Hirudin inhibits glioma growth through mTOR-regulated autophagy.

Glioma is the most common primary malignant brain tumour, and survival is poor. Hirudin has anticancer pharmacological effects through suppression of glioma cell progression, but the molecular target and mechanism are poorly understood. In this study, we observed that hirudin dose- and time-dependently inhibited glioma invasion, migration and proliferation. Mechanistically, hirudin activated LC3-II but not Caspase-3 to induce the autophagic death of glioma cells by decreasing the phosphorylation of mTOR and its downstream substrates ULK1, P70S6K and 4EBP1. Furthermore, hirudin inhibited glioma growth and induced changes in autophagy in cell-derived xenograft (CDX) nude mice, with a decrease in mTOR activity and activation of LC3-II. Collectively, our results highlight a new anticancer mechanism of hirudin in which hirudin-induced inhibition of glioma progression through autophagy activation is likely achieved by inhibition of the mTOR signalling pathway, thus providing a molecular basis for hirudin as a potential and effective clinical drug for glioma therapy.

[Study on the correlation between combined detection of RACK1 and M2/M1 in oral squamous cell carcinoma and prognosis of the patients].

PURPOSE: To explore the relationship between combined detection of tissue active protein kinase C receptor 1 (RACK1) and M2/M1 and the prognosis of oral squamous cell carcinoma (OSCC). METHODS: 129 OSCC patients admitted to the Affiliated Hospital of Nantong University were selected as the research subjects from February 2017 to May 2018. RACK1 and M2/M1 in cancer tissues and adjacent tissues of patients were detected and compared. The relationship between RACK1 expression, M2/M1 and clinicopathological factors, and relationship between RACK1 expression, M2/M1 and prognosis in cancer tissue were analyzed. SPSS 18.0 software package was used for data analysis. RESULTS: The positive expression rate of RACK1 and the value of M2/M1 in cancer tissues were significantly higher than those in adjacent tissues (P<0.05). The positive expression rate of RACK1 in cancer tissues of patients with stage Ⅲ-Ⅳ, cervical lymph node metastasis, and vascular involvement were significantly higher than those with stage Ⅰ-Ⅱ, no lymph node metastasis, and no vascular involvement(P<0.05). M2/M1 values in cancer tissues of patients with stage Ⅲ-Ⅳ, poor differentiation, and cervical lymph node metastasis were significantly higher than those with stage Ⅰ-Ⅱ, moderate differentiation, and no cervical lymph node metastasis(P<0.05). Cox regression analysis showed that the stage of tumor, cervical lymph node metastasis, positive expression rate of RACK1, and M2/M1 were independent prognostic factors affecting overall survival (P<0.05). ROC curve showed that AUC of cancer tissue RACK1, M2/M1 and combination of the two to predict the prognosis of OSCC patients were 0.743, 0.718 and 0.875, respectively. The survival rate of patients with positive expression of RACK1 was 62.24%, and the survival rate of patients with negative expression of RACK1 was 92.31%. The overall survival rate of patients with positive expression of RACK1 and patients with negative expression were compared, and the difference was statistically significant(P<0.05). The survival rate of patients with M2/M1≥2.06 was 61.70%, the survival rate of patients with M2/M1<2.06 was 88.24%, and the overall survival curve of patients with M2/M1≥2.06 was compared with that of patients with M2/M1<2.06, and the difference was statistically significant(P<0.05). CONCLUSIONS: The expression of RACK1 and M2/M1 is abnormally high in cancer tissues of OSCC patients. The expression of RACK1 and M2/M1 is related to the patient's pathology and prognosis. The combination of the two has certain efficacy in predicting the prognosis of OSCC patients.

Concurrent binding to DNA and RNA facilitates the pluripotency reprogramming activity of Sox2.

Some transcription factors that specifically bind double-stranded DNA appear to also function as RNA-binding proteins. Here, we demonstrate that the transcription factor Sox2 is able to directly bind RNA in vitro as well as in mouse and human cells. Sox2 targets RNA via a 60-amino-acid RNA binding motif (RBM) positioned C-terminally of the DNA binding high mobility group (HMG) box. Sox2 can associate with RNA and DNA simultaneously to form ternary RNA/Sox2/DNA complexes. Deletion of the RBM does not affect selection of target genes but mitigates binding to pluripotency related transcripts, switches exon usage and impairs the reprogramming of somatic cells to a pluripotent state. Our findings designate Sox2 as a multi-functional factor that associates with RNA whilst binding to cognate DNA sequences, suggesting that it may co-transcriptionally regulate RNA metabolism during somatic cell reprogramming.

Dental pulp stem cells expressing SIRT1 improve new bone formation during distraction osteogenesis.

Distraction osteogenesis (DO) is one of the most promising reconstructive methods for repairing large craniofacial defects or growth deficiencies through bone regeneration, but it is also a challenge because of an undesirably long process and its complications, which limit its application in clinical practice. The transplantation of mesenchymal stem cells (MSCs) is regarded as an innovative approach to accelerate bone regeneration. Dental pulp stem cells (DPSCs) have shown some advantages over other human adult MSCs, and DPSCs have been regarded as one of the most promising cell sources used in the endogenous tissue engineering. Furthermore, using stem cells modified by gene engineering in DO has been reported in previous studies. It has been shown that Sirtuin-1 (SIRT1) can directly regulate the differentiation of MSCs into osteoblasts. In this study, DPSCs expressing SIRT1 were prepared and their effects on the new bone formation were further investigated in rabbits with tibia. Rabbits were injected with the adenovirus (Adv)-SIRT1-green fluorescent protein (GFP)-transfected DPSCs (overexpression group, Group OE), Adv-GFP transfected DPSCs (negative control group, Group NC) or physiologic saline (control group, Groups CON) into the distraction gap. The new bone tissues in the distraction gap were harvested 8 weeks later, and subjected to by radiographic examination, micro-CT evaluation, and histological and mechanical testing. The better bone formation, the highest bone mineral density (BMD) and the highest bone mineral content (BMC) were observed in the OE group. In conclusion, SIRT1-modified DPSCs in DO was more effective to promote new bone formation during DO, which provides evidence for further investigation about the role of of SIRT1 in the DO.

[Mesenchymal stem cells modified with Runt-related transcription factor 2 promote bone regeneration in rabbit mandibular distraction osteogenesis].

OBJECTIVE: This work investigated mesenchymal stem cells (MSCs) modified with Runt-related transcription factor 2 (Runx2) therapy for bone regeneration in rabbit mandibular distraction osteogenesis. METHODS: Forty-eight New Zealand mature white rabbits were randomly divided into three groups after the rabbit model of mandibular distraction osteogenesis was established: reconstruction plasmid modified with Runx2 (group A), plasmid without Runx2 (group B), and the same dose of saline as control (group C). At the fifth day of distraction phase, MSCs with reconstruction plasmid modified with adv-hRunx2-gfp were injected into the distraction gap of group A. MSCs with reconstruction plasmid modified with adv-gfp was injected into the distraction gap of group B, whereas group C was injected with the same dose of saline. At 8 weeks after injection, all animals were sacrificed, and the distracted mandibles were harvested. The general imaging histological observation and three-point bending test were used for evaluation. RESULTS: CT plain scan and histological analysis confirmed that the amount of new bone forming in the distraction gap of group A was significantly higher than those in groups B and C. Dual-energy X ray and three-point bending test results also showed that the bone mineral density, bone mineral content, and maximum load of the distraction gap of group A were significantly higher than those of groups B and C (P<0.01). CONCLUSION: Runx2-ex vivo gene therapy based on MSCs can effectively promote the bone regeneration in rabbit mandibular distraction osteogenesis and shorten the stationary phase. Therefore, reconstruction of craniofacial fracture would be a valuable strategy

Runx2 modified dental pulp stem cells (DPSCs) enhance new bone formation during rapid distraction osteogenesis (DO).

Distraction osteogenesis (DO) remains a major challenge in orthopedic and craniofacial surgery. The transplantion of mesenchymal stem cells (MSCs) could reduce the treatment period and the associated complications by increasing new bone formation during long-bone DO. Runt-related transcription factor 2 (Runx2) encodes a nuclear protein that is a pivotal regulator of osteoblast differentiation. It significantly stimulates calcium accumulation and alkaline phosphatase (ALP) activity in dental pulp stem cells (DPSCs). In this study, we investigated the effects of gene therapy using Runx2 on new bone formation during tibia DO of rabbits. The distraction gap of the rabbits was injected with adenovirus (Adv)-Runx2-green fluorescent protein (GFP)-transfected DPSCs (overexpression group, Group OE) or Adv-GFP-transfected DPSCs (negative control group, Group NC). Rabbits in the control group (Groups CON) were injected with physiologic saline. The generation of new bone tissue in the distraction gap was studied by radiographic examination, micro-computed tomography (CT) evaluation, histological analyze, and Mechanical testing at weeks 8 in the consolidation period. Excellent bone formation in the distracted callus was observed in Group OE and Group NC. Moreover, the OE group showed better bone formation and the highest bone mineral density (BMD) and bone mineral content (BMC). Group CON animals showed inadequate bone formation in the distracted callus compared to the other groups. The results suggest that gene therapy using Runx2-modified DPSCs was more effective during bone deposition and new bone formation in tibia DO.