Mechanism underlying circularRNA_014301-mediated regulation of neuronal cell inflammation and apoptosis.

Spinal cord injury (SCI) causes damage to the spinal cord owing to trauma or disease and myelinated fiber tracts that transmit sensation and motor signals to and from the brain. Circular RNAs (circRNAs) are a recently discovered class of regulatory molecules, and their roles in SCI are still unknown. circRNA_014301 was indicated to be differentially expressed in the spinal cord at the site of SCI in a rat model. To analyze the role of circRNA_014301 in SCI, we exposed rat adrenal pheochromocytoma PC12 cells were exposed to increasing concentrations of lipopolysaccharide (LPS) and to construct a PC12 cell inflammatory model. Cell Counting Kit-8 assay was used to analyze cell viability. Reverse transcription-quantitative PCR and ELISA were used to detect the expression of inflammatory factors (IL-1ß, IL-6 and TNF-α). Annexin V-FITC/PI double staining was employed to detect cell apoptosis, and western blotting was performed to detect the expression of apoptotic proteins (Bax/Bcl-2/cleaved caspase-3) and NF-κB. The results demonstrated that LPS induced inflammation in PC12 cells as evidenced by the reduced cell proliferation and enhanced expression of inflammatory and apoptotic factors under increasing LPS concentrations. Western blotting analyses indicated that circRNA_014301 induced the expression of p-NF-κB/NF-κB, Bax and cleaved caspase-3, and decreased the expression of Bcl-2 following LPS-induced inflammation, and this apoptosis-promoting effect was relieved by small interfering-RNA-mediated knockdown of circRNA_014301. Thus, circRNA_014301 silencing alleviated apoptosis and inflammation in PC12 cells. SCI is invariably associated with spinal cord inflammation, and LPS was used to stimulate apoptosis and inflammatory injury in PC12 cells, and create a cell model of SCI. By promoting PC12 cell apoptosis under inflammatory conditions, it was indicated that circRNA_014301 may suppress SCI. Therefore, circRNA_014301 may represent a potential target for SCI diagnosis and therapy.

Exploration of Immune-Related Gene Expression in Osteosarcoma and Association With Outcomes.

Importance: Host immune dysregulation is associated with initiation and development of osteosarcoma. In addition, immunotherapy for osteosarcomas requires some knowledge of the immune state of patients. Objective: To perform an immunogenomic landscape analysis based on The Cancer Genome Atlas (TCGA) project, which provides osteosarcoma samples with clinical information. Design, Setting, and Participants: This genetic association study was conducted from July 20, 2020, to September 20, 2020, as a secondary analysis of public data. Cox regression and risk score analyses were used to construct signatures of immune-related genes (IRGs) in 84 patients with osteosarcoma from TCGA with corresponding clinical information. Patients were divided into high- and low-risk groups with 42 individuals in each group according to their risk scores. Data were analyzed from July 20 to September 20, 2020. Main Outcomes and Measures: Differentially expressed genes (DEGs) were analyzed between groups, and potential molecular mechanisms, expression regulation, and immune cell infiltration were also explored using bioinformation methods. A prognostic model based on independent risk factors selected from multivariate Cox hazard ratio regression was established to estimate 1-year overall survival. Results: In this genetic association study based on 84 samples from patients with osteosarcoma from TCGA (mean [SD] age, 15.0 [4.8] years; 47 [56.0%] men; mean [SD] follow-up time, 4.1 [2.8] years), a total of 14 survival-associated IRGs were identified. Patients assigned to the high-risk group had worse survival than patients from the low-risk group (1 death [2.4%] vs 26 deaths [61.9%%]; P < .001). The protein digestion and absorption pathway was one of the associated pathways in the functional enrichment analysis (gene ratio, 2:8; P < .001). The prognostic model based on metastases at diagnosis and risk score performed well in 1-year overall survival estimations (area under the curve, 0.947; 95% CI, 0.832-0.972). The risk score was correlated with immune cell infiltration (B cells: r = 0.331; P = .002; macrophages: r = 0.410; P < .001; CD8 T cells: r = 0.230; P = .04). Conclusions and Relevance: This genetic association study developed a prognostic modeling tool for osteosarcoma based on IRG expression profiles, which could result in improved survival rates through more individualized therapies. Further research on IRG expression profiles could provide potential targets for future studies on immune treatment for osteosarcoma.

Platelet-Rich Plasma Promotes Spinal Ligament Healing after Injury.

BACKGROUND: Spinal ligament injury is often a comorbidity in spine fracture. Although the spinal ligament is important to maintain spinal stability, little is done to improve its healing after injury. Platelet-rich plasma (PRP) has been shown effective in treating many tendon and ligament disorders, but its role in spinal ligament injury remains to be evaluated. METHODS: Supraspinous ligament and interspinous ligament were cut in rabbits to simulate spinal ligament injury after spine fracture. After the injury, the administration of autologous PRP was performed and compared with saline injection control. Morphology and histological analysis were utilized to assess PRP effect and compare it to the saline control group. To understand potential molecular mechanisms of PRP on ligamentous healing, bioinfor-matics analysis of the microarray dataset (GSE70918) from the Gene Expression Omnibus database was conducted. RESULTS: The PRP group was more likely to have a better appearance both morphologically and histologically than did the saline control group. Pathway analysis determined that IL-17 signaling pathway and TNF signaling pathway were the two significantly induced signaling pathways in tendon fibroblasts treated by PRP. In the protein-protein interaction network analysis, interleukin 6 had the highest degrees of connectivity. CONCLUSIONS: PRP improves spinal ligament healing through the activation of pathways related to inflammation. Further studies are required to determine the dosing and timing of PRP administration to achieve better longterm treatment outcome.

A Network Pharmacology-Based Study on the Anti-Lung Cancer Effect of Dipsaci Radix.

OBJECTIVE: Dipsaci Radix (DR) has been used to treat fracture and osteoporosis. Recent reports have shown that myeloid cells from bone marrow can promote the proliferation of lung cancer. However, the action and mechanism of DR has not been well defined in lung cancer. The aim of the present study was to define molecular mechanisms of DR as a potential therapeutic approach to treat lung cancer. METHODS: Active compounds of DR with oral bioavailability ≥30% and drug-likeness index ≥0.18 were obtained from the traditional Chinese medicine systems pharmacology database and analysis platform. The potential target genes of the active compounds and bone were identified by PharmMapper and GeneCards, respectively. The compound-target network and protein-protein interaction network were built by Cytoscape software and Search Tool for the Retrieval of Interacting Genes webserver, respectively. GO analysis and pathway enrichment analysis were performed using R software. RESULTS: Our study demonstrated that DR had 6 active compounds, including gentisin, sitosterol, Sylvestroside III, 3,5-Di-O-caffeoylquinic acid, cauloside A, and japonine. There were 254 target genes related to these active compounds as well as to bone. SRC, AKT1, and GRB2 were the top 3 hub genes. Metabolisms and signaling pathways associated with these hub genes were significantly enriched. CONCLUSIONS: This study indicated that DR could exhibit the anti-lung cancer effect by affecting multiple targets and multiple pathways. It reflects the traditional Chinese medicine characterized by multicomponents and multitargets. DR could be considered as a candidate for clinical anticancer therapy by regulating bone physiological functions.

Mechanism of lung adenocarcinoma spine metastasis induced by CXCL17.

PURPOSE: Spine metastases are common in patients with lung adenocarcinoma (LUAD). Improving the clinical outcome of spine metastasis LUAD patients requires knowledge on the mechanism underlying the metastatic process. Here, we sought to decipher the effect and mechanism of C-X-C motif chemokine ligand 17 (CXCL17) on LUAD spine metastasis. METHODS: Clinical tumor tissue samples, lung cancer cell lines and a TCGA dataset were used for CXCL17 expression analyses. A transwell invasion assay was used to assess the chemotaxis capacity of mononuclear macrophages induced by CXCL17. Western blotting was performed to explore the mechanism of mononuclear macrophage chemotaxis towards CXCL17. A cell counting kit-8 assay was employed to investigate the effect of conditioned medium from M1 and M2 macrophages on the proliferation of lung cancer cells. RESULTS: We found that the expression of CXCL17 was higher in clinical LUAD samples and LUAD cell lines than in lung squamous cell carcinoma (LUSC) samples and cell lines. Moreover, we found that CXCL17 increased the migration of THP-1 mononuclear macrophages by activating the Src/FAK pathway. In addition, we found that conditioned medium from M2 macrophages promoted the proliferation of LUAD cells. CONCLUSIONS: From our data we conclude that CXCL17 is a key regulator of LUAD spine metastasis. CXCL17 and its downstream Src/FAK pathway may serve as clinical intervention targets.

DUSP19 mediates spinal cord injury-induced apoptosis and inflammation in mouse primary microglia cells via the NF-kB signaling pathway.

Objective: Spinal cord injury (SCI) is a common injury that seriously threatens human health. NF-κB may be involved in the secondary injury of SCI that is mediated by inflammation and aggravates damage. Our study was aimed to investigate the role of NF-κB signaling in DUSP19-mediated cleaved Caspase-3 expression and the release of inflammatory factors in vivo and in vitro.Materials and Methods: DUSP19 mRNA expression and the content of IL-6 and IL-8 in patients with traumatic SCI (TSCI) were measured by real-time PCR and ELISA, respectively. The levels of p-NF-κBp65, NF-κBp65 and cleaved Caspase-3 expression and the concentrations of IL-6 and IL-8 were measured by western blotting and ELISA, respectively.Results: Patients with TSCI showed lower DUSP19 expression and higher concentration of IL-6 and IL-8 compared with healthy controls. DUSP19 overexpression inhibited p-NF-κBp65 level, cleaved Caspase-3 expression, and production of IL-8 and IL-6 in the mice induced by TSCI. DUSP19 silencing increased p-NF-κBp65 level, cleaved Caspase-3 expression, and concentration of IL-6 and IL-8 in mouse primary microglia cells. DUSP19 overexpression had an inverse effect. Importantly, DUSP19 silencing and overexpression mediated p-NF-κBp65 level, cleaved Caspase-3 expression, and concentration of IL-6 and IL-8 in mouse primary microglia cells were reversed by NF-κB inhibitor pyrrolidine dithiocarbamate (PDTC) and NF-κB activator 12-myristate 13-acetate (PMA), respectively.Conclusion: These results suggested that DUSP19-mediated SCI-induced apoptosis and inflammation via NF-κB signaling and might therefore serve as a potential therapeutic target for SCI.

Homeobox C10 knockdown suppresses cell proliferation and promotes cell apoptosis in osteosarcoma cells through regulating caspase 3.

AIM: Homeobox (HOX) genes and their protein products have been found to function as oncogenes in the progression of many cancers. But the role of Homeobox C10 (HOXC10) in osteosarcoma (OS) still remains less understood. In this study, we firstly determine the biologic functions of HOXC10 in OS. MATERIALS AND METHODS: We examined the expression of HOXC10 in OS tissues by quantitative real-time polymerase chain reaction and Western blot assays. We investigated the effects of HOXC10 on cell proliferation, apoptosis and caspase 3 activity in three OS cell lines by RNA interference, Cell Counting Kit-8, flow cytometry and colorimetric assays. RESULTS: We found that HOXC10 was elevated in OS tissues. Silencing HOXC10 significantly inhibited cell proliferation, induced cell apoptosis and increased the expression and activity of caspase 3. The resistance assay further suggested that HOXC10 affected cell growth and apoptosis through regulating the expression and activity of caspase 3. CONCLUSION: HOXC10 might function as an oncogene in OS by regulating the expression and activity of caspase 3.

Effects of combined c-myc and Bmi-1 siRNAs on the growth and chemosensitivity of MG-63 osteosarcoma cells.

Osteosarcoma is the most common form of primary malignant bone tumor. Patients who are insensitive to chemotherapy treatment often have a poor prognosis. According to our previous study, recombinant adenovirus (Myc-AS) in combination with caffeine enhances the induction of apoptosis and the chemotherapeutic effects of cisplatin (CDDP) in MG-63 osteosarcoma cells. The present study aimed to investigate the combinational effects of the small interfering RNAs (siRNAs) c-myc and Bmi-1 on the growth and chemosensitivity of MG-63 osteosarcoma cells. The results indicated that the cell growth inhibition rates of MG-63 cells gradually increased with increasing concentrations of CDDP (P<0.05). This observation was consistent in the single and combined siRNA groups. At a concentration of 5.0 µg/ml CDDP, the growth inhibition rates were 53.3±5.2, 42.7±6.3 and 40.9±4.7% in the combined, c-myc and Bmi-1 siRNA groups, respectively. The cell growth inhibition rate in the combined siRNA group was higher than that observed in the two single siRNA groups (P<0.05). The cell apoptotic rate was 37.3±4.9% in the combined siRNA group, which was significantly higher than that observed in the c-myc (24.8±5.6%) and Bmi-1 siRNA groups (22.7±6.1%; P<0.05). These results suggest that the chemosensitivity of MG-63 cells to CDDP may be markedly enhanced in the siRNA combination group. A decrease in cell proliferation and increased cell apoptosis were also observed in the siRNA combination group. The present study may provide novel insights to further elucidate the pathogenesis and drug resistance mechanisms involved in osteosarcoma. It may also improve our understanding of the underlying mechanisms involved in chemotherapeutic sensitivity, and thus aid the development of future therapeutic strategies for the treatment of osteosarcoma.

Enhancement effect of adenovirus-mediated antisense c-myc and caffeine on the cytotoxicity of cisplatin in osteosarcoma cell lines.

AIMS: Studies on cancer biology have shown that overexpression of oncogenes (with or without functional loss of tumor suppressor genes), which is responsible for the progression of human malignancies via a multistep process, may be reduced by antisense technology. Caffeine enhances the effect of cisplatin (CDDP) chemotherapy on osteosarcoma cells. We constructed the recombinant adenovirus (Myc-AS) encoding the antisense c-myc fragment and investigated the synergic effect of caffeine and Myc-AS on the in vitro sensitivity of osteosarcoma MG-63 cells to cisplatin. METHODS: The recombinant adenovirus (Myc-AS) encoding the antisense c-myc fragment was constructed by cloning c-myc cDNA of about 750 bp in a reverse direction into adenovirus vector, then undergoing recombination, amplification and complementation in vivo. Myc-AS and caffeine were used either alone or in combination with CDDP to treat osteosarcoma MG-63 cells in vitro. Western blot, MTT, flow cytometry (FCM) and electron microscopy were used to evaluate the expression of c-myc protein, tumor cell proliferation in vitro and apoptosis and to perform cell cycle analysis. RESULTS: Myc-AS encoding antisense c-myc fragment was obtained with a titer of 2 x 10(9) pfu/ml. Myc-AS downregulated the expression of c-myc protein after transfecting MG-63 cells for 48 h, induced tumor cell apoptosis and inhibited tumor cell proliferation in vitro. Myc-AS or caffeine can enhance the cytotoxic effects of 2.0 and 5.0 microg/ml CDDP on MG-63 cells. Moreover, the significantly enhancing effect of the Myc-AS-caffeine combination on CDDP chemotherapy of MG-63 cells was not restricted to apoptosis but also decreased tumor cell proliferation in vitro. Expression of the apoptosis-associated bcl-2 gene was downregulated and bax was upregulated, with no changes in E2F-1 expression. FCM analysis showed that CDDP treatment induced a block in S phase, and caffeine reversed this block and accelerated cell progression through the S phase. CONCLUSIONS: Myc-AS can induce obvious G2/M phase arrest in transfected cells. Myc-AS combined with caffeine can enhance apoptosis induction and chemotherapeutic effects of CDDP on osteosarcoma MG-63 cells.

[The treatment of knee joint peripheral fractures and/or dislocations with vascular injury].

OBJECTIVE: To investigate the effect and influence factors on knee joint peripheral fractures and/or dislocations with an associated vascular injury through retrospectively study. METHODS: From March 2002 to November 2007 31 patients with knee joint peripheral fractures and/or dislocations with an associated vascular injury were treated, including 24 males and 7 females with a mean age of 41 years (range from 21 to 62 years). Definite diagnosis of vascular injury by combining colored ultrasonic, CTA, operative exploration with clinical signs, fixing fractures and/or dislocations with fixators, plates and screws, reconstructing blood circulation based on the condition of the vascular injury by vascular repair, homograft vein or artificial vascular grafting separately and analysing the effects of PSI, diagnosis and treatment methods on salvage lower extremities. RESULTS: Successful reconstruction was carried out in 31 cases, however there were 1 death because of mult-fractures and brain injury and 6 amputation, 24 cases successful salvage followed up mean 24.2 months, 6 cases bone nonunion and infected bone defect were cured by delayed bone planting or bone transportation. Ligaments repair reconstruction of 7 cases knee joint dislocation were done in delayed 3 or 4 weeks after first operation, the good functional rate was 71.4%. CONCLUSIONS: The patients of PSI under 10 grades in knee joint peripheral fractures and/or dislocations with an associated vascular injury should been carried out treatment, early definite diagnosis and blood circulation reconstruction are the key factors of successful salvage treatment.

Recombinant antisense C-myc adenovirus increase in vitro sensitivity of osteosarcoma MG-63 cells to cisplatin.

C-myc is an oncogene with the important role of cell proliferation controller. It has been found to be amplified and overexpressed in osteosarcoma. Moreover, it can promote cell transformation and induce metastatic features. Some studies showed that overexpression of c-myc could induce resistance in response to antineoplastic agents. Currently, we constructed the recombinant adenovirus (Ad-Asc-myc) encoding antisense c-myc fragment and investigated its effect on the in vitro sensitivity of osteosarcoma MG-63 cells to cisplatin(CDDP). The osteosarcoma MG-63 cells were transfected by the Ad-Asc-myc in vitro, and Western Blot, MTT assay, RT-PCR, flow cytometry (FCM), and transmission electron microscopy (TEM) were used to study expression of c-myc and caspase-3 protein, tumor cell proliferation in vitro, cell apoptotic morphology and cell cycle change. Ad-Asc-myc encoding antisense c-myc fragment was obtained with the titer of 2.0 x 10(9) pfu/ml. Ad-Asc-myc downregulated the expression of c-myc protein after transfected MG-63 cells for 48 hours, combined with the treatment of 2.0, 5.0 microg/ml cisplatin for 2 hours can inhibited tumor cells proliferation in vitro by 33.4 and 54.2 percent, respectively, which had significant difference compared with control recombinant adenovirus (Ad-LacZ) groups (P < 0.05). RT-PCR revealed that Ad-Asc-myc downregulated expression of bcl-2 and upregulated expression of Bax, and no appreciable changes were observed in the expression of E2F-1. Detection of caspase-3 protein TEM, and FCM analysis showed that Ad-Asc-myc could induce apoptosis of transfected cells, which was enhanced by the treatment of cisplatin. Cell cycle analysis showed that obvious G(2)/M phase arrested in transfected cells. In conclusion, Ad-Asc-myc increased the in vitro sensitivity of osteosarcoma MG-63 cells to cisplatin as well as induced apoptosis.

[Adenovirus mediated antisense c-myc gene on the chemotherapy sensitivity of osteosarcoma cells to cisplatin].

OBJECTIVE: To construct the recombinant adenovirus encoding antisense c-myc fragment and to investigate its effect on the chemotherapy sensitivity of osteosarcoma MG-63 cells to cisplatin. METHODS: The recombinant adenovirus (Ad-Asc-myc) encoding antisense c-myc fragment was constructed by cloning c-myc cDNA of about 720 base pairs in a reverse direction into adenovirus vector, then undergoing recombination, amplifying and being complemented in vivo. The osteosarcoma MG-63 cells were transfected by the Ad-Asc-myc in vitro, and Wright staining, Acridine Orange staining, Western Blot, MTT, Flow Cytometry (FCM) were used to study cell morphology, expression of c-myc protein, tumor cell proliferation in vitro, apoptosis and cell cycle change. RESULTS: Ad-Asc-myc encoding antisense c-myc fragment was obtained with the titer of 2 x 10(9) pfu/ml. Ad-Asc-myc down-regulated the expression of c-myc protein after transfected MG-63 cells for 48 h, combined with the treatment of 2.0, 5.0 microg/ml cisplatin for 2 h could inhibit tumor cells proliferation in vitro by 33.4% and 54.2% respectively, which were significantly difference compared with control recombinant adenovirus (Ad-LacZ) groups (P < 0.05). Acridine Orange staining and FCM analysis showed that Ad-Asc-myc could induce apoptosis of transfected cells, which was enhanced by the treatment of cisplatin cell. Cycle analysis showed that obvious G2/M phase arrested in transfected cells. CONCLUSION: Ad-Asc-myc increases the chemotherapy sensitivity of osteosarcoma MG-63 cells to cisplatin as well as induced apoptosis.

[Construction of antisense c-myc recombinant adenovirus and its anti-tumor effects on osteosarcoma cell lines MG-63 and U2OS].

BACKGROUND & OBJECTIVE: c-myc, an oncogene, plays an important role in regulation of cell proliferation, and has been found to be amplified and overexpressed in osteosarcoma. Moreover, it can promote cell transformation, and induce metastasis. This study was to construct recombinant adenovirus encoding antisense c-myc, and to investigate its effects on osteosarcoma cell lines MG-63 (p53-deficient) and U2OS (with wild type p53). METHODS: Recombinant adenovirus Ad-As-c-myc encoding antisense c-myc was constructed by gene reconstruction technique, defined by polymerase chain reaction (PCR), and transfected into human osteosarcoma cell lines MG-63 and U2OS. Western blot, acridine orange staining, reverse transcription-PCR (RT-PCR), and flow cytometry (FCM) were used to detect expression of c-myc, proliferation, apoptosis, and cell cycle of MG-63 and U2OS cells. RESULTS: Ad-As-c-myc encoding antisense c-myc was obtained with the titer of 2x10(9) pfu/ml. Ad-As-c-myc significantly inhibited proliferation of both cell lines, while U2OS with wild type p53 was more susceptible to Ad-As-c-myc. Expression of c-myc mRNA was down-regulated in 2 cell lines 48 h after transfection of Ad-As-c-myc. Acridine orange staining and FCM analysis showed that Ad-As-c-myc induced apoptosis of both cell lines, cell cycle analysis showed obvious G(2)/M phase arrest in MG-63 cells,and G1 phase arrest in U2OS cells after transfection of Ad-As-c-myc. CONCLUSION: Ad-As-c-myc could induce apoptosis through both P53-dependent and P53-independent pathways, and inhibit proliferation of osteosarcoma cells.

[Inhibitory effects of survivin antisense oligonucleotide on drug-resistant human ovarian cancer cell COC1/DDP].

BACKGROUND & OBJECTIVE: Recent studies have shown that survivin is an anti-apoptosis gene, which plays an important role in the carcinogenesis and drug resistance of ovarian cancer. This study was designed to explore the effects of liposome-survivin antisense oligonucleotide (Lip-ASODN) on the growth,apoptosis,and cell cycle distribution of drug-resistant human ovarian cancer cell line COC1/DDP. METHODS: Survivin-ASODN were transfected into COC1/DDP cells mediated by lipofectin. The proliferation of COC1/DDP cells was assessed by cyto-dynamics and MTT assay. The mRNA expression of survivin was determined by reverse transcription- polymerase chain reaction (RT-PCR). The caspase-3 protein activity was measured by Western blot analysis. The apoptotic rate and cell cycle distribution were estimated by flow cytometry (FCM). RESULTS: Compared with Lip-SODN and Lip alone groups, the proliferation of COC1/DDP cells after cultured with Lip-ASODN has been significantly inhibited, its inhibitory rate was (68.3+/-6.2)% after cultured for 72 hours (P< 0.05). The mRNA expression of survivin in Lip-ASODN group was significantly decreased, while the caspase-3 activity increased in a time-dependent manner as compared with Lip-SODN and Lip alone groups (P< 0.05). Cell cycle distribution significantly changed in Lip-ASODN group, many cells have been blocked in G0/G1 phase (79.21%), while G2/M phase (4.92%) and S phase (15.87%) decreased. The apoptotic rate of Lip-ASODN group reached 33.18%, which was much higher than those of Lip-SODN and Lip alone groups (P< 0.05). CONCLUSION: Survivin ASODN can inhibit COC(1)/DDP cell proliferation, reduce the mRNA expression of survivin, and induce cell apoptosis.