MiR-92b inhibits proliferation and invasion of lung cancer by targeting EZH2.

OBJECTIVE: To verify that miR-92b inhibits proliferation and invasion of lung cancer by targeting EZH2. MATERIALS AND METHODS: The expression levels of miR-92b and EZH2 in human bronchial epithelial cell line BEAS-2B and human lung cancer cell line (A549, NCI-H23, NCI-H358, NCI-H1975, PC-9) were detected, and miR-92b mimic, sh-EZH2 expression vector, and plasmid blank vector (blank group) were constructed. Blank group, miR-92b mimic, miR-92b mimic+sh-EZH2 group (combined group) were set up, MTT and transwell were used to detect the proliferation and invasion ability of A549 and NCI-H23 cells, and fluorescein report verified the regulatory relationship of miR-92b to EZH2. RESULTS: The expression level of miR-92b in A549, NCI-H23, NCI-H358, NCI-H1975, and PC-9 cells was lower than that in BEAS-2B cells (p<0.05). The expression level of EZH2 was higher than that of BEAS-2B cells (p<0.05). A549 and NCI-H23 cells were selected for transfection. After that, the expression level of miR-92 in miR-92b mimic, combined group A549 and NCI-H23 cells was higher than that in blank group (p<0.05), and miR-92b mimic had no difference with joint group (p>0.05). The expression level of EZH2 in cells of miR-92b mimic, blank group A549, and NCI-H23 was lower than that of combined group (p<0.05), and miR-92b mimic was lower than that of blank group (p<0.05). After the overexpression of miR-92b, pmirGLO-EZH2-3'UT Wt luciferase activity decreased significantly (p<0.05) but had no effect on pmirGLO-EZH2-3'UTR Mut Luciferase activity (p>0.05). Cell proliferation ability and invasion ability of A549 cells and NCI-H23 cells in miR-92b mimic group were lower than those in blank group (p<0.05), while those in combined group were higher than those in miR-92b mimic group (p<0.05). CONCLUSIONS: MiR-92b inhibits proliferation and invasion of lung cancer cells through targeted inhibition of EZH2, which is a potential target for future treatment of lung cancer.

[The relationship between non-alcoholic fatty liver disease and metabolic syndrome in patients with latent autoimmune diabetes in adults].

Objective: To investigate the relationship between non-alcoholic fatty liver disease (NAFLD) and metabolic syndrome (MetS) in patients with latent autoimmune diabetes in adults (LADA). Methods: A total of 223 hospitalized patients were recruited between January 2007 and June 2009 in Department of Endocrinology and Metabolism of the Sixth People's Hospital Affiliated to Shanghai Jiaotong University. Finally, 142 patients with complete clinical data and without history of drinking were included in this study. According to the Chinese Medical Association's Guidelines of NAFLD, based on the result of ultrasound, all subjects were divided into two groups including patients with LADA and NAFLD (n=37) and patients with LADA but without NAFLD (n=105). Clinical data including diabetes duration, history of smoking and medications, height, weight, blood pressure, blood lipids, blood glucose, C-peptide, and liver and kidney function were collected. The prevalence and components of MetS were compared between two groups. The association between MetS and NAFLD was also explored. Results: After adjusting for age and sex, compared with the subjects without NAFLD, the subjects with NAFLD were older and had higher percentage of hypertension, and had higher body mass index[(26.5±3.7) kg/m(2) vs (21.9±3.1) kg/m(2)], waist-hip ratio(0.92±0.06 vs 0.86±0.07), low density lipoprotein cholesterol[(3.26±0.72) mmol/L vs (2.70±0.87) mmol/L], C-reactive protein, fasting C-peptide, 2 h postprandial C-peptide, systolic blood pressure, diastolic blood pressure, alanine aminotransferase and triglyceride (all P<0.05). But they had lower high-density lipoprotein cholesterol[(1.17±0.43) mmol/L vs (1.35±0.40) mmol/L]and HbA1c[(8.83±2.14) % vs (10.02±2.79)%](both P<0.05). In addition, after adjusting for age and sex, compared with the patients with LADA but without NAFLD, the prevalence of MetS in the patients with LADA and NAFLD was obviously higher (97.3% vs 47.6%, P<0.001), and the proportion of the patients with four (32.4% vs 16.2%, P<0.001) and five (43.2% vs 5.7%, P<0.001) components of MetS in the patients with LADA and NAFLD was also significantly increased than that in the patients with LADA but without NAFLD. Binary regression analysis showed that NAFLD was an independent factor associated with MetS in the patients with LADA after correcting other confounding factors (P<0.001). Conclusions: Compared with the patients with LADA but without NAFLD, the prevalence of MetS was obviously higher, and had more serious metabolic disorder in the patients with LADA and NAFLD. The presence of NAFLD was an independent factor associated with MetS in the patients with LADA.

Sarcopenia, as defined by low muscle mass, strength and physical performance, predicts complications after surgery for colorectal cancer.

AIM: Recent studies have shown that sarcopenia is associated with negative postoperative outcomes. However, none of these studies analysed muscle strength or physical performance, which are also important components of sarcopenia. The present study aimed to investigate whether sarcopenia itself, as defined by low muscle mass, strength and physical performance, would predict complications after surgery for colorectal cancer. METHOD: We conducted a prospective study of patients who underwent surgery for colorectal cancer at our department between August 2014 and February 2015. Sarcopenia was diagnosed by a combination of third lumbar vertebra muscle index (L3 MI), handgrip strength and 6-m usual gait speed. Univariate and multivariate analyses evaluating the risk factors for postoperative complications were performed. Only complications classified as Grade II or above according to the Clavien-Dindo classification were analysed in this study. RESULTS: A total of 142 patients were included in the study, and 17 patients were diagnosed as having sarcopenia. Postoperative complications of Grade II or above occurred in 40 patients, including 10 with sarcopenia and 30 without sarcopenia. Multivariate analysis showed that sarcopenia and previous abdominal surgery were independent risk factors for postoperative complications. Patients with sarcopenia also had an obvious tendency to a higher incidence of infectious complications. By comparing two logistic regression models, sarcopenia showed a better predictive power for postoperative complications than did low muscle mass. CONCLUSION: Sarcopenia and previous abdominal surgery are independent risk factors for complications after surgery for colorectal cancer. Including a functional aspect to the definition of sarcopenia may result in a better prediction of postoperative complications.

Loss of STAG2 causes aneuploidy in normal human bladder cells.

The aim of this study was to determine how the function of human stromal antigen 2 (STAG2) plays an important role in proper chromosome separation. STAG2 mRNA in normal bladder cells and bladder tumor cells was evaluated by RT-PCR. The protein levels of STAG2 in normal bladder cells and bladder tumor cells were determined by western blot. A cell proliferation assay was used to measure the growth of tumor cells and STAG2-inhibited normal cells, and STAG2- inhibited normal cells were subjected to karyotype analysis. Both STAG-2 mRNA and protein expression levels were lower in bladder cancer cells compared to the controls. Knockdown of STAG2 caused aneuploidy in normal bladder cells, leading to a decreased expression of the cohesin complex components SMC1, SMC3 and RAD21, but there was no obvious effect of STAG2 knockdown on cell proliferation. Our study indicated that abnormal expression of STAG2 could cause aneuploidy in normal bladder cells.

Composite artificial semi-knee joint system.

OBJECTIVES: The purpose of the study was to investigate the clinical implantation protocol of custom-made artificial semi-knee joint based on computer-aided design so as to improve the limb salvage efficiency. MATERIALS AND METHODS: The custom-made artificial semi-knee joint was designed and manufactured based on rapid prototyping technology. The repeated modifications were carried out in the design and manufacture of the semi-knee joint, together with the operation protocol. Clinical trial was conducted on 2 cases of osteosarcoma, one receiving allograft prosthesis composite transplantation, and the other receiving synthetic bone graft prosthesis composite transplantation. The clinical outcomes of the 2 patients were evaluated. RESULTS: The custom-made artificial semi-knee joint met the clinical customization needs. In clinical trial, 18-month follow-up demonstrated the satisfactory knee joint function recovery in near future. CONCLUSIONS: The custom-made artificial semi-knee joint based on computer-aided design can afford satisfactory knee joint function recovery following allograft bone transplantation.

Mapping codon usage in sequence regions flanking cleavage positions in the hepatitis A virus polyprotein.

To analyze the synonymous codon usage patterns of sequence regions flanking cleavage sites in the hepatitis A virus (HAV) polyprotein, the codon usage bias at codon positions and the synonymous codon usage in the target contexts of 30 virus strains were estimated by two simple methods that were based on the values for relative synonymous codon usage. In addition, the pattern of synonymous codon usage was compared between the genomic sequences in HAV and those of its human host. Our results indicated that HAV adopts a combination of coincidence and antagonism with the synonymous codon usage in humans. This characteristic may help HAV to efficiently use the translational machinery in its human host. We also observed that codon usage exhibited a strong bias in some specific positions in these contexts, and that the underrepresented synonymous codons, CUA for Leu, ACG for Thr, GUA for Val, and UCG for Ser, are preferentially used in these positions. These underrepresented synonymous codons likely play roles in regulating the rate of protein translation and influencing the secondary structure of the sequence regions flanking the cleavage sites.

Activated human hydroxy-carboxylic acid receptor-3 signals to MAP kinase cascades via the PLC-dependent PKC and MMP-mediated EGFR pathways.

BACKGROUND AND PURPOSE 3-Hydroxy-octanoate, recently identified as a ligand for, the orphan GPCR, HCA(3), is of particular interest given its ability to treat lipid disorders and atherosclerosis. Here we demonstrate the pathway of HCA(3)-mediated activation of ERK1/2. EXPERIMENTAL APPROACH Using CHO-K1 cells stably expressing HCA(3) receptors and A431 cells, a human epidermoid cell line with high levels of endogenous expression of functional HCA(3) receptors, HCA(3)-mediated activation of ERK1/2 was measured by Western blot. KEY RESULTS HCA(3)-mediated activation of ERK1/2 was rapid, peaking at 5 min, and was Pertussis toxin sensitive. Our data, obtained by time course analyses in combination with different kinase inhibitors, demonstrated that on agonist stimulation, HCA(3) receptors evoked ERK1/2 activation via two distinct pathways, the PLC/PKC pathway at early time points (≤ 2 min) and the MMP/ epidermal growth factor receptor (EGFR) transactivation pathway with a maximum response at 5 min. Furthermore, our present results also indicated that the ßγ-subunits of the G(i) protein play a critical role in HCA(3)-activated ERK1/2 phosphorylation, whereas ß-arrestins and Src were not required for ERK1/2 activation. CONCLUSIONS AND IMPLICATIONS We have described the molecular mechanisms underlying the coupling of human HCA(3) receptors to the ERK1/2 MAP kinase pathway in CHO-K1 and A431 cells, which implicate the G(i) protein-initiated, PLC/PKC -and platelet-derived growth factor receptor/EGFR transactivation-dependent pathways. These observations may provide new insights into the pharmacological effects and the physiological functions modulated by the HCA(3)-mediated activation of ERK1/2.

Structural determinants in the second intracellular loop of the human cannabinoid CB1 receptor mediate selective coupling to G(s) and G(i).

BACKGROUND AND PURPOSE: The cannabinoid CB(1) receptor is primarily thought to be functionally coupled to the G(i) form of G proteins, through which it negatively regulates cAMP accumulation. Here, we investigated the dual coupling properties of CB(1) receptors and characterized the structural determinants that mediate selective coupling to G(s) and G(i). EXPERIMENTAL APPROACH: A cAMP-response element reporter gene system was employed to quantitatively analyze cAMP change. CB(1)/CB(2) receptor chimeras and site-directed mutagenesis combined with functional assays and computer modelling were used to determine the structural determinants mediating selective coupling to G(s) and G(i). KEY RESULTS: CB(1) receptors could couple to both G(s)-mediated cAMP accumulation and G(i)-induced activation of ERK1/2 and Ca(2+) mobilization, whereas CB(2) receptors selectively coupled to G(i) and inhibited cAMP production. Using CB(1)/CB(2) chimeric receptors, the second intracellular loop (ICL2) of the CB(1) receptor was identified as primarily responsible for mediating G(s) and G(i) coupling specificity. Furthermore, mutation of Leu-222 in ICL2 to either Ala or Pro switched G protein coupling from G(s) to G(i), while to Ile or Val led to balanced coupling of the mutant receptor with G(s) and G(i) . CONCLUSIONS AND IMPLICATIONS: The ICL2 of CB(1) receptors and in particular Leu-222, which resides within a highly conserved DRY(X)(5) PL motif, played a critical role in G(s) and G(i) protein coupling and specificity. Our studies provide new insight into the mechanisms governing the coupling of CB(1) receptors to G proteins and cannabinoid-induced tolerance.

Treatment of osteonecrosis of the femoral head with hBMP-2-gene-modified tissue-engineered bone in goats.

The efficacy of beta-tricalcium phosphate (beta-TCP) loaded with bone morphogenetic protein-2 (BMP-2)-gene-modified bone-marrow mesenchymal stem cells (BMSCs) was evaluated for the repair of experimentally-induced osteonecrosis of the femoral head in goats. Bilateral early-stage osteonecrosis was induced in adult goats three weeks after ligation of the lateral and medial circumflex arteries and delivery of liquid nitrogen into the femoral head. After core decompression, porous beta-TCP loaded with BMP-2 gene- or beta-galactosidase (gal)-gene-transduced BMSCs was implanted into the left and right femoral heads, respectively. At 16 weeks after implantation, there was collapse of the femoral head in the untreated group but not in the BMP-2 or beta-gal groups. The femoral heads in the BMP-2 group had a normal density and surface, while those in the beta-gal group presented with a low density and an irregular surface. Histologically, new bone and fibrous tissue were formed in the macropores of the beta-TCP. Sixteen weeks after implantation, lamellar bone had formed in the BMP-2 group, but there were some empty cavities and residual fibrous tissue in the beta-gal group. The new bone volume in the BMP-2 group was significantly higher than that in the beta-gal group. The maximum compressive strength and Young's modulus of the repaired tissue in the BMP-2 group were similar to those of normal bone and significantly higher than those in the beta-gal group. Our findings indicate that porous beta-TCP loaded with BMP-2-gene-transduced BMSCs are capable of repairing early-stage, experimentally-induced osteonecrosis of the femoral head and of restoring its mechanical function.

Effects of methylselenocysteine on PKC activity, cdk2 phosphorylation and gadd gene expression in synchronized mouse mammary epithelial tumor cells.

Methylselenocysteine (MSC), an organic selenium compound is an effective chemopreventive agent against mammary cell growth both in vivo and in vitro but its mechanism of action is still not understood. We have previously demonstrated that MSC is able to inhibit growth in a synchronized TM6 mouse mammary epithelial tumor cell line at 16 h time point followed by apoptosis at 48 h. The decrease in cdk2 kinase activity was coincident with prolonged arrest of cells in S-phase. The present set of experiments showed that cdk2 phosphorylation was reduced by 72% in the MSC-treated cells at 16 h time point. Expression for gadd34, 45 and 153 was elevated 2.5 to 7 fold following MSC treatment only after 16 h time point. In order to investigate a possible upstream target for MSC, we analyzed protein kinase C (PKC) in this model. Total PKC activity was reduced in TM6 cells by MSC (50 microM) within 30 min of treatment, both in cytosolic (55.4 and 77.6%) and membrane (35.2 and 34.1%) fractions for calcium-dependent and independent PKCs, respectively. PMA significantly elevated the PKC activity in membrane fraction (P < 0.01) and MSC inhibited this activation by more than 57%. The effect of MSC was selenium specific as selenomethionine and sulfurmethyl-L-cysteine (SMC) did not alter PKC activity either in cytosolic or membrane fraction. Immunoblot analysis showed that PKC-alpha was translocated to the membrane by PMA and MSC did not alter this translocation. PKC-delta was faintly detectable in membrane fractions of control and MSC-treated cells. MSC treatment slightly reduced levels of PKC-e (in cytosolic and membrane fractions) and PKC-zeta (cytosolic fractions). The data presented herein suggest that PKC is a potential upstream target for MSC that may trigger one or all of the downstream effects; i.e. the decrease of cdk2 kinase activity, decreased DNA synthesis, elevation of gadd gene expression and finally apoptosis.

Diagnosis of hepatocellular carcinoma by quantitative detection of hepatoma-specific bands of serum gamma-glutamyltransferase.

Tissues of hepatocellular carcinoma (HCC) can express hepatoma-specific gamma-glutamyltransferase (GGT) and secrete GGT into circulating blood. Serum GGT was separated into several bands (up to 11), including hepatoma-specific bands (HSBs, I', II, and II') by a vertical slab electrophoresis assay of polyacrylamide stage gradient gel. In the present study, the HSBs of serum GGT were separated, and the HSB activity was quantitatively measured in 91 patients with HCC and compared with that of 106 patients with benign liver disease, 16 patients with extrahepatic tumors, and 30 healthy control subjects. Significant differences of the HSB activity were observed between the HCC group and each study group. An HSB activity greater than 5.5 U/L seems to be diagnostic of HCC. The quantitative method has a sensitivity of 85.3%, a specificity of 97.2%, a positive predictive value of 95.1%, a negative predictive value of 91.2%, and an accuracy of 92.5% for detecting HCC. No correlation was found between HSB activity and the serum alpha-fetoprotein (AFP) level or tumor size in patients with HCC. The quantitative analysis of HSB activity of GGT is superior to detection of the AFP concentration and is useful in early diagnosis of small HCC or AFP-negative HCC.

Treatment of acute promyelocytic leukemia with all-trans retinoic acid. A five-year experience.

From January 1986 to April 1991, 107 consecutive patients with acute promyelocytic leukemia (APL) were treated with retinoic acid (RA) at an oral dose of 45-60mg/m2/d, alone or in combination with chemotherapy. In 91 cases treated with RA alone, 74 (81.3%) achieved complete remission (CR). The CR rate was 75% in 16 cases treated with combined therapy. Among 50 patients closely followed for a median of 36 months (4-60), 10 received RA as continuation therapy (Group A), 10 received chemotherapy (Group B) and 30 were treated with RA and chemotherapy alternately in regular sequence (Group C). The mean survival time was 8.4, 9.7 and 21.6 months, respectively, for the 29 cases who died. The survival probability was higher in Group C than in Group A and B (P < 0.01). RA did not provoke or aggravate DIC, it did not cause marrow hypoplasia or aplasia. The side effects were relatively mild as compared with chemotherapy. CFU-GM markedly reduced before treatment was restored to normal level after CR, while the result for L-CFU was reversed. In 40 cases examined for in vitro induction of differentiation, 39 responders were culminating in CR. Aberrant karyotype t (15; 17) was positive in all 47 cases examined prior to the treatment. It disappeared in all of the 20 cases studied after achieving CR, and reappeared in 3 cases following relapse. The best regimen to maintain a longer CR duration and survival time in this study was to use RA and chemotherapy alternately as continuation therapy.

Problems existing in differentiation therapy of acute promyelocytic leukemia (APL) with all-trans retinoic acid (ATRA).

A large number of acute promyelocytic leukemia (APL) patients, treated with all-trans retinoic acid (ATRA) and chemotherapy, were studied. The results of the studies are as follows: (1) Among 65 patients investigated for the postremissional therapy, the 5-year survival probabilities were 0.20 +/- 0.13 (mean +/- SE) in the group treated with ATRA alone, 0.47 + 0.10 (mean +/- SE) in the group using chemotherapy alone and 0.42 +/- 0.09 (mean +/- SE) in the group treated with chemotherapy and ATRA. (2) The main severe adverse effects in the ATRA treatment include retinoic acid syndrome, renal failure, and thrombosis. These sequelae were observed more frequently in cases with persistent, marked elevation of white blood cell count without significant maturation of leukemic promyelocytes. (3) APL is not a homogeneous disease in that among 50 patients studied at the molecular level, although a PML-RARA fusion gene was detected in 45 cases, one had a variant translocation t(11;17) bearing fusion gene PLZF-RARA, one presented no obvious structural alteration of the PML gene while the RARA gene was rearranged, and three patients had no rearrangement of either PML or RARA genes. (4) Using RT/PCR to detect minimal residual disease, we found positive rates of 22%, 18.4%, and 11.5%, respectively, 12, 24, and 36 months after CR. This observation justifies the use of chemotherapy for at least 3 years after CR induced by ATRA. (5) It seems likely that the fusion gene PML-RARA plays an important role in APL leukemogenesis and in its response to the ATRA treatment.

Treatment of acute promyelocytic leukemia with all-trans retinoic acid in China.

Seventy patients with acute promyelocytic leukemia (APL) were entered in this study. Fifty-seven cases received all-trans retinoic acid (RA) as sole agent and 48 cases (84.2%) attained complete remission (CR) in about 42 days. While the other 8 cases (14%) obtained partial remission (PR) during some time. Thirteen patients received combinations of RA and chemotherapy. 9 cases (69.2%) attained CR in about two months and 4 other cases (30.8%) got PR. Total CR rate reached 95.9% after continuing the treatment. Fifty cases were closely followed after CR. Three kinds of continuation therapy were administered including RA as sole agent (Group A), chemotherapy alone (Group B) and RA and chemotherapy alternatively (Group C). The results showed Group C gave the longest duration of CR and longest duration of survival.

Use of all-trans retinoic acid in the treatment of acute promyelocytic leukemia.

Twenty-four patients with acute promyelocytic leukemia (APL) were treated with all-trans retinoic acid (45 to 100 mg/m2/day). Of these, eight cases had been either nonresponsive or resistant to previous chemotherapy; the other 16 cases were previously untreated. All patients attained complete remission without developing bone marrow hypoplasia. Bone marrow suspension cultures were studied in 15 of the 24 patients. Fourteen of these patients had morphological maturation in response to the retinoic acid (1 mumol/L). Chloroacetate esterase and alpha-naphthyl acetate esterase staining as well as electronmicroscopic examination confirmed that retinoic acid-induced cells differentiated to granulocytes with increased functional maturation (as measured by nitroblue tetrazolium reduction, NBT). The single nonresponder to retinoic acid in vitro was resistant to treatment with retinoic acid but attained complete remission after addition of low-dose cytosine arabinoside (ara-C). During the course of therapy, none of the patients showed any abnormalities in the coagulation parameters we measured, suggesting an absence of any subclinical disseminated intravascular coagulation. The only side effects consisted of mild dryness of the lips and skin, with occasional headaches and digestive symptoms. Eight patients have relapsed after 2 to 5 months of complete remission. The others remain in complete remission at 1+ to 11+ months and are still being followed up. We conclude that all-trans retinoic acid is an effective inducer for attaining complete remission in APL.