Discoidin domain receptor 2 activation of p38 mitogen-activated protein kinase as an important pathway for osteonectin-regulating osteoblast mineralization

Objective The present study aimed to determine the role of the discoidin domain receptor 2 (DDR2) in the osteonectin (ON) regulation of osteoblast mineralization through the activation of p38 mitogen-activated protein kinase (MAPK). Methods Four groups were established: the ON group, the inhibitor group, the Ddr2-small interfering ribonucleic acid (siRNA) group, and the control group. Osteoblasts from the parietal bones of neonatal Sprague–Dawley rats were isolated and cultured. In the ON group, 1 µg/mL ON was added to the osteoblasts. The gene expressions of collagen 1 (Col 1) and Ddr2 were detected using reverse transcription-quantitative polymerase chain reaction (RT-qPCR). In the inhibitor group, the osteoblasts were added to WRG-28 (a specific DDR2 inhibitor), and in the Ddr2-siRNA group, the osteoblasts were transfected with Ddr2-siRNA. The gene and protein expressions of DDR2, bone sialoprotein, osteocalcin, osteopontin, and p38 MAPK were determined using RT-qPCR and western blot analysis. Alizarin red staining and transmission electron microscopy were used to detect mineralization. Results The results showed that ON enhanced the osteoblast Col 1 and Ddr2 gene expressions, while the use of a Ddr2-siRNA/DDR2-blocker decreased the OPN, BSP, OCN, and P38 gene and protein expressions and reduced osteoblast cellular activity and mineralized nodules. Conclusion The present study demonstrated that DDR2 activation of p38 MAPK is an important approach to ON-regulating osteoblast mineralization.

Effect of Sirtuin1 (Sirt1) on Bone Marrow Mesenchymal Stem Cells (BMSCs) Osteogenesis/Adipogenesis via β-Catenin/The Transcription Factor T Cell Factor 1 (TCF1)/Runt-Related Transcription Factor 2 (RUNX2)

Bone marrow mesenchymal stem cells (BMSCs) have self-renewal potential. Sirt1 regulates cell differentiation and apoptosis. However, Sirt1’s effect on BMSCs osteogenic/adipogenic differentiation has not been fully elucidated. SD rats were randomly divided into Osteoporosis (OP) group and sham operation group. OP rat BMSCs were isolated and assigned into control group, NC group and Sirt1 siRNA group followed by analysis of Sirt1 level by Real-time PCR, cell proliferation by MTT assay, expression of OC, OPN and FABP4 level by real time PCR, and β-Catenin/TCF1/Runx2 protein expression by Western blot. In OP group, Sirt1 expression was significantly increased and BMSCs proliferation was decreased along with reduced OC and OPN mRNA expression, increased FABP4 expression and reduced β-Catenin/TCF1/Runx2 expression compared with sham operation group (P < 0.05). In Sirt1 siRNA group, Sirt1 expression was significantly reduced, BMSCs proliferation was increased, OC and OPN mRNA expression was increased, FABP4 expression was decreased, and β-Catenin/TCF1/Runx2 expression was increased compared to OP group (P < 0.05). Sirt1 is increased in osteoporosis. Down-regulating Sirt1 in osteoporotic BMSCs can regulate β-Catenin/TCF1/Runx2 signaling and promote BMSCs osteogenic differentiation and inhibit adipogenic differentiation.

Influences of S100A8 and S100A9 on Proliferation of Nasopharyngeal Carcinoma Cells through PI3K/Akt Signaling Pathway

Objective. To investigate the effects of S100A8 and S100A9 on proliferation in nasopharyngeal carcinoma cells and the regulatory effects of PI3K/Akt signaling pathway. Methods. Nasopharyngeal carcinoma cells (CNE1) were cultured and randomly divided into three groups: control group, S100A8/S100A9 overexpression group, and siRNA S100A8/S100A9 group. CCK-8 method was used to detect the effect of S100A8 and S100A9 on the viability of nasopharyngeal carcinoma cells. The effects of S100A8 and S100A9 on the colony forming ability of nasopharyngeal carcinoma cells were detected by colony forming assay. The effects of S100A8 and S100A9 on the proliferation of nasopharyngeal carcinoma cells were detected by EdU staining. The mRNA levels of PI3K and Akt were detected by RT-PCR. The expression levels of PI3K and Akt in NPC cells were detected by Western blot. Wortmannin, an inhibitor of PI3K/Akt pathway, was used to inhibit the activation of PI3K/Akt pathway. Results. Compared with the control group, the cell viability, the number of plate clones, the positive rate of EdU staining, and the mRNA and protein levels of PI3K and Akt were increased in the overexpression group. Compared with the control group, the cell viability, the number of plate clones, the positive rate of EdU staining, and the mRNA and protein levels of PI3K and Akt were decreased in the siRNA group. After inhibiting the activation of PI3K/Akt pathway, the viability of NPC cells in the overexpression group decreased significantly at 48 h and 72 h, while that in the siRNA group increased significantly. Conclusion. SiRNA S100A8 and S100A9 could inhibit the proliferation of nasopharyngeal carcinoma cells, and the underlying mechanism may be related to the inhibition of PI3K/Akt signaling pathway.

Inhibitory Effect of Centipede Scolopendra Extract on Gallbladder Carcinoma via Up-regulation of PUMA

Background: To observe the effect of centipede scolopendra extract on gallbladder carcinoma (GBC) and further investigate its underlying mechanism.Methods: The GBC cell line GBC-SD was purchased and cultured. Small interfering RNA (siRNA) was constructed. The mRNA expression levels of PUMA were measured by reverse transcription-quantitative PCR (RT-qPCR) and protein expression levels of p53, PUMA, Bax and Bcl-2 were measured by western blotting respectively. Viability and proliferation were detected using MTT and colony formation assays respectively. The apoptosis rate was determined by Annexin-V-FITC/PI double staining. Results: The GBC-SD cell line was successfully obtained and cultured. MTT and colony formation assays demonstrated that the viability and proliferation of GBC-SD cells could be markedly inhibited by centipede scolopendra extract in a concentration-dependent manner in the limited concentration range. Following pretreatment with centipede scolopendra extract, RT-qPCR demonstrated that the expression levels of PUMA were markedly increased in the GBC-SD cells, and western blotting showed that the high expression of PUMA was accompanied by upregulation of Bax and downregulation of p53 and Bcl-2 in the GBC-SD cells. However, this effect has proved hard to reproduce after PUMA-siRNA. Flow cytometry revealed that the apoptosis rate was 9.8±2.2%, 25.3±3.6%, 10.6±2.0%, and 13.5±2.4% in control group, centipede scolopendra extract group, PUMA-siRNA group, and centipede scolopendra extract combined with PUMA-siRNA group respectively. Conclusions: Centipede scolopendra extract could induce the apoptosis of GBC-SD cells by promoting the PUMA-Bax signaling pathway. It could serve as a potential novel therapy for GBC in clinical practice.

CDK-associated Cullin 1 promotes Cell Proliferation and inhibits Cell Apoptosis in Human Glioblastoma

Background: Glioma is the most common intracranial primary tumour of adult humans, and its pathological mechanism and molecular characteristics are under investigation. CDK-associated cullin 1 (CACUL1) has been shown to regulate colorectal carcinoma, lung cancer and gastric cancer development. Objective: This study aims to explore the role of CACUL1 in the pathogenesis of human glioma. Methods: CACUL1 levels in human glioma tissue microarrays were detected by immunohistochemistry analysis. Two glioblastoma cell lines, namely, U87 and U251, were transfected with CACUL1 siRNA, and cell proliferation, cell cycle, cell apoptosis and regulating molecules including cyclin E1, cyclin A2, CDK2, p21, Bcl2 and Bax were assessed by CCK8, flow cytometry and Western blot. Results: CACUL1 expression in glioma tissue was significantly higher than that in normal brain tissue. CACUL1 knockdown impeded cell proliferation, induced cell apoptosis and caused G1/S transition arrest in glioblastoma cells. The cell cycle-related proteins CDK2, cyclin E1 and cyclin A2 were dramatically decreased in the CACUL1 siRNA group compared to the non-targeting siRNA group in both U87 and U251 cells, while the CDK inhibitory protein p21 was increased in U87 cells. Additionally, the Bcl-2/Bax ratio was significantly decreased. Conclusion: CACUL1 can promote cell proliferation and suppress apoptosis of glioma cells and might serve as a potential oncogene for gliomas.

Study on the correlation between the expression of basic transcription factor 3 (BTF3) and the efficacy and prognosis of patients with malignant melanoma (MM).

e21554 Background: BTF3 is an RNA polymerase II transcription factor, studies have confirmed that BTF3 is abnormally expressed in several types of tumors and closely related to the proliferation and prognosis of gastrointestinal tumors. However, the function of BTF3 in MM remains unknown. The present study aim to detect the expression and proliferation of BTF3，to explore the correlation between the BTF3 expression and the efficacy and prognosis of MM patients. Methods: We explored the effect of BTF3 through silencing BTF3 expression by siRNA transfection with lentivirus in human melanoma cell lines. The cell proliferation, cell cycle and apoptosis were determined by methyl-thaizolyl-tetrazolium assay, flow cytometry and western blot, respectively. Then we investigated the effect of BTF3 in nude mice by tumor formation experiment. The expression of BTF3 was determined by immunohistochemistry (IHC) from 32 patients with different subtypes of MM during October 2014 to October 2020. Among them, 28 cases could be resected (stage I-III), 4 cases could not be resected (stage III-IV). Cutaneous MM 7 cases, Acral MM 14 cases, mucosal MM 9 cases, unknown primary MM 2 cases. The correlation of BTF3 expression and RFS (recurrence-free survival) /OS (overall survival) in 28 patients with resected MM was explored by Student’s t test. Results: The increased viability of negative control virus infection group was 4.706 times than that of BTF3-siRNA group. Compared with the former, BTF3-siRNA group had more cells in G1 and S phase (P< 0.05) and more apoptotic cells (P< 0.05). BTF3-siRNA reduced tumor formation in the nude mice (P<0.01). The relative IHC scores of BTF3 was 5.50±3.19 in tumor tissues (the higher the score, the higher the expression of BTF3), 6.52±4.13 in adjacent noncancerous tissues. The expression of BTF3 in different subtypes of MM tissues are shown in the table. In the median follow-up time of 27 months (6-68 months), 13 cases survived without recurrence or metastasis, 10 cases survived with recurrence or metastasis, 8 cases died. In 28 resectable MM patients, the median RFS/OS of high BTF3 expression (IHC score>5) and low expression (IHC score:0-4) were 18.57 / 24.62 months and 20.42 / 22.78 months. The corresponding RFS/OS P values of patients with high and low expression were 0.323 and 0.607. Conclusions: The study suggests that BTF3 play a role in the proliferation, cell cycle regulation, apoptosis and tumorigenicity of MM. Preliminary small-sample retrospective clinical study did not suggest that BTF3 was related to the efficacy and prognosis of patients, which needs to be further verified by large-sample prospective studies.[Table: see text]

Effect of Down-Regulation of Long-Chain Non-Coding RNAs Myocardial Infarction Associated Transcript 2 Expression on Osteoarthritis Chondrocytes

Osteoarthritis (OA) is featured as articular cartilage degradation. LncRNA Mirt2 involves in inflammation, but its role in osteoarthritis is unclear. Our study intends to assess LncRNA Mirt2’s role in OA chondrocytes. The chondrocytes of OA patients (OA group) and healthy controls (control group) were isolated to measure LncRNA Mirt2 expression by Real time PCR. Chondrocytes were assigned into control group, LPS group, LPS + si-NC group, LPS + Mirt2 siRNA group followed by analysis of LncRNA Mirt2 level by real time PCR, cell proliferation by MTT assay, cell apoptosis by flow cytometry, expression of COL2A1, MMP13, ADAMTS-5, MEK1/2, Erk1/2 and phosphorylated Erk1/2 by western blot. LncRNA Mirt2 level was increased in OA chondrocytes. Under LPS stim-ulation, Mirt2 expression was significantly increased in chondrocytes and chondrocyte proliferation was decreased, along with significantly increased apoptosis and upregulated COL2A1, MMP13, ADAMTS-5, MEK1/2 and Erk1/2 and phosphorylated Erk1/2 (P < 0.05). Transfection of Mirt2 siRNA down-regulated its expression in chondrocytes stimulated by LPS, which significantly reversed the above changes (P < 0.05). LncRNA Mirt2 expression is increased in OA chondrocytes. Downregulation of LncRNA Mirt2 can regulate COL2A1, MMP13 and ADAMTS-5 level via MAPK/ERK signaling pathway, promote OA chondrocytes proliferation and inhibit apoptosis.

Effect of siRNA Interference with Upregulated Gene 11 Expression on the Biological Functions of Osteosarcoma Cell Line MG63 and Their Mechanisms

This study investigated the effect of siRNA interference with URG11 expression on biological function of osteosarcoma cell line MG63 and its mechanism. MG63 cells cultured in vitro were apportioned to make up a control group (not transfected), NC-siRNA group (transfected nonspecific NC-siRNA), and URG11-siRNA group (transfected URG11-siRNA). The expression of URG11 was detected through reverse-transcription polymerase chain reaction (RT-PCR). The metastasis, infiltration, and apoptosis of MG63 cells were examined through CCK-8, Transwell, and flow cytometry techniques, respectively. The expressions of URG11 protein in each group of cells and of proteins β-catenin, c-Myc, cyclin D1, MMP-2, and survivin, which are related to the Wnt/β-catenin signal pathway, were visualized by the Western blot method. In comparison with the control group, transfected URG11-siRNA may reduce the expression levels of URG11 and URG11 mRNA, β-catenin, CyclinD1, c-Myc, MMP-2, and survivin proteins in MG63 cells, and may decrease the metastasis and infiltration of MG63 cells and enhance apoptosis; nevertheless, there was no obvious change in MG63 cells after NC-siRNA transfection. The proliferation and infiltration of MG63 cells can be inhibited by interference with URG11 expression, and the apoptosis of MG63 cells can be promoted by interference with URG11 expression. The mechanism of interference with URG11 expression may relate to inhibiting the activation of the Wnt/β-catenin pathway.

Electroacupuncture Alleviates Cerebral Ischemia/Reperfusion Injury in Rats by Histone H4 Lysine 16 Acetylation-Mediated Autophagy

Background: Electroacupuncture (EA) treatment in ischemic stroke has been highlighted recently; however, the specific mechanism is still elusive. Autophagy is considered a new target for cerebral ischemia/reperfusion (I/R), but whether it plays a role of protecting or causing rapid cell apoptosis remains unclear. Studies have reported that the reduction in lysine 16 of histone H4 acetylation coheres with autophagy induction. The primary purpose of the study was to explore whether EA could alleviate I/R via autophagy-mediated histone H4 lysine 16 acetylation in the middle cerebral artery occlusion (MCAO) rat model.Methods: One hundred and twenty male Sprague-Dawley rats were divided into five groups: control group, MCAO group, MCAO+EA group, MCAO+EA+hMOF siRNA group, and MCAO+EA+Sirt1 inhibitor group. EA was applied to “Baihui” (Du20) and “Renzhong” (Du26) at 5 min after modeling and 16 h after the first EA intervention. The structure and molecular markers of the rat brain were evaluated.Results: EA significantly alleviated I/R injury by upregulating the expressions of Sirt1, Beclin1, and LC3-II and downregulating the expressions of hMOF and H4K16ac. In contrast, the Sirt1 inhibitor lowered the increase in Sirt1, Beclin1, and LC3-II and enhanced the level of hMOF and H4K16ac expressions associated with EA treatment. Besides, ChIP assay revealed that the binding of H4K16ac in the Beclin1 promoter region of the autophagy target gene was significantly raised in the MCAO+EA group and MCAO+EA+hMOF siRNA group.Conclusions: EA treatment inhibited the H4K16ac process, facilitated autophagy, and alleviated I/R injury. These findings suggested that regulating histone H4 lysine 16 acetylation-mediated autophagy may be a key mechanism of EA at Du20 and Du26 to treat I/R.

Hyperbranched Cationic Glycogen Derivative-Mediated IκBα Gene Silencing Regulates the Uveoscleral Outflow Pathway in Rats

The role of the IκB/NF-κB signaling pathway in the uveoscleral outflow pathway was investigated with IκBα gene silencing mediated by the 3-(dimethylamino)-1-propylamine-conjugated glycogen (DMAPA-Glyp) derivative. The IκBα-siRNA-loaded DMAPA-Glyp complex was transfected into the ciliary muscles of rats by intracameral injection (labeled as the DMAPA-Glyp+siRNA group). The Lipofectamine™ 2000 (Lipo)/siRNA complex and the naked siRNA were set as the controls. The mRNA and protein expression of IκBα, NF-κBp65, and MMP-2 were analyzed by real-time PCR, western blotting, and in situ gelatin zymography. Nuclear translocation of NF-κBp65 was analyzed by immunofluorescence. Rat intraocular pressure (IOP) was monitored pre- and postinjection. Gene transfection efficiency and toxicity of the DMAPA-Glyp derivative were also evaluated. After RNA interference (RNAi), IκBα mRNA and protein expression were significantly inhibited. NF-κBp65 mRNA and protein expression showed no significant differences. Nevertheless, nuclear translocation of NF-κBp65 occurred in the DMAPA-Glyp+siRNA group. Both mRNA expression and activity of MMP-2 increased, with the largest increase in the DMAPA-Glyp+siRNA group. IOP in the DMAPA-Glyp+siRNA group fell to the lowest level on day 3 after RNAi. The levels of Cy3-siRNA in the ciliary muscle of the DMAPA-Glyp+siRNA group did not significantly decrease over time. At 7 and 14 d after RNAi, no significant pathological damage was detectable in the eyes injected with the DMAPA-Glyp derivative or the DMAPA-Glyp/siRNA complex. Taken together, our results suggest that downregulation of IκBα expression in the ciliary muscle plays a crucial role in reducing the IOP values of rats. IκBα may become a new molecular target for lowering IOP in glaucoma. The DMAPA-Glyp derivative is safe and feasible as an effective siRNA vector in rat eyes.