Circular RNA hsa_circ_0075323 promotes glioblastoma cells proliferation and invasion via regulation of autophagy.

BACKGROUND: Protein p62 (sequestosome 1) encoded by gene SQSTM1 plays a vital role in mediating protectively selective autophagy in tumor cells under stressed conditions. CircSQSTM1 (hsa_circ_0075323) is a circular transcript generated from gene SQSTM1 (chr5:179260586-179260782) by back-splicing. However, the potential role of hsa_hsa_circ_0075323 in glioblastoma (GBM) remains unclear. Here, we aimed to explore the biological function of hsa_circ_0075323 in GBM and its relationship with autophagy regulation. RESULTS: Hsa_circ_0075323 is highly expressed in GBM cells and mainly locates in the cytoplasm. Inhibition of hsa_circ_0075323 in U87-MG and T98G cells attenuated proliferation and invasion ability significantly, while upregulation of has_ circ_0075323 enhanced proliferation and migration of U251-MG and A172 cells. Mechanistically, depletion of hsa_circ_0075323 in GBM cells resulted in impaired autophagy, as indicated by increased expression of p62 and decreased expression of LC3B. CONCLUSIONS: Hsa_circ_0075323 regulates p62-mediated autophagy pathway to promote GBM progression and may serve as a prognostic biomarker potentially.

Application and thinking of minimally invasive transforaminal lumbar interbody fusion in degenerative lumbar diseases.

Background: This study sought to investigate the clinical efficacy and safety of minimally invasive transforaminal lumbar interbody fusion (MIS-TLIF) in the treatment of lumbar degenerative diseases. Methods: The clinical data of 55 patients with lumbar degenerative diseases treated at our hospital from January 2018 to January 2020 were analyzed retrospectively. Of the 55 patients, 35 who underwent MIS-TLIF were included in the MIS-TLIF group, and 20 who underwent posterior lumbar interbody fusion (PLIF) were included in the PLIF group. The visual analogue scale (VAS) score, Oswestry disability index (ODI) score, operation time, incision length, intraoperative bleeding, postoperative drainage, postoperative landing time, postoperative hospital stay, postoperative interbody fusion rate, and complications were compared between the two groups. Results: The patients in both groups were followed-up for at least 1.5 years (range, 18-30 months; with an average of 27.5±2.6 months). There was no significant difference in the operation time, incision length, intraoperative bleeding, VAS score for low back and leg pain, ODI score, interbody fusion rate, hospitalization expenses, and complication rate between the two groups (P>0.05). One patient had nail failure in the MIS-TLIF group, 1 patient in each group had nerve root irritation, and 1 patient in each group had superficial incision infection and local suture dehiscence. The postoperative drainage volume, postoperative landing time, and postoperative hospital stay of the MIS-TLIF group were less than those of the PLIF group (P<0.05). Conclusions: Compared to PLIF, the use of MIS-TLIF in the treatment of lumbar degenerative diseases has a number of advantages, including more complete intraoperative hemostasis, less postoperative drainage, earlier landing, and faster discharge, and also significantly improves postoperative lumbar discomfort.

Comparison of the differentiation abilities of bone marrow-derived mesenchymal stem cells and adipose-derived mesenchymal stem cells toward nucleus pulposus-like cells in three-dimensional culture.

Nucleus pulposus cell (NPC) transplantation can be a potential therapeutic approach for intervertebral disc degeneration (IDD). However, low cell viability has restricted the therapeutic capacity of NPCs, and sources of natural NPCs are limited. Bone marrow-derived mesenchymal stem cells (BMSCs) and adipose-derived mesenchymal stem cells (ADSCs) can be differentiated toward NPC-like cells. However, it is unknown whether there are differences in the abilities of these two cell types to differentiate into NPC-like cells, or which cell type exhibits the best differentiation ability. The present study compared the abilities of BMSCs and ADSCs to differentiate toward NPC-like cells with or without a 3D culture system to lay a foundation for stem cell transplantation therapy for IDD. BMSCs were isolated from the rat whole bone marrow cell using the repeated adherent culture method. ADSCs were isolated from rat adipose tissues in the subcutaneous inguinal region using the enzyme digestion method. Cells were identified using flow cytometry. Cell viability was assessed via Cell Counting Kit-8 assays, and reverse transcription-quantitative PCR and western blotting were carried out to evaluate the expression of NPC markers and chondrocyte-specific genes. Glycosaminoglycans (GAGs) and proteoglycans were examined via Alcian blue and safranin O staining, respectively. ADSCs in 3D culture displayed the highest cell proliferative ability, compared with the 2D culture system and BMSC culture. In addition, ADSCs in 3D culture exhibited increased GAG and proteoglycan synthesis than BMSCs. Compared with BMSCs in 3D culture, ADSCs in 3D culture exhibited higher mRNA and protein expression of NPC marker genes (hypoxia-inducible factor 1-α, glucose transporter 1) and chondrocyte-specific genes (Sox-9, aggrecan and type II collagen). The present findings indicated that ADSCs exhibited a better ability to differentiate into NPC-like cells in 3D culture compared with BMSCs, which may be of value for the regeneration of intervertebral discs using cell transplantation therapy.

Both p53 codon 72 Arg/Arg and pro/Arg genotypes in glioblastoma multiforme are associated with a better prognosis in bevacizumab treatment.

BACKGROUND: It has previously been shown that bevacizumab, when added to chemotherapy, improved overall survival in several cancers. In glioblastoma multiforme (GBM), bevacizumab increased progression-free survival and it is widely used for tumor recurrence, though it has failed to improve overall survival (OS) in controlled trials. However, an effective biomarker for predicting the prognosis of bevacizumab treatment has yet to be identified. This study, therefore, aimed to retrospectively analyze the polymorphisms of p53 codon 72 and the clinical characteristics of GBM specimens from Taiwanese patients. METHODS: The polymorphisms of p53 codon 72 in 99 patients with GBM treated at Taichung Veterans General Hospital in Taiwan from 2007 to 2017 were analyzed using direct DNA sequencing and PCR-RFLP analysis. RESULTS: We found that among these GBM patients, the distribution of codon 72 polymorphisms was 28.3% for proline homozygotes (Pro/Pro), 38.4% for arginine homozygotes (Arg/Arg), and 33.3% for proline/arginine heterozygotes (Pro/Arg). Although the polymorphisms of p53 codon 72 were not directly associated with the overall survival of GBM, both the Arg/Arg and Arg/Pro genotypes were associated with significant benefits in terms of overall survival in patients treated with CCRT plus bevacizumab compared to patients treated with CCRT alone. CONCLUSIONS: This pilot study suggests that both the Arg/Arg and Arg/Pro genotypes of p53 codon 72 polymorphism may have value as independent prognostic or predictive parameters for bevacizumab treatment response and failure. Relatedly, the results of the study further demonstrate the utility of stratifying GBM patients according to bevacizumab sensitivity.

AHIF promotes glioblastoma progression and radioresistance via exosomes.

Glioblastoma multiforme (GBM) has the highest mortality rate among patients with brain tumors, and radiotherapy forms an important part of its treatment. Thus, there is an urgent requirement to elucidate the mechanisms conferring GBM progression and radioresistance. In the present study, it was identified that antisense transcript of hypoxia­inducible factor­1α (AHIF) was significantly upregulated in GBM cancerous tissues, as well as in radioresistant GBM cells. The expression of AHIF was also upregulated in response to radiation. Knockdown of AHIF in GBM cells decreased viability and invasive capacities, and increased the proportion of apoptotic cells. By contrast, overexpression of AHIF in GBM cells increased viability and invasive capacities, and decreased the proportion of apoptotic cells. Furthermore, exosomes derived from AHIF­knockdown GBM cells inhibited viability, invasion and radioresistance, whereas exosomes derived from AHIF­overexpressing GBM cells promoted viability, invasion and radioresistance. Further biochemical analysis identified that AHIF regulates factors associated with migration and angiogenesis in exosomes. To the best of our knowledge, the present study is the first to establish that AHIF promotes glioblastoma progression and radioresistance via exosomes, which suggests that AHIF is a potential therapeutic target for GBM.

Methylation-mediated silencing of miR-124 facilitates chondrogenesis by targeting NFATc1 under hypoxic conditions.

Chondrogenic differentiation of mesenchymal stem cells is regulated by many different pathways. Recent studies have established that hypoxia and epigenetic alterations potently affect expression of chondrogenesis marker genes. Sox9 is generally regarded as a master regulator of chondrogenesis and microRNA-124 (miRNA-124) regulates gene expression in murine bone marrow-derived mesenchymal stem cells. Therefore, in this study we investigated whether epigenetic regulation of miRNA-124 could affect the expression of Sox9 and thereby regulate chondrogenesis. A cell pellet culture model was used to induce chondrogenesis in C3H10T1/2 cells under hypoxic conditions (2% O2) to determine the effects of hypoxia on miR-124 expression and DNA methylation. The expression of miR-124 was significantly downregulated under hypoxic conditions compared to normoxic conditions (21% O2). The expression of chondrogenesis marker genes was significantly increased under hypoxic conditions. Bisulfite sequencing of the CpG islands in the promoter region of miR-124-3 showed that CpG methylation was significantly increased under hypoxic conditions. Treating the cells with the DNA demethylating agent 5'-AZA significantly increased miR-124 expression and decreased expression of markers of chondrogenesis. Overexpressing miR-124 under hypoxic conditions inhibited NFATc1 reporter activity. NFATc1 was shown to bind to the promoter region of Sox9. Taken together, our data provide evidence that miR-124 acts as an inhibitor of NFATc1. Under hypoxic conditions when miR-124 is downregulated by methylation of CpG islands in the promoter, NFATc1 can bind to the Sox9 promoter and induce the expression of Sox9 leading to chondrogenesis. These results support the role of epigenetic regulation in establishing and maintaining a chondrogenic phenotype.

[HYPOXIA INDUCIBLE FACTOR lα/2α GENES EXPRESSION IN CHONDROGENIC DIFFERENTIATION OF HUMAN BONE MARROW MESENCHYMAL STEM CELLS].

OBJECTIVE: To observe the genes expression of hypoxia inducible factor lα (HIF-1α) and HIF- 2α by inducing chondrogenic differentiation of human bone marrow mesenchymal stem cells (hBMSCs) so as to provide a fundamental basis for HIF involving in the mechanism of chondrogenesis. METHODS: High density pellet of hBMSCs was obtained by centrifugation and cultured with H-DMEM medium containing 2% fetal bovine serum (control group) and with chondrogenic medium (chondrogenic induction group) under hypoxia (2% O2) for 3 weeks. Immunohistochemistry staining was utilized to identify extracellular proteoglycan and collagen type II at 3 weeks after culture. Western blot was applied for measuring HIF-1α and HIF-2α protein levels at 1 week after culture. Real-time quantitative PCR was performed to detect the genes expressions of HIF-1α, HIF-2α, Sox-9, collagen type II, collagen type X, and Aggrecan at 1, 2, and 3 weeks after culture. RESULTS: Toluidine blue staining showed sparse nucleus in the control group, and dense nucleus in the chondrogenic induction group; extracellular matrix staining was deeper in the chondrogenic induction group than the control group. Immunohistochemical staining for collagen type II was positive in cytoplasm; when compared with the chondrogenic induction group, the control group showed sparse and light-coloured nucleus. At 1 week after culture, the protein expression levels of HIF-1α and HIF-2α in the chondrogenic induction group were significantly lower than those in the control group (t = 8.345, P = 0.001; t = 7.683, P = 0.002). When compared with control group, the HIF-1α mRNA expression was significantly down-regulated at 1 week and significantly up-regulated at 2 weeks in chondrogenic induction group (P < 0.05), but no significant difference was found at 3 weeks between the 2 groups (P > 0.05). And the mRNA expression of HIF-2α was significantly down-regulated and mRNA expression of Sox-9 was significantly up-regulated after chondrogenic differentiation when compared with the control group (P < 0.01). The mRNA expressions of collagen type II and collagen type X were significantly up-regulated at 2 and 3 weeks after chondrogenic differentiation when compared with the control group (P < 0.05). And the mRNA expression of Aggrecan was significantly up-regulated at each time point after chondrogenic differentiation (P < 0.05). CONCLUSION: HIF-1α may involve the hBMSCs chondrogenic differentiation under hypoxia, while HIF-2α expression is depressed throughout the period and may have negative effect on differentiation.

Induction of chondrogenic differentiation of mouse embryonic mesenchymal stem cells through an in vitro pellet model.

This study employed transforming growth factor beta 3 (TGF-ß3) induction of the C3H10T1/2 mesenchymal stem cell (MSC) line to construct an in vitro chondrogenic differentiation model for MSCs. A C3H10T1/2 MSC cell line was cultured, amplified, and the seventh generation of cells was centrifuged to construct pellets, which were divided into a non-induced group and an induced group (treated with TGF-ß3, vitamin C, dexamethasone, and ITS). Specimens were taken after 7, 14, 21, and 28 days under non-induced and induced culture to compare these two groups by Alcian Blue staining, collagen type II immunohistochemical staining, and transmission election microscopy (TEM) on days 21 and 42. Cell pellets in the non-induced group were smaller than those in the induced group on days 7, 14, 21, and 28 with the pellet morphology of the induced group being more regular and nearly spherical. Alcian blue staining in the induced group was consistently stronger than that in non-induced group across all time points, and type II collagen immunohistochemical IOD values were significantly higher in the induced group over the non-induced group across all time points. On days 21 and 42, TEM revealed that the induced group displayed greater karyokinesis and a higher euchromatin ratio compared to the non-induced group. This specially constructed pellet model treated with TGF-ß3-containing chondrogenic medium can effectively promote chondrogenic differentiation of C3H10T1/2 MSC cells in vitro. This in vitro pellet model should be of value in providing a preliminary cell model reference for further studies of the mechanism of chondroblast differentiation of stem cells.

Malignant transformation of craniopharyngioma with detailed follow-up.

A 29-year-old male patient was admitted into hospital with the main complaint of progressive visual disturbance. Both CT SCAN and MRI demonstrated a cystic-solid contrast-enhancing sellar-suprasellar mass with obvious calcification. Histopathological examination of the first resected specimen showed a typical appearance of adamantinomatous craniopharyngioma. The patient received gamma knife therapy after his first operation because of partial tumor removal. He experienced two relapses in the subsequent 2 years, for which only surgical resection was performed. The later histopathology presented malignant appearance with tumor cells moderate to severe pleomorphism, hyperchromasia, increased nuclear cytoplastic ratio, high mitotic activity (30/10 high power fields) and focal coagulative necrosis. The patient died 9 months after identification of histologic malignancy. Clinical and histopathological features, biological behavior of one case of malignant craniopharyngioma were discussed, with a brief review of the relevant literature.

Comparison of artificial cervical arthroplasty versus anterior cervical discectomy and fusion for one-level cervical degenerative disc disease: a meta-analysis of randomized controlled trials.

PURPOSE: The aim of the study was to evaluate whether there is a superior clinical effect of artificial cervical arthroplasty compared with anterior cervical discectomy and fusion (ACDF) for the treatment of one-level cervical degenerative disc disease (CDDD). METHODS: A comprehensive literature search of multiple databases, including PubMed, ScienceDirect, Scopus, Embase, Cochrane Central Register of Controlled Trials, was conducted to identify studies that met the inclusion criteria. Methodological quality was assessed and relevant data were extracted, and if appropriate, meta-analysis was performed. RESULTS: Thirteen randomized controlled trials were identified. At 24 months post-operatively, total disc replacement (TDR) was demonstrated to be more beneficial for patients compared with ACDF for the following outcomes: neurological success [odds ratio (OR) 1.92; 95% confidence interval (CI) 1.47-2.49; p < 0.00001], range of motion [mean differences (MD), 6.67; 95% CI 4.82-8.53; p < 0.00001], secondary surgical procedures (OR 0.50; 95% CI 0.37-0.68; p < 0.00001), and visual analogue scale neck pain scores (MD -5.99; 95% CI -10.54 to -1.45; p = 0.001) and visual analogue scale arm pain scores (MD -3.23; 95% CI -6.48 to 0.02; p = 0.004). Other outcomes, including length of the hospital stay (MD -0.03; 95% CI -0.18 to 0.12; p = 0.68), blood loss (MD 6.92 mL; 95% CI -3.09 to 16.92 mL; p = 0.18), Neck Disability Index scores (MD -1.00; 95% CI -5.28 to 3.28; p = 0.65) and rate of adverse events [risk ratio (RR), 0.93; 95% CI 0.76-1.15; p = 0.52] demonstrated no differences between the 2 groups. Although the TDR group had a significantly longer operation time than the ACDF group, it was not considered clinically important. CONCLUSIONS: For patients with one-level CDDD, TDR was found to be more superior than ACDF in terms of neurological success, secondary surgical procedures, visual analogue scale pain scores and range of motion at 24 months post-operatively. Therefore, cervical arthroplasty is a safe and effective surgical procedure for treating one-level CDDD. We suggest adopting TDR on a large scale; with failure of TDR, ACDF would be performed.

Shock absorbing function study on denucleated intervertebral disc with or without hydrogel injection through static and dynamic biomechanical tests in vitro.

Hydrogel injection has been recently proposed as a novel therapy for disc degenerative diseases, with the potential to restore the spine motion and the intervertebral disc height. However, it remains unknown whether the new technique could also maintain the shock absorbing property of the treated intervertebral disc. In this study, 18 porcine lumbar bone-disc-bone specimens were collected and randomly divided into three groups: the normal with intact intervertebral discs, the mimic for the injection of disulfide cross-linked hyaluronan hydrogels following discectomy, and the control disc with discectomy only. In the static compression test, specimens in the mimic group exhibited displacements similar to those in the normal discs, whereas the control group showed a significantly larger displacement range in the first two steps (P < 0.05). With the frequency increasing, all specimens generally displayed an increasing storage modulus, decreasing loss modulus, and tanδ. At any frequency point, the control group exhibited the largest value in all the three parameters among three groups while the normal group was the lowest, with the mimic group being mostly close to the normal group. Therefore, the hydrogel injection into the intervertebral discs greatly restored their shock absorbing function, suggesting that the technique could serve as an effective approach to maintaining biomechanical properties of the degenerative intervertebral disc.

1,25-Dihydroxyvitamin D3 activates MMP13 gene expression in chondrocytes through p38 MARK pathway.

Osteoarthritis (OA) is the most prevalent degenerative joint disease. The highly regulated balance of matrix synthesis and degradation is disrupted in OA, leading to progressive breakdown of articular cartilage. The molecular events and pathways involved in chondrocyte disfunction of cartilage in OA are not fully understood. It is known that 1,25-dihydroxyvitamin D3(1,25-(OH)2D3) is synthesized by macrophages derived from synovial fluid of patients with inflammatory arthritis. Vitmain D receptor is expressed in chondrocytes within osteoarthritic cartilage, suggesting a contributory role of 1,25-(OH)2D3 in the aberrant behavior of chondrocytes in OA. However, the physiological function of 1,25-(OH)2D3 on chondrocytes in OA remains obscure. Effect of 1,25-(OH)2D3 on gene expression in chondrocytes was investigated in this study. We found that 1,25-(OH)2D3 activated MMP13 expression in a dose-dependent and time-dependent manner, a major enzyme that targets cartilage for degradation. Interestingly, a specific mitogen-activated protein kinase p38 inhibitor SB203580, but not JNK kinase inhibitor SP600125, abrogated 1,25-(OH)2D3 activation of MMP13 expression. 1,25-(OH)2D3-induced increase in MMP13 protein level was in parallel with the phosphorylation of p38 in chondrocytes. To further address the effect of 1,25-(OH)2D3 on MMP13 expression, transfection assays were used to show that 1,25-(OH)2D3 activated the MMP13 promoter reporter expression. MMP13 is known to target type II collagen and aggrecan for degradation, two major components of cartilage matrix. We observed that the treatment of 1,25-(OH)2D3 in chondrocytes results in downregulation of both type II collagen and aggrecan while MMP13 was upregulated. Taken together, we provide the first evidence to demonstrate that 1,25-(OH)2D3 activates MMP13 expression through p38 pathway in chondrocytes. Since MMP13 plays a major role in cartilage degradation in OA, we speculate that the ability of 1,25-(OH)2D3 to potentiate MMP13 expression might facilitate cartilage erosion at the site of inflammatory arthritis.

Inhibition of lymphoma cell proliferation by peroxisomal proliferator-activated receptor-γ ligands via Wnt signaling pathway.

Peroxisome proliferator-activated receptor gamma (PPARγ) plays an important role in regulating energy balance, glucose and lipid metabolisms and inflammation. PPARγ also exerts multiple anti-cancer effects including tumor growth and angiogenesis inhibition, induction of cell differentiation, and apoptosis. Perturbed Wnt/ß-catenin signaling likely plays a key role in tumorigenesis and the interaction between PPARγ and the transcriptional regulator ß-catenin maybe important in this process. Phosphorylation of ß-catenin by GSK-3ß inactivates it and suppresses tumor cell proliferation and self-renewal of tumor stem cells. In combination with Frizzled, Wnt suppresses GSK-3ß and causes degradation of ß-catenin and activation of many tumor proliferation factors. In the present study, we investigated the effects of PPARγ agonist rosiglitazone (RGZ) and PPARγ antagonist GW9662 on the growth, mitotic cycle, and apoptosis of human lymphoma cell line, Raji cells. We also studied the influence of PPARγ ligands on the expression of ß-catenin and GSK-3ß in Raji cells to reveal whether Wnt/GSK-3ß/ß-catenin signaling pathways are involved in PPARγ ligands triggered Raji cell apoptosis. Results showed that both RGZ and GW9662 can inhibit the growth of Raji cells by inducing apoptosis and arresting cell cycle; however, there was no correlation between these effects and expression of PPARγ. Both the PPARγ ligands, RGZ and GW9662, appear to reciprocally regulate the mRNA and protein expressions of GSK-3ß, which promotes apoptosis, and of ß-catenin, which blocks apoptosis. These results suggest that PPARγ ligands mediate their effects via Wnt/GSK-3ß/ß-catenin signaling on Raji cell proliferation and survival.

Drug or vaccine?: selecting the appropriate treatment for malignant glioma patients.

Malignant gliomas are the most common and aggressive form of brain tumour. Current combinations of aggressive surgical resection, radiation therapy and chemotherapy regimens do not significantly improve long-term patient survival for these cancers. Therefore, investigative therapies including tumour vaccines have targeted this devastating condition. This article reviews evidence and data on chemotherapy and immunotherapy for a personalized medicine approach in order to enable physicians to select the appropriate treatment for glioma patients. Dendritic cell- and peptide-based therapy for gliomas seems to be safe and without major adverse effects. Gene-modified vaccines have also shown promise in the treatment of malignant gliomas. The concept of 'personalized medicine' is currently important in oncology treatment development. Using a personalized medicine approach, it may be necessary to evaluate the molecular genetic abnormalities in individual patient tumours, and such findings should be the mainstay of immunotherapeutic strategies designed for the individual patient.

Analysis and prevention of cerebral infarction in patients with traumatic brain injury.

We reviewed the clinical data of 32 cases of cerebral infarction after traumatic brain injury, aiming to explore the pathogenesis and assess the prognosis of the condition so as to find effective prevention and treatment measures. According to Glasgow Outcome Scale (GOS) scores, 12 patients were classified as having good recovery, 5 moderate neurological deficit, 4 severe neurological deficit, and 3 in vegetative state. Death occurred in 8 cases. These results indicate the mortality and morbidity of this condition are relatively high, and early diagnosis and treatment may improve prognosis, which should await further investigation.