MMP-9 Research Articles

IL-17 induces NSCLC cell migration and invasion by elevating MMP19 gene transcription and expression through the interaction of p300-dependent STAT3-K631 acetylation and its Y705-phosphorylation.

The cancer cell metastasis is a major death reason for patients with non-small cell lung cancer (NSCLC). Although researchers have disclosed that interleukin 17 (IL-17) can increase matrix metalloproteinases (MMPs) induction causing NSCLC cell metastasis, the underlying mechanism remains unclear. In the study, we found that IL-17 receptor A (IL-17RA), p300, p-STAT3, Ack-STAT3, and MMP19 were up-regulated both in NSCLC tissues and NSCLC cells stimulated with IL-17. p300, STAT3 and MMP19 overexpression or knockdown could raise or reduce IL-17-induced p-STAT3, Ack-STAT3 and MMP19 level as well as the cell migration and invasion. Mechanism investigation revealed that STAT3 and p300 bound to the same region (-544 to -389 nt) of MMP19 promoter, and p300 could acetylate STAT3-K631 elevating STAT3 transcriptional activity, p-STAT3 or MMP19 expression and the cell mobility exposed to IL-17. Meanwhile, p300-mediated STAT3-K631 acetylation and its Y705-phosphorylation could interact, synergistically facilitating MMP19 gene transcription and enhancing cell migration and invasion. Besides, the animal experiments exhibited that the nude mice inoculated with NSCLC cells by silencing p300, STAT3 or MMP19 gene plus IL-17 treatment, the nodule number, and MMP19, Ack-STAT3, or p-STAT3 production in the lung metastatic nodules were all alleviated. Collectively, these outcomes uncover that IL-17-triggered NSCLC metastasis involves up-regulating MMP19 expression via the interaction of STAT3-K631 acetylation by p300 and its Y705-phosphorylation, which provides a new mechanistic insight and potential strategy for NSCLC metastasis and therapy.

Inhibition of angiotensin II type 1 receptor by candesartan reduces tumor growth and ameliorates fibrosis in colorectal cancer.

Colorectal cancer (CRC) is an important cause of cancer-related mortality. Aberrant activation of the renin-angiotensin system (RAS) is reported to be associated with poor clinical outcomes in patients with CRC. This study was designed to explore the anti-tumor effects of the angiotensin receptor blocker Candesartan either alone or in combination with 5-FU in in vitro and in vivo models of CRC. The cytotoxic effects of Candesartan were assessed using the MTT assay in two colorectal cancer cell lines (CT-26 and SW-480). To investigate the potential regulatory role of Candesartan on tumor growth, apoptosis, and migration, the expression levels of Cyclin D1, Survivin, MMP3, MMP9, and E-cadherin mRNAs were evaluated. The oxidant/antioxidant balance was also examined by determining the levels of MDA, thiols, SOD, and CAT. We used a xenograft model of colon cancer to investigate the effects of Candesartan alone, or in combination with 5-FU, on tumor growth following histological staining (Hematoxylin & Eosin and Masson trichrome staining) and biochemical studies as well as gene expression analyses by RT-PCR and western blotting. Candesartan suppressed tumor cell proliferation and migration by modulating Cyclin D1, MMP3/9, and E-cadherin. Treatment with Candesartan either alone, or in combination with 5-FU decreased tumor size in the mouse model, and also increased the level of oxidative markers MDA and reduced CAT, SOD, and thiols. Histological evaluation showed that Candesartan increased tumor necrosis, reduced tumor density and attenuated collagen deposition reducing tumor fibrosis in tumor xenograft. Candesartan, an inhibitor of the RAS, when used in combination with 5-FU reduced tumor growth by inhibiting fibrosis and inducing ROS production, supporting further clinical studies on this therapeutic approach for treatment of CRC.

Immune stimulating and galectin-3 inhibitory effects of a hydrophilic polysaccharide nutraceutical from Tinospora cordifolia

Tinospora cordifolia is an important ayurvedic herb known for its immune stimulating effect. In this investigation, we describe the galectin-3 inhibitory and immune stimulating properties of a novel water-soluble nutraceutical made from Tinospora cordifolia stem. This proprietary extract (T2CA) had a total yield of 6% and contained 86.9% carbohydrates with a high proportion of patented (1,4)-α-D-glucan in it. The T2CA activated T-cytotoxic and NK cells significantly contributing to its immune stimulating effect. Moreover, T2CA induced the activation of functional NK cells in human lymphocyte cultures that in turn contributed to the death of K562 leukemic cells in a dose-dependent manner in co-culture experiments. T2CA inhibited the expression of phosphorylated galectin-3 expression in human glioblastoma cell line which in turn inhibits the activity of MMP2 and MMP9 proteins in zymography investigations and inhibits migration of cancer cells in in vitro scratch assay. Western blot hybridization studies showed that T2CA downregulates the expression of total gal 3 proteins and extended the survival of AKR/J mice injected with Ehrlich ascites tumor cells in the Kepler-Meier survival curve analysis. The inhibitory effect of T2CA on phopho-galectin-3 expression and the activation of T-cytotoxic and NK cells suggested its protective effects against pathogenic infections and human malignancies via its immune stimulating mechanisms.

Siyah üzüm ekstresinin meme kanseri hücrelerinde MMP-9 gen ekspresyonuna etkisi

Meme kanseri sağ kalım oranındaki düşüş ve tedavisindeki başarısızlığın en önemli etkenleri arasında metastaz görülmektedir. Meme kanseri metastazının moleküler yolaklarının aydınlatılması büyük bir öneme sahiptir. Son yıllarda yapılan araştırmalar, bitkilerde bulunan polifenolik bileşiklerin anti-invaziv ve anti-metastatik yeteneklerine sahip olduğunu göstermektedir. Bol miktarda polifenol içeren siyah üzüm ekstresinin meme kanseri ve metastazı üzerindeki etkisi ve moleküler mekanizması henüz tam olarak bilinmemektedir. Çalışmamızda, insan meme kanseri hücre dizisine uygulanan siyah üzüm ektresinin hücre canlılığı ve adezyonuna etkisi ile metastazda rolü olabileceği düşünülen MMP-9 gen ekspresyonuna etkisinin belirlenmesi amaçlanmıştır. MCF-7 meme kanser hücre dizilerinde tripan blue yöntemi ile hücre canlılığı, XTT yöntemi ile hücre adezyonu ve qRT-PCR ile MMP-9 gen ekspresyonu belirlenmiştir. Çalışmamızda, siyah üzüm ekstresinin MMP-9 gen ekspresyonunu, hücre canlılığını ve adezyonunu (p>0.05) azalttığı bulunmuştur. Sonuç olarak polifenoller gibi doğal kanser önleyici ajanların kanserin oluşmasını ve yayılmasını önleyebileceği fikri desteklenmiştir. Bununla birlikte polifenollerin etkilediği moleküler mekanizmaların aydınlatılabilmesi için ileri çalışmalara ihtiyaç vardır.

In Vitro Action of Matrix Metalloproteinases 2 and 9 Inhibitors on Na + /K + -ATPase, H + /K + -ATPase and PMCA Activities in the Osmoregulatory Epithelia of Climbing Perch ( Anabas testudineus Bloch)

Matrix metalloproteinases 2 and 9 (MMP2 and MMP9) are involved in the extracellular matrix (ECM) remodeling. We tested the short-term in vitro action of inhibitors of MMP2 and MMP9 on P-type ion transporter function in organ explants of climbing perch ( Anabas testudineus ) to understand how these ECM remodeling components influence the ion transporter function in the osmoregulatory epithelia of fish. Graded doses (10 -8 , 10 -7 and 10 -6 M) of inhibitors of MMP2 and MMP9 were administered in vitro to explants of gills, kidney and intestine, kept for either 15 or 30 min and the activities of P-type ATPase such as Na + /K + -ATPase (NKA), H + /K + -ATPase (HKA) and plasma membrane Ca 2+ -ATPase (PMCA) were quantified. We found that the inhibitors of MMP2 and MMP9 produced dose- and time-dependent modulation in the activities of NKA, HKA and PMCA in the tested tissue explants. Incubation of MMP2 and 9 inhibitors at the highest dose (10 -6 M) for 15 and 30 min produced substantial rise in NKA activity. Likewise, HKA activity that showed significant rise after incubation of 10 -7 and 10 -8 M inhibitors in gills and kidney explants, decreased at the lowest dose (10-8 M) of inhibitors. The lower doses of both inhibitors, while increasing PMCA activity in kidney and intestinal explants inhibited its activity in gill explant. These differential tissue-responsive actions of MMP2 and MMP9 inhibitors indicate that these ECM remodeling components can modify the function of the membrane-bound P-type ion transporters in the osmoregulatory tissues of fish.

Effect of natalizumab treatment on metalloproteinases and their inhibitors in a mouse model of multiple sclerosis.

Matrix metalloproteinases (MMPs) regulated by their tissue inhibitors (TIMPs) play a significant role in the pathogenesis of multiple sclerosis (MS) and its mouse model, experimental autoimmune encephalomyelitis (EAE), as they degrade extracellular matrix including vascular basal laminae and by damaging blood-brain barrier (BBB) facilitate transmigration of immune cells into the central nervous system. MMPs are also involved in destruction of myelin sheaths, leading to axonal and neuronal loss. The aim of the present study was to assess whether natalizumab, a transmigration-inhibiting monoclonal antibody against α4β1 integrin, influences expression of MMPs and TIMPs in the central nervous system of mice with EAE. MMP-2 and MMP-9, their respective inhibitors TIMP-2 and TIMP-1 and laminin were assessed by quantitative immunohistochemistry in the spinal cord cryosections of C57BL/6 mice with EAE in the successive phases of the disease (onset, peak and chronic). The percentage of immunopositive areas were calculated in sections encompassing the whole spinal cord cross-sectional area occupied by the gray and white matter. Results obtained in animals administered with 5 mg/kg natalizumab were compared with those collected from control mice receiving 5 mg/kg IgG. Both studied MMPs and both TIMPs were upregulated in control EAE mice. Natalizumab treatment significantly reduced expression of MMPs and increased expression of TIMPs in the peak and chronic phases of the disease. This effect was accompanied by inhibition of laminin degradation in the vascular basal laminae and reduction of inflammatory infiltration. Results of this study demonstrate that in addition to its well known anti-integrin activity counteracting transmigration of immune cells into the central nervous system, natalizumab strengthens this effect by its probably indirect influence on MMPs and TIMPs leading to protection of blood-brain barrier integrity.

CircTMBIM6 promotes osteoarthritis-induced chondrocyte extracellular matrix degradation via miR-27a/MMP13 axis.

Osteoarthritis is a degenerative disease characterized by degeneration of articular cartilage, but the current mechanism is unclear. Circular RNA (circRNA) plays a significant role in a series of biological processes related to osteoarthritis, but its mechanism remains unclear. The purpose of this study was to investigate the role of the circTMBIM6/miR-27a/MMP13 axis in osteoarthritis. The expression levels of circTMBIM6, miR-27a and MMP13 in cartilage of osteoarthritis patients and normal human cartilage were detected by reverse transcription polymerase chain reaction (RT-PCR). Osteoarthritis cell model was induced by IL-1β and TNF-α, and the expression changes of circTMBIM6, miR-27a and MMP13 in the in vitro model were detected. In addition, the in vitro regulation of circTMBIM6 and miR-27a in osteoarthritis was verified by transfection of circTMBIM6 and miR-27a plasmids, and the regulation of miR-27a on MMP13 was also verified. The dimethylmethylene blue (DMMB) method was used to analyze the secretion and formation of soluble glycosaminoglycan sulfate (sGAG), and the effects of circTMBIM6 and miR-27a chondrocytes were evaluated. The expression levels of circTMBIM6 and MMP13 in cartilage tissue of patients with osteoarthritis were higher than that of normal group, while the expression level of miR-195 in cartilage tissue of patients with osteoarthritis was lower. After IL-1β and TNF-α treatment, the expression of circTMBIM6 and MMP13 in chondrocytes increased, while the expression of miR-27a decreased. CircTMBIM6 overexpression reduced miR-27a expression but increased MMP13 expression. The circTMBIM6 gene knockout showed the opposite effect. CircTMBIM6 promotes osteoarthritis-induced chondrocyte extracellular matrix degradation via miR-27a/MMP13 axis.

Effect of CPEB4 on Migration and Cycle of Chronic Myeloid Leukemia Cell

To investigate the effects of CPEB4 on the migration and cycle of K562 cells and the changes of protein molecules that may be involved in the regulatory mechanism. Western blot was used to detect the expression of CPEB4 in normal leukocytes and K562 cells. The overexpression plasmid pcDNA3.1(+)-His-CPEB4, silencing plasmid pPLK+Puro-CPEB4 shRNA were transfected into K562 cells by electroporation so as to change CPEB4. The transfection efficiency was detected by Western blot. Finally, the migration and cycle of different cells were detected by Transwell chamber and flow cytometry.Western blot was used to detect the expression changes of MMP2, MMP9, CDK4, CyclinD1 and P21 proteins. Compared with normal white blood cells, the expression of CPEB4 protein in K562 cells was significantly enhanced (P＜0.01); Compared with the control group, CPEB4-silenced K562 cells showed that the cell migration ability was significantly enhanced (P＜0.01); G0/G1 phase cell ratio reduced, G2/M phase cell ratio increased, and cell cycle progression accelerated（P＜0.01）, The expression levels of MMP2 (P＜0.05), MMP9 (P＜0.05), CDK4 (P＜0.01), CyclinD1 (P＜0.01) proteins increased significantly. The expression level of P21 protein significantly decreased (P＜0.01). The migration ability of K562 cells after CPEB4 overexpression was decreased (P＜0.01), the cell ratio of G0/G1 phase in the cell cycle increased, the cell proportion of S phase decreased and the cell cycle progression was arrested at G0/G1 phase (P＜0.01). The expression of P21 protein increased, MMP2 , MMP9, CDK4, CyclinD1 protein expression decreased significantly（P＜0.05-0.01）. CPEB4 can inhibit the migration of K562 cells and arrest cell cycle progression at G0/G1 phase. Its mechanism may be related with regulating the exprossion of MMP2, MMP9, CDK4, CyclinD1 and P21 proteins.

Matrilin-3 alleviates extracellular matrix degradation of nucleus pulposus cells via induction of IL-1 receptor antagonist.

The intervertebral disc contains abundant extracellular matrix (ECM) imbued with proteoglycans, collagens, and water. With the development of intervertebral disc degeneration (IVDD), the ECM undergoes changes characterized by loss of water content, proteoglycans, and collagen content. The purpose of this study was to explore the vital role of Matrilin-3, an ECM protein involved in the progress of IVDD. NP cells were isolated from the patients' disc samples and exposed to recombinant human (rh)-Matrilin-3 protein (MATN3), and IL-1β is used as a reducer of nucleus pulposus (NP) cells degeneration. Matrilin-3 and IL-1 receptor antagonist (IL-1Ra) were knocked down by siRNA transfection. Messenger RNA expressions of IL-1Ra, Collagen II, aggrecan, MMP-13, and ADAMTS-5 were determined using Real-Time quantitative Polymerase Chain Reaction (RT-qPCR). Later, the protein levels of IL-Ra, Collagen II, and aggrecan were also detected by Western blot. The IL-1Ra, MMP-13, and ADAMTS-5 dose of the supernatants in the culture medium was determined by enzyme linked immunosorbent assay (ELISA). Finally, immunofluorescence was used to expose the expression of Collagen II, aggrecan, and Collagen X. It was found that the expression of IL-1Ra was markedly increased in the present of MATN3 or IL-1β, especially these two at once. Besides, MATN3 could upregulate Collagen II and aggrecan expressions, as well as inhibit the MMP-13 and Collagen X production of NP cells. However, the protective effects of Collagen II and aggrecan were abolished after Matrilin-3 silenced. Furthermore, IL-1β downregulated the Collagen II and aggrecan but promoted the MMP-13 and Collagen X levels of NP cells, which were antagonized by the action of MATN3. Surprisingly, silencing of IL-1Ra significantly abolished the MATN3-induced the protective effects of ECM in NP cells. This study provides a novel viewpoint of Matrilin-3 in the ECM stability of NP due to its ability to activate IL-1Ra. It is considered that MATN3 efficiently protects ECM degeneration of human NP cells related to maintain the content of Collagen II and aggrecan, as well as inflammatory inhibition.

Expression of lncRNA AK058003 in esophageal carcinoma and analysis of its intervention effect.

The aim of this study was to investigate the expression of long non-coding ribonucleic acid (lncRNA) AK058003 in esophageal carcinoma (EC) tissues, and to analyze its intervention effect. The expression of lncRNA AK058003 in EC tissues and para-carcinoma tissues from 130 EC patients was detected via quantitative Polymerase Chain Reaction (qPCR). EC cell lines were selected for exogenous interference in lncRNA AK058003. Subsequently, the expression of lncRNA AK058003 in normal esophageal epithelial cell line (Het-1A) and EC cell lines (EC109, EC9706, KYSE-150, KYSE-30, and TE-1) was detected by qPCR. EC9706 cell lines with the highest expression of lncRNA AK058003 were selected and transfected with lncRNA AK058003 siRNA and lncRNA AK058003 control, respectively. After transfection, the expression of lncRNA AK058003 was determined using PCR. The changes in cell growth and proliferation were analyzed via cell growth curve and cell cycle assay. Meanwhile, the changes in cell migration and invasion were analyzed through wound healing assay. Protein expressions of matrix metalloproteinase-1 (MMP1) and MMP2 were determined by Western blot. Clinical data were collected from EC patients, and the association between lncRNA AK058003 expression and tumor-node-metastasis (TNM) stage was finally analyzed. LncRNA AK058003 was highly expressed EC tissues compared with para-carcinoma tissues (p<0.01). Compared with Het-1A cells, the expression of lncRNA AK058003 was significantly higher in EC109, EC9706, KYSE-150, KYSE-30, and TE-1 cells, with highest level in EC9706 cells (p<0.05). The expression of lncRNA AK058003 remarkably declined in lncRNA AK058003 siRNA group compared with lncRNA AK058003 control group (p<0.001). Compared with lncRNA AK058003 control group, the proliferation of EC cells was significantly weakened in lncRNA AK058003 siRNA group, with the greatest difference at 3 d. Flow cytometry results revealed that cell cycle was arrested in G0/G1 phase in lncRNA AK058003 siRNA group. Wound healing assay indicated that the intercellular distance became large, and cell migration ability was evidently enhanced in lncRNA AK058003 siRNA group with time (p<0.05). Besides, the protein expressions of MMP1 and MMP2 were remarkably lower in lncRNA AK058003 siRNA group than those in lncRNA AK058003 control group. This indicated remarkably declined invasion and metastasis ability. In addition, the postoperative prognosis was significantly worse in patients with higher expression of lncRNA AK058003 (p<0.05). All these findings suggested that lncRNA AK058003 could serve as a biomarker for EC prognosis. LncRNA AK058003 is highly expressed in EC patients, which promotes proliferation, migration, invasion, and metastasis of EC cells. In addition, the postoperative prognosis of EC patients with high expression of lncRNA AK058003 is relatively poor.

Preliminary study on prevention and treatment of experimental autoimmune uveitis by blocking CD73 detachment from the surface of retinal pigment epithelial cells with matrix metalloprotein-9 inhibitor

Objective To preliminarily investigate the mechanism of MMP-9 blocking CD73 detachment from RPE cells surface and preventing and treating experimental autoimmune pigment membranitis(EAU). Methods RPE cells isolated from wild-type C57BL/6 and CD73 gene knockout (CD73-/-) mice were cultured in vitro, and treated with lipopolysaccharide and TNF-α to induce CD73 detachment from RPE surface. According to whether MMP-9 inhibitor CTK8G1150 was added at the same time (the final concentration was 5.0 mol/L) or not, RPE cells cultured in the two types of mice were respectively set as MMP-9 inhibitor intervention group and non-intervention control group. The cells in each group were treated with the intervention of a solvent, 1 μmol/Ladenosine monophosphate (AMP), 1 μmol/L AMP, and 3 μmol/L 5' -α,β-methylene adenosine diphosphate (APCP) (AMP+APCP). The stimulating effect of RPE cells in different groups on CD4+ T cell proliferation was detected by tritiated thymidine incorporation. Adoptive immune induced EAU in wild-type B6 mice and CD73-/- mice, respectively. The receptor mice were randomly divided into the MMP-9 inhibitor intervention group and the non-intervention control group, and CTK8G1150 or the solvent were injected into the subretinal cavity 4, 7 and 10 days after adoptive immunity. CD73 mRNA and protein expression in RPE cells of recipient mice were detected by real-time quantitative PCR (RT-PCR) and Western blot. One-way ANOVA was used to analyze all experimental data. Results When the stimulation mode was AMP, the proliferation of CD4+ T cells in the C57BL/6 MMP-9 inhibitor intervention group decreased significantly compared with the nonintervention group (F=13.28, P 0.05). There was no statistically significant difference in the proliferation capacity of CD4+ T cells between the CD73-/- MMP-9 inhibitor intervention group and the non-intervention group (F=5.24, 6.12, 7.04; P>0.05). RT-PCR results showed that there was no statistically significant difference in the relative expression of CD73 mRNA in RPE cells between the MMP-9 inhibitor group and the non-intervention control group(F=6.54, P>0.05). Western blot results showed that the expression of CD73 protein in RPE cells in the MMP-9 inhibitor group of B6 receptor mice was significantly increased compared with the control group (F=15.24, P<0.01). Conclusion MMP-9 inhibitor blocks CD73 detachment from RPE cells surface and has a protective effect on EAU. Key words: Matrix metalloproteinase 9/antagonists & inhibitors; 5'-nucleotidase; Retinal pigment epithelium; Cells, cultured; Uveitis/physiopathology; Animal experimentation

SIRT2 inhibits oxidative stress and inflammatory response in diabetic osteoarthritis.

Diabetes mellitus is involved in inflammation, immunity, and metabolism during osteoarthritis (OA). It destroys the normal synthesis and degradation balance of chondrocytes (CHs) and extracellular matrix (ECM). The purpose of this study was to explore the possible way of SIRT2 influencing the progress of diabetic OA. Proteins of diabetic OA and normal OA cartilage samples were extracted from patients undergoing knee joint operation. CHs were also isolated from the cartilage exempted from diabetes for cell culture. Glucose was used to treat CHs for imitating the microenvironment of diabetes. The expressions of SIRT2, acetylated H3K9, H3K14, and H3K56 protein were determined by Western blotting. SIRT2, 8-hydroxy-2' deoxyguanosine (8-OH), and MMP-13 expressions were analyzed using immunofluorescence. RT-PCR was performed to measure the mRNA levels of SOD1, SOD2, CAT, MMP-13, ADAMTS-4, and ADAMTS-5. Total ROS level was performed by flow cytometry assay. SIRT2 expression was reduced, whereas acetylated H3K9, H3K14, and H3K56 were upregulated in diabetic cartilage compared to normal. High glucose suppressed the expression of SIRT2 but accelerated the acetylation of H3K9, H3K14, and H3K56. Besides, high glucose promoted the expression of 8-OH, and inhibited SOD1, SOD2, and CAT mRNA expressions, resulting in the up-regulated ROS level of CHs. In addition, high glucose activated the inflammatory response by upregulation of MMP-13, ADAMTS-4, and ADAMTS-5 expressions. SirReal2 suppressed SIRT2 and resulted in several acetylations of H3, more ROS, less antioxidant enzymes, and stronger inflammatory response caused by high glucose. However, supplied rh-SIRT2 reversed these negative effects of high glucose in CHs. SIRT2 expression is reduced along with the diabetic OA process with increased acetylation of H3, oxidative stress, and inflammatory response. Suppression of SIRT2 accelerates the progress of diabetic OA and upregulation of SIRT2 alleviates diabetic OA development by suppressing oxidative stress and inflammatory response that are likely to be related to the deacetylation of H3.

Clinical effect of monosialotetrahexosyl ganglioside in the treatment of neonatal intracranial hemorrhage

Objective To investigate the clinical effect of monosialotetrahexosyl ganglioside in the treatment of neonatal intracranial hemorrhage. Methods From January 2016 to December 2018, 142 neonates with intracranial hemorrhage admitted to the Maternal and Child Health Hospital of Zhoushan were randomly divided into observation group (71 cases) and control group (71 cases) according to the digital table.The control group was treated with routine treatment, while the observation group was treated with ganglioside needle on the basis of the control group.Both two groups were treated for 14 days.The therapeutic effects, muscle tone recovery time, reflex recovery time and consciousness recovery time were compared.The changes of neurobehavioral assessment score (NABA score), TNF-α, IL-1β, MMP-2, TIMP-1 and NSE levels before and after treatment were compared. Results The total effective rate of the observation group (92.96%) was higher than that of the control group (77.47%) (χ2=6.762, P<0.05). The recovery time of muscle tension, reflex and consciousness in the observation group [(7.68±1.29)d, (6.83±1.20)d and (8.34±1.54)d] were shorter than those in the control group [(10.25±2.31)d, (9.17±1.86)d and (10.53±1.08)d] (t=8.185, 8.908, 9.811, all P<0.05). After treatment, the NABA score of the observation group[(40.37±0.65)points] was higher than that of the control group[(37.16±0.93)points](t=23.838, P<0.05). The serum levels of TNF-α [(26.37±4.25)pg/L], IL-1β [(16.74±3.24)ng/L], MMP-2 [(78.39±16.57)g/L], TIMP-1 [(179.32±17.65)ng/mL] and NSE [(13.52±2.19)g/L] in the observation group were lower than those in the control group [(53.21±7.39)pg/L, (28.93±5.64)ng/L, (97.42±12.63)g/L, (238.63±28)ng/mL and (21.43±2.89)μg/L] (t=26.529, 15.792, 7.696, 14.938, 18.381, all P<0.05). Conclusion Ganglioside has good therapeutic effect on neonatal intracranial hemorrhage.It can reduce the serum levels of TNF-α, IL-1β, MMP-2, TIMP-1 and NSE, and improve the neurobehavioral function of neonates. Key words: Intracranial hemorrhages; Tumor necrosis factor-alpha; Interleukin-1beta; Matrix Metalloproteinase 2; Monosialotetrahexosyl ganglioside; Infant, newborn

Suppression of p38/HBP1 pathway alleviates hyperosmotic stress-induced senescent progression of chondrocyte senescence.

This study aims to explore the effect of p38 mitogen-activated protein kinase and its downstream target HMG-box transcription factor 1 (HBP1) in the chondrocyte (CH) senescence caused by hyperosmotic stress. Human cartilage tissue with or without osteoarthritis (OA) were collected to detect the differential expression of p38 and HBP1 by Western blot. CHs were isolated from cartilage without OA and used the hyperosmotic medium to accelerate CH senescence in vitro. A p38 inhibitor and siRNA were used to mediate the expression of p38 and HBP1. The viability of CHs was determined by cell counting kit 8 (CCK8) assay. CH-related mRNA expression was analyzed by quantitative real-time polymerase chain reaction (RT-PCR). Immunofluorescence was also used to detect collagen II and beta-galactosidase expression. Senescent cells were increased in both OA cartilage and hyperosmotic stress treatment with a marked upregulation of p38 and HBP1. Suppression of p38 activation reversed the hyperosmotic stress-induced CH senescence and led to an inhibition of HBP1, p16, Runx-2, MMP-13, collagen X expression, and an upregulation of collagen II and SOX-9 expression. Moreover, the silencing of HBP1 also played a protective effect on CH senescence. The suppression of the p38/HBP1 pathway alleviates the hyperosmotic stress-induced senescent progression of CHs.

Cyclooxygenase-2 inhibitor rofecoxib prevents chondrocytes against hypertrophy via Wnt/β-catenin pathway.

Previous reports indicated that specific cyclooxygenase-2 (COX-2) inhibitor suppresses osteoarthritis (OA). This study aimed to further explore the possible mechanism of Rofecoxib as a COX-2 inhibitor on the inhibition of chondrocyte (CH) hypertrophic development and tested the optimal treatment of Rofecoxib on CH. Basically, IL-1β was used as a stimulus to establish a degenerated CH model. Immunofluorescence, Western blot, and RT-PCR were performed to determine the gene expression of Axin2, β-catenin, GSK3β, collagen X, collagen II, COX-2, PGE-2, SOX-9, Runx-2, and MMP- 13 expression. Cell Counting Kit (CCK-8) assay was used to analyze the viability of CHs. The data indicated that Rofecoxib significantly inhibited COX-2 expression and had less harmful effects on CH viability. Rofecoxib reversed the IL-1β-induced upregulation of collagen X, COX-2, PGE-2, Runx-2, and MMP-13 expression, and promoted the viability of collagen II, SOX-9 expression of CHs. Furthermore, Rofecoxib suppressed Axin2, β-catenin, and GSK3β expression of the Wnt pathway, which was activated by IL-1β or human recombinant Wnt-1 protein treatment. Therefore, Rofecoxib is an effective COX-2 inhibitor that protects CHs from hypertrophy by suppression of the Wnt/β-catenin pathway.

Estimation of Salivary MMP-8, MMP-9, MMP-13 and TIMP-1 in Chronic Periodontitis in Mosul

The aim of the study was to estimate salivary concentrations of MMP-8, MMP-9, MMP-13, and TIMP-1in chronic periodontitis and control patients, and correlate periodontal parameters with salivary biomarkers in chronic periodontitis patients. The study group consisted of 44 patients suffering from chronic periodontitis, aging between ≤ 16-45 years old and 40 control samples collected from healthy individuals ranged between 16-40 years old. Chronic periodontitis was assessed on the basis of several periodontal parameters, including probing pocket depth (PPD), clinical attachment loss (CAL), bleeding on probing (BOP) and plaque index (PI). 5ml of unstimulated saliva was collected from patients and control groups to measure salivary biomarkers by ELISA technique. All periodontal clinical parameters were significantly higher in chronic periodontitis patients compared to control group (p ≤ 0.000). Salivary MMP-8, MMP-9 and MMP-13 concentrations showed significant increase in chronic periodontitis (p ≤ 0.000), while salivary TIMP-1 level showed significant increase in control group comparing with periodontitis (p ≤ 0.000). Salivary MMP-13and MMP-9 showed significant difference in concentrations with different PPD, and CAL levels, while MMP-8 showed no significant difference, whereas all salivary biomarkers showed no significant difference with BOP index. According to Plaque index, MMP-8 concentration showed highly significant difference with different PI values.

Regulation and its mechanism of MeCP2 on biological behavior and epithelial-mesenchymal transition of LECs

Objective To investigate the role of methyl-CpG-binding protein 2 (MeCP2) in the regulation and epithelial-mesenchymal transition (EMT) of human lens epithelial cells (LECs) and its possible mechanism. Methods Human LEC lines (SRA01/04) were divided into MeCP2-mimic group, MeCP2-NC group and small interferening RNA-MeCP2 (si-MeCP2) group, and MeCP2 analog plasmid, blank plasmid and MeCP2 si-RNA plasmid was used respectively to transfect the cells.The expression of MeCP2 mRNA in the cells was detected by using real-time PCR 24 hours after transfection.At 48 hours after transfection, the migration rate of the cells was evaluated by scratching test, and the expression of Wnt3a protein in the cells was detected by immunofluorescence stainning.The relative expressions of β-catenin, E-cadherin, Vimentin, matrix metallo proteinase (MMP)-9, MMP-7 and secreted frizzled-related protein 5 (SFRP5) proteins in the cells were detected by Western blot. Results After 24 hours of transfection, the relative expression of MeCP2 mRNA in the cells was significantly different among the MeCP2-mimic group, MeCP2-NC group and si-MeCP2 group (F=4 773.00, P<0.00 1). The migrating rate of the cells in the MeCP2-mimic group, MeCP2-NC group and si-MeCP2 group was (57.45±5.20)%, (32.71±10.02)% and (17.77±9.22)%, respectively, showing a significant difference among the three groups (F=124.00, P<0.001), and the migrating rate of the cells in the si-MeCP2 group was significantly lower than that of the MeCP2-mimic group or MeCP2-NC group (both at P<0.001). The relative expressing intensity (absorbance) of Wnt3a in the cells of the MeCP2-mimic group, MeCP2-NC group and si-MeCP2 group was 75.92±6.10, 52.03±5.22 and 28.75±3.39, respectively, with a significant difference among three the groups (F=221.30, P<0.001), and the relative expressing intensity (absorbance) of Wnt3a in the cells was significantly lower in the si-MeCP2-mimic group than that of the MeCP2-NC group and MeCP2-mimic group (both at P<0.001). The relative expressing level of E-cadherin protein was significantly elevated and the expressions of β-catenin, Vimentin, MMP-9 and MMP-7 were significantly reduced in the si-MeCP2 group compared with the MeCP2-mimnic group and MeCP2-NC group (all at P<0.01). The relative expressing level of SFRP5 protein in the MeCP2-mimic group, MeCP2-NC group and si-MeCP2 group was 27.19±0.03, 47.54±0.05 and 74.93±0.05, respectively, showing a statistical difference among the three groups (F=183.49, P<0.001), and the relative expressing level of SFRP5 in the si-MeCP2 group was significantly higher than that in the MeCP2-mimic group and MeCP2-NC group (both at P<0.001). Conclusions MeCP2C can promote EMT of human LECs by down-regulating the expression of SFRP5 and therefore activating the Wnt3a/β-catenin signal pathway. Key words: Methyl-CpG-binding protein 2; Lens epithelial cells, human; Epithelial-mesenchymal transition; Signal pathway

Study on the mechanism of long non-coding RNA migration inhibitory factor antisense RNA1 in regulating proliferation, invasion and metastasis of non-small cell lung cancer

Objective To explore the molecular mechanism by which long non-coding RNA (lncRNA) migration inhibitory factor antisense RNA1 (MIF-AS1)/microRNA (miRNAM miR)-370-3p/mitogen activated protein kinase 9 (MAP3K9) molecular axis regulates proliferation, invasion and migration of non-small cell lung cancer (NSCLC). Methods Twenty cancer samples of NSCLC patients were collected from Henan Provincial People’s Hospital from May 2017 to January 2019. There were 12 males and 8 females, aged (52.37±10.34) years. Real-time quantitative reverse transcriptase-polymerase chain reaction (RT-qPCR) and Western blotting were used to detect the expression levels of MIF-AS1, miR-370-3p and MAP3K9 in cancer tissues and 9 cell lines. NSCLC A549 cells were transfected with si-MIF-AS1, si-NC, si-MIF-AS1+ anti-miR-370-3p or si-MIF-AS1+ pcDNA-MAP3K9, respectively. Methyl thiazol tetrazolium (MTT) assay and Transwell assay were used to detect the proliferation, migration and invasion of transfected cells. Western blotting was used to detect the expression of Cyclin D1, p21, p27, matrix metalloproteinase (MMP)-2, MMP-9 and MMP-14 proteins. The dual luciferase reporter gene verified the targeting regulation of MIF-AS1 on miR-370-3p and miR-370-3p on MAP3K9. Results The expression of MIF-AS1 and MAP3K9 in NSCLC tissues (2.49±0.25 and 2.24±0.22) and A549 cells (2.54±0.24 and 2.31±0.23), H1299 cells (2.11±0.21 and 2.44±0.24) and PC-9 cells (2.26±0.23 and 2.16±0.22) was significantly higher than that in adjacent tissues (1.00±0.09 and 1.02±0.09) and normal lung epithelial cells BEAS-2B (1.01±0.09 and 1.00±0.09) (t=25.078, P<0.05; F=94.367, P<0.05). The expression of miR-370-3p in NSCLC tissues (0.42±0.04), A549 cells (0.34±0.03), H1299 cells (0.53±0.05), and PC-9 cells (0.44±0.04) was significantly lower than that in adjacent tissues (1.01±0.08) and BEAS-2B cells (1.00±0.08) (t=29.500, P<0.05; F=269.552, P<0.05). Transfection with si-MIF-AS1 significantly inhibited NSCLC cell proliferation (24 h: 0.27±0.03, 48 h: 0.36±0.03, 72 h: 0.48±0.04), invasion (24.33±3.14), migration (32.46±3.34) ability (P<0.01), up-regulated p21 (0.64±0.06), p27 (0.77±0.07) protein expression (t=17.441, P<0.05; t=17.726, P<0.05) and down-regulated Cyclin D1 (0.29±0.03), MMP-2 (0.25±0.03), MMP-9 (0.34±0.03) and MMP-14 (0.29±0.03) protein expression (t=17.732, P<0.05). The dual luciferase reporter gene confirmed that MIF-AS1 was bond to miR-370-3p and miR-370-3p was bond to MAP3K9. Transfection with si-MIF-AS1+ anti-miR-370-3p or si-MIF-AS1+ pcDNA-MAP3K9 inhibited the effects of si-MIF-AS1 on proliferation, invasion and migration of NSCLC cells. Conclusion LncRNA MIF-AS1 promotes proliferation, migration and invasion of NSCLCs by regulating miR-370-3p/MAP3K9 molecular axis. Key words: Non-small cell lung cancer; Long non-coding RNA migration inhibitory factor antisense RNA1; MicroRNA-370-3p; Mitogen activated protein kinase 9; Proliferation; Migration; Invasion

Expression of prostate cancer-associated transcript 6 in serum of patients with liver cancer and the effect of its interference on biological function of liver cancer cells

Objective To investigate the expression of prostate cancer-associated transcript 6 (PCAT6) in the serum of hepatocarcinoma patients and its effect on the proliferation, migration, invasion and apoptosis of Hep3B cells. Methods The qPCR method was used to detect the level of long non-coding RNA (lncRNA) PCAT6 in serum and THLE-3, Hep3B and HepG2 cells. The Hep3B cells were randomly divided into blank control group (NC group), negative empty vector transfection group (si-con group) and lncRNA PCAT6 silencing group (si-PCAT6 group). Western blotting was used to detect the expression of matrix metalloproteinase (MMP)-2, MMP-9, cleaved cysteinyl aspartate-specific protease (Caspase)-3, cleaved Caspase-9, phosphatidylinositol 3 kinase (PI3K), phosphorylated-PI3K (p-PI3K), protein kinase B (Akt), and phosphorylated Akt (p-Akt) proteins. The cell proliferation was measured by methyl thiazol tetrazolium (MTT) assay. The apoptosis was examined by flow cytometry, and the migration and invasion was tested detected by Transwell chamber method. The SPSS 21.0 software was used for statistical analysis, and the measurement data were expressed as mean±standard deviation (SD). Results The expression level of lncRNA PCAT6 in patients with moderate or high differentiation (60.94%) was significantly higher than that in patients with low differentiation (6.25%) (χ2=22.968, P<0.01). The percentage of patients with high expression of lncRNA PCAT6 in T-stage Ⅱ-Ⅳ (57.81%) was significantly higher than that in stage Ⅰ (9.38%) (χ2=8.529, P<0.01). The expression of lncRNA PCAT6 in Hep3B cells (3.72±0.67) and HepG2 cells (3.38±0.53) was significantly higher than that in THLE-3 cells (1.03±0.14) (t=9.322 and 8.144, P<0.05). The expression of lncRNA PCAT6 (0.21±0.11) in si-PCAT6 group was significantly lower than that in si-CON group (0.96±0.15) (t=9.915, P<0.05), and the cell viability was significantly reduced at 24 h, 48 h and 72 h (t=3.280, 6.144 and 6.373, P<0.05). Western blotting showed that the expression of cleaved Caspase-3, cleaved Caspase-9, p-pi3k and p-Akt increased significantly (t=11.408, 14.628, 8.683 and 9.585, P<0.01), and the expression of MMP-2 and MMP-9 decreased significantly in si-PCAT6 group (t=10.568 and 10.814, P<0.01). The apoptosis rate of Hep3B cells increased significantly (t=21.075, P<0.01), and the migration and invasion of Transwell cells decreased significantly (t=12.816 and 12.707, P<0.01). Conclusion The serum lncRNA PCAT6 is highly expressed in HCC patients, which is related to the degree of HCC differentiation and T stage. Silencing lncRNA PCAT6 can inhibit the biological behaviors of HCC probably by inhibiting the Akt signaling pathway activation. Key words: Long non-coding RNA; Prostate cancer-associated transcript 6; Liver cancer; Biological function

CHCHD2 decreases docetaxel sensitivity in breast cancer via activating MMP2.

This study aims to clarify the influences of CHCHD2 and MMP2 on docetaxel resistance in breast cancer. Differential levels of CHCHD2 in breast cancer and para-tumor tissues were detected. The relationship between CHCHD2 and prognosis in breast cancer patients was analyzed. After generating Docetaxel-resistant breast cancer cell lines (MCF-7/DTX and SKBR3/DTX), the regulatory effects of CHCHD2 on proliferative and migratory potentials were assessed by Cell Counting Kit-8 (CCK-8) and transwell assay, respectively. The interaction between CHCHD2 and MMP2 was tested by Western blot and Pearson correlation test. At last, the involvement of MMP2 in CHCHD2-regulated proliferation and migration in Docetaxel-resistant breast cancer cells was analyzed. CHCHD2 was upregulated in breast cancer tissues. It predicted high incidence of distant metastasis and poor prognosis in breast cancer patients. Proliferation inhibition rate was lower in MCF-7/DTX and SKBR3/DTX cells compared with their parental cells. After knockdown of CHCHD2 in MCF-7/DTX and SKBR3/DTX cells, their proliferative and migratory potentials were markedly reduced. MMP2 was upregulated in breast cancer tissues, and its level was positively regulated by CHCHD2. Overexpression of MMP2 could reverse the regulatory effects of CHCHD2 on proliferative and migratory potentials in MCF-7/DTX and SKBR3/DTX cells. Upregulated CHCHD2 in breast cancer is related to distant metastasis rate and poor prognosis. CHCHD2 and MMP2 are positively correlated to each other. CHCHD2 stimulates proliferative and migratory potentials in Docetaxel-resistant breast cancer cells by upregulating MMP2.