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.

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.

Effects of benazepril on renal function and kidney expression of matrix metalloproteinase-2 and tissue inhibitor of metalloproteinase-2 in diabetic rats

Background Excessive deposition of extraceUular matrix （ECM） in the kidney is the hallmark of diabetic nephropathy. Increased matrix synthesis has been well documented but the effects of diabetes on degradative pathways, particularly in the in vivo setting. The renal protective effect of these pathways on matrix accumulation has not been fully elucidated. The present study was understaken to investigate the activity of matrix metalloproteinase-2 （MMP-2）, the expression of MMP-2 and tissue inhibitor of metalloproteinase-2 （TIMP-2） in kidney tissues of diabetic rats, and to explore the degradative pathway of type Ⅳ collagen （Ⅳ-C） and the renal protective effects of ACE inhibition- benazepril.

Effects of irbesartan on the expression of matrix metalloproteinase-2/ tissue inhibitor of metalloproteinase-2 in streptozotocin-induced diabetic rat kidney

Glomerular hypertrophy and progressive expansion of extracelluar matrix (ECM) have been regarded as the early feature of diabetic nephropathy (DN), which leads to accumulation of ECM and thickening of glomerular basement membrane, and subsequently induces renal fibrosis. Both increasing synthesis and decreasing degradation of matrix components are responsible for matrix accumulation. The later may play more important role in DN that involves a number of matrix metalloproteinases (MMPs). MMPs are a family of proteolytic enzymes whose activity is tightly regulated by tissue inhibitor of metalloproteinases (TIMPs), and the MMP/TIMP ratio is critical for coordinating matrix production and degradation. 1 Recently, considerable evidence suggests that the intrarenal renin-angiotensin system plays an important role in the development of DN. 2 Blockade of the renin-angiotensin system (RAS) by angiotensin-coverting enzyme inhibitor (ACEI) or angiotensin Ⅱ receptor antagonist (AIIRA) delays the progression of renal injury associated with diabetes. 3 AIIRA has been regarded as the first line choice for DN therapy. However, the potential mechanism for AIIRA in the ECM degradative pathway has not been fully elucidated. The present study is to investigate the effect of Irbesartan (Irb), a newly developed AIIRA, on renal expression of MMP-2 and TIMP-2 in streptozotocin (STZ)-induced diabetic rats.

Correlations between papillary thyroid cancer and peripheral blood levels of matrix metalloproteinase-2, matrix metalloproteinase-9, tissue inhibitor of metalloproteinase-1, and tissue inhibitor of metalloproteinase-2

Background The relationship between the presence of metalloproteinases and thyroid cancer remains unknown, and many controversies still exist in this field. The objective of this study was to investigate the correlations between papillary thyroid cancer and peripheral blood levels of matrix metalloproteinase-2, matrix metalloproteinase-9, tissue inhibitor of metalloproteinase-1, and tissue inhibitor of metall0Proteinase-2. Methods The correlations were studied bY detecting the levels of matrix metalloproteinase-2, matrix metalloproteinase-9, tissue inhibitor of metalloProteinase-1, and tissue inhibitor of metalloproteinase-2 by enzyme-linked immunosorbant assay and reverse-transcription polymerase chain reaction in the peripheral blood of 30 patients with papillary thyroid carcinoma, 27 patients with benign thyroid disease, and 25 hea !hy vo!unteers. Results The leve!s of matrix metalloproteinase-2, matrix metalloproteinase-9, tissue inhibitor of metalloproteinase-1, and tissue inhibitor of metalloproteinase-2 in the peripheral blood of patients with papillary thyroid carcinoma were significantly higher than those in the peripheral blood of patients with benign thyroid disease and healthy volunteers （P 〈0.05）. However, there were no significant differences between patients with benign thyroid disease and healthy volunteers （P 〉0.05）. The accuracy of detection by both enzyme-linked immunosorbant assay and reverse-transcription polymerase chain reaction in the papillary thyroid cancer group was 83.33%. Conclusions The levels of matrix metalloproteinase-2, matrix metalloproteinase-9, tissue inhibitor of metalloproteinase-1, and tissue inhibitor of metalloproteinase-2 in the peripheral blood are helpful in identifying thyroid carcinoma and aid in preoperative assessment.

Duodenal-jejunal bypass surgery on type 2 diabetic rats reduces the expression of matrix metalloproteinase-9 and tissue inhibitor of matrix metalloproteinase-1 in the thoracic aorta

Background Bariatric surgery offers a productive resolution of type 2 diabetes mellitus （T2DM）.The development of T2DM vasculopathy is due to chronic inflammation,which increases matrix metalloproteinase-9 （MMP-9） and tissue inhibitor of matrix metalloproteinase-1 （TIMP-1） expression.This study sought to examine MMP-9 and TIMP-1 expression in the thoracic aorta after duodenal-jejunal bypass （DJB） surgery on a T2DM rat model induced by a high-fat diet and low dose streptozotocin （STZ）.Methods Twenty-one T2DM Wistar rats induced by high-fat diet and low dose STZ were randomly divided into DJB and sham duodenal-jejunal bypass （S-DJB） groups.Ten Wistar rats were fed a normal diet as a control.Recovery of gastrointestinal function post-operation and resumption of a normal diet completed the experiment.Body weight,blood glucose,blood lipid levels,and MMP-9 and TIMP-1 expression levels in aortic endothelial cells were measured throughout.Results DJB rats showed significant weight loss 2 weeks post-operation compared with S-DJB rats.After surgery,DJB rats showed significant improvement and steady glycemic control with improved insulin sensitivity and glucose tolerance.They also exhibited improved lipid metabolism with a decrease in fasting free fatty acids （FFAs） and triglycerides （all P ＜0.05）.Immunohistochemistry showed decreased MMP-9 and TIMP-1 expression 12 weeks after surgery （P ＜ 0.01）.Conclusions DJB surgery on an induced T2DM rat model improves blood glucose levels and lipids,following a high-fat diet and low dose STZ treatment.In addition,DJB decreased MMP-9 and TIMP-1 expression in vascular endothelial cells,which may play an important role in delaying the development of T2DM vascular disease.

Cloning and expressing a recombinant human tissue inhibitor of metalloproteinase-2(TIMP-2)in methylotrophic yeast Pichia pastoris and its characterizations

To obtain human tissue inhibitor of metalloproteinase-2(TIMP-2)cDNA and the secretory expression of TIMP-2 gene in Pichia pastoris,we designed and synthesized a 618 base pairs artificial gene coding for the TIMP-2 with a computer-aided design method using a standard chemical synthesis technique,which was composed of frequently used codons in the highly expressed Pichia pastoris genes.Then the synthetic gene encoding TIMP-2 was checked by means of dideoxynucleotide sequencing.The verified gene of TIMP2 was cloned to the Escherichia coli-yeast shuttle vector of pPIC9 to construct a recombinant plasmid pPIC9-T2.The plasmid was transformed into GS115 cells of the methylotrophic yeast,Pichia pastoris by electroporation,and we got the expression cell through phenotype selection and induction with methanol.Separation,purification,and bioactivity analysis of the expressed products were performed.

肝纤维化时基质金属蛋白酶-2及其组织抑制因子的表达

目的 :了解基质金属蛋白酶 - 2 (MMP - 2 )及其组织抑制因子 (TIMP - 2 )在不同病因致人肝纤维化不同病理阶段中的变化 ,分析MMP - 2 /TIMP - 2与肝病理损害的关系。方法 :用免疫组织化学 (LsAB法 ) ,在正常肝组织、慢活肝伴早期肝纤维化、肝癌伴早期肝纤维化、胆汁性肝硬化、坏死后性肝硬化病例中MMP - 2及TIMP - 2的联合检测、行半定量研究。结果 :正常肝组织MMP - 2 /TIMP - 2的比值高于病肝组 ;早期肝纤维化组MMP - 2 /TIMP - 2比值高于肝硬化组 ;早期肝纤维化组组间、肝硬化组组间MMP - 2 /TIMP - 2比值无显著差异。结论 :早期肝纤维化MMP - 2表达相对增高 ,对肝纤维化的始动和进展起重要作用 ,肝硬化时TIMP - 2表达明显增高 ,抑制MMP - 2 ,细胞外基质 (ECM)降解障碍而大量沉积 。

Atorvastatin reduces myocardial fibrosis in a rat model with post- myocardial infarction heart failure by increasing the matrix metaHoproteinase-2/tissue matrix metalloproteinase inhibitor-2 ratio

Background The cholesterol-lowering statin drugs have some non-lipid-lowering effects, such as inhibiting myocardial remodeling. However, the underlying mechanism is still unclear. Methods The left anterior descending coronary artery was ligated to establish a rat model of heart failure, and the rats were divided into a sham operation （SO） group, myocardial infarction model （MI） group, and MI-atorvastatin group. Changes in hemodynamic parameters were recorded after the final drug administration. Histological diagnosis was made by reviewing hematoxylin and eosin （HE） stained tissue. Real-time quantitative polymerase chain reaction （PCR） was performed to determine the expressions of type I and type III collagen, matrix metalloproteinase-2 （MMP-2）, and tissue matrix metalloproteinase inhibitor-2 （TIMP-2）. Further, primary rat cardiac fibroblasts were cultured and the MTT assay was performed to determine the effect of atorvastatin on cardiac fibroblast proliferation. Results The model of heart failure was established and the results of HE staining and Masson＇s trichrome staining revealed that the rats in the heart failure group showed obvious hyperplasia of fibrotic tissue, which was significantly reduced in the atorvastatin group. Real-time quantitative PCR showed that the MI group showed a significantly increased expression of type I and type III coltagen, MMP-2, and TIMP-2, but a significantly reduced MMP-2/T＇IMP- 2 ratio. Compared with the MI group, the atorvastatin group showed significantly reduced expression of type I and III collagen, unchanged expression of MMP-2, significantly reduced expression of TIMP-2, and an increased MMP-2/ TIMP-2 ratio. We further found that atorvastatin significantly inhibited the Ang II-induced fibroblast proliferation and the expression of type I and type III collagen in cardiac fibroblasts while increasing the MMP-2/TIMP-2 ratio. Conclusions These data suggest that atorvastatin can inhibit cardiac fibroblast proliferation and enhance collagen degradation by increasing the MMP-2/TIMP-2 ratio, thereby inhibiting the formation of myocardial fibrosis in rats with heart failure after myocardial infarction.

Lichong decoction reduces Matrix Metalloproteinases-2 expression but increases Tissue Inhibitors of Matrix Metalloproteinases-2 ex-pression in a rat model of uterine leiomyoma

OBJECTIVE:To study the effect of Lichong decoction(LD) on expression of matrix metalloproteinase-2(MMP-2) and metalloproteinase-2(TIMP-2) in a rat model of uterine leiomyoma(UL).METHODS:UL was induced in rats using exogenous estrogen and progesterone.LD was administered(p.o.) for 4 weeks,and mifepristone(RU-486)used as a control.To observe the effect of LD on the uterine coefficient and uterine transverse diameter,a radioimmunoassay method was used to detect serum levels of sex hormones.Light microscopic analyses of pathologic changes in the tissues of UL rats were evaluated.Expression of the proteins of matrix metalloproteinases(MMPs) and tissue inhibitors of metalloproteinases(TIMPs) in uterine tissues was assessed by immunohistochemical staining and western blotting.RESULTS:A UL model in rats was established successfully.LD reduced uterine weight,uterine coefficient,and uterine transverse diameter compared with untreated controls.LD reduced levels of estradiol,progesterone,follicle-stimulating hormone,and luteinizing hormone in our UL models.LD improved the pathologic condition of uterine muscle.Expression of MMP-2 protein decreased to varying extents in LD-treated groups,but TIMP-2 levels were enhanced.LD appears to reduce MMP-2 expression and increase TIMP-2 expression in UL tissue.CONCLUSION:These data suggest that the mechanism of action of LD on ULs may involve reduction of MMP-2 expression and increase in TIMP-2 expression in rats.

Inhibition of Metastatic Progression of SSTR2 Gene Transfection Mediated by Adenovirus in Human Pancreatic Carcinoma Cells

The inhibition of metastatic progression of Somatostatin receptor type 2 （SSTR2） gene transfection mediated by adenovirus in human pancreatic carcinoma cells and the mechanisms involved in this effect were studied. The full-length human SSTR2 cDNA was introduced into the pancreatic cancer cell line BXPC-3 by adenovirus-mediated transfection. Stable expression of mRNAs and protein of SSTR2 was detected by RT-PCR and Western-blot. The Matrigel-coated Transwell was used to detect the migratory and invasive ability of SSTR2-expressing cells, Adv-GFP control cells and mock control cells. Furthermore, the expression of matrix metalloproteinase-2 （MMP-2） and tissue inhibitor of metalloproteinase-2 （TIMP-2） was detected by RT-PCR in these cells. The stable expression of SSTR2 was detected in BXPC-3 transfected by Adv-GFP-SSTR2. A dramatic decrease of BXPC-3 expressing sst2 cells migrating through a Matrigel-coated filter was observed, as compared with Adv-GFP control and mock control cells （P〈0. 01）. Moreover, the expression of MMP-2 mRNA was significantly reduced in the SSTR2-expressing cells and converse- ly the expression of TIMP-2 mRNA was significantly increased in the SSTR2-expressing cells when compared with the Adv-GFP control and mock control （P〈0. 01）. The expression of reintroduced human SSTR2 gene in BXPC-3 cells by Adv-GFP-SSTR2 had the anti-migratory and anti-invasive effects, and the mechanisms involved in this effect may be due to the down-regulated expression of MMP-2 and up-regulated expression of TIMP-2.

The ratio of MMP-2 to TIMP-2 in hilar cholangiocarcinoma:a semi-quantitative study

BACKGROUND: Hilar cholangiocarcinoma is associated with low resectability and poor survival. The aim of this study was to evaluate the roles of matrix metalloproteinases- 2 (MMP-2 ) and its tissue inhibitor of metalloproteinase-2 (TIMP-2) in tumor invasion or as a prognostic factor in patients with human hilar cholangiocarcinoma. METHODS: The expressions of MMP-2 and TIMP-2 were investigated in patients. Paraffinized tissue sections ob- tained from 50 patients with human hilar cholangiocarcino- ma were analysed. The expressions of MMP-2 and TIMP-2 were examined immunohistochemically. Image analysis with image-pro plus analysis software was used to semi- quantitatively determine the ratio of MMP-2 to TIMP-2. RESULTS: The expression levels of MMP-2 and TIMP-2 were strongly associated with tumor hepatic invasion in pa- tients with hilar cholangiocarcinoma. Significant diffe- rences in the ratio of MMP-2 to TIMP-2 between some pathologic factors were observed in patients with hilar cholangiocarcinoma. CONCLUSIONS: MMP-2 plays an essential role in tumor invasion and metastasis,while TIMP-2 is shown to strongly inhibit cancer invasion and metastasis. The ratio of MMP-2 to TIMP-2 may be a prognostic indicator for patients with hilar cholangiocarcinoma.

Effects of an Engineered Anti-HER2 Antibody chA21 on Invasion of Human Ovarian Carcinoma Cell In Vitro

Objective： HER-2 plays an important role in the development and progression of ovarian carcinoma. A number of monoclonal antibodies （MAbs） and engineered antibody fragments （such as scFvs） against the subdomain II or IV of HER-2 extracellular domain （ECD） have been developed. We investigated the effect of chA21, an engineered anti-HER-2 antibody that bind primarily to subdomain I, on ovarian carcinoma cell invasion in vitro, and explored its possible mechanisms. Methods： Growth inhibition of SK-OV-3 cells was assessed using a Methyl thiazolyl tetrazolium （MTT） assay. The invasion ability of SK-OV-3 was determined by a Transwell invasion assay. The expression of matrix metalloproteinase-2 （MMP-2） and its tissue inhibitors （TIMP-2） was detected by immunocytochemical staining, and the expression of p38 and the phosphorylation of p38 were assayed by both immunocytochemistry and Western blot. Results： After treatment with chA21, the invasion of human ovarian cancer SK-OV-3 cells was inhibited in dose- and time-dependent manners. Simultaneously the expression of p38, phospho-p38, MMP-2 and the MMP-2/TIMP-2 ratio decreased, while TIMP-2 expression increased. Additionally, the decrease in phospho-p38 was much greater than that of p38. Conclusion： chA21 may inhibit SK-OV-3 cell invasion via the signal transduction pathway involving MMP-2, TIMP-2, p38 and the activation of p38MAPK.