT venography and CT pulmonary angiography are recommended to confirm thromboembolic disease.

Morphological analysis of the cervical pedicles, lateral masses, and laminae in developmental canal stenosis.

Study Design. Retrospective cross-sectional study. Objective. This study aimed to elucidate the relationship between developmental spinal canal stenosis (DCS) and morphologic features in the cervical spine by comparing the features between DCS and nondevelopmental spinal canal stenosis (NDCS). Summary of Background Data. DCS is an important predisposing factor for cervical myelopathy. Further, various posterior cervical spinal instrumentations have been developed. However, no study has specifically addressed the cervical posterior morphology of DCS. Methods. A total of 52 consecutive patients underwent cervical spine computed tomography myelography. Axial images of the largest pedicle diameter were selected from C3 to C7 vertebrae and 260 images were analyzed. The following parameters were measured: spinal canal longitudinal diameter (SCLD), spinal canal transverse diameter, osseous spinal canal area, dural sac area, spinal cord area, pedicle outer width, pedicle axis length, pedicle transverse angulation, lateral mass longitudinal diameter, lateral mass transverse diameter, lamina outer width, and lamina axis length. The participants were classified into 2 groups: DCS group (SCLD <12 mm at any level) and NDCS group (SCLD ≧12 mm at all levels). Results. The mean osseous spinal canal area and dural sac area at C3–C5 in the DCS group were less than those in the NDCS group. The mean spinal cord area did not differ significantly at C3–C7 between the groups. The mean pedicle outer width at C6 and C7 in the DCS group was less than that in NDCS group. The mean lateral mass transverse diameter at C5 and mean lateral mass longitudinal diameter at C3, C5, and C6 in the DCS group were less than those in the NDCS group. Conclusion. Myelopathy is expected to progress in patients with DSC and these patients with severe neurologic symptoms may need cervical operation. However, posterior screw insertions should be considered more carefully than in NDCS patients.

Tumor suppressive microRNA-138 inhibits metastatic potential via the targeting of focal adhesion kinase in Ewing's sarcoma cells

Short non-coding RNAs, called microRNAs (miRNAs), regulate cell biology by affecting the expression of target genes. However, we know little about the miRNAs regulating the growth and progression of Ewings sarcoma (ES). To identify possible oncogenic factors in ES, we used a microarray-based approach to profile the changes in the expression of miRNAs and the downstream mRNAs in five ES cell lines. One miRNA, miR‑138, was significantly downregulated, whereas the expression of focal adhesion kinase (FAK) was significantly upregulated in all tested ES cells. When miR‑138 was transfected into ES cell lines, the expression of FAK in these cells was greatly suppressed and inhibited the proliferation and mobility of ES cells. Overexpression of miR‑138 in vitro resulted in further inhibition of the cell cycle at the G1 phase and in the induction of anoikis, in a dose- and time-dependent manner. Moreover, miR‑138 overexpression in ES cells significantly suppressed the number of distant metastases in vivo. The data in the present study demonstrates for the first time a novel mechanism that regulates the expression of FAK via miR‑138 in ES cells.

MicroRNA-301a promotes cell proliferation via PTEN targeting in Ewing's sarcoma cells

MicroRNAs (miRNAs) regulate cell proliferation and differentiation by affecting gene expression at the post-transcriptional level by binding to complementary sequences within mRNAs in cancer cells, indicating that miRNAs can function as tumor suppressors or oncogenes. Recent studies showed that dysregulation of miRNA expression was associated with increased tumorigenicity and poor prognosis in several types of cancers, including Ewings sarcoma (ES). To explore possible oncogenic factors in ES, we conducted microarray-based investigation and profiled the changes in miRNA expression and their effects on downstream mRNAs in five ES cell lines and human mesenchymal stem cells (hMSCs). miR-301a was significantly upregulated, while the phosphatase and tensin homolog (PTEN) expression was significantly downregulated in all tested ES cells as compared to hMSCs. When anti-miR-301a was transfected into ES cell lines, PTEN expression was significantly enhanced, suggesting that PTEN might be a target of miR-301a in ES cells. The expression of protein kinase B (Akt), which is inversely correlated with PTEN expression, was significantly downregulated in anti-miR-301a-transfected cells. Additionally, the transfection of anti-miR-301a inhibited ES cell proliferation and cell cycle progression. Furthermore, downregulation of miR-301a in ES cells significantly suppressed tumor growth in vivo. Our results demonstrated the novel mechanism controlling PTEN expression via miR-301a in ES cells. Given that PTEN is a pivotal phosphatase factor that regulates cell cycle progression, apoptosis, and proliferation, these results might lead to development of new ES-related therapeutic targets.