Long noncoding RNAs HAND2-AS1 ultrasound microbubbles suppress hepatocellular carcinoma progression by regulating the miR-873-5p/tissue inhibitor of matrix metalloproteinase-2 axis

BACKGROUND Increasing data indicated that long noncoding RNAs(lncRNAs)were directly or indirectly involved in the occurrence and development of tumors,including hepatocellular carcinoma(HCC).Recent studies had found that the expression of lncRNA HAND2-AS1 was downregulated in HCC tissues,but its role in HCC progression is unclear.Ultrasound targeted microbubble destruction mediated gene transfection is a new method to overexpress genes.AIM To study the role of ultrasound microbubbles(UTMBs)mediated HAND2-AS1 in the progression of HCC,in order to provide a new reference for the treatment of HCC.METHODS In vitro,we transfected HAND2-AS1 siRNA into HepG2 cells by UTMBs,and detected cell proliferation,apoptosis,invasion and epithelial-mesenchymal transition(EMT)by cell counting kit-8 assay,flow cytometry,Transwell invasion assay and Western blotting,respectively.In addition,we transfected miR-837-5p mimic into UTMBs treated cells and observed the changes of cell behavior.Next,the UTMBs treated HepG2 cells were transfected together with miR-837-5p mimic and tissue inhibitor of matrix metalloproteinase-2(TIMP2)overexpression vector,and we detected cell proliferation,apoptosis,invasion and EMT.In vivo,we established a mouse model of subcutaneous transplantation of HepG2 cells and observed the effect of HAND2-AS1 silencing on tumor formation ability.RESULTS We found that UTMBs carrying HAND2-AS1 restricted cell proliferation,invasion,and EMT,encouraged apoptosis,and HAND2-AS1 silencing eliminated the effect of UTMBs.Additionally,miR-873-5p targets the gene HAND2-AS1,which also targets the 3’UTR of TIMP2.And miR-873-5p mimic counteracted the impact of HAND2-AS1.Further,miR-873-5p mimic solely or in combination with pcDNA-TIMP2 had been transformed into HepG2 cells exposed to UTMBs.We discovered that TIMP2 reversed the effect of miR-873-5p mimic caused by the blocked signalling cascade for matrix metalloproteinase(MMP)2/MMP9.In vivo results showed that HAND2-AS1 silencing significantly inhibited tumor formation in mice.CONCLUSION LncRNA HAND2-AS1 promotes TIMP2 expression by targeting miR-873-5p to inhibit HepG2 cell growth and delay HCC progression.

Expression of matrix metalloproteinase-1 and tissue inhibitor of metalloproteinase-1 in ulcerative colitis

AIM: To examine the expression of metalloproteinase-1 (MMP-1) and tissue inhibitor of metalloproteinase-1 (TIMP-1) in the colonic mucosa of patients with ulcer- ative colitis (UC). METHODS: Reverse transcription-polymerase chain re- action (RT-PCR) and immunohistochemistry were used to study the expression of MMP-1 and TIMP-1 at both mRNA and protein levels in patients with UC and con- trols. The relationship between MMP-1 mRNA, TIMP-1 mRNA, MMP-1 mRNA/TIMP-1 mRNA ratio and the sever- ity of clinical symptoms of the patients with UC were also analyzed. RESULTS: The expression of MMP-1 mRNA and TIMP-1 mRNA in the ulcerated and inflamed colonic mucosa was signifi cantly higher than that in the non-inflamed colonic mucosa (P < 0.001), but there was no statistically signif i- cant difference in the non-inflamed colonic mucosa of UC patients and normal controls (P > 0.05). The mRNA ex- pression of MMP-1 and TIMP-1 in ulcerated colonic mu- cosa of UC patients was increased by 80-fold and 2.2-fold, respectively when compared with the normal controls. In the inflamed colonic mucosa, the increase was 30-fold and 1.6-fold, respectively. Immunohistochemical analy- sis showed that among the ulcerated, inflamed, and non-inflamed colonic mucosae of UC patients and the normal controls, the positive rate of MMP-1 expression was 87%, 87%, 40% and 35% respectively, and the positive rate of TIMP-1 expression was 89%, 89%, 80% and 75%, respectively. Furthermore, the expression of MMP-1 mRNA, TIMP-1 mRNA and the MMP-1 mRNA/ TIMP-1 mRNA ratio were correlated with the severity of clinical symptoms (P <0.05).CONCLUSION: Excessive expression of MMP-1 in the diseased colonic mucosa causes excessive hydrolysis of the extracellular matrix (ECM) and ulceration in UC pa-tients. MMP-1 mRNA, TIMP-1 mRNA and MMP-1 mRNA/ TIMP-1 mRNA ratio can be used as biomarkers to judge the severity of clinical symptoms in patients with UC. Exogenous TIMP-1 or MMP-1 inhibitor therapy is a novel treatment for patients with UC.

Levels of matrix metalloproteinase-1 and tissue inhibitors of metalloproteinase-1 in gastric cancer

AIM:To evaluate the levels of preoperative serum matrix metalloproteinase-1 (MMP-1) and tissue inhibitor of metalloproteinase-1 (TIMP-1) in gastric cancer.METHODS:One hundred gastric cancer patients who underwent gastrectomy were enrolled in this study.The serum concentrations of MMP-1 and TIMP-1 in these patients and in fifty healthy controls were determined using an enzyme-linked immunosorbent assay.RESULTS:Higher serum MMP-1 and TIMP-1 levels were observed in patients than in controls (P < 0.001).Serum MMP-1 and TIMP-1 levels were positively associated with morphological appearance,tumor size,depth of wall invasion,lymph node metastasis,liver metastasis,perineural invasion,and pathological stage.They were not significantly associated with age,gender,tumor location,or histological type.CONCLUSION:Increased MMP-1 and TIMP-1 were associated with gastric cancer.Although these markers are not good markers for diagnosis,these markers show in advanced gastric cancer.

Matrix metalloproteinase-9 and tissue inhibitor of metalloproteinase-1 expression in early focal cerebral infarction following urokinase thrombolysis in rats

Activity of matrix metalloproteinase-9 increases following cerebral ischemia/reperfusion, and is associated with cerebral microvascular permeability, blood-brain barrier destruction, inflammatory cell infiltration and brain edema. Matrix metalloproteinase-9 also likely participates in thrombolysis. A rat model of middle cerebral artery infarction was established by injecting autologous blood clots into the internal carotid artery. At 3 hours following model induction, urokinase was injected into the caudal vein. Decreased neurological severity score, reduced infarct volume, and increased expression of matrix metalloproteinase-9 and tissue inhibitor of metalloproteinase-1 were observed in the cerebral cortex 24 hours after urokinase thrombolysis. These results suggest that urokinase can suppress damage in the acute-early stage of cerebral infarction.

Expression of matrix metalloproteinase-1 and tumor necrosis factor-α in ulcerative colitis

AIM： To examine the expression of matrix metalloproteinase-1 （MMP-1） and tumor necrosis factor-α （TNF-~~） in the colon mucosa of patients with ulcerative colitis （UC）.METHODS： Reverse transcription-polymerase chain reaction （RT-PCR） and immunohistochemistry were used to examine the expression of MMP-1 and TNF-α at both mRNA and protein levels in the colon mucosa of patients with UC. Correlation between MMP-1 and TNF-α and their correlation with the severity of the disease were also analyzed statistically.RESULTS： The expression of MMP-1 and TNF-α in the ulcerated and inflamed colon mucosa of patients with UC was significantly higher than that in the non-inflamed mucosa of normal controls at both mRNA and protein levels. Furthermore, the expression of MMP-1 and TNF-α in the ulcerated area was significantly higher than that in the inflamed area of patients with UC （0.9797 ± 0.1433 vs 0.6746 ± 0.0373, 0.8669 ± 0.0746 vs 0.5227 ± 0.0435, P 〈 0.05）. There was no statistically significant difference in the non-inflamed area of normal controls. There was a significant correlation between MMP-1 and TNF-c~ expression （0.9797 ± 0.1433 vs 0.8669 ± 0.0746, P 〈 0.05）, the correlating factor was 0.877. MMP-1 and TNF-α showed a significant correlation with the severity of the disease （0.0915 ：e 0.0044 vs 0.0749 vs 0.0032, 0.0932 ± 0.0019 vs 0.0724 ± 0.0043, P 〈 0.05）, their correlating factors were 0.942 and 0.890, respectively.CONCLUSION： Excessively expressed MMP-1 directly damages the colon mucosa by degrading extracellular matrix （ECM） in patients with UC. While damaging colon mucosa, excessively expressed TNF-α stimulates MMPs secreting cells to produce more MMP-1 and aggravates the mucosa damage. MMP-1 promotes secretion of TNF-α in a positive feedback manner to cause further injury in the colon mucosa. MMP-1 and TNF-α correlate well with the severity of the disease, and therefore, can be used clinically as biological markers to judge the severity of UC.

Macrophage-derived matrix metalloproteinase-1 enhances aortic aneurysm formation in transgenic rabbits

Increased expression of matrix metalloproteinase-1(MMP-1)has been observed in the lesions of atherosclerosis and aneurysms;however,it is not fully understood whether macrophage-derived MMP-1 affects these diseases.To investigate whether macrophage-derived MMP-1 participates in the development of vascular diseases,we generated transgenic(Tg)rabbits expressing human MMP-1 in the monocyte/macrophage lineage under the control of the human scavenger receptor enhancer/promoter.Tg rabbits exhibited no visible abnormalities throughout their bodies.Western blotting analysis revealed that the amount of MMP-1 proteins in the conditioned media secreted from peritoneal macrophages of Tg rabbits was up to 3-fold higher than that in non-Tg rabbits.For the first experiment,Tg and non-Tg rabbits were fed a cholesterol diet for 16 weeks,and aortic and coronary atherosclerosis were evaluated.The gross lesion area of aortic atherosclerosis in Tg rabbits was not significantly different from that in non-Tg rabbits,but Tg rabbits had marked destruction of the medial elastic lamina of the aortic lesions on microscopic examination.For the second experiment,we generated aortic aneurysms by incubating with elastase.Compared with non-Tg rabbits,Tg rabbits exhibited a significantly greater aortic dilation.Increased macrophage-derived MMP-1 led to increased medial destruction in both aortic atherosclerosis and aneurysms.These results demonstrate that MMP-1 plays a different role in the pathogenesis of atherosclerosis and aneurysms.

Imbalance of matrix metalloproteinase-9 and matrix metalloproteinase tissue inhibitor-1 may contribute to hemorrhage in cerebellar arteriovenous malformations

In this study, we determined the expression levels of matrix metalloproteinase-2 and -9 and matrix metalloproteinase tissue inhibitor-1 and -2 in brain tissues and blood plasma of patients undergoing surgery for cerebellar arteriovenous malformations or primary epilepsy （control group）. Immunohistochemistry and enzyme-linked immunosorbent assay revealed that the expression of matrix metalloproteinase-9 and matrix metalloproteinase tissue inhibitor-1 was significantly higher in patients with cerebellar arteriovenous malformations than in patients with primary epilepsy. The ratio of matrix metalloproteinase-9 to matrix metalloproteinase tissue inhibitor-1 was significantly higher in patients with hemorrhagic cerebellar arteriovenous malformations compared with those with non-hemorrhagic malformations. Matrix metalloproteinase-2 and matrix metalloproteinase tissue inhibitor-2 levels were not significantly changed. These findings indicate that an imbalance of matrix metalloproteinase-9 and matrix metalloproteinase tissue inhibitor-I, resulting in a relative overabundance of matrix metalloproteinase-9, might be the underlying mechanism of hemorrhage of cerebellar arteriovenous malformations.

Expressions of matrix metalloproteinase-9 and tissue inhibitor of metalloproteinase-1 in malignant peripheral nerve sheath tumor

BACKGROUND： Matrix metalloproteinase-9 （MMP-9） can degrade collagen IV （the main structural ingredient of basilar membrane）, and it also plays an important role in tumor vascularization, tumor cell progression, formation of metastatic focus, etc. Tissue inhibitor of metalloproteinase-1 （T1MP-1） can bind with MMP-9 to form 1 ： 1 compound and inhibit its activity, and can negatively regulate the tumor progression and metastasis. OBJECTIVE： To analyze the relationship of MMP-9 and T1MP-1 expressions with the pathological grade, metastasis and prognosis of malignant peripheral nerve sheath tumor （MPNST）. DESIGN： An observational comparative experiment. SETTING： Heze Medical College. PARTICIPANTS： Fifty-eight surgical pathological samples, which were clearly diagnosed to be MPNST, were collected from the pathological laboratory archives in the Department of Pathology, Heze Municipal Hospital from January 1988 to December 2003. The MPNST pathological types were common tumor in 53 cases, malignant triton tumor in 2 cases, epithelial MPNST in 2 cases and MPNST with gland differentiation in 1 case. The pathological grade was grade 1 in 11 cases, grade 2 in 24 cases and grade 3 in 23 cases. Besides, the resected tumor samples of 20 patients with benign peripheral nerve tumor （10 cases of nerve sheath tumor and 10 cases of neurofibromatosis） and the normal peripheral nerves （by-products of some surgeries） of 5 patients were also collected. The samples were used with the approval of the patients. Rat-anti-human MMP-9, TIMP-1 monoclonal antibody and S-P kit were purchased from Fuzhou Maixin Biotechnology, Co.,Ltd. METHODS： The documented paraffin blocks were again prepared to sections of 5 lJ m. The expressions of MMP-9 and TIMP-1 in the samples were detected with mmunohistochemical S-P method. The relationships of the MPNST severity, recurrence, metastasis and survival rate with the expressions of MMP-9 and TIMP-1 were analyzed. MAIN OUTCOME MEASURES： Relationships of MMP-9 and TIMP-1 expressions with the MPNST severity and prognosis. RESULTS： ①Expressions of MMP-9 and TIMP-1 in three tissues： MMP-9 and TIMP-1 stainings were mainly observed in cytoplasm. Among the 58 MPNST patients, the MMP-9 expression was significantly higher than those in normal peripheral nerve and benign tumor （P 〈 0.05）, while the TIMP-1 expression in MPNST was lower than those in normal peripheral nerve and benign tumor （P 〈 0.05）. ②Relationship of MMP-9 and TIMP-1 expressions with the severity and prognosis of MPNST： The expressions of both proteins were observed in the four subtypes. The positive expression of MMP-9 in the MPNST patients of grades 2 - 3 was significantly higher than that in the MPNST patients of grade 1 （P 〈 0.05）, while the expression of MMP-9 was significantly lower than that in the MPNST patients of grade 1 （P 〈 0.05）. The metastatic rate was positively correlated with MMP-9 expression （r =1.696, P 〈 0.05）, but negatively correlated with TIMP-1 expression （r = - 2.125, P 〈 0.05）. CONCLUSION： MMP-9 and TIMP-1 are associated with MPNST pathological grades and metastasis, and can be used as the indicators for judging the severity and orognosis of MPNST.

Rosiglitazone uppresses lipopolysaccharide-induced matrix metalloproteinase-2 activity in rat aortic endothelial cells via rasMEK1/2 signaling

ix metalloproteinase(MMPs) plays a key role in the pathogenesis of chronic inflammatory disease,such as atherosclerosis.Among MMPs,MMP-2 is regarded as a major proteinase in atherosclerotic plaque lesions.Peroxisome proliferator activated receptor-gamma(PPARg) ameliorates oxidative stress and the inflammatory response.The aim of the present study was to evaluate the effect of Rosiglitazone on Lipopolysaccharide(LPS)-induced MMP-2 activation as well as its possible mechanism.LPS-induced MMP-2 activity was inhibited by Rosiglitazone(PPARg agonist) in the rat aortic endothelial cells(RAEC).LPS-induced MMP-2 activation was diminished no matter exposure to NF-kB Activation Inhibitor II(JSH-23)or Ras inhibitor,farnesylthiosalicylic acid(FTS). Further study shows that LPS-induced activation of Phospho-Rho A and Phospho-MEKl/2 were significantly inhibited by Rosiglitazone.The activation of NF-kB p65 in the nuclear extract of cells was also significantly suppressed by Rosiglitazone, moreover,the expression of NF-κB p65 was partly activated by GW9662(PPARg antagonist).NF-kB DNA binding activity was also demolished by Rosiglitazone.In summary,our data showed that PPARg agonist,Rosiglitazone suppresses LPS-activated MMP-2 secretion via Ras-MEK1/2 signaling pathways and NF-kB activation.PPARg agonist and Ras-MEK1/2 pathway may be another potential therapeutic target for the disease induced by chronic inflammation.

MMP-1 Over-expression Promotes Malignancy and Stem-Like Properties of Human Osteosarcoma MG-63 Cells In Vitro

Osteosarcoma is the most common primary malignant bone tumor in childhood,and it maintains a high level of recurrence.Matrix metalloproteinase-1(MMP-1)was found to contribute to cancer progression.The present study was to investigate the in vitro effects of MMP-1 over-expression on the proliferation,invasion,metastasis and stem-like properties of osteosarcoma MG-63 cells.The MG-63 cells were cultured and had a full length MMP-1 cDNA inserted by the tentiviral vector (MG-63^MMP-1+).MG-63 negative control and MG-63 blank control groups were established as well.MMP-1 expression was detected in MG-63^MMP-1+,MG-63 negative control and MG-63 blank control cells using qPCR,Western blotting and immunofluorescence after 24h of culture. The cell proliferation assay was performed with a camera attached to a bioreactor,which was programmed to photograph five regions of each well every 10 min over a period of 48 h.The cell invasion assay was conducted with Matrigel to assess the invasive potential,of MG-63 cells over 24h,the qPCR analysis to measure stem cell markers,including Oct4, Sox-2,Nanog,and Pax-7,and Western blot analysis to detect invasive and metastatic potential markers TIMP-1,VEGF and BMP2/4,after 24h of culture.Immunofluorescence was used to investigate the presence of the stem cell marker Pax-7 after 24-h culture. The results showed that over-expression of MMP-1 after transfection could significantly increase minor cell proliferation and invasion (P<0.05,MG-63^MMP-1+ versus controls).Pax-7 was highly expressed in MG-63^MMP-1+ cells,with no significant changes of Oct-4,Sox-2, and Nanog observed (P<0.05).MG-63^MMP-1+ cells showed higher expression of VEGF and BMP 2/4 proteins and lower expression of TIMP-1 protein than controls (P<0.05).It was concluded that MMP-1 over-expression in MG-63 cells contributed to the proliferation, invasion,metastasis and stem-like properties of osteosarcoma cells.Future studies should focus on in vivo effects of MMP-1 over-expression and the application of MMP-1 and Pax-7 inhibition in vivo to osteosarcoma theraoies.

Multifactor Regulation on Expressions of MMP-9 and TIMP-1 in Endometrial Stromal Cells

Objective To investigate the regulatory effect of multifactor on the matrix metalloproteinases-9 （MMP-9） and the tissue inhibitor of metalloproteinase-1 （TIMP-1） in endometrial stromal cells. Methods The endometrial stromal cells separated from the proliferative endometrial tissues were incubated with medium alone, 17-β estradiol （E2,10^-8 mol/L）, medroxyprogesterone acetate （MPA, 10^-6 mol/L）, E2（10^-8 mol/L）＋MPA （10^-6 mol/L）, E2 （10^-8 mol/L）＋MPA （10^-6 mol/L）＋RU486 （10^-5 mol/L） or HB-EGF （10 ng/ml） for 48 h respectively. The expressions of MMP-9 and TIMP-1 were detected by in situ hybridization, immunocytochemistry, reverse transcriptase-polymerase chain reaction （RT-PCR） and Western blotting. Results Compared with control group [mRNA, 0. 729 ± 0. 090 （MMP-9） and 1.056± 0.154 （TIMP-1）; protein, 0.545 ±0.086 （MMP-9） and 0.745 ±0.154 （TIMP-1）], expressions of MMP-9 and TIMP-1 in E2 alone, progestin alone or E2 combined with progestin group were respectively：mRNA, 0.413 ± 0.069, 0.402 ± 0.073 and 0.407 ± 0.039; 0.487 ± 0.093, 0.503 ± 0.093 and 0.468 ± 0.075：protein, 0.294 ± 0.076, 0.331 ±0.064 and 0.265 ±0.049; 0.425 ±0.085, 0.397 ±0.065 and 0.435 ± 0.099. RU486 weakened the expression level of down-regulation, while HB-EGF elevated the level of MMP-9 and TIMP-1 after 48 h treatment （mRNA, 0.955 ± 0.068 and 1.396 ± 0.238; protein, 0. 780 ± 0.109 and 0.985 ± 0.165）. Conclusions 1） Both E2 and progestin can down-regulate the expressions of MMP-9 and TIMP-1 in endometrial stromal cells, but RU486 can inhibit the effect. 2） HB-EGF can elevate the level of MMP-9 and TIMP-1. 3） E2, progestin and HB-EGF have effect on the ratio of MM-P/TIMP-1.

Increased Expression and Activity of MMP-9 in C-reactive Protein-induced Human THP-1 Mononuclear Cells Is Related to Activation of Nuclear Factor Kappa-B

The relation between the expression and activity of MMP-9 in C-reactive protein （CRP）-induced human THP-1 mononuclear cells and the activation of nuclear factor kappa-B （NF-κB） was studied to investigate the possible role of CRP in plaque destabilization. Human THP-1 cells were incubated in the presence of CRP at 0 （control group）, 25, 50 and 100 μg/mL （CRP groups） for 24 h. In PDTC （a specific NF-κB inhibitor） group, the cells were pre-treated with PDTC at 10 μmol/L and then with 100 μg/mL CRP. The conditioned media （CM） and human THP-1 cells in different groups were harvested. MMP-9 expression in CM and human THP-1 cells was measured by ELISA and Western blotting. MMP-9 activity was assessed by fluorogenic substrates. The expression of NF-κB inhibitor α （IκB-α） and NF-κB p65 was detected by Western blotting and ELISA respectively. The results showed that CRP increased the expression and activity of MMP-9 in a dose-dependent manner in the human THP-1 cells. Western blotting revealed that IiB-α expression was decreased in the cells with the concentrations of CRP and ELISA demonstrated that NF-κB p65 expression in the CRP-induced cells was increased. After pre-treatment of the cells with PDTC at 10 μmol/L, the decrease in IκB-α expression and the increase in NF-κB p65 expression in the CRP-induced cells were inhibited, and the expression and activity of MMP-9 were lowered too. It is concluded that increased expression and activity of MMP-9 in CRP-induced human THP-1 cells may be associated with activation of NF-κB. Down-regulation of the expression and activity of MMP-9 may be a new treatment alternative for plaque stabilization by inhibiting the NF-κB activation.

Association and prognostic significance of alpha-L-fucosidase-1 and matrix metalloproteinase 9 expression in esophageal squamous cell carcinoma

BACKGROUND Alpha-L-fucosidase-1(FUCA1)has been demonstrated to play opposing regulatory roles in adenocarcinoma and non-adenocarcinoma.Moreover,recent studies reported that FUCA1 could decrease the invasion capability by downregulating matrix metalloproteinase 9(MMP-9)expression.However,the potential role and prognostic significance of FUCA1 in esophageal squamous cell carcinoma(ESCC)have not yet been explored.AIM To evaluate the status,association,and prognostic value of FUCA1 and MMP-9 expression in ESCC.METHODS Patients who underwent esophagectomy for ESCC between January 1,2014,and December 31,2014 at Sun Yat-Sen University Cancer Center were enrolled.The expression status of FUCA1 and MMP-9 in cancerous tissues was detected using immunohistochemistry.In addition,the expression profiles of the FUCA1 and MMP-9 genes in non-metastatic ESCC were extracted from The Cancer Genome Atlas(TCGA)database.RESULTS High expression of FUCA1 and MMP-9 was found in 90 patients(75.6%)and 62 patients(52.1%),respectively.In the high FUCA1 expression group,the constituent ratios of patients with stage III disease(61.1%vs 37.9%,P=0.029),lymphatic invasion(62.2%vs 31.0%,P=0.003),and high MMP-9 expression(60.0%vs 27.6%,P=0.002)were significantly higher than those in the low FUCA1 expression group.In Kaplan-Meier univariate analysis,advanced tumor-nodemetastasis stage(III,P=0.001),positive regional lymph node metastasis(N+,P=0.002),high FUCA1 expression(P=0.001),and high MMP-9 expression(P=0.002)were potential predictors of shorter overall survival(OS),which was similar to the results analyzed based on the TCGA database.Further Cox multivariate regression analyses still demonstrated that FUCA1 and MMP-9 expression levels were independent prognostic factors of OS[hazard ratio(HR):0.484,95%confidence interval(CI):0.239-0.979;P=0.044;and HR:0.591,95%CI:0.359-0.973,P=0.039,respectively].CONCLUSION FUCA1 cooperation with MMP-9 may have a major role in affecting the ESCC invasion and metastatic capability,and serve as a valuable prognostic biomarker in ESCC.

The serum levels of MMP-9, sICAM-1, CRP and WBC increased in patients with acute coronary syndrome

Objective：To evaluate the association of the peripheral levels of the defined inflammatory markers with different types of acute coronary syndrome （ACS） and stable angina, and the role inflammation played in the pathogenesis of ACS. Methods ：For understanding the variation of serum concentrations of matrix metalloproteinase-9 （MMP-9）, soluble intercellular adhesion molecule-1 （sICAM-1） ,C-reactive protein （CRP）, and white blood cell count （WBC） and their association with ACS, 90 patients with coronary heart disease （CHD） and 30 healthy volunteers were recruited. The enrolled people were assigned into four equal groups, including acute myocardial infarction （AMI） group, unstable angina pectoris（UAP） group, stable angina pectoris（SAP） group and healthy control group. The serum levels of MMP-9 and sICAM-1 were measured with ELISA kits, CRP were measured with im munoturbidimetric assay, and WBC number were assessed all before any treatment was administrated. Results： （1）The serum levels of MMP-9 ,sICAM-1 ,CRP and WBC in the patients with ACS were significantly higher than those in the control group （P 〈 0.01 ）. （2）Compared with control group, patients with SAP only had higher serum level of sICAM-1 （P 〈 0.01 ）. While the levels of MMP-9, CRP, and WBC had no significant difference between them （P 〉 0.05 all）. （3）Significant positive correlation between the serum levels of MMP-9 and sICAM-1 and CRP and WBC all were observed in the patients with ACS （P 〈 0.05）. Conclusion： The elevation of serum concentrations of inflammatory markers including MMP-9, slCAM-1, CRP and WBC were associated with initiation and progression of ACS, and they may help predicting cardiovascular events.

携TIMP-1基因RNAi慢病毒载体感染大鼠HSC-T6细胞抑制目的基因表达

目的验证构建成功的携基质金属蛋白酶组织抑制因子-1(TIMP-1)基因特异性小干扰RNA的慢病毒载体对目的基因表达的抑制效应。方法以慢病毒质粒感染HSC-T6细胞,在感染6天和8天后,观察慢病毒感染效率;采用定量PCR法检测TIMP-1 mRNA水平变化;采用免疫印迹法检测TIMP-1蛋白表达变化。结果在慢病毒载体感染6天时,TIMP-1 mRNA水平较正常对照组下降约0.668倍[2-ΔΔCt为(0.668±0.046)],下降程度明显高于阴性对照组[2-ΔΔCt为(1.001±0.041),(P<0.05)];在慢病毒感染6天和8天时,RNAi组对TIMP-1蛋白表达具有明显的抑制作用,且以感染第6天为显著。结论构建成功的携TIMP-1基因特异性RNAi慢病毒载体能有效感染HSC-T6细胞,并抑制目的基因的表达。

KISS-1 inhibits the proliferation and invasion of gastric carcinoma cells

AIM:To investigate the function of the KISS-1 gene in gastric carcinoma cells and to explore its potential mechanism.METHODS:A KISS-1 eukaryotic expression vector was constructed and transfected into BGC-823 cells.Resistant clones were obtained through G418 selection.reverse transcription-polymerase chain reaction and western blotting were used to detect KISS-1 and matrix metalloproteinase-9(MMP-9)expression in transfected cells.The growth of transfected cells was investigated by 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide(MTT)proliferation assays,and the cells'invasive potential was analyzed by basement membrane(Matrigel)invasion assays.The anti-tumor effects of KISS-1 were tested in vivo using allografts in nude mice.RESULTS:The expression level of KISS-1 mRNA andprotein in BGC-823/KISS-1 transfected cells were significantly higher than in BGC-823/pcDNA3.1 transfected cells(P<0.05)or the parental BGC-823 cell line(P< 0.05).The expression level of MMP-9 mRNA and protein in BGC-823/KISS-1 were significantly less than in BGC-823/pcDNA3.1(P<0.05)or BGC-823 cells(P< 0.05).MTT growth assays show the proliferation of BGC-823/KISS-1 cells at 48 h(0.642±0.130)and 72 h(0.530±0.164)were significantly reduced compared to BGC-823/pcDNA3.1(0.750±0.163,0.645±0.140)(P<0.05)and BGC-823 cells(0.782±0.137,0.685± 0.111)(P<0.05).Invasion assays indicate the invasive potential of BGC-823/KISS-1 cells(16.50±14.88)is significantly reduced compared to BGC-823/pcDNA3.1(20.22±14.87)(P<0.05)and BGC-823 cells after 24 h(22.12±16.12)(P<0.05).In vivo studies demonstrate the rate of pcDNA3.1-KISS-1 tumor growth is significantly slower than pcDNA3.1 and control cell tumor growth in nude mice.Furthermore,tumor volume of pcDNA3.1-KISS-1 tumors(939.38±82.08 mm3)was significantly less than pcDNA3.1(1250.46±44.36 mm3) and control tumors(1284.36±55.26 mm3)(P<0.05).Moreover,the tumor mass of pcDNA3.1-KISS-1 tumors(0.494±0.84 g)was significantly less than pcDNA3.1(0.668±0.55 g)and control tumors(0.682±0.38 g)(P <0.05).CONCLUSION:KISS-1 may inhibit the proliferation and invasion of gastric carcinoma cells in vitro and in vivo through the downregulation of MMP-9.

S100A4 siRNA Inhibits Human Pancreatic Cancer Cell Invasion In Vitro

Objective Pancreatic cancer is one of the most deadly cancers, which is characterized by its high metastatic potential. S100A4 is a major prometastatic protein involved in tumor invasion and metastasis which precise role in pancreatic cancer has not been fully investigated. We knocked down the S100A4 gene in the Bxpc-3 pancreatic cancer cell line via RNA interference to study the changes in cell behavior. Methods Real-time polymerase chain reaction and western blotting were used to detect mRNA and protein expression levels of S100A4, matrix metalloproteinase （MMP）-2, E-cadherin and thrombospondin （TSP）-I. Transwell chambers were used to detect the migration and invasion abilities; a cell adhesion assay was used to detect adhesion ability; colony forming efficiency was used to detect cell proliferation; flow cytometry was used to detect apoptosis. Results S100A4 mRNA expression was reduced to 17% after transfection with SIOOA4-siRNA, and protein expression had a similar trend, mRNA and protein expression of MMP-2 was reduced and that of E-cadherin and TSP-1 was elevated, indicating that S100A4 affects their expression. S100A4-silenced cells exhibited a marked decrease in migration and invasiveness and increased adhesion, whereas overall proliferation and apoptosis were not overtly altered. Conclusion S100A4 and its downstream factors play important roles in pancreatic cancer invasion, and silencing AIOOA4 can significantly contain the invasiveness of pancreatic cancer.

Therapeutic effect of interleukin-10 on CCI_4-induced hepatic fibrosis in rats

AIM： To study the therapeutic effect of exogenous interleuldn-10 on CCl4-induced hepatic fibrosis in rats and its passible mechanisms. METHODS： Fourty-seven SD rats were randomly divided into control group （group N） and CCl4-induced hepatic fibrosis model group （group C）. After CCl4 was given for 9 wk, the model group was divided into three groups. Rats in group H were put to death immediately, rats in group T were treated with IL-10 for another three wk and then put to death, rats in group R recovered after three weeks and were then killed. The degree of hepatic fibrosis was measured by HE staining and histological activity index （HAI）. Histological activity index （HAI）, change of collagen types Ⅰ and Ⅲ were measured by Picrosirius staining. The expression of TNF-α, HHP-2 and TIMP-1 in liver tissue was measured by S-P immunohis tochemistry.RESULTS： CCl4- induced experimental rat hepatic fibrosis model was established successfully. The degree of hepatic fibrosis was markedly lower in group T than in groups H and R, and there was no difference between the two groups. The expression of collagen types I and III was significantly suppressed in group T and was slightly suppressed in groups H and R. The positive levels of TNF-α, HHP-2 and TIHP-1 in group H increased significantly compared to those in group N （P〈0.01）. The positive signals decreased significantly in groups T and R （P〈0.01）, but positive score was significantly lower in group T than in group R （P〈 0.01）. CONCLUS10N： Exogenous IL-10 can reverse CCl4-induced hepatic fibrosis in rats. IL-10 may exert its reversible effects on hepatic fibrosis by blocking CCl4-induced inflammation, inhibiting expression of HHP-2 and TIMP-1 and promoting resolution of collagen types Ⅰ and Ⅲ.

Increased expression of matrix metalloproteinase-9 associated with gastric ulcer recurrence

AIM: To compare matrix metalloproteinase (MMP)-9 and tissue inhibitor of metalloproteinase (TIMP)-1 in gastric ulcer (GU) and chronic superficial gastritis (CSG). METHODS: This study enrolled 63 patients with GU and 25 patients with CSG. During upper gastroduodenal endoscopy, we took samples of gastric mucosa from the antrum and ulcer site from patients with GU, and samples of antral mucosa from patients with CSG. Mucosal biopsy tissues were cultured for 24 h, and the culture supernatant was measured for levels of MMP-9 and TIMP-1. After receiving eradication therapy for Helicobacter pylori (H. pylori ) and 8 wk proton-pump inhibitor therapy for GU, follow-up endoscopy examination was performed after 6 mo and whenever severe symptoms occurred. RESULTS: Levels of MMP-9 and TIMP-1 at the ulcer site or in the antrum were significantly higher in GU than CSG patients. MMP-9 levels at the ulcer site were significantly higher than in the antrum in GU patients, and had a significantly positive correlation with TIMP-1. MMP-9 levels were significantly higher in H. pylori -positive than H. pylori -negative GU and CSG patients. Levels of MMP-9 or TIMP-1 at the ulcer site were associated with the histological severity of activity and inflammation. About 57 GU patients were followed up, and seven had GU recurrence. H. pyloriinfection and MMP-9 levels were risk factors for the recurrence of GU adjusted for age and sex by multiple logistic regression analysis. CONCLUSION: MMP-9 may perform an important function in gastric ulcer formation and recurrence.