Objective To investigate the role of chemokine receptor CXCR7 in the development and progression of pancreatic carcinoma. Methods The short hairpin RNA (shRNA) targeting CXCR7 was designed and delivered into AsPC-1 pancreatic carcinoma cells to knock down CXCR7 expression. The cell proliferation, cell cycle, and apoptosis after CXCR7 knockdown was determined by MTT and flow cytometry, respectively. The invasive ability of pancreatic carcinoma cells was evaluated by using the Transwell system. Results Compared with the blank control group (BC group), transfection of AsPC-1 cells with CXCR7-shRNA resulted in a significantly decreased expression of CXCR7 at both mRNA and protein levels (P<0.05), and the ability of proliferation and invasion significantly decreased (P<0.05). Knockdown of CXCR7 also significantly increase apoptosis (P<0.05), induce cell cycle arrest at G0/G1 phase (P<0.05). Conclusions Taken together, the present study showes that the knockdown of CXCR7 expression may play an important role in pancreatic carcinoma development, invasion, and metastasis, CXCR7 may be a potential therapeutic target for the treatment of pancreatic carcinoma.
ObjectiveTo investigate the expression and biological function of centromere protein F (CENPF) in non-small cell lung cancer (NSCLC) and the association with prognosis.MethodsThrough retrieving and analyzing the bioinformatics data such as Oncomine database, Human Protein Atlas (HPA), Kaplan-Meier Plotter, STRING and DAVID database, the expression of CENPF in both normal tissues and cancer tissues of lung cancer patients was identified, and the protein interaction network analysis, functional annotation and pathway analysis of CENPF with its associated genes were carried out.ResultsCENPF was overexpressed in lung adenocarcinoma tissues, but not in normal tissues. The median overall survival (OS) of NSCLC patients with low expression of CENPF was significantly longer than that of patients with high expression of CENPF. Further sub-analysis showed that low expression group from lung adenocarcinoma patients had longer median disease-free survival and OS compared with high expression group patients. CENPF and its associated hub genes mainly affected the protein K11-linked ubiquitination in biological process, anaphase-promoting complex (APC) in cell composition, ATP binding in molecular function, and cell cycle in KEGG pathway.ConclusionCENPF is regulated in tumorigenesis and progression of NSCLC, and its protein expression level has the value of early diagnosis and prognosis evaluation in lung adenocarcinoma. It is suggested that CENPF gene can be a potential target for molecular targeted therapy of NSCLC.
Objective To explore the molecular mechanism of miR-515-5p in inhibiting chondrocyte apoptosis and alleviating inflammatory response in osteoarthritis (OA). Methods Human cartilage cell line C28/I2 was cultured in vitro and treated with 10 ng/mL interleukin 1β (IL-1β) for 24 hours to construct an in vitro OA model. C28/I2 cells were transfected with miR mimics, mimics negative control (NC), over expression (oe)-NC, and oe-Toll-like receptor 4 (TLR4), respectively, and then treated with 10 ng/mL IL-1β for 24 hours to establish OA model. Cell proliferation capacity was detected by cell counting kit 8 and 5-Ethynyl-2’-deoxyuridine, cell apoptosis and cell cycle were detected by flow cytometry, and B-cell lymphoma 2 protion (Bcl-2), Bcl-2-associated X protein (Bax), cleaved-Caspase-3, TLR4, myeloid differentiation primary response gene 88 (MyD88), p65 and phosphorylated p65 (p-p65) protein expression levels were detected by Western blot. Real-time fluorescence quantitative PCR was used to detect mRNA expression levels of miR-515-5p and TLR4, and ELISA was used to detect pro-inflammatory factor prostaglandin E2 (PGE2), tumor necrosis factor α (TNF -α), and IL-6 levels in cell supernatant. The potential binding sites between miR-515-5p and TLR4 were predicted by BiBiServ2 database, and the targeting relationship between miR-515-5p and TLR4 was verified by dual luciferase reporting assay. Results After the treatment of C28/I2 cells with IL-1β, the expressions of miR-515-5p and Bcl-2 protein and the proliferation ability of C28/I2 cells significantly reduced. The expression levels of Bax and cleaved-Caspase-3 protein, the levels of pro-inflammatory factors (PGE2, TNF-α, IL-6) in the supernatant of C28/I2 cells, and the apoptosis of C28/I2 cells significantly increased. In addition, the proportion of the cells at S phase and G2 phase decreased significantly, and the proportion of cells at G1 phase increased significantly, suggesting that the cell cycle was blocked after IL-1β treatment. After transfection with miR mimics, the expression level of miR-515-5p in the cells significantly up-regulated, partially reversing the apoptosis of OA chondrocytes induced by IL-1β, and alleviating the cycle arrest and inflammatory response of OA chondrocytes. After treating C28/I2 cells with IL-1β, the mRNA and protein levels of TLR4 significantly increased. Overexpression of miR-515-5p targeted inhibition of TLR4 expression and blocked activation of MyD88/nuclear factor κB (NF-κB) pathway. Overexpression of TLR4 could partially reverse the effect of miR mimics on IL-1β-induced apoptosis and inflammation of OA chondrocytes. ConclusionmiR-515-5p negatively regulates the expression of TLR4, inhibits the activation of MyD88/NF-κB pathway and apoptosis of OA chondrocytes, and effectively alleviates the inflammatory response of the cells.
Objective To investigate the effect of circadian rhythm disorder on rat knee cartilage and the mechanism of basic helix-loop-helix ARNT like 1 (Bmal1) on the regulation of cell cycle-related genes. Methods Forty rats were randomly divided into normal group, circadian rhythm disorder group (disorder group), Bmal1 overexpression lentivirus infection circadian rhythm disorder group (Bmal1 up-regulated group) and Bmal1 overexpression lentivirus negative infection circadian rhythm disorder group (Bmal1 negative infection group), with 10 rats in each group. Saffron fast green staining, TdT-mediated dUTP nick-end labeling staining, immunohistochemical staining, reverse transcription polymerase chain reaction and Western blotting were used to compare the pathological changes of cartilage tissue, the apoptosis of chondrocytes, and the relative mRNA expression levels of Bmal1, WEE1 G2 checkpoint kinase (Wee1), cyclin-dependent kinase 1 (Cdk1), cyclin B1 (Ccnb1), BCL2-associated X protein (Bax), apoptosis regulator 2 (Bcl2), interleukin 1 (Il1), interleukin 6 (Il6), tumor necrosis factor (Tnf) and matrix metallopeptidase 13 (Mmp13) among different groups. The relative expression levels of BMAL1, WEE1, CDK1, CCNB1, BAX and BCL2 proteins were detected, and correlation analysis was performed according to the relative expression of mRNA. Results Safranine fast green staining showed that the thickness of cartilage matrix in the normal group was normal and uniform red. The cartilage matrix in the disorder group and the Bmal1 negative infection group was destroyed, and the proteoglycan was lost obviously, showing uneven red. The thickness of cartilage matrix in the Bmal1 up-regulated group was basically normal, and the proteoglycan was not lost obviously, and the red was slightly less uniform. Compared with those of the normal group, the positive rates of apoptotic cells in articular cartilage of the disorder group and the Bmal1 negative infection group increased significantly, the mRNA and protein expression levels of Bmal1, Wee1, and Bcl2 were down-regulated, the mRNA and protein expression levels of Cdk1, Ccnb1, and Bax were up-regulated, the mRNA expression levels of Il1, Il6, Tnf and Mmp13 were up-regulated, the differences were statistically significant (P<0.05); there was no significant change in the positive rate of apoptotic cells in the articular cartilage of the Bmal1 up-regulated group, and there was no significant difference in the mRNA or protein expression of Bmal1, Wee1, Bcl2, Cdk1, Ccnb1 or Bax, nor the mRNA expression of Il1, Il6, Tnf or Mmp13 (P>0.05). Correlation analysis showed that Bmal1 was positively correlated with Wee1 and Bcl2 (r=0.84, 0.44; P<0.01), and negatively correlated with Cdk1, Ccnb1 and Bax (r=–0.55, –0.72, –0.41; P<0.01). Conclusion Chronic circadian rhythm disorder can cause the increase of chondrocyte apoptosis and osteoarthritis-like changes of articular cartilage through the expression changes of circadian clock gene Bmal1 and cell cycle-related genes and proteins.