Regulation Of Cell Proliferation Research Articles

Notch3 as a novel therapeutic target for the treatment of ADPKD by regulating cell proliferation and renal cyst development

Autosomal dominant polycystic kidney disease (ADPKD) is a common monogenic kidney disease. Emerging research indicates that the Notch signaling pathway plays an indispensable role in the pathogenesis of numerous kidney diseases, including ADPKD. Herein, we identified that Notch3 but not other Notch receptors was overexpressed in renal tissues from mice with ADPKD and ADPKD patients. Inhibiting Notch3 with γ-secretase inhibitors, which blocks a proteolytic cleavage required for Notch3 activation, or shRNA knockdown of Notch3 significantly delayed renal cyst growth in vitro and in vivo. Subsequent mechanistic study elucidated that the cleaved intracellular domain of Notch3 (N3ICD) and Hes1 could bind to the PTEN promoter, leading to transcriptional inhibition of PTEN. This further activated the downstream PI3K-AKT-mTOR pathway and promoted renal epithelial cell proliferation. Overall, Notch3 was identified as a novel contributor to renal epithelial cell proliferation and cystogenesis in ADPKD. We envision that Notch3 represents a promising target for ADPKD treatment.

Construction of ceRNA network mediated by circRNAs screening from microarray and identification of novel biomarkers for myasthenia gravis

BackgroundRecent studies have revealed that circRNA can serve as ceRNA to participate in multiple autoimmune diseases. Our study aims to explore the key circRNA as ceRNA and biomarker for MG. Methods: We used circRNA microarray to explore differentially expressed circRNAs (DECs) from MG and compare with control. Then, we predicted the target miRNA associated with DECs and screened miRNAs by the algorithm of random walk with restart (RWR). Next, we constructed the circRNA-miRNA-mRNA ceRNA regulated network (CMMC) to identify the hub objects. Following, we detected the expression of hub-circRNAs by RT-PCR. We verify has_circ_0004183 (circFRMD4) sponging miR-145-5p regulate cells proliferation using luciferase assay and CCK-8. Results: We found that the expression level of circFRMD4 and has_circ_0035381 (circPIGB) were upregulated and has_circ_0089153(circ NUP214) had the lowest expression level in MG. Finally, we proved circFRMD4 sponging miR-145-5p regulate Jurkat cells proliferation. CircFRMD4 take part in the genesis and development of MG via circFRMD4/miR145-5p axis. Conclusions: We found that circFRMD4, circPIGB and circNUP214 can be considered as valuable potential novel biomarkers for AchR + MG. CircFRMD4 participate in the development of AchR + MG via targeting binding with miR-145-5p.

GSK0660 enhances antitumor immunotherapy by reducing PD-L1 expression

Blockade of PD-1/PD-L1 immune checkpoint is wildly used for multiple types of cancer treatment, while the low response rate for patients is still completely unknown. As nuclear hormone receptor, PPARδ (peroxisome-proliferator-activated receptor) regulates cell proliferation, inflammation, and tumor progression, while the effect of PPARδ on tumor immune escape is still unclear. Here we found that PPARδ antagonist GSK0660 significantly reduced colon cancer cell PD-L1 protein and gene expression. Luciferase analysis showed that GSK0660 decreased PD-L1 gene transcription activity. Moreover, reduced PD-L1 expression in colon cancer cells led to increased T cell activity. Further analysis showed that GSK0660 decreased PD-L1 expression in a PPARδ dependent manner. Implanted tumor model analysis showed that GSK0660 inhibited tumor immune escape and the combined PD-1 antibody with GSK0660 effectively enhanced colorectal cancer immunotherapy. These findings suggest that GSK0660 treatment could be an effective strategy for cancer immunotherapy.

The emerging role of DOT1L in cell proliferation and differentiation: Friend or foe.

Cell proliferation and differentiation are the basic physiological activities of cells. Mistakes in these processes may affect cell survival, or cause cell cycle dysregulation, such as tumorigenesis, birth defects and degenerative diseases. In recent years, it has been found that histone methyltransferase DOT1L is the only H3 lysine 79 methyltransferase, which plays an important role in the process of cell fate determination through monomethylation, dimethylation and trimethylation of H3K79. DOT1L has a pro-proliferative effect in leukemia cells; however, loss of heart-specific DOT1L leads to increased proliferation of cardiac tissue. Additionally, DOT1L has carcinogenic or tumor suppressive effects in different neoplasms. At present, some DOT1L inhibitors for the treatment of MLL-driven leukemia have achieved promising results in clinical trials, but completely blocking DOT1L will also bring some side effects. Thus, this uncertainty suggests that DOT1L has a unique function in cell physiology. In this review, we summarize the primary findings of DOT1L in regulating cell proliferation and differentiation. Correlations between DOT1L and cell fate specification might suggest DOT1L as a therapeutic target for diseases.

OsMADS6-OsMADS32 and REP1 control palea cellular heterogeneity and morphogenesis in rice

Precise regulation of cell proliferation and differentiation is vital for organ morphology. Rice palea, serving as sepal, comprises two distinct regions: the marginal region (MRP) and body of palea (BOP), housing heterogeneous cell populations, which makes it an ideal system for studying organ morphogenesis. We report that the transcription factor (TF) REP1 promotes epidermal cell proliferation and differentiation inthe BOP,resulting in hard silicified protrusion cells, by regulating the cyclin-dependent kinase gene, OsCDKB1;1. Conversely, TFs OsMADS6 and OsMADS32 are expressed exclusively in the MRP, where they limit celldivision rates by inhibiting OsCDKB2;1 expression and promote endoreduplication, yielding elongatedepidermal cells. Furthermore, reciprocal inhibition between the OsMADS6-OsMADS32 complex andREP1 fine-tunes the balance between cell division and differentiation during palea morphogenesis. Wefurther show the functional conservation of these organ identity genes in heterogeneous cell growthin Arabidopsis, emphasizing a critical framework for controlling cellular heterogeneity in organ morphogenesis.

A scenario of unhealthy life cycle: The role of circadian rhythms in health.

Circadian rhythms are oscillations in physiology and behavior caused by the circadian regulator. Cryptochromes, Periods, and Bmal1 are circadian clock genes that have been linked to aging and cancer. Human pathologies alter circadian clock gene expression, and transgenic rats with clock gene defects progress to cancer and age prematurely. In the growth of age-linked pathologies and carcinogenesis, cell proliferation and genome integrity play critical roles. The relationship concerning the cell cycle regulation and circadian clock is discussed in this article. The circadian clock controls the behavior and countenance of many main cell cycle and cell cycle check-point proteins, and cell cycle-associated proteins, in turn, control the activity and expression of circadian clock proteins. The circadian clock can be reset by DNA disruption, providing a molecular mechanism for mutual control amid the cell cycle and the clock. This circadian clock-dependent regulation of cell proliferation, composed with other circadian clock-dependent physiological functions including metabolism control, genotoxic and oxidative stress response, and DNA repair, unlocks new avenues for studying the processes of aging and carcinogenesis.

Long non-coding RNA MALAT1 in hematological malignancies and its clinical applications.

Metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) is a well-established oncogenic long non-coding RNA, the higher expression of which is strongly correlated with cancer events such as tumorigenesis, progression, metastasis, drug resistance, and treatment outcome in solid cancers. Recently, a series of studies has highlighted its potential role in hematological malignancies in terms of these events. Similar to solid cancers, MALAT1 can regulate various target genes via sponging and epigenetic mechanisms, but the miRNAs sponged by MALAT1 differ from those identified in solid cancers. In this review, we systematically describe the role and underlying mechanisms of MALAT1 in multiple types of hematological malignancies, including regulation of cell proliferation, metastasis, stress response, and glycolysis. Clinically, MALAT1 expression is related to poor treatment outcome and drug resistance, therefore exhibiting potential prognostic value in multiple myeloma, lymphoma, and leukemia. Finally, we discuss the evaluation of MALAT1 as a novel therapeutic target against cancer in preclinical studies.

MiR-199a-5p targets DUSP14 to regulate cell proliferation, invasion and stemness in non-small cell lung cancer

BackgroundNon-small cell lung cancer (NSCLC) shows the highest morbidity among malignant tumors worldwide. Despite improvements of diagnosis and treatment, patient prognosis remains unfavorable. Therefore, there is a need to discover a novel treatment strategy for NSCLC. DUSP14 is related to various cancers as the regulatory factor for cellular processes. However, its specific roles in NSCLC and the upstream modulator remain largely unclear. MethodsDUSP14 expression patterns within the lung cancer patient cohort from TCGA database were analyzed using UALCAN online tool. Different databases including miRDB, starbase, and Targetscan were employed to screen the upstream regulator of DUSP14. DUSP14 and miR-199a-5p expression was determined by qRT-PCR and Western blot techniques. To confirm binding interaction of DUSP14 with miR-199a-5p, we conducted a dual-luciferase reporter assay. Cell viability, migration, and stemness properties were assessed using CCK-8, EdU (5-ethynyl-2′-deoxyuridine) incorporation, transwell invasion, and sphere formation assays. The effect of DUSP14 silencing on tumorigenesis was assessed with the NSCLC cell xenograft mouse model. ResultsOur study discovered that DUSP14 exhibited high expression within NSCLC tumor samples, which is related to the dismal prognostic outcome in NSCLC patients. Silencing DUSP14 impaired NSCLC cell proliferation, migration, and tumor sphere formation. Besides, we identified miR-199a-5p as the upstream regulatory factor for DUSP14, and its expression was negatively related to DUSP14 level within NSCLC tissues. Introducing miR-199a-5p recapitulated the function of DUSP14 silencing in NSCLC cell aggressiveness and stemness. Moreover, knocking down DUSP14 efficiently inhibited tumor formation in NSCLC cells of the xenograft model. ConclusionsOur study suggests that DUSP14 is negatively regulated by miR-199a-5p within NSCLC, whose overexpression is required for sustaining NSCLC cell proliferation, invasion and stemness.

Cardiac-specific deletion of heat shock protein 60 induces mitochondrial stress and disrupts heart development in mice

Congenital heart diseases are the most common birth defects around the world. Emerging evidence suggests that mitochondrial homeostasis is required for normal heart development. In mitochondria, a series of molecular chaperones including heat shock protein 60 (HSP60) are engaged in assisting the import and folding of mitochondrial proteins. However, it remains largely obscure whether and how these mitochondrial chaperones regulate cardiac development. Here, we generated a cardiac-specific Hspd1 deletion mouse model by αMHC-Cre and investigated the role of HSP60 in cardiac development. We observed that deletion of HSP60 in embryonic cardiomyocytes resulted in abnormal heart development and embryonic lethality, characterized by reduced cardiac cell proliferation and thinner ventricular walls, highlighting an essential role of cardiac HSP60 in embryonic heart development and survival. Our results also demonstrated that HSP60 deficiency caused significant downregulation of mitochondrial ETC subunits and induced mitochondrial stress. Analysis of gene expression revealed that P21 that negatively regulates cell proliferation is significantly upregulated in HSP60 knockout hearts. Moreover, HSP60 deficiency induced activation of eIF2α-ATF4 pathway, further indicating the underlying mitochondrial stress in cardiomyocytes after HSP60 deletion. Taken together, our study demonstrated that regular function of mitochondrial chaperones is pivotal for maintaining normal mitochondrial homeostasis and embryonic heart development.

Integration analysis of cell division cycle-associated family genes revealed potential mechanisms of gliomagenesis and constructed an artificial intelligence-driven prognostic signature

Cell division cycle-associated (CDCA) gene family members are essential cell proliferation regulators and play critical roles in various cancers. However, the function of the CDCA family genes in gliomas remains unclear. This study aims to elucidate the role of CDCA family members in gliomas using in vitro and in vivo experiments and bioinformatic analyses. We included eight glioma cohorts in this study. An unsupervised clustering algorithm was used to identify novel CDCA gene family clusters. Then, we utilized multi-omics data to elucidate the prognostic disparities, biological functionalities, genomic alterations, and immune microenvironment among glioma patients. Subsequently, the scRNA-seq analysis and spatial transcriptomic sequencing analysis were carried out to explore the expression distribution of CDCA2 in glioma samples. In vivo and in vitro experiments were used to investigate the effects of CDCA2 on the viability, migration, and invasion of glioma cells. Finally, based on ten machine-learning algorithms, we constructed an artificial intelligence-driven CDCA gene family signature called the machine learning-based CDCA gene family score (MLCS). Our results suggested that patients with the higher expression levels of CDCA family genes had a worse prognosis, more activated RAS signaling pathways, and more activated immunosuppressive microenvironments. CDCA2 knockdown inhibited the proliferation, migration, and invasion of glioma cells. In addition, the MLCS had robust and favorable prognostic predictive ability and could predict the response to immunotherapy and chemotherapy drug sensitivity.

The RNA secondary structure of androgen receptor-FL and V7 transcripts reveals novel regulatory regions.

The androgen receptor (AR) is a ligand-dependent nuclear transcription factor belonging to the steroid hormone nuclear receptor family. Due to its roles in regulating cell proliferation and differentiation, AR is tightly regulated to maintain proper levels of itself and the many genes it controls. AR dysregulation is a driver of many human diseases including prostate cancer. Though this dysregulation often occurs at the RNA level, there are many unknowns surrounding post-transcriptional regulation of AR mRNA, particularly the role that RNA secondary structure plays. Thus, a comprehensive analysis of AR transcript secondary structure is needed. We address this through the computational and experimental analyses of two key isoforms, full length (AR-FL) and truncated (AR-V7). Here, a combination of in-cell RNA secondary structure probing experiments (targeted DMS-MaPseq) and computational predictions were used to characterize the static structural landscape and conformational dynamics of both isoforms. Additionally, in-cell assays were used to identify functionally relevant structures in the 5' and 3' UTRs of AR-FL. A notable example is a conserved stem loop structure in the 5'UTR of AR-FL that can bind to Poly(RC) Binding Protein 2 (PCBP2). Taken together, our results reveal novel features that regulate AR expression.

The Identification of a Novel Nucleomodulin MbovP467 of Mycoplasmopsis bovis and Its Potential Contribution in Pathogenesis.

Mycoplasmopsis bovis is a causative agent of crucial diseases in both dairy and beef cattle leading to substantial economic losses. However, limited control measures for M. bovis-related diseases exist due to a lack of understanding about the virulence factors of this pathogen, a common challenge in mycoplasma research. Consequently, this study aimed to characterize a novel nucleomodulin as a virulence-related factor of M. bovis. Employing bioinformatic tools, we initially predicted MbovP467 to be a secreted protein with a nuclear localization signal based on SignalP scores and the cNLS (Nuclear Localization Signal) Mapper, respectively. Subsequently, the MbovP467 gene was synthesized and cloned into a pEGFP plasmid with EGFP labeling to obtain a recombinant plasmid (rpEGFP-MbovP467) and then was also cloned in pET-30a with a consideration for an Escherichia coli codon bias and expressed and purified for the production of polyclonal antibodies against the recombinant MbovP467 protein. Confocal microscopy and a Western blotting assay confirmed the nuclear location of MbovP467 in bovine macrophages (BoMacs). RNA-seq data revealed 220 up-regulated and 20 down-regulated genes in the rpEGFP-MbovP467-treated BoMac group compared to the control group (pEGFP). A GO- and KEGG-enrichment analysis identified associations with inflammatory responses, G protein-coupled receptor signaling pathways, nuclear receptor activity, sequence-specific DNA binding, the regulation of cell proliferation, IL-8, apoptotic processes, cell growth and death, the TNF signaling pathway, the NF-κB signaling pathway, pathways in cancer, and protein families of signaling and cellular processes among the differentially expressed up-regulated mRNAs. Further experiments, investigating cell viability and the inflammatory response, demonstrated that MbovP467 reduces BoMac cell viability and induces the mRNA expression of IL-1β, IL-6, IL-8, TNF-α, and apoptosis in BoMac cells. Further, MbovP467 increased the promoter activity of TNF-α. In conclusion, this study identified a new nucleomodulin, MbovP467, for M. bovis, which might have an important role in M. bovis pathogenesis.

Expression and function analysis of Rspo1 in oocyte primary growth of Qi River crucian carp (Carassius auratus)

RSPO1, also referred to R-spondin1, plays a crucial role in female sex determination and differentiation in both mammals and teleosts. Nevertheless, limited documentation exists regarding its contribution to the subsequent ovarian development in teleosts. In this research, Qi River crucian carp (Carassius auratus) was employed as a model organism to investigate the function of Rspo1 during the initial stages of ovarian growth. To clarify the expression patterns, a polyclonal antibody targeting Rspo1 was generated. According to Western blot analysis, Rspo1 was able to identify a band that matched the size of Rspo1 in adult Qi River crucian carp. Furthermore, during the development of the ovary, the expression of Rspo1 peaked at 90 days after hatching (dah), a critical period for the ovary development in Qi River crucian carp, then decreased. Immunohistochemical (IHC) analysis revealed the widespread presence of Rspo1 in oogonia, stage I and II oocytes, as well as certain stage III oocytes, stromal cells, and granulosa cells throughout the ovary. To further confirm the essential roles of Rspo1 in early ovary development, we used dsRNA to suppress its expression for a duration of 30 days starting at 90 dah. Following this, there was a significant reduction in the number of oocytes. Additionally, the expression of genes associated with female development, such as β-catenin, foxl2, cyp19a1a, and figlα, exhibited a noticeable decline. Conversely, the expression of apoptosis-promoting factors caspase3 and p53 increased significantly. The gene ki-67, serving as a marker for cell proliferation, experienced a decrease. These findings suggest that Rspo1 may exert influence on ovarian development through the regulation of germ cell proliferation. The inhibition of Rspo1 expression during the crucial phase of ovarian growth in Qi River crucian carp is anticipated to induce somatic cell apoptosis and perturb the structural integrity of the ovary. Our investigation offers significant elucidation on the molecular mechanisms governing the involvement of Rspo1 in the initial stages of ovarian development in teleosts.

Functional analysis of RRAS2 pathogenic variants with a Noonan-like phenotype.

Introduction: RRAS2, a member of the R-Ras subfamily of Ras-like low-molecular-weight GTPases, is considered to regulate cell proliferation and differentiation via the RAS/MAPK signaling pathway. Seven RRAS2 pathogenic variants have been reported in patients with Noonan syndrome; however, few functional analyses have been conducted. Herein, we report two patients who presented with a Noonan-like phenotype with recurrent and novel RRAS2 pathogenic variants (p.Gly23Val and p.Gly24Glu, respectively) and the results of their functional analysis. Materials and methods: Wild-type (WT) and mutant RRAS2 genes were transiently expressed in Human Embryonic Kidney293 cells. Expression of RRAS2 and phosphorylation of ERK1/2 were confirmed by Western blotting, and the RAS signaling pathway activity was measured using a reporter assay system with the serum response element-luciferase construct. WT and p.Gly23Val RRAS2 were expressed in Drosophila eye using the glass multiple reporter-Gal4 driver. Mutant mRNA microinjection into zebrafish embryos was performed, and the embryo jaws were observed. Results: No obvious differences in the expression of proteins WT, p.Gly23Val, and p.Gly24Glu were observed. The luciferase reporter assay showed that the activity of p.Gly23Val was 2.45 ± 0.95-fold higher than WT, and p.Gly24Glu was 3.06 ± 1.35-fold higher than WT. For transgenic flies, the p.Gly23Val expression resulted in no adults flies emerging, indicating lethality. For mutant mRNA-injected zebrafish embryos, an oval shape and delayed jaw development were observed compared with WT mRNA-injected embryos. These indicated hyperactivity of the RAS signaling pathway. Discussion: Recurrent and novel RRAS2 variants that we reported showed increased in vitro or in vivo RAS signaling pathway activity because of gain-of-function RRAS2 variants. Clinical features are similar to those previously reported, suggesting that RRAS2 gain-of-function variants cause this disease in patients.

Analysis of the Expression of LSF Transcription Factor in the Regulation of Transcription and TSG101 during the Neoplastic Transformation of Endometrial Cells.

Previous research indicates that carcinogenesis involves disrupting the functions of numerous genes, including factors involved in the regulation of transcription and cell proliferation. For these reasons, in endometrial carcinogenesis, we decided to investigate the expression of TSG101 (a suppressor of tumor transformation) and LSF (a transcription factor involved in numerous cellular processes, such as cell cycle regulation, cell growth, development, and apoptosis). LSF may be involved in the regulation of TSG101 expression. The research material consisted of endometrial cancer samples from 60 patients. The control group consisted of normal endometrium samples donated by 60 women undergoing surgery for benign diseases of the female reproductive organs. The samples were subjected to immunohistochemical staining with antibodies specific to TSG101 and LSF. Specific antibodies were used to identify TSG101 and LSF in the examined histopathological preparations. An approximately 14-fold lower risk of endometrial cancer development was observed in patients with TSG expression in more than 75% of the assessed cells (4% vs. 36%; OR = 0.07; p = 0.0182). There was a four-fold lower risk of endometrial cancer development in patients with LSF expression in more than 50% of the assessed cells (32% vs. 64%; OR = 0.26; p = 0.0262). A more than three-fold lower risk of endometrial cancer development was observed in patients with LSF expression in more than 75% of the assessed cells (24% vs. 52%; OR = 0.29; p = 0.0454). Endometrial cancer was diagnosed in those with a lower level of TSG101 expression than in those with a cancer-free endometrium. Decreased expression of TSG101 may be a marker of endometrial cancer, and increased expression of LSF when diagnosed with endometrial cancer may indicate greater advancement of the disease. These markers might be used as diagnostic and prognostic markers-however, there is a lack of a correlation between them.

Matrix metalloproteinase-1 as a potential biomarker for early gastric cancer detection and its effect on gastric cancer cell proliferation and migration

The present study aimed to investigate the association between matrix metalloproteinase-1 (MMP-1) and early gastric cancer (EGC), while also evaluating the effect of MMP-1 on gastric cancer cell proliferation and migration. Transcriptome RNA sequencing and database analysis were conducted to assess the relationship between MMP-1 expression and EGC. Differences in MMP-1 expression between clinical EGC samples and paracancerous tissues were detected using fluorescence quantitative polymerase chain reaction (PCR). In N87 gastric cancer cells, changes in proliferation- and migration-related indicator expression were determined. Gene sequencing revealed differential expression of MMP-1 in early and advanced gastric cancers. Furthermore, enhanced MMP-1 expression was observed in early and advanced gastric cancer tissues, exhibiting a positive correlation with the malignant phenotype in gastric cancer cell lines. Fluorescence quantitative PCR revealed considerably higher MMP-1 expression in EGC tissues than in paracancerous tissues. CCK8 and EdU assays demonstrated a significant increase in N87 cell proliferation on MMP-1 upregulation and a decrease on its downregulation. The scratch assay results demonstrated a corresponding enhancement in N87 cell migratory capacity with MMP-1 upregulation, which was attenuated on its downregulation. Western blot experiments revealed a decrease in the expression of the epithelial-mesenchymal transition-related protein E-cadherin after MMP-1 upregulation, while vimentin expression significantly increased. Conversely, the downregulation of MMP-1 led to opposite outcomes. Overall, MMP-1 emerges as a potential biomarker for EGC diagnosis and plays a crucial role in the regulation of N87 gastric cancer cell proliferation and migration.

Impact of media supplements FGF2, LIF and IGF1 on the genome activity of porcine embryos produced in vitro

In this article, we focused on the impact of precisely chemically modified FLI maturation medium enriched with fibroblast growth factor 2 (FGF2), leukemia inhibitory factor (LIF), insulin-like growth factor 1 (IGF1), and polyvinyl alcohol (PVA) and its potential to improve the efficiency of in vitro production of porcine embryos. We hypothesized that enhancing the composition of the maturation medium could result in an elevated production of embryos in vitro and can affect EGA. FLI medium resulted in a significantly higher rate of oocyte blastocyst maturation and formation compared to the control DMEM medium. In addition, immunocytochemical labelling confirmed the detection of UBF in 4-cell FLI parthenogenic embryos, suggesting similarities with natural embryo development. Through RNAseq analysis, upregulated genes present in 4-cell FLI embryos were found to play key roles in important biological processes such as cell proliferation, cell differentiation, and transcriptional regulation. Based on our findings, we demonstrated the positive influence of FLI medium in the evaluation of in vitro embryo production, EGA detection, transcriptomic and proteomic profile, which was confirmed by the positive activation of the embryonal genome in the 4-cell stage of parthenogenetically activated embryos.

Sperm-Associated Antigen 5 Knockout Reduces Doxorubicin and Docetaxel Resistance in Triple-Negative Breast Cancer MDA-MB-231 and BT549 Cells.

Sperm-associated antigen 5 (SPAG5), also known as Astrin, was previously demonstrated as a biomarker for cellular resistance to major breast cancer therapies, including chemo-, endocrine- and targeted therapy. However, the contribution of SPAG5 to anthracycline- and taxane-based chemotherapy in triple-negative breast cancer (TNBC) remains controversial. In the present study, the SPAG5 knockout cell model was established by using clustered regularly interspaced palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9) system in MDA-MB-231 and BT549 TNBC cell lines. The knockout of SPAG5 was confirmed on both gene and protein levels using genomic PCR, DNA sequencing and western blotting. The functional loss of SPAG5 was determined by colony-formation assay. SPAG5-regulated doxorubicin- and docetaxel-resistance was assessed by MTT and apoptosis assays. The results indicated that all the SPAG5 knockout MDA-MB-231 and BT549 clones were biallelic, where one allele was replaced by the donor template, and the other allele had the same "T" insertion (indel) adjacent to the cutting sites of gRNAs at the exon 1 boundary, irrespective of the gRNAs and cell lines. The locus of indel interrupted the SPAG5 transcription by damaging the GT-AG mRNA processing rule. Deletion of SPAG5 decreased clonogenicity in both MDA-MB-231 and BT549 cells. SPAG5 was able to regulate the resistance and the drug-induced apoptosis of both doxorubicin and docetaxel. In conclusion, recombinant plasmid-based CRISPR-Cas9 technology can be used to delete the SPAG5 gene in the TNBC cell lines. SPAG5 has an important role in regulating cell proliferation and doxorubicin- and docetaxel-resistance in MDA-MB-231 and BT549 cells.

Parkinson's Disease and MicroRNAs: A Duel Between Inhibition and Stimulation of Apoptosis in Neuronal Cells.

Parkinson's disease (PD) is one of the most prevalent diseases of central nervous system that is caused by degeneration of the substantia nigra's dopamine-producing neurons through apoptosis. Apoptosis is regulated by initiators' and executioners' caspases both in intrinsic and extrinsic pathways, further resulting in neuronal damage. In that context, targeting apoptosis appears as a promising therapeutic approach for treating neurodegenerative diseases. Non-coding RNAs-more especially, microRNAs, or miRNAs-are a promising target for the therapy of neurodegenerative diseases because they are essential for a number of cellular processes, including signaling, apoptosis, cell proliferation, and gene regulation. It is estimated that a substantial portion of coding genes (more than 60%) are regulated by miRNAs. These small regulatory molecules can have wide-reaching consequences on cellular processes like apoptosis, both in terms of intrinsic and extrinsic pathways. Furthermore, it was recommended that a disruption in miRNA expression levels could also result in perturbation of typical apoptosis pathways, which may be a factor in certain diseases like PD. The latest research on miRNAs and their impact on neural cell injury in PD models by regulating the apoptosis pathway is summarized in this review article. Furthermore, the importance of lncRNA/circRNA-miRNA-mRNA network for regulating apoptosis pathways in PD models and treatment is explored. These results can be utilized for developing new strategies in PD treatment.

Abstract 1799: Regulation of epidermal stem cell proliferation and adhesion by ACSS2

Abstract Acetyl-CoA Synthetase Short-Chain Family Member 2 (ACSS2) is an enzyme that converts acetate into acetyl-CoA, a crucial metabolite required for protein acetylation and lipogenesis. Acetyl-CoA is membrane-impermeable and must be synthesized in the compartment in which it will be used. Among ACSS enzymes, ACSS2 alone is found in the nucleocytoplasmic compartment rather than the mitochondria. This enzyme shows dynamic localization dependent on cell nutrient status, with nutrient stress promoting nuclear localization where ACSS2 activity promotes cell growth and survival. Emerging evidence suggests ACSS2 may also play roles in promoting metastasis, and has been proposed as a potential therapeutic target. However, the role of ACSS2 in controlling normal tissue homeostasis is understudied. Addressing this knowledge gap is critical to ensure safety and efficacy of cancer therapeutics. As 80% to 90% of human cancers originate from epithelial tissues, we have leveraged human epidermal stem cells to further interrogate the role of ACSS2. As progenitor keratinocytes differentiate, they detach from the basal lamina and migrate from the metabolically privileged basal layer to the more challenging environment at the surface of the epidermis. Using shRNA-mediated knockdown, we first found ACSS2 knockdown dramatically decreases progenitor keratinocyte clonogenicity, abolishing proliferative capacity. Analysis of RNA sequencing data following ACSS2 knockdown with five constructs showed significant increases in expression of genes related to cell adhesion, extracellular matrix organization, collagen catabolism, and cell migration, in addition to keratinocyte differentiation. Downregulated genes were associated with cell division, DNA repair, and DNA replication. Live cell imaging revealed knockdown cells show striking differences in cell adhesion compared to control. These results indicate a role for ACSS2 in regulating keratinocyte proliferation and adhesion during the maintenance of epidermal homeostasis. Citation Format: Nicholas K. Chamberlain, Xiaomin Bao. Regulation of epidermal stem cell proliferation and adhesion by ACSS2 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1799.