Proto-oncogene Protein Research Articles

Kinome Profiling to Predict Sensitivity to MAPK Inhibition in Melanoma and to Provide New Insights into Intrinsic and Acquired Mechanism of Resistance.

Mitogen-activated protein kinase (MAPK) inhibition with the combination of BRAF (Rapidly Accelerated Fibrosarcoma) and MEK (Mitogen-activated protein kinase kinase) inhibitors has become the standard of first-line therapy of metastatic melanoma harbouring BRAF V600 mutations. However, about half of the patients present with primary resistance while the remaining develop secondary resistance under prolonged treatment. Thus, there is a need for predictive biomarkers for sensitivity and/or resistance to further refine the patient population likely to benefit from MAPK inhibitors. In this study, we explored a top-down approach using a multiplex kinase assay, first, to discover a kinome signature predicting sensitivity, intrinsic and acquired resistance to MAPK inhibitors in melanoma, and second, to understand the mechanism of resistance using cell lines. Pre-dose tissues from patients (four responders and three non-responders to BRAFi monotherapy) were profiled for phosphotyrosine kinase (PTK) and serine-threonine kinase (STK) activities on a PamChip® peptide microarray in the presence and absence of ex vivo BRAFi. In addition, molecular studies were conducted on four sensitive parental lines, their offspring with acquired resistance to BRAFi and two lines with intrinsic resistance. PTK and STK activities in cell lysates were measured in the presence and absence of ex vivo BRAFi and/or MEKi. In tissue lysates, concentration-dependent ex vivo inhibition of STK and PTK activities with dabrafenib was stronger in responders than in non-responders. This difference was confirmed in cell lines comparing sensitive and resistant ones. Interestingly, common features of resistance were increased activity of receptor tyrosine kinases, Proto-oncogene tyrosine-protein kinase Src (Src) family kinases and protein kinase B (PKB, AKT) signalling. These latter results were confirmed by Western blots. While dabrafenib alone showed an inhibition of STK and PTK activities in both tissues and cell lines, the combination of dabrafenib and trametinib showed an antagonism on the STK activities and a synergism on PTK activities, resulting in stronger inhibitions of overall tyrosine kinase activities. Altogether; these data reveal that resistance of tumours and cell lines to MAPK inhibitors can be predicted using a multiplex kinase assay and is associated with an increase in specific tyrosine kinase activities and globally to AKT signalling in the patient’s tissue. Thus, such a predictive kinome signature would help to identify patients with innate resistance to MAPK double inhibition in order to propose other therapies.

T-type calcium channel inhibition restores sensitivity to MAPK inhibitors in de-differentiated and adaptive melanoma cells

BackgroundDrug resistance remains as one of the major challenges in melanoma therapy. It is well known that tumour cells undergo phenotypic switching during melanoma progression, increasing melanoma plasticity and resistance to mitogen-activated protein kinase inhibitors (MAPKi).MethodsWe investigated the melanoma phenotype switching using a partial reprogramming model to de-differentiate murine melanoma cells and target melanoma therapy adaptation against MAPKi.ResultsHere, we show that partially reprogrammed cells are a less proliferative and more de-differentiated cell population, expressing a gene signature for stemness and suppressing melanocyte-specific markers. To investigate adaptation to MAPKi, cells were exposed to B-Raf Proto-Oncogene (BRAF) and mitogen-activated protein kinase kinase (MEK) inhibitors. De-differentiated cells became less sensitive to MAPKi, showed increased cell viability and decreased apoptosis. Furthermore, T-type calcium channels expression increased in adaptive murine cells and in human adaptive melanoma cells. Treatment with the calcium channel blocker mibefradil induced cell death, differentiation and susceptibility to MAPKi in vitro and in vivo.ConclusionIn summary, we show that partial reprogramming of melanoma cells induces de-differentiation and adaptation to MAPKi. Moreover, we postulated a calcium channel blocker such as mibefradil, as a potential candidate to restore sensitivity to MAPKi in adaptive melanoma cells.

Triterpene-Enriched Olive Extract as an Immunopotentiator in Black Sea Bream (Acanthopagrus schlegelii)

Recently, the use of natural immunopotentiators, such as plant extracts, is predicted to offer high disease-prevention potential in aquaculture. However, few reports on the immunomodulatory impacts of olive extract (OE) on teleost are available. Therefore, the effects of dietary intake of triterpene-enriched OE on black sea bream ( Acanthopagrus schlegelii ) were investigated in this study. Our data showed that total blood cell counts, in vivo content of lysozyme, activities of antiprotease and myeloperoxidase, and contents of IL-4 and IL-6 were significantly up-regulated by dietary intake of triterpene-enriched OE. Additionally, the hampered immune response induced by cadmium exposure was significantly mitigated by the administration of OE as were indicated by partially or completely rescued immune-relating parameters. Furthermore, the expressions of immune-related genes encoding NF-κB inhibitor alpha (IkBα), tumor necrosis factor α (TNFα), cyclooxygenase-2 (COX-2), and proto-oncogene protein c-fos (FOS) were found to be significantly up-regulated by the dietary intake of OE. In general, the results suggested that the dietary intake of triterpene-enriched OE has immune enhancing effect in black sea bream. Such effect may be realized by 1) increasing the total counts of diverse blood cells; 2) activating nonspecific immune biomolecules; and 3) affecting signaling pathways such as IKK and ERK and subsequently inducing IL-4 and IL-6 biosyntheses.

Expression and significance of phosphorylated protein kinase B and spleen tyrosine protein kinase in different TNM stages of gastric cancer patients

Objective To investigate the expression of phosphorylated protein kinase B (PKB/AKT) and spleen tyrosine protein kinase (Syk) in different tumor node metastasis (TNM) stages of gastric cancer patients. Methods From January 2015 to April 2018, 82 patients with gastric cancer confirmed by gastroscopy and surgical pathology were enrolled in this study. All patients were selected for cancer tissue, and 30 patients were randomly selected from normal gastric mucosa at 5 cm adjacent to the tumor. Immunohistochemistry was used to detect the expression of PKB/AKT and Syk protein. To compare the expression of PKB/AKT and Syk in gastric cancer and adjacent normal tissues, and to analyze the relationship between PKB/AKT and Syk expression and clinicopathological features in gastric cancer tissues, and the correlation between PKB/AKT and Syk and TNM staging of gastric cancer patients. Results The positive expression rate of PKB/AKT in gastric cancer tissues was higher than that in adjacent tissues (P<0.05). The positive expression rate of Syk was lower than that in adjacent tissues (P<0.05). PKB/Akt and Syk gene expression in gastric cancer were related to histological grade, tumor infiltration, TNM staging, lymph node metastasis and distant metastasis (P<0.05), and the expression of PKB/AKT was significantly increased with the increase of TNM staging in gastric cancer patients, and the positive expression of Syk was significantly decreased (P<0.05). Conclusions PKB/AKT is positively correlated with TNM staging of gastric cancer patients. Syk is negatively correlated with TNM staging of gastric cancer patients. The clinical expression of PKB/AKT and Syk can be used to determine the TNM staging of gastric cancer, which provides a strong basis for tumor treatment. It is of great significance in treatment. Key words: Stomach neoplasms; Neoplasm staging; Phosphorylation; Proto-oncogene proteins c-akt; Syk kinase

Involvement of proliferative and apoptotic factors in the development of hindgut in rat fetuses with ethylenethiourea-induced anorectal malformations

BackgroundAnorectal malformations (ARMs) are common congenital malformations of the terminal digestive tract, but little is known regarding their pathogenesis. Aberrant cell proliferation/apoptosis are believed to be involved in ARMs. However, there are no studies on proliferation/apoptosis-related genes. PurposeWe aimed to investigate the spatiotemporal expression patterns of two proliferation/apoptosis-related genes (MYC proto-oncogene and tumor protein p53) and explore their potential functions in the hindguts of ethylene thiourea-induced ARMs rat fetuses. MethodsMYC and p53 expression was evaluated using immunohistochemical staining, western blotting, and quantitative real-time polymerase chain reaction (RT-qPCR). Terminal deoxynucleotidyl transferase mediated dUTP nick end labeling (TUNEL) and p53 costaining were performed to assay the colocalization of apoptotic and p53-expressing cells. ResultsRat fetuses with ARMs displayed fusion failure of the urogenital septum and cloacal membrane. In the control group, MYC was persistently expressed from gestational day (GD)14 to GD16 and distributed throughout the hindgut, while p53 was weakly detected in the terminal segment of the urethra and hindgut; in the ARMs group, MYC expression was obviously reduced, while p53 was widely and highly expressed in the urethra and hindgut. Western blotting and RT-qPCR confirmed the decrease in MYC and increase in p53 expression in ARMs. TUNEL and p53 co-staining revealed considerable overlap between apoptotic and p53-expressing cells. ConclusionThe expression patterns of c-myc and p53 were disrupted in ARMs rat embryos, and the downregulation of c-myc and upregulation of p53 might be related to the development of ARMs at the key time points of ARMs morphogenesis.

Research progress of BRAFV600E and TERT promoter mutations coexistence in papillary thyroid cancer

Papillary thyroid cancer (PTC) is the most common pathological type of thyroid cancer, and its morbidity raises rapidly in the global world. V-raf murine sarcoma viral oncogene homolog B1 (BRAF)V600E mutation and telomerase reverse transcriptase (TERT) promoter mutations are the most common molecular markers of PTC. Nowadays, more and more studies find that the coexistence of BRAFV600E and TERT promoter mutations plays a synergistic role in the invasion and prognosis of PTC. This article reviews the recent advance in the synergism of BRAFV600E and TERT promoter mutations in PTC. Key words: Thyroid neoplasms; Mutation; Proto-oncogene proteins B-raf; Telomerase; Trends

MYC gene associated polymorphisms and Wilms tumor risk in Chinese children: a four-center case-control study.

Wilms tumor (WT) is a common embryonal malignancy in the kidney, ranking fourth in childhood cancer worldwide. MYC, a critical proto-oncogene, plays an important role in tumorigenesis. Single nucleotide polymorphisms in the MYC gene may lead to the deregulation of MYC proto-oncogene protein and thereby promote the initiation and development of tumors. Here, we assessed the association between MYC gene associated polymorphisms and WT susceptibility by performing a case-control study with 355 cases and 1070 controls. Two MYC gene associated polymorphisms (rs4645943 C > T, rs2070583 A > G) were genotyped by TaqMan technique. Odds ratios (ORs) and 95% confidence intervals (CIs) were used for evaluating the association between these two polymorphisms and WT susceptibility. No significant association was detected between the selected polymorphisms and WT risk in the overall analysis as well as stratification analysis. These results indicate that neither of two selected MYC gene associated polymorphisms might affect WT susceptibility in the Chinese population. Large well-designed studies with diverse ethnicities are warranted to verify these results.

Mechanism underlying the anti-apoptotic activity of pORF5 plasmid protein in Chlamydia trachomatis via high-mobility group box 1 protein: a preliminary study

Objective To explore the molecular mechanism underlying the anti-apoptotic activity of pORF5 plasmid protein of Chlamydia trachomatis, so as to provide an experimental basis for further clarifying the pathogenesis of Chlamydia trachomatis. Methods HeLa cells were divided into two groups: carbonyl cyanide m-chlorophenyl hydrazone (CCCP, an apoptosis inducer) group was stimulated by CCCP for 30 minutes, and pORF5 + CCCP group was pretreated with pORF5 plasmid protein for 18 hours followed by CCCP for 30 minutes. Then, Western blot analysis was performed to determine the expression of apoptosis-related proteins Bcl-2, Bax and caspase-3, JC-1 fluorescent probe was used to detect changes in the mitochondrial membrane potential in HeLa cells, and cytochrome c release from mitochondria was analyzed by indirect immunofluorescence assay. To analyze whether high-mobility group box 1 (HMGB1) protein participated in the anti-apoptotic role of pORF5 plasmid protein, HMGB1 shRNA and control RNA were separately transfected into the HeLa cells, which were then stimulated by pORF5 plasmid protein and CCCP. Then, the protein expression of Bcl-2, Bax, activated caspase-3 was determined, and cytochrome c release was analyzed. Data were compared between two groups by using paired t test. Results pORF5 plasmid protein could antagonize the CCCP-induced decrease of mitochondrial membrane potential, and the red/green fluorescence intensity ratio was significantly lower in the CCCP group (0.4 ± 0.1) than in the pORF5 + CCCP group (1.7 ± 0.3; t = 6.95, P < 0.01) . The protein expression of Bcl-2 in the HeLa cells in the pORF5 + CCCP group was 5.3 ± 0.6 times more than that in the CCCP group (t = 8.62, P < 0.01) , while the protein expression of Bax and activated caspase-3 in the pORF5 + CCCP group significantly decreased by 79% ± 10% (t = 9.23, P < 0.01) and 75% ± 8% (t = 4.26, P < 0.05) respectively compared with the CCCP group. Compared with the control RNA transfection group, the HMGB1 shRNA transfection group showed significantly decreased mitochondrial membrane potential in the HeLa cells (t = 11.23, P < 0.01) , increased cytochrome c release, decreased Bcl-2 expresson (t = 7.19, P < 0.05) and increased Bax expression (t = 13.06, P < 0.01) after stimulation with pORF5 and CCCP. Conclusion Chlamydia trachomatis plasmid protein pORF5 plays an anti-apoptosis role by blocking the mitochondrial apoptotic pathway through HMGB1 protein. Key words: Chlamydia trachomatis; Apoptosis; Proto-oncogene proteins c-bcl-2; Bcl-2-associated X protein; Caspase-3; High mobility group box protein 1; pORF5 plasmid protein

Identifying crucial genes for prognosis in septic patients: Gene integration study based on PRISMA guidelines.

Background:Sepsis is a serious clinical condition with a poor prognosis, despite improvements in diagnosis and treatment.Therefore, novel biomarkers are necessary that can help with estimating prognosis and improving clinical outcomes of patients with sepsis.Methods:The gene expression profiles GSE54514 and GSE63042 were downloaded from the GEO database. DEGs were screened by t test after logarithmization of raw data; then, the common DEGs between the 2 gene expression profiles were identified by up-regulation and down-regulation intersection. The DEGs were analyzed using bioinformatics, and a protein-protein interaction (PPI) survival network was constructed using STRING. Survival curves were constructed to explore the relationship between core genes and the prognosis of sepsis patients based on GSE54514 data.Results:A total of 688 common DEGs were identified between survivors and non-survivors of sepsis, and 96 genes were involved in survival networks. The crucial genes Signal transducer and activator of transcription 5A (STAT5A), CCAAT/enhancer-binding protein beta (CEBPB), Myc proto-oncogene protein (MYC), and REL-associated protein (RELA) were identified and showed increased expression in sepsis survivors. These crucial genes had a positive correlation with patients’ survival time according to the survival analysis.Conclusions:Our findings indicate that the genes STAT5A, CEBPB, MYC, and RELA may be important in predicting the prognosis of sepsis patients.

From the Editor’s desk…: August 2019

From the Editor’s desk…: August 2019

Mesenchymal stem cell therapy improves erectile dysfunction in experimental spinal cord injury.

The aim of this study is to investigate the therapeutic potential of adipose-derived mesenchymal stem cell (AD-MSC) from brown adipose tissue on erectile dysfunction (ED) in experimentally induced spinal cord injury in rats. 24 male Wistar rats were divided into 3 groups; control, spinal cord injury (SCI) + vehicle, and SCI + AD-MSC. To induce SCI, a standard weight-drop method that induced a moderate to severe injury (100 g/cm force) at T7-T10, was used. AD-MSC (3 × 105 cells /5 μL) was applied by local transplantation into the region of injury. At the end of four-weeks, rats underwent neurological examinations and then intracavernosal and mean arterial pressures (ICP and MAP) measurements. After decapitation, spinal cord and cavernosal tissue samples were taken to analyze neuronal nitric oxide synthase (n-NOS), proto-oncogene protein c-FOS and nerve growth factor (NGF). Tissues were also examined histologically. Spinal cord injury caused decrease on NGF and n-NOS levels while c-FOS was increased. The ICP/MAP value in vehicle-treated SCI rats was found to be significantly higher than the control group. On the other hand, in SCI + AD-MSC group, all these parameters were reversed back to control levels. AD-MSC therapy may be beneficial against erectile dysfunction in experimentally induced SCI by ameliorating neuronal damage.

Gastrointestinal Stromal Tumors of Small Intestine.

Gastrointestinal stromal tumor (GIST) is defined as mesenchymal tumors of the gastrointestinal tract expressing proto-oncogene protein CD117. They are the most common sarcomatous tumors of the gastrointestinal tract. GISTs are presumed to arise from interstitial cells of Cajal or gastrointestinal pacemaker cells which control gut motility. They have unpredictable biological behavior. Prognosis is dependent on tumor size as well as mitotic count. Radical surgical excision is the treatment of choice. They rarely metastasize to lymph nodes. Imatinib therapy is used as an adjuvant therapy. The follow-up of patients postsurgery is not standardized.

A comparison between cDNA and DNA samples for the detection of c-KIT exon 17 mutation in acute myeloid leukemia patients

Objective To investigate the impact of sample typeon the detection of c-KIT exon 17 mutation in acute myeloid leukemia (AML) patients. Methods A retrospective study was conducted on 51 bone marrow samples collected from 37 AML patients [17 maleand 20 female, with a median age of 33 (range from 1 to 82)] at diagnosis or after treatment from June 2016 to August 2018. Of the 37 cases of AML, 24 were t(8; 21) AML, 11 were inv(16)/t(16;16) AML and 2 were non-CBF-AML. RNA and DNA were simultaneously extracted from every sample. PCR followed by Sanger sequencing were used to screen c-KIT exon 17 mutation, and the comparisons were made between paired cDNA and DNAsamples. Results (1) Of the 51 paired samples, 14 pairs were simultaneously detected positive for c-KITmutation in both of cDNA and DNA samples, but 17 pairs were detected negative in both, and the remaining 20 pairswere only detected positive for the mutation in cDNA but not in DNA, with an inconsistency rate of 39.2%. The positive rate of detecting c-KITmutation was significantly higher in cDNA than in DNA samples (66.7% vs 27.5%,P=0.000 073). (2)Inconsistent mutation results between paired cDNA and DNA samples occurred in t(8;21)AML, inv(16)AML and non-CBF-AML patients with the inconsistency rate of 36.4% (12/33), 27.2% (3/11) and 71.4% (5/7), respectively. (3)The inconsistency rate was significantly higher in samples collected after treatment compared with those collected at diagnosis (72.7% vs 13.8%, P=0.000 03). (4) All 5 serially monitored patients with c-KITmutation had inconsistency in mutation detection between cDNA and DNA samples during follow up. Conclusion cDNA improves the detection of c-KIT exon 17 mutation in AML patients compared with DNA, which is especially common after treatment. Key words: Leukemia, myeloid, acute; Proto-oncogene proteins c-kit; Exons; Mutation; DNA,Complementary; DNA

Fitness of unregulated human Ras mutants modeled by implementing computational mutagenesis and machine learning techniques

Ras proteins play a pivotal role as oncogenes by participating in diverse signaling events, including those linked to cell growth, differentiation, and proliferation. Using experimental fitness data and implementing artificial intelligence and a computational mutagenesis technique, we developed models that reliably predict fitness for all single residue mutants of H-ras proto-oncogene protein p21. The computational mutagenesis generated a feature vector of protein structural changes for each variant, and these data correlated well with fitness. Random forest classification and tree regression machine learning algorithms were implemented for training predictive models. Cross-validations were used to evaluate model performance, and control experiments were performed to assess statistical significance. Classification models revealed a balanced accuracy rate as high as 82%, with a Matthew's correlation of 0.63, and an area under ROC curve of 0.90. Similarly, regression models displayed Pearson's correlation reaching 0.79. On the other hand, control data sets led to performance values consistent with random guessing. Comparisons with several related state-of-the-art methods reflected favorably on our trained models. This H-Ras proof-of-principle study suggests a complementary approach for understanding mechanisms with which other proteins are involved in oncogenesis, including related Ras isoforms, and for providing useful insights into designing future diagnostic and treatment modalities.

Transcriptome analysis revealed ameliorative effect of probiotic Lactobacillus johnsonii BS15 against subclinical necrotic enteritis induced hepatic inflammation in broilers

Subclinical necrotic enteritis (SNE) broadly occurs in boilers, which reduces the growth performance by causing serious economic and social problems. The following study was conducted to better understand the molecular mechanism of the SNE on liver inflammation and to examine the innovative prevention of Lactobacillus johnsonii BS15 upon SNE. The research was based on the regulatory molecular mechanism of Lactobacillus johnsonii BS15, and its effect on liver inflammatory pathways in the broiler with SNE infection. Day old one hundred and eighty (Cobb 500) broiler chickens were distributed into 3 groups (control, SNE and BS15 group) and reared for 28 days. RNA sequencing was used for the analysis of gene expression extracted from liver samples. Gene expression was detected with the help of quantitative real-time PCR (qRT-PCR). RNA-Seq analysis revealed altered expressions of genes involved in liver inflammatory pathway. A total number of 385 genes were found as differentially expressed (DEGs) in the liver samples that belonged to SNE group as compared with the control liver samples (p < 0.05). Out of those 385 genes, 117 were down-regulated and 268 were up-regulated. The DEGs related to liver inflammation between control group and SNE group or SNE and BS15 groups, included cluster of differentiation 80 (CD80), Interleukin 1 beta (IL1B), Phosphoinositide 3- Kinase regulatory subunit 5 (PIK3R5), Toll-like receptor 4 (TLR4), Toll-like receptor 2 A (TLR2A), and proto-oncogene protein (FOS). The RNA-Seq analysis provided DEGs expression and this result was validated by qRT-PCR. Results confirmed that these genes are essential in the regulation of liver inflammation in the SNE infected chickens. Findings of current research indicated that the hepatic inflammation could be induced by SNE in broilers. Simultaneously, effects of SNE infection on liver could be subsided by improved TLRs signaling pathway with the naturally present prophylactic strategy as BS15.

Expression patterns and immunohistochemical analysis of ERK, HRAS and MEK1 proteins during ovarian prehierarchical follicular development in Zi geese (Anser cygnoides)

The objective of this study was to investigate the spatiotemporal expression levels and protein localization of extracellular regulated MAP kinase (ERK), HRas proto-oncogene, GTPase (HRAS), and mitogen-activated protein kinase kinase 1 (MEK1) genes in ovarian prehierarchical follicles of geese. The prehierarchical follicles from healthy laying geese (n=6) at the age of 35 to 37 weeks were harvested. The relative expression levels of ERK, HRAS, and MEK1 in various sized prehierarchical follicles were detected by real-time quantitative polymerase chain reaction (RT-qPCR), western blotting, and follicular wall localization was investigated by using immunohistochemistry. The results revealed that the candidate genes were expressed differently at mRNA and protein levels at five stages of prehierarchical follicle development. These results suggest that ERK, HRAS, and MEK1 might be associated to the key biological mechanisms regulating Zi geese folliculogenesis.

DNA Methylation and Transcription Factors Competitively Regulate SIRT4 Promoter Activity in Bovine Adipocytes: Roles of NRF1 and CMYB.

Sirtuin 4 (SIRT4) belongs to the mitochondrial sirtuin protein family, a class of NAD+-dependent protein deacylases that remove post-translational acyl modifications from cellular substrates during the regulation of various biological pathways. SIRT4 has been shown to regulate lipid homeostasis. However, the mechanism by which the bovine SIRT4 gene is transcriptionally regulated remains unknown. To explore the molecular mechanism of SIRT4 expression, we obtained a 400-kb fragment of the 5'-regulatory region of bovine SIRT4 by molecular cloning, which contained a CpG island. Electrophoretic mobility shift assays and luciferase reporter gene assays identified the nuclear respiratory factor 1 (NRF1) and myb proto-oncogene protein (CMYB) binding sites as transcriptional repression and activation sites in the SIRT4 promoter region, respectively. We further verified that NRF1 and CMYB bind to the SIRT4 promoter using chromatin immunoprecipitation assays. In addition, from DNA methylation and reporter gene assays, results revealed that SIRT4 promoter activity was enhanced by demethylation. Further, NRF1-mediated transcriptional inhibition and CMYB-mediated transcriptional activation of SIRT4 expression were strengthened by demethylation during bovine adipocyte differentiation. Taken together, our results shed light on the mechanism underlying the promoter methylation and transcriptional regulation of SIRT4 expression in bovine adipocytes.

MMP-1 is overexpressed in triple-negative breast cancer tissues and the knockdown of MMP-1 expression inhibits tumor cell malignant behaviors in vitro.

Matrix metalloproteinase 1 (MMP-1) is a member of the zinc-dependent endopeptidase family, which cleaves the extracellular matrix. The present study investigated the functional role of MMP-1 in breast cancer ex vivo and in vitro in order to determine the underlying molecular mechanisms. The levels of MMP-1 were analyzed in 99 breast cancer specimens using immunohistochemistry and western blotting. A stable short hairpin RNA (shRNA) knockdown of MMP-1 expression was performed in MCF-7 and MDA-MB-231 breast cancer cells, and the effects were examined using MTT and colony formation assays, as well as migration and invasion assays, while western blotting was used to detect the activation of intracellular signaling. The MMP-1 protein was more highly expressed in triple-negative breast cancer tissues than in estrogen receptor(+) and human epidermal growth factor 2 receptor(3+) breast cancer tissues (P<0.05). Furthermore, the MMP-1 levels were significantly higher in the tumor and tumor stromal cells of lymph node metastatic breast cancer tissues than in those of non-metastatic tissues. The knockdown of MMP-1 expression in MCF-7 and MDA-MB-231 cells using MMP-1 shRNA significantly inhibited cell proliferation, migration and invasion, and the expression of the Myc proto-oncogene protein, phosphorylated and total RAC-α serine/threonine-protein kinase 1, and B-cell lymphoma 2, but increased the protein levels of apoptosis regulator BAX and caspase 3. In conclusion, the data suggest that MMP-1 serves an important role in breast cancer development and metastasis. Future studies should assess MMP-1 as a prognostic marker for patients with breast cancer and its inhibition as a novel strategy for controlling breast cancer.

Prognostic impact of MYD88 mutation, proliferative index and cell origin in diffuse large B cell lymphoma

BackgroundDiffuse large B-cell lymphoma, among non-Hodgkin lymphomas, is one of the most frequent subtypes. Clinical laboratory data and post-treatment outcomes are scarce in the Brazilian population. ObjectiveThe main objective of this retrospective study was to assess the impact of tumor markers, including the Myeloid differentiation primary response 88 (MYD88) mutation. MethodEighty-three patients were included and treated with R-CHOP or R-CHOP-like regimens. ResultsMedian age was 64-years old and 58% were female patients. The median follow-up was 42 months. The progression free survival (PFS) at this time was 63% and overall survival (OS), 66%. In the patients with tumors expressing Myc proto-oncogene protein (MYC) and B-cell lymphoma 2 (BCL2), assessed by immunohistochemistry (IHC), known as dual protein expressers, median post-progression survival was 31 (15–45) months. An increased proliferative index were associated with a high rate of progression (hazard ratio 2.31 [95% confidence interval [1.05–5.12]; p=0.04). The cell of origin (COO), identified by IHC, was not able to predict PFS (p=0.76). The MYD88 L265P mutation was present in 10.8% (9/83) of patients and did not show a prognostic correlation. ConclusionIn conclusion, the MYD88 mutation, although an important tool for diagnosis and a possible target drug, presented at a low frequency and was not a prognostic marker in this population.

Coordinate regulation by transcription factors and DNA methylation in the core promoter region of SIRT6 in bovine adipocytes

Sirtuin6 (SIRT6) is an ADP-ribosyltransferase and NAD+-dependent deacylase of acetyl groups and long-chain fatty acyl groups, and has been shown as a regulator of insulin secretion, glucose metabolism, lipid metabolism, and cancer. In this study, we determined that the bovine SIRT6 showed higher levels of mRNA expression in the testis, longissimus thoracis, and subcutaneous fat tissue. To elucidate the molecular regulation mechanism of bovine SIRT6 expression, we obtained a 2-kb fragment containing the 5′-regulatory region, and the functional proximal minimal promoter of bovine SIRT6 was identified in the −472/−73 bp region. The CCAAT enhancer binding protein beta (CEBPβ), paired box 6 (PAX6), Kruppel-like factor 2 (KLF2), myb proto-oncogene protein (CMYB), nuclear respiratory factor 1 (NRF1), and E2F transcription factor 1 (E2F1) binding sites, as transcriptional activators or repressors in the core promoter region of SIRT6, were determined by electrophoretic mobility shift assay (EMSA) experiments and luciferase reporter assays. In addition, the results from methylation assay and luciferase report assay showed that the bovine SIRT6 promoter activity was coordinately regulated by methylation and NRF1 or E2F1 during bovine adipocyte differentiation. Taken together, this study illuminated the underlying mechanism of methylation and transcription regulation of SIRT6 expression in bovine adipocytes.