Enhancing the productivity and proliferation of CHO-K1 cells by oncoprotein YAP (Yes-associated protein).

CHO cells are extensively employed in biological drug industry to manufacture therapeutic proteins. Nevertheless, production of biopharmaceuticals faces obstacles such as limited growth and inadequate productivity. Employing host cell engineering techniques for CHO cells serves as a valuable approach to address the constraints encountered in biologics manufacturing. Despite advancements, most techniques focus on specific genes to address individual cellular challenges. The significance of YAP, transcriptional co-activator, cannot be overstated due to its involvement in regulating organ size and tumor formation. YAP's influence extends to various cellular processes and is regulated by kinase cascade in the Hippo pathway, which phosphorylates serine residues in specific LATS recognition motifs. Activation of YAP has been observed to impact both the size and quantity of cells. This research investigates the effects of YAP5SA on proliferation, apoptosis, and productivity in CHO-K1 cells. YAP5SA, with mutations in all five LATS-target sites, is selected for its heightened activity and resistance to repression through the Hippo-LATS1/2 kinase signaling pathway. Plasmid harboring YAP5SA was transfected into EPO-CHO and the influence of YAP5SA overexpression was investigated. According to our findings, transfection of EPO-CHO cells with YAP5SA exhibited a substantial enhancement in CHO cell productivity, resulting in a 3-fold increase in total protein and EPO, as well as a 1.5-fold increase in specific productivity. Additionally, it significantly contributes in augmenting viability, size, and proliferation. Overall, the findings of this study exemplify the potential of utilizing YAP5SA to impact particular cellular mechanisms, thereby presenting an avenue for customizing cells to fulfill production demands. KEY POINTS: • YAP5SA in CHO cells boosts growth, reduces apoptosis, and significantly improves productivity. • YAP5SA regulates genes involved in proliferation, survival, and mTOR activation. • YAP5SA increases productivity by improving cell cycle, c-MYC expression, and mTOR pathway.

Blood miRNAs miR-549a, miR-552, and miR-592 serve as potential disease-specific panels to diagnose colorectal cancer.

Introduction: miRNAs originating from colorectal cancer (CRC) tissue receive significant focus in the early diagnosis of CRC due to their stability in body fluids. However, if these miRNAs originated from alternative organs, their prognostic value will diminish. Thus, in this study, we aim to identify disease-specific miRNAs for colorectal cancer (CRC) by employing bioinformatics and experimental methodologies. Method: To identify CRC-specific miRNAs, we retrieved miRNA profiles of CRC and normal tissues from the Cancer Genome Atlas (TCGA) database. Subsequently, computational strategies were utilized to select potential candidate miRNAs. Following this, the expression levels of the potent miRNAs were assessed through RT-qPCR in both CRC tissue and serum samples from patients (N = 46), as well as healthy individuals (N = 46). Additionally, the associations between clinicopathological characteristics, survival outcomes, and diagnostic accuracy were evaluated. Results: A total of 8893 RNA-seq expression data were acquired from TCGA, comprising 8250 data from 19 distinct cancer types and 643 corresponding healthy samples. Based on the computational methodology, miR-549a, miR-552, and miR-592 were identified as the principal expressed miRNAs in colorectal cancer (CRC). Within these miRNAs, miR-552 displayed a substantial association with tumors at the N and T stages. miR-549a and miR-592 were observed to be linked exclusively to the invasion of tumor depth and tumor stage (TNM), respectively. The receiver operating characteristic (ROC) analysis conducted on the miRNA expression in serum samples revealed that all miRNAs exhibited an area under the ROC curve (AUC) of up to 0.86, thereby indicating their high diagnostic accuracy. Conclusion: Considering the strong associations of these three identified miRNAs with CRC, they can collectively serve as a panel for specific discrimination of CRC from other types of cancer within the body. Although this study focused solely on CRC, this approach can potentially be applied to other cancer types as well.

Enhancing protein production and growth in chinese hamster ovary cells through miR-107 overexpression.

Chinese Hamster Ovary (CHO) cells are widely employed as host cells for biopharmaceutical production. The manufacturing of biopharmaceuticals poses several challenges, including restricted growth potential and inadequate productivity of the host cells. MicroRNAs play a crucial role in regulating gene expression and are considered highly promising tools for cell engineering to enhance protein production. Our study aimed to evaluate the effects of miR-107, which is recognized as an onco-miR, on erythropoietin-producing CHO cells (CHO-hEPO). To assess the impact of miR-107 on CHO cells, a DNA plasmid containing miR-107 was introduced to CHO-hEPO cells through transfection. Cell proliferation and viability were assessed using the trypan blue dye exclusion method. Cell cycle analysis was conducted by utilizing propidium iodide (PI) staining. The quantification of EPO was determined using an immunoassay test. Moreover, the impact of miR-107 on the expression of downstream target genes was evaluated using qRT-PCR. Our findings highlight and underscore the substantial impact of transient miR-107 overexpression, which led to a remarkable 2.7-fold increase in EPO titers and a significant 1.6-fold increase in the specific productivity of CHO cells (p < 0.01). Furthermore, this intervention resulted in significant enhancements in cell viability and growth rate (p < 0.05). Intriguingly, the overexpression of miR­107 was linked to the downregulation of LATS2, PTEN, and TSC1 genes while concurrently driving upregulation in transcript levels of MYC, YAP, mTOR, and S6K genes within transgenic CHO cells. In conclusion, this study collectively underscores the feasibility of utilizing cancer-associated miRNAs as a powerful tool for CHO cell engineering. However, more in-depth exploration is warranted to unravel the precise molecular intricacies of miR-107's effects in the context of CHO cells.

Enhancing productivity of Chinese hamster ovary (CHO) cells: synergistic strategies combining low-temperature culture and mTORC1 signaling engineering.

Introduction: The growing demand for recombinant proteins in medicine has prompted biopharmaceutical companies to seek ways to maximize the manufacturing process. Despite its known negative impact on cell growth, temperature shift (TS) has emerged as a cost-effective strategy to enhance protein quantity and quality in Chinese Hamster Ovary cells (CHO). As cells adapt their growth and protein synthesis rate to the environment through influencing mTOR complex 1 (mTORC1), here we evaluated the potential of mTORC1 signaling engineering to improve the production of granulocyte-macrophage colony-stimulating factor (GM-CSF) protein in stable CHO cells at low temperature. Methods: First, the expression of genes that negatively control mTORC1 functions in response to environmental fluctuations, including TSC1, AMPK, MAPKAPK5, and MARK4 genes, was assessed via real-time qPCR in CHO-K1 after a temperature shift from 37°C to 30°C. Then, plasmids harboring the shRNAs targeting these genes were constructed into the PB513B-1 plasmid with expression driven by either the constitutive CMV promoter or the cold-inducible HSP90 promoter. Finally, the impact of transient gene downregulation was evaluated on GM-CSF and mTOR proteins productivity in GM-CSF-producing CHO-K1 cells using ELISA and Western-blot assays, respectively. The growth rate of the transfected cells at the two temperatures was evaluated using flow cytometry. Results: Hypothermic conditions promote the upregulation of mTORC1 inhibitor genes, especially TSC1 and MAPKAPK5, while downregulating S6K, a key effector of the mTORC1 signaling pathway, in CHO-K1 cells. Transcription and protein levels of mTOR increased upon transfection, "pB513-b CMV-P/4shRNAs/GFP" plasmid, "pB513-bHSP90-P/4sh-RNAs/GFP" and pB513B-1 plasmid as mock group in GM-CSF-producing CHO-K1 cells (approximately 60%), along with a high transcript level of S6K. Cell growth-related characteristics were improved, albeit with distinct effects at different temperatures. Notably, these changes were more efficient at 30°C when utilizing the HSP90 promoter, resulting in a three-fold increase in GM-CSF production after 3 days. Conclusion: This study highlights the importance of temperature regulation and mTORC1 modulation in CHO cellular processes, particularly in recombinant protein production. Understanding these mechanisms paves the way for developing innovative strategies to enhance cell growth, protein synthesis, and overall bioprocess performance, particularly in manufacturing human therapeutic proteins.

Positive effect of miR-2392 on fibroblast to cardiomyocyte-like cell fate transition: An in silico and in vitro study.

INTRODUCTION: Somatic cell fate transition is now gained great importance in tissue regeneration. Currently, research is focused on heart tissue regeneration by reprogramming diverse cells into cardiomyocyte-like cells. Here, we examined the possible effect of miRNAs on the transdifferentiation of fibroblasts into cardiomyocyte-like cells. METHODS: First heart-specific miRNAs were identified by comparing the gene expression profiles of heart tissue to other body tissues using bioinformatic techniques. After identifying heart-specific miRNAs, their cellular and molecular functions were studied using the miRWalk and miRBase databases. Then the candidate miRNA was cloned into a lentiviral vector. Following, human dermal fibroblasts were cultured and treated with compounds forskolin, valproic acid, and CHIR99021. After 24 h, the lentivector harboring miRNA gene was transfected into the cells to initiate the transdifferentiation process. Finally, after a two-week treatment period, the efficiency of transdifferentiation was examined by inspecting the appearance of the cells and measuring the expression levels of cardiac genes and proteins using RT-qPCR and immunocytochemistry techniques. RESULTS: Nine miRNAs were identified with higher expression in the heart. The miR-2392 was nominated as the candidate miRNA due to its function and specific expression in the heart. This miRNA has a direct connection with genes involved in cell growth and differentiation; e.g., MAPK and Wnt signaling pathways. According to in vitro results cardiac genes and proteins demonstrated an increase in expression in the fibroblasts that simultaneously received the three chemicals and miR-2392. CONCLUSION: Considering the ability of miR-2392 to induce the expression of cardiac genes and proteins in fibroblast cells, it can induce fibroblasts to differentiate into cardiomyocyte-like cells. Therefore, miR-2392 could be further optimized for cardiomyocyte regeneration, tissue repair, and drug design studies.

In silico drug repurposing for the treatment of heart diseases using gene expression data and molecular docking techniques.

Heart diseases are known as the most primary causes of mortality worldwide. Although many therapeutic approaches and medications are proposed for these diseases, the identification of novel therapeutics in fatal heart conditions is promptly demanded. Besides, the interplay between gene expression data and molecular docking provides several novel insights to discover more effective and specific drugs for the treatment of the diseases. This study aimed to discover potent therapeutic drugs in the heart diseases based on the expression profile of heart-specific genes exclusively. Initially, the heart-specific and highly expressed genes were identified by comparing the gene expression profile of different body tissues. Subsequently, the druggable-genes were identified using in silico techniques. The interaction between these druggable genes with more than 1600 FDA approved drugs was then investigated using the molecular docking simulation. By comprehensively analyzing RNA-sequencing data obtained from 949 normal tissue samples, 48 heart-specific genes were identified in both the heart development and function. Notably, of these, 24 heart-specific genes were capable to be considered as druggable genes, among which only MYBPC3, MYLK3, and SCN5A genes entered the molecular docking process due to their functions. Afterward, the pharmacokinetics properties of top 10 ligands with the highest binding affinity for these proteins were studied. Accordingly, methylergonovine, fosaprepitant, pralatrexate, daunorubicin, glecaprevir, digoxin, and venetoclax drugs were competent, in order to interact with the target proteins perfectly. It was shown that these medications can be used as specific drugs for the treatment of heart diseases after fulfilling further experiments in this regard.

WDR81 Gene Silencing Can Reduce Exosome Levels in Human U87-MG Glioblastoma Cells.

Glioblastoma is a very invasive and prevalent brain tumor that affects 15 in 100,000 persons over the age of 70 years. Studies have shown that the expression of the WD repeat domain 81 (WDR81) gene, which is effective in vesicular transport and inhibition of autophagy, is increased in glioblastoma. The decreased autophagy was found to be related to the increased production of exosomes, which is a major factor in the pathogenesis of glioblastoma. The PI-3kinase complex is a pre-autophagic complex that is highly active in the absence of WDR81. The WDR81 gene, as a negative regulator of PI3K activity, prevents autophagy and increases exosome secretion by preventing the formation of the class III PI3K complex. Therefore, targeted reduction of exosomes can be considered an effective strategy for reducing the pathogenesis of glioblastoma. This study aimed to assess the effect of WDR81 gene silencing with siRNA on exosome levels in a U87-MG cell line. Culturing of U87-MG cells was carried out in Dulbecco's modified Eagle medium (DMEM) containing 5% FBS and 1% penicillin/streptomycin. Thereafter, silencing of WDR81 was performed using WDR81 siRNA, whose gene expression level was determined via real-time qRT-PCR. Cell viability was evaluated using the MTT assay. The exosomes were extracted from a cell culture using the Exocib kit. The size accuracy of the exosomes was confirmed by dynamic light scattering (DLS). Finally, the protein content and RNA of the exosomes were assessed. WDR81 gene expression of siRNA-transfected cells was decreased to 82% after 24 h compared to the non-transfected control cells. The analysis of the exosomes showed that the concentration of exosomes and their RNA and protein content in the siRNA-transfected cells decreased significantly compared to the non-transfected control cells. No considerable difference was observed in cell viability after transfection with either WDR81-specific siRNAs or scrambled control siRNAs. Our findings showed that silencing the WDR81 gene could reduce the level of exosomes in human U87-MG glioblastoma cells. Therefore, the reduced exosome content may be suggested as a new gene therapy strategy for targeted therapy of glioblastoma by increasing autophagy via activation of PI3KIII. However, more studies are needed in this regard.

Novel Study of Model-Based Clustering Time Series Gene Expression in Different Tissues: Applications to Aging Process.

BACKGROUND: Aging is an organized biological process that is regulated by highly interconnected pathways between different cells and tissues in the living organism. Identification of similar genes between tissues in different ages may also help to discover the general mechanism of aging or to discover more effective therapeutic decisions. OBJECTIVE: According to the wide application of model-based clustering techniques, the aim is to evaluate the performance of the Mixture of Multivariate Normal Distributions (MMNDs) as a valid method for clustering time series gene expression data with the Mixture of Matrix-Variate Normal Distributions (MMVNDs). METHODS: In this study, the expression of aging data from NCBI's Gene Expression Omnibus was elaborated to utilize proper data. A set of common genes which were differentially expressed between different tissues were selected and then clustered together through two methods. Finally, the biological significance of clusters was evaluated, using their ability to find genes in the cell using Enricher. RESULTS: The MMVNDs is more efficient to find co-express genes. Six clusters of genes were observed using the MMVNDs. According to the functional analysis, most genes in clusters 1-6 are related to the B-cell receptors and IgG immunoglobulin complex, proliferating cell nuclear antigen complex, the metabolic pathways of iron, fat, and body mass control, the defense against bacteria, the cancer development incidence, and the chronic kidney failure, respectively. CONCLUSION: Results showed that most biological changes of aging between tissues are related to the specific components of immune cells. Also, the application of MMVNDs can increase the ability to find similar genes.

Induced pluripotent stem cell-derived extracellular vesicles: A novel approach for cell-free regenerative medicine.

In recent years, induced pluripotent stem cells (iPSCs) have been considered as a promising approach in the field of regenerative medicine. iPSCs can be generated from patients' somatic cells and possess the potential to differentiate, under proper conditions, into any cell type. However, the clinical application of iPS cells is restricted because of their tumorigenic potential. Recent studies have indicated that stem cells exert their therapeutic benefit via a paracrine mechanism, and extracellular vesicles have been demonstrated that play a critical role in this paracrine mechanism. Due to lower immunogenicity, easier management, and presenting no risk of tumor formation, in recent years, researchers turned attention to exosomes as potential alternatives to whole-cell therapy. Application of exosomes derived from iPSCs and their derived precursor provides a promising approach for personalized regenerative medicine. This study reviews the physiological functions of extracellular vesicles and discusses their potential therapeutic benefit in regenerative medicine.

Rn7SK small nuclear RNA is involved in neuronal differentiation.

Rn7SK-mediated global transcriptional regulation, key function of this small nuclear RNA (snRNA), is mediated by inhibition of the positive transcription elongation factor b (P-TEFb). Recently, we have identified a potential anti-proliferative and tumor-suppressive function of Rn7SK. However, its possible regulatory role in development and cell programming has not been investigated so far. Here, we examined transcriptional levels of Rn7SK in different mouse organs. Interestingly, an increased expression level of the RNA was observed in the brain. Furthermore, we could demonstrate that Rn7SK has a dynamic expression pattern during brain development from embryo to adult: 7SK snRNA expression was particularly high at embryonic day (E) 18.5 and adult stages, while a low level of this non-coding RNA was detected at E11.5. Moreover, a decreased transcription level was identified in proliferating progenitors whereas a strong upregulation of Rn7SK was observed during neural differentiation in vivo. Similar to the in vivo situation, in vitro neuronal differentiation experiments employing embryonic stem cells (ESCs) demonstrated the same expression pattern of 7SK with high expression levels in differentiating neurons. Neuronal differentiation of ESCs was compromised when we knocked down Rn7SK, indicating an important role of 7SK in the acquisition of a neural fate.

7SK small nuclear RNA transcription level down-regulates in human tumors and stem cells.

The small nuclear noncoding RNA (snRNA) 7SK is a highly conserved noncoding RNA of 331 nucleotides in animals, which is present in a nuclear ribonucleoprotein complex with proteins such as methylphosphate capping enzyme (MePCE), hexamethylene bisacetamide-inducible proteins 1 and 2 (HEXIM1 and HEXIM2) and La-related protein 7 (Larp7). Regulating the activity of the positive transcription elongation factor b (P-TEFb) is the key function of 7SK noncoding RNA. Recently, we have shown that 7SK snRNA over-expression reduces human embryonic kidney 293T cell line viability. Here, we attempt to monitor the expression level of 7SK snRNA in different human cell lines and cancer tissues. Examination of 7SK transcription either in cell lines or in different malignant tissues including blood (CML), breast and colon showed that 7SK expression significantly down-regulated in cancer. Similar to human cancer tissues and cell lines, 7SK transcriptional level decreased in stem cells in comparison with differentiated cell types. In this regard, over-expression of 7SK snRNA might be a powerful tool for blocking cancer progression by controlling the activity of P-TEFb.

A novel treatment for weight reduction by the recombinant "Pichia pastoris" yeast expressing the hybrid protein of "irisin-furin-transferrin".

Obesity is a major health problem across the world, but there are few ways to effectively treat or manage it in the long term. Researchers are searching for more convenient, cost-effective and noninvasive therapies for overweight and obese people. Recent studies have illustrated that the microbiome of the body's different organs can be used as a vehicle for in-situ gene therapy. We suggest that the recombinant form of "Pichia pastoris" yeast expressing the hybrid protein of "irisin-furin-transferrin" under the control of the enolase 1 promoter is a new nutraceutical strategy to absorb fewer calories from intestinal nutrients, and induce a higher metabolic rate to expend more calories, similar to that from engaging in physical activity. By comparison, this method can be a long-term, noninvasive treatment and can be used for obese patients who have movement limitations.

Effects of disruption of the nucleotide pattern in CRID element and Kozak sequence of interferon ß on mRNA stability and protein production.

Interferon ß (IFNß) is the most important drug that has been used frequently for multiple sclerosis treatment. This study has tried to improve the IFNß production by introducing mutations in the coding region of IFNß, while its amino acid sequence is intact. Two recombinant vectors IFNß(K) and IFNß(K+CRID )were designed by site-directed mutagenesis. The IFNß(K) and IFNß(K+CRID) have two substitutions in Kozak sequence and four substitutions in CRID sequence, respectively. The Chinese hamster ovary (CHO) cell codon usage optimization was also performed for both of them. They were transiently transfected to CHO-dhfr(-) cell line using Lipofectamine kit (Invitrogen, Grand Island, NY). The amount of mRNA and protein was determined by real time PCR and ELISA. The results of this study indicate that the amount of IFNß protein produced by CHO cells containing IFNß(K) has been elevated up to 3.5-fold. On the other hand, enormous amounts of IFNß mRNA and protein were produced by cells containing IFNß(K+CRID) construct; more than 4.6-fold and 6-fold, respectively. It could be concluded that disruption of AT pattern in CRID element increase RNA and protein production, improve IFNß mRNA stability and, may also enhance mRNA half-life. In a similar way, more proteins are produced by modification of Kozak sequence.

Adiponectin as a potential biomarker of vascular disease.

The increasing prevalence of diabetes and its complications heralds an alarming situation worldwide. Obesity-associated changes in circulating adiponectin concentrations have the capacity to predict insulin sensitivity and are a link between obesity and a number of vascular diseases. One obvious consequence of obesity is a decrease in circulating levels of adiponectin, which are associated with cardiovascular disorders and associated vascular comorbidities. Human and animal studies have demonstrated decreased adiponectin to be an independent risk factor for cardiovascular disease. However, in animal studies, increased circulating adiponectin alleviates obesity-induced endothelial dysfunction and hypertension, and also prevents atherosclerosis, myocardial infarction, and diabetic cardiac tissue disorders. Further, metabolism of a number of foods and medications are affected by induction of adiponectin. Adiponectin has beneficial effects on cardiovascular cells via its antidiabetic, anti-inflammatory, antioxidant, antiapoptotic, antiatherogenic, vasodilatory, and antithrombotic activity, and consequently has a favorable effect on cardiac and vascular health. Understanding the molecular mechanisms underlying the regulation of adiponectin secretion and signaling is critical for designing new therapeutic strategies. This review summarizes the recent evidence for the physiological role and clinical significance of adiponectin in vascular health, identification of the receptor and post-receptor signaling events related to the protective effects of the adiponectin system on vascular compartments, and its potential use as a target for therapeutic intervention in vascular disease.

The long term oral regulation of blood glucose in diabetic patients by using of Escherichia coli Nissle 1917 expressing CTB-IGF-1 hybrid protein.

Regarding to the high prevalence and comorbidities of chronic high blood glucose in diabetic patients and the limited efficacy and current painful treatments. It is necessary to improve new treatments that are non-invasive and long-term for controlling blood glucose. Recent studies have shown that the healthy microflora in different body organs can perform as the gene vectors for expressing different types of gene therapies in situ. We have proposed that by constructing a recombinant Escherichia coli Nissle 1917 that expresses CTB-IGF-1 hybrid gene under control of ompC glucose sensitive promoter, the intestinal glucose level can be regulated. This method in comparison with other methods is a non-invasive way to control the blood glucose orally and it can be used for all types of diabetes.