Analysis of androgen receptor expression and activity in the mouse brain.

Androgen deprivation therapy (ADT) is the core treatment for advanced prostate cancer (PCa), with a proven survival benefit. ADT lowers circulating testosterone levels throughout the body, but with it comes a variety of reported side effects including fatigue, muscle wastage, weight gain, hot flushes and importantly cognitive impairment, depression, and mood swings. Testosterone has a key role in brain masculinization, but its direct effects are relatively poorly understood, due both to the brain's extreme complexity and the fact that some of testosterone activities are driven via local conversion to oestrogen, especially during embryonic development. The exact roles, function, and location of the androgen receptor (AR) in the adult male brain are still being discovered, and therefore the cognitive side effects of ADT may be unrecognized or under-reported. The age of onset of several neurological diseases overlap with PCa, therefore, there is a need to separate ADT side effects from such co-morbidities. Here we analysed the activity and expression level of the AR in the adult mouse brain, using an ARE-Luc reporter mouse and immunohistochemical staining for AR in all the key brain regions via coronal slices. We further analysed our data by comparing to the Allen Mouse Brain Atlas. AR-driven luciferase activity and distinct nuclear staining for AR were seen in several key brain areas including the thalamus, hypothalamus, olfactory bulb, cerebral cortex, Purkinje cells of the cerebellum and the hindbrain. We describe and discuss the potential role of AR in these areas, to inform and enable extrapolation to potential side effects of ADT in humans.

BRCA Mutations and MicroRNA Expression Patterns in the Peripheral Blood of Breast Cancer Patients.

Breast cancer (BC) persists as the predominant malignancy globally, standing as the foremost cause of cancer-related mortality among women. Despite notable advancements in prevention and treatment, encompassing the incorporation of targeted immunotherapies, a continued imperative exists for the development of innovative methodologies. These methodologies would facilitate the identification of women at heightened risk, enhance the optimization of therapeutic approaches, and enable the vigilant monitoring of emergent treatment resistance. Circulating microRNAs (miRNAs), found either freely circulating in the bloodstream or encapsulated within extracellular vesicles, have exhibited substantial promise for diverse clinical applications. These applications range from diagnostic and prognostic assessments to predictive purposes. This study aimed to explore the potential associations between BRCA mutations and specific miRNAs (miR-21, miR-155, miR-126, and miR-200c) expression that are known to be dysregulated in BC patient samples. Our findings indicate a robust correlation between miRNA expression status and disease subtypes. We found a correlation between the expression status of miRNAs and distinct disease subtypes. Intriguingly, however, no significant associations were discerned between disease status, subtypes, or miRNA expression levels and the presence of BRCA mutations. To advance the validation of miRNAs as clinically relevant biomarkers, additional investigations within larger and meticulously selected patient cohorts are deemed imperative. These microRNA entities hold the potential to emerge as groundbreaking and readily accessible tools, poised for seamless integration into the landscape of clinical practice.

Impact of Hypoxia-Induced miR-210 on Pancreatic Cancer.

Pancreatic cancer (PC) poses significant clinical challenges, with late-stage diagnosis and limited therapeutic options contributing to its dismal prognosis. A hallmark feature of PC is the presence of a profoundly hypoxic tumour microenvironment, resulting from various factors such as fibrotic stroma, rapid tumour cell proliferation, and poor vascularization. Hypoxia plays a crucial role in promoting aggressive cancer behaviour, therapeutic resistance, and immunosuppression. Previous studies have explored the molecular mechanisms behind hypoxia-induced changes in PC, focusing on the role of hypoxia-inducible factors (HIFs). Among the myriad of molecules affected by hypoxia, microRNA-210 (miR-210) emerges as a central player. It is highly responsive to hypoxia and regulated by HIF-dependent and HIF-independent pathways. miR-210 influences critical cellular processes, including angiogenesis, metastasis, and apoptosis, all of which contribute to PC progression and resistance to treatment. Understanding these pathways provides insights into potential therapeutic targets. Furthermore, investigating the role of miR-210 and its regulation in hypoxia sheds light on the potential development of early diagnostic strategies, which are urgently needed to improve outcomes for PC patients. This review delves into the complexities of PC and introduces the roles of hypoxia and miR-210 in the progression of PC.

miR-34a-FOXP1 Loop in Ovarian Cancer.

Ovarian cancer (OC) is the main cause of gynecological cancer mortality in most developed countries. microRNA (miR) expression dysregulation has been highlighted in human cancers, and miR-34a is found to be downregulated and associated with inhibition of tumor growth and invasion in several malignancies, including OC. The winged helix transcription factor forkhead box P1 (FOXP1) is reported as either an oncogene or tumor suppressor in various cancers. This study aimed to elucidate potential clinical and biological associations of miR-34a and transcription factor FOXP1 in OC. We investigated nine OC patients' blood samples and two OC cell lines (SKOV-3 and OVCAR-3) using quantitative real-time reverse transcription polymerase chain reaction (RT-qPCR) to determine both miR-34a and FOXP1 expressions. We have found that miR-34a and FOXP1 are reversely correlated in both in vitro and in vivo. Inhibition of miR-34a transiently led to upregulation of FOXP1 mRNA expression and increased cellular invasion in vitro. Our data indicate that miR-34a could be a potential biomarker for improving the diagnostic efficiency of OC, and miR-34a overexpression may reduce OC pathogenesis by targeting FOXP1.

Prohibitin Links Cell Cycle, Motility and Invasion in Prostate Cancer Cells.

Prohibitin (PHB) is a tumour suppressor gene with several different molecular activities. PHB overexpression leads to G1/S-phase cell cycle arrest, and PHB represses the androgen receptor (AR) in prostate cancer cells. PHB interacts with and represses members of the E2F family in a manner that may also be AR-linked, therefore making the AR:PHB:E2F interaction axis highly complex. PHB siRNA increased the growth and metastatic potential of LNCaP mouse xenografts in vivo. Conversely, PHB ectopic cDNA overexpression affected several hundred genes in LNCaP cells. Furthermore, gene ontology analysis showed that in addition to cell cycle regulation, several members of the WNT family were significantly downregulated (WNT7B, WNT9A and WNT10B), as well as pathways for cell adhesion. Online GEO data studies showed PHB expression to be decreased in clinical cases of metastatic prostate cancer, and to be correlated with higher WNT expression in metastasis. PHB overexpression reduced prostate cancer cell migration and motility in wound-healing assays, reduced cell invasion through a Matrigel layer and reduced cellular attachment. In LNCaP cells, WNT7B, WNT9A and WNT10B expression were also upregulated by androgen treatment and downregulated by androgen antagonism, indicating a role for AR in the control of these WNT genes. However, these WNTs were strongly cell cycle regulated. E2F1 cDNA ectopic expression and PHB siRNA (both cell cycle promoting effects) increased WNT7B, WNT9A and WNT10B expression, and these genes were also upregulated as cells were released from G1 to S phase synchronisation, indicating further cell cycle regulation. Therefore, the repressive effects of PHB may inhibit AR, E2F and WNT expression and its loss may increase metastatic potential in human prostate cancer.

Inhibiting CDK4/6 in pancreatic ductal adenocarcinoma via microRNA-21.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive malignancies, with a 5-year survival rate of 5-10 %. The high mortality rate is due to the asymptomatic progression of clinical features in metastatic stages of the disease, which renders standard therapeutic options futile. PDAC is characterised by alterations in several genes that drive carcinogenesis and limit therapeutic response. The two most common genetic aberrations in PDAC are the mutational activation of KRAS and loss of the tumour suppressor CDK inhibitor 2A (CDKN2A), which culminate the activation of the cyclin-dependent kinase 4 and 6 (CDK4/6), that promote G1 cell cycle progression. Therapeutic strategies focusing on the CDK4/6 inhibitors such as palbociclib (PD-0332991) may potentially improve outcomes in this malignancy. MicroRNAs (miRs/miRNAs) are small endogenous non-coding RNA molecules associated with cellular proliferation, invasion, apoptosis, and cell cycle. Primarily, miR-21 promotes cell proliferation and a higher proportion of PDAC cells in the S phase, while knockdown of miR-21 has been linked to cell cycle arrest at the G2/M phase and inhibition of cell proliferation. In this study, using a CRISPR/Cas9 loss-of-function screen, we individually silenced the expression of miR-21 in two PDAC cell lines and in combination with PD-0332991 treatment, we examined the synergetic mechanisms of CDK4/6 inhibitors and miR-21 knockouts (KOs) on cell survival and death. This combination reduced cell proliferation, cell viability, increased apoptosis and G1 arrest in vitro. We further analysed the mitochondrial respiration and glycolysis of PDAC cells; then assessed the protein content of these cells and revealed numerous Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways associated with PD-0332991 treatment and miR-21 knocking out. Our results demonstrate that combined targeting of CDK4/6 and silencing of miR-21 represents a novel therapeutic strategy in PDAC.

microRNA 1307 Is a Potential Target for SARS-CoV-2 Infection: An in Vitro Model.

microRNAs (miRs) are proposed as critical molecular targets in SARS-CoV-2 infection. Our recent in silico studies identified seven SARS-CoV-2 specific miR-like sequences, which are highly conserved with humans, including miR-1307-3p, with critical roles in COVID-19. In this current study, Vero cells were infected with SARS-CoV-2, and miR expression profiles were thereafter confirmed by qRT-PCR. miR-1307-3p was the most highly expressed miR in the infected cells; we, therefore, transiently inhibited its expression in both infected and uninfected cells. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) cell proliferation assay assessed cell viability following SARS-CoV-2 infection, identifying that miR-1307 expression is inversely correlated with cell viability. Lastly, changes in miR-1307-dependent pathways were analyzed through a detailed miRNOME and associated in silico analysis. In addition to our previously identified miRs, including miR-1307-3p, the upregulation of miR-193a-5p, miR-5100, and miR-23a-5p and downregulation of miR-130b-5p, miR34a-5p, miR-505-3p, miR181a-2-3p, miR-1271-5p, miR-598-3p, miR-34c-3p, and miR-129-5p were also established in Vero cells related to general lung disease-related genes following SARS-CoV-2 infection. Targeted anti-miR-1307-3p treatment rescued cell viability in infection when compared to SARS CoV-2 mediated cell cytotoxicity only. We furthermore identified by in silico analysis that miR-1307-3p is conserved in all SARS-CoV-2 sequences/strains, except in the BA.2 variant, possibly contributing to the lower disease severity of this variant, which warrants further investigation. Small RNA seq analysis was next used to evaluate alterations in the miRNOME, following miR-1307-3p manipulation, identifying critical pathobiological pathways linked to SARS-CoV-2 infection-mediated upregulation of this miR. On the basis of our findings, miRNAs like miR-1307-3p play a critical role in SARS-CoV-2 infection, including via effects on disease progression and severity.

microRNA expression in acute myeloid leukaemia: New targets for therapy?

Recent studies have shown that short non-coding RNAs, known as microRNAs (miRNAs) and their dysregulation, are implicated in the pathogenesis of acute myeloid leukaemia (AML). This is due to their role in the control of gene expression in a variety of molecular pathways. Therapies involving miRNA suppression and replacement have been developed. The normalisation of expression and the subsequent impact on AML cells have been investigated for some miRNAs, demonstrating their potential to act as therapeutic targets. Focussing on miRs with therapeutic potential, we have reviewed those that have a significant impact on the aberrant biological processes associated with AML, and crucially, impact leukaemic stem cell survival. We describe six miRNAs in preclinical trials (miR-21, miR-29b, miR-126, miR-181a, miR-223 and miR-196b) and two miRNAs that are in clinical trials (miR-29 and miR-155). However none have been used to treat AML patients and greater efforts are needed to develop miRNA therapies that could benefit AML patients in the future.

3D Disease Modelling of Hard and Soft Cancer Using PHA-Based Scaffolds.

Tumour cells are shown to change shape and lose polarity when they are cultured in 3D, a feature typically associated with tumour progression in vivo, thus making it significant to study cancer cells in an environment that mimics the in vivo milieu. In this study we established hard (MCF7 and MDA-MB-231, breast cancer) and soft (HCT116, colon cancer) 3D cancer tumour models utilizing a blend of P(3HO-co-3HD) and P(3HB). P(3HO-co-3HD) and P(3HB) belong to a group of natural biodegradable polyesters, PHAs, that are synthesised by microorganisms. The 3D PHA scaffolds produced, with a pore size of 30 to 300 µm, allow for nutrients to diffuse within the scaffold and provide the cells with the flexibility to distribute evenly within the scaffold and grow within the pores. Interestingly, by Day 5, MDA-MB-231 showed dispersed growth in clusters, and MCF7 cells formed an evenly dispersed dense layer, while HCT116 formed large colonies within the pockets of the 3D PHA scaffolds. Our results show Epithelial Mesenchymal Transition (EMT) marker gene expression profiles in the hard tumour cancer models. In the 3D-based PHA scaffolds, MDA-MB-231 cells expressed higher levels of Wnt-11 and mesenchymal markers, such as Snail and its downstream gene Vim mRNAs, while MCF7 cells exhibited no change in their expression. On the other hand, MCF7 cells exhibited a significantly increased E-Cadherin expression as compared to MDA-MB-231 cells. The expression levels of EMT markers were comparative to their expression reported in the tumour samples, making them good representative of cancer models. In future these models will be helpful in mimicking hypoxic tumours, in studying gene expression, cellular signalling, angiogenesis and drug response more accurately than 2D and perhaps other 3D models.

Nickel's Role in Pancreatic Ductal Adenocarcinoma: Potential Involvement of microRNAs.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal cancer types with a limited overall survival rate due to the asymptomatic progression of symptoms in metastatic stages of the malignancy and the lack of an early reliable diagnostic biomarker. MicroRNAs (miRs/miRNAs) are small (~18-24 nucleotides), endogenous, non-coding RNAs, which are closely linked to the development of numerous malignancies comprising PDAC. Recent studies have described the role of environmental pollutants such as nickel (Ni) in PDAC, but the mechanisms of Ni-mediated toxicity in cancer are still not completely understood. Specifically, Ni has been found to alter the expression and function of miRs in several malignancies, leading to changes in target gene expression. In this study, we found that levels of Ni were significantly higher in cancerous tissue, thus implicating Ni in pancreatic carcinogenesis. Hence, in vitro studies followed by using both normal and pancreatic tumor cell lines and increasing Ni concentration increased lethality. Comparing LC50 values, Ni-acetate groups demonstrated lower values needed than in NiCl2 groups, suggesting greater Ni-acetate. Panc-10.05 cell line appeared the most sensitive to Ni compounds. Exposure to Ni-acetate resulted in an increased phospho-AKT, and decreased FOXO1 expression in Panc-10.05 cells, while NiCl2 also increased PTEN expression in Panc-10.05 cells. Specifically, following NiCl2 exposure to PDAC cells, the expression levels of miR-221 and miR-155 were significantly upregulated, while the expression levels of miR-126 were significantly decreased. Hence, our study has suggested pilot insights to indicate that the environmental pollutant Ni plays an important role in the progression of PDAC by promoting an association between miRs and Ni exposure during PDAC pathogenesis.

microRNA-21 Regulates Stemness in Pancreatic Ductal Adenocarcinoma Cells.

Pancreatic ductal adenocarcinoma (PDAC) is the most common and aggressive type of pancreatic cancer (PCa) with a low survival rate. microRNAs (miRs) are endogenous, non-coding RNAs that moderate numerous biological processes. miRs have been associated with the chemoresistance and metastasis of PDAC and the presence of a subpopulation of highly plastic "stem"-like cells within the tumor, known as cancer stem cells (CSCs). In this study, we investigated the role of miR-21, which is highly expressed in Panc-1 and MiaPaCa-2 PDAC cells in association with CSCs. Following miR-21 knockouts (KO) from both MiaPaCa-2 and Panc-1 cell lines, reversed expressions of epithelial-mesenchymal transition (EMT) and CSCs markers were observed. The expression patterns of key CSC markers, including CD44, CD133, CX-C chemokine receptor type 4 (CXCR4), and aldehyde dehydrogenase-1 (ALDH1), were changed depending on miR-21 status. miR-21 (KO) suppressed cellular invasion of Panc-1 and MiaPaCa-2 cells, as well as the cellular proliferation of MiaPaCa-2 cells. Our data suggest that miR-21 is involved in the stemness of PDAC cells, may play roles in mesenchymal transition, and that miR-21 poses as a novel, functional biomarker for PDAC aggressiveness.

Role of microRNAs in response to cadmium chloride in pancreatic ductal adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most fatal and aggressive malignancies with a 5-year survival rate less than 9%. Early detection is particularly difficult due to the lack of symptoms even in advanced stages. microRNAs (miRs/miRNAs) are small (~ 18-24 nucleotides), endogenous, non-coding RNAs, which are involved in the pathogenesis of several malignancies including PDAC. Alterations of miR expressions can lead to apoptosis, angiogenesis, and metastasis. The role of environmental pollutants such as cadmium (Cd) in PDAC has been suggested but not fully understood. This study underlines the role of miRs (miR-221, miR-155, miR-126) in response to cadmium chloride (CdCl2) in vitro. Lethal concentration (LC50) values for CdCl2 resulted in a toxicity series of AsPC-1 > HPNE > BxPC-3 > Panc-1 = Panc-10.5. Following the treatment with CdCl2, miR-221 and miR-155 were significantly overexpressed, whereas miR-126 was downregulated. An increase in epithelial-mesenchymal transition (EMT) via the dysregulation of mesenchymal markers such as Wnt-11, E-cadherin, Snail, and Zeb1 was also observed. Hence, this study has provided evidence to suggest that the environmental pollutant Cd can have a significant role in the development of PDAC, suggesting a significant correlation between miRs and Cd exposure during PDAC progression. Further studies are needed to investigate the precise role of miRs in PDAC progression as well as the role of Cd and other environmental pollutants.

The Role of CDK4 in the Pathogenesis of Pancreatic Cancer.

Pancreatic cancer (PC) continues to have the lowest overall survival and the lack of effective early diagnosis. Cyclin-dependent kinase 4 (CDK4) plays a fundamental role in the orderly progression of the cell cycle, binding to cyclin D to promote the progression through the G1/2 transition. The inhibition of CDK4/6 has therefore gained substantial interest in the hope of new and effective therapeutics in multiple cancers, such as advanced metastatic breast cancer. While the use of these agents is encouraging, their potential is yet to be fully explored. In this study we used the GLOBOCAN database to understand the most recent epidemiology of PC, Human Protein Atlas and KEGG to highlight the role, prevalence, and significance on patient survival of CDK4 in PC. We found that CDK4 cannot be used as prognostic in PC and no significant differences were observed between CDK4 expression and the patient's clinical status, though larger studies, especially concerning CDK4 protein expressions, are required for a more thorough understanding. The use of CDK4/6 inhibitors in PC is still in clinical trials. However, due to only modest improvements observed in the use of single-agent therapies, efforts have focused on combinatorial approaches.

In Vitro Investigations of miR-33a Expression in Estrogen Receptor-Targeting Therapies in Breast Cancer Cells.

(1) Background: Increased fatty acid synthesis leads to the aggressive phenotype of breast cancer and renders efficiency of therapeutics. Regulatory microRNAs (miRNAs) on lipid biosynthesis pathways as miR-33a have potential to clarify the exact mechanism. (2) Methods: We determined miR-33a expression levels following exposure of MCF-7 and MDA-MB-231 breast cancer cells to estrogen receptor (ER) activator (estradiol-17ß, E2) or anti-estrogens (ICI 182,780, Fulvestrant, FUL) at non-cytotoxic concentrations. We related miR-33a expression levels in the cells to cellular lipid biosynthesis-related pathways through immunoblotting. (3) Results: miR-33a mimic treatment led to significantly downregulation of fatty acid synthase (FASN) in MCF-7 cells but not in MDA-MB-231 cells in the presence of estradiol-17ß (E2) or Fulvestrant (FUL). In contrast to the miR-33a inhibitor effect, miR-33a mimic co-transfection with E2 or FUL led to diminished AMP-activated protein kinase α (AMPKα) activity in MCF-7 cells. E2 increases FASN levels in MDA-MB-231 cells regardless of miR-33a cellular levels. miR-33a inhibitor co-treatment suppressed E2-mediated AMPKα activity in MDA-MB-231 cells. (4) Conclusions: The cellular expression levels of miR-33a are critical to understanding differential responses which include cellular energy sensors such as AMPKα activation status in breast cancer cells.

Non-coding RNAs in pancreatic ductal adenocarcinoma: New approaches for better diagnosis and therapy.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive malignancies with a 5-year survival rate less than 8%, which has remained unchanged over the last 50 years. Early detection is particularly difficult due to the lack of disease-specific symptoms and a reliable biomarker. Multimodality treatment including chemotherapy, radiotherapy (used sparingly) and surgery has become the standard of care for patients with PDAC. Carbohydrate antigen 19-9 (CA 19-9) is the most common diagnostic biomarker; however, it is not specific enough especially for asymptomatic patients. Non-coding RNAs are often deregulated in human malignancies and shown to be involved in cancer-related mechanisms such as cell growth, differentiation, and cell death. Several micro, long non-coding and circular RNAs have been reported to date which are involved in PDAC. Aim of this review is to discuss the roles and functions of non-coding RNAs in diagnosis and treatments of PDAC.

Prostate Cancer Cell Extracellular Vesicles Increase Mineralisation of Bone Osteoblast Precursor Cells in an In Vitro Model.

Skeletal metastases are the most common form of secondary tumour associated with prostate cancer (PCa). The aberrant function of bone cells neighbouring these tumours leads to the devel-opment of osteoblastic lesions. Communication between PCa cells and bone cells in bone envi-ronments governs both the formation/development of the associated lesion, and growth of the secondary tumour. Using osteoblasts as a model system, we observed that PCa cells and their conditioned medium could stimulate and increase mineralisation and osteoblasts' differentiation. Secreted factors within PCa-conditioned medium responsible for osteoblastic changes included small extracellular vesicles (sEVs), which were sufficient to drive osteoblastogenesis. Using MiR-seq, we profiled the miRNA content of PCa sEVs, showing that miR-16-5p was highly ex-pressed. MiR-16 was subsequently higher in EV-treated 7F2 cells and a miR-16 mimic could also stimulate mineralisation. Next, using RNA-seq of extracellular vesicle (EV)-treated 7F2 cells, we observed a large degree of gene downregulation and an increased mineralisation. Ingenuity® Pathway Analysis (IPA®) revealed that miR-16-5p (and other miRs) was a likely upstream effec-tor. MiR-16-5p targets in 7F2 cells, possibly involved in osteoblastogenesis, were included for val-idation, namely AXIN2, PLSCR4, ADRB2 and DLL1. We then confirmed the targeting and dow-regulation of these genes by sEV miR-16-5p using luciferase UTR (untranslated region) reporters. Conversely, the overexpression of PLSCR4, ADRB2 and DLL1 lead to decreased osteoblastogene-sis. These results indicate that miR-16 is an inducer of osteoblastogenesis and is transmitted through prostate cancer-derived sEVs. The mechanism is a likely contributor towards the for-mation of osteoblastic lesions in metastatic PCa.

Peptidylarginine Deiminase Inhibitor Application, Using Cl-Amidine, PAD2, PAD3 and PAD4 Isozyme-Specific Inhibitors in Pancreatic Cancer Cells, Reveals Roles for PAD2 and PAD3 in Cancer Invasion and Modulation of Extracellular Vesicle Signatures.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive malignancies with limited survival rate. Roles for peptidylarginine deiminases (PADs) have been studied in relation to a range of cancers with roles in epigenetic regulation (including histone modification and microRNA regulation), cancer invasion, and extracellular vesicle (EV) release. Hitherto though, knowledge on PADs in PDAC is limited. In the current study, two PDAC cell lines (Panc-1 and MiaPaCa-2) were treated with pan-PAD inhibitor Cl-amidine as well as PAD2, PAD3, and PAD4 isozyme-specific inhibitors. Effects were assessed on changes in EV signatures, including EV microRNA cargo (miR-21, miR-126, and miR-221), on changes in cellular protein expression relevant for pancreatic cancer progression and invasion (moesin), for mitochondrial housekeeping (prohibitin, PHB), and gene regulation (deiminated histone H3, citH3). The two pancreatic cancer cell lines were found to predominantly express PAD2 and PAD3, which were furthermore expressed at higher levels in Panc-1, compared with MiaPaCa-2 cells. PAD2 isozyme-specific inhibitor had the strongest effects on reducing Panc-1 cell invasion capability, which was accompanied by an increase in moesin expression, which in pancreatic cancer is found to be reduced and associated with pancreatic cancer aggressiveness. Some reduction, but not significant, was also found on PHB levels while effects on histone H3 deimination were variable. EV signatures were modulated in response to PAD inhibitor treatment, with the strongest effects observed for PAD2 inhibitor, followed by PAD3 inhibitor, showing significant reduction in pro-oncogenic EV microRNA cargo (miR-21, miR-221) and increase in anti-oncogenic microRNA cargo (miR-126). While PAD2 inhibitor, followed by PAD3 inhibitor, had most effects on reducing cancer cell invasion, elevating moesin expression, and modulating EV signatures, PAD4 inhibitor had negligible effects and pan-PAD inhibitor Cl-amidine was also less effective. Compared with MiaPaCa-2 cells, stronger modulatory effects for the PAD inhibitors were observed in Panc-1 cells, which importantly also showed strong response to PAD3 inhibitor, correlating with previous observations that Panc-1 cells display neuronal/stem-like properties. Our findings report novel PAD isozyme regulatory roles in PDAC, highlighting roles for PAD isozyme-specific treatment, depending on cancer type and cancer subtypes, including in PDAC.

MiR-21 Is Required for the Epithelial-Mesenchymal Transition in MDA-MB-231 Breast Cancer Cells.

Breast cancer (BCa) is one of the leading health problems among women. Although significant achievements have led to advanced therapeutic success with targeted therapy options, more efforts are required for different subtypes of tumors and according to genomic, transcriptomic, and proteomic alterations. This study underlines the role of microRNA-21 (miR-21) in metastatic MDA-MB-231 breast cancer cells. Following the knockout of miR-21 from MDA-MB-231 cells, which have the highest miR-21 expression levels compared to MCF-7 and SK-BR-3 BCa cells, a decrease in epithelial-mesenchymal transition (EMT) via downregulation of mesenchymal markers was observed. Wnt-11 was a critical target for miR-21, and the Wnt-11 related signaling axis was altered in the stable miR-21 knockout cells. miR-21 expression was associated with a significant increase in mesenchymal markers in MDA-MB-231 BCa cells. Furthermore, the release of extracellular vesicles (EVs) was significantly reduced in the miR-21 KO cells, alongside a significant reduction in relative miR-21 export in EV cargo, compared with control cells. We conclude that miR-21 is a leading factor involved in mesenchymal transition in MDA-MB-231 BCa. Future therapeutic strategies could focus on its role in the treatment of metastatic breast cancer.

MicroRNAs for Virus Pathogenicity and Host Responses, Identified in SARS-CoV-2 Genomes, May Play Roles in Viral-Host Co-Evolution in Putative Zoonotic Host Species.

Our recent study identified seven key microRNAs (miR-8066, 5197, 3611, 3934-3p, 1307-3p, 3691-3p, 1468-5p) similar between SARS-CoV-2 and the human genome, pointing at miR-related mechanisms in viral entry and the regulatory effects on host immunity. To identify the putative roles of these miRs in zoonosis, we assessed their conservation, compared with humans, in some key wild and domestic animal carriers of zoonotic viruses, including bat, pangolin, pig, cow, rat, and chicken. Out of the seven miRs under study, miR-3611 was the most strongly conserved across all species; miR-5197 was the most conserved in pangolin, pig, cow, bat, and rat; miR-1307 was most strongly conserved in pangolin, pig, cow, bat, and human; miR-3691-3p in pangolin, cow, and human; miR-3934-3p in pig and cow, followed by pangolin and bat; miR-1468 was most conserved in pangolin, pig, and bat; while miR-8066 was most conserved in pangolin and pig. In humans, miR-3611 and miR-1307 were most conserved, while miR-8066, miR-5197, miR-3334-3p and miR-1468 were least conserved, compared with pangolin, pig, cow, and bat. Furthermore, we identified that changes in the miR-5197 nucleotides between pangolin and human can generate three new miRs, with differing tissue distribution in the brain, lung, intestines, lymph nodes, and muscle, and with different downstream regulatory effects on KEGG pathways. This may be of considerable importance as miR-5197 is localized in the spike protein transcript area of the SARS-CoV-2 genome. Our findings may indicate roles for these miRs in viral-host co-evolution in zoonotic hosts, particularly highlighting pangolin, bat, cow, and pig as putative zoonotic carriers, while highlighting the miRs' roles in KEGG pathways linked to viral pathogenicity and host responses in humans. This in silico study paves the way for investigations into the roles of miRs in zoonotic disease.

Specific c-Jun N-Terminal Kinase Inhibitor, JNK-IN-8 Suppresses Mesenchymal Profile of PTX-Resistant MCF-7 Cells through Modulating PI3K/Akt, MAPK and Wnt Signaling Pathways.

Paclitaxel (PTX) is a widely used chemotherapeutic agent in the treatment of breast cancer, and resistance to PTX is a common failure of breast cancer therapy. Therefore, understanding the effective molecular targets in PTX-resistance gains importance in identifying novel strategies in successful breast cancer therapy approaches. The aim of the study was to investigate the functional role of PTX resistance on MCF-7 cell survival and proliferation related to PI3K/Akt and MAPK pathways. The generated PTX-resistant (PTX-res) MCF-7 cells showed enhanced cell survival, proliferation, and colony formation potential with decreased cell death compared to wt MCF-7 cells. PTX-res MCF-7 cells exhibited increased motility profile with EMT, PI3K/Akt, and MAPK pathway induction. According to the significant SAPK/JNK activation in PTX-res MCF-7 cells, specific c-Jun N-terminal kinase inhibitor, JNK-IN-8 is shown to suppress the migration potential of cells. Treatment of JNK inhibitor suppressed the p38 and SAPK/JNK and Vimentin expression. However, the JNK inhibitor further downregulated Wnt signaling members in PTX-res MCF-7 cells. Therefore, the JNK inhibitor JNK-IN-8 might be used as a potential therapy model to reverse PTX-resistance related to Wnt signaling.