Short-term stimulation with histone deacetylase inhibitor trichostatin a induces epithelial-mesenchymal transition in nasopharyngeal carcinoma cells without increasing cell invasion ability.

BACKGROUND: Epithelial-mesenchymal transition (EMT) may be one of the reasons for the failure in some clinical trials regarding histone deacetylase inhibitors (HDACIs)-treated solid tumors. We investigated the effects of a pan-HDACI trichostatin A (TSA) on the proliferation and EMT of nasopharyngeal carcinoma (NPC) cells. METHODS: Poorly-differentiated NPC cell line CNE2 and undifferentiated C666-1 were treated with various concentrations of TSA, the cell viability was assessed by CCK-8 assay, the morphology was photographed, and the mRNA level of HDACs was assessed by semiquantitative PCR. After determination the cell cycle distributions, cells were subjected to western blotting analysis of cell cycle and EMT-associated genes expression. And the changes in migration ability were assessed by transwell migration assay and scratch wound healing assay. Finally, histone deacetylases activator ITSA-1 was used to assess the reverse of TSA-induced changes in NPC cells. RESULTS: TSA inhibited the proliferation of CNE2 and C666-1 cells in a concentration-dependent manner and arrested the cell cycle at G1 phases. TSA reduced PCNA, cyclin D1, cyclin E1, CDK2, p16 and p21 expressions and stimulated CDK6 levels. TSA stimulation for 48 h could effectively induce the EMT in CNE2 and C666-1 cells, which showed an increase of spindle-like cells and promoted expression of Vimentin and Snail1 expression in a concentration-dependent manner. Surprisingly, this short period of TSA treatment that induced EMT also impeded the migration ability of CNE2 and C666-1 cells. Interestingly, ITSA-1 rescued TSA-impeded CNE2 and C666-1 cells' proliferation, migration and HDACs expression, also re-induced the cells to turn into epithelial cell phenotypes. CONCLUSIONS: These results indicate that short-term stimulation of TSA effectively inhibits cell proliferation and induce EMT-like changes in NPC cells but not increase its invasion ability.

SETD4 in the Proliferation, Migration, Angiogenesis, Myogenic Differentiation and Genomic Methylation of Bone Marrow Mesenchymal Stem Cells.

Epigenetic modification is a crucial mechanism affecting the biological function of stem cells. SETD4 is a histone methyltransferase, and its biological role in bone marrow mesenchymal stem cells (BMSCs) is currently unknown. In this study, we employed CRISPR/Cas9 technology edited mouse model and found that SETD4 knockout significantly promoted the proliferation of BMSCs, impaired BMSCs migration and differentiation potentials of lineages of cardiacmyocyte and smooth muscle cell, and even the angiogenesis via paracrine of VEGF. Through Reduced Representation Bisulfite Sequencing (RRBS) method, we verified that the overall genomic methylation of BMSCs in the SETD4 knockout group only was decreased by 0.47 % compared with wild type. However, the changed genomic methylation covers a total of 96,331 differential methylated CpG sites and 8,692 differential methylation regions (DMRs), with part of them settled in promoter regions. Bioinformatic analysis revealed that differential CpG islands and DMRs in promoter impacted 270 GO functions and 34 KEGG signaling pathways, with some closely related to stem cell biology. Mechanismly, SETD4 knockout inhibited sets of monomethylases and dimethylases for histone lysine, along with significant changes in some factors including Nkx2.5, Gata4, Gli2, Grem2, E2f7, Map7, Nr2f2 and Shox2 that associated with stem cell biology. These results are the first to reveal that even though SETD4 changes the genome's overall methylation to a limited extent in BMSCs, it still affects the numerous cellular functions and signaling pathways, implying SETD4-altered genomic methylation serves a crucial molecular role in BMSCs' biological functions.

Ingenuity pathway analysis of differentially expressed genes involved in signaling pathways and molecular networks in RhoE gene­edited cardiomyocytes.

RhoE/Rnd3 is an atypical member of the Rho superfamily of proteins, However, the global biological function profile of this protein remains unsolved. In the present study, a RhoE­knockout H9C2 cardiomyocyte cell line was established using CRISPR/Cas9 technology, following which differentially expressed genes (DEGs) between the knockout and wild­type cell lines were screened using whole genome expression gene chips. A total of 829 DEGs, including 417 upregulated and 412 downregulated, were identified using the threshold of fold changes ≥1.2 and P<0.05. Using the ingenuity pathways analysis system with a threshold of ­Log (P­value)>2, 67 canonical pathways were found to be enriched. Many of the detected signaling pathways, including that of oncostatin M signaling, were found to be associated with the inflammatory response. Subsequent disease and function analysis indicated that apart from cardiovascular disease and development function, RhoE may also be involved in other diseases and function, including organismal survival, cancer, organismal injury and abnormalities, cell­to­cell signaling and interaction, and molecular transport. In addition, 885 upstream regulators were enriched, including 59 molecules that were predicated to be strongly activated (Z­score >2) and 60 molecules that were predicated to be significantly inhibited (Z­scores <­2). In particular, 33 regulatory effects and 25 networks were revealed to be associated with the DEGs. Among them, the most significant regulatory effects were 'adhesion of endothelial cells' and 'recruitment of myeloid cells' and the top network was 'neurological disease', 'hereditary disorder, organismal injury and abnormalities'. In conclusion, the present study successfully edited the RhoE gene in H9C2 cells using CRISPR/Cas9 technology and subsequently analyzed the enriched DEGs along with their associated canonical signaling pathways, diseases and functions classification, upstream regulatory molecules, regulatory effects and interaction networks. The results of the present study should facilitate the discovery of the global biological and functional properties of RhoE and provide new insights into role of RhoE in human diseases, especially those in the cardiovascular system.

[Quantitative proteomics and differential signal enrichment in nasopharyngeal carcinoma cells with or without SETD2 gene knockout].

OBJECTIVE: To analyze the effects of alterations in the expressions of methyltransferase SETD2 on protein expression profiles in human nasopharyngeal carcinoma (NPC) cells and enrich the differential signaling pathways. METHODS: The total protein was extracted from SETD2-knockout cell line CNE1SETD2-KO and the wild-type cell line CNE1WT, and the differentially expressed proteins were screened by tandem mass tag (TMT) labeled protein quantification technique and tandem mass spectrometry. GO analysis was used to annotate and enrich the differentially expressed proteins, and the KEGG database was used to enrich and analyze the pathways of the differential proteins. RESULTS: With a fold change (FC)≥1.2 and P < 0.05 as the screening standard, 2049 differentially expressed proteins were identified in CNE1SETD2-KO cells, among which 904 were up-regulated and 1145 were down-regulated. GO functional annotation results indicated that SETD2 knockout caused characteristic changes in multiple biological processes (cell processes and regulation, cell movement, metabolic processes, and biosynthesis of cellular components), molecular functions (catalytic activity and molecular binding, transcription factor activity), and cellular components (cell membrane, organelle, macromolecular complex). KEGG analysis showed that the differentially expressed proteins were involved in an array of signaling pathways closely related to tumors, including MAPK, PI3K-Akt, Ras, Rap1, mTOR, Hippo, HIF-1, Wnt, AMPK, FoxO, ErbB, P53 and JAK-STAT. CONCLUSIONS: SETD2 knockout significantly changes the protein expression characteristics of NPC cells and affects a number of signal pathways closely related to tumors. The results provide evidence for investigation of the pathogenesis and therapeutic target screening of NPC.

Epstein-Barr virus-encoded LMP1 regulated Pim1 kinase expression promotes nasopharyngeal carcinoma cells proliferation.

BACKGROUND: Epstein-Barr virus-encoded LMP1 plays a critical role in the carcinogenesis of nasopharyngeal carcinoma (NPC), but the mechanism remains elusive. We aimed to analyze the expression and clinical pathological significance of provirus integration site for Moloney murine leukemia virus 1 (Pim1) in clinical NPC, and to elucidate the effect of LMP1 on Pim1 expression and its mechanism. METHODS: Immunohistochemical staining was used to detect the expression of Pim1 in clinical NPC tissues and control nasopharyngeal chronic inflammation (NPI) tissues, and the correlation between Pim1 and clinical parameters of NPC patients was analyzed. The LMP1 stable expression cell line CNE1-LMP1-OV was constructed through infecting the well-differentiated nasopharyngeal carcinoma cells CNE1 with LMP1 overexpressing lentivirus. Then the in vivo experiments were conducted. RESULTS: Among 89 NPC patients, 48 cases (53.93%) were positive for Pim1, while only one case was Pim1 positive in 15 NPI controls (6.67%). Pim1 expression was not correlated with gender, age, smoking status and clinical classification of NPC patients, but positively correlated with T, N and M classification. CNE1-LMP1-OV cell line was successfully established, which displayed a higher cell proliferation ability and Pim1 expression. NF-κB inhibitor PDTC, PKC inhibitor GF109203X and STAT3 inhibitor Stattic significantly attenuated LMP1-induced Pim1 expression, and while AP-1 inhibitor SR11302 showed no inhibitory effect. Interestingly, Pim1 inhibitor quercetagetin significantly inhibited the proliferation of CNE1-LMP1-OV cells. CONCLUSION: LMP1 mediates Pim1 expression through NF-κB, PKC and STAT3 signaling, which promotes the proliferation of NPC cells and participate in the clinical progression of NPC.