USP33 promotes nonalcoholic fatty acid disease-associated fibrosis in gerbils via the c-myc signaling.

Nonalcoholic fatty acid disease (NAFLD) is a common complication of obesity associated with liver fibrosis. The underlying molecular mechanisms involved in the progression from normal to fibrosis remain unclear. Liver tissues from the liver fibrosis model identified the USP33 gene as a key gene in NAFLD-associated fibrosis. USP33 knockdown inhibited hepatic stellate cell activation and glycolysis in gerbils with NAFLD-associated fibrosis. Conversely, overexpression of USP33 caused a contrast function on hepatic stellate cell activation and glycolysis activation, which was inhibited by c-Myc inhibitor 10058-F4. The copy number of short-chain fatty acids-producing bacterium Alistipes sp. AL-1, Mucispirillum schaedleri, Helicobacter hepaticus in the feces, and the total bile acid level in serum were higher in gerbils with NAFLD-associated fibrosis. Bile acid promoted USP33 expression and inhibiting its receptor reversed hepatic stellate cell activation in gerbils with NAFLD-associated fibrosis. These results suggest that the expression of USP33, an important deubiquitinating enzyme, is increased in NAFLD fibrosis. These data also point to hepatic stellate cells as a key cell type that may respond to liver fibrosis via USP33-induced cell activation and glycolysis.

EBP50 suppresses the proliferation of MCF-7 human breast cancer cells via promoting Beclin-1/p62-mediated lysosomal degradation of c-Myc.

c-Myc, a key activator of cell proliferation and angiogenesis, promotes the development and progression of breast cancer. Ezrin-radixin-moesin-binding phosphoprotein-50 (EBP50) is a multifunctional scaffold protein that suppresses the proliferation of breast cancer cells. In this study we investigated whether the cancer-suppressing effects of EBP50 resulted from its regulation of c-Myc signaling in human breast cancer MCF-7 cells in vitro and in vivo. We first found a significant correlation between EBP50 and c-Myc expression levels in breast cancer tissue, and demonstrated that EBP50 suppressed cell proliferation through decreasing the expression of c-Myc and its downstream proteins cyclin A, E and Cdc25A in MCF-7 cells. We further showed that EBP50 did not regulate c-Myc mRNA expression, but it promoted the degradation of c-Myc through the autophagic lysosomal pathway. Moreover, EBP50 promoted integration between c-Myc and p62, an autophagic cargo protein, triggering the autophagic lysosomal degradation of c-Myc. In EBP50-silenced MCF-7 cells, activation of autophagy by Beclin-1 promoted the degradation of c-Myc and inhibited cell proliferation. These results demonstrate that the EBP50/Beclin-1/p62/c-Myc signaling pathway plays a role in the proliferation in MCF-7 breast cancer cells: EBP50 stimulates the autophagic lysosomal degradation of c-Myc, thereby inhibits the proliferation of MCF-7 cells. Based on our results, promoting the lysosomal degradation of c-Myc might be a promising new strategy for treating breast cancer.

Pterostilbene suppresses the growth of esophageal squamous cell carcinoma by inhibiting glycolysis and PKM2/STAT3/c-MYC signaling pathway.

Pterostilbene (PTS) is a dietary phytochemical that has shown antitumor activity in many types of cancer, but the molecular mechanism remains unclear. It has also not been adequately studied on PTS against esophageal squamous cell carcinoma (ESCC). Thus, this study investigated the effect of PTS on ESCC in vitro and in vivo and explored the underlying molecular mechanism. We found that PTS can inhibit the proliferation, colony formation, and migration of ESCC cells. According to the bioinformatics analysis of proteomics, PTS had a great influence on the metabolic process of ESCC cells. KEGG analysis showed that PTS down-regulated the pyruvate metabolism pathway. Moreover, PTS can inhibit the PK activity, glucose consumption, and lactate production in ESCC cells. By administration of PTS into xenograft mice, experiment results demonstrated that PTS can suppress tumor progress and the PKM2/STAT3/c-MYC signaling pathway. We found that PTS inhibited the PKM2/STAT3/c-MYC signaling pathway by targeting PKM2 in ESCC cells. Collectively, this study revealed that PTS inhibited ESCC growth by suppressing PKM2 mediated aerobic glycolysis and PKM2/STAT3/c-MYC signaling pathway, which enriching the anti-tumor molecular mechanism of PTS and providing a theoretical basis for its clinical application.

The splicing factor proline and glutamine rich promotes the growth of osteosarcoma via the c-Myc signaling pathway.

Splicing factor proline- and glutamine-rich (SFPQ) regulates transcripts in skeletal muscle metabolism and tumorigenesis. As osteosarcoma (OS) is the most common malignant bone tumor characterized by genome instability, such as MYC amplification, this study aimed to investigate the role and mechanism of SFPQ in OS. Expression of SFPQ in OS cell lines and human OS tissues was detected using quantitative real-time PCR, western blot, and fluorescence in situ hybridization (FISH) analyses. The oncogenic role of SFPQ in OS cells and murine xenograft models and the underlying mechanism of SFPQ on the c-Myc signaling pathway were assessed in vitro and in vivo. Results showed that SFPQ expression was upregulated and correlated with poor prognosis in OS patients. SFPQ overexpression promoted the malignant biological behavior of OS cells, while its knockdown markedly reduced the oncogenic function of OS. Additionally, depletion of SFPQ inhibited OS growth and bone destruction in nude mice. SFPQ overexpression induced malignant biological behaviors, which could be rescued by the depletion of c-Myc. These results suggest an oncogenic role of SFPQ in OS, possibly through the c-Myc signaling pathway.

DARS2 promotes the occurrence of lung adenocarcinoma via the ERK/c-Myc signaling pathway.

BACKGROUND: DARS2 expression is upregulated in lung adenocarcinoma (LUAD) which correlates with tumor patient stage and prognosis. The mechanism of DARS2 involvement in LUAD still needs to be further explored. METHODS: In this study, we found that DARS2 expression in LUAD tissue was significantly higher than that in normal tissue. At the same time, the Kaplan-Meier curve showed that the survival prognosis of LUAD patients with high expression of DARS2 was significantly worse than low expression of DARS2. The expression of DARS2 was detected in LUAD and adjacent normal tissues by IHC staining, histochemical scoring and a survival curve was generated. In addition, we demonstrated that the knockdown and overexpression of DARS2 significantly affected the proliferation, invasion, and migration of LUAD cells in vitro and in vivo. Finally, western blot and rescue assay were performed on LUAD cells to further explore and verify the signaling pathway. RESULTS: DARS2 expression was significantly upregulated in LUAD tissues and cell lines. What is more, the increased expression of DARS2 was closely related to proliferation, invasion and metastasis. The tumorigenic assay in nude mice further showed that the tumorigenic ability of nude mice was significantly improved with the increase in DARS2 expression. Finally, we determined that DARS2 plays its role in LUAD by targeting the ERK/c-Myc signaling pathway. CONCLUSION: Our data revealed the oncogenic role of DARS2 in LUAD, indicating that DARS2 may be a predictive biomarker and novel therapeutic target for LUAD.

Knockdown of FGFR3 inhibits the proliferation, migration and invasion of intrahepatic cholangiocarcinoma.

The FGF/FGFR signaling axis deregulation of the fibroblast growth factor receptor (FGFR) family is closely related to tumorigenesis, tumor progression and drug resistance to anticancer therapy. And fibroblast growth factor receptor 3 (FGFR3) is one member of this family. In this study, we aimed to investigate the effect of siRNA-induced knockdown of FGFR3 on the biological behaviors of intrahepatic cholangiocarcinoma (ICC). The expression levels of FGFR3 were determined in three intrahepatic cholangiocarcinoma cell lines RBE, HUCCT1 and HCCC9810 cell lines by Western blot. FGFR3 expression in RBE cell line was knocked down by siRNA. Our study found that knockdown of FGFR3 inhibited the migration, invasion and proliferation of ICC cells using Wound healing assay, Transwell migration and invasion assays and Cell proliferation assay. And significantly down-regulated the protein expression levels of MMP2, cyclinD1, and NCadherin, but had no significant effect on MMP9, cyclinD3, vimentin, E-cadherin protein. In addition, we found that ERK/c-Myc presumably is its signaling pathway by bioinformatics analysis and Western blot verification. To sum up, knockdown of FGFR3 inhibited the migration, invasion and proliferation of ICC cells. It demonstrated that FGFR3 probably becomes a therapeutic target for ICC and increases the proportion of potentially curable intrahepatic cholangiocarcinoma patients treated with FGFR inhibitors.

[High expression of MYH9 inhibits apoptosis of non-small cell lung cancer cells through activating the AKT/c-Myc pathway].

OBJECTIVE: To investigate the role of myosin heavy chain 9 (MYH9) in regulation of cell proliferation, apoptosis, and cisplatin sensitivity of non-small cell lung cancer (NSCLC). METHODS: Six NSCLC cell lines (A549, H1299, H1975, SPCA1, H322, and H460) and a normal bronchial epithelial cell line (16HBE) were examined for MYH9 expression using Western blotting. Immunohistochemical staining was used to detect MYH9 expression in a tissue microarray containing 49 NSCLC and 43 adjacent tissue specimens. MYH9 knockout cell models were established in H1299 and H1975 cells using CRISPR/Cas9 technology, and the changes in cell proliferation cell were assessed using cell counting kit-8 (CCK8) and clone formation assays; Western blotting and flow cytometry were used to detect apoptosis of the cell models, and cisplatin sensitivity of the cells was evaluated using IC50 assay. The growth of tumor xenografts derived from NSCLC with or without MYH9 knockout was observed in nude mice. RESULTS: MYH9 expression was significantly upregulated in NSCLC (P < 0.001), and the patients with high MYH9 expression had a significantly shorter survival time (P=0.023). In cultured NSCLC cells, MYH9 knockout obviously inhibited cell proliferation (P < 0.001), promoted cell apoptosis (P < 0.05), and increased their chemosensitivity of cisplatin. In the tumor-bearing mouse models, the NSCLC cells with MYH9 knockout showed a significantly lower growth rate (P < 0.05). Western blotting showed that MYH9 knockout inactivated the AKT/c- Myc axis (P < 0.05) to inhibit the expression of BCL2- like protein 1 (P < 0.05), promoted the expression of BH3- interacting domain death agonist and the apoptosis regulator BAX (P < 0.05), and activated apoptosis-related proteins caspase-3 and caspase-9 (P < 0.05). CONCLUSION: High expression of MYH9 contributes to NSCLC progression by inhibiting cell apoptosis via activating the AKT/c-Myc axis.

MicroRNA-378a-3p prevents initiation and growth of colorectal cancer by fine tuning polyamine synthesis.

BACKGROUND: Inhibitors of ornithine decarboxylase (ODC) are effective at preventing colorectal cancer (CRC). However, their high toxicity limits their clinical application. This study was aimed to explore the potential of microRNAs (miRNAs) as an inhibitor of ODC. METHODS: miRNA array was used to identify dysregulated miRNAs in CRC tumors of mice and patients. Azoxymethane (AOM)/Dextran Sodium Sulfate (DSS) were used to induce CRC in mice. miRNA function in carcinogenesis was determined by soft-agar colony formation, flow cytometry, and wound healing of CRC cells. Mini-circle was used to deliver miRNA into colons. RESULTS: MiRNA profiling identified miR-378a-3p (miR-378a) as the most reduced miRNA in CRC tumors of patients and mice treated with AOM/DSS. Pathway array analysis revealed that miR-378a impaired c-MYC and ODC1 pathways. Further studies identified FOXQ1 (forkhead box Q1) and ODC1 as two direct targets of miR-378a. FOXQ1 activated transcription of c-MYC, a transcription activator of ODC1. In addition to directly targeting ODC1, miR-378a also inhibited expression of ODC1 via the FOXQ1-cMYC axis, thereby inhibiting polyamine synthesis in human CRC cells. Phenotypically, by reducing polyamine synthesis, miR-378a induced apoptosis and inhibited proliferation and migration of CRC cells, while disrupting the association of miR-378a with FOXQ1 and ODC1 offset the effects of miR-378a, suggesting that FOXQ1 and ODC1 were required for miR-378a to inhibit CRC cell growth. MiR-378a treatment robustly prevented growth of HCC by inhibiting polyamine synthesis in AOM/DSS mice. CONCLUSION: MiR-378a prevents CRC by inhibiting polyamine synthesis, suggesting its use as a novel ODC inhibitor against CRC.

DEAD-box helicase 56 functions as an oncogene promote cell proliferation and invasion in gastric cancer via the FOXO1/p21 Cip1/c-Myc signaling pathway.

DEAD-box helicase (DDX) family exerts a critical effect on cancer initiation and progression through alternative splicing, transcription and ribosome biogenesis. Increasing evidence has demonstrated that DEAD-box helicase 56 (DDX56) is over-expressed in several cancers, which plays an oncogenic role. Till the present, the impact of DDX56 on gastric cancer (GC) remains unclear. We conducted high-throughput sequencing (RNA-seq) to demonstrate aberrant DDX56 levels within 10 GC and matched non-carcinoma tissue samples. DDX56 levels were detected through qRT-PCR, western blotting (WB) and immunochemical staining in GC patients. We conducted gain- and loss-of-function studies to examine DDX56's biological role in GC development. In vitro, we carried out 5­Ethynyl­2­deoxyuridine (EdU), scratch, Transwell, and flow cytometry (FCM) assays for detecting GC cell growth, invasion, migration and apoptosis. Additionally, gene set enrichment analysis (GSEA), WB assay, and Encyclopedia of RNA Interactomes (ENCORI) were carried out for analyzing DDX56-regulated downstream genes and signaling pathways. In vivo, tumor xenograft experiment was performed for investigating how DDX56 affected GC development within BALB/c nude mice. Functionally, DDX56 knockdown arrested cell cycle at G1 phase, invasion and migration of AGS and MKN28 cells, and enhanced their apoptosis. Ectopic DDX56 expression enhanced the cell growth, migration and invasion, and inhibited apoptosis. Knockdown of DDX56 suppressed GC growth in the tumor models of BALB/c nude mice. Mechanistically, DDX56 post-transcriptionally suppressed FOXO1/p21 Cip1 protein expression, which could activate its downstream cyclin E1/CDK2/c-Myc signaling pathways. This sheds lights on the GC pathogenic mechanism and offers a potential anti-cancer therapeutic target.

A-kinase interacting protein 1 regulates the cell proliferation, invasion, migration and angiogenesis of clear cell renal cell carcinoma cells and affects the ERK/c-Myc signaling pathway by binding to Rac1.

A-kinase interacting protein 1 (AKIP1) has previously been demonstrated to be overexpressed in clear cell renal cell carcinoma (ccRCC) tissues and is associated with patient prognosis. The aim of the present study was to explore whether AKIP1 can affect the proliferation, invasion, migration and angiogenesis of ccRCC cells via its interaction with Rac1. Furthermore, the influence of AKIP1 and therefore Rac1 on the expression of the downstream ERK/cellular (c)-Myc signaling pathway was explored. The interaction between AKIP1 and Rac1 was determined using co-immunoprecipitation. The mRNA and protein expression levels of AKIP1 and Rac1 in normal renal epithelial cell lines and ccRCC cell lines were detected using reverse transcription-quantitative PCR (RT-qPCR) and western blotting, respectively. The transfection efficiency of small interfering RNA-AKIP1 and the Rac1 overexpression vector were also confirmed using RT-qPCR and western blotting. The viability, proliferation, invasion and migration of ccRCC cells following transfection were analyzed using the Cell Counting Kit-8, 5-ethynyl-2'-deoxyuridine staining, Transwell and wound healing assays, respectively. The tube formation ability of HUVECs was assessed using the tube formation assay. The protein expression levels of proliferation, invasion, migration and tube-formation-associated proteins as well as proteins associated with the ERK/c-Myc signaling pathway, were detected via western blotting. The results demonstrated that AKIP1 expression levels were increased in ccRCC cell lines. AKIP1 knockdown inhibited the proliferation, invasion and migration of ccRCC cells and HUVEC tube-formation. In addition, AKIP1 was demonstrated to bind to Rac1 in ccRCC cells and AKIP1 downregulation inhibited Rac1 expression. Furthermore, Rac1 overexpression reversed the effects of AKIP1 knockdown on ccRCC cells. AKIP1 knockdown also suppressed the ERK/c-Myc signaling pathway, which was reversed by Rac1 overexpression. In conclusion, AKIP1 knockdown potentially suppressed the proliferation, invasion, migration and angiogenesis of ccRCC cells and inhibited the ERK/c-Myc signaling pathway by binding to Rac1.

Compound Kushen Injection intervenes metabolic reprogramming and epithelial-mesenchymal transition of HCC via regulating ß-catenin/c-Myc signaling.

BACKGROUND: Hepatocellular carcinoma (HCC) is one of the most extensive and most deadly cancers worldwide. The invasion and metastasis characteristics of HCC dramatically affect the prognosis and survival of HCC patients. Compound Kushen Injection (CKI) is a GMP produced, proverbially applied traditional Chinese medicine formula in China to treat cancer-associated pains, and used as an adjunctive therapy for HCC. Until so far, whether CKI could suppress the metastasis of HCC through regulation of epithelial-mesenchymal transition or metabolic reprogramming is still ambiguous. PURPOSE: In this study, the anti-metastasis effects of CKI were clarified and its pharmacological mechanisms were systematically explored. METHODS: Cell invasion and cell adhesion assay were performed in SMMC-7721 cells to assess the anti-metastasis role of CKI, and the histopathological evaluation and biochemical detection were utilized in DEN-induced HCC rats to verify the anti-HCC effect of CKI. Serum and liver samples were analyzed with 1H NMR metabolomics approach to screen the differential metabolites and further target quantification the content of key metabolites. Finally, western blotting and immunofluorescence assay were applied to verify the crucial signaling pathway involved in metabolites. RESULTS: CKI markedly repressed the invasion and adhesion in SMMC-7721 cells and significantly improved the liver function of DEN-induced HCC rats. CKI significantly regulated the expression of epithelial-mesenchymal transition (EMT) markers (Vimentin and E-cadherin). Metabolomics results showed that CKI regulated the metabolic reprogramming of HCC by inhibiting the key metabolites (citrate and lactate) and enzymes (HK and PK) in glycolysis process. Importantly, we found that c-Myc mediates the inhibitory effect of CKI on glycolysis. We further demonstrated that CKI inhibits c-Myc expression through modulating Wnt/ß-catenin pathway in SMMC-7721 cells and DEN-induced HCC rats. Furthermore, through activating Wnt/ß-catenin pathway with LiCl, the inhibitory effects of CKI on HCC were diminished. CONCLUSION: Together, this study reveals that CKI intervenes metabolic reprogramming and epithelial-mesenchymal transition of HCC via regulating ß-catenin/c-Myc signaling pathway. Our research provides a new understanding of the mechanism of CKI against invasion and metastasis of HCC from the perspective of metabolic reprogramming.

Expression of microRNA-6768-5p in lung cancer tissue and its effect on malignant biological behavior of lung cancer cells / 国际检验医学杂志

Objective To investigate the expression of miR-6768-5p in lung cancer tissue and its effect on the proliferation and invasion of lung cancer cells through targeted regulation of carboxypeptidase A4(CPA4).Methods The expression of miR-6768-5p in lung cancer tissues and adjacent tissues was analyzed u-sing the TCGA database.Quantitative real-time PCR(qPCR)was used to detect the expression of miR-6768-5p in human lung cancer cell lines(HCC1588,H1650,H1299,A549,HCC827)and normal alveolar epithelial cells(HPAEpiC cells).Lung cancer cells were transfected with NC mimics and miR-6768-5p mimics,respec-tively,and divided into NC group and miR-6768-5p group.The MTS assay and Matrigel invasion assay were used to detect the cell proliferation and invasion ability of each group,respectively.The putative binding sites of miR-6768-5p and CPA4 were verified using RNAhybrid software and dual-luciferase reporter gene experi-ment.The expression of CPA4 mRNA in each group of cells was detected by qPCR.The expression of AKT/c-MYC signaling pathway proteins in the cells of each group was analyzed by Western blot.Results Com-pared with the adjacent tissues,the relative expression level of miR-6768-5p in lung cancer tissues was signifi-cantly decreased,and the difference was statistically significant(P＜0.05).Compared with HPAEpiC cells,the relative expression level of miR-6768-5p was significantly decreased in lung cancer cell lines,and the differ-ence was statistically significant(P＜0.05).Compared with the NC group,the cell proliferation rate of miR-6768-5p group was significantly decreased(P＜0.05).The number of invasive cells in NC group and miR-6768-5p group was(131.30±12.55)and(37.45±7.77),respectively,and the number of invasive cells in miR-6768-5p group was significantly lower than that in NC group(P＜0.05).The relative expression level of CPA4 mRNA in H1299 cells of miR-6768-5p group was significantly lower than that in NC group(t=4.93,P＜0.05).Compared with the NC group,the expressions of AKT/c-myC signaling pathway proteins p-AKT,p-mTORC1,XIAP,MDM2 and C-myC proteins in miR-6768-5p group were significantly decreased.Conclusion The expression of miR-6768-5p is decreased in lung cancer tissues,and miR-6768-5p may inhibit the activation of AKT/c-MYC signaling pathway by targeting CPA4,and reduce the proliferation and invasion ability of lung cancer H1299 cells.

High expression of MYH9 inhibits apoptosis of non-small cell lung cancer cells through activating the AKT/c-Myc pathway / 南方医科大学学报

OBJECTIVE@#To investigate the role of myosin heavy chain 9 (MYH9) in regulation of cell proliferation, apoptosis, and cisplatin sensitivity of non-small cell lung cancer (NSCLC).@*METHODS@#Six NSCLC cell lines (A549, H1299, H1975, SPCA1, H322, and H460) and a normal bronchial epithelial cell line (16HBE) were examined for MYH9 expression using Western blotting. Immunohistochemical staining was used to detect MYH9 expression in a tissue microarray containing 49 NSCLC and 43 adjacent tissue specimens. MYH9 knockout cell models were established in H1299 and H1975 cells using CRISPR/Cas9 technology, and the changes in cell proliferation cell were assessed using cell counting kit-8 (CCK8) and clone formation assays; Western blotting and flow cytometry were used to detect apoptosis of the cell models, and cisplatin sensitivity of the cells was evaluated using IC50 assay. The growth of tumor xenografts derived from NSCLC with or without MYH9 knockout was observed in nude mice.@*RESULTS@#MYH9 expression was significantly upregulated in NSCLC (P < 0.001), and the patients with high MYH9 expression had a significantly shorter survival time (P=0.023). In cultured NSCLC cells, MYH9 knockout obviously inhibited cell proliferation (P < 0.001), promoted cell apoptosis (P < 0.05), and increased their chemosensitivity of cisplatin. In the tumor-bearing mouse models, the NSCLC cells with MYH9 knockout showed a significantly lower growth rate (P < 0.05). Western blotting showed that MYH9 knockout inactivated the AKT/c- Myc axis (P < 0.05) to inhibit the expression of BCL2- like protein 1 (P < 0.05), promoted the expression of BH3- interacting domain death agonist and the apoptosis regulator BAX (P < 0.05), and activated apoptosis-related proteins caspase-3 and caspase-9 (P < 0.05).@*CONCLUSION@#High expression of MYH9 contributes to NSCLC progression by inhibiting cell apoptosis via activating the AKT/c-Myc axis.