BPH-1 induces aromatase expression in prostatic stromal cells through paracrine of PGE2

AIM: To characterize the effect of prostatic epithelial cell paracrine on aromatase expression in prostatic stromal cells.METHODS: Conditioned medium (CM) of prostatic epithelial cell lines (BPH-1, LNCap, DU-145 and PC3) were collected and used to treat prostatic stromal cells. Expression of aromatase was determined by real-time RT-PCR and Western blotting. Expression of cyclooxygenase-2 mRNA in prostatic epithelial cell lines and prostaglandin (PGE2) in CMs were examined by real-time RT-PCR and ELISA, respectively. The CM of BPH-1 cells cultured with NS-398, specific inhibitor of cyclooxygenase-2, were collected, and the effect of NS-398 and PGE2 on aromatase expression was analyzed.RESULTS: CM of human benign prostate hyperplasia epithelial cell line (BPH-1) stimulated expression of aromatase mRNA and protein in stromal cells. But CM of prostate cancer epithelial cell lines (LNCap, DU145, PC3) had no effect on aromatase expression. COX-2 mRNA level in BPH-1 was much higher than that of other cell lines and PGE2 concentration in BPH-1 CM was much higher than that of other CMs. PGE2 concentration of the CM from BPH-1 cultured with NS-398 significantly decreased. CM from BPH-1 cultured with NS-398 failed to stimulate aromatase expression, while PGE2 induced aromatase expression in prostatic stromal cells.CONCLUSION: BPH-1 could induce aromatase expression in prostatic stromal cells through paracrine of PGE2.     

ST14/MT-SP1 influences expression of MT2-MMP and TIMP-2 to promote invasiveness of colorectal cancer cells

AIM: To evaluate how ST14/MT-SP1 overexpression alters invasiveness of colorectal cancer cells.METHODS: Human full length ST14/MT-SP1 gene was transiently transfected into colorectal cancer (RKO) cell lines.The expression products were purified by chromatography on Ni2+-affinity resin column and analyzed for gelatinase activity by gelatin zymography.Cell invasion and migration were determined by ECM invasion assay in vitro.RNA was isolated from stable ST14-transfected RKO cells and a cDNA microarray was utilized to detect the change in expression of MMPs and TIMPs.Real-time quantitative PCR was used to validate the change of expression.RESULTS: The full length ST14/MT-SP1 was transfected and expressed in RKO cells.The expressed protein showed a gelatinase activity.In addition,invasiveness of RKO was significantly enhanced by ST14/MT-SP1 overexpression (P<0.01).Blockage of ST14/MT-SP1 resulted in a decrease in the invasiveness of SW480 and SW620 (P<0.01).Furthermore,MT2-MMP (MMP-15) expression was significantly up-regulated (2.5-fold change) and TIMP2 down-regulated (0.35-fold change) by ST14/MT-SP1 overexpression in RKO.CONCLUSION: ST14/MT-SP1 overexpression results in an increase in invasiveness of colorectal cancer RKO cells.Increased invasiveness is due to an increase in the gelatinase activity of ST14/MT-SP1 and a change in up-regulated MT2-MMP and down-regulated TIMP-2 expression.Therefore,ST14/MT-SP1 overexpression enhances invasiveness of colorectal cancer cells.     

MMP-7,MMP-10 and TIMP-4 expression in ventricular remodeling of human heart failure

AIM:To sieve matrix metalloproteinases (MMPs) and the tissue inhibitors of matrix metalloproteinases (TIMPs) closely associated with ventricular remodeling of human heart failure using antibody chip technology.METHODS:We performed cytokine-specific antibody array analysis using individual left ventricular myocardial samples from 6 patients with heart failure due to arrythmogenic right ventricular cardiomyopathy (ARVC) undergoing transplantation and matched samples from 6 non-failing subjects to screen differentially expressed MMPs and TIMPs associated with the ventricular remodeling of heart failure.The results were further validated by ELISA and immunohistochemical analysis.RESULTS:We identified high expression of MMP-7 and MMP-10 and low expression of TIMP-4 in ARVC failing hearts compared to non-failing hearts by hybridization with the cytokine-specific antibody arrays containing 17 MMPs and 4 TIMPs on the chips.ELISA and immunohistochemical analyses further confirmed that differentially expressed levels of MMP-7, MMP-10, and TIMP-4 were observed not only in ARVC failing heart, but also in failing hearts due to ischemic (ICM) and dilated cardiomyopathy (DCM).CONCLUSION:Highly expressed MMP-7 and MMP-10 and lowly expressed TIMP-4 may be involved in the ventricular remodeling of heart failure derived from cardiomyopathy of different etiology.     

Expression of SAL-like 4 protein in human prostate cancer tissue/cell lines and its relationship with clinical outcome

AIM:To evaluate the expression of SAL-like 4 (SALL4) protein in human prostate cancer cell lines and tissues, and to analyze the relationship between SALL4 expression and the clinicopathological parameters. METHODS:Immunofluorescence, RT-PCR and Western blotting were performed to detect the expression of SALL4 at mRNA and protein levels in 3 common prostate cancer cell lines LNCaP, DU145 and PC-3. The normal prostate epithelial cell line RWPE-1 was used for control. The protein levels of SALL4 in the tissues of benign prostate hyperplasia and prostate cancer tissues were determined by the method of immunohistochemistry. RESULTS:The SALL4 protein was predominantly expressed in the cytoplasm of the cells. The protein levels of SALL4 in 3 common prostate cancer cell lines were significantly higher than that in RWPE-1 cells. However, the mRNA level of SALL4 had no obvious difference among the 4 cell lines. Immunohistochemistry results showed that the expression level of SALL4 in the cancerous tissues was significantly higher than that in noncancerous (benign and normal) prostatic tissues. In addition, we found that the expression level of SALL4 in prostate cancer was significantly correlated with the Gleason score, clinical stage, prognosis estimation and tissue prostate-specific antigen (PSA) expression, but not associated with age, the level of serum total PSA, prostate volume and the expression of androgen receptor in the tissues of the patients. CONCLUSION: The over-expression of SALL4 protein may play an important role in the pathogenesis and progression of prostate cancer, and provides some reference indexes for estimating the malignancy, progression and prognosis of prostate cancer.     

Imbalance between matrix metalloproteinases and tissue inhibitor of metalloproteinases during wound healing in diabetic rats

AIM: To investigate the imbalance between the expression of metalloproteinases (MMPs) and that of tissue inhibitors of metalloproteinase (TIMPs) during wound healing in diabetic rats. METHODS: Diabetic rats were induced with streptozotocin. All rats were maintained for 6 weeks. A full-thickness excisional wound was created on the back of each rat. Every group was randomly divided into 3 subgroups of 7 rats: 3 d group, 7 d group, 14 d group and animals were killed at 3rd, 7th and 14th day. Routine pathological examination, Masson′s trichrome staining and immunohistochemistry were made to calculate the score of epidermal and dermal regeneration, granulation tissue thickness, angiogenesis, matrix density, and infiltrated cells at different time points. RT-PCR and Western blotting were used to detect the expression of mRNA and protein of MMP-9 and TIMP-1 in the skin at those time points. RESULTS: Six weeks after streptozotocin treatment, Three days after injury, the wound healing rate of normal rats was faster than that of diabetic rats. From 3rd to 14th day, there were a lot of fibroblast and macrophage in normal skin, while few such cells were observed in diabetic skin. The other histological scores in normal skin were higher than those in diabetic rats at 7th and 14th day. Both MMP-9 and TIMP-1 had minimally detectable levels before wounding but exhibited rapid, significantly large increases within 3 d after wounding. Subsequently, they showed a rapid decline by 14 d. The relative values of expression of MMP-9 mRNA and protein in diabetic group were higher than those in normal group at different time points. However, the values of TIMP-1 mRNA and protein in diabetic group were significantly lower than those in control group. Significant difference was observed between two groups with the ratio of MMP-9/TIMP-1, higher in diabetic group than that in normal group. CONCLUSION: Abnormal reepithelialization, angiogenesis, inflammatory cell infiltration, collagen fibers generation, granulation tissue deposition, seem to be the basic histopathology that delays wound healing. The imbalance between MMPs and TIMPs in diabetic skin tissue before and after injury may be one of the important reasons of these alterations of histopathology.     

Regulation by reactive oxygen species of matrix metalloproteinase-1, 3 and tissue inhibitor of matrix metalloproteinase-1 in smooth muscle cells

AIM: To understand whether reactive oxygen species promote the rupture of atherosclerotic plaques by regulating the balance of matrix metalloproteinase-1, 3 (MMP-1, 3) and tissue inhibitor of matrix metalloproteinase-1 (TIMP-1) in smooth muscle cells. METHODS: Aortic smooth muscle cells from 4-6months-healthy abortive fetuses were incubated for 24 hours with xanthine (100 μmol/L) and xanthine oxidase (5 U/L) in vitro . MMP-1, 3 and TIMP-1 in the concentrated culture media were measured by Western blotting ( n =3 independent experiments). RESULTS: Incubation with xanthine/xanthine oxdiase decreased the amount of MMP-1 in the aortic smooth muscle cells (21.2%±5.5% of the control group), and pro-MMP-1 was activated completely. Reactive oxygen species (ROS) also activated pro-MMP-3, and increased the production of MMP-3 in the aortic smooth muscle cells. On the other hand, ROS inhibited the production of TIMP-1 in the aortic smooth muscle cells. CONCLUSION: It is complicated that ROS regulates the balance of MMPs and TIMPs. ROS may contribute to matrix degradation and the rupture in the atherosclerotic plaques.     

Regulation of MMP-2 and TIMP-3 expression by uPA signal transduction system in human bone giant cell tumor

AIM: To study effects of urokinase-type plasminogen activator (uPA) signal transduction on expression of matrix metalloproteinase-2 (MMP-2) and tissue inhibitor of matrix metalloproteinase-3 (TIMP-3) in giant cell tumor of bone (GCT). METHODS: Expression of uPAR, MMP-2 and TIMP-3 in GCT tissue was detected by immunohistochemistry. Phosphorylation level of mitogen-activated protein kinase (p44) in uPA/uPAR signal pathway in cultured GCT cells was detected by immunoprecipitation. The expression of MMP-2 and TIMP-3 in cultured cells after treatment with uPA-ATF or anti-uPAR antibody was also detected by Western blotting. RESULTS: 1) Urokinase-type plasminogen activator receptor (uPAR) was positive on the cell membrane and in cytoplasm of some mononuclear stromal cells (MSCs) and multinucleated giant cells (MGCs); 2) MMP-2 was positive in the cytoplasm and on the cell membrane of almost all of MSCs and some of MGCs. The polar distribution of MMP-2 in the cytoplasm of MGCs was especially obvious; 3) The expression of TIMP-3 of some MSCs and MGCs in GCT was much lower than MMP-2. The positive signal also showed a prominent polarity; 4) After treatment with uPA-ATF, the phosphorylation level of p44 in GCT cultured cells was much higher than the control. Addition of anti-uPAR antibody in the cells remarkably down-regulated the phosphorylation level of p44 as compared with the control group, suggesting that uPA-ATF participates cell signal transduction and this reaction can be inhibited by anti-uPAR antibody; 5) uPA-ATF cell signal pathway up-regulated expression of MMP-2 and TIMP-3, while anti-uPAR antibody down-regulated the expression of MMP-2 and TIMP-3. CONCLUSION: These results demonstrate for the first time that uPA-ATF directly regulates the expression of MMP-2 and TIMP-3 by signal transduction pathway, and the over-expression of MMP-2 and TIMP-3 may play an important role in local osteolysis of GCT.     

Effect of abnormal expression of PDK4 on glycolysis and proliferation in prostate cancer cells

AIM To investigate the expression of pyruvate dehydrogenase kinase 4 （PDK4） in prostate cancer tissue and its effect on glycolysis and growth of prostate cancer cells. METHODS Immunohistochemistry was used to compare the expression differences of PDK4 protein in benign prostatic hyperplasia （BPH） and prostate cancer tissues. The expression levels of PDK4 in normal prostatic epithelial cells （RWPE-1） and different prostate cancer cell lines （PC3， LNCaP， DU145 and C4-2） were detected by RT-qPCR and Western blot. Recombinant plasmid carrying PDK4-shRNA was constructed， and the expression of PDK4 in prostate cancer PC3 cells was down-regulated by transfection with PDK4-shRNA. The changes in glycolysis level of PC3 cells before and after transfection were determined by cell glycolysis kit， and the effects of PDK4 on the viability and cell cycle distribution of PC3 cells were detected by CCK-8 assay and flow cytometry. RESULTS In prostate cancer tissues， the expression level of PDK4 protein was significantly higher than that in BPH tissues （P<0.05）， and the analysis of immunohistochemical score showed that prostate cancer tissues with high Gleason score displayed significantly higher PDK4 expression than those with low Gleason score （P<0.05）. Compared with normal prostatic epithelial cells， RT-qPCR and Western blot results indicated that the expression level of PDK4 was also significantly increased in prostate cancer cell lines （P<0.05）. In addition， CCK-8 assay results showed that the viability of prostate cancer PC3 cells was significantly decreased after knockdown of PDK4 expression （P<0.05）. The results of flow cytometry demonstrated that knockdown of PDK4 expression in PC3 cells resulted in a notable increase in G₀/G₁ phase arrest （P<0.05）. CONCLUSION PDK4 is highly expressed in prostate cancer tissues and cell lines， and significantly increases in prostate cancer with high Gleason score. In addition， down-regulation of PDK4 expression significantly inhibits glycolysis and growth of prostate cancer cells， resulting in cell cycle arrest at G₀/G₁ phase.     

Mechanism of miR-30c over-expression inhibiting malignant phenotypes of cervical cancer cells

AIM:To investigate the molecule mechanism of microRNA (miR)-30c over-expression inhibiting malignant phenotypes of cervical cancer cells. METHODS:Cervical cancer cell lines C33A, HeLa, SiHa and CaSki were transfected with pGenesil-1-miR-30c plasmid using Lipofectamine 2000 kit, and the expression of miR-30c was determined by TaqMan real-time PCR. The cell viability inhibition rate, colony formation ability, migration rate and apoptotic rate were measured by MTT assay, colony formation assay, Transwell experiment, and flow cytometry with Annexin V-FITC staining. The protein expression of Bax, Bcl-2, matrix metalloproteinase (MMP)-9, MMP-13 and tissue inhibitor of metalloprotei-nase-1 (TIMP-1) was detected by Western blot. RESULTS:The expression levels of miRNA-30c in the cervical cancer cell lines transfected with pGenesil-1-miR-30c plasmid were significantly higher than those in negative control groups (cell lines transfected with pGenesil-1 plasmid) (P<0.01). Significantly increased cell viability inhibition rate, and decreased colony formation ability and migration rate were found in the cervical cancer cell lines over-expressing miR-30c as compared with negative control groups (P<0.05). The apoptotic rate in the cervical cancer cell lines over-expressing miR-30c was dramatically higher than that in control groups (P<0.05). Over-expression of miR-30c in cervical cancer cells promoted the protein expression of Bax and TIMP-1, and decreased the protein expression of Bcl-2 and MMP-13 (P<0.05 or P<0.01). CONCLUSION:Over-expression of miR-30c significantly inhibits the viability and migration, and induces apoptosis of cervical cancer cells. The mechanism may be related to activating apoptosis pathway and inhibiting MMP-13 protein expression.     

miR-496 over-expression inhibits growth and metastasis in colon cancer cells

AIM: To investigate the effect of miR-496 over-expression on the growth and metastasis of colon cancer cells and its molecular mechanism.METHODS: The proteins interacting with miR-496 were screened by bioinformatic method. The levels of miR-496, CTNNB1 mRNA and β-catenin protein in colon cancer cell lines, HT29, HCT116 and SW480, and normal colonic epithelial cell line NCM460 were detected by real-time PCR and Western blot. HT29, HCT116 and SW480 cells were transfected with miR-496 mimics using Lipofectamine 2000 and named as HT29-miR-496 mimics, HCT116-miR-496 mimics and SW480-miR-496 mimics cells, respectively, and the cells transfected with the scramble served as negative control. The cell viability, lactate dehydrogenase (LDH) leakage, and colony formation and metastatic abilities were determined by MTT assay, LDH assay, colony formation assay and Transwell method, respectively. The promoter activity of miR-496 was measured using luciferase reporter gene assay. The protein levels of β-catenin, eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1), p-4E-BP1, low-density lipoprotein receptor-related protein 6(LRP6), p-LRP6, MMP-7, MMP-9, MMP-13 and TIMP-2 were monitored by Western blot.RESULTS: Endogenous miR-406 interacted with β-catenin was found in the colon cancer cells. Low miR-496 expression in the HT29, HCT116 and SW480 cells and high miR-496 expression in NCM460 cells were detected. In contrast, high β-catenin expression was found in the HT29, HCT116 and SW480 cells and low β-catenin expression was observed in the NCM460 cells. Compared with control group, the cell viability, colony formation rate and the number of metastatic cells remarkably decreased in the HT29-miR-496 mimics, HCT116-miR-496 mimics and SW480-miR-496 mimic cells (P<0.05). The promoter activity of miR-496 was significantly increased in colon cancer cells transfected with miR-496 mimics, and was 1.75, 2.04 and 1.61 times as high as control group. miR-496 over-expression inhibited β-catenin levels, and p-4E-BP1 and p-LRP6 protein levels were also reduced. siRNA- or over-expressed miR-496-mediated β-catenin down-regulation inhibited MMP-7 and MMP-9 expression, but promoted TIMP-2 expression.CONCLUSION: The expression level of miR-496 in the colon cancer cells is low, but in the normal colonic epithelial cells is high. miR-496 over-expression inhibits the protein levels of MMP-7 and MMP-9, and promotes the protein expression of TIMP-2 via inhibiting Wnt/β-catenin pathway, thus suppressing malignant phenotype in the colon cancer cells.     

Effects of furin inhibitor on metastasis of human breast cancer MCF-7 cells

AIM：To investigate the mechanism underlying breast cancer metastasis and to provide theoretical data for studying the pathogenesis of breast cancer onset and development. METHODS：Human breast cancer MCF-7 cells were treated with different concentrations of furin inhibitor α1-PDX for 48 h. Wound healing assay and Transwell assay were applied to detect the migration and invasion abilities of the MCF-7 cells. The expression of cell migration-associated proteins, including membrane-type 1 matrix metalloproteinase (MT1-MMP), vascular endothelial growth factor (VEGF)-C and VEGF-D, was determined by Western blotting. The protein levels of MMP2 and MMP9 in the supernatant were measured by ELISA. RESULTS：Compared with control group, 200 nmol/L of furin inhibitor exerted significant inhibitory effects on the cell migration (P<0.05). The expression of cell migration-associated proteins MT1-MMP, VEGF-C and VEGF-D was significantly inhibited after treated with α1-PDX (P<0.05). Significant inhibitory effects of α1-PDX on the expression of MMP9 and MMP2 (P<0.05) in the supernatant were observed. CONCLUSION：Furin inhibitor suppresses the metastasis of MCF-7 cells via down-regulating the expression of MMPs and VEGFs.     

Molecular mechanism of miR-29a inhibiting malignant phenotypes in prostate cancer cells

AIM:To investigate the biological functions of microRNA-29a (miR-29a) in prostate cancer and the molecular mechanism of miR-29a over-expression inhibiting malignant phenotypes of prostate cancer cells. METHODS:The levels of miR-29a expression in the prostate cancer tissues and cells were detected and analyzed using gene microarray and bioinformatics. The expression levels of miR-29a and lysine (K)-specific demethylase 4B (KDM4B) mRNA in prostate cancer tissues, paracarcinomatous tissues, 4 prostate cancer cell lines (PC3, DU145, LNCaP and ArCaP) and normal prostate epithelial cell line (RWPE-1) were measured by real-time PCR. PC3, DU145, LNCaP and ArCaP cells were transfected with pGenesil-1-miR-29a plasmid using transient transfection. The cell viability, colony formation rate and apoptotic rate were analyzed by MTT assay, colony formation assay and flow cytometry with Annexin V-FITC/PI staining, respectively. The protein expression of KDM4B was determined by Western blot. RESULTS:The results of gene microarray and bioinformatic analysis indicated that differential expression of miR-29a was found in the prostate cancer tissues and the paracarcinomatous tissues. The levels of miR-29a in the prostate cancer tissues and prostate cancer cells were significantly decreased, while the mRNA levels of KDM4B were notably increased compared with the paracarcinomatous tissues and RWPE-1 cells, respectively (P<0.05). Compared with negative control (pGenesil-1) group, the cell viability and colony formation rate were significantly decreased, the apoptotic rate was significantly increased, and the protein expression of KDM4B was notably inhibited in the prostate cancer cells with miR-29a over-expression (P<0.05). The cell viability was significantly enhanced, and the apoptosis was significantly inhibited in the prostate cancer cells with KDM4B over-expression (P<0.05). CONCLUSION:Low expression of miR-29a was found in the prostate cancer tissues and cells. miR-29a over-expression inhibits the growth of prostate cancer cells and induces apoptosis. The mechanism may be associated with inhibiting the protein expression of KDM4B.     

Effects of low-molecular weight heparin on MMP-2, TIMP-2 expression and invasiveness of cytotrophoblastic cells

AIM: To investigate the hypothesis that low-molecular weight heparin (LMWH) regulates in vitro cytotrophoblast invasiveness and production of metalloproteinases-2 (MMP-2), tissue inhibitor of metalloproteinas-2 (TIMP-2). METHODS: Chorionic villi tissue of normal 6-8 weeks pregnancy was obtained. Trophoblastic cells were collected by trypsin-collagenase digestion and Percoll gradient centrifugation. The cytotrophoblastic cells were cultured for 24 h and divided into 4 groups according to the concentrations (1.0×102 IU/L, 1.0×103 IU/L or 1.0×104 IU/L) of LMWH adding into the medium. The contents of MMP-2 and TIMP-2 in cell culture supernatants were measured by the method of ELISA. Cytotrophoblast invasiveness was determined by Transwell chamber assay. RESULTS: With the increasing concentrations of LMWH, the invasion activity of cytotrophoblastic cells and MMP-2 secretion were increased. At concentration of 1.0×103IU/L, LMWH greatly enhanced cytotrophoblast invasiveness and the expression of MMP-2 (P<0.05). The levels of TIMP-2 were decreased after intervention with LMWH. At concentration of 1.0×103IU/L or 1.0×104 IU/L, LMWH induced a significant decrease in TIMP-2 expression. No significant difference between group 1×103IU/L and group 1.0×104 IU/L was observed (P>0.05). CONCLUSION: LMWH might regulate cytotrophoblast invasiveness in vitro by influencing the expression of MMP-2 and TIMP-2 in cytotrophoblastic cells.     

Oxysterols downregulate tissue inhibitor of metalloproteinase -1 expression in vascular smooth muscle cells

AIM:To investigate the effects of oxysterols on matrix metalloproteinase (MMP) and tissue inhibitor of metalloproteinase-1 (TIMP-1) expression in vascular smooth muscle cells. METHODS:Rabbit aortic vascular smooth muscle cells were culturedin vitroand incubated with cholesterol, Triol and 25-hydroxycholesterol (25-OH), respectively. Slot blot was used to detect the mRNA expression level of TIMP-1 in vascular smooth muscle cells (VSMCs); meanwhile the protein expression level of MMP-9 and TIMP-1 was detected by immunohistology. RESULTS:Triol and 25-OH inhibited the expression of TIMP-1 compared with control and cholesterol, but have no effect on expression of MMP-9. CONCLUSION:Both Triol and 25-OH downregulated TIMP-1 expression in VSMCs.     

Expression of matrix metalloproteinase and tissue inhibitor of metalloproteinase in lung tissue of hypercapnia rat model

AIM: To study the expression of matrix metalloproteinase-9(MMP-9), matrix metalloproteinase-2(MMP-2) and the tissue inhibitor of metalloproteinase(TIMP-1) in the lung tissue of the hypercapnia rat.METHODS: Forty Wistar rats were randomly divided into a control group (group A, n=20) and hypercapnia group (group B, n=20). Group B received mix gas exposure (6% CO2, 21% O2, 72% N2) 7 h daily for 4 weeks. The parameters we would examine were as follow: arterial blood gas; the mean pulmonary artery pressure;MMP-2,MMP-9, TIMP-1, and NE activity in lung tissue. Masson pigmentation of elasticity fibre was analyzed by computer image analyzer. Histopathological changes of lung tissue were observed under light microscope. The protein expression of MMP (MMP-2, MMP-9) and TIMP (TIMP-1) in lung tissue were determined by immunocytochemistry.RESULTS: Decompensate respiratory acidosis (pH=7.20±0.04, PaCO2=7.84±0.15) developed in group B. The mean pulmonary artery pressure were similar between groups B and A (P>0.05). Tissue edema in the lung, endothelial cell damage of the small blood vessels, pulmonary micro thrombus formations and increased pulmonary capillary permeability were observed in group B. NE activity increased significantly (P<0.01). However, no significant change of MMP-2, MMP-9, TIMP-1 activity was found in group B and group A (P>0.05). There was significant decrease in the relative content of elasticity fibre in lung tissue in group B compared to group A (P<0.01). The expression of MMP-2 protein in the lung tissue of group B was lower than that in group A (P<0.01), but the expression of both MMP-9 and TIMP-1 proteins in the lung tissue in group B were higher than those in group A (P<0.01).CONCLUSION: Hypercapnia rat model is successfully reproduced by exposure of animals to the mix gas exposure (6% CO2, 21% O2, and 72% N2). The pulmonary artery pressure is not affected by hypercapnia. High concentration of CO2 causes increase of NE activity and decrease in the relative content of elasticity fibre. High concentration of CO2 causes the increase of MMP-2 protein expression and decrease in the MMP-9 and TIMP-1 protein expression.     

Analysis of different methylation patterns of histone H3 between androgen-dependent and androgen-independent prostate cancer cells by heavy methyl SILAC

AIM：To study the epigenetic mechanisms involved in the evolution of prostate cancer from an androgen-dependent state to an androgen-independent state, and the global difference of histone H3 methylation between androgen-dependent and -independent prostate cancer cells. METHODS：The methylation sites and patterns of histone H3 in androgen-dependent prostate cancer cell line LNCaP and androgen-independent prostate cancer cell line DU145 were analyzed by heavy methyl stable isotope labeling with amino acids in cell culture (SILAC) coupled with 2D LC-MS/MS. Western blotting was used to verify the results from MS. The differential expression of related methylases and demethylases was tested by real-time PCR. RESULTS：Five methylation sites on histone H3 were found in both cell lines, the patterns of which were as follows: H3K14me2, H3R17me1, H3K36me1, H3K36me2, H3K36me3, H3R72me2, H3K79me1 and H3K79me2. There were 2 different peptides both containing methylated H3K36, “KSAPATGGVKKPHR” and “KSAPSTGGVKKPHR”, which were different from the 31th amino acid residue “A” and “S”. The former peptide belonging to histone H3 variants, H31T, H31 and H32, was mainly identified in DU145 cells, the total peptide counts of which were much more than that of the latter peptide belonging to histone H3 variant H31T, suggesting that these 2 cell lines expressed different histone H3 variants. Mono- and dimethylation of H3K36 were not different between these 2 cell lines, but the trimethylation was significantly higher in DU145 cells than that in LNCaP cells. Many H3K36 demethyltransferases were decreased in DU145 cells compared with LNCaP cells. CONCLUSION：The differential expression of histone H3 variants and H3K36 demethyltransferases may result in up-regulation of H3K36 tri-methylation during the evolution of prostate cancer from an androgen-dependent state to an androgen-independent state.     

Construction of human prostate-specific membrane antigen expression vector and identification of tissue specificity

AIM: To construct a prostate-specific expression vector of promoter and enhancer of human prostate-specific membrane antigen (PSMA).METHODS: The promoter and enhancer of PSMA were amplified by PCR separately. The two segments were cloned into the expression vector pGL3-Basic, a prostate-specific expression vector pGL3-PSMP-PSME was constructed. The vector was transfected into prostatic carcinoma cell PC-3M and four kinds of non-prostatic carcinoma cells by lipofectamine. The activity of luciferase of transfected cells and tissue specificity of vector was examined at 48 hours after transfection. RESULTS: With DNA sequence of the vector pGL3-PSMP-PSME, the segment of clone was proved correct. The activity of luciferase of the pGL3-PSMP-PSME was expressed distinctly in PC-3M and was not expressed in other nonprostate cell lines. CONCLUSION: The prostate-specific expression vector was constructed successfully. It lays foundation for studying target gene therapy of the prostate cancer.