Aryl hydrocarbon receptor: Linking environment to aging process in elderly patients with asthma / 中华医学杂志(英文版)

Aging is a significant risk factor for various diseases, including asthma, and it often leads to poorer clinical outcomes, particularly in elderly individuals. It is recognized that age-related diseases are due to a time-dependent accumulation of cellular damage, resulting in a progressive decline in cellular and physiological functions and an increased susceptibility to chronic diseases. The effects of aging affect not only the elderly but also those of younger ages, posing significant challenges to global healthcare. Thus, understanding the molecular mechanisms associated with aging in different diseases is essential. One intriguing factor is the aryl hydrocarbon receptor (AhR), which serves as a cytoplasmic receptor and ligand-activated transcription factor and has been linked to the aging process. Here, we review the literature on several major hallmarks of aging, including mitochondrial dysfunction, cellular senescence, autophagy, mitophagy, epigenetic alterations, and microbiome disturbances. Moreover, we provide an overview of the impact of AhR on these hallmarks by mediating responses to environmental exposures, particularly in relation to the immune system. Furthermore, we explore how aging hallmarks affect clinical characteristics, inflammatory features, exacerbations, and the treatment of asthma. It is suggested that AhR signaling may potentially play a role in regulating asthma phenotypes in elderly populations as part of the aging process.

Human ESC-derived vascular cells promote vascular regeneration in a HIF-1α dependent manner

Hypoxia-inducible factor (HIF-1α), a core transcription factor responding to changes in cellular oxygen levels, is closely associated with a wide range of physiological and pathological conditions. However, its differential impacts on vascular cell types and molecular programs modulating human vascular homeostasis and regeneration remain largely elusive. Here, we applied CRISPR/Cas9-mediated gene editing of human embryonic stem cells and directed differentiation to generate HIF-1α-deficient human vascular cells including vascular endothelial cells, vascular smooth muscle cells, and mesenchymal stem cells (MSCs), as a platform for discovering cell type-specific hypoxia-induced response mechanisms. Through comparative molecular profiling across cell types under normoxic and hypoxic conditions, we provide insight into the indispensable role of HIF-1α in the promotion of ischemic vascular regeneration. We found human MSCs to be the vascular cell type most susceptible to HIF-1α deficiency, and that transcriptional inactivation of ANKZF1, an effector of HIF-1α, impaired pro-angiogenic processes. Altogether, our findings deepen the understanding of HIF-1α in human angiogenesis and support further explorations of novel therapeutic strategies of vascular regeneration against ischemic damage.

Unveiling the role of osteoblastic micro RNAs in the skeleton: from biological functions to therapeutic potential / Rol de los micro-ARN osteoblásticos en el esqueleto: desde las funciones biológicas al potencial terapéutico

MicroRNAs (miRNAs) are small non-coding RNA molecules that play critical roles in post-transcriptional gene regulation. They function by binding to target messenger RNA (mRNA) molecules, leading to their degradation or inhibiting their translation into proteins. In the context of skeletal diseases, such as osteoporosis, osteoarthritis, and bone metastasis, there is growing evidence osteoblastic miRNAs, are involved in the regulation of bone formation and maintenance.Osteoblasts are bone-forming cells responsible for synthesizing and depositing the extracellular matrix, which ultimately mineralizes to form bone tissue. Osteoblastic miRNAs modulate various aspects of osteoblast function, including proliferation, differentiation, mineralization, and apoptosis. Dysregulation of these miRNAs can disrupt the balance between bone formation and resorption, leading to skeletal diseases.The therapeutic implications of targeting osteoblastic miRNAs in skeletal diseases are significant. Modulating the expression levels of specific miRNAs holds promise for developing novel therapeutic strategies to enhance bone formation, prevent bone loss, and promote bone regeneration. Potential therapeutic approaches include the use of synthetic miRNA mimics to restore miRNA expression in diseases associated with miRNA downregulation or the use of anti-miRNA oligonucleotides to inhibit miRNA function in diseases associated with miRNA upregulation.miRNA-based therapies are still in the early stages of development, and further research is needed to fully understand the complexity of miRNA networks. Additionally, the delivery of miRNAs to specific target tissues and cells remains a challenge that needs to be addressed for effective clinical translation. Nonetheless, targeting osteoblastic miRNAs represents a promising avenue for future therapeutic interventions in skeletal diseases. (AU)

Los micro-ARNs (miARNss) son pequeños ARN no codificantes que desempeñan un papel fundamental en la regulación génica postranscripcional. Ejercen su función al unir-se a moléculas de ARN mensajero (ARNm), promoviendo su degradación e inhibiendo su traducción en proteínas. En el contexto de las enfermedades esqueléticas, como la osteoporosis, la osteoartritis y la metástasis ósea existe evidencia de que los miARNs osteoblásticos están involucrados en la regulación de la formación y del mantenimiento óseo. Los osteoblastos son células formadoras de hueso responsables de sintetizar y depositar la matriz extracelular, que finalmente se mineraliza para formar el hueso. Los miARNs derivados de osteoblastos modulan varios aspectos de la función de estas células, incluida la proliferación, diferenciación, mineralización y la apoptosis. La desregulación de estos miARNs puede alterar el equilibrio entre la formación y la resorción ósea, lo que lleva a enfermedades óseas. Las implicaciones terapéuticas de los miARNs osteoblásticos en enfermedades esqueléticas son significativas. La modulación de los niveles de expresión de miARNs específicos es prometedora para desarrollar nuevas estrate-gias terapéuticas a fin de mejorar la formación, prevenir la pérdida y promover la regeneración ósea. Los enfoques terapéuticos potenciales incluyen el uso de miméticos de miARNs para restaurar la expresión de miARNs o el uso de oligonucleótidos anti-miARNs para inhibir su función. Las terapias basadas en miARNs aún se encuentran en las primeras etapas de desarrollo. La administración de miARNs a las células y los tejidos específicos sigue siendo un desafío para lograr una aplicación clínica eficaz. (AU)

Research Advances on Strategies to Promote Homing and Engraftment of Hematopoietic Stem Cells--Review / 中国实验血液学杂志

The homing and engraftment of hematopoietic stem cells (HSC) into bone marrow is the first critical step for successful clinical hematopoietic stem cell transplantation (HSCT). SDF-1 / CXCR4 is considered to be a very promising target to promote HSC homing. In recent years, with the in-depth research on the HSC homing, a variety of new strategies for promoting HSC homing and engraftment have been explored, such as nuclear hormone receptor, histone deacetylase inhibitor, prostaglandin and metabolic regulation, so as to increase the success rate of HSCT and improve the survival of patients. In this review, the recent research advances in the mechanism of HSC homing and strategies to promote HSC homing and engraftment were summarized and discussed.

Research progress on genetic control of host susceptibility to tuberculosis / 浙江大学学报·医学版

The "Lübeck disaster", twins studies, adoptees studies, and other epidemiological observational studies have shown that host genetic factors play a significant role in determining the host susceptibility to Mycobacterium tuberculosis infection and pathogenesis of tuberculosis. From linkage analyses to genome-wide association studies, it has been discovered that human leucocyte antigen (HLA) genes as well as non-HLA genes (such as SLC11A1, VDR, ASAP1 as well as genes encoding cytokines and pattern recognition receptors) are associated with tuberculosis susceptibility. To provide ideas for subsequent studies about risk prediction of MTB infection and the diagnosis and treatment of tuberculosis, we review the research progress on tuberculosis susceptibility related genes in recent years, focusing on the correlation of HLA genes and non-HLA genes with the pathogenesis of tuberculosis. We also report the results of an enrichment analysis of the genes mentioned in the article. Most of these genes appear to be involved in the regulation of immune system and inflammation， and are also closely related to autoimmune diseases.

Analysis of WRKY transcription factor family based on full-length transcriptome sequencing in Polygonatum cyrtonema / 中国中药杂志

WRKY transcription factor family plays an important role in plant growth and development, secondary metabolite synthesis, and biotic and abiotic stress responses. The present study performed full-length transcriptome sequencing of Polygonatum cyrtonema by virtue of the PacBio SMRT high-throughput platform, identified the WRKY family by bioinformatics methods, and analyzed the physicochemical properties, subcellular localization, phylogeny, and conserved motifs. The results showed that 30.69 Gb nucleotide bases and 89 564 transcripts were obtained after redundancy removal. These transcripts had a mean length of 2 060 bp and an N50 value of 3 156 bp. Based on the full-length transcriptome sequencing data, 64 candidate proteins were selected from the WRKY transcription factor family, with the protein size of 92-1 027 aa, the relative molecular mass of 10 377.85-115 779.48 kDa, and the isoelectric point of 4.49-9.84. These WRKY family members were mostly located in the nucleus and belonged to the hydrophobic proteins. According to the phylogenetic analysis of WRKY family in P. cyrtonema and Arabidopsis thaliana, all WRKY family members were clustered into seven subfamilies and WRKY proteins from P. cyrtonema were distributed in different numbers in these seven subgroups. Expression pattern analysis confirmed that 40 WRKY family members had distinct expression patterns in the rhizomes of 1-and 3-year-old P. cyrtonema. Except for PcWRKY39, the expression of 39 WRKY family members was down-regulated in 3-year-old samples. In conclusion, this study provides abundant reference data for genetic research on P. cyrtonema and lays a foundation for the in-depth investigation of the biological functions of the WRKY family.

Strategies for exogenous RNA delivery in RNAi-mediated pest management / 生物工程学报

Plant diseases and insect pests threaten the safety of crop production greatly. Traditional methods for pest management are challenged by the problems such as environmental pollution, off-target effects, and resistance of pathogens and insects. New biotechnology-based strategies for pest control are expected to be developed. RNA interference (RNAi) is an endogenous process of gene regulation, which has been widely used to study the gene functions in various organisms. In recent years, RNAi-based pest management has received increasing attention. The effective delivery of the exogenous interference RNA into the targets is a key step in RNAi-mediated plant diseases and pest control. Considerable advances were made on the mechanism of RNAi, and various RNA delivery systems were developed for efficient pest control. Here we review the latest advances on mechanisms and influencing factors of RNA delivery, summarize the strategies of exogenous RNA delivery in RNAi-mediated pest control, and highlight the advantages of nanoparticle complexes in dsRNA delivery.

Advances in epigenetic regulation of Chinese hamster ovary cells / 生物工程学报

Chinese hamster ovary (CHO) cells play an irreplaceable role in biopharmaceuticals because the cells can be adapted to grow in suspension cultures and are capable of producing high quality biologics exhibiting human-like post-translational modifications. However, gene expression regulation such as transgene silencing and epigenetic modifications may reduce the recombinant protein production due to the decrease of expression stability of CHO cells. This paper summarized the role of epigenetic modifications in CHO cells, including DNA methylation, histone modification and miRNA, as well as their effects on gene expression regulation.

CRISPR-assisted transcription activation by phase-separation proteins

The clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 system has been widely used for genome engineering and transcriptional regulation in many different organisms. Current CRISPR-activation (CRISPRa) platforms often require multiple components because of inefficient transcriptional activation. Here, we fused different phase-separation proteins to dCas9-VPR (dCas9-VP64-P65-RTA) and observed robust increases in transcriptional activation efficiency. Notably, human NUP98 (nucleoporin 98) and FUS (fused in sarcoma) IDR domains were best at enhancing dCas9-VPR activity, with dCas9-VPR-FUS IDR (VPRF) outperforming the other CRISPRa systems tested in this study in both activation efficiency and system simplicity. dCas9-VPRF overcomes the target strand bias and widens gRNA designing windows without affecting the off-target effect of dCas9-VPR. These findings demonstrate the feasibility of using phase-separation proteins to assist in the regulation of gene expression and support the broad appeal of the dCas9-VPRF system in basic and clinical applications.

Proapoptotic protein Bim regulates the suppressive function of Treg cells / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

Regulatory T (Treg) cells are a special immunosuppressive subset of cluster of differentiation 4-positive (CD4+‍)‍-T lymphocytes and play a pivotal role in the establishment of immune homeostasis in vivo (Zhang et al., 2021). The transcription factor forkhead box protein P3 (Foxp3) is the master marker of Treg cells, which is highly expressed in Treg cells and is also essential for their suppressive function (Hori et al., 2003). In addition to Foxp3, other regulators of Treg cells have been discovered (Wu et al., 2017, 2022; Wu and Sun, 2023a, 2023b); however, a deeper understanding of the regulation of these cells is required.

Research Progress of microRNA-7/124/155 in Parkinson's Disease / 中国医学科学院学报

Parkinson's disease(PD)is the second most common neurodegenerative disease after Alzheimer's disease,with high morbidity and high disability rate.Since the early symptoms of PD are not typical and often similar to those of normal aging or other diseases.It is easy to missed diagnosis and misdiagnosis,which seriously affects the diagnosis and treatment of this disease and aggravetes the burden on the patients' life.MicroRNAs(miRNA)are a class of endogenous non-coding RNAs that are involved in post-transcriptional regulation by binding to target messenger RNAs(mRNA).They are highly conserved,short,easy to obtain,and can stably exist in peripheral body fluids.They have been used as biomarkers for a variety of diseases.Recent studies have demonstrated that miRNA play an important role in the development of PD.This paper reviews the recent research progress of miR-7/124/155,three mature miRNA in PD,aiming to provide reference for clarifying the pathogenesis and guiding the diagnosis and treatment of PD.

Cloning, identification and functional analysis of the goat transcription factor c-fos / 生物工程学报

C-fos is a transcription factor that plays an important role in cell proliferation, differentiation and tumor formation. The aim of this study was to clone the goat c-fos gene, clarify its biological characteristics, and further reveal its regulatory role in the differentiation of goat subcutaneous adipocytes. We cloned the c-fos gene from subcutaneous adipose tissue of Jianzhou big-eared goats by reverse transcription-polymerase chain reaction (RT-PCR) and analyzed its biological characteristics. Using real-time quantitative PCR (qPCR), we detected the expression of c-fos gene in the heart, liver, spleen, lung, kidney, subcutaneous fat, longissimus dorsi and subcutaneous adipocytes of goat upon induced differentiation for 0 h to 120 h. The goat overexpression vector pEGFP-c-fos was constructed and transfected into the subcutaneous preadipocytes for induced differentiation. The morphological changes of lipid droplet accumulation were observed by oil red O staining and bodipy staining. Furthermore, qPCR was used to test the relative mRNA level of the c-fos overexpression on adipogenic differentiation marker genes. The results showed that the cloned goat c-fos gene was 1 477 bp in length, in which the coding sequence was 1 143 bp, encoding a protein of 380 amino acids. Protein structure analysis showed that goat FOS protein has a basic leucine zipper structure, and subcellular localization prediction suggested that it was mainly distributed in the nucleus. The relative expression level of c-fos was higher in the subcutaneous adipose tissue of goats (P < 0.05), and the expression level of c-fos was significantly increased upon induced differentiation of subcutaneous preadipocyte for 48 h (P < 0.01). Overexpression of c-fos significantly inhibited the lipid droplets formation in goat subcutaneous adipocytes, significantly decreased the relative expression levels of the AP2 and C/EBPβ lipogenic marker genes (P < 0.01). Moreover, AP2 and C/EBPβ promoter are predicted to have multiple binding sites. In conclusion, the results indicated that c-fos gene was a negative regulatory factor of subcutaneous adipocyte differentiation in goats, and it might regulate the expression of AP2 and C/EBPβ gene expression.

H19 recruited N 6 -methyladenosine (m 6 A) reader YTHDF1 to promote SCARB1 translation and facilitate angiogenesis in gastric cancer / 中华医学杂志(英文版)

BACKGROUND@#Angiogenesis is described as a complex process in which new microvessels sprout from endothelial cells of existing vasculature. This study aimed to determine whether long non-coding RNA (lncRNA) H19 induced the angiogenesis of gastric cancer (GC) and its possible mechanism.@*METHODS@#Gene expression level was determined by quantitative real-time polymerase chain reaction and western blotting. Cell counting kit-8, transwell, 5-Ethynyl-2'-deoxyuridine (EdU), colony formation assay, and human umbilical vein endothelial cells (HUVECs) angiogenesis assay as well as Matrigel plug assay were conducted to study the proliferation, migration, and angiogenesis of GC in vitro and in vivo . The binding protein of H19 was found by RNA pull-down and RNA Immunoprecipitation (RIP). High-throughput sequencing was performed and next Gene Ontology (GO) as well as Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis was conducted to analyze the genes that are under H19 regulation. Methylated RIP (me-RIP) assay was used to investigate the sites and abundance among target mRNA. The transcription factor acted as upstream of H19 was determined through chromatin immunoprecipitation (ChIP) and luciferase assay.@*RESULTS@#In this study, we found that hypoxia-induced factor (HIF)-1α could bind to the promoter region of H19, leading to H19 overexpression. High expression of H19 was correlated with angiogenesis in GC, and H19 knocking down could inhibit cell proliferation, migration and angiogenesis. Mechanistically, the oncogenic role of H19 was achieved by binding with the N 6 -methyladenosine (m 6 A) reader YTH domain-containing family protein 1 (YTHDF1), which could recognize the m 6 A site on the 3'-untransated regions (3'-UTR) of scavenger receptor class B member 1 (SCARB1) mRNA, resulting in over-translation of SCARB1 and thus promoting the proliferation, migration, and angiogenesis of GC cells.@*CONCLUSION@#HIF-1α induced overexpression of H19 via binding with the promoter of H19, and H19 promoted GC cells proliferation, migration and angiogenesis through YTHDF1/SCARB1, which might be a beneficial target for antiangiogenic therapy for GC.

Emerging role of long non-coding RNA JPX in malignant processes and potential applications in cancers / 中华医学杂志(英文版)

Long non-coding RNAs (lncRNAs) reportedly function as important modulators of gene regulation and malignant processes in the development of human cancers. The lncRNA JPX is a novel molecular switch for X chromosome inactivation and differentially expressed JPX has exhibited certain clinical correlations in several cancers. Notably, JPX participates in cancer growth, metastasis, and chemoresistance, by acting as a competing endogenous RNA for microRNA, interacting with proteins, and regulating some specific signaling pathways. Moreover, JPX may serve as a potential biomarker and therapeutic target for the diagnosis, prognosis, and treatment of cancer. The present article summarizes our current understanding of the structure, expression, and function of JPX in malignant cancer processes and discusses its molecular mechanisms and potential applications in cancer biology and medicine.

Advances in the role of natural products in human gene expression / 中国天然药物

Natural products (NPs), especially those from traditional herbal medicines, can evidently modulate human gene expression at multiple levels, leading to a wide diversity of bioactivities. Although numerous bio-functions of NPs for human body have been found, there is little understanding about how NPs achieve it, as less attention was drawn to the definite mechnism by which NPs regulate gene expression. Furthermore, based on the rapidly advancing knowledge of mechanisms for gene regulation in recent years, newly-understood mechanisms, such as post-transcriptional regulation, are found to be involved in NP-elicited bio-effects, providing a new perspective on understanding the role of NPs in gene expression. Therefore, in the current review, we summarize the function of NPs in gene expression from the perspectives of transcriptional, post-transcriptional, and post-translational regulation, which will reinforce the understanding of NP-induced effects in gene expression and facilitate the exploration of more NPs with potential therapeutic effects.

Expression, functional mechanism and therapy application of long noncoding RNA in β-thalassemia / 中南大学学报(医学版)

β-thalassemia (β-thal) is one of the most common genetic diseases in the world, its pathogenesis is extremely complex and there is no effective treatment at present. The birth of children with moderate and severe β-thal brings economic pressure to families, social medical and health services. Long noncoding RNA (lncRNA) is a type of noncoding protein transcripts with a length greater than 200 nucleotides, which is involved in a variety of biological processes, such as cell proliferation, differentiation and chromosome variation and plays an important role in the epigenetic and post-transcriptional regulation of genes. It has potential value in the diagnosis, prevention and treatment of β-thal. LncRNA possesses the characteristics such as tissue specificity, cell specificity, developmental stage specificity, space-time specificity and disease specificity, and its complex interaction network has become a challenge to translate research results into clinical practice. Taking lncRNA as an entry point, in-depth understanding of the function of lncRNA in β-thal and explanation of its related regulatory mechanisms will provide theoretical basis for targeting treatment of β-thal, which can improve the diagnosis and treatment of β-thal.

Cloning and tissue expression analysis of the LepROT gene of Rana dybowskii / 生物工程学报

Leptin receptor overlapping transcript (LepROT) plays multiple roles in the regulation of immune systems. However, very little information is available about the anti-infectious mechanisms of amphibians LepROT. In this study, the cDNA sequence of the Rana dybowskii LepROT gene was determined by using RT-PCR and bioinformatics analysis. Then, the Aeromonas hydrophila (Ah) and lipopolysaccharides (LPS) infected models of R. dybowskii was constructed to obtain histopathological characteristics. Constitutive expression of LepROT mRNA and NF-κB signaling pathway were detected by real-time quantitative PCR. The full-length cDNA of LepROT gene was 396 bp and encoded 131 amino acids. Amino acid sequence analysis revealed LepROT shares 93.74% and 86.39% identity with homologues from other amphibians and mammals respectively, and the LepROT gene was quite conserved among different species. After infection, the relative expression levels of LepROT, NF-κB, IKKα and IKKβ mRNA were all significantly upregulated (P < 0.01), but showed a diverse temporal pattern of up-regulation in different tissues. Therefore, it was proposed that the LepROT gene of R. dybowskii might activate the NF-κB signaling pathway to exert anti-infectious effects, thus providing evidence for further extending the biological function of LepROT.

Optimization of the Light-On system in a lentiviral platform to a light-controlled expression of genes in neurons

BACKGROUND: Molecular brain therapies require the development of molecular switches to control gene expression in a limited and regulated manner in time and space. Light-switchable gene systems allow precise control of gene expression with an enhanced spatio-temporal resolution compared to chemical inducers. In this work, we adapted the existing light-switchable Light-On system into a lentiviral platform, which consists of two modules: (i) one for the expression of the blue light-switchable transactivator GAVPO and (ii) a second module containing an inducible-UAS promoter (UAS) modulated by a light-activated GAVPO. RESULTS: In the HEK293-T cell line transfected with this lentiviral plasmids system, the expression of the reporter mCherry increased between 4 to 5 fold after light induction. A time expression analysis after light induction during 24 h revealed that mRNA levels continuously increased up to 9 h, while protein levels increased throughout the experiment. Finally, transduction of cultured rat hippocampal neurons with this dual Light-On lentiviral system showed that CDNF, a potential therapeutic trophic factor, was induced only in cells exposed to blue light. CONCLUSIONS: In conclusion, the optimized lentiviral platform of the Light-On system provides an efficient way to control gene expression in neurons, suggesting that this platform could potentially be used in biomedical and neuroscience research, and eventually in brain therapies for neurodegenerative diseases.

CRISPR-Cas, el editor de genes / CRISPR, gene editor

El término CRISPR, por su acrónimo en inglés refiere a Clustered Regularly Interspaced Short Palindromic Repeats, es decir, repeticiones palindrómicas cortas, agrupadas y regularmente esparcidas, por sus características en el genoma, pertenece naturalmente al sistema de defensa de bacterias y arqueas. Este ha sido adaptado biotecnológicamente para la edición del ADN de células eucariotas, incluso de células humanas. El sistema CRISPR-Cas para editar genes consta, en forma generalizada, de dos componentes: una proteína nucleasa (Cas) y un ARN guía (sgRNA). La simplicidad del complejo lo hace una herramienta molecular reprogramable capaz de ser dirigida y de editar cualquier sitio en un genoma conocido. Su principal foco son las terapias para enfermedades hereditarias monogénicas y para el cáncer. Sin embargo, además de editor de genes, la tecnología CRISPR se utiliza para edición epigenética, regulación de la expresión génica y método de diagnóstico molecular. Este artículo tiene por objetivo presentar una revisión de las aplicaciones de la herramienta molecular CRISPR-Cas, particularmente en el campo biomédico, posibles tratamientos y diagnósticos, y los avances en investigación clínica, utilizando terapia génica con CRISPR/Cas más relevantes hasta la fecha. (AU)

CRISPR are Clustered Regularly Interspaced Short Palindromic Repeats, which naturally belong to the defense system of bacteria and archaea. It has been biotechnologically adapted for editing the DNA of eukaryotic cells, including human cells. The CRISPR-Cas system for editing genes generally consists of two components, a nuclease protein (Cas) and a guide RNA (sgRNA). The simplicity of the complex makes it a reprogrammable molecular tool capable of being targeted and editing any site in a known genome. Its main focus is therapies for monogenic inherited diseases and cancer. However, in addition to gene editor, CRISPR technology is used for epigenetic editing, regulation of gene expression, and molecular diagnostic methods. This article aims to present a review of the applications of the CRISPR-Cas molecular tool, particularly in the biomedical field, possible treatments and diagnoses, and the advances in clinical research, using the most relevant CRISPR-Cas gene therapy to date. (AU)

Deciphering the functional role of EGR1 in Prostaglandin F2 alpha induced luteal regression applying CRISPR in corpus luteum of buffalo

BACKGROUND: PGF2α is essential for the induction of the corpus luteum regression which in turn reduces progesterone production. Early growth response (EGR) proteins are Cys2-His2-type zinc-finger transcription factor that are strongly linked to cellular proliferation, survival and apoptosis. Rapid elevation of EGR1 was observed after luteolytic dose of PGF2α. EGR1 is involved in the transactivation of many genes, including TGFß1, which plays an important role during luteal regression. METHODS: The current study was conducted in buffalo luteal cells with the aim to better understand the role of EGR1 in transactivation of TGFß1 during PGF2α induced luteal regression. Luteal cells from mid stage corpus luteum of buffalo were cultured and treated with different doses of PGF2α for different time durations. Relative expression of mRNAs encoding for enzymes within the progesterone biosynthetic pathway (3ßHSD, CYP11A1 and StAR); Caspase 3; AKT were analyzed to confirm the occurrence of luteolytic event. To determine if EGR1 is involved in the PGF2α induced luteal regression via induction of TGFß1 expression, we knocked out the EGR1 gene by using CRISPR/Cas9. RESULT: The present experiment determined whether EGR1 protein expression in luteal cells was responsive to PGF2α treatment. Quantification of EGR1 and TGFß1 mRNA showed significant up regulation in luteal cells of buffalo at 12 h post PGF2α induction. In order to validate the role of PGF2α on stimulating the expression of TGFß1 by an EGR1 dependent mechanism we knocked out EGR1. The EGR1 ablated luteal cells were stimulated with PGF2α and it was observed that EGR1 KO did not modulate the PGF2α induced expression of TGFß1. In PGF2α treated EGR1 KO luteal cell, the mRNA expression of Caspase 3 was significantly increased compared to PGF2α treated wild type luteal cells maintained for 12 h. We also studied the influence of EGR1 on steroidogenesis. The EGR1 KO luteal cells with PGF2α treatment showed no substantial difference either in the progesterone concentration or in StAR mRNA expression with PGF2α-treated wild type luteal cells. CONCLUSION: These results suggest that EGR1 signaling is not the only factor which plays a role in the regulation of PGF2α induced TGFß1 signaling for luteolysis.