TP53 Expression Research Articles

Single-Cell and Spatial Transcriptomics Revealing HPV-Cervical Cancer Tumor Microenvironment Heterogeneity and Implications to Radiation and Immunotherapy

<h3>Purpose/Objective(s)</h3> Cervical cancer (CC) is a common gynecological tumor and one of the leading causes of cancer death in women that is primarily cured by radiation-based therapy. However, 10%-40% of patients failed treatment. The high heterogeneity of tumors and their microenvironment (TME) is one of the main reasons for treatment failure which has been demonstrated recently through single-cell transcriptome (SCRNAseq) of the HPV+ CC. Since the tumor and TME of HPV- and HPV+ CC are significantly different, this study aimed to comprehensively understand the tumor and TME of HPV- CC at the single-cell level. <h3>Materials/Methods</h3> The primary method was single-cell and spatial transcriptome sequencing (STRNAseq) of tumor tissues. Newly diagnosed HPV- CC patients were eligible for testing of the tumor and tumor microenvironment (TME) heterogeneity. Fresh tumor tissue samples were obtained from HPV- CC patients by biopsy and divided into two parts: one was prepared as a single-cell suspension for SCRNAseq using the 10 × Genomics, and the other was frozen and embedded for STRNAseq. Seurat 4.0 was used to cluster and annotate cell clusters and map SCRNAseq data to the STRNAseq data. The CNV of epithelial cells (ECs) was estimated by InferCNV. CellChat and TCGA datasets were used to analyze cell interaction and survival, respectively. <h3>Results</h3> A total of 9800 cells were clustered 15 cell subsets in HPV- CC tissues, including 6 ECs subsets, 2 macrophage subsets, 2 fibroblasts subsets, 3 T cells subsets, plasma-B cell, and endothelial cell. Notably, ECs make up 90% of the entire tumor tissue. In addition, all ECs had high CNV compared with reference cells and high expression of TP63, the marker of malignancy, so all ECs were defined as tumor cells. The C1 and C4 tumor cells had 95% in G2M/S phase. According to pathological morphology and gene function enrichment of each cluster, tumor clusters were spatially mapped and divided into mature areas and naive areas with high proliferation and migration ability, and hypermetabolic tumor areas. A tight intercellular matrix was seen between tumor cells and fibroblasts through a variety of glycoprotein and collagen ligand-receptor pairs (LR). Moreover, we found that tumor cells recruited macrophages to migrate to the tumor periphery by secreting MIF, and macrophages interacted with tumor cells or fibroblasts through the SPP1/CD44 LR to form cell adhesion. The RNA expression of SPP1 in HPV- CC from the TCGA database showed that SPP1 high expression patients had poor survival (p = 0.08). <h3>Conclusion</h3> This study comprehensively analyzed the tumor and TME of HPV- CC at the single-cell transcriptional scale and the gene expression and functional characteristics of different tumor clusters, indicating the high heterogeneity and 'cold' tumor signature of HPV-CC TME. The high percentage of G2/M cells may indicate the sensitivity of this tumor to radiation. The ‘Cold' signature and the tight adhesion between cells in TME may explain the low infiltration of immune cells.

Down-regulation of EVA1A by miR-103a-3p promotes hepatocellular carcinoma cells proliferation and migration

BackgroundEVA1A (Eva-1 homolog A), a novel protein involved in autophagy and apoptosis, functions as a tumor suppressor in some human primary cancers, including hepatocellular carcinoma (HCC). While it is consistently downregulated in several cancers, its involvement in hepatocarcinogenesis is still largely unknown.MethodsWe first detected the expression of EVA1A in HCC tissues and cell lines using RT‒qPCR, immunohistochemistry and western blotting and detected the expression of miR-103a-3p by RT‒qPCR. Then, bioinformatics prediction, dual-luciferase reporter gene assays and western blotting were used to screen and identify the upstream microRNA of EVA1A. After manipulating the expression of miR-103a-3p or EVA1A, wound healing, invasion, proliferation, colony formation, apoptosis, autophagy, mitosis and mitochondrial function assays, including mitochondrial membrane potential, ROS and ATP production assays, were performed to investigate the functions of miR-103a-3p targeting EVA1A in HCC cells. Apoptosis-related proteins were assessed by RT‒qPCR (TP53) or western blotting (TP53, BAX, Bcl-2 and caspase-3). Autophagy level was evaluated by observing LC3 puncta and examining the protein levels of p62, Beclin1 and LC3-II/I.ResultsWe found that EVA1A expression was decreased while miR-103a-3p expression was increased in HCC tissues and cell lines and that their expression was inversely correlated in HCC patients. The expression of miR-103a-3p was associated with HCC tumor stage and poor prognosis. miR-103a-3p could target EVA1A through direct binding to its 3'-UTR and suppress its expression. Overexpression of miR-103a-3p significantly downregulated the expression of EVA1A, TP53 and BAX, upregulated the JAK2/STAT3 pathway and promoted HCC cell migration, invasion and proliferation, while repression of miR-103a-3p dramatically upregulated the expression of EVA1A, TP53, BAX and cleaved-caspase-3, inhibited HCC cell migration, invasion and proliferation, and caused mitochondrial dysfunction and apoptosis. Overexpression of EVA1A significantly attenuated the cancer-promoting effects of miR-103a-3p in HCC cells, while knockdown of EVA1A alleviated the mitochondrial dysfunction and apoptosis caused by miR-103a-3p inhibition. Overexpression of EVA1A did not induce significant changes in autophagy levels, nor did it affect G2/M transition or mitosis.ConclusionThese findings indicate that the downregulation of the tumor suppressor EVA1A by miR-103a-3p potentially acts as a key mediator in HCC progression, mainly by inhibiting apoptosis and promoting metastasis. The miR-103a/EVA1A/TP53 axis provides a new potential diagnostic and therapeutic target for HCC treatment.

Network pharmacology-based analysis of the mechanism of Guben Sanjie Pill in the treatment of lung cancer

BackgroundLung cancer (LC) is a malignant tumor with the highest morbidity and mortality in the world. A traditional Chinese medicine formula, Guben Sanjie Pill (GBSJP), is an effective method for the clinical treatment of LC patients, but its molecular mechanism is unclear. Here, the aim was to detect the potential molecular mechanism of GBSJP in LC. MethodsComponents and potential targets of GBSJP were obtained from TCMSP databases. Targets related to LC were obtained from the databases. The potential biological effects for targets of GBSJP were analyzed by the R Bioconductor package on the basis of GO analysis and KEGG pathway enrichment. The compound-target network, target-pathway network, and PPI network were constructed by using Cytoscape 3.3.0 software. The underlying mechanism of GBSJP in the treatment of LC was assessed through molecular docking analyses. Finally, the molecular mechanism was validated experimentally. Results50 active ingredients and 158 common targets in GBSJP were identified to be closely associated with LC. The PPI network revealed that AKT1, IL-6, and TP53 were potential core targets. KEGG pathway analysis indicated that these targets were mostly involved in PI3K-Akt signaling pathway in LC. Molecular docking analysis found that quercetin, the main active ingredient of GBSJP had high binding activity with AKT1, IL-6, and TP53. A molecular study demonstrated that high-dose quercetin treatment markedly decreased the expression of AKT1, IL-6, and TP53. ConclusionGBSJP may regulate the expression of AKT1, IL-6, and TP53 to influence biological processes as a molecular mechanism for the treatment of LC.

Evaluating the effect of LPS from periodontal pathogenic bacteria on the expression of senescence-related genes in human dental pulp stem cells.

The human dental pulp stem cells (hDPSCs) are one of the readily available sources of multipotent mesenchymal stem cells (MSCs) and can be considered as a type of tool cells for cell-based therapies. However, the main limitation in the clinical use of these cells is DPSC senescence, which can be induced by lipopolysaccharide (LPS) of oral pathogenic bacteria. Up to now, far little attention has been paid to exploring the molecular mechanisms of senescence in DPSCs. So, the current study aimed to investigate the underlying molecular mechanism of senescence in hDPSCs stimulated with Porphyromonas gingivalis (P. gingivalis) and Escherichia coli (E. coli)-derived LPSs, by evaluating both mRNA and protein expression of four important senescence-related genes, including TP53, CDKN1A, CDKN2A and SIRT1. To this purpose, hDPSCs were stimulated with different LPSs for 6, 24 and 48 h and then the gene expression was evaluated using quantitative real-time polymerase chain reaction (qPCR) and western blotting. Following stimulation with P. gingivalis and E. coli-derived LPSs, the relative mRNA and protein expression of all genes were significantly up-regulated in a time-dependent manner, as compared with unstimulated hDPSCs. Moreover, the hDPSCs stimulated with P. gingivalis LPS for 6 and 24 h had the highest mRNA expression of CDKN1A and SIRT1, respectively (p &lt; 0.0001), whereas the highest mRNA expression of CDKN2A and TP53 was seen in hDPSCs stimulated with E. coli LPS for 48 h (p &lt; 0.0001). In summary, because DPSCs have been reported to have therapeutic potential for several cell-based therapies, targeting molecular mechanisms aiming at preventing DPSC senescence could be considered a valuable strategy.

Inhibition of miR-652-3p Regulates Lipid Metabolism and Inflammatory Cytokine Secretion of Macrophages to Alleviate Atherosclerosis by Improving TP53 Expression.

Purpose The aim was to elucidate the regulatory function of miR-652-3p on lipid metabolism and inflammatory cytokine secretion of macrophages in atherosclerosis. Methods miR-652-3p level in atherosclerosis patients, ox-LDL-treated macrophages, and their controls were monitored by Q-PCR. After ox-LDL treatment and miR-652-3p mimic, si-TP53 and their controls transfection, ELISA, and Q-PCR assays were used to detect IL-1ß, IL-6, and TNF-α levels. oil red O staining was processed to verify cholesterol accumulation. CE/TC and lipid metabolism were also detected. The protein levels of ABCA1, ABCG1, PPARα, CRT1, ADRP, and ALBP were detected by western blot assay. Based on the TargetScan database, the TP53 3′UTR region had complementary bases with miR-652-3p, which was also verified by dual-luciferase reporter gene assay. Finally, the regulation of miR-652-3p and TP53 was confirmed by rescue assay in atherosclerosis. Results miR-652-3p is highly expressed in atherosclerosis, miR-652-3p inhibitor decreased IL-1β, IL-6, and TNF-α expression after ox-LDL treatment. Knockdown of miR-652-3p reduces foam formation in ox-LDL-treated macrophages. miR-652-3p inhibitor ameliorates cholesterol accumulation and lipid metabolism disorder. miR-652-3p negatively regulated TP53 in atherosclerosis. Si-TP53 rescued the effect of miR-652 inhibitor in atherosclerosis. Conclusion miR-652-3p regulates the lipid metabolism of macrophages to alleviate atherosclerosis by inhibiting TP53 expression. It might be a potential target for atherosclerosis treatment.

Effects of polyphenolic-rich extracts from Citrus hystrix on proliferation and oxidative stress in breast and colorectal cancer

Abstract Objectives The anti-proliferative effects of Citrus hystrix have been reported. However, information on breast and colorectal cancer is limited especially the mechanistic aspects. In this study, the antioxidant activities of hexane, ethyl acetate, methanol and water extracts of C. hystrix leaves and their growth inhibitory effects on colorectal (HCT 116) and breast cancer (MCF 7, MDA-MB 231 and HCC 1937) cells were analysed. Methods Antioxidant and oxidative stress status were measured using non-cellular and cellular assays. Caspase and gene expression were utilized to determine anti-proliferative effects. Polyphenolic content was analysed using LC-IT-TOF/MS. Results The water extract showed the highest polyphenolic content and antioxidant activities (FRAP, DPPH, ABTS, superoxide anion radical scavenging, ferrous ion chelation, cellular antioxidant assay). The ethyl acetate extracts of C. hystrix (CH-EA) demonstrated the highest anti-proliferative activity against all cancer cell lines (IC50&lt;100 μg/mL). Increase in ROS was observed in CH-EA-treated HCT 116, MDA-MB 231 and HCC 1937 cells (p&lt;0.05). Increase in caspase activities and upregulation of Bax, Bcl-2, Cdk-1, TP53 and TNF-α expression in HCT 116 cells indicated activation of apoptosis by CH-EA. LC-IT-TOF/MS analysis indicated presence of quercetin and rutin in CH-EA. Conclusions CH-EA showed anti-proliferative effects, possibly through modulation of oxidative stress and apoptosis.

Molecular Characterization of CF33 Canine Cell Line and Evaluation of Its Ability to Respond against Infective Stressors in Sight of Anticancer Approaches.

Simple SummaryCanine mammary cancer is very common and has many similarities with human breast cancer. Risk factors, physiological and pathological behaviors, and the clinical course in dogs are very similar to humans. Several molecular similarities have also been reported, such as overexpression of EGF, proliferation markers, metalloproteinase and cyclooxygenase, TP53 mutations, and CXCR4/SDF1 axis activation. These common characteristics make these breast tumors resistant to conventional therapies. It is therefore necessary to study therapeutic alternatives. Cell lines could be helpful to test in vitro immunomodulant anti-cancer therapies, allowing a reduction of laboratory animals’ involvement in the preliminary tests and achieving results in a shorter time. Although the canine mammary carcinoma cell line CF33 has been widely used in many studies on dog mammary cancer, characterization of its gene expression profile and of the influence of infective stressors of this cell line is poor. Our study shows the interaction of CF33 and Salmonella Typhimurium (ST) as an infective stressor, indicating that these cells may represent an in vitro model for assessing novel therapeutic approaches using bacteria.Spontaneous mammary tumors are the most frequent neoplasms in bitches and show similarities with human breast cancer in risk factors, clinical course, and histopathology. The poor prognosis of some cancer subtypes, both in human and dog, demands more effective therapeutic approaches. A possible strategy is the new anticancer therapy based on immune response modulation through bacteria or their derivatives on canine mammary carcinoma cell lines. The aim of the present study was to analyze the CF33 cell line in terms of basal expression of immune innate genes, CXCR4 expression, and interaction with infectious stressors. Our results highlight that CF33 maintains gene expression parameters typical of mammary cancer, and provides the basal gene expression of CF33, which is characterized by overexpression of CXCR4, CD44, RAD51, LY96, and a non-continuous expression of TP53 and PTEN. No mutations appeared in the CXCR4 gene until the 58th passage; this may represent important information for studying the CXCR4 pathway as a therapeutic target. Moreover, the CF33 cell line was shown to be able to interact with Salmonella Typhimurium (ST) (an infective stressor), indicating that these cells could be used as an in vitro model for developing innovative therapeutic approaches involving bacteria.

Anti-Cancer Properties of Clove Bud Essential Oil in Colon Cancer Cell Line

Colon cancer is a type of cancer that begins in the large intestine (colon). Its aggression is owing to late diagnosis so poor prognosis and higher mortality rate are reported. Colon cancer has become a thorny research region that requires more examination in cellular pathways involved in its emergency. Here we aim to investigate the possible anticancer properties of the clove oil as natural and popular oil in colon cancer CaCo-2 cells. Accordingly, cell viability rate and number of survived cells were monitored in response to the clove oil treatment compared with the black seed oil and DMSO-treatment. Lactate dehydrogenase (LDH) production was also considered as an indicator for necrotic event following treatment. The relative gene expression of Raf-1, MEK, and ERK was detected as proliferation effectors that linked to the expression profile of the tumor suppressor genes PTEN and TP53 using quantitative real-time PCR (qRT-PCR). Interestingly, here we identified Raf-1 pathway as a potential targeted singling by the clove oil resulted in controlling colon cancer cell proliferation. The current study elucidates the anticancer activities of the clove oil in colon cancer division through restoring the relative gene expression of PTEN and TP53 accompanied by over-production of interleukin 1 alpha (IL-1α) and IL-1β. Accordingly, treatment of the CaCo-2 cells with different concentrations of the clove oil increased programmed cells cell death (PCD) and inhibited the proliferation singling when compared with the black seed oil. Collectively, our results demonstrate that controlling of Raf-1 activity and PCD by clove oil treatment provide evidence for its anticancer effect in colon cancer cells.

Modeling lung diseases using reversibly immortalized mouse pulmonary alveolar type 2 cells (imPAC2)

BackgroundA healthy alveolar epithelium is critical to the gas exchange function of the lungs. As the major cell type of alveolar epithelium, alveolar type 2 (AT2) cells play a critical role in maintaining pulmonary homeostasis by serving as alveolar progenitors during lung injury, inflammation, and repair. Dysregulation of AT2 cells may lead to the development of acute and chronic lung diseases and cancer. The lack of clinically relevant AT2 cell models hampers our ability to understand pulmonary diseases. Here, we sought to establish reversibly immortalized mouse pulmonary alveolar type 2 cells (imPAC2) and investigate their potential in forming alveolar organoids to model pulmonary diseases.MethodsPrimary mouse pulmonary alveolar cells (mPACs) were isolated and immortalized with a retroviral expression of SV40 Large T antigen (LTA). Cell proliferation and survival was assessed by crystal violet staining and WST-1 assays. Marker gene expression was assessed by qPCR, Western blotting, and/or immunostaining. Alveolar organoids were generated by using matrigel. Ad-TGF-β1 was used to transiently express TGF-β1. Stable silencing β-catenin or overexpression of mutant KRAS and TP53 was accomplished by using retroviral vectors. Subcutaneous cell implantations were carried out in athymic nude mice. The retrieved tissue masses were subjected to H & E histologic evaluation.ResultsWe immortalized primary mPACs with SV40 LTA to yield the imPACs that were non-tumorigenic and maintained long-term proliferative activity that was reversible by FLP-mediated removal of SV40 LTA. The EpCAM+ AT2-enriched subpopulation (i.e., imPAC2) was sorted out from the imPACs, and was shown to express AT2 markers and form alveolar organoids. Functionally, silencing β-catenin decreased the expression of AT2 markers in imPAC2 cells, while TGF-β1 induced fibrosis-like response by regulating the expression of epithelial-mesenchymal transition markers in the imPAC2 cells. Lastly, concurrent expression of oncogenic KRAS and mutant TP53 rendered the imPAC2 cells a tumor-like phenotype and activated lung cancer-associated pathways. Collectively, our results suggest that the imPAC2 cells may faithfully represent AT2 populations that can be further explored to model pulmonary diseases.

In Vitro effects of alternative smoking devices on oral cells: Electronic cigarette and heated tobacco product versus tobacco smoke

ObjectiveTo investigate the biological effects of electronic cigarette (e-cigarette) and heated tobacco product extracts respect to tobacco smoke extract on human gingival fibroblasts and human oral keratinocytes analysing cell viability, morphology, migration, apoptosis, cell cycle and epithelial-mesenchymal transition (EMT). DesignHuman gingival fibroblasts and human oral keratinocytes viability was analysed by MTT assay, cell morphology using scanning electron microscope (SEM) and cell migration by Scratch assay, a method that mimics the cell migration during wound healing in vivo. Apoptosis and cell cycle were analysed with flow cytometry and the related-gene expression of TP53, BCL2, CDKN2A and CDKN1A was indagated using real-time polymerase chain reaction. EMT process was analysed through expression of specific markers: CDH1, SNAI2, TWIST1, MMP2, FN1 and VIM. All investigations were evaluated after 24 h an in vitro exposure. ResultsUndiluted tobacco smoke extract induced significant inhibition of cell viability and cell migration, caused morphological alterations and induced an increase in cell death. No alterations or damage were observed after treatment with e-cigarette extracts. Heated tobacco product extract induced proliferation as highlighted by an increase of cell viability, cell migration and alterations of cycle analysis. ConclusionsComparing the different cigarette extracts, tobacco smoke turns out to be the most harmful, e-cigarette did not determine morphological and functional alterations and heated tobacco product must be carefully investigated for its possible clinical effects on oral cell populations.

Vitamin D alters the transcriptional profile of blood cells in patients with primary hypothyroidism

Our work aimed to study the influence of cholecalciferol on neurogenesis, neurotrophins and their receptors pathway-focused genes expression in patients with autoimmune thyroiditis and hypothyroidism. The pathway-specific PCR array (Neurotrophins and Receptors RT2 Profiler PCR Array, “QIAGEN”, Germany) was used to identify and validate the neurogenesis regulatory genes expression in patients with thyroid pathology and control group. The results showed that in patients with autoimmune thyroiditis-caused primary hypothyroidism, the expression of BDNF, FAS, FGF2, HSPB1, IL10, NGF, NGFR, NRG1, NTF3, NTF4, TGFB1, and TP53 were increased after treatment with cholecalciferol and L-thyroxine. The mRNA levels of BAX, GDNF, CD40, FOS, GDNF, GFRA1, IL1B, and STAT3 were significantly decreased in patients with autoimmune thyroiditis-caused primary hypothyroidism after treatment with cholecalciferol and Lthyroxine compared to the patients who received only L-thyroxine. Therefore, the addition of vitamin D to standard therapy alters the transcriptional profile of blood cells in patients with primary hypothyroidism.

Evaluation of TP53 and HER-2/neu Genes Expression Levels in Gestational Trophoblastic Diseases Cases and Determining Their Predictive Value in Diagnosis of Malignancy and Disease Progression

Background: Gestational trophoblastic disease (GTD) is a general term that includes several types of pathologically different diseases, which range from hydatidiform mole to choriocarcinoma. Early differentiation of these types is important to determine treatment strategy and prognosis. Objectives: Genetic markers such as TP53 and HER-2/neu expression are recently shown to have diagnostic and prognostic values. The aim of this study was to evaluate its significance. Methods: We enrolled 62 patients diagnosed with GTD referred to Imam Hossein Hospital in Tehran, Iran between 2012 and 2017. Endometrial pathologic specimens were stained, using the immunohistochemistry (IHC) method for the expression of TP53 and HER-2/neu genes. Expression levels determined by IHC were compared between final pathologic diagnoses, using one-way ANOVA test (analysis of variances), which detects significant differences between the means of 3 or more independent groups. Results: Out of 62 participants, 32 and 24 cases were diagnosed as partial and complete hydatidiform mole, respectively. Four cases had invasive hydatidiform mole and only 2 cases were diagnosed as choriocarcinoma. Analysis using ANOVA demonstrated that expression levels of both TP53 and HER-2/neu genes are significantly higher among patients with invasive form and choriocarcinoma compared with non-invasive hydatidiform mole (P &lt; 0.05 for both genes). The receiver operating characteristic (ROC) curve for each gene showed that more than 55% positive staining for the TP53 gene can differentiate non-invasive hydatidiform mole from invasive form and choriocarcinoma with 100% sensitivity, and 92.9% specificity. Conclusions: TP53 expression might serve as a potential diagnostic aid to differentiate benign and malignant GTDs and a future target for adjusting treatment based on the expression levels.

Aspirin Mediates Its Antitumoral Effect Through Inhibiting PTTG1 in Pituitary Adenoma

DNA demethylation and inhibitory effects of aspirin on pituitary cell proliferation have been demonstrated. Our aim was to clarify the molecular mechanisms behind the aspirin-related effects in pituitary cells. DNA methylome and whole transcriptome profile were investigated in RC-4B/C and GH3 pituitary cell lines upon aspirin treatment. Effects of aspirin and a demethylation agent, decitabine, were further tested in vitro. PTTG1 expression in 41 human PitNET samples and whole genome gene and protein expression data of 76 PitNET and 34 control samples (available in Gene Expression Omnibus) were evaluated. Aspirin induced global DNA demethylation and consequential transcriptome changes. Overexpression of Tet enzymes and their cofactor Uhrf2 were identified behind the increase of 5-hydroxymethylcytosine (5hmC). Besides cell cycle, proliferation, and migration effects that were validated by functional experiments, aspirin increased Tp53 activity through p53 acetylation and decreased E2f1 activity. Among the p53 controlled genes, Pttg1 and its interacting partners were downregulated upon aspirin treatment by inhibiting Pttg1 promoter activity. 5hmC positively correlated with Tet1-3 and Tp53 expression, and negatively correlated with Pttg1 expression, which was reinforced by the effect of decitabine. Additionally, high overlap (20.15%) was found between aspirin-regulated genes and dysregulated genes in PitNET tissue samples. A novel regulatory network has been revealed, in which aspirin regulated global demethylation, Tp53 activity, and Pttg1 expression along with decreased cell proliferation and migration. 5hmC, a novel tissue biomarker in PitNET, indicated aspirin antitumoral effect in vitro as well. Our findings suggest the potential beneficial effect of aspirin in PitNET.

LncRNA FGF7-5 and lncRNA GLRX3 together inhibits the formation of carotid plaque via regulating the miR-2681-5p/ERCC4 axis in atherosclerosis

ABSTRACT Atherosclerotic plaques belong to the common vascular disease in the aged, which rupture will lead to acute thromboembolic diseases, the leading cause of fatal cardiovascular events. Accumulating evidence indicates that the lncRNAs-miRNAs-mRNA regulatory network plays a critical role in atherosclerosis. Based on RNA sequencing (GSE207252), we constructed expression profiles of lncRNAs, microRNAs, and mRNA in the carotid plaque of atherosclerosis patients and analyzed differentially expressed genes (DEGs). We identified three candidate lncRNAs using two algorithms (LASSO and SVM-RFE): lnc_GLRX3, lnc_FGF7-5, and DISC1FP1). LNCipedia, TargetScan, and miRDB databases were used to predict target miRNAs of lncRNAs and target genes of miRNAs. Gene ontology (GO) functional annotation, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis, and Gene Set Enrichment Analysis (GSEA) analysis of DEGs was carried out using the R package clusterProfiler. A PPI network was constructed using the STRING website and visualized by Cytoscape. According to the “MCC” method of the plug-in cytoHubba in Cytoscape, ERCC4 was the top hub gene of the PPI network. We constructed a lncRNA_FGF7-5/lncRNA_GLRX3-miR-2681-5p-ERCC4 regulatory network for carotid plaque using lncRNA-miRNA and miRNA-mRNA pairs. Next, lncRNA_FGF7-5 and lncRNA_GLRX3 targeted miR-2681-5p directly to upregulate ERCC4 expression. Silencing of lncRNA_FGF7-5 and lncRNA_GLRX3 promoted apoptosis and TP53 expression in HUVECs treated with ox-LDL; however, these effects were reversed by ERCC4-overexpression. Taken together, these findings indicated that lncRNA_FGF7-5 and lncRNA_GLRX3 together reduced atherosclerosis-induced apoptosis of HUVECs via targeting miR-2681-5p to increase ERCC4 expression, thereby preventing the formation of carotid plaque and finally inhibiting atherosclerosis progression.

Transcription Factors and Regulators Pathway-focused Genes Expression Analysis in Patients with Different Forms of Thyroid Pathology.

Autoimmune thyroiditis (AIT), a T cell-mediated organ-specific disorder, and transcription factors have a critical role in the regulation of immune responses, especially in the fate of T-helper cells. This study aims to investigate changes in the gene expression profile of transcription factors and regulators in patients with different forms of thyroid pathology. We used the pathway-specific real-time PCR array (Neurotrophins and Receptors RT2 Profiler PCR Array, QIAGEN, Germany) to identify and verify transcription factors and regulators pathway-focused genes expression in peripheral white blood cells of patients with postoperative hypothyroidism, hypothyroidism as a result of AIT and AIT with elevated serum and antithyroglobulin (anti-Tg) and anti-thyroid peroxidase (anti-TPO) antibodies. It was shown that in patients with postoperative hypothyroidism FOS, NR1I2, STAT4, and TP53 significantly increased their expression, whereas the expression of STAT1, STAT2, and STAT3 decreased. In patients with hypothyroidism as a result of AIT, we have found increased expression of NR1I2, STAT2, and STAT3. In contrast, the expression of STAT1 and TP53 decreased. FOS and STAT4 mRNAs did not change their expression. In patients with AIT and elevated serum anti-Tg and anti-TPO antibodies, the expression of FOS and NR1I2 reduced, whereas the mRNA level of STAT3 increased. STAT1, STAT2, and STAT4 mRNAs did not change their expression. MYC did not change its expression in all groups of patients. The results of this study demonstrate that autoimmune thyroiditis and hypothyroidism affect the mRNA-level expression of transcription factors and regulators genes in a gene-specific manner and that these changes to genes expression can be one of the triggers of autoimmune inflammation progression in the thyroid gland.

Clinical significance of p53 protein expression and TP53 variation status in colorectal cancer

In human colorectal cancer (CRC), TP53 is one of the most important driver genes. Immunohistochemistry (IHC) has been used most often to assess the variational status of TP53. Recently, next-generation sequencing (NGS) of the TP53 gene has increased. However, to our knowledge, a comparison between TP53 status evaluated by IHC and NGS has not been studied. Therefore, the primary aim of this study was to compare the clinical effect of TP53 status evaluated by IHC and NGS in patients with CRC. The secondary aim was to investigate the correlation between expression of p53 by IHC and variational status of TP53 by NGS. We performed immunohistochemical staining of p53 and sequencing of TP53 by NGS in 204 human samples of CRC. We then analyzed the correlation between variational status of TP53 and p53 expression, along with their prognostic impact in CRC patients. There was significant correlation between p53 expression and TP53 variation, TP53 variation and higher N stage, and positive p53 expression and higher N stage. Positive IHC expression of p53 was significantly associated with overall survival (OS) of CRC patients by univariate analysis and was revealed as an independent prognostic factor by multivariate analysis. Additionally, the nonsense/frameshift p53 expression pattern showed a significantly better prognosis than the wild type and missense p53 expression patterns. However, the variational status of TP53 was not significant in OS of CRC patients. These results suggest that IHC expression of p53 protein correlates with variation status of TP53 and expression of p53 protein rather than variation status of TP53 has more significant impact on the OS of CRC patients.

BCL6 inhibition ameliorates resistance to ruxolitinib in CRLF2-rearranged acute lymphoblastic leukemia.

Philadelphia chromosome-like acute lymphoblastic leukemia (Ph-like ALL) is an intractable disease and most cases harbor genetic alterations that activate JAK or ABL signaling. The commonest subtype of Ph-like ALL exhibits a CRLF2 gene rearrangement that brings about JAK1/2-STAT5 pathway activation. However, JAK1/2 inhibition alone is insufficient as a treatment, so combinatorial therapies targeting multiple signals are needed. To better understand the mechanisms underlying the insufficient efficacy of JAK inhibition, we explored gene expression changes upon treatment with a JAK1/2 inhibitor (ruxolitinib) and found that elevated BCL6 expression was one such mechanism. Upregulated BCL6 suppressed the expression of TP53 along with its downstream cell cycle inhibitor p21 (CDKN2A) and pro-apoptotic molecules, such as FAS, TNFRSF10B, BID, BAX, BAK, PUMA, and NOXA, conferring cells some degree of resistance to therapy. BCL6 inhibition (with FX1) alone was able to upregulate TP53 and restore the TP53 expression that ruxolitinib had diminished. In addition, ruxolitinib and FX1 concertedly downregulated MYC. As a result, FX1 treatment alone had growth-inhibitory and apoptosis- sensitizing effects, but the combination of ruxolitinib and FX1 more potently inhibited leukemia cell growth, enhanced apoptosis sensitivity, and prolonged the survival of xenografted mice. These findings provide one mechanism for the insufficiency of JAK inhibition for the treatment of CRLF2-rearranged ALL and indicate BCL6 inhibition as a potentially helpful adjunctive therapy combined with JAK inhibition.

Biological Functions and Cross-Kingdom Host Gene Regulation of Small RNAs in Lactobacillus plantarum-Derived Extracellular Vesicles.

Extracellular vesicle-mediated transfer of microRNAs is a novel mode of cell-to-cell genetic transmission. Extracellular vesicles produced by microbes have been shown to contain significant quantities of physiologically active molecules such as proteins, lipids, and RNA, which could be transported to host cells and play a key role in both inter-kingdom signaling and physiological responses. In this study, we identified sRNAs by sequencing small RNAs (sRNAs) from Lactobacillus plantarum-derived extracellular vesicles (LDEVs) and detected the expression levels of vesicular sRNAs using quantitative reverse transcription-polymerase chain reaction (RT-PCR), which demonstrated the presence of microRNA-sized RNAs (msRNAs) within these vesicles. We chose sRNA71, a highly expressed msRNA, for further investigation, predicted its potential target genes for the human genome, and indicated that it could be translocated into mammalian cells. The biological functions of this sRNA71 were subsequently explored through cellular proteomics, western blot, and luciferase reporter assay. According to the findings, transfection with synthetic sRNA71 mimics substantially reduced Tp53 expression in HEK293T cells and suppressed the gene expression through binding to the 3' UTR of Tp53 mRNA. In conclusion, it is hypothesized that microbial-derived extracellular vesicles serve as carriers of functional molecules such as sRNAs, which play an essential role in regulating microbial-host communication.

Context‐specific roles of diphthamide deficiency in hepatocellular carcinogenesis

Diphthamide biosynthesis protein 1 (DPH1) is biochemically involved in the first step of diphthamide biosynthesis, a post-translational modification of eukaryotic elongation factor 2 (EEF2). Earlier studies showed that DPH1, also known as ovarian cancer-associated gene 1 (OVCA1), is involved in ovarian carcinogenesis. However, the role of DPH1 in hepatocellular carcinoma (HCC) remains unclear. To investigate the impact of DPH1 in hepatocellular carcinogenesis, we performed data mining from The Cancer Genome Atlas Liver Hepatocellular Carcinoma (TCGA-LIHC) dataset. We found that reduced DPH1 levels were associated with advanced stages and poor survival of patients with HCC. Also, we generated hepatocyte-specific Dph1-deficient mice and showed that diphthamide-deficient EEF2 resulted in a reduced translation elongation rate in the hepatocytes and led to mild liver damage with fatty accumulation. After N-diethylnitrosamine (DEN)-induced acute liver injury, p53-mediated pericentral hepatocyte death was increased, and compensatory proliferation was reduced in Dph1-deficient mice. Consistent with these effects, Dph1 deficiency decreased the incidence of DEN-induced pericentral-derived HCC and revealed a protective effect against p53 loss. In contrast, Dph1 deficiency combined with Trp53- or Trp53/Pten-deficient hepatocytes led to increased tumor loads associated with KRT19 (K19)-positive periportal-like cell expansion in mice. Further gene set enrichment analysis also revealed that HCC patients with lower levels of DPH1 and TP53 expression had enriched gene-sets related to the cell cycle and K19-upregulated HCC. Additionally, liver tumor organoids obtained from 6-month-old Pten/Trp53/Dph1-triple-mutant mice had a higher frequency of organoid re-initiation cells and higher proliferative index compared with those of the Pten/Trp53-double-mutant. Pten/Trp53/Dph1-triple-mutant liver tumor organoids showed expression of genes associated with stem/progenitor phenotypes, including Krt19 and Prominin-1 (Cd133) progenitor markers, combined with low hepatocyte-expressed fibrinogen genes. These findings indicate that diphthamide deficiency differentially regulates hepatocellular carcinogenesis, which inhibits pericentral hepatocyte-derived tumors and promotes periportal progenitor-associated liver tumors. © 2022 The Pathological Society of Great Britain and Ireland.

HDAC inhibitor chidamide synergizes with venetoclax to inhibit the growth of diffuse large B-cell lymphoma via down-regulation of MYC, BCL2, and TP53 expression.

Diffuse large B-cell lymphoma (DLBCL) is an aggressive type of non-Hodgkin's lymphoma. A total of 10%‒15% of DLBCL cases are associated with myelocytomatosis viral oncogene homolog(MYC) and/or B-cell lymphoma-2 (BCL2) translocation or amplification. BCL2 inhibitors have potent anti-tumor effects in DLBCL; however, resistance can be acquired through up-regulation of alternative anti-apoptotic proteins. The histone deacetylase (HDAC) inhibitor chidamide can induce BIM expression, leading to apoptosis of lymphoma cells with good efficacy in refractory recurrent DLBCL. In this study, the synergistic mechanism of chidamide and venetoclax in DLBCL was determined through in vitro and in vivo models. We found that combination therapy significantly reduced the protein levels of MYC, TP53, and BCL2 in activated apoptotic-related pathways in DLBCL cells by increasing BIM levels and inducing cell apoptosis. Moreover, combination therapy regulated expression of multiple transcriptomes in DLBCL cells, involving apoptosis, cell cycle, phosphorylation, and other biological processes, and significantly inhibited tumor growth in DLBCL-bearing xenograft mice. Taken together, these findings verify the in vivo therapeutic potential of chidamide and venetoclax combination therapy in DLBCL, warranting pre-clinical trials for patients with DLBCL.