Long non-coding RNA00461 regulates apoptosis in triple-negative breast cancer cells by combining with the proto-oncogene c-Myc / 中华内分泌外科杂志

Objective:To investigate the effect of long non-coding RNA00461 (LINC00461) on apoptosis of triple negative breast cancer cells.Methods:RT-qPCR was used to detect the expression of LINC00461 in normal breast epithelial cells MCF-10A, TNBC cell lines MDA-MB-231 and MDA-MB-468. The apoptosis rate was detected by Annexin V-FITC/PI double staining cell apoptosis kit. Western blot was used to detect apoptosis-associated protein. The localization of LINC00461 in cells was detected by FISH. RNA pulldown and RIP were used to detect the interaction between LINC00461 and c-Myc. The effect of LINC00461 on TNBC apoptosis was verified by subcutaneous tumorigenesis in nude mice.Results:Compared with MCF-10A, LINC00461 expression in MDA-MB-231 and MDA-MB-468 cells was significantly increased. Interference with LINC00461 significantly reduced the expression of LINC00461 and c-Myc protein. Overexpression of LINC00461 significantly decreased the expression level of apoptosis-related protein. Overexpression of LINC00461 significantly reduced the apoptosis rate by 50%-70%, with a statistically significant difference. FISH results showed that LINC00461 was mainly localized in the cytoplasm. RNA pulldown results showed that LINC00461 interacted with c-Myc. RIP results showed that the concentration of LINC00461 in c-Myc group was more than 400%, and the difference was statistically significant. In addition, overexpression of c-Myc reduced the promotion effect of knockdown LINC00461 on the apoptosis rate of 30%-50%. In animal experiments, overexpression of LINC00461 significantly increased tumor volume (50%-80%) and tumor weight (30%-50%) .Conclusion:LINC00461, mainly located in the cytoplasm, regulates the apoptosis of TNBC cells, and its molecular mechanism may be that LINC00461 interacts with c-Myc protein to play an important role in regulating the apoptosis of TNBC cells.

Effects of Decitabine Combined with Anlotinib on Proliferation and Apoptosis of Multiple Myeloma Cells / 中国实验血液学杂志

OBJECTIVE@#To investigate the biological effects and its relative mechanism of decitabine combined with anlotinib on multiple myeloma cells.@*METHODS@#The human MM cell lines and primary cells were treated with different concentrations of decitabine, anlotinib, and decitabine+anlotinib, respectively. The cell viability was detected and combination effect was calculated by CCK-8 assay. The apoptosis rate was measured by flow cytometry and the level of c-Myc protein was determined by Western blot.@*RESULTS@#Both decitabine and anlotinib could effectively inhibit the proliferation and induce the apoptosis of MM cell lines NCI-H929 and RPMI-8226. The effect of combined treatment on the inhibition of cell proliferation and induction of apoptosis was stronger than that of single-drug treatment. The combination of the two drugs also showed strong cytotoxicity in primary MM cells. Decitabine and anlotinib could down-regulate the level of c-Myc protein in MM cells and the c-Myc level in the combination group was the lowest.@*CONCLUSION@#Decitabine combined with anlotinib can effectively inhibit the proliferation and induce apoptosis of MM cells, which provides a certain experimental basis for the treatment of human MM.

The differential effect of AICAR on c-Myc expression and proliferation in specific tumor cell lines / 中国药理学通报

Aim To investigate the effect of AICAR on the expression of the proto-oncogene c-Myc and cell proliferation rates in specific cancer cell lines. Methods The mRNA levels of c-Myc were evaluated using fluorescence-based qRT-PCR to examine the effect of AICAR treatment on c-Myc mRNA expression levels. Western blot was used to evaluate the protein levels of c-Myc following AICAR treatment. RNA interference was employed to determine whether the regulatory effect of AICAR on c-Myc was dependent on AMPK and the downstream metabolic enzymes relating to AICAR. Actinomycin D and cycloheximide were used to assess the effect of AICAR on the stability of cMyc mR-NA and protein. Western blot was used to examine the regulatory effect of AICAR on c-Myc in various cancer cell lines. The MTT assay was used to determine the effect of AICAR on cell viability in these cell lines. Results AICAR significantly up-regulated c-Myc at both mRNA and protein levels. The protein level of c-Myc reached a plateau 12 h after the AICAR treatment. The up-regulatory effect of c-Myc induced by AICAR was not dependent on either the AMPK signaling pathway or the downstream metabolites of AICAR. AICAR could significantly enhance the mRNA stability of c-Myc but did not affect the protein stability. The up-regulation of c-Myc induced by AICAR was cell-type specific. AICAR up-regulated c-Myc in SW1990, 786-0, and A549, while down-regulated c-Myc in HepG2, MCF7, and U20S. In HepG2 cells, AICAR treatment decreased cell viability. However, in SW1990 and A549 cells, AICAR treatment did not lead to any significant difference in cell viability. AICAR decreased the cell viability only when c-Myc was knocked down in SW1990 and A549 cells. Conclusions AICAR directly up-regulates c-Myc expression in an AMPK-independent manner. The up-regulation effect is cell-type dependent. The regulation of c-Myc expression by AICAR is linked to the inhibitory effect of AICAR on tumor cell proliferation.

Correlation between expression of Lin28B and C-myc in patients with laryngeal squamous cell carcinoma and clinicopathological features and prognosis / 中南大学学报(医学版)

OBJECTIVES@#Laryngeal squamous cell carcinoma (LSCC) is a common malignant tumor of head and neck. Screening of target genes for malignant tumor therapy is one of the focuses of cancer research, with proto-oncogene and tumor suppressor gene as the breakthrough. It has become an urgent need to find the target gene related to the treatment and prognosis of LSCC.This study aims to explore the role of Lin28B and C-myc in LSCC by detecting the expressions of these two proteins and analyze the correlation between the expression of Lin28B and C-myc and clinicopathological features and prognosis of LSCC.@*METHODS@#We detected the expression of Lin28B and C-myc proteins in 102 specimens of LSCC and 90 specimens of adjacent tissues by immunochemistry, and analyzed the correlation between Lin28B and C-myc protein expressions in LSCC as well as the correlation between the expressions of the two proteins and the clinicopathological features of LSCC. At the same time, the Kaplan-Meier method was used to analyze the relation between Lin28B and C-myc protein levels with the postoperative survival rate of LSCC patients.@*RESULTS@#The protein levels of Lin28B and C-myc in the LSCC tissnes were significantly higher than those in the adjacent tissues (both P<0.05),and there was a positive correlation between the expression of Lin28B and C-myc in LSCC (r＝0.476, P<0.05). The expression of Lin28B protein was closely related to age, lymph node metastasis, clinical stage, tumor size, and pathological differentiation of LSCC patients (all P<0.05). while the expression of C-myc protein was closely related to lymph node metastasis, clinical stage, tumor size, and pathological differentiation of LSCC patients (all P<0.05). A relevant survival analysis showed that in patients with higher level of Lin28B (P＝0.001) or C-myc protein (P<0.001), the postoperative survival rate was relatively low.@*CONCLUSIONS@#Lin28B and C-myc proteins are highly expressed in LSCC with a positive correlation. Furthermore, they are closely related to lymph node metastasis, clinical stage, tumor size, pathological differentiation and prognosis, suggesting that both Lin28B and C-myc might be involved in the occurrence and development of LSCC.

Study on the Biological Function of Abemecilib in Inhibiting the Proliferation, Invasion and Migration of Small Cell Lung Cancer with High c-Myc Expression / 中国肺癌杂志

BACKGROUND@#Small cell lung cancer (SCLC) with high c-Myc expression is prone to relapse and metastasis, leading to extremely low survival rate. Cyclin-dependent kinases 4 and 6 (CDK4/6) inhibitor Abemaciclib plays a key role in the treatment of tumors, but the effects and mechanisms on SCLC remain unclear. This study was to analyze the effect and molecular mechanism of Abemaciclib in inhibiting proliferation, migration and invasion of SCLC with high c-Myc expression, with a view to expanding a new direction for reducing the recurrence and metastasis.@*METHODS@#Proteins interacting with CDK4/6 were predicted using the STRING database. The expressions of CDK4/6 and c-Myc in 31 cases of SCLC cancer tissues and paired adjacent normal tissues were analyzed by immunohistochemistry. The effects of Abemaciclib on the proliferation, invasion and migration of SCLC were detected by CCK-8, colony formation assay, Transwell and migration assay. Western blot was used to detect the expressions of CDK4/6 and related transcription factors. Flow cytometry was used to analyze the effects of Abemaciclib on the cell cycle and checkpoint of SCLC.@*RESULTS@#The expression of CDK4/6 was associated with c-Myc by STRING protein interaction network. c-Myc can directly modalize achaete-scute complex homolog 1 (ASCL1), neuronal differentiation 1 (NEUROD1) and Yes-associated protein 1 (YAP1). Moreover, CDK4 and c-Myc regulate the expression of programmed cell death ligand 1 (PD-L1). Immunohistochemistry showed that the expressions of CDK4/6 and c-Myc in cancer tissues were higher than those in adjacent tissues(P<0.0001). CCK-8, colony formation assay, Transwell and migration assay verified that Abemaciclib could effectively inhibit the proliferation, invasion and migration of SBC-2 and H446OE(P<0.0001). Western blot analysis further showed that Abemaciclib not only inhibited CDK4 (P<0.05) and CDK6 (P<0.05), but also affected c-Myc (P<0.05), ASCL1 (P<0.05), NEUROD1 (P<0.05) and YAP1 (P<0.05), which are related to SCLC invasion and metastasis. Flow cytometry showed that Abemaciclib not only inhibited the cell cycle progression of SCLC cells (P<0.0001), but also significantly increased PD-L1 expression on SBC-2 (P<0.01) and H446OE (P<0.001).@*CONCLUSIONS@#Abemaciclib significantly inhibits the proliferation, invasion, migration and cell cycle progression of SCLC by inhibiting the expressions of CDK4/6, c-Myc, ASCL1, YAP1 and NEUROD1. Abemaciclib can also increase the expression of PD-L1 in SCLC.

Atypical MYC rearrangement pattern of 3? deletion and 5? amplification along with independent IGH rearrangement: A case study

A spectrum of Cellular homolog of the v-myc oncogene (cMYC) alterations such as translocation, overexpression, mutation, and amplification plays an important role in lymphomagenesis, particularly in high-grade lymphomas, and are associated with prognostic significance. Accurate identification of cMYC gene alteration is important for diagnostic, prognostic, and therapeutic implications. With the application of different FISH (fluorescence in situ hybridization) probes that helped overcome the analytical diagnostic challenges as a result of variant patterns, we report rare, concomitant, and independent gene alterations in cMYC and Immunoglobulin heavy-chain gene (IGH) with detailed characterization of its variant rearrangement. Short-term follow-up post R-CHOP (rituximab plus cyclophosphamide, doxorubicin, vincristine, and prednisone) therapy seemed to be favorable. Accumulation of many more literature studies on such cases with their therapeutic implications would lead to the categorization of these cases as a separate subclass in large B-cell lymphomas followed by molecular targeted therapy.

c-MYC-mediated TRIB3/P62+ aggresomes accumulation triggers paraptosis upon the combination of everolimus and ginsenoside Rh2

The mammalian target of rapamycin (mTOR) pathway is abnormally activated in lung cancer. However, the anti-lung cancer effect of mTOR inhibitors as monotherapy is modest. Here, we identified that ginsenoside Rh2, an active component of Panax ginseng C. A. Mey., enhanced the anti-cancer effect of the mTOR inhibitor everolimus both in vitro and in vivo. Moreover, ginsenoside Rh2 alleviated the hepatic fat accumulation caused by everolimus in xenograft nude mice models. The combination of everolimus and ginsenoside Rh2 (labeled Eve-Rh2) induced caspase-independent cell death and cytoplasmic vacuolation in lung cancer cells, indicating that Eve-Rh2 prevented tumor progression by triggering paraptosis. Eve-Rh2 up-regulated the expression of c-MYC in cancer cells as well as tumor tissues. The increased c-MYC mediated the accumulation of tribbles homolog 3 (TRIB3)/P62+ aggresomes and consequently triggered paraptosis, bypassing the classical c-MYC/MAX pathway. Our study offers a potential effective and safe strategy for the treatment of lung cancer. Moreover, we have identified a new mechanism of TRIB3/P62+ aggresomes-triggered paraptosis and revealed a unique function of c-MYC.

Juglone induces proliferation inhibition and apoptosis of cervical cancer cells via promoting c-Myc ubiquitination / 南方医科大学学报

OBJECTIVE@#To observe the expression of c-Myc protein in cervical cancer HeLa cells and explore the effect of juglone on the proliferation and apoptosis of HeLa cells by affecting c-Myc ubiquitination.@*METHODS@#HeLa cells treated with different concentrations (0, 10, 20, or 50 μmol/L) of juglone or with 20 μmol/L juglone for different time lengths were examined for expression of c-Myc protein with Western blotting. The half-life of c-Myc protein was determined using cycloheximide (CHX) and c-Myc protein degradation was detected using coimmunoprecipitation. We also assessed the effects of 20 μmol/L juglone combined with 0, 1.0 or 2.0 μmol/L MG132 (a proteasome inhibitor) on c-Myc expression. The effects of 20 μmol/L juglone on the proliferation and apoptosis of HeLa cells with RNA interference-mediated knockdown of c-Myc were evaluated with MTT assay and flow cytometry.@*RESULTS@#Treatment with juglone significantly lowered c-Myc protein expression in HeLa cells in a concentration-and time-dependent manner (P < 0.05). Juglone obviously shortened the half-life of c-Myc protein, and the addition of MG132 significantly up-regulated the expression level of c-Myc protein (P < 0.05). Juglone treatment also promoted ubiquitination of c-Myc protein in HeLa cells. Compared with the cells transfected with a negative control construct, the cells transfected with si-c-Myc showed significantly decreased proliferation inhibition and a lowered cell rate with early apoptosis after juglone treatment (P < 0.05).@*CONCLUSION@#Juglone inhibits proliferation and promotes apoptosis of HeLa cells by affecting the ubiquitination of c-Myc protein.

Expression Level and Correlation of miR-211, miR-155, C-myc in Acute T Lymphocytic Leukemia / 中国实验血液学杂志

OBJECTIVE@#To investigate the expression and correlation of miR-211, miR-155, and C-myc in acute T lymphocytic leukemia (T-ALL), aiming to provide evidence for the diagnosis and treatment.@*METHODS@#A total of 96 T-ALL patients who were diagnosed and treated in People's Hospital of Zhengzhou from June 2014 to June 2017 were selected, and 69 healthy volunteers who had a physical examination were selected as control group in the same period. Real-time fluorescent quantitative PCR (RT-PCR) was used to determine the expression levels of miR-211, miR-155, and C-myc in peripheral blood mononuclear cells in each group. Kaplan-Meier was used to analyze the survival of T-ALL patients and correlation of miR-211, miR-155, and C-myc with prognosis. Pearson correlation analysis was used to evaluate the correlation of miR-211, miR-155, and C-myc with disease risk.@*RESULTS@#The expression levels of miR-211 mRNA, miR-155 mRNA, and C-myc mRNA in T-ALL group were higher than those in the control group (P<0.01), those in non-remission group were higher than those in remission group (P<0.01), and those in high-risk group were also higher than those in low-risk group and intermediate-risk group (P<0.01). The survival time of T-ALL patients with low miR-211 expression was longer than that with high miR-211 expression (P<0.01), that with low miR-155 expression was longer than that with high miR-155 expression (P<0.01), and that with low C-myc expression was also longer than that with high C-myc expression (P<0.01). The high expression of miR-211, miR-155, and C-myc was linearly positively correlated with high risk of disease (r=0.749, 0.781, 0.804).@*CONCLUSION@#The expressions of miR-211, miR-155, and C-myc are up-regulated in T-ALL patients, closely related to prognosis, and linearly positively correlated with disease risk.

Cancer Treatment Strategies by Targeting the Oncoprotein c-Myc / 中国生物化学与分子生物学报

c-Myc is a transcription factor involved in the Myc/Max/Mxd signal regulatory network. c-Myc plays important roles in human development and the oncogenesis or progression of different types of tumors. Current studies have shown that c-Myc mutations or expression changes are present in more than 70% of tumors. Therefore, c-Myc-targeted inhibitors might become a new strategy for tumor therapy. Currently, there is no clinical treatment for targeting c-Myc. With the continuous study aiming at the c-Myc targeted clinical application, Omomyc has become a representative c-Myc inhibitor by direct inhibition of c-Myc in tumors, which may be a feasible clinical treatment strategy. Although targeting c-Myc has broad prospects in cancer treatment, direct inhibition of c-Myc still has many risks and challenges. Therefore, in this review, firstly, we will summarize the regulatory network and biological functions of c-Myc in cells. Secondly, the potential significance of targeting c-Myc or its homologues in tumor therapy will be discussed. Additionally, the challenges faced by c-Myc as a potential therapeutic target in clinical application will be summarized. Finally, we will also discuss the advantages and disadvantages of some c-Myc inhibitors that have been discovered to date, such as small molecule inhibitors and protein and peptide inhibitors, therefore providing the theoretical basis for c-Myc targeted clinical therapy in cancer.

Effects of MRE11 on Apoptosis and Proliferation of Esophageal Squamous Cancer Cells / 肿瘤防治研究

Objective To investigate the effect of MRE11 on the proliferation and apoptosis of esophageal squamous cancer cells and its molecular mechanism. Methods MRE11 expression was downregulated by MRE11 siRNA transfection in esophageal squamous cancer cells. The AKT agonist SC79 (0, 0.1, 0.5, 1, 1.5, 1.8, 2 μg/ml) were used to treat cells with MRE11 inhibition for 24 h. Overexpression vector pcDNA.3.1-c-myc was constructed and co-transfected cells with MRE11 siRNA. Western blot method was used to detect the protein expressions of MRE11, p-AKT and c-myc in esophageal squamous cancer cells Ec9706 and TE-1. The Annexin-V FITC/PI kit was used to detect the apoptosis of Ec9706 and TE-1 cells; the activity of caspase-3 was detected by the Caspase-3 activity detection kit; the proliferation of Ec9706 and TE-1 cells was tested by the BrdU method. Results The protein expressions of MRE11 in Ec9706 and TE-1 cells were significantly increased, compared with human esophageal epithelial Het-1A cells. After MRE11 siRNA transfection, AKT phosphorylation and the protein expressions of MRE11 and c-myc were significantly decreased in esophageal squamous cancer cells. MRE11 inhibition significantly promoted the apoptosis and caspase-3 activity in Ec9706 and TE-1 cells, while inhibited the proliferation of Ec9706 and TE-1 cells. SC79 (1.5, 1.8 and 2 μg/ml) significantly increased AKT phosphorylation in MRE11-suppressed esophageal squamous cancer cells, and reversed the inhibitory effects of MRE11 inhibition on c-myc protein expression and cell proliferation and the promoting effect on cell apoptosis. Overexpression of c-myc inhibited the inhibitory effect of MRE11 down-regulation on cell proliferation and the promotion on caspase-3 activity. Conclusion MRE11 inhibition could effectively inhibit the proliferation of esophageal squamous cancer cells and promote cell apoptosis by regulating AKT and c-myc.

Are stem cell marker expression and cd133 analysis relevant to differentiate colorectal cancer? / A expressão de marcadores de células-tronco e a análise do cd133 são relevantes na diferenciação do câncer colorretal?

ABSTRACT Background: CD133 and AXL have been described as cancer stem cell markers, and c-MYC as a key regulatory cellular mechanism in colorectal cancer (CRC). Aim: Evaluate the prognostic role of the biomarkers CD133, AXL and c-MYC and their association with clinicopathologic characteristics in colorectal adenocarcinomas and adenomas. Methods: A total of 156 patients with UICC stage I-IV adenocarcinomas (n=122) and adenomas (n=34) were analyzed. Tissue microarrays (TMA) from primary tumors and polyps for CD133, c-MYC and AXL expression were performed and analyzed for their significance with clinicopathologic characteristics. Results: Poorly differentiated adenocarcinomas and disease progression were independent risk factors for poor overall survival. The median overall survival time was 30 months. Positive CD133 expression (35.9% of all cases), particularly of right-sided CRCs (44.8% of the CD133+ cases), was negatively correlated with death in the univariate analysis, which did not reach significance in the multivariate analysis. c-MYC (15.4% of all cases) was predominantly expressed in advanced-stage patients with distant (non-pulmonary/non-hepatic) metastasis. AXL expression was found only occasionally, and predominantly dominated in adenomas, with less penetrance in high-grade dysplasia. Conclusions: CD133 expression was not associated with inferior overall survival in CRC. While AXL showed inconclusive results, c-MYC expression in primary CRCs was associated with distant metastasis.

RESUMO Racional: CD133 e AXL são descritos na literatura como marcadores de células-tronco tumorais, e c-MYC cumpre papel chave como mecanismo de regulação celular no câncer colorretal (CCR). Objetivo: Avaliar o papel prognóstico dos biomarcadores CD133, AXL e c-MYC e sua associação com características clinicopatológicas de adenocarcinomas e adenomas colorretais. Métodos: Um total de 156 pacientes com adenocarcinomas de estádio UICC I-IV (n=122) e adenomas (n=34) colorretais foram avaliados. Microarranjos teciduais (TMA) dos tumores primários e adenomas foram realizados em busca de expressão de CD133, c-MYC e AXL, com posterior análise de relação significativa com características clinicopatológicas. Resultados: Adenocarcinomas pobremente diferenciados e progressão de doença foram fatores de risco independentes para má sobrevida global. A taxa mediana de sobrevida global foi de 30 meses. Expressão positiva de CD133 (35,9% dos casos), particularmente em cânceres de cólon direito (44,8% dos casos CD133+), correlacionou-se negativamente com óbito na análise univariada, sem significância estatística na análise multivariada. c-MYC (15,4% dos casos) teve predomínio de expressão em pacientes com estádio avançado com metástases distantes (não-pulmonares/não-hepáticas). Expressão de AXL foi pouco encontrada, com predomínio em adenomas, com menor penetrância em displasia de alto grau. Conclusão: Expressão de CD133 não se associou com sobrevida global inferior em CCR. Enquanto AXL demonstrou resultados inconclusivos, expressão de c-MYC em tumores primários se associou-se à metástases à distância.

De novo double-hit B-cell precursor leukemia/lymphoma - an unusual presentation as peritoneal lymphomatosis

Peritoneal lymphomatosis (PL) is a rare presentation of extranodal precursor leukemia/lymphoma. The presentation is often non-specific, leading to delayed diagnosis and treatment. In this case, though the preliminary diagnosis was established on ascitic fluid cytology, the disease progressed rapidly, leading to demise before initiating chemotherapy. Immunophenotyping and molecular studies, performed later, established a diagnosis of de novo B-cell precursor leukemia/lymphoma with MYC, BCL2 rearrangements (Double-hit lymphoma). MYC, BCL2 rearrangements are rarely reported in precursor B-lymphoma/leukemia which carry dismal prognosis. In this report, we illustrate autopsy findings of PL in an elderly gentleman who presented with ascites for evaluation.

Expressions of long non-coding RNA CASC11 and proto-oncogene c-myc in colorectal cancer and their correlation with recurrence and metastasis / 肿瘤研究与临床

Objective:To investigate the expressions of long non-coding RNA (lncRNA) CASC11 and proto-oncogene c-myc in colorectal cancer and their correlation with recurrence and metastasis.Methods:The cancer tissues and paracancerous tissues of 90 colorectal cancer patients in Tangshan Union Medical College Hospital of Hebei Province from February 2016 to July 2018 were collected. Normal colon epithelial cell lines CCD841 and colorectal cancer cell lines SW480, SW620, HCT116, HT29, DLD-1 were cultured in vitro. Separated by the average value of relative expression level of CASC11 or c-myc mRNA in cancer tissues, patients were divided into the high expression and low expression of CASC11 or c-myc. The protein expressions of CASC11 and c-myc mRNA and c-myc were detected by using real-time fluorescence quantitative polymerase chain reaction (qRT-PCR) and Western blot. Cell lines with the highest protein expressions of CASC11, c-myc mRNA and c-myc were used to do the subsequent experiments. The association of the expression levels of CASC11 and c-myc mRNA with the clinicopathological features was analyzed. Pearson correlation test was used to analyze the relationship between CASC11 and c-myc mRNA of cancer tissues. JASPAR software was used to analyze whether there were binding sites of CASC11 and c-myc gene. The wild-type and mutant-type CASC11 recombinant plasmids were constructed, and the relationship between c-myc and CASC11 was confirmed by using dual luciferase reporter gene assay. Cell lines with the highest expressions of CASC11 and c-myc were transfected with c-myc interference sequence plasmid (Sh-c-myc group) or the negative control sequence plasmid (Sh-NC group), and the conventional cultured blank control group (NC group). The proliferation of cells was detected by using methyl thiazolyl tetrazolium (MTT) method, the invasion and migration abilities of cells were detected by using Tanswell test, and the protein expressions of CASC11, c-myc mRNA and c-myc in cells of all groups were detected by using qRT-PCR and Western blot.Results:The protein expression levels of CASC11, c-myc mRNA and c-myc protein in cancer tissues were increased compared with those in paracancerous tissues, and the differences were statistically significant (all P < 0.05). Compared with CCD841 cells, the protein expression levels of CASC11, c-myc mRNA and c-myc in all colorectal cancer cell lines were increased, and the differences were statistically significant (all P < 0.05); the highest protein expressions of CASC11, c-myc mRNA and c-myc were found in SW480 cell lines which were used to do the subsequent experiments. Pearson correlation analysis showed that there was a positive correlation between CASC11 mRNA and c-myc mRNA in cancer tissues ( r = 0.494, P < 0.05). The high expression rate of CASC11 and c-myc mRNA in cancer tissues for patients with lymph node metastasis was higher than that for those without lymph node metastasis [73.7% (28/38) vs. 26.9% (14/52), 84.2% (32/38) vs. 23.1% (12/52)], the high expression rate of CASC11 and c-myc mRNA in cancer tissues for patients with TNM stage Ⅲ-Ⅳ was higher than that for those with TNM stage Ⅰ-Ⅱ [76.0% (38/50) vs. 10.0% (4/40), 72.0% (36/50) vs. 20.0% (8/40)], and the differences were statistically significant (all P < 0.05). JASPAR software showed that the binding sites were detected in CASC11 promoter region and c-myc gene; dual luciferase reporter gene assay results showed that the relative activity of SW480 cells co-transfected with Sh-c-myc and wild-type CASC11 plasmid was lower compared with that of SW480 cells co-transfected with Sh-NC and wild-type CASC11 plasmid ( P < 0.05). Compared with NC group and Sh-NC group, the expression level of CASC11 mRNA, the number of invasive and migratory cells of SW480 cells in Sh-c-myc group were decreased, while cell proliferation inhibition rate was increased, and the differences were statistically significant (all P < 0.05). Conclusions:CASC11 and c-myc mRNA are highly expressed in colorectal cancer tissues and cell lines, and binding sites can be detected in CASC11 promoter region and c-myc gene. The expressions of both have a correlation, and the down regulation of c-myc can inhibit the invasion and migration of colorectal cancer cells by inhibiting the expression of CASC11.

Advances in the research on mechanism of tumor metabolism regulated by c-Myc / 中国药科大学学报

@#The transcription factor c-Myc regulates the proliferation, differentiation, metabolism and other key processes of normal cells extensively.The unleashed MYC oncogene frequently produces abundant c-Myc protein, which directly regulates the gene expression of key metabolic enzymes, or tumor-related metabolic pathways by inhibiting microRNA, leading to abnormal metabolism characterized by heightened nutrients uptake, enhanced glycolysis and glutaminolysis, and elevated fatty acid and nucleotide synthesis.This paper briefly summarizes how c-Myc regulated metabolism on glycolysis, glutamine metabolism, tricarboxylic acid cycle, lipid metabolism and nucleotide synthesis in cancer cell,which provides some theoretical reference for the development of antitumor targets and drugs involving c-Myc.

Targeting "undruggable" c-Myc protein by synthetic lethality / 医学前沿

Synthetic lethal screening, which exploits the combination of mutations that result in cell death, is a promising method for identifying novel drug targets. This method provides a new avenue for targeting "undruggable" proteins, such as c-Myc. Here, we revisit current methods used to target c-Myc and discuss the important functional nodes related to c-Myc in non-oncogene addicted network, whose inhibition may cause a catastrophe for tumor cell destiny but not for normal cells. We further discuss strategies to identify these functional nodes in the context of synthetic lethality. We review the progress and shortcomings of this research field and look forward to opportunities offered by synthetic lethal screening to treat tumors potently.

Regulatory effect of annexin A5 on glioma cell invasion andmigration and its mechanisms / 西安交通大学学报(医学版)

【Objective】 To investigate the regulatory effect of annexin A5 on glioma cell invasion and migration and its mechanism. 【Methods】 The expression of annexin A5 in 100 cases of glioma tissues and 20 cases of normal brain tissues was detected by immunohistochemistry. The expression of annexin A5 was downregulated by transfection with siRNA targeting annexin A5 (si-Annexin A5) in human glioma cell line (U251). The expression of annexin A5 was confirmed by RT-PCR and Western blot analysis. The proliferation ability of U251 cells was detected by MTT test and colony formation test, the apoptosis of U251 cells was detected by flow cytometry and Hoechst 33258 staining, and the migration and invasion ability of U251 cells was examined by wound healing test and Matrigel Transwell invasion test. The expressions of Raf, p-Raf, MEK1/2, p-MEK1/2, ERK1/2, p-ERK1/2, c-Myc and E-Cadherin in U251 cells were analyzed by Western blot. 【Results】 Compared with those of normal brain tissues, the mRNA and protein expression levels of Annexin A5 in glioma tissues increased by 2.45 times and 2.87 times, respectively (P<0.05). Pearson correlation analysis showed that with the increase of tumor grade, the positive rate of Annexin A5 gradually increased, and the tumor grade and positive rate were significantly positively correlated (r=1.000, P=0.000). The cell viability of U251 cells in the si-Annexin A5 group after 48 h and 72 h of culture was significantly reduced by 29.46% and 40.43%, respectively, compared with that in the control group (P<0.05). Compared with the control group, in the si-Annexin A5 group the colony formation rate was reduced by 68.58%, while the apoptosis rate was increased by 24.41 times (P<0.05); the cell migration rate and invasion rate were reduced by 65.35% and 68.80% (P<0.05). The protein expression of p-Raf, p-MEK1/2, p-ERK1/2 and c-Myc in the si-Annexin A5 group were significantly reduced by 54.67%, 70.37%, 60.26% and 54.95%, respectively, and that of E-Cadherin was increased by 3.58 times (P<0.05). 【Conclusion】 Downregulation of Annexin A5 inhibits the growth and motility of glioma cells and induces cell apoptosis by inhibiting the Raf/MEK/ERK signaling pathway.

Inhibition of YAP-c-Myc signaling pathway involved effect of ORI on anti-glioma / 中国药理学通报

Aim To investigate the effects of oridonin on proliferation, migration and apoptosis of U87 glioma cells and to explore the involvement of the mechanism in the inhibition of Yes-associated protein (YAP)-c - Myc signaling pathway. Methods The effect of oridonin on U87 viability was measured by MTT assay; the migration and invasiveness of cells were measured by transwell assays; the apoptotic rates of cells were assessed by flow cytometry; the caspase-3, B c l - 2, Bax, YAP, c - MycmRNA expression in U87 glioma cells was detected by real-time quantitative P C R; the caspase-3, Bcl-2, Bax, YAP, p-YAP (Seri 27), c-Myc protein expressions were detected by Western blot. Results The proliferation of U87 cells was significantly inhibited by oridonin in a dose-dependent manner (P < 0. 05), and the ability of cell migration and invasion was weakened (P <0. 01), cell apoptosis rate in flow cytometry analysis increased significantly (P <0. 01), the protein and mRNA expression of caspase-3 increased (P < 0. 05), the mRNA and protein expression of Bcl-2/Bax decreased (P < 0. 05), the mRNA and protein expression of Y A P and c-Myc decreased (P < 0. 05), and the protein expression of p - Y A P increased (P < 0. 05). Conclusions Oridonin can significantly inhibit the proliferation and migration of U87 glioma cells and promote the transformation apoptosis of glioma cells; the mechanism may be related to the inhibition of YAP-c-Myc signaling pathway.

Are stem cell marker expression and cd133 analysis relevant to differentiate colorectal cancer? / A expressão de marcadores de células-tronco e a análise do cd133 são relevantes na diferenciação do câncer colorretal?

ABSTRACT Background: CD133 and AXL have been described as cancer stem cell markers, and c-MYC as a key regulatory cellular mechanism in colorectal cancer (CRC). Aim: Evaluate the prognostic role of the biomarkers CD133, AXL and c-MYC and their association with clinicopathologic characteristics in colorectal adenocarcinomas and adenomas. Methods: A total of 156 patients with UICC stage I-IV adenocarcinomas (n=122) and adenomas (n=34) were analyzed. Tissue microarrays (TMA) from primary tumors and polyps for CD133, c-MYC and AXL expression were performed and analyzed for their significance with clinicopathologic characteristics. Results: Poorly differentiated adenocarcinomas and disease progression were independent risk factors for poor overall survival. The median overall survival time was 30 months. Positive CD133 expression (35.9% of all cases), particularly of right-sided CRCs (44.8% of the CD133+ cases), was negatively correlated with death in the univariate analysis, which did not reach significance in the multivariate analysis. c-MYC (15.4% of all cases) was predominantly expressed in advanced-stage patients with distant (non-pulmonary/non-hepatic) metastasis. AXL expression was found only occasionally, and predominantly dominated in adenomas, with less penetrance in high-grade dysplasia. Conclusions: CD133 expression was not associated with inferior overall survival in CRC. While AXL showed inconclusive results, c-MYC expression in primary CRCs was associated with distant metastasis.

RESUMO Racional: CD133 e AXL são descritos na literatura como marcadores de células-tronco tumorais, e c-MYC cumpre papel chave como mecanismo de regulação celular no câncer colorretal (CCR). Objetivo: Avaliar o papel prognóstico dos biomarcadores CD133, AXL e c-MYC e sua associação com características clinicopatológicas de adenocarcinomas e adenomas colorretais. Métodos: Um total de 156 pacientes com adenocarcinomas de estádio UICC I-IV (n=122) e adenomas (n=34) colorretais foram avaliados. Microarranjos teciduais (TMA) dos tumores primários e adenomas foram realizados em busca de expressão de CD133, c-MYC e AXL, com posterior análise de relação significativa com características clinicopatológicas. Resultados: Adenocarcinomas pobremente diferenciados e progressão de doença foram fatores de risco independentes para má sobrevida global. A taxa mediana de sobrevida global foi de 30 meses. Expressão positiva de CD133 (35,9% dos casos), particularmente em cânceres de cólon direito (44,8% dos casos CD133+), correlacionou-se negativamente com óbito na análise univariada, sem significância estatística na análise multivariada. c-MYC (15,4% dos casos) teve predomínio de expressão em pacientes com estádio avançado com metástases distantes (não-pulmonares/não-hepáticas). Expressão de AXL foi pouco encontrada, com predomínio em adenomas, com menor penetrância em displasia de alto grau. Conclusão: Expressão de CD133 não se associou com sobrevida global inferior em CCR. Enquanto AXL demonstrou resultados inconclusivos, expressão de c-MYC em tumores primários se associou-se à metástases à distância.

Estudos funcionais e biológicos associados à interação de DUSP3 com a proteína HNRNPC1/C2 / Functional and biological studies associated to DUSP3 and HNRNPC1/C2 protein inte ractions

A regulação da fosforilação/desfosforilação das proteínas é o eixo central de muitas cascatas de sinalização. A fosfatase DUSP3, constituída apenas por um único domínio catalítico, desempenha papéis fundamentais na proliferação e senescência celular. Nas células HeLa, submetidas ao estresse genotóxico, o DUSP3 interage fisicamente com as proteínas HNRNPC, mas o efeito dessa função molecular ainda é desconhecido. Aqui demostramos que a ausência de DUPS3 mantem a proteína HNRNPC1/C2 num estado hiperfosforilado. Para entender melhor o envolvimento da interação DUSP3-HNRNPC nas funções biológicas da HNRNPC1/C2, foram estudadas células de fibroblasto deficientes de DUSP3. Foi analisado o efeito da deficiência de DUSP3 na biogênese dos ribossomos através do ensaio de perfil de polirribossomos e quantificação dos rRNAs com RT-qPCR. Os resultados mostraram que a deficiência de DUSP3 não afeta a maturação das subunidades ribossômicas, mas teria um impacto na transcrição dos pré-rRNAs e no acumulo das espécies 47S/45S. A expressado de genes contendo sequencias IRES foi analisado através do RT-qPCR e sua tradução ao longo do ciclo e em condições de estresse. Da expressão, não existe nenhuma diferença nos níveis de transcrição dos genes c-myc e xiap nas células normais e deficientes de DUSP3 em condições basais. Embora a síntese destas proteínas é maior nas células deficientes, mantendo um nível maior de tradução ao longo de todo o ciclo. Sob condições de estresse, esta duas proteínas sempre mantem uma maior expressão nas células Knockdown para DUSP3. Neste trabalho também foi estabelecido a presença de DUSP3 nos complexos da subunidade 40S, através do analise das frações obtidas do ensaio de polirribossomos e interação in vitro (Co-IP). A presença de DUSP3 nas subunidades 40S, os monossomas 80S e polissomos poderia ser através da interação direta com proteínas que possuem um domínio RRM e seria dependente dos complexos formados pelas proteínas e seus RNAs alvos. Aqui mostramos a interação in vitro de DUSP3 com a proteína PABP (com quatro domínios RRM), proteína que tem um papel importante na manutenção da taxa global de tradução, esta interação é enfraquecida na ausência de RNAs. A deficiência de DUSP3 também teria um impacto na interação das proteínas HNRNPC1/C2 e P53 in vitro. A ausência de DUSP3 diminui a interação HNRNPC-P53 através da hiperfosforilação da proteina HNRNPC1/C2. A perda desta interação, aumentaria os níveis da proteína P53 na célula deficiente de DUSP3 e poderia gerar parada no ciclo celular. Através de ensaios de imunofluorescência, se observo uma maior taxa de transcrição global na célula deficiente de DUSP3. Por fim, aqui demostramos que a interação direta de DUSP3 e HNRNPC1/C2 vai permitir a regulação das funções biológicas desta proteína, e a ausência de DUSP3 vai ter efeitos pleiotrópicos na homeostase da célula

inglêsProtein phosphorylation/dephosphorylation regulation is a central axis of many signaling cascades. DUSP3 phosphatase, consisting only of a single catalytic domain, plays key roles in cell proliferation and senescence. In HeLa cells subjected to genotoxic stress, DUSP3 physically interacts with HNRNPC proteins, but the effect of this molecular function is still unknown. Here we demonstrate that the absence of DUPS3 keeps the HNRNPC1/C2 proteins in a hyperphosphorylated state. To better understand the involvement of DUSP3- HNRNPC interaction on the biological functions of HNRNPC1/C2, DUSP3 deficient fibroblast cells were studied. The effect of DUSP3 deficiency on ribosome biogenesis was analyzed by polyribosome profile assay and RT-qPCR for rRNA quantification. The results showed that DUSP3 deficiency does not affect ribosomal subunit maturation, but would have an impact on transcription of pre-rRNAs and accumulation of 47S / 45S species. The expression of genes containing IRES sequences was analyzed by RT-qPCR and their translation throughout the cycle and under stress conditions. From expression, there is no difference in transcriptional levels of c-myc and xiap genes in normal and DUSP3 deficient cells under basal conditions. Although, the synthesis of these proteins is higher in deficient cells and these maintain a higher level of translation throughout the cell cycle. Under stress conditions, these two proteins always maintain higher expression in Knockdown cells for DUSP3. In this work, the presence of DUSP3 in the 40S ribosomal subunit complexes was also established by analyzing the fractions obtained from the polyribosome assay and in vitro interaction (CoIP). The presence of DUSP3 in the 40S subunits, 80S monosomes and polysomes could be through direct interaction with proteins that have an RRM domain and would be dependent on the complexes formed by the proteins and their target RNAs. Here we show the in vitro interaction of DUSP3 with PABP protein (with four RRM domains), a protein that plays an important role in maintaining the overall translation rate, this interaction is weakened in the absence of RNAs. DUSP3 deficiency would also have an impact on the interaction of HNRNPC1/C2 and P53 proteins in vitro. The absence of DUSP3 decreases HNRNPC-P53 interaction through hyperphosphorylation of the HNRNPC1/C2 proteins. Loss of this interaction would increase P53 protein levels in the DUSP3 deficient cell and could lead to cell cycle arrest. Through immunofluorescence assays, a higher overall transcription rate is observed in the DUSP3 deficient cell. Finally, we demonstrate that the direct interaction of DUSP3 and HNRNPC1/C2 will allow the regulation of the biological functions of this protein, and the absence of DUSP3 will have pleiotropic effects on cell homeostasis