Kaempferol Can Reverse the 5-Fu Resistance of Colorectal Cancer Cells by Inhibiting PKM2-Mediated Glycolysis

Resistance to 5-Fluorouracil (5-Fu) chemotherapy is the main cause of treatment failure in the cure of colon cancer. Therefore, there is an urgent need to explore a safe and effective multidrug resistance reversal agent for colorectal cancer, which would be of great significance for improving clinical efficacy. The dietary flavonoid kaempferol plays a key role in the progression of colorectal cancer and 5-Fu resistance. However, the molecular mechanism of kaempferol in reversing 5-Fu resistance in human colorectal cancer cells is still unclear. We found that kaempferol could reverse the drug resistance of HCT8-R cells to 5-Fu, suggesting that kaempferol alone or in combination with 5-Fu has the potential to treat colorectal cancer. It is well known that aerobic glycolysis is related to tumor growth and chemotherapy resistance. Indeed, kaempferol treatment significantly reduced glucose uptake and lactic acid production in drug-resistant colorectal cancer cells. In terms of mechanism, kaempferol promotes the expression of microRNA-326 (miR-326) in colon cancer cells, and miR-326 could inhibit the process of glycolysis by directly targeting pyruvate kinase M2 isoform (PKM2) 3′-UTR (untranslated region) to inhibit the expression of PKM2 or indirectly block the alternative splicing factors of PKM mRNA, and then reverse the resistance of colorectal cancer cells to 5-Fu. Taken together, our data suggest that kaempferol may play an important role in overcoming resistance to 5-Fu therapy by regulating the miR-326-hnRNPA1/A2/PTBP1-PKM2 axis.     

hnRNPA2B1-Mediated Extracellular Vesicles Sorting of miR-122-5p Potentially Promotes Lung Cancer Progression

Extracellular vesicles (EVs) released by tumor cells play important roles on the remodeling of the tumor–stromal environment and on promoting tumor metastasis. Our earlier studies revealed that miR-122-5p, a type of small non-coding RNA, was dysregulated in non-small cell lung cancer (NSCLC) cell-derived EVs. In this study, we found that miR-122-5p was selectively sorted and secreted into lung cancer EVs through binding to RNA-binding protein hnRNPA2B1. In addition, we found that hnRNPA2B1 interacted with miR-122-5p through the EXO-motif. The delivering of lung cancer EVs-miR-122-5p promoted the migration of liver cells, which may play roles in establishing a pre-metastatic micro-environment and hepatic metastasis of lung cancer. Importantly, our findings revealed the molecular mechanism that RNA-binding protein controls the selective sorting of tumor-derived EV miR-122-5p, which potentially promotes lung cancer progression.     

The Pro-Apoptotic Role of the Regulatory Feedback Loop between miR-124 and PKM1/HNF4α in Colorectal Cancer Cells

Accumulating evidence indicates that miRNA regulatory circuits play important roles in tumorigenesis. We previously reported that miR-124 is correlated with prognosis of colorectal cancer due to PKM-dependent regulation of glycolysis. However, the mechanism by which miR-124 regulates apoptosis in colorectal cancer remains largely elusive. Here, we show that miR-124 induced significant apoptosis in a panel of colorectal cancer cell lines. The mitochondrial apoptosis pathway was activated by miR-124. Furthermore, the pro-apoptotic role of miR-124 was dependent on the status of PKM1/2 level. PKM1 was required for miR-124-induced apoptosis. Via direct protein-protein interaction, PKM1 promoted HNF4α binding to the promoter region of miR-124 and transcribing miR-124. Moreover, HNF4α or PKM1 had a more dramatic effect on colorectal cancer cell apoptosis in the presence of miR-124. However, inhibition of miR-124 blocked cell apoptosis induced by HNF4α or PKM1. These data indicate that miR-124 not only alters the expression of genes involved in glucose metabolism but also stimulates cancer cell apoptosis. In addition, the positive feedback loop between miR-124 and PKM1/HNF4α plays an important role in colorectal cancer cell apoptosis; it suggests that disrupting this regulatory circuit might be a potential therapeutic tool for colorectal cancer treatment.     

Matched Analyses of Brain Metastases versus Primary Non-Small Cell Lung Cancer Reveal a Unique microRNA Signature

Distant spreading of tumor cells to the central nervous system in non-small cell lung cancer (NSCLC) occurs frequently and poses major clinical issues due to limited treatment options. RNAs displaying differential expression in brain metastasis versus primary NSCLC may explain distant tumor growth and may potentially be used as therapeutic targets. In this study, we conducted systematic microRNA expression profiling from tissue biopsies of primary NSCLC and brain metastases from 25 patients. RNA analysis was performed using the nCounter Human v3 miRNA Expression Assay, NanoString technologies, followed by differential expression analysis and in silico target gene pathway analysis. We uncovered a panel of 11 microRNAs with differential expression and excellent diagnostic performance in brain metastasis versus primary NSCLC. Five microRNAs were upregulated in brain metastasis (miR-129-2-3p, miR-124-3p, miR-219a-2-3p, miR-219a-5p, and miR-9-5p) and six microRNAs were downregulated in brain metastasis (miR-142-3p, miR-150-5p, miR-199b-5p, miR-199a-3p, miR-199b-5p, and miR-199a-5p). The differentially expressed microRNAs were predicted to converge on distinct target gene networks originating from five to twelve core target genes. In conclusion, we uncovered a unique microRNA profile linked to two target gene networks. Our results highlight the potential of specific microRNAs as biomarkers for brain metastasis in NSCLC and indicate plausible mechanistic connections.     

Adventitial Tertiary Lymphoid Organs as Potential Source of MicroRNA Biomarkers for Abdominal Aortic Aneurysm

Abdominal aortic aneurysm (AAA) is an inflammatory disease associated with marked changes in the cellular composition of the aortic wall. This study aims to identify microRNA (miRNA) expression in aneurysmal inflammatory cells isolated by laser microdissection from human tissue samples. The distribution of inflammatory cells (neutrophils, B and T lymphocytes, mast cells) was evaluated in human AAA biopsies. We observed in half of the samples that adventitial tertiary lymphoid organs (ATLOs) with a thickness from 0.5 to 2 mm were located exclusively in the adventitia. Out of the 850 miRNA that were screened by microarray in isolated ATLOs (n = 2), 164 miRNAs were detected in ATLOs. The three miRNAs (miR-15a-3p, miR-30a-5p and miR-489-3p) with the highest expression levels were chosen and their expression quantified by RT-PCR in isolated ATLOs (n = 4), M1 (n = 2) and M2 macrophages (n = 2) and entire aneurysmal biopsies (n = 3). Except for the miR-30a-5p, a similar modulation was found in ATLOs and the two subtypes of macrophages. The modulated miRNAs were then evaluated in the plasma of AAA patients for their potential as AAA biomarkers. Our data emphasize the potential of miR-15a-3p and miR-30a-5p as biomarkers of AAA but also as triggers of ATLO evolution. Further investigations will be required to evaluate their targets in order to better understand AAA pathophysiology.     

Glycolytic Reprogramming in Silica-Induced Lung Macrophages and Silicosis Reversed by Ac-SDKP Treatment

Glycolytic reprogramming is an important metabolic feature in the development of pulmonary fibrosis. However, the specific mechanism of glycolysis in silicosis is still not clear. In this study, silicotic models and silica-induced macrophage were used to elucidate the mechanism of glycolysis induced by silica. Expression levels of the key enzymes in glycolysis and macrophage activation indicators were analyzed by Western blot, qRT-PCR, IHC, and IF analyses, and by using a lactate assay kit. We found that silica promotes the expression of the key glycolysis enzymes HK2, PKM2, LDHA, and macrophage activation factors iNOS, TNF-α, Arg-1, IL-10, and MCP1 in silicotic rats and silica-induced NR8383 macrophages. The enhancement of glycolysis and macrophage activation induced by silica was reduced by Ac-SDKP or siRNA-Ldha treatment. This study suggests that Ac-SDKP treatment can inhibit glycolytic reprogramming in silica-induced lung macrophages and silicosis.     

MiR-7-5p Is Involved in Ferroptosis Signaling and Radioresistance Thru the Generation of ROS in Radioresistant HeLa and SAS Cell Lines

In cancer therapy, radioresistance or chemoresistance cells are major problems. We established clinically relevant radioresistant (CRR) cells that can survive over 30 days after 2 Gy/day X-ray exposures. These cells also show resistance to anticancer agents and hydrogen peroxide (H₂O₂). We have previously demonstrated that all the CRR cells examined had up-regulated miR-7-5p and after miR-7-5p knockdown, they lost radioresistance. However, the mechanism of losing radioresistance remains to be elucidated. Therefore, we investigated the role of miR-7-5p in radioresistance by knockdown of miR-7-5p using CRR cells. As a result, knockdown of miR-7-5p increased reactive oxygen species (ROS), mitochondrial membrane potential, and intracellular Fe²⁺ amount. Furthermore, miR-7-5p knockdown results in the down-regulation of the iron storage gene expression such as ferritin, up-regulation of the ferroptosis marker ALOX12 gene expression, and increases of Liperfluo amount. H₂O₂ treatment after ALOX12 overexpression led to the enhancement of intracellular H₂O₂ amount and lipid peroxidation. By contrast, miR-7-5p knockdown seemed not to be involved in COX-2 and glycolysis signaling but affected the morphology of CRR cells. These results indicate that miR-7-5p control radioresistance via ROS generation that leads to ferroptosis.     

PKM2 Modulation in Head and Neck Squamous Cell Carcinoma

The enzyme pyruvate kinase M2 (PKM2) plays a major role in the switch of tumor cells from oxidative phosphorylation to aerobic glycolysis, one of the hallmarks of cancer. Different allosteric inhibitors or activators and several posttranslational modifications regulate its activity. Head and neck squamous cell carcinoma (HNSCC) is a common disease with a high rate of recurrence. To find out more about PKM2 and its modulation in HNSCC, we examined a panel of HNSCC cells using real-time cell metabolic analysis and Western blotting with an emphasis on phosphorylation variant Tyr105 and two reagents known to impair PKM2 activity. Our results show that in HNSCC, PKM2 is commonly phosphorylated at Tyrosine 105. Its levels depended on tyrosine kinase activity, emphasizing the importance of growth factors such as EGF (epidermal growth factor) on HNSCC metabolism. Furthermore, its correlation with the expression of CD44 indicates a role in cancer stemness. Cells generally reacted with higher glycolysis to PKM2 activator DASA-58 and lower glycolysis to PKM2 inhibitor Compound 3k, but some were more susceptible to activation and others to inhibition. Our findings emphasize the need to further investigate the role of PKM2 in HNSCC, as it could aid understanding and treatment of the disease.     

Prenatal Exposure to Delta-9-tetrahydrocannabinol (THC) Alters the Expression of miR-122-5p and Its Target Igf1r in the Adult Rat Ovary

As cannabis use during pregnancy increases, it is important to understand its effects on the developing fetus. Particularly, the long-term effects of its psychoactive component, delta-9-tetrahydrocannabinol (THC), on the offspring’s reproductive health are not fully understood. This study examined the impact of gestational THC exposure on the miRNA profile in adult rat ovaries and the possible consequences on ovarian health. Prenatal THC exposure resulted in the differential expression of 12 out of 420 evaluated miRNAs. From the differentially expressed miRNAs, miR-122-5p, which is highly conserved among species, was the only upregulated target and had the greatest fold change. The upregulation of miR-122-5p and the downregulation of its target insulin-like growth factor 1 receptor (Igf1r) were confirmed by RT-qPCR. Prenatally THC-exposed ovaries had decreased IGF-1R-positive follicular cells and increased follicular apoptosis. Furthermore, THC decreased Igf1r expression in ovarian explants and granulosa cells after 48 h. As decreased IGF-1R has been associated with diminished ovarian health and fertility, we propose that these THC-induced changes may partially explain the altered ovarian follicle dynamics observed in THC-exposed offspring. Taken together, our data suggests that prenatal THC exposure may impact key pathways in the developing ovary, which could lead to subfertility or premature reproductive senescence.     

A Signature of Four Circulating microRNAs as Potential Biomarkers for Diagnosing Early-Stage Breast Cancer

Breast cancer (BC) is the most predominant type of cancer among women. The aim of this study is to find new biomarkers that can help in early detection of BC, especially for those who are too young to be screened using mammography as per guidelines. Using microRNA microarray, we previously showed dysregulation of 74 microRNAs in tumors from early BC patients as compared with normal adjacent tissues, which we were interested in studying in blood circulation. In this study, we investigated the expression of 12 microRNA (miR-21/miR-155/miR-23a/miR-130a/miR-145/miR-425-5p/miR-139-5p/miR-451/miR-195/miR-125b/miR-100, and miR-182) in the plasma of 41 newly diagnosed Lebanese BC patients with early invasive ductal carcinoma as compared with 32 healthy controls. Total RNA was extracted from plasma, and expression levels of miRNA of interest were measured using RT-qPCR followed by statistical analysis; miR-21, miR-155, miR-23a, miR-130a, miR-145, miR-425-5p, and miR-139-5p were significantly upregulated and miR-451 was significantly downregulated, in the plasma of BC patients as compared with healthy controls. The positively correlated miR-23a, miR-21, and miR-130a had a high diagnostic accuracy (86%). Importantly, the combination of miR-145/miR-425-5p/miR-139-5p/miR-130a scored the highest diagnostic accuracy of 95% with AUC = 0.97 (sensitivity 97% and specificity 91%). MicroRNAs are promising non-invasive diagnostic biomarkers for early-stage BC with the panel of miR-145/miR-425-5p/miR-139-5p/miR-130a having the highest diagnostic accuracy.     

Circulating miRNA Expression Profiles and Machine Learning Models in Association with Response to Irinotecan-Based Treatment in Metastatic Colorectal Cancer

Colorectal cancer represents a leading cause of cancer-related morbidity and mortality. Despite improvements, chemotherapy remains the backbone of colorectal cancer treatment. The aim of this study is to investigate the variation of circulating microRNA expression profiles and the response to irinotecan-based treatment in metastatic colorectal cancer and to identify relevant target genes and molecular functions. Serum samples from 95 metastatic colorectal cancer patients were analyzed. The microRNA expression was tested with a NucleoSpin miRNA kit (Machnery-Nagel, Germany), and a machine learning approach was subsequently applied for microRNA profiling. The top 10 upregulated microRNAs in the non-responders group were hsa-miR-181b-5p, hsa-miR-10b-5p, hsa-let-7f-5p, hsa-miR-181a-5p, hsa-miR-181d-5p, hsa-miR-301a-3p, hsa-miR-92a-3p, hsa-miR-155-5p, hsa-miR-30c-5p, and hsa-let-7i-5p. Similarly, the top 10 downregulated microRNAs were hsa-let-7d-5p, hsa-let-7c-5p, hsa-miR-215-5p, hsa-miR-143-3p, hsa-let-7a-5p, hsa-miR-10a-5p, hsa-miR-142-5p, hsa-miR-148a-3p, hsa-miR-122-5p, and hsa-miR-17-5p. The upregulation of microRNAs in the miR-181 family and the downregulation of those in the let-7 family appear to be mostly involved with non-responsiveness to irinotecan-based treatment.     

Tumor Suppressive Role of miR-342-5p in Human Chondrosarcoma Cells and 3D Organoids

Chondrosarcomas are malignant bone tumors. Their abundant cartilage-like extracellular matrix and their hypoxic microenvironment contribute to their resistance to chemotherapy and radiotherapy, and no effective therapy is currently available. MicroRNAs (miRNAs) may be an interesting alternative in the development of therapeutic options. Here, for the first time in chondrosarcoma cells, we carried out high-throughput functional screening using impedancemetry, and identified five miRNAs with potential antiproliferative or chemosensitive effects on SW1353 chondrosarcoma cells. The cytotoxic effects of miR-342-5p and miR-491-5p were confirmed on three chondrosarcoma cell lines, using functional validation under normoxia and hypoxia. Both miRNAs induced apoptosis and miR-342-5p also induced autophagy. Western blots and luciferase reporter assays identified for the first time Bcl-2 as a direct target of miR-342-5p, and also Bcl-xL as a direct target of both miR-342-5p and miR-491-5p in chondrosarcoma cells. MiR-491-5p also inhibited EGFR expression. Finally, only miR-342-5p induced cell death on a relevant 3D chondrosarcoma organoid model under hypoxia that mimics the in vivo microenvironment. Altogether, our results revealed the tumor suppressive activity of miR-342-5p, and to a lesser extent of miR-491-5p, on chondrosarcoma lines. Through this study, we also confirmed the potential of Bcl-2 family members as therapeutic targets in chondrosarcomas.     

MiR-320-3p Regulates the Proliferation and Differentiation of Myogenic Progenitor Cells by Modulating Actin Remodeling

Skeletal myogenesis is essential for the maintenance of muscle quality and quantity, and impaired myogenesis is intimately associated with muscle wasting diseases. Although microRNA (miRNA) plays a crucial role in myogenesis and relates to muscle wasting in obesity, the molecular targets and roles of miRNAs modulated by saturated fatty acids (SFA) are largely unknown. In the present study, we investigated the role of miR-320-3p on the differentiation of myogenic progenitor cells. Palmitic acid (PA), the most abundant dietary SFA, suppressed myogenic factors expression and impaired differentiation in C2C12 myoblasts, and these effects were accompanied by CFL2 downregulation and miR-320-3p upregulation. In particular, miR-320-3p appeared to target CFL2 mRNA directly and suppress the expression of CFL2, an essential factor for filamentous actin (F-actin) depolymerization. Transfection of myoblasts with miR-320-3p mimic increased F-actin formation and nuclear translocation of Yes-associated protein 1 (YAP1), a key component of mechanotransduction. Furthermore, miR-320-3p mimic increased myoblast proliferation and markedly impeded the expression of MyoD and MyoG, consequently inhibiting myoblast differentiation. In conclusion, our current study highlights the role of miR-320-3p on CFL2 expression, YAP1 activation, and myoblast differentiation and suggests that PA-inducible miR-320-3p is a significant mediator of muscle wasting in obesity.     

Cardiovascular Disease-Associated MicroRNAs as Novel Biomarkers of First-Trimester Screening for Gestational Diabetes Mellitus in the Absence of Other Pregnancy-Related Complications

We assessed the diagnostic potential of cardiovascular disease-associated microRNAs for the early prediction of gestational diabetes mellitus (GDM) in singleton pregnancies of Caucasian descent in the absence of other pregnancy-related complications. Whole peripheral venous blood samples were collected within 10 to 13 weeks of gestation. This retrospective study involved all pregnancies diagnosed with only GDM (n = 121) and 80 normal term pregnancies selected with regard to equality of sample storage time. Gene expression of 29 microRNAs was assessed using real-time RT-PCR. Upregulation of 11 microRNAs (miR-1-3p, miR-20a-5p, miR-20b-5p, miR-23a-3p, miR-100-5p, miR-125b-5p, miR-126-3p, miR-181a-5p, miR-195-5p, miR-499a-5p, and miR-574-3p) was observed in pregnancies destinated to develop GDM. Combined screening of all 11 dysregulated microRNAs showed the highest accuracy for the early identification of pregnancies destinated to develop GDM. This screening identified 47.93% of GDM pregnancies at a 10.0% false positive rate (FPR). The predictive model for GDM based on aberrant microRNA expression profile was further improved via the implementation of clinical characteristics (maternal age and BMI at early stages of gestation and an infertility treatment by assisted reproductive technology). Following this, 69.17% of GDM pregnancies were identified at a 10.0% FPR. The effective prediction model specifically for severe GDM requiring administration of therapy involved using a combination of these three clinical characteristics and three microRNA biomarkers (miR-20a-5p, miR-20b-5p, and miR-195-5p). This model identified 78.95% of cases at a 10.0% FPR. The effective prediction model for GDM managed by diet only required the involvement of these three clinical characteristics and eight microRNA biomarkers (miR-1-3p, miR-20a-5p, miR-20b-5p, miR-100-5p, miR-125b-5p, miR-195-5p, miR-499a-5p, and miR-574-3p). With this, the model identified 50.50% of GDM pregnancies managed by diet only at a 10.0% FPR. When other clinical variables such as history of miscarriage, the presence of trombophilic gene mutations, positive first-trimester screening for preeclampsia and/or fetal growth restriction by the Fetal Medicine Foundation algorithm, and family history of diabetes mellitus in first-degree relatives were included in the GDM prediction model, the predictive power was further increased at a 10.0% FPR (72.50% GDM in total, 89.47% GDM requiring therapy, and 56.44% GDM managed by diet only). Cardiovascular disease-associated microRNAs represent promising early biomarkers to be implemented into routine first-trimester screening programs with a very good predictive potential for GDM.     

Global Lysine Acetylome Analysis of LPS-Stimulated HepG2 Cells Identified Hyperacetylation of PKM2 as a Metabolic Regulator in Sepsis

Sepsis-induced liver dysfunction (SILD) is a common event and is strongly associated with mortality. Establishing a causative link between protein post-translational modification and diseases is challenging. We studied the relationship among lysine acetylation (Kac), sirtuin (SIRTs), and the factors involved in SILD, which was induced in LPS-stimulated HepG2 cells. Protein hyperacetylation was observed according to SIRTs reduction after LPS treatment for 24 h. We identified 1449 Kac sites based on comparative acetylome analysis and quantified 1086 Kac sites on 410 proteins for acetylation. Interestingly, the upregulated Kac proteins are enriched in glycolysis/gluconeogenesis pathways in the Kyoto Encyclopedia of Genes and Genomes (KEGG) category. Among the proteins in the glycolysis pathway, hyperacetylation, a key regulator of lactate level in sepsis, was observed at three pyruvate kinase M2 (PKM2) sites. Hyperacetylation of PKM2 induced an increase in its activity, consequently increasing the lactate concentration. In conclusion, this study is the first to conduct global profiling of Kac, suggesting that the Kac mechanism of PKM2 in glycolysis is associated with sepsis. Moreover, it helps to further understand the systematic information regarding hyperacetylation during the sepsis process.     

Quantitative Prediction of Steatosis in Patients with Non-Alcoholic Fatty Liver by Means of Hepatic MicroRNAs Present in Serum and Correlating with Hepatic Fat

Non-alcoholic fatty liver disease (NAFLD) is the most prevalent form of chronic liver disease worldwide, but a reliable non-invasive method to quantify liver steatosis in primary healthcare is not available. Circulating microRNAs have been proposed as biomarkers of severe/advanced NAFLD (steatohepatitis and fibrosis). However, the use of circulating miRNAs to quantitatively assess the % of liver fat in suspected NAFLD patients has not been investigated. We performed global miRNA sequencing in two sets of samples: human livers from organ donors (n = 20), and human sera from biopsy-proven NAFLD patients (n = 23), both with a wide range of steatosis quantified in their liver biopsies. Partial least squares (PLS) regression combined with recursive feature elimination (RFE) was used to select miRNAs associated with steatosis. Moreover, regression models with only 2 or 3 miRNAs, with high biological relevance, were built. Comprehensive microRNA sequencing of liver and serum samples resulted in two sets of abundantly expressed miRNAs (418 in liver and 351 in serum). Pearson correlation analyses indicated that 18% of miRNAs in liver and 14.5% in serum were significantly associated with the amount of liver fat. PLS-RFE models demonstrated that 50 was the number of miRNAs providing the lowest error in both liver and serum models predicting steatosis. Comparison of the two miRNA subsets showed 19 coincident miRNAs that were ranked according to biological significance (guide/passenger strand, relative abundance in liver and serum, number of predicted lipid metabolism target genes, correlation significance, etc.). Among them, miR-10a-5p, miR-98-5p, miR-19a-3p, miR-30e-5p, miR-32-5p and miR-145-5p showed the highest biological relevance. PLS regression models with serum levels of 2–3 of these miRNAs predicted the % of liver fat with errors <5%.