Upstream Regulatory Gene Research Articles

The M6A Methyltransferase METTL14 Promotes Myelodysplastic Syndromes Development Via PI3K/Akt Signaling Pathway

Background: RNA N6-methyladenosine (m6A) plays a critical role in normal and malignant hematopoiesis. The m6A methyltransferases METTL3 and METTL14 were reported as oncogenes in acute myeloid leukemia. Epigenetic dysregulation is a prominent biological feature of myelodysplastic syndromes (MDS), however, whether RNA methylation dysregulation exists in MDS, and what's the functions of m6A methyltransferases are largely unknown. Methods: The global m6A modification level of RNA in bone marrow was detected in MDS patients by colorimetric method, and the expression level of METTL14 in bone marrow of MDS patients was detected by q-PCR. Both gain- and loss-of-function experiments were performed to study the role of METTL14 in MDS. M6A sequencing and RNA sequencing were examined to explore the potential signaling pathways and target genes that mediated METTL14 function in MDS. Results: We detected obvious RNA m6A dysregulation in bone marrow of MDS patients compared with normal controls, and found that the up-regulation of the global m6A modification is closely related to higher-risk category and worse survival of MDS patients. By analyzing the MDS public database (GSE_58831) and q-PCR data of MDS patients in our center, we found that among 23 classical m6A related genes, m6A methyltransferase METTL14 was the key factor causing the m6A dysregulation in MDS: high expression of METTL14 was associated with higher disease risk stratification, more bone marrow blasts, poorer prognosis, and higher response rates to hypomethylating agents (HMAs). In order to explore the functions of METTL14 in MDS, we performed gain- and loss-of-function experiments in MDS cell line MDSL in vitro. It was observed that the expression of METTL14 promoted the cell proliferation and clone formation ability of MDS cells by inducing cell apoptosis and differentiation. More interestingly, we found that HMAs down-regulated the level of global m6A modification by inhibiting METTL14 expression, and the expression of METTL14 could increase the sensitivity of MDS cells to HMAs in MDS cells. To search for upstream regulatory genes of METTL14, we analyzed the ChIPBase online database and identified ELF1 might be the potential upstream transcription factor. The relationship between ELF1 and METTL14 was further confirmed by ELF1-overexpressed and ELF1-knowndowned MDSL cell lines. To explore the relationship between gene mutations and METTL14, we analyzed the correlations between next generation sequencing (NGS) data and METTL14 expression level in MDS patients in our center, and found that the METTL14 expression level was significantly increased in MDS patients carrying TP53 gene mutations (P<0.05). The increased METTL14 and ELF1 expression were all observed in MDS cell lines with introduction of TP53 missense mutations (M237I and Y220C) or TP53 knockdown. Mechanistically, m6A sequencing and RNA-seq sequencing revealed METTL14 promoted MDS development via PI3K/Akt signaling pathway, and the potential target genes involved in PI3K/Akt signaling pathway were ANGPT1, CDK6, FGFR2, GNG11, IGF1R et al. Conclusion： Herein, we reported RNA m6A methylation dysregulation did exist in MDS, and m6A methyltransferase METTL14 was an important regulatory factor. Higher METTL14 expression was associated with higher-risk stratification, poorer survival, and better response to HMAs in MDS patients. METTL14 was required for the cell growth and response to HMAs in MDS cells. TP53 mutations were significantly correlated with the up-regulation of METTL14 expression level, and TP53 mutations could induce METTL14 overexpression by regulating transcription factor ELF1. Mechanistically, METTL14 exerts its oncogenic role via PI3K/Akt signaling pathway. Our findings highlighted that METTL14 could potentially be a novel therapeutic target in MDS especially those with TP53 mutations. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Alleviative mechanism and effect of Bifidobacterium animalisA6 on dextran sodium sulfate-induced ulcerative colitis in mice.

Probiotics have been increasingly investigated for their role in alleviating symptoms of ulcerative colitis (UC), but the specific mechanism involved remains unclear. We investigated the alleviating effect of Bifidobacterium animalis A6 (BAA6) in UC through a mouse dextran sulfate sodium (DSS) model. When treated with a high dose of BAA6 (1 × 1010 cfu/ml), it was found that colitis symptoms were significantly alleviated, and mucosal damages experienced obvious relief. Moreover, a high dose of BAA6 effectively upregulated free fatty acid receptors 2 and 3 (FFAR2 and FFAR3) expression and butyric acid metabolism specifically. Furthermore, the supplement of BAA6 significantly suppressed pro-inflammatory cytokines levels (interleukin-13) and the expression of pore-forming protein claudin-2. The upstream regulatory genes of claudin-2, such as STAT6, GATA4, Cdx2, were also significantly inhibited by BAA6. Collectively, this study concludes that BAA6 attenuated DSS-induced colitis by increasing the levels of intestinal butyric acid, activating the butyric acid-FFAR pathway, suppressing excessive proinflammatory response, and protecting the function of the colon epithelial barrier.

Identification of key genes associated with cancer stem cell characteristics in Wilms’ tumor based on bioinformatics analysis

Nephroblastoma, also known as Wilms' tumor (WT), remains one of the major causes of tumor-related deaths worldwide in children. Cancer stem cells (CSCs) are considered to be the main culprits in cancer resistance and disease recurrence, which are reported in multiple types of tumors. However, the research on CSCs in WT is limited. Therefore, our study aimed to identify the key genes related to CSCs in WT to provide new ideas for treating WT. The RNA-seq and clinical data of WT samples were obtained from the University of California Santa Cruz (UCSC) Xena database, which included 120 WT and six para-cancerous tissues. The mRNA stemness index (mRNAsi) based on mRNA expression was calculated to evaluate tumor stem cell characteristics in WT patients. A Kaplan-Meier (KM) analysis was performed to explore the clinical characteristics of the mRNAsi in WT. A weighted gene co-expression network analysis (WGCNA) was used to identify the key modules and genes related to the mRNAsi. A Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis was performed to explore the signaling pathways based on the key genes. The expression levels of the key genes were validated by the Gene Expression Omnibus (GEO) database. Further, the important upstream genes were identified by DisNor and gene co-expression analyses. The mRNAsi was significantly upregulated in WT (P=7.2e-05) and showed an upward trend in line with the pathological stage. Patients with lower mRNAsi scores had better overall survival (OS) than those with higher mRNAsi scores (P=0.0087). Eleven genes were defined as the key genes associated with the mRNAsi based on our WGCNA analysis [cor.MM (correlation. Module membership) >0.8 and cor.GS (correlation. Gene significance) >0.45] and were closely related to cell proliferation-related signaling pathways (P<0.05). Moreover, using protein interaction analysis, we identified ATM and CDKN1A as the key upstream regulatory genes of the 11 key genes. Our study showed that the mRNAsi score was a potential prognostic factors in WT and identified the upstream genes ATM and CDKN1A and 11 genes closely related to the mRNAsi, which may provide new insights for CSC-targeted therapy in WT and improve clinical outcomes for WT patients.

The upstream regulatory mechanism of BplMYB46 and the function of upstream regulatory factors that mediate resistance to stress in Betula platyphylla.

Previously, we have shown that the transcription factor BplMYB46 in Betula platyphylla can enhance tolerance to salt and osmotic stress and promote secondary cell wall deposition, and we characterized its downstream regulatory mechanism. However, its upstream regulatory mechanism remains unclear. Here, the promoter activity and upstream regulatory factors of BplMYB46 were studied. Analyses of β-glucuronidase (GUS) staining and activity indicated that BplMYB46 promoter was specific temporal and spatial expression, and its expression can be induced by salt and osmotic stress. We identified three upstream regulatory factors of BplMYB46: BpDof1, BpWRKY3, and BpbZIP3. Yeast-one hybrid assays, GUS activity, chromatin immunoprecipitation, and quantitative real-time polymerase chain reaction revealed that BpDof1, BpWRKY3, and BpbZIP3 can directly regulate the expression of BplMYB46 by specifically binding to Dof, W-box, and ABRE elements in the BplMYB46 promoter, respectively. BpDof1, BpWRKY3, and BpbZIP3 were all localized to the nucleus, and their expressions can be induced by stress. Overexpression of BpDof1, BpWRKY3, and BpbZIP3 conferred the resistance of transgenic birch plants to salt and osmotic stress. Our findings provide new insights into the upstream regulatory mechanism of BplMYB46 and reveal new upstream regulatory genes that mediate resistance to adverse environments. The genes identified in our study provide novel targets for the breeding of forest tree species.

PFKFB4 modulated by miR-195-5p can boost the malignant progression of cervical cancer cells

PFKFB4 is dysregulated in varying tumors and has the biological function of regulating tumor progression. However, its biological function in cervical cancer is poorly understood. We obtained the upstream regulatory gene (miR-195-5p) of PFKFB4 through bioinformatics analysis. Then, experiments were introduced to measure expression and targeting relationship of miR-195-5p and PFKFB4 in cervical cancer cells, in order to evaluate their influence on proliferation, migration, invasion and angiogenesis of cervical cancer cells. As expressed in results, PFKFB4 was abnormally increased and boosted malignant progression of cervical cancer cells. Besides, miR-195-5p was markedly decreased and restrained PFKFB4 in cervical cancer. While tumor-suppressive effect of miR-195-5p was partially restored by overexpressing PFKFB4, indicating that miR-195-5p and PFKFB4 may be new therapeutic targets for cervical cancer patients.

Insulin peptides and their receptors regulate ovarian development and oviposition behavior in Diaphorina citri.

Diaphorina citri is an important vector of Citrus Huanglongbing (HLB) disease. After feeding on young host plant shoots, the population of D.citri can increase significantly. Females also only lay eggs on young shoots. However, there are few studies on the mechanism of this phenomenon. Exogenous nutrient signals can affect the insulin signaling system of D.citri after feeding on young shoots. In this study, the expression of upstream factors DcILP1, DcILP2, and DcIR in the insulin signaling system of D.citri was upregulated after feeding on young shoots. After being silenced by RNA interference technology, the results showed that the number of oviposited eggs of D.citri was significantly decreased and the ovarian development was inhibited with severe vacuolation. In addition, detection using quantitative reverse transcription-polymerase chain reaction showed that the upstream regulatory gene DcRheb of the target of rapamycin (TOR) pathway and the downstream reproduction-related DcVg gene were also significantly downregulated. These results suggest that feeding upon young shoots may upregulate the expression levels of upstream factors DcILP1, DcILP2, and DcIR in the insulin signaling system. The signal will be through upregulating the expression of DcRheb, an upstream gene of the TOR signaling pathway. This in turn influences yolk metabolism, which eventually causes the ovaries of female D.citri to mature and therefore initiate oviposition behavior.

MicroRNA-873-5p suppresses cell malignant behaviors of thyroid cancer via targeting CXCL5 and regulating P53 pathway

ABSTRACT We aimed to examine the roles of microRNA-873-5p and CXCL5 in thyroid cancer (TC) cells. qRT-PCR was adopted to measure the expression levels of CXCL5 mRNA and microRNA-873-5p in TC cells, and western blot was adopted to evaluate the CXCL5 protein expression level. Bioinformatics analysis was done to predict the upstream gene of CXCL5. Dual-luciferase assay was applied to validate the binding relationship of CXCL5 and the upstream regulatory gene. Cell experiments were done to detect the effects of microRNA-873-5p targeting CXCL5 on malignant progression of cancer cells. Western blot was adopted to demonstrate the phosphorylation level of P53 pathway related-proteins. CXCL5 was upregulated in TC cells and tissues. The results of in vitro assays displayed that CXCL5 downregulation dramatically suppressed the malignant behaviors of TC cells. MicroRNA-873-5p suppressed CXCL5 expression, but the suppressive effect of microRNA-873-5p on TC cells was abolished through CXCL5 overexpression. Additionally, microRNA-873-5p could mediate p53 pathway and thereby inhibit the malignant behaviors of TC cells through targeting CXCL5. In summary, we proved that microRNA-873-5p repressed the malignant behaviors of TC cells through targeting CXCL5 and P53 pathway, indicating that microRNA-873-5p can be a biomarker for TC.

Decay of TRPV3 as the genomic trace of epidermal structure changes in the land-to-sea transition of mammals.

The epidermis plays an indispensable barrier function in animals. Some species have evolved unique epidermal structures to adapt to different environments. Aquatic and semi‐aquatic mammals (cetaceans, manatees, and hippopotamus) are good models to study the evolution of epidermal structures because of their exceptionally thickened stratum spinosum, the lack of stratum granulosum, and the parakeratotic stratum corneum. This study aimed to analyze an upstream regulatory gene transient receptor potential cation channel, subfamily V, member 3 (TRPV3) of epidermal differentiation so as to explore the association between TRPV3 evolution and epidermal changes in mammals. Inactivating mutations were detected in almost all the aquatic cetaceans and several terrestrial mammals. Relaxed selective pressure was examined in the cetacean lineages with inactivated TRPV3, which might contribute to its exceptionally thickened stratum spinosum as the significant thickening of stratum spinosum in TRPV3 knock‐out mouse. However, functional TRPV3 may exist in several terrestrial mammals due to their strong purifying selection, although they have “inactivating mutations.” Further, for intact sequences, relaxed selective constraints on the TRPV3 gene were also detected in aquatic cetaceans, manatees, and semi‐aquatic hippopotamus. However, they had intact TRPV3, suggesting that the accumulation of inactivating mutations might have lagged behind the relaxed selective pressure. The results of this study revealed the decay of TRPV3 being the genomic trace of epidermal development in aquatic and semi‐aquatic mammals. They provided insights into convergently evolutionary changes of epidermal structures during the transition from the terrestrial to the aquatic environment.

MiR-139-5p Targeting CCNB1 Modulates Proliferation, Migration, Invasion and Cell Cycle in Lung Adenocarcinoma.

Lung adenocarcinoma (LUAD) is the most frequent histological subtype of non-small cell lung cancer. Cyclin B1 (CCNB1) is the vital initiator and controller of mitosis. Studies have indicated that CCNB1 overexpression is closely associated with cell proliferation and tumorigenesis in many cancers. Thus, discovery of molecular mechanism of CCNB1 in LUAD is conducive to developing new diagnostic or therapeutic targets for LUAD. We acquired mature miRNA and mRNA expression information of LUAD from TCGA database, as well as related clinical data. CCNB1 expression in normal and LUAD tissue was analyzed. Relationship between CCNB1 and patient's survival and clinical stage was analyzed. Upstream regulatory gene miRNA of CCNB1 was predicted. qRT-PCR and western blot examined expression levels of CCNB1 and miR-139-5p in cells. CCK-8 tested cell proliferation. Scratch healing and Transwell determined cell migration and invasion. Flow cytometry analyzed the cell cycle. Dual-luciferase verified targeting relationship between the two genes. Compared to controls, CCNB1 expression was prominently high in LUAD patient samples, and associated with advanced tumor stages and shorter overall survival. MiR-139-5p expressed an evidently negative correlation with CCNB1 and was predicted to target CCNB1. MiR-139-5p mimics reduced CCNB1 mRNA and protein expression, and suppressed luciferase activity in a target-specific manner, as confirmed by a control construct with a mutated miR-139-5p binding site. CCNB1 overexpression fostered progression of LUAD cells. Mechanistically, miR-139-5p might negatively regulate CCNB1 in LUAD, thereby suppressing cell proliferation, migration, invasion and cell cycle.

MiR-671-5p Promotes Cell Proliferation, Invasion, and Migration in Hepatocellular Carcinoma through Targeting ALDH2.

We explored the mechanism of acetaldehyde dehydrogenase 2 (ALDH2) in modulating cell behaviors in hepatocellular carcinoma (HCC), and provided fresh ideas for targeted treatment of HCC. The target messenger RNA (mRNA) was determined by The Cancer Genome Atlas (TCGA) analysis, and the upstream regulatory gene miRNA was obtained by further analysis. The expression of ALDH2 mRNA and miR-671-5p in HCC cell lines was assayed by quantitative reverse transcription polymerase chain reaction (qRT-PCR), and protein expression was assessed by Western blot. The impact of ALDH2 on biological functions of HCC cells was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), wound-healing, and Transwell assays. Bioinformatics method was utilized to predict binding site of miR-671-5p and ALDH2, and their targeted relationship was detected by dual-luciferase gene assay, qRT-PCR and Western blot. ALDH2 expression was reduced in HCC tissue and cell lines. ALDH2 worked as a tumor inhibitor in HCC. Overexpressing ALDH2 could hinder proliferation, migration and invasion of HCC cells. miR-671-5p was the upstream regulatory gene of ALDH2, and it presented remarkably high expression in HCC. A negative modulatory relationship existed between miR-671-5p and ALDH2. The rescue experiments further illustrated the effects of the two on the malignant behaviors of HCC cells. Forced expression of miR-671-5p fostered the proliferation, migration and invasion of HCC cells by restraining ALDH2.

NRF-1 directly regulates TFE3 and promotes the proliferation of renal cancer cells.

The role of transcription factor binding to IGHM enhancer 3 (TFE3) in renal cell carcinoma (RCC) is not well understood. Nuclear respiratory factor 1 (NRF-1) may be the positive upstream regulatory gene of TFE3. The aim of the present study was to determine whether NRF-1 could directly regulate the expression of TFE3 and regulate tumorigenesis and progression of RCC through TFE3. Short hairpin RNA (shRNA) was used to silence the expression of NRF-1 in the 786-O human kidney adenocarcinoma cell line and the 293T human embryonic kidney cell line. Luciferase reporter assays were used to determine the relationship between NRF-1 and TFE3. The CHIP experiment was used to verify the actual binding of NRF-1 and TFE3 promoter regions. MitoTimer staining was used to measure mitochondrial biosynthesis. Flow cytometry was used to detect cell cycle and apoptosis. The 786-O and 293T cells were used to examine the underlying mechanism of action. The results demonstrated that NRF-1 could bind to the promoter region of the TFE3 gene and directly regulate the expression of TFE3. Following NRF-1 knockdown, the protein levels of phosphorylated (p)-AKT and p-S6 of mTOR pathway was inhibited, cell cycle progression was blocked, the levels of apoptosis increased, and mitochondrial generation was reduced. Following overexpression of TFE3, the levels of mTOR-associated markers were restored in NRF-1 knockdown cells. These findings suggest that NRF-1 may regulate the mTOR pathway through TFE3 and regulate the energy metabolism, proliferation and growth of cancer cells by directly regulating the expression of TFE3.

MiR-30a-5p Regulates Viability, Migration, and Invasion of Lung Adenocarcinoma Cells via Targeting ECT2.

Objective The abnormal expression of epithelial cell transforming sequence 2 (ECT2) is often considered the driving factor for the growth and invasion of tumors. This study was performed to investigate the regulatory effect of miR-30a-5p and ECT2 on lung adenocarcinoma (LUAD), which provides a basis for the effective clinical treatment of LUAD. Methods The mature miRNAs, expression data of mRNAs, and clinical data of LUAD were downloaded from The Cancer Genome Atlas (TCGA). The expression levels of ECT2 mRNA and miR-30a-5p in cancer cell lines were detected by qRT-PCR. Western blot was performed to test the expression of ECT2 protein. The targeting relationship between miR-30a-5p and ECT2 was verified by dual-luciferase assay. The CCK-8 method and Transwell assay were conducted to test the viability, migratory, and invasive abilities of cells. Results ECT2 expression was upregulated in LUAD and was significantly correlated with the LUAD clinical stage and pathologic T stage, and the expression of its upstream regulatory gene miR-30a-5p was downregulated. miR-30a-5p targeted ECT2 in LUAD. Downregulation of ECT2 could inhibit the viability, migration, and invasion of LUAD cells, which could be reversed by simultaneously suppressing the expression of miR-30a-5p. Conclusion Our results suggested that miR-30a-5p repressed the malignant progression of LUAD via downregulating ECT2. miR-30a-5p and ECT2 may be effective targets for LUAD patients.

MiR-18a-5p Targets FBP1 to Promote Proliferation, Migration, and Invasion of Liver Cancer Cells and Inhibit Cell Apoptosis.

Liver cancer is one of the most aggressive malignant tumors. It is significant to understand the molecular mechanism of liver cancer cells to develop new treatment plans. Studies have identified that FBP1 serves as a cancer inhibitor gene. To research the effect mechanism of FBP1 in liver cancer cells, bioinformatics analysis was performed to study its expression in liver cancer tissue. Survival analysis was also performed. Moreover, starBase database was applied to predict upstream regulatory genes of FBP1. Dual-luciferase assay was performed to testify their targeted relationship. The mRNA and protein expression levels of FBP1 in liver cancer cells were detected by qRT-PCR and western blot, respectively. Cell viability was analyzed by CCK-8 assay. The migratory and invasive abilities of cells were analyzed by Transwell assay. The apoptosis of liver cancer cells was detected by flow cytometry. The results showed that the expression of FBP1 was downregulated in liver cancer tissue and cells. FBP1 low expression was correlated with the poor prognosis of patients. miR-18a-5p could inhibit FBP1 expression. Overexpression of FBP1 could inhibit the progression of liver cancer cells and promote cell apoptosis. Overexpressing miR-18a-5p could promote the progression of liver cancer cells and inhibit cell apoptosis. However, overexpressing FBP1 simultaneously could reverse the effect. miR-18a-5p and FBP1 are expected to be candidates for liver cancer treatment.

The Role of N-myc Downstream-Regulated Gene Family in Glioma Based on Bioinformatics Analysis.

Glioma is the most common type of primary tumor in the central nervous system, and the molecular mechanisms remain elusive. N-myc downstream-regulated gene (NDRG) family is reported to take part in the pathogenesis of various diseases, including some preliminary exploration in glioma. However, there has been no bioinformatics analysis of NDRG family in glioma yet. Herein, we focused on the expression changes of NDRGs with their value in predicting patients' prognoses, upstream regulatory mechanisms (DNA mutation, DNA methylation, transcription factors, and microRNA regulation) and gene enrichment analysis based on co-expressed genes with data from public databases. Furthermore, the expression pattern of NDRGs was verified by the paired glioma and peritumoral samples in our institute. It was suggested that NDRGs were differentially expressed genes in glioma. In particular, the lower expression of NDRG2 or NDRG4 could serve as a predictor of higher grade tumor and poorer prognosis. Also, NDRGs might play a crucial role in signal transduction, energy metabolism, and cross-talk among cells in glioma, under the control of a complex regulatory network. This study enables us to better understand the role of NDRGs in glioma and with further research, it may contribute to the development of glioma treatment.

Reconstruction of a Context-Specific Model Based on Genome-Scale Metabolic Simulation for Identification of Prochloraz Resistance Mechanisms in Penicillium digitatum.

Penicillium digitatum is the most destructive postharvest pathogen of citrus fruits, causing substantial economic losses. Prochloraz-resistant strains have emerged due to overuse of imidazole fungicides in agriculture. To study the prochloraz resistance mechanisms at the system level, a genome-scale metabolic model (GEM, iPD1512) of P. digitatum was reconstructed and constrained based on context-specific transcriptome data of the prochloraz-resistant strain, PdF6, from our previous work, a newly sequenced, context-specific transcriptome result of the major facilitator superfamily transporter-encoding gene mfs2 knockout mutant PdF6Δmfs2, and experimentally derived growth rate data. Through the model, iPD1512, the processes of prochloraz resistance in P. digitatum were well simulated. In detail, the growth rates of both wild-type and mutant P. digitatum under different prochloraz concentrations were simulated using constraint-based reconstruction and analysis. The growth rates of the mutant strains (sterol regulatory element-binding protein-encoding gene sreA knockout mutant PdF6ΔsreA and PdF6Δmfs2) were calculated and confirmed to be consistent with the simulation results. Furthermore, correlations between genes and prochloraz resistance were predicted and showed a great difference when compared with correlation results based on p-values from the hypothesis testing used by comparative transcriptomics. To sum up, in contrast to traditional transcriptome analysis, the GEM provides a systemic and dynamic drug resistance mechanism, which might help to detect some key upstream regulatory genes, but with small expression changes, and might provide more efficient targets to control prochloraz-resistant P. digitatum.

Si-SNHG5-FOXF2 inhibits TGF-\u03b21-induced fibrosis in human primary endometrial stromal cells by the Wnt/\u03b2-catenin signalling pathway

BackgroundIntrauterine adhesions (IUAs) are manifestations of endometrial fibrosis characterized by inflammation and fibrinogen aggregation in the extracellular matrix (ECM). The available therapeutic interventions for IUA are insufficiently effective in the clinical setting for postoperative adhesion recurrence and infertility problems. In this study, we investigated whether si-SNHG5-FOXF2 can serve as a molecular mechanism for the inhibition of IUA fibrosis ex vivo.MethodsFOXF2, TGF-β1 and collagen expression levels were measured by microarray sequencing analysis in three normal endometrium groups and six IUA patients. We induced primary human endometrial stromal cells (HESCs) into myofibroblasts (MFs) to develop an IUA cell model with various concentrations of TGF-β1 at various times. Downstream target genes of FOXF2 were screened by chromatin immunoprecipitation combined with whole-genome high-throughput sequencing (ChIP-seq). We investigated ECM formation, cell proliferation and Wnt/β-catenin signalling pathway-related proteins in primary HESCs with FOXF2 downregulation by quantitative reverse transcription-polymerase chain reaction (qRT-PCR), western blotting (WB), immunohistochemistry (IHC), flow cytometry, ethylenediurea (EdU) and CCK8 assays. We identified long noncoding RNAs (lncRNA) SNHG5 as the upstream regulatory gene of FOXF2 through RNA immunoprecipitation (RIP), RNA pulldown and fluorescence in situ hybridization (FISH). Finally, we examined FOXF2 expression, ECM formation, cell proliferation and Wnt/β-catenin signalling pathway-related proteins in primary HESCs upon FOXF2 downregulation.ResultsFOXF2 was highly expressed in the endometrium of patients with IUA. Treatment of primary HESCs with 10 ng/ml TGF-β1 for 72 h was found to be most effective for developing an IUA cell model. FOXF2 regulated multiple downstream target genes, including collagen, vimentin (VIM) and cyclin D2/DK4, by ChIP-seq and ChIP-PCR. FOXF2 downregulation inhibited TGF-β1-mediated primary HESC fibrosis, including ECM formation, cell proliferation and Wnt/β-catenin signalling pathway-related protein expression. We identified lncRNA SNHG5 as an upstream gene that directly regulates FOXF2 by RIP-seq, qRT-PCR, WB and FISH. SNHG5 downregulation suppressed FOXF2 expression in the IUA cell model, resulting in synergistic repression of the Wnt/β-catenin pathway, thereby altering TGF-β1-mediated ECM aggregation in endometrial stromal cells ex vivo.ConclusionsRegulation of the Wnt/β-catenin signalling pathway and ECM formation by si-SNHG5-FOXF2 effectively inhibited the profibrotic effect of TGF-β1 on primary HESCs. This finding can provide a molecular basis for antagonizing TGF-β1-mediated fibrosis in primary HESCs.

The role of Virostatic genes in modulating Immune Checkpoints in Epstein-Barr Virus associated Tumors

Abstract Epstein-Barr Virus (EBV) is a complex oncovirus that infects around 90% of adults. Most primary infections remain asymptomatic but result in lifelong infection. EBV can drive lethal malignancies of both lymphoid (e.g. Burkitt lymphoma) and epithelial origins (e.g. Stomach Adenocarcinoma). Despite decades of research, the exact molecular mechanisms of EBV and host immune system interactions that lead to tumorigenesis remain elusive. Through systematic analyses of high-throughput sequencing data from &amp;gt;1000 patients with different cancer types, we identified twelve EBV transcripts, including LMP1 and LMP2, which when overexpressed, significantly suppressed viral reactivation. These ‘virostatic genes’ had frequent missense and nonsense variations in cancer patients, disrupting their ability to restrict EBV reactivation. We also found that viral reactivation by classical chemical inducers up-regulates the expression of Programmed Death-Ligand 1 (PD-L1). Further analyses of the same RNA-seq dataset classified EBV+ tumors into two molecular subtypes based on expression pattern of upstream regulatory immune-related genes and Immune Checkpoint (IC) markers, including PD-L1. Overexpression of virostatic genes in lymphoblastic cells significantly repressed cellular PD-L1 transcription, leading us to hypothesize that a combination of IC and virostatic gene modulation may have synergistic benefits for immunotherapy of EBV-associated cancers.

Carnitine palmitoyltransferase 1C contributes to progressive cellular senescence.

Stable transfection manipulation with antibiotic selection and passaging induces progressive cellular senescence phenotypes. However, the underlying mechanisms remain poorly understood. This study demonstrated that stable transfection of the empty vector induced PANC-1 cells into cellular senescence. Metabolomics revealed several acylcarnitines and their upstream regulatory gene, carnitine palmitoyltransferase 1C (CPT1C) involved in fatty acid β-oxidation in mitochondria, were strikingly decreased in senescent PANC-1 cells. Low CPT1C expression triggered mitochondrial dysfunction, inhibited telomere elongation, impaired cell survival under metabolic stress, and hindered the malignance and tumorigenesis of senescent cells. On the contrary, mitochondrial activity was restored by CPT1C gain-of-function in senescent vector PANC-1 cells. PPARα and TP53/CDKN1A, crucial signaling components in cellular senescence, were downregulated in senescent PANC-1 cells. This study identifies CPT1C as a key regulator of stable transfection-induced progressive PANC-1 cell senescence that inhibits mitochondrial function-associated metabolic reprogramming. These findings confirm the need to identify cell culture alterations after stable transfection, particularly when cells are used for metabolomics and mitochondria-associated studies, and suggest inhibition of CPT1C could be a promising target to intervene pancreatic tumorigenesis.

EWS promotes cell proliferation and inhibits cell apoptosis by regulating miR-199a-5p/Sox2 axis in osteosarcoma.

Osteosarcoma is one of the most common malignant bone tumors which mainly occurs in children and adolescents. It is characterized by high malignancy and high metastasis rate, resulting in high mortality and disability. Accumulating studies have validated that long noncoding RNAs (lncRNAs) exerted vital roles in multiple cancer progression by regulating the expression of specific genes. This work aimed to explore the potential molecular mechanism of EWS in osteosarcoma. In this study, we discovered that both EWS and Sox2 were highly expressed in osteosarcoma tissue samples. In addition, the expression of EWS was positively associated with Sox2 level. We conducted a series of functional assays and observed that Sox2 overexpression could significantly overturned the enhancement of cell proliferation and the decline of cell apoptosis induced by EWS knockdown in osteosarcoma. Moreover, we found a key upstream regulatory gene of Sox2: miR-199a-5p. Through molecular biology studies and rescue assays, we further demonstrated that EWS promotes tumor growth through the miR-199a-5p/Sox2 signaling axis in osteosarcoma. These findings may provide an important theoretical basis for the clinical diagnosis and treatment of osteosarcoma.

Citrus Polyamines: Structure, Biosynthesis, and Physiological Functions.

Polyamines (PAs) are ubiquitous biogenic amines found in all living organisms from bacteria to Archaea, and Eukaryotes including plants and animals. Since the first description of putrescine conjugate, feruloyl-putrescine (originally called subaphylline), from grapefruit leaves and juice, many research studies have highlighted the importance of PAs in growth, development, and other physiological processes in citrus plants. PAs appear to be involved in a wide range of physiological processes in citrus plants; however, their exact roles are not fully understood. Accordingly, in the present review, we discuss the biosynthesis of PAs in citrus plants, with an emphasis on the recent advances in identifying and characterizing PAs-biosynthetic genes and other upstream regulatory genes involved in transcriptional regulation of PAs metabolism. In addition, we will discuss the recent metabolic, genetic, and molecular evidence illustrating the roles of PAs metabolism in citrus physiology including somatic embryogenesis; root system formation, morphology, and architecture; plant growth and shoot system architecture; inflorescence, flowering, and flowering-associated events; fruit set, development, and quality; stomatal closure and gas-exchange; and chlorophyll fluorescence and photosynthesis. We believe that the molecular and biochemical understanding of PAs metabolism and their physiological roles in citrus plants will help citrus breeding programs to enhance tolerance to biotic and abiotic stresses and provide bases for further research into potential applications.