OBJECTIVE: To explore the effect of miR-143 regulating matrix metalloproteinase(MMP)-13 expression on migration and invasion of osteosarcoma cells. METHODS: The mouse osteosarcoma cell line 143B cells were cultured in 96-well plates, and blank group, negative group, positive group, and intervention group were set up. Then, the blank group did no treatment 50 μg miR-143 mimic was added to positive group, negative group added equal mimic NC (control sequence of miR-143 mimic), the intervention group was added 50 μg miR-143 mimic and 10 μg MMP-13 protein, all groups continued to culture for 3 to 6 hours, and finally the serum was aspirated to treat for half an hour. The protein expressions of miR-143 and MMP-13 in each group were measured by fluorescence quantitative PCR experiment and Western blot experiment, respectively, and the invasion and migration abilities of cells were measured by Transwell and scratch experiments. RESULTS: The expression of MMP-13 protein in the positive group and the intervention group was significantly lower than that in the blank group, and the positive group was lower than the intervention group (P<0.05);The mean numbers of invasive cells in blank group, negative group, positive group and intervention group were (1 000.01±44.77), (959.25±46.32), (245.04±4.33), (634.06±33.78) cells/field, respectively;the scratch healing rate of the positive group and the intervention group was significantly lower than that of the blank group, and the positive group was lower than the intervention group (P<0.05). CONCLUSION MMP-13 is a target of miR-143, which can reduce the migration and invasion ability of osteosarcoma cells by inhibiting the expression of MMP-13.
Osteosarcoma/pathology* ; MicroRNAs/genetics* ; Matrix Metalloproteinase 13/genetics* ; Neoplasm Invasiveness ; Animals ; Mice ; Cell Line, Tumor ; Cell Movement

OBJECTIVETo investigate whether the functional polymorphisms in the promoter region of MMP-12 (-82A/G) and MMP-13(-77A/G) are associated with epithelial ovarian carcinoma (EOC).

METHODSThe MMP-12 -82A/G and MMP-13 -77A/G were genotyped by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) in 300 epithelial ovarian carcinoma patients and 300 control women.

RESULTSThe A/G genotype frequency of the MMP-12 gene was significantly higher in the patients than in the controls (P= 0.003); similarly, the frequency of MMP-12 -82G allele was higher in the patient group (P= 0.004). Compared with the A/A genotype, the A/G genotype carriers significantly increased the risk of EOC development (OR= 2.81, 95%CI: 1.38-5.74). No overall association between the MMP-13 -77A/G polymorphism and EOC(P= 0.15) was observed. However, the A/A genotype carriers in the MMP-13 -77A/G locus had significantly higher risk of developing serous-papillary and mucinous ovarian cancer (OR= 1.93, 95% CI: 1.05-3.53; OR= 5.16, 95% CI: 1.62-16.44, respectively), comparing with the G/G genotype carriers. Combining the two SNPs, the haplotype distributions in patients were not significantly different from that in control women (P= 0.06).

CONCLUSIONThese results suggested that individuals with MMP-12 -82A/G and MMP-13 -77A/A might have higher risk of overall or special histological type of EOC development.

Adult ; Aged ; Female ; Gene Frequency ; Genetic Predisposition to Disease ; Genotype ; Humans ; Matrix Metalloproteinase 12 ; genetics ; Matrix Metalloproteinase 13 ; genetics ; Middle Aged ; Neoplasms, Glandular and Epithelial ; genetics ; Ovarian Neoplasms ; genetics ; Polymorphism, Single Nucleotide ; Promoter Regions, Genetic ; genetics ; Young Adult

BACKGROUND: Histone deacetylase 4 (HDAC4) regulates chondrocyte hypertrophy and bone formation. The aim of the present study was to explore the effects of HDAC4 on Interleukin 1 beta (IL-1β)-induced chondrocyte extracellular matrix degradation and whether it is regulated through the WNT family member 3A (WNT3A)/β-catenin signaling pathway. METHODS: Primary chondrocytes (CC) and human chondrosarcoma cells (SW1353 cells) were treated with IL-1β and the level of HDAC4 was assayed using Western blotting. Then, HDAC4 expression in the SW1353 cells was silenced using small interfering RNA to detect the effect of HDAC4 knockdown on the levels of matrix metalloproteinase 3 (MMP3) and MMP13 induced by IL-1β. After transfection with HDAC4 plasmids, the overexpression efficiency was examined using Real-time quantitative polymerase chain reaction (qRT-PCR) and the levels of MMP3 and MMP13 were assayed using Western blotting. After incubation with IL-1β, the translocation of β-catenin into the nucleus was observed using immunofluorescence staining in SW1353 cells to investigate the activation of the WNT3A/β-catenin signaling pathway. Finally, treatment with WNT3A and transfection with glycogen synthase kinase 3 beta (GSK3β) plasmids were assessed for their effects on HDAC4 levels using Western blotting. RESULTS: IL-1β downregulated HDAC4 levels in chondrocytes and SW1353 cells. Furthermore, HDAC4 knockdown increased the levels of MMP3 and MMP13, which contributed to the degradation of the extracellular matrix. Overexpression of HDAC4 inhibited IL-1β-induced increases in MMP3 and MMP13. IL-1β upregulated the levels of WNT3A, and WNT3A reduced HDAC4 levels in SW1353 cells. GSK-3β rescued IL-1β-induced downregulation of HDAC4 in SW1353 cells. CONCLUSION HDAC4 exerted an inhibitory effect on IL-1β-induced extracellular matrix degradation and was regulated partially by the WNT3A/β-catenin signaling pathway.
Cell Line, Tumor ; Cells, Cultured ; Chondrocytes/metabolism* ; Glycogen Synthase Kinase 3 beta/genetics* ; Histone Deacetylases/genetics* ; Humans ; Interleukin-1beta/pharmacology* ; Matrix Metalloproteinase 13/metabolism* ; Matrix Metalloproteinase 3 ; Repressor Proteins ; Wnt Signaling Pathway ; Wnt3A Protein/genetics* ; beta Catenin/metabolism*

The study investigated the effects of adenovirus-mediated gene transfection of basic fibroblast growth factor (bFGF), bFGF combined with interleukin-1 receptor antagonist protein (IL-Ra) and/or insulin-like growth factor-1 (IGF-1) both in human osteoarthritis (OA) chondrocytes and rabbits OA model. Human OA chondrocytes were delivered by adenovirus-mediated bFGF, IL-Ra and IGF-1 vectors, respectively. Chondrocyte proliferation, glycosaminoglycan (GAG) content, expression of type II collagen, ADAMTS-5, MMP-13, MMP-3 and TIMP-1 were determined. Rabbit OA model was induced by anterior cruciate ligament transaction (ACLT) in knees. Adenoviral vectors encoding human bFGF, IL-Ra and IGF-1 were injected intraarticularly into the knee joints after ACLT. The effects of adenovirus- mediated gene transfection on rabbit OA were evaluated. In vitro, the transfected genes were expressed in cell supernatant of human OA chondrocytes. AdbFGF group significantly promoted chondrocyte proliferation, and increased GAG and type II collagen synthesis than in the OA group. As two or three genes were transfected in different combinations, there was significant enhancement on the GAG content, type II collagen synthesis, and TIMP-1 levels, while ADAMTS-5, MMP-13, and MMP-3 levels were reduced. In vivo, the transfected genes were expressed in synovial fluid of rabbits. Intraarticular delivery of bFGF enhanced the expression of type II collagen in cartilage and decreased cartilage Mankin score compared with the OA control group (P = 0.047; P < 0.01, respectively). Multiple-gene transfection in different combinations showed better results than bFGF transfection alone. This study suggests that bFGF gene transfection is effective in treating experimental OA. Multiple gene transfection has better biologic effects on OA.
Adenoviridae/*genetics ; Animals ; Chondrocytes/drug effects/*metabolism ; Collagen Type II/genetics/metabolism ; Fibroblast Growth Factor 2/*genetics ; Gene Therapy/methods ; Genetic Vectors/administration & dosage/*genetics ; Humans ; Insulin-Like Growth Factor I/*genetics/metabolism ; Interleukin 1 Receptor Antagonist Protein/*genetics/metabolism ; Interleukin-1/genetics/metabolism ; Matrix Metalloproteinase 13/genetics/metabolism ; Matrix Metalloproteinase 3/genetics/metabolism ; Osteoarthritis/*therapy ; Rabbits ; Tissue Inhibitor of Metalloproteinase-1/genetics ; Transfection

OBJECTIVESTo demonstrate the gene expression of MMP-13 in the progressive phase of ethanol-induced experimental liver fibrosis in rats.

METHODS34 SD rats were randomized into two groups. The rats in experimental group (n=24) were given ethanol (44%, 7g/kg) every day, and the rats in control group (n=10) were given equality normal saline. Liver samples were harvested from experimental rats at the 4th, 12th and 24th weeks respectively. The dynamic expression of MMP-13 mRNA was assayed by semi-quantity reverse transcription-polymerase chain reaction (RT-PCR).

RESULTSIn normal rat liver, a faint band of MMP-13 mRNA was observed by RT-PCR (0.24+/-0.41). The gene expression of MMP-13 increased in the livers of rats treated with ethanol for 4 weeks (0.62+/-0.54), but it was not considered statistically, when compared with that in normal rats livers. And the livers from 12-week-treated rats showed a markedly MMP-13 mRNA expression (1.65+/-0.47, t=-4.363, P<0.01). Once the fibrosis became prominent (24 weeks), a faint band of MMP-13 mRNA was observed (0.39+/-0.25).

CONCLUSIONMMP-13 participates in the degradation of newly-formed matrix in the early phase of rat liver fibrosis induced by ethanol, but it expresses in a distinct time frame

Animals ; Collagenases ; genetics ; metabolism ; Disease Progression ; Ethanol ; Gene Expression ; Liver Cirrhosis ; chemically induced ; metabolism ; Matrix Metalloproteinase 13 ; Rats ; Rats, Sprague-Dawley

OBJECTIVETo study the effect of laminarin polysaccharide (LP) on the activity of matrix metalloproteinase of photoaging skins.

METHODKunming SPF mice were prepared with back hair shaved, and randomly divided into the control group, the model group, the LP low does group (LP-L, 1 mg x kg(-1)), the LP high dose group (LP-H, 5 mg x kg(-1)) and the Vit E (100 mg x kg(-1)) group. They were abdominally injected with drugs twice on a daily basis. Except for the control group, all groups were exposed to ultraviolet rays for 1 hour every day, five times on a weekly basis, with accumulated exposure dose of UVB being 21.60 J x cm(-2) and accumulated exposure dose of UVA being 84.02 J x cm(-2). Eight weeks later, exposed back skins were collected to detect thickness of dermis by HE stain, content of hydroxyproline (Hyp) by chemical colorimetry, and serum MMP-1 and TIMP-1 content by ELISA. In addition, matrix metalloproteinase-1 (MMP-1) mRNA and relative content of tissue inhibitor of metalloproteinase-1 (TIMP1) mRNA was analyzed with Real-time PCR.

RESULTCompared with the model group, the LP-H group could significantly increase the thickness of dermis, skin Hyp content and serum TIMP-1 level, and decrease relative content of MMP-1 mRNA in skin and MMP-1 content in serum.

CONCLUSIONLP can regulate the metabolism of collagen photoaging skins by adjusting the activity of matrix metalloproteinase.

Animals ; Female ; Glucans ; Matrix Metalloproteinase 13 ; biosynthesis ; genetics ; metabolism ; Mice ; Plant Extracts ; chemistry ; pharmacology ; Plants, Medicinal ; chemistry ; Polysaccharides ; chemistry ; pharmacology ; RNA, Messenger ; genetics ; metabolism ; Skin Aging ; drug effects ; physiology ; radiation effects ; Tissue Inhibitor of Metalloproteinase-1 ; biosynthesis ; genetics ; metabolism ; Ultraviolet Rays

OBJECTIVETo compare the effects of matrix metalloproteinase (MMP) inhibitor doxycycline, losartan, and their combination on the expression of MMP-8, 13, tissue inhibitor of MMP-1, 2 (TIMP-1, 2), and collagen remodeling in the noninfarcted myocardium after acute myocardial infarction (AMI) in rats.

METHODSTwo hundred and fifty-four AMI rats, induced by left coronary ligation, were randomly assigned to the following groups: (1) AMI controls group (n = 64); (2) doxycycline group (30 mg x kg(-1) x d(-1), n = 63); (3) losartan group (10 mg x kg(-1) x d(-1), n = 62); (4) concomitant doxycycline and losartan group (30 and 10 mg x kg(-1) x d(-1) respectively, n = 65); and (5) Sham-operated rats (n = 30), which were randomly selected to serve as noninfarction controls. Each group was further divided into three subgroups of 1, 2, and 4 weeks that received treatment. After the completion of treatment, the rats were killed. The mRNA and protein expression of MMPs and TIMPs in the noninfarcted myocardium were quantified by RT-PCR and Western blot, respectively. The type I and type III collagen volume fraction (CVF) of the noninfarced myocardium were assessed immunohistochemically.

RESULTSNo significant difference existed in myocardial infarction sizes among the 12 subgroups of AMI controls and the three treatment groups (42%-48%, all P > 0.05). Compared with sham operated rats, the mRNA and protein expression of MMP-8 and 13 significantly increased by 39%-183% in all three subgroups of AMI controls (all P < 0.05), except both of their mRNA expressions in 2-week subgroups; the mRNA and protein levels of TIMP-1 increased only in 1-week subgroup of AMI controls by 104% and 67%, respectively (both P < 0.05); the mRNA of TIMP-2 increased in all 1, 2, and 4-week subgroups by 144%-232% (all P < 0.05), but its protein expression lagged and only enhanced in 2 and 4-week subgroups of AMI controls by 231% and 332%, respectively (both P < 0.05). Meanwhile, both type I and type III CVF of noninfarcted myocardium significantly increased in all three subgroups of AMI controls (type I CVF: 3.01%-5.64% vs 1.53%-1.67%, P < 0.01-0.001; type III CVF: 2.19%-4.42% vs 1.46%-1.59%, P < 0.05-0.001), with type I CVF being higher in 4-week than in 1 and 2-week subgroups (5.64% vs 3.01% and 3.02% respectively, all P < 0.05). Compared with AMI controls, all three kinds of treatment significantly reduced the increased mRNA and protein expressions of MMP-8, 13 and TIMP-1, 2 after AMI by 14%-60% (all P < 0.05), as well as type I/III CVF in their 2 and 4-week subgroups (type I CVF: 1.56%-2.38% vs 3.02%-5.64%, P < 0.05-0.001; type III CVF: 1.92%-2.65% vs 4.19%-4.42%, P < 0.05-0.01), except for doxycycline's effect on type III CVF in any of its three subgroups (all P > 0.05). Among the three treatment groups, significant differences existed in the above mentioned indicators only at some subgroup levels (all P < 0.05).

CONCLUSIONSLike losartan, doxycycline can also suppress the enhanced mRNA and protein expression of MMP-8, 13 and TIMP-1, 2, and reduce type I collagen deposition in the noninfarcted myocardium after AMI in rats. However, it has no effect on type III collagen deposition.

Angiotensin II Type 1 Receptor Blockers ; pharmacology ; Animals ; Collagen Type I ; biosynthesis ; genetics ; Collagenases ; biosynthesis ; genetics ; Doxycycline ; pharmacology ; Drug Synergism ; Female ; Losartan ; pharmacology ; Matrix Metalloproteinase 13 ; Matrix Metalloproteinase 8 ; biosynthesis ; genetics ; Matrix Metalloproteinase Inhibitors ; Myocardial Infarction ; metabolism ; Myocardium ; metabolism ; RNA, Messenger ; biosynthesis ; genetics ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Tissue Inhibitor of Metalloproteinase-1 ; biosynthesis ; genetics ; Tissue Inhibitor of Metalloproteinase-2 ; biosynthesis ; genetics ; Tissue Inhibitor of Metalloproteinases ; biosynthesis ; genetics

OBJECTIVETo establish a reliable model for drug screening and therapy by culturing rat femoral head and inducing cartilage degeneration quickly in vitro.

METHODSThe femoral heads from the same SD rats of two-month old were divided into control group and experimental group respectively. They were cultured with DMEM medium plus 10% fetal bovine serum or DMEM medium plus 10% fetal bovine serum plus 50 ng/ml IL-1β for three days. Femoral heads were fixed in 4% paraformaldehyde, decalcified, dehydrated, embedded in paraffin and cut into slices. Specimens were stained with Toluidine blue and Safranine O-Fast Green FCF. The protein expression levels of type II collagen, MMP13, Sox9 and ADAMTS5 were analyzed by immunofluorescence.

RESULTSBoth the Toluidine blue and Safranine O staining were pale in the margin of femoral heads which were stimulated with IL-1β for three days compared to that in control group. The Fast Green FCF staining was positive at the edge of the femoral head in experimental group, which indicated that cartilage became degenerated. The expression levels of both type H collagen and Sox9 were decreased significantly while the expression levels of MMP13 and ADAMTS5 were increased in experimental group.

CONCLUSIONThe model of cartilage degeneration is established by culturing and inducing the degeneration of the femoral heads quickly in vitro.

Animals ; Cartilage Diseases ; genetics ; metabolism ; Collagen Type II ; genetics ; metabolism ; Disease Models, Animal ; Femur Head ; metabolism ; Humans ; In Vitro Techniques ; Interleukin-1beta ; genetics ; metabolism ; Male ; Matrix Metalloproteinase 13 ; genetics ; metabolism ; Rats ; Rats, Sprague-Dawley ; SOX9 Transcription Factor ; genetics ; metabolism

BACKGROUNDThere is a difficulty in evaluating the in vivo functionality of individual chondrocytes, and there is much heterogeneity among cartilage affected by osteoarthritis (OA). In this study, in vitro cultured chondrocytes harvested from varying stages of degeneration were studied as a projective model to further understand the pathogenesis of osteoarthritis.

METHODSCartilage of varying degeneration of end-stage OA was harvested, while cell yield and matrix glycosaminoglycan (GAG) content were measured. Cell morphology, proliferation, and gene expression of collagen type I, II, and X, aggrecan, matrix metalloproteinase 13 (MMP-13), and ADAMTS5 of the acquired chondrocytes were measured during subsequent in vitro culture.

RESULTSBoth the number of cells and the GAG content increased with increasing severity of OA. Cell spreading area increased and gradually showed spindle-like morphology during in vitro culture. Gene expression of collagen type II, collagen type X as well as GAG decreased with severity of cartilage degeneration, while expression of collagen type I increased. Expression of MMP-13 increased with severity of cartilage degeneration, while expression of ADAMTS-5 remained stable. Expression of collagen type II, X, GAG, and MMP-13 substantially decreased with in vitro culture. Expression of collagen type I increased with in vitro cultures, while expression of ADAMTS 5 remained stable.

CONCLUSIONSExpression of functional genes such as collagen type II and GAG decreased during severe degeneration of OA cartilage and in vitro dedifferentiation. Gene expression of collagen I and MMP-13 increased with severity of cartilage degeneration.

ADAM Proteins ; ADAMTS5 Protein ; Cartilage ; pathology ; Cell Differentiation ; genetics ; physiology ; Cells, Cultured ; Chondrocytes ; metabolism ; Collagen Type II ; genetics ; Collagen Type X ; genetics ; Glycosaminoglycans ; metabolism ; Humans ; Matrix Metalloproteinase 13 ; genetics ; Osteoarthritis ; genetics ; pathology

OBJECTIVETo study the effects of Yiqi Huayu Bushen Recipe (YHBR) and its disassembled recipes on mRNA expressions of collagen I, III, X, matrix metalloproteinase (MMPs) and tissue inhibitor of metalloproteinase (TIMP) in extracellular matrix of cervical disc in model rats of cervical vertebral disc degeneration.

METHODSThe mRNA expressions of collagens, MMP-13 and TIMP-1 were detected by RT-PCR. The strips were scanned by gel imaging system scanner, and the optical density was autocalculated by computer.

RESULTSCompared with those of the sham-operative group, the mRNA expressions of collagen I , Ill and X and MMP-13 of the model rats increased markedly (P < 0.01), which were lowered by YHBR and its disassembled recipes (P < 0.01 or P < 0.05), and the levels after YHBR treatment were significantly different to those after Western medicine treatment. However, no remarkable change was found in TIMP-1 mRNA expression in the model rats (P > 0.05).

CONCLUSIONIn the degenerated intervertebral disc the mRNA expressions of collagen I , III, X and MMP-13 increased, TIMP-1 mRNA expression decreased and the proportion of MMPs/TIMP was in imbalance. YHBR and its disassembled recipes could postpone the degeneration of intervertebral disc through regulating mRNA expressions of collagens and their correlated metabolic enzymes.

Animals ; Cervical Vertebrae ; Collagen ; biosynthesis ; genetics ; Drugs, Chinese Herbal ; pharmacology ; therapeutic use ; Extracellular Matrix ; drug effects ; metabolism ; Intervertebral Disc ; drug effects ; metabolism ; pathology ; Intervertebral Disc Displacement ; drug therapy ; genetics ; metabolism ; Male ; Matrix Metalloproteinase 13 ; biosynthesis ; genetics ; Phytotherapy ; RNA, Messenger ; biosynthesis ; genetics ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Reverse Transcriptase Polymerase Chain Reaction ; Tissue Inhibitor of Metalloproteinase-1 ; biosynthesis ; genetics