Chip Analysis Research Articles

AIMP1 promotes multiple myeloma malignancy through interacting with ANP32A to mediate histone H3 acetylation

Multiple myeloma (MM) is the second most common hematological malignancy. An overwhelming majority of patients with MM progress to serious osteolytic bone disease. Aminoacyl-tRNA synthetase-interacting multifunctional protein 1 (AIMP1) participates in several steps during cancer development and osteoclast differentiation. This study aimed to explore its role in MM. The gene expression profiling cohorts of MM were applied to determine the expression of AIMP1 and its association with MM patient prognosis. Enzyme-linked immunosorbent assay, immunohistochemistry, and Western blotting were used to detect AIMP1 expression. Protein chip analysis, RNA-sequencing, and chromatin immunoprecipitation and next-generation sequencing were employed to screen the interacting proteins and key downstream targets of AIMP1. The impact of AIMP1 on cellular proliferation was determined using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay in vitro and a xenograft model in vivo. Bone lesions were evaluated using tartrate-resistant acid phosphatase staining in vitro. A NOD/SCID-TIBIA mouse model was used to evaluate the effect of siAIMP1-loaded exosomes on bone lesion formation in vivo. AIMP1 expression was increased in MM patients and strongly associated with unfavorable outcomes. Increased AIMP1 expression promoted MM cell proliferation in vitro and in vivo via activation of the mitogen-activated protein kinase (MAPK) signaling pathway. Protein chip assays and subsequent experiments revealed that AIMP1 interacted with acidic leucine-rich nuclear phosphoprotein 32 family member A (ANP32A) to regulate histone H3 acetylation. In addition, AIMP1 increased histone H3 acetylation enrichment function of GRB2-associated and regulator of MAPK protein 2 (GAREM2) to increase the phosphorylation of extracellular-regulated kinase 1/2 (p-ERK1/2). Furthermore, AIMP1 promoted osteoclast differentiation by activating nuclear factor of activated T cells c1 (NFATc1) in vitro. In contrast, exosome-coated small interfering RNA of AIMP1 effectively suppressed MM progression and osteoclast differentiation in vitro and in vivo. Our data demonstrate that AIMP1 is a novel regulator of histone H3 acetylation interacting with ANP32A in MM, which accelerates MM malignancy via activation of the MAPK signaling pathway.

PRC1-independent binding and activity of RYBP on the KSHV genome during de novo infection.

Kaposi’s sarcoma-associated herpesvirus (KSHV) is an oncogenic virus that causes lifelong infection in humans by establishing latency after primary infection. Latent infection is a prerequisite for both persistent infection and the development of KSHV-associated cancers. While viral lytic genes are transiently expressed after primary infection, their expression is significantly restricted and concomitant with the binding of host epigenetic repressors Polycomb Repressive Complex 1 and 2 (PRC1 and PRC2) to lytic genes. PRC1 and PRC2 mediate the repressive histone marks H2AK119ub and H3K27me3, respectively, and maintain heterochromatin structure on KSHV lytic genes to inhibit their expression. In contrast to PRC2, little is known about the recruitment and role of PRC1 factors on the KSHV genome following de novo infection. Thus, the goal of this study was to examine the function of PRC1 factors in the establishment of KSHV latency. To address this question, we performed an shRNA screen targeting 7 different components of the canonical and non-canonical PRC1 complexes during primary KSHV infection. We found that RYBP, a main subunit of the non-canonical PRC1 complexes, is a potent repressor of KSHV lytic genes that can bind to the viral genome and inhibit lytic genes as early as 4 hours post infection. Surprisingly, our ChIP analyses showed that RYBP binds to lytic viral gene promoters in a PRC1-independent manner, does not affect PRC1 activity on the KSHV genome, and can reduce the level of histone marks associated with transcription elongation. Our data also suggest that RYBP can repress the viral lytic cycle after primary infection by inhibiting the transcription elongation of the lytic cycle inducer KSHV gene RTA. Based on our results we propose that RYBP uses a PRC1-independent mechanism to block KSHV RTA expression thereby promoting the establishment of KSHV latency following de novo infection.

A dimer-monomer switch controls CHIP-dependent substrate ubiquitylation and processing

The high substrate selectivity of the ubiquitin/proteasome system is mediated by a large group of E3 ubiquitin ligases. The ubiquitin ligase CHIP regulates the degradation of chaperone-controlled and chaperone-independent proteins. To understand how CHIP mediates substrate selection and processing, we performed a structure-function analysis of CHIP and addressed its physiological role in Caenorhabditis elegans and human cells. The conserved function of CHIP in chaperone-assisted degradation requires dimer formation to mediate proteotoxic stress resistance and to prevent protein aggregation. The CHIP monomer, however, promotes the turnover of the membrane-bound insulin receptor and longevity. The dimer-monomer transition is regulated by CHIP autoubiquitylation and chaperone binding, which provides a feedback loop that controls CHIP activity in response to cellular stress. Because CHIP also binds other E3 ligases, such as Parkin, the molecular switch mechanism described here could be a general concept for the regulation of substrate selectivity and ubiquitylation by combining different E3s.

DNA methylation influences the CTCF-modulated transcription of RASSF1A in lung cancer cells.

Ras-association domain family 1A (RASSF1A) is one of the most methylated genes in lung cancer (LC). We investigate whether the high DNA methylation level of RASSF1A can relieve the resistance of RASSF1A to LC by inhibiting RASSF1A's transcription factor binding to RASSF1A. RASSF1A expression in tissues and cells was tested utilizing quantitative real-time polymerase chain reaction (qRT-PCR), and Western blot. RASSF1A expression and RASSF1A methylation level in LC cells exposed to 5-Aza-dc were assessed by qRT-PCR and quantitative methylation-specific PCR. The association between CTCF and RASSF1A was assessed using hTFtarget, ChIP, and luciferase reporter gene analysis. The effects of 5-Aza-dc, CTCF, and RASSF1A on cell biological behaviors and epithelial-mesenchymal transition (EMT)-related markers were assessed by cell function experiments and Western blot. Moreover, we constructed the xenograft tumor and pulmonary nodule metastasis models, and assessed tumor volume and weight. RASSF1A expression and pulmonary nodule metastasis were tested utilizing qRT-PCR, Western blot, and H&E staining. RASSF1A was under-expressed in LC tissues and cells. 5-Aza-dc enhanced RASSF1A level and weakened RASSF1A methylation level in LC cells. RASSF1A silencing neutralized 5-Aza-dc-mediated repressing effects on LC cell biological function and EMT. The loss of CTCF binding to RASSF1A in LC cells was associated with DNA methylation. The effect of 5-Aza-dc on RASSF1A level, LC cell malignant behaviors, and EMT-related factors were strengthened by CTCF upregulation. RASSF1A overexpression suppressed LC tumor growth and pulmonary nodule metastasis in vivo. DNAmethylation blocked the modulation of RASSF1A expression by CTCF and relieved the resistance of RASSF1A to LC.

Targeted Long-Read Bisulfite Sequencing Identifies Differences in the TERT Promoter Methylation Profiles between TERT Wild-Type and TERT Mutant Cancer Cells.

Simple SummaryTERT promoter methylation is enriched in cancers lacking TERT genetic alterations (wild-type cancers), but its functional impact on TERT transcription remains elusive. We developed a long-read bisulfite-sequencing platform to characterize the TERT promoter methylation profile at a single-molecule level. In wild-type cancer cell lines, both epialleles were hypermethylated symmetrically on the TERT distal promoter. In the core and proximal promoter, by contrast, the transcribed epialleles were significantly more hypomethylated than the silent epialleles. Decitabine-therapy reduced the core and proximal (not the distal) promoter methylation and reactivated the silent allele. We showed that TERT allele-specific expression is amenable to in vitro epigenetic manipulation in wild-type cancers.Background: TERT promoter methylation, located several hundred base pairs upstream of the transcriptional start site, is cancer specific and correlates with increased TERT mRNA expression and poorer patient outcome. Promoter methylation, however, is not mutually exclusive to TERT activating genetic alterations, as predicted for functionally redundant mechanisms. To annotate the altered patterns of TERT promoter methylation and their relationship with gene expression, we applied a Pacific Biosciences-based, long-read, bisulfite-sequencing technology and compared the differences in the methylation marks between wild-type and mutant cancers in an allele-specific manner. Results: We cataloged TERT genetic alterations (i.e., promoter point mutations or structural variations), allele-specific promoter methylation patterns, and allele-specific expression levels in a cohort of 54 cancer cell lines. In heterozygous mutant cell lines, the mutant alleles were significantly less methylated than their silent, mutation-free alleles (p < 0.05). In wild-type cell lines, by contrast, both epialleles were equally methylated to high levels at the TERT distal promoter, but differentially methylated in the proximal regions. ChIP analysis showed that epialleles with the hypomethylated proximal and core promoter were enriched in the active histone mark H3K4me2/3, whereas epialleles that were methylated in those regions were enriched in the repressive histone mark H3K27me3. Decitabine therapy induced biallelic expression in the wild-type cancer cells, whereas the mutant cell lines were unaffected. Conclusions: Long-read bisulfite sequencing analysis revealed differences in the methylation profiles and responses to demethylating agents between TERT wild-type and genetically altered cancer cell lines. The causal relation between TERT promoter methylation and gene expression remains to be established.

Role of the WNT/β-catenin/ZKSCAN3 Pathway in Regulating Chromosomal Instability in Colon Cancer Cell lines and Tissues.

Zinc finger protein with KRAB and SCAN domains 3 (ZKSCAN3) acts as an oncogenic transcription factor in human malignant tumors, including colon and prostate cancer. However, most of the ZKSCAN3-induced carcinogenic mechanisms remain unknown. In this study, we identified ZKSCAN3 as a downstream effector of the oncogenic Wnt/β-catenin signaling pathway, using RNA sequencing and ChIP analyses. Activation of the Wnt pathway by recombinant Wnt gene family proteins or the GSK inhibitor, CHIR 99021 upregulated ZKSCAN3 expression in a β-catenin-dependent manner. Furthermore, ZKSCAN3 upregulation suppressed the expression of the mitotic spindle checkpoint protein, Mitotic Arrest Deficient 2 Like 2 (MAD2L2) by inhibiting its promoter activity and eventually inducing chromosomal instability in colon cancer cells. Conversely, deletion or knockdown of ZKSCAN3 increased MAD2L2 expression and delayed cell cycle progression. In addition, ZKSCAN3 upregulation by oncogenic WNT/β-catenin signaling is an early event of the adenoma–carcinoma sequence in colon cancer development. Specifically, immunohistochemical studies (IHC) were performed using normal (NM), hyperplastic polyps (HPP), adenomas (AD), and adenocarcinomas (AC). Their IHC scores were considerably different (61.4 in NM; 88.4 in HPP; 189.6 in AD; 246.9 in AC). In conclusion, ZKSCAN3 could be responsible for WNT/β-catenin-induced chromosomal instability in colon cancer cells through the suppression of MAD2L2 expression.

A Study on the Expression of Messenger RNAs and Long Noncoding RNA in Keloid Fibroblasts Based on Gene Expression Omnibus Microarray Data Mining.

The purpose of this study was to find the coding RNA [messenger RNA (mRNA)] and long noncoding RNA (lncRNA) expressed in keloid through the analysis of Gene Expression Omnibus microarray chip of keloid fibroblasts. Gene Expression Omnibus database GSE7890 database was downloaded with selection of keloids and normal scar group data. The data were analyzed by R language combined with online database. The log2FC>1, P value <0.01 was chosen as screening criteria, and the differentially expressed mRNAs were screened for GO and KEGG function analysis. One hundred fifty-five mRNA expression in the keloid group was significantly different from that in the normal group, including 31 groups with upregulated mRNA expression and 124 groups with down-regulated mRNA expression. Meanwhile, 8 lncRNAs were changed in the keloid group, including 3 upregulated (Rp11-420a23.1, Rp11-522b15.3, and Rp11-706j10.1) and 5 down-regulated (LINC00511, LINC00327, Hoxb-as3, Rp11-385n17.1, and Rp3-428l16.2). Quantitative polymerase chain reaction analysis of DElncRNAs in keloid fibroblasts showed that the expression of all DElncRNAs except for RP11-385N17.1 was increased in the keloid group compared with the control group. Moreover, the differences in LINC00511 and RP11-706J10.1 were statistically significant. The noncoding RNA information of Gene Expression Omnibus chip data can be deeply mined through bioinformatics, and the potential epigenomic mechanism affecting keloid formation can be found from the existing database.

Maternal high-fat diet modifies epigenetic marks H3K27me3 and H3K27ac in bone to regulate offspring osteoblastogenesis in mice

ABSTRACT Studies from both humans and animal models indicated that maternal chronic poor-quality diet, especially a high fat diet (HFD), is significantly associated with reduced bone density and childhood fractures in offspring. When previously studied in a rat model, our data suggested that maternal HFD changes epigenetic marks such as DNA methylation and histone modifications to control osteoblast metabolism. In mouse embryonic and postnatal offspring bone samples, a ChIP-sequencing (ChIP-Seq)-based genome-wide method was used to locate the repressive histone mark H3K27me3 (mediated via the polycomb histone methyltransferase, Ezh2) and expressive histone mark H3K27ac (p300/CBP mediated) throughout the genome. Using isolated mouse embryonic cells from foetal calvaria (osteoblast-like cells), H3K27me3 ChIP-Seq showed that 147 gene bodies and 26 gene promoters in HFD embryotic samples had a greater than twofold increase in H3K27me peaks compared to controls. Among the HFD samples, Pthlh and Col2a1 that are important genes playing roles during chondro- and osteogenesis had significantly enriched levels of H3K27me3. Their decreased mRNA expression was confirmed by real-time PCR and standard ChIP analysis, indicating a strong association with Ezh2 mediated H3K27me3 epigenetic changes. Using embryonic calvaria osteoblastic cells and offspring bone samples, H3K27ac ChIP-Seq analysis showed that osteoblast inhibitor genes Tnfaip3 and Twist1 had significantly enriched peaks of H3K27ac in HFD samples compared to controls. Their increased gene expression and association with H3K27ac were also confirmed by real-time PCR and standard ChIP analysis. These findings indicate that chronic maternal HFD changes histone trimethylation and acetylation epigenetic marks to regulate expression of genes controlling osteoblastogenesis.

Exploring the pharmacological mechanism of compound kushen injection in the treatment of breast cancer using in vitro experiments: Coupling network pharmacology with GEO database.

BackgroundBreast cancer (BC) is one of the most common malignant tumors in women and poses a serious threat to their health. Compound Kushen injection (CKI) has shown therapeutic effects on a variety of cancers, including BC, and it can significantly improve the lives of patients. However, the underlying mechanism remains unclear and needs to be fully elucidated.MethodsThe active constituents of CKI were identified through a literature review, and the anti-BC targets of CKI were determined using multiple databases and a ChIP data analysis. Subsequently, the target was analyzed on the DAVID database through GO and KEGG to identify the key pathway that CKI affects to exhibit anti-BC activity. In addition, MCF-7 and MDA-MB-231 cells were treated with CKI for 24 and 48 hours at five concentrations, and the effects of CKI on cell proliferation and apoptosis were measured using MTT and annexin V/propidium iodide staining assays, respectively. The genes and protein identified to be involved in this pathway were verified using real-time quantitative PCR (qPCR) and western blot(WB) in BC cells.ResultsTwelve CKI anti-BC targets were obtained by a comprehensive analysis of the targets collected in the databases and results from the ChIP analysis. Bioinformatics analysis was performed for 12 targets. KEGG analysis showed that the 12 targets were mainly related to the VEGF, ErbB, and TNF signaling pathways. We focused our study on the VEGF signaling pathway as the p-value for the VEGF signaling pathway was the lowest among the three pathways. In vitro experiments showed that CKI significantly inhibited the proliferation of BC cells and induced apoptosis. Furthermore, qPCR and WB experiments showed that the expression of VEGF signaling pathway genes PIK3CA and NOS3 were significantly increased meanwhile SRC was significantly decreased after CKI intervention.ConclusionCKI significantly inhibited the proliferation of BC cells and induced apoptosis. The main mechanism for the anti-BC effect of CKI may be that it regulates the VEGF signaling pathway by increasing the expression of PIK3CA, SRC, and NOS3. Macrozamin and lamprolobine may be the main active components of CKI against BC.

Set2 family regulates mycotoxin metabolism and virulence via H3K36 methylation in pathogenic fungus Aspergillus flavus

ABSTRACT Aspergillus flavus infects various crops with aflatoxins, and leads to aspergillosis opportunistically. Though H3K36 methylation plays an important role in fungal toxin metabolism and virulence, no data about the biological function of H3K36 methylation in A. flavus virulence has been reported. Our study showed that the Set2 histone methyltransferase family, AshA and SetB, involves in morphogenesis and mycotoxin anabolism by regulating related transcriptional factors, and they are important for fungal virulence to crops and animals. Western-blotting and double deletion analysis revealed that AshA mainly regulates H3K36me2, whereas SetB is mainly responsible for H3K36me3 in the nucleus. By construction of domain deletion A. flavus strain and point mutation strains by homologous recombination, the study revealed that SET domain is indispensable in mycotoxin anabolism and virulence of A. flavus, and N455 and V457 in it are the key amino acid residues. ChIP analysis inferred that the methyltransferase family controls fungal reproduction and regulates the production of AFB1 by directly regulating the production of the transcriptional factor genes, including wetA, steA, aflR and amylase, through H3K36 trimethylation in their chromatin fragments, based on which this study proposed that, by H3K36 trimethylation, this methyltransferase family controls AFB1 anabolism through transcriptional level and substrate utilization level. This study illuminates the epigenetic mechanism of the Set2 family in regulating fungal virulence and mycotoxin production, and provides new targets for controlling the virulence of the fungus A. flavus. AUTHOR SUMMARY The methylation of H3K36 plays an important role in the fungal secondary metabolism and virulence, but no data about the regulatory mechanism of H3K36 methylation in the virulence of A. flavus have been reported. Our study revealed that, in the histone methyltransferase Set2 family, AshA mainly catalyzes H3K36me2, and involves in the methylation of H3K36me1, and SetB mainly catalyzes H3K36me3 and H3K36me1. Through domain deletion and point mutation analysis, this study also revealed that the SET domain was critical for the normal biological function of the Set2 family and that N455 and V457 in the domain were critical for AshA. By ChIP-seq and ChIP-qPCR analysis, H3K36 was found to be trimethylation modified in the promotors and ORF positions of wetA, steA, aflR and the amylase gene (AFLA_084340), and further qRT-PCR results showed that these methylation modifications regulate the expression levels of these genes. According to the results of ChIP-seq analysis, we proposed that, by H3K36 trimethylation, this methyltransferase family controls the metabolism of mycotoxin through transcriptional level and substrate utilization level. All the results from this study showed that Set2 family is essential for fungal secondary metabolism and virulence, which lays a theoretical groundwork in the early prevention and treatment of A. flavus pollution, and also provides an effective strategy to fight against other pathogenic fungi.

Thermomechanical Reliability Investigation of Carbon Nanotube Off-Chip Interconnects for Electronic Packages

This article investigates the mechanical reliability benefits of carbon nanotube (CNT) off-chip interconnects for future electronic packages. Finite-element analysis (FEA) of a silicon chip attached to an organic substrate showed that CNT off-chip interconnects with an isotropic modulus of 1 MPa reduced the principal stresses in the silicon chip ~71%–93% compared with lead-free solder balls without underfill. In addition to reducing chip stresses, chip warpage was essentially eliminated by utilizing CNT interconnects compared with solder balls. Parameters of importance for the implementation of CNTs as interconnects such as CNT modulus and height were investigated with finite-element analysis. It was found that chip stresses increase with increasing CNT modulus, but for the modulus range reported in the literature and examined here (0.1–500 MPa), chip stresses were much less than packages utilizing solder balls. Due to the structure and morphology of CNT forests, the different material model types of fully isotropic versus transversely isotropic that can be used to model CNT interconnects were investigated. While the models yielded similar mechanical behavior in the finite-element analyses, the isotropic CNT model was mechanically conservative and simpler to implement and, therefore, sufficient for future thermomechanical reliability studies. To further demonstrate the thermomechanical reliability benefits, silicon chips with CNT off-chip interconnects were fabricated and assembled to organic substrates and subjected to cyclic thermal shock testing. During testing, the daisy chain circuit containing CNT interconnects survived 1660 cycles, demonstrating that CNT interconnects mechanically decouple the chip from the substrate and, therefore, reduce chip stresses caused by coefficient of thermal expansion (CTE) mismatch.

CD13 downregulation mediated by ubenimex inhibits autophagy to overcome 5-FU resistance by disturbing the EMP3/FAK/NF-κB pathway in gastric cancer cells.

BackgroundGastric cancer (GC) is one of the most common malignant tumours in China, but the efficacy of chemotherapy on GC is significantly reduced due to the occurrence of drug resistance. Some studies have shown that the expression level of CD13 is associated with tumour resistance, but whether ubenimex, as a CD13 inhibitor, reverses GC drug resistance and the underlying mechanism remain unclear.MethodsHerein, resistance to 5-fluorouracil (5-FU) was reversed in GC by ubenimex, and the underlying mechanism was determined using Cell Counting Kit-8 (CCK-8) assays, gene chip analysis, high content screening (HCS), transmission electron microscopy, flow cytometry, immunofluorescence and western blot assays.ResultsFlow cytometry, transmission electron microscopy and immunofluorescence analyses indicated that ubenimex, an inhibitor of CD13, regulated the autophagy and apoptosis of SGC7901/5-FU cells by downregulating CD13 expression. In addition, Gene chip analysis and HCS demonstrated that epithelial membrane protein 3 (EMP3)/focal adhesion kinase (FAK) was a putative signalling pathway downstream of CD13. Furthermore, western blot analyses showed that ubenimex not only inhibited EMP3, FAK and nuclear factor-κB (NF-κB) expression but also suppressed GC autophagy and activated apoptosis by targeting CD13. These findings indicated a potential mechanism via the CD13/EMP3/FAK/NF-κB pathway and that the activity of which was restrained.ConclusionsUbenimex affects autophagy and apoptosis to reverse GC cell resistance by targeting the CD13/EMP3/FAK/NF-κB pathway. These results showed that ubenimex is a promising agent that may inhibit GC autophagy to improve chemotherapeutic drug sensitivity and thereby reverse drug resistance.

Establishment of esophageal squamous carcinoma cell lines with different invasive metastatic potential and differential gene screening.

The present study aimed to establish esophageal squamous carcinoma cell (ESCC) sublines with different invasive and metastatic potentials. Gene microarrays were used for differential gene screening with the establishment of ESCC invasive metastatic gene expression profiles. Some differential gene expressions were validated. Parent line Eca109-T0 was screened in a Transwell chamber to establish Eca109-T4 with high invasion and metastasis. The migrative and proliferative capacities of ESCCs were compared. The Eca109-T0 and Eca109-T4 cell lines were taken as the research objects and were hybridized with gene chips to obtain cell sublines for the screening of differential genes of ESCCs with varying invasive and metastatic potentials. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) clustering analyses were conducted. Some differential genes (HSP90AA1, ANXA1, YWHAB, CXCR7, SDC2, and TNFRSF10D messenger ribonucleic acid) were validated by qualitative real-time polymerase chain reaction and Western blot analysis. As a result, some Eca109-T4 ESCC sublines with high invasive and metastatic potential were screened in a Transwell chamber. The gene chip analysis screened out 326 differential genes, of which 123 were upregulated and 203 were downregulated by Eca109-T4. The GO cluster analysis indicated that the genes were in the cytoplasm, nucleus, and cytosol. The molecular functions of these genes involved the binding of proteins and metal ions and participation in biological processes, including cell signal transduction, transcription, and apoptosis. The KEGG clustering showed that these genes were mainly involved in signaling pathways, such as actin cytoskeleton regulation, the mitogen-activated protein kinase pathway, and the cancer pathway. The validation results were basically consistent with the gene microarray screening results. In Eca109-T4 and CE81T-4, HSP90AA1, YWHAB, and CXCR7 were highly expressed, while the expression of ANXA1 was low. In conclusion esophageal squamous carcinoma cell models with different invasive and metastatic potentials were established. The establishment of differential gene expression profiles for invasion and metastasis together with a bioinformatics analysis provided rich information for studies related to ESCC invasion and metastasis. HSP90α, 14-3-3β, and CXCR7 were highly expressed in ESCCs with high invasion and metastasis, while Annexin A1 was highly expressed in ESCCs with low metastasis.

MEN1 silencing triggers the dysregulation of mTORC1 and MYC pathways in ER+ breast cancer cells.

Menin, encoded by the MEN1 gene, has been identified as a critical factor regulating ESR1 transcription, playing an oncogenic role in ER+ breast cancer (BC) cells. Here, we further dissected the consequences of menin inactivation in ER+ BC cells by focusing on factors within two major pathways involved in BC, mTOR and MYC. MEN1 silencing in MCF7 and T-47D resulted in an increase in phosphor-p70S6K1, phosphor-p85S6K1 and phosphor-4EBP1 expression. The use of an AKT inhibitor inhibited the activation of S6K1 and S6RP triggered by MEN1 knockdown (KD). Moreover, MEN1 silencing in ER+ BC cells led to increased formation of the eIF4E and 4G complex. Clinical studies showed that patients with menin-low breast cancer receiving tamoxifen plus everolimus displayed a trend toward better overall survival. Importantly, MEN1 KD in MCF7 and T-47D cells led to reduced MYC expression. ChIP analysis demonstrated that menin bound not only to the MYC promoter but also to its 5' enhancer. Furthermore, E2-treated MEN1 KD MCF7 cells displayed a decrease in MYC activation, suggesting its role in estrogen-mediated MYC transcription. Finally, expression data mining in tumors revealed a correlation between the expression of MEN1 mRNA and that of several mTORC1 components and targets and a significant inverse correlation between MEN1 and two MYC inhibitory factors, MYCBP2 and MYCT1, in ER+ BC. The current work thus highlights altered mTORC1 and MYC pathways after menin inactivation in ER+ BC cells, providing insight into the crosstalk between menin, mTORC1 and MYC in ER+ BC.

A truncated derivative of FGFR1 kinase cooperates with FLT3 and KIT to transform hematopoietic stem cells in syndromic and de novo AML

BackgroundMyeloid and lymphoid malignancies associated with chimeric FGFR1 kinases are the hallmark of stem cell leukemia and lymphoma syndrome (SCLL). In all cases, FGFR1 kinase is constitutively phosphoactivated as a result of chromosome translocations, which lead to acquisition of dimerization motifs in the chimeric proteins. Recently, we demonstrated that these chimeric kinases could be cleaved by granzyme B to generate a truncated derivative, tnFGFR1, which localized exclusively into the nucleus and was not phosphorylated.MethodsStem cell transduction and transplantation in syngeneic mice was used to assess the transforming ability of tnFGFR1 in bone marrow stem cells, and RPPA and RNA-Seq was used to examine the related signaling pathways and regulated target genes.ResultsFor the first time, we show that this non-classical truncated form of FGFR1 can independently lead to oncogenic transformation of hematopoietic stem cells in an animal model in vivo. These leukemia cells show a mixed immunophenotype with a B-cell B220 + Igm- profile in the majority of cells and Kit+ in virtually all cells, suggesting a stem cell disease. tnFGFR1, however, does not activate classic FGFR1 downstream signaling pathways but induces a distinct profile of altered gene expression with significant upregulation of transmembrane signaling receptors including FLT3 and KIT. We further show that de novo human AML also express tnFGFR1 which correlates with upregulation of FLT3 and KIT as in mouse leukemia cells. ChIP analysis demonstrates tnFGFR1 occupancy at the Flt3 and Kit promoters, suggesting a direct transcriptional regulation. Cells transformed with tnFGFR1 are insensitive to FGFR1 inhibitors but treatment of these cells with the Quizartinib (AC220) FLT3 inhibitor, suppresses in vitro growth and development of leukemia in vivo. Combined treatment with FGFR1 and FLT3 inhibitors provides increased survival compared to FGFR1 inhibition alone.ConclusionsThis study demonstrates a novel model for transformation of hematopoietic stem cells by chimeric FGFR1 kinases with the combined effects of direct protein activation by the full-length kinases and transcriptional regulation by the truncated nuclear tnFGFR1 derivative, which is associated with GZMB expression levels. Genes significantly upregulated by tnFGFR1 include Flt3 and Kit which promote a leukemia stem cell phenotype. In human AML, tnFGFR1 activation leads to increased FLT3 and KIT expression, and higher FLT3 and GZMB expression levels are associated with an inferior prognosis. These observations provide insights into the relative therapeutic value of targeting FGFR1 and FLT3 in treating AML with this characteristic gene expression profile.

Molecular investigation of candidate genes for pyroptosis-induced inflammation in diabetic retinopathy.

BackgroundDiabetic retinopathy is a diabetic microvascular complication. Pyroptosis, as a way of inflammatory death, plays an important role in the occurrence and development of diabetic retinopathy, but its underlying mechanism has not been fully elucidated. The purpose of this study is to identify the potential pyroptosis-related genes in diabetic retinopathy by bioinformatics analysis and validation in a diabetic retinopathy model and predict the microRNAs (miRNAs) and long non-coding RNAs (lncRNAs) interacting with them. Subsequently, the competing endogenous RNA (ceRNA) regulatory network is structured to explore their potential molecular mechanism.MethodsWe obtained mRNA expression profile dataset GSE60436 from the Gene Expression Omnibus (GEO) database and collected 51 pyroptosis-related genes from the PubMmed database. The differentially expressed pyroptosis-related genes were obtained by bioinformatics analysis with R software, and then eight key genes of interest were identified by correlation analysis, Gene Ontology (GO) enrichment analysis, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis, and protein–protein interaction (PPI) network analysis. Then, the expression levels of these key pyroptosis-related genes were validated with quantitative real-time polymerase chain reaction (qRT-PCR) in human retinal endothelial cells with high glucose incubation, which was used as an in vitro model of diabetic retinopathy. Western blot was performed to measure the protein levels of gasdermin D (GSDMD), dasdermin E (GSDME) and cleaved caspase-3 in the cells. Moreover, the aforementioned genes were further confirmed with the validation set. Finally, the ceRNA regulatory network was structured, and the miRNAs and lncRNAs which interacted with CASP3, TLR4, and GBP2 were predicted.ResultsA total of 13 differentially expressed pyroptosis-related genes were screened from six proliferative diabetic retinopathy patients and three RNA samples from human retinas, including one downregulated gene and 12 upregulated genes. A correlation analysis showed that there was a correlation among these genes. Then, KEGG pathway and GO enrichment analyses were performed to explore the functional roles of these genes. The results showed that the mRNA of these genes was mainly related to inflammasome complex, interleukin-1 beta production, and NOD-like receptor signaling pathway. In addition, eight hub genes—CASP3, TLR4, NLRP3, GBP2, CASP1, CASP4, PYCARD, and GBP1—were identified by PPI network analysis using Cytoscape software. High glucose increased the protein level of GSDMD and GSDME, as critical effectors of pyroptosis, in retinal vascular endothelial cells. Verified by qRT-PCR, the expression of all these eight hub genes in the in vitro model of diabetic retinopathy was consistent with the results of the bioinformatics analysis of mRNA chip. Among them, CASP4, GBP1, CASP3, TLR4, and GBP2 were further validated in the GSE179568 dataset. Finally, 20 miRNAs were predicted to target three key genes—CASP3, GBP2, and TLR4, and 22 lncRNAs were predicted to potentially bind to these 20 miRNAs. Then, we constructed a key ceRNA network that is expected to mediate cellular pyroptosis in diabetic retinopathy.ConclusionThrough the data analysis of the GEO database by R software and verification by qRT-PCR and validation set, we successfully identified potential pyroptosis-related genes involved in the occurrence of diabetic retinopathy. The key ceRNA regulatory network associated with these genes was structured. These findings might improve the understanding of molecular mechanisms underlying pyroptosis in diabetic retinopathy.

Exploring the Regulation of Jiangtang Tiaozhi Formula on the Biological Network of Obese T2DM Complicated With Dyslipidemia Based on Clinical Transcriptomics

ObjectiveTo use systems biology to explore the biomolecular network mechanism of the Jiangtang Tiaozhi Recipe (JTTZR) in the intervention of obese Type 2 diabetes (T2DM) patients with dyslipidemia.MethodsTwelve patients with obese type 2 diabetes mellitus and dyslipidemia (traditional Chinese medicine syndrome differentiation was excess heat syndrome of the stomach and intestines) were treated with JTTZR for 24 weeks, and 12 patients were included in the healthy control group. First, blood samples from 6 patients in each group (disease group before treatment, disease group after treatment, and healthy control group) were collected for RNA microarray analysis. Quantitative polymerase chain reaction (qPCR) was used to validate these target lncRNAs and mRNAs. Finally, a detailed analysis of the differences in the disease group before treatment vs. the healthy control group and the disease group after treatment vs. the disease group before treatment was undertaken. In addition, we focused on disease-related pathways and analyzed the correlation between the differential expression of target lncRNAs and clinical indicators. Results(1) Disease group before treatment vs. healthy control group: There were 557 up-regulated lncRNAs, 273 down-regulated lncRNAs, 491 up-regulated mRNAs, and 1639 down-regulated mRNAs. GO analysis and pathway analysis showed that T2DM may be related to cell proliferation in the forebrain, post-embryonic organ development, calcium signaling pathway. qPCR validation showed that the expression of XLOC-005590 and HNF1A-AS1 as target lncRNAs increased, and this was verified by gene chip analysis. (2) Disease group after treatment vs. disease group before treatment: 128 lncRNAs were upregulated, 32 lncRNAs were downregulated, 45 mRNAs were upregulated, and 140 mRNAs were downregulated. GO analysis and pathway analysis showed that JTTZR may treat T2DM through endosome transport, the insulin signaling pathway, and glycine, serine, and threonine metabolism. qPCR validation showed that in the healthy control group, XLOC_005590 was upregulated, whereas the downstream gene (ECI2) was downregulated in the disease group before treatment. However, after 24 weeks of intervention with JTTZR, XLOC_005590 was downregulated and ECI2 was upregulated compared with the disease group before treatment (0 weeks) (P <0.05).ConclusionJTTZR may interfere in patients with obese T2DM with dyslipidemia by regulating pathways such as fatty acid degradation, glycolysis/gluconeogenesis, and pyruvate metabolism.

Clinical immunofluorescence comparison analysis of microfluidic chips for highly efficient detection of CTCs with colorectal cancer and other cancers

Clinical immunofluorescence comparison analysis of microfluidic chips for highly efficient detection of CTCs with colorectal cancer and other cancers

FBXL17/spastin axis as a novel therapeutic target of hereditary spastic paraplegia

BackgroundSpastin significantly influences microtubule regulation in neurons and is implicated in the pathogenesis of hereditary spastic paraplegia (HSP). However, post-translational regulation of the spastin protein remains nebulous. The association between E3 ubiquitin ligase and spastin provides a potential therapeutic strategy.ResultsAs evidenced by protein chip analysis, FBXL17 inversely correlated with SPAST-M1 at the protein level in vitro and, also in vivo during embryonic developmental stage. SPAST-M1 protein interacted with FBXL17 specifically via the BTB domain at the N-terminus of SPAST-M1. The SCFFBXL17 E3 ubiquitin ligase complex degraded SPAST-M1 protein in the nuclear fraction in a proteasome-dependent manner. SPAST phosphorylation occurred only in the cytoplasmic fraction by CK2 and was involved in poly-ubiquitination. Inhibition of SCFFBXL17 E3 ubiquitin ligase by small chemical and FBXL17 shRNA decreased proteasome-dependent degradation of SPAST-M1 and induced axonal extension. The SPAST Y52C mutant, harboring abnormality in BTB domain could not interact with FBXL17, thereby escaping protein regulation by the SCFFBXL17 E3 ubiquitin ligase complex, resulting in loss of functionality with aberrant quantity. Although this mutant showed shortening of axonal outgrowth, low rate proliferation, and poor differentiation capacity in a 3D model, this phenotype was rescued by inhibiting SCFFBXL17 E3 ubiquitin ligase.ConclusionsWe discovered that a novel pathway, FBXL17-SPAST was involved in pathogenicity of HSP by the loss of function and the quantitative regulation. This result suggested that targeting FBXL17 could provide new insight into HSP therapeutics.

TBX3 activating PVT1 accelerates proliferation, migration, and invasion by modulating the miR-30a/LOX axis in anaplastic thyroid carcinoma.

Anaplastic thyroid carcinoma (ATC) is a nearly chemo-resistant malignancy with high invasion and mortality. Long non-coding RNAs (lncRNAs) have been demonstrated to be dysregulated and play a crucial role in the development and process of ATC. The present study aimed to explore the mechanism of PVT1 dysregulation in ATC. The mRNA levels of PVT1 and T-box3 (TBX3), and the protein levels of TBX3 in ATC and paracancerous tissues, and FRO and Nthy-ori 3-1 cells were determined by quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) and western blot, respectively. The transcriptional factor binding site was predicted and validated between TBX3 and PVT1 promoter through the JASPAR website, and ChIP and luciferase analysis. The proliferation, migration, and invasion of FRO cells were assessed by MTT, colony formation, and transwell assays. PVT1 expression was upregulated in ATC, which was positively correlative with the level of transcription factor TBX3. Downregulation of PVT1 inhibited the proliferation, migration, and invasion of FRO cells. Moreover, TBX3 targeting the promoter region of PVT1 promoted the expression level of PVT1 and modulated the downstream signalling axis of PVT1, miR-30a/LOX. Also, interference of PVT1 reversed the stimulative role of overexpression of TBX3 in the progress of FRO cells. TBX3 enhanced proliferation, migration, and invasion of ATC cells via activation of PVT1 and modulation of the miR-30a/LOX signalling axis.