Cell Counting Kit-8 Test Research Articles

CircRNA_002178 promotes the proliferation and migration of oral squamous cell carcinoma cells by activating the Akt/mTOR pathway.

We aimed to investigate the biological effects of circRNA_002178 in oral squamous cell carcinoma (OSCC) tissues and to analyze its potential mechanism. CircRNA_002178 expression in 50 pairs of OSCC tissues and adjacent ones was studied by quantitative polymerase chain reaction (qPCR) analysis, and the correlations of circRNA_002178 with clinicopathological indicators, as wells as prognosis of OSCC patients were analyzed. qPCR was used to verify circRNA_002178 expression in OSCC cell lines. Subsequently, circRNA_002178 knockdown models were constructed using lentivirus in OSCC cell lines, and the impacts of circRNA_002178 on the function of OSCC cells were assessed by cell counting kit-8 (CCK-8) test, plate cloning experiment, transwell assay and nude mouse tumor formation experiments. Finally, rescue experiments in vitro were used to explore the potential mechanism of circRNA_002178 activating Akt/mTOR pathway. Our data showed that circRNA_002178 expression in OSCC tissue specimens was remarkably higher than that in adjacent ones. In comparison to patients in low circRNA_002178 expression group, patients in high expression group showed higher incidences of advanced pathological stage and distant metastasis, and a lower overall survival rate. Cell functional experiments revealed that knockdown of circRNA_002178 markedly attenuated the proliferation and migration ability of OSCC cells compared to the sh-NC group, and the consistent results were observed in the nude mouse experiment. In addition, Western blot suggested that the expression of key proteins in Akt/mTOR signaling pathway was remarkably reduced after downregulation of circRNA_002178 in OSCC cells. Meanwhile, Akt activator SC79 reversed the inhibitory effect of circRNA_002178 on the metastasis and proliferation of OSCC cells. CircRNA_002178, over-expressed in OSCC tissues and cell lines, may promote the malignant progression of OSCC through activating the Akt/mTOR signaling pathway.

CircABCB10 promotes the proliferation and migration of lung cancer cells through down-regulating microRNA-217 expression.

We aimed at studying the role and molecular mechanism of circular RNA circABCB10 in the progression of lung cancer (LCa). We collected LCa tissues using quantitative real-time polymerase chain reaction (qRT-PCR) technology to determine circABCB10 expression and performed survival analysis based on the clinical data of LCa patients. At the same time, the specific effects of circABCB10 on the biological function of LCa cell lines were determined by certain cell function experiments, including cell counting kit-8 (CCK-8) test, plate cloning experiment, transwell and cell wound healing assays. The downstream key gene microRNA-217 of circABCB10 was predicted through bioinformatics analysis and the potential regulation between them was confirmed by luciferase assay. microRNA-217 was knocked down in LCa cell lines to verify its important role in the progression of LCa. CircABCB10 showed abnormally high expression in LCa tissues and cell lines and was related to the poor prognosis of patients. In vitro cell experiments demonstrated that knocking down circABCB10 remarkably suppressed the proliferation and migration ability of LCa cells. In addition, circABCB10 can specifically bind to microRNA-217 and negatively regulate its expression of microRNA-217 in LCa cells. Finally, cell functional experiments showed that microRNA-217 is a key downstream gene that mediates the regulation of circABCB10 on LCa cell function. CircABCB10, abnormally highly expressed in LCa tissues, is able to induce the malignant progression of this cancer.

RETRACTED ARTICLE: Two novel nanostructured La(III) coordination polymers were synthesized by sonochemical: crystal structures along with anticancer activity on human nasopharyngeal carcinoma cells

The preparation and the structural characterization of two La(III)-based coordination polymers was described in this study, in which {[La(H2O)(BTCTB)]·2H2O}n (1, H3BTCTB = 3,3′,3′′-[1,3,5‐benzenetriyltris(carbonyl amino)] three benzoic acid) and [(La2(TATAB)2)·4DMF·4H2O]n (2, H3TATAB = 4,4,4″s-triazine-1,3,5-triethyl-m-aminobenzoic acid) were prepared by means of two flexible tricarboxylic acid ligands with different joints and functional backbones. Moreover, a sonochemical synthesis way has been carried out to produce the nanostructures of complexes 1 and 2. In addition, their anticancer activity was evaluated on nasopharyngeal carcinoma cells. Firstly, Cell Counting Kit-8 test kit was used to test the cancer cells proliferation after treated with nano 1 or 2. Then, the Annexin V-FITC/PI test was performed and the percentage of the apoptotic cancer cells was determined after nano 1 and 2 treatment. Next, the ROS accumulation after nano 1 and 2 treatment was detected by DCFH-DA detection kit. Finally, the expression level of FHIT in nasopharyngeal carcinoma cells treated with nano 1 and 2 was evaluated via western blot.

In vitro and in vivo evaluations of nano-hydroxyapatite/polyamide 66/yttria-stabilized zirconia as a novel bioactive material for bone screws: Biocompatibility and bioactivity.

Zirconia and its derivatives have been receiving increased levels of attention with regard to their potential application in bone tissue engineering. These materials are of particular interest because of their excellent characteristics, such as superior biological and mechanical properties. In this study, yttria-stabilized tetragonal zirconia (YTZ)-reinforced nanohydroxyapatite/polyamide 66 (nHA/PA66) bone screws were prepared. The biocompatibility and bioactivity of nHA/PA66/YTZ were evaluated in vitro using MC3T3-E1 cells. Biocompatibility and bioactivity experiments (cell counting kit-8 tests, cell immunofluorescence analysis, and polymerase chain reaction) showed that nHA/PA66/YTZ could facilitate the biological functions of MC3T3-E1 cells. The attachment, proliferation, spreading, and expression of genes associated with osteogenesis (collagen 1, osteopontin, and osteocalcin) in cells cultured with the nHA/PA66/YTZ composite were all superior compared with the control groups (P < 0.05). In addition, nHA/PA66/YTZ bone screws were implanted into the femoral condyles of rabbits, and titanium screws were employed as a control group; postoperative histology and blood analysis revealed no obvious damage to the liver, kidneys, or any other major organs in either of the experimental groups. Moreover, nHA/PA66/YTZ screws resulted in significantly better bone-implant contact interfaces and enhanced formation of trabecular bone (P < 0.05); these characteristics were markedly better than those in the group that received titanium screws. These observations indicate that YTZ-reinforced nHA/PA66 composites have significant potential for applications in bone tissue engineering.

METTL3 promotes the progression of nasopharyngeal carcinoma through mediating M6A modification of EZH2.

The aim of this study was to investigate whether METTL3 promoted the progression of nasopharyngeal carcinoma (NPC) by silencing CDKN1C through EZH2. Quantitative Real Time-Polymerase Chain Reaction (qRT-PCR) was performed to examine the expression level of METTL3 in 48 pairs of NPC tissues and adjacent normal tissues. METTL3 expression in patients with different tumor lymph node metastasis (TNM) stages was detected by qRT-PCR as well. The Kaplan-Meier method was used to analyze the interplay between METTL3 expression and the prognosis of patients with NPC. At the same time, METTL3 expression in normal epithelial cell line (BEAS-2B) and NPC cell lines (SUNE-1 and C666-1) was examined using qRT-PCR. After METTL3 was knocked down in SUNE-1 cells, cell viability and migration abilities were analyzed by cell counting kit-8 (CCK-8) test and wound healing assay, respectively. The mRNA and protein expressions of EZH2 were detected by qRT-PCR and Western blot, respectively. RNA immunoprecipitation (RIP) assay was applied to detect the binding of METTL3 to EZH2 mRNA and the m6A modification on EZH2 mRNA. After knockdown of EZH2 in SUNE-1 cells, qRT-PCR was used to detect the mRNA expression of CDKN1C. Meanwhile, chromatin immunoprecipitation (ChIP) assay was conducted to analyze the binding of EZH2 to the CDKN1C promoter region. After down-regulation of METTL3 in SUNE-1 cells, the protein expressions of EZH2 and CDKN1C were detected using Western blot. After simultaneous knockdown of METTL3 and CDKN1C in SUNE-1 cells, CCK8 assay and wound healing assay were applied to examine cell viability and migration abilities. METTL3 expression in NPC tissues was remarkably higher than that of adjacent normal tissues. Meanwhile, METTL3 expression in T3 and T4 tumors was significantly higher than that of T1 and T2 tumors. In patients with lymph node metastasis, the expression of METTL3 was remarkably higher than those without metastasis. Survival analysis demonstrated that patients with higher expression of METTL3 exhibited significantly longer overall survival time than those with lower METTL3 expression. QRT-PCR revealed that METTL3 was highly expressed in NPC cell lines, including SUNE-1 and C666-1. After knock-down of METTL3 in SUNE-1 cells, cell viability and migration abilities were both markedly weakened. Meanwhile, the protein expression of EZH2 was remarkably reduced. However, no significant changes were observed in EZH2 mRNA level. RIP assay revealed that METTL3 could bind to EZH2 mRNA, and a m6A modification was verified on EZH2 mRNA. After knockdown of EZH2, the mRNA level of CDKN1C in SUNE-1 cells was significantly up-regulated. CHIP assay indicated that EZH2 could bind to CDKN1C. Western blot showed that, after interfering with METTL3 in SUNE-1 cells, the protein expression of EZH2 decreased significantly, while CDKN1C was up-regulated. In addition, simultaneous downregulation of METTL3 and CDKN1C in SUNE-1 cells reversed the influence of METTL3 on cell viability and migration abilities. METTL3 was highly expressed in NPC tissues, which might inhibit EZH2 expression by mediating M6A modification of EZH2 mRNA. Furthermore, CDKN1C could increase the malignancy of NPC cells and promote the progression of NPC.

Combined usage of monosaccharides with polysaccharides may decelerate tumor growth and malignance versus solely using a certain kind of saccharide

Saccharides used in clinical nutrients and the hyperglycemia status may facilitate tumor growth and aggravate cancer patients’ outcome. As glucose, fructose and maltodextrin are widely used in clinical practice, various effects on tumor progress among them remain unknown. Six kinds of tumor cell lines were included in this study. We evaluated the discrepant effects of sugars on tumor growth by nude mice xenograft model, wound-healing assay and cell counting kit-8 test were for measuring the migration and proliferation capability in vitro, and oral gavage on C57BL/6 N mice was applied to assess the fluctuation of blood glucose level. Results showed that though tumor cells presented discrepant sensitivity to different saccharides, the combined usage of glucose, fructose with maltodextrin has milder effect on tumor progression and moderate effect on blood glucose fluctuation, which may indicate a brighter option on saccharides selection for tumor patients.

LINC00511 can promote the proliferation, migration and invasion of esophageal cancer cells through regulating microRNA-150-5p.

The aim of this study was to investigate the potential role of LINC00511 in esophageal cancer (ECa), and to explore its underlying mechanism through in vitro cell experiments. LINC00511 expression in ECa was analyzed by GEPIA database and verified by real-time fluorescence quantitative polymerase chain reaction (qPCR). The bioinformatics website was used to analyze the miRNAs that can bind to LINC00511, and the regulatory relationship between them was verified through Luciferase assay, qPCR as well as Western blotting analysis. Then, the impacts of LINC00511 and microRNA-150-5p on the proliferation or invasiveness of ECa cell lines Kyse30 and ECA109 were investigated by cell counting kit-8 (CCK-8) test and transwell experiment, respectively. Meanwhile, cell cycle and apoptosis were detected by flow cytometry. Analysis results of the GEPIA database revealed that LINC00511 had a significant high expression in ECa tissue samples in comparison with normal control ones, which is consistent with qPCR results. Meanwhile, a significant negative correlation was found between LINC00511 and microRNA-150-5p. In brief, LINC00511 was able to bind to microRNA-150-5p and inhibited its expression. Besides, overexpression of LINC00511 enhanced ECa cell proliferation and migration, accelerated cell cycle, and suppressed cell apoptosis, while transfection with microRNA-150-5p mimics caused the opposite effects. This study shows for the first time that LINC00511 modulates the progression of ECa by binding to microRNA-150-5p.

The evaluation of leukocyte-platelet rich fibrin as an anti-inflammatory autologous biological additive. A novel in vitro study.

Objectives:To investigate the use of leukocyte-platelet rich fibrin on suppressing the porphyromonas gingivalis (PG-LPS)-induced secretion of proinflammatory cytokines.Methods:This quantitative experimental study was conducted at the School and Hospital of Stomatology, Jilin University, Changchun, China, between September 2017 and January 2019. A modified technique was used to obtain human gingival fibroblast cells (HGFCs). 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and Cell Counting Kit-8 tests were established to determine the proliferation rate. Human gingival fibroblast cells were treated by PG-LPS at different periods and the isolated mRNA was subjected to reverse transcription polymerase chain reaction and real time quantitative polymerase chain reaction. The release of platelet-derived growth factor and transforming-growth factor-β1 at various time intervals was observed.Results:We successfully established a modified technique for the production of HGFCs culture. One µg/mL PG-LPS was the recommended concentration to inhibit fibroblast proliferation. The expression of the pro-inflammatory cytokines messenger ribnucleic acid was notably raised at 3 and 6 hours post-PG-LPS treatment. The cumulative release of growth factors peaked during the first 24 hours and the production continued for 10 days. However, the fibroblast expression of cytokines was significantly suppressed after treatment with leucocyte- and platelet-rich fibrin (L-PRF).Conclusion:This study provided a novel way of obtaining HGFCs and greater understanding of the clinical impacts through the assessment of the anti-inflammatory properties of L-PRF in vitro.

Carnosine Protects Mouse Podocytes from High Glucose Induced Apoptosis through PI3K/AKT and Nrf2 Pathways

Diabetic nephropathy is the complication of diabetes mellitus that can lead to chronic renal failure. Reactive oxygen species (ROS) production plays an important role in its pathological process. Previous studies showed that carnosine may reduce diabetic nephropathy by antioxidant effect. However, the molecular mechanism of its antioxidant was not fully understood. In the current study, we developed high glucose containing different concentrations of carnosine to reduce ROS levels and podocytes apoptosis, and Cell Counting Kit-8 test was used to observe the cell viability. Carnosine (5-20mM) was found to protect mouse podocytes (MPC5) cells from HG-induced injury. Quantitative real-time PCR, Western blotting, and immunofluorescence staining revealed that high glucose induced ROS levels and podocytes apoptosis were downregulated by PI3K/AKT and Nrf2 signaling pathways. The current findings suggest that carnosine may reduce ROS levels and MPC5 cells apoptosis by PI3K/AKT and Nrf2 signaling pathways activation.

Electrospun polycaprolactone/collagen type Ⅰnanofibers oriented patch for rotator cuff repairing

Electrospinning technique was used to manufacture polycaprolactone (PCL)/collagen typeⅠ nanofibers orientated patches and to study their physical and chemical characterization, discussing their feasibility as synthetic patches for rotator cuff repairing. PCL patches were prepared by electrospinning with 10% PCL electrospinning solution (control group) and PCL/collagen typeⅠorientated nanofibers patches were prepared by electrospinning with PCL electrospinning solution with 25% collagen type Ⅰ(experimental group). The morphology and microstructure of the two patches were observed by gross and scanning electron microscopy, and the diameter and porosity of the fibers were measured; the mechanical properties of the patches were tested by uniaxial tensile test; the composition of the patches was analyzed by Fourier transform infrared spectroscopy; and the contact angle of the patch surface was measured. Two kinds of patch extracts were co-cultured with the third generation of rabbit tendon stem cells. Cell counting kit 8 (CCK-8) was used to detect the toxicity and cell proliferation of the materials. Normal cultured cells were used as blank control group. Rabbit tendon stem cells were co-cultured with the two patches and stained with dead/living cells after 3 days of in vitro culture, and laser confocal scanning microscopy was used to observe the cell adhesion and activity on the patch. Gross and scanning electron microscopy showed that the two patch fibers were arranged in orientation. The diameter of patch fibers in the experimental group was significantly smaller than that in the control group ( t=26.907, P=0.000), while the porosity in the experimental group was significantly larger than that in the control group ( t=2.506, P=0.032). The tensile strength and Young's modulus of the patch in the experimental group were significantly higher than those in the control group ( t=3.705, P=0.029; t=4.064, P=0.034). Infrared spectrum analysis showed that PCL and collagen type Ⅰ were successfully mixed in the experimental group. The surface contact angle of the patch in the experimental group was (73.88±4.97)°, which was hydrophilic, while that in the control group was (128.46±5.10) °, which was hydrophobic. There was a significant difference in the surface contact angle between the two groups ( t=21.705, P=0.002). CCK-8 test showed that with the prolongation of culture time, the cell absorbance ( A) value increased gradually in each group, and there was no significant difference between the experimental group and the control group at each time point ( P>0.05). Laser confocal scanning microscopy showed that rabbit tendon stem cells could adhere and grow on the surface of both patches after 3 days of culture. The number of cells adhered to the surface of the patches in the experimental group was more than that in the control group, and the activity was better. PCL/ collagen type Ⅰ nanofibers orientated patch prepared by electrospinning technology has excellent physical and chemical properties, cell adhesion, and no cytotoxicity. It can be used as an ideal scaffold material in tendon tissue engineering for rotator cuff repair in the future.

Mechanical properties and cell compatibility of a chitosan-graphene oxide guided bone regeneration composite membrane

Objective: To explore the feasibility of graphene oxide (GO)-chitosan (CS) guided bone regeneration composite membrane being used as a new guided bone regeneration (GBR) membrane by testing its tensile strength and its effect on the proliferation of human gingival fibroblasts (HGF). Methods: The CS solution and GO solution were mixed in following ratios, ultrasonically crushed and dispersed, and formed into membranes by self-evaporation. Composite membrane with 0.5%, 1.0%, 2.0%, 4.0%, 6.0%, 8.0% GO in CS was prepared. The tensile strength of the composite membranes was tested by mechanical universal testing machine (n=6). The microstructure of the composite membrane with the best tensile strength was observed by scanning electron microscopy and transmission electron microscopy. X-ray diffraction was used to characterize the ingredients of the material. A group of samples was prepared again with the proportion of the highest tensile strength composite membrane, and were immersed in a sodium hydroxide solution for acid removal. The tensile strength of the new group of samples was tested. The newly extracted impacted teeth were collected from the Department of Oral and Maxillofacial Surgery, Stomatologic Hospital and College, Anhui Medical University, and the gingival tissues remained on the teeth were taken for primary HGF and were cultured to P2 generation, which were identified by immunocytochemical staining methods. Cell counting kit-8 (CCK-8) was applied to detect cell counts in the blank control group, the pure CS membrane group, and the composite membrane group with the highest tensile strength (n=5) and all groups co-cultured with the HGF for 24 h and 48 h. Results: After adding GO, the cross-section of the composite membrane was in an ordered layer structure. X-ray diffraction analysis showed that GO could be found in the composite membrane. The tensile strength increased with the increase of GO ratio. When the mass fraction of GO in CS was 4.0%, the tensile strength reached (134.8±7.3) MPa and the tensile strength reached (144.6±8.1) MPa after deacidification of the composite membrane. The CCK-8 test of HGF showed that there was no significant difference in absorbance between the pure CS membrane group and the GO/CS composite membrane group when they were compared with the blank control group (P>0.05). Conclusions: When the mass fraction of GO in CS is 4.0%, the composite membrane has the best tensile strength. The composite membrane showes good cytocompatibility, which lays a foundation for further in vivo experiments and the development of a new generation of GBR membrane.

Versatile Pt NCs-based chemotherapeutic agents significantly induce the apoptosis of cisplatin-resistant non-small cell lung cancer

Recently, the incidence of lung cancer is generally rising along with air pollution and smoking, and non-small cell lung cancer (NSCLC) accounts for nearly 85% among all lung cancer diagnoses. With the development of chemotherapy, the drug resistance rate of common platinum-based chemotherapeutic drugs (like cisplatin) is gradually increased, which seriously affects the chemotherapy efficiency and survival rate of patients. In this study, polyethylenimine caged platinum nanoclusters (PEI-caged Pt NCs) were proposed as a new chemotherapeutic agent to apply in the treatment of NSCLC, choosing the classical cisplatin-resistant A549/DDP cells and normal A549 cells as targets. It was found that our Pt NCs-based chemotherapeutic drugs showed its preferable therapeutic effect in cisplatin-resistant NSCLC through the results of confocal microscopic images, cell counting kit-8 test, cell apoptosis assay and western blot. Most importantly, in the cisplatin-resistance A549/DDP cells, this kind of agents could enter the nucleus obviously, and emerged a superior inhibitory and apoptotic effects than A549 via activating p53 protein and the related signaling pathways. Comparing with the traditional chemotherapy drugs, these Pt NCs-based chemotherapeutic agents exhibit great potential and advantages in the treatment and diagnosis of NSCLC regardless of the therapeutic effect or toxic side effects, especially the drug resistance.

Effect of RKIP on proliferation and migration of malignant melanoma cells and potential mechanism

Objective: To explore the effect and mechanism of Rafkinase inhibitor protein (RKIP) on proliferation and migration of malignant melanoma cells in vitro. Methods: The RKIP overexpression and down-regulated stable transfected strains of mouse malignant melanoma cell line B16 were constructed by recombinant lentiviral transfection technique and established as RKIP overexpression group and RKIP down-regulation group, the mouse malignant melanoma B16 cells without any treatment were used as a blank control group, and the proliferation activity and migration ability of each group were detected by cell counting kit-8 (CCK-8) and cell scratch test. The relative expression levels of CyclinD1, Calcium-dependent cell adhesin, Ki-67, Matrix metalloproteinase (MMP)-9, MMP-13, MMP-2 and Phosphatidylethanolamine binding protein (PEBP-1) were detected by quantitative real-time polymerase chain reaction (qPCR). Western blot was used to detect the difference of RKIP expression and the protein expression level of nuclear factor-kappa B (NF-κB) signaling pathway in each group. Results: Comparison of RKIP overexpression group and blank control group shown cell proliferation and migration were significantly inhibited in RKIP overexpression group (0.794±0.038 vs 1.200±0.081) (P<0.001). However, there was no significant difference in cell proliferation between RKIP down-regulation group and blank control group (1.077±0.084 vs 1.200±0.081) (P>0.05), and the cell migration ability of RKIP down-regulation group was significantly higher than that of the blank control group (P<0.001). In addition, there was no significant difference between the RKIP down-regulation group and the blank control group in PEBP-1 expression (P>0.05), while the expression levels of the remaining genes in the RKIP overexpression group were significantly lower than those in the blank control group, and the expression levels in the RKIP down-regulation group were significantly higher than those in the blank control group (P<0.001). Furthermore, the protein level of phosphorylated P65 (p-P65) in RKIP overexpression group was significantly lower than that in blank control group (0.080±0.000 vs 0.236±0.000), and RKIP down-regulation group was significantly higher than that in blank control group (1.139±0.001 vs 0.236±0.000) (both P<0.001). Conclusion: RKIP overexpression can inhibit the proliferation and migration of malignant melanoma cells, which may be related to the regulation of NF-κB signaling pathway-related protein p-P65.

Substance P inhibits high urea-induced apoptosis through the AKT/GSK-3β pathway in human corneal epithelial cells.

To investigate the effect of substance P (SP) on human corneal epithelial cells (HCECs) that have been stressed by a high urea environment and to determine the relationship between SP and the protein kinase B (AKT)/glycogen synthase kinase-3β (GSK-3β) signaling pathway. An in vitro model of chronic renal failure (CRF)-related dry eye was used to study HCECs that were treated with high urea concentrations. Cell proliferation was assayed using a cell counting kit-8 test. Besides, cell apoptosis was evaluated by flow cytometry. Furthermore, the effects of SP and the AKT inhibitor perifosine on the urea-treated HCECs were examined using immunofluorescence, quantitative real time polymerase chain reaction (qRT-PCR), and Western blot analysis. SP markedly reduced the number of apoptotic HCECs and decreased the cleaved caspase-3 expression levels while contributing to increased cellular proliferation (P < 0.05). The Western blot analysis and qRT-PCR experiments revealed that SP significantly increased the expression of p-AKT and p-GSK-3β (P < 0.05); additionally, these increases were attenuated after the perifosine inhibition of the AKT signaling pathway (P < 0.05). These in vitro experiments demonstrated that SP may protect against the apoptotic damage of HCECs caused by the high urea condition. The underlying mechanism may be related to the activation of the AKT/GSK-3β signaling pathway.

MT1‑MMP promotes the proliferation and invasion of gastric carcinoma cells via regulating vimentin and E‑cadherin.

The present study aimed to explore the possible effects of membrane-type 1 matrix metalloproteinase (MT1-MMP) on gastric carcinoma cells proliferation and invasion. Immunohistochemistry analysis was conducted to measure MT1-MMP expression level in 15 patients with gastric carcinoma. Subsequently, recombinant short hairpin RNA (shRNA) vectors targeting MT1-MMP were constructed to silence the expression of MT1-MMP in gastric carcinoma cells. Then, the inhibitive efficiency was verified via reverse transcription quantitative polymerase chain reaction (RT-qPCR) and western blot analysis. The effects of MT1-MMP silencing on cell proliferation and invasion were detected through Cell Counting Kit-8 test and Transwell assays. The expression levels of vimentin and epithelial cadherin (E-cadherin) were detected by RT-qPCR. The immunohistochemistry analysis results revealed that MT1-MMP expression in gastric carcinoma tissues was markedly overexpressed compared with non-cancerous adjacent tissues. The MT1-MMP expression level in cancer-derived cell line AGS cells was also significantly increased compared with that in non-cancer-derived GES-1 cells. In addition, the MT1-MMP expression level in AGS cells was significantly decreased via shRNA transfection. Cell proliferation and invasion were markedly inhibited following knockdown of MT1-MMP level in AGS cells. These inhibitory effects were associated with the decreased expression of vimentin and increased expression of E-cadherin. MT1-MMP was overexpressed in gastric carcinoma cells, and silencing of MT1-MMP inhibited the proliferation and invasion of cells via regulating the expression of vimentin and E-cadherin.

SPARC promotes the proliferation and metastasis of oral squamous cell carcinoma by PI3K/AKT/PDGFB/PDGFRβ axis.

Oral squamous cell carcinoma (OSCC) is a highly lethal cancer in the world, and the prognosis of OSCC is poor with a 60% 5-year survival rate in recent decades. Here, we introduced a novel secretory and acid glycoprotein with cysteine rich (secreted protein acidic and rich in cysteine, SPARC), which is correlated with the worst pattern of invasion (WPOI) and prognosis of OSCC. SPARC expression levels were measured in OSCC tissues and normal tissues using quantitative polymerase chain reaction and immunohistochemistry. The influence of SPARC on cell proliferation was examined by cell counting kit-8, colony formation, and Edu tests. Then, the effect of SPARC on the metastasis of OSCC cells was detected by wound healing and transwell migration assays. Next, the biologic characteristics of SPARC shared by STRING were analyzed. Furthermore, the underlying mechanisms were confirmed by western blot analysis. SPARC revealed higher expression in OSCC tissues than nontumor tissues. Higher SPARC expression was correlated with poorer tumor differentiation, poorer WPOI pattern, and significantly and shorter overall survival. Knockdown SPARC significantly restrained OSCC cell growth, migration, and invasion. In addition, bioinformatics analysis found SPARC had a coexpression network with the platelet-derived growth factor-B (PDGFB) and PI3K/AKT signaling pathways with minimal false discovery rate. Furthermore, SPARC promotes OSCC cells metastasis by regulating the expressions of PDGFB, PDGFRβ, p-PDGFRβ , and the PI3K/AKT pathway. Higher SPARC expression was positively correlated with poor WPOI and differentiation in OSCC. SPARC activates the PI3K/AKT/PDGFB/PDGFRβ axis to promote proliferation and metastasis by OSCC cell lines. Therefore, SPARC may be a potential therapeutic target for patients with OSCC.

D-RADA16-RGD-Reinforced Nano-Hydroxyapatite/Polyamide 66 Ternary Biomaterial for Bone Formation.

Nano-hydroxyapatite/polyamide 66 (nHA/PA66) is a composite used widely in the repair of bone defects. However, this material is insufficient bioactivity. In contrast, D-RADA16-RGD self-assembling peptide (D-RADA16-RGD sequence containing all D-amino acids is Ac-RADARADARADARADARGDS-CONH2) shows admirable bioactivity for both cell culture and bone regeneration. Here, we describe the fabrication of a favorable biomaterial material (nHA/PA66/D-RADA16-RGD). Proteinase K and circular dichroism spectroscopy were employed to test the stability and secondary structural properties of peptide D-RADA16-RGD respectively. Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) were used to characterize the surface of these materials. Confocal laser scanning (CLS), cell counting kit-8 tests (CCK-8), alizarin red S staining, cell immunofluorescence analysis and Western blotting were involved in vitro. Also biosafety and bioactivity of them have been evaluated in vivo. Proteinase K and circular dichroism spectroscopy demonstrated that D-RADA16-RGD in nHA/PA66 was able to form stable-sheet secondary structure. SEM and TEM showed that the D-RADA16-RGD material was 7-33 nm in width and 130-600 nm in length, and the interwoven pore size ranged from 40 to 200 nm. CLS suggests that cells in nHA/PA66/D-RADA16-RGD group were linked to adjacent cells with more actin filaments. CCK-8 analysis showed that nHA/PA66/D-RADA16-RGD revealed good biocompatibility. The results of Alizarin-red S staining and Western blotting as well as vivo osteogenesis suggest nHA/PA66/D-RADA16-RGD exhibits better bioactivity. This study demonstrates that our nHA/PA66/D-RADA16-RGD composite exhibits reasonable mechanical properties, biocompatibility and bioactivity with promotion of bone formation.

Mechanism of LncRNA FOXC2-AC1 promoting lung cancer metastasis by regulating miR-107.

Previous studies have shown that long non-coding RNA (lncRNA) FOXC2-AC1 is one of cancer-promoting genes. However, the role of FOXC2-AC1 in lung cancer (LCa) has not been reported. This study aimed to investigate the expression characteristics of FOXC2-AC1 in LCa, and to further explore the mechanism by which it accelerates the metastasis of LCa. Quantitative Real-time polymerase chain reaction (qRT-PCR) was used to detect the level of FOXC2-AC1 in 62 pairs of LCa tissues and adjacent normal tissues, and the relationship between FOXC2-AC1 and LCa pathological parameters as well as the prognosis of patients were analyzed. Meanwhile, FOXC2-AC1 level was further verified in LCa cells by qRT-PCR. In addition, FOXC2-AC1 knockdown and overexpression models were constructed using lentivirus in LCa cell lines including H1299 and SPCA1, and the effect of FOXC2-AC1 on the biological function of LCa cells was analyzed by cell counting kit-8 (CCK-8) test along with transwell invasion and migration assay. Finally, the potential mechanism was explored using Western blotting assay. In this study, qRT-PCR results indicated that the expression level of FOXC2-AC1 in LCa was considerably higher than that in normal tissues, with statistically significant differences. Compared with patients with low expression of FOXC2-AC1, patients with high expression of FOXC2-AC1 had higher incidence of distant metastasis and lower overall survival rate. Compared with the control group, the cell proliferation, invasion and metastasis capacities of FOXC2-AC1 overexpressing group were considerably enhanced, while opposite results were observed in the FOXC2-AC1 silencing group. In addition, miR-107 expression was found significantly reduced no matter in LCa cell lines or in tissues and showed a negative correlation with FOXC2-AC1. Subsequently, luciferase reporter gene assay demonstrated that overexpression of miR-107 significantly attenuated the luciferase activity of the wild-type FOXC2-AC1 vector without reducing the activity of the mutant vector or empty vector, further proving that FOXC2-AC1 could be targeted by miR-107 through this binding site. In addition, rescue experiment also found that FOXC2-AC1 and miR-107 have mutual regulation, which jointly affected the malignant progression of LCa. These studies indicate that LncRNA FOXC2-AC1 is notably upregulated in LCa and is significantly correlated with LCa distant metastasis as well as poor prognosis. Therefore, it is suggested that lncRNA FOXC2-AC1 may promote malignant progression of LCa through the mutual regulation of miR-107.

CD36- and obesity-associated granulosa cells dysfunction.

Emerging evidence indicates that obesity impairs granulosa cell (GC) function, but the underlying mechanisms remain unclear. Gene expression profiles in GC of non-polycystic ovary syndrome (PCOS) obese (NPO), PCOS obese (PO), PCOS normal weight (PN) and non-PCOS normal weight (NPN) patients were analysed by microarray analysis. Compared with the NPN group, there were 16, 545 and 416 differently expressed genes in the NPO, PO and PN groups respectively. CD36 was the only intersecting gene, with greater than two fold changes in expression between the NPO versus NPN and PO versus NPN comparisons, and was not present in the PN versus NPN comparison. In addition, levels of CD36 protein were higher in GC from obese than normal weight patients. Furthermore, CD36 overexpression in a GC line inhibited cell proliferation, as determined by the cell counting kit-8 (CCK8) test, promoted cell apoptosis, as determined by flow cytometry, and inhibited the secretion of oestradiol by depositing triglyceride in cells and increasing cellular lipid peroxide levels. These adverse effects were reduced by sulfo-N-succinimidyloleate, a specific inhibitor of CD36. Together, the findings of this study suggest that obesity with and without PCOS should be regarded as separate entities, and that CD36 overexpression in GC of obese patients is one of the mechanisms by which obesity impairs GC function.

Effect of natural hirudin on angiogenesis of human microvascular endothelial cells

To explore the effect of natural hirudin on proliferation of human microvascular endothelial cells (HMVECs) and its preliminary mechanism of promoting angiogenesis. Three-dimensional culture models of HMVECs were established in vitro and observed by inverted phase contrast microscopy after 24 hours of culturing. Then, the three-dimensional culture models of HMVECs were treated with different concentrations (1, 4, and 7 ATU/mL) of the natural hirudin, respectively, and Dulbecco's modified Eagle's medium containing 10% fetal bovine serum as control. The cell proliferations of 4 groups were detected by cell counting kit 8 (CCK-8) method at 24, 48, and 72 hours; the angiogenesis of 4 groups were observed by tube formation assay at 24 hours; the expressions of vascular endothelial growth factor (VEGF) and Notch1 of HMVECs in 4 groups were observed by immunofluorescence staining at 24 hours. The observation of cells in three-dimensional culture models showed that HMVECs attached to Matrigel well, and the cells formed tube structure completely after 24 hours. The results of CCK-8 test showed that the absorbance ( A) value of 1 and 4 ATU/mL groups were higher than that of control group at each time point ( P<0.05), and A value of 4 ATU/mL group was the highest. The A value of 7 ATU/mL group was significantly lower than those of 1 and 4 ATU/mL groups and control group ( P<0.05). The tube formation assay showed that the tube structure was more in 1 and 4 ATU/mL groups than in 7 ATU/mL group and control group, and in 4 ATU/mL group than in 1 ATU/mL group, showing significant differences ( P<0.05). There was no significant difference between 7 ATU/mL group and control group ( P>0.05). The results of immunofluorescence staining showed that compared with control group, the Notch1 expression was higher in 1 and 4 ATU/mL groups and lower in 7 ATU/mL group; and there was significant difference between 4 and 7 ATU/mL groups and control group ( P<0.05). The VEGF expression was higher in 1, 4, and 7 ATU/mL groups than in control group, in 4 ATU/mL group than in 1 and 7 ATU/mL groups, showing significant differences ( P<0.05). Natural hirudin can promote angiogenesis at low and medium concentrations, but suppress angiogenesis at high concentrations. Its mechanism may be related to the VEGF-Notch signal pathway.