Peripherical Blood hsa-miR-335-5p Quantification as a Prognostic, but Not Diagnostic, Marker of Gastric Cancer.

Gastric cancer (GC) is a leading cause of death, and this pathology often receives a diagnosis in an advanced stage. The development of a less invasive and cost-effective test for detection is essential for decreasing the mortality rate and increasing the life expectancy of GC patients. We evaluated the potential targeting of CD54/ICAM1, a marker of gastric cancer stem cells, with miRNAs to detect GC in blood samples. The analyses included 79 blood samples, 38 from GC patients and 41 from healthy donors, who attended INCan, México City. The total RNA was obtained from the blood plasma, and RT-PCR and qPCR were performed to obtain the relative expression of each miRNA. Hsa-miR-335-5p was detected in the plasma of GC patients and healthy donors at the same levels. The ROC curve analyses indicated that this miRNA was not a candidate for the molecular diagnosis of GC. We did not observe a correlation between the expression of hsa-miR-335-5p and clinical variables; however, the Kaplan-Meier analyses indicated that, in patients who survived more than 12 months, a lower expression of hsa-miR-335-5p was correlated with a better prognosis. It would be convenient to evaluate a larger panel of miRNAs, including miRNAs expressed in a limited number of cell types or with a low number targets, to obtain more specific candidates for developing a robust test for the diagnosis/prognosis of GC.

CircROBO1 knockdown improves the radiosensitivity of hepatocellular carcinoma by regulating RAD21.

INTRODUCTION AND OBJECTIVES: Radioresistance is a common problem in the treatment of many cancers, including hepatocellular carcinoma (HCC). Previous studies have shown that circROBO1 is highly expressed in HCC tissues and acts as a cancer promoter to accelerate the malignant progression of HCC. However, the role and mechanism of circROBO1 in HCC radioresistance remain unclear. MATERIALS AND METHODS: CircROBO1, microRNA (miR)-136-5p and RAD21 expression levels were analyzed by quantitative real-time PCR. Cell function and radioresistance were evaluated by colony formation assay, cell counting kit 8 assay, EdU assay and flow cytometry. Protein expression was determined using western blot analysis. RNA interaction was analyzed by dual-luciferase reporter assay and RNA pull-down assay. In vivo experiments were performed by constructing mice xenograft models. RESULTS: CircROBO1 was highly expressed in HCC, and its knockdown inhibited HCC cell proliferation and promoted apoptosis to enhance cell radiosensitivity. On the mechanism, circROBO1 could serve as miR-136-5p sponge to positively regulate RAD21. MiR-136-5p inhibitor or RAD21 overexpression reversed the regulation of circROBO1 knockdown on the radiosensitivity of HCC cells. Also, circROBO1 interference improved the radiosensitivity of HCC tumors in vivo. CONCLUSIONS: CircROBO1 might be a promising target for treating HCC radioresistance.

LncRNA HCG18 affects aortic dissection through the miR-103a-3p/HMGA2 axis by modulating proliferation and apoptosis of vascular smoothing muscle cells.

BACKGROUND: Aortic Dissection (AD) is a vascular disease with a high mortality rate and limited treatment strategies. The current research analyzed the function and regulatory mechanism of lncRNA HCG18 in AD. METHODS: HCG18, miR-103a-3p, and HMGA2 levels in the aortic tissue of AD patients were examined by RT-qPCR. After transfection with relevant plasmids, the proliferation of rat aortic Vascular Smoothing Muscle Cells (VSMCs) was detected by CCK-8 and colony formation assay, Bcl-2 and Bax was measured by Western blot, and apoptosis was checked by flow cytometry. Then, the targeting relationship between miR-103a-3p and HCG18 or HMGA2 was verified by bioinformation website analysis and dual luciferase reporter assay. Finally, the effect of HCG18 was verified in an AD rat model induced by ß-aminopropionitrile. RESULTS: HCG18 and HMGA2 were upregulated and miR-103a-3p was downregulated in the aortic tissues of AD patients. Downregulating HCG18 or upregulating miR-103a-3p enhanced the proliferation of VSMCs and limited cell apoptosis. HCG18 promoted HMGA2 expression by competing with miR-103a-3p and restoring HMGA2 could impair the effect of HCG18 downregulation or miR-103a-3p upregulation in mediating the proliferation and apoptosis of VSMCs. In addition, down-regulation of HCG18 could improve the pathological injury of the aorta in AD rats. CONCLUSION: HCG18 reduces proliferation and induces apoptosis of VSMCs through the miR-103a-3p/HMGA2 axis, thus aggravating AD.

Glucocorticoid modulates oxidative and thermogenic function of rat brown adipose tissue and human brown adipocytes.

Chronic and excessive glucocorticoid (GC) exposure can cause Cushing's syndrome, resulting in fat accumulation in selected body areas. Particularly in the brown adipose tissue (BAT), GC acts negatively, resulting in whitening of the tissue. We hypothesized that dysregulation of microRNAs by GC could be an additional mechanism to explain its negative actions in BAT. Male Wistar rats were divided into two groups: (1) Control sham and (2) GC group that was administered dexamethasone 6.25 mg/200 µL via osmotic pump implantation over 28 days. After this period, the animals were euthanized and BAT tissue was properly stored. Human fat cells treated with dexamethasone were used to translate the experimental results found in animals to human biology. GC-treated rat BAT presented with large lipid droplets, severely impaired thermogenic activation, and reduced glucose uptake measured by 18F-FDG PET/CT. GC exposure induced a reduction in the mitochondrial OXPHOS system and oxygen consumption. MicroRNA profiling of BAT revealed five top-regulated microRNAs and among them miR-21-5p was the most significantly upregulated in GC-treated rats compared to the control group. Although upregulation of miR-21-5p in the tissue, differentiated primary brown adipocytes from GC-treated rats had decreased miR-21-5p levels compared to the control group. To translate these results to the clinic, human brown adipocytes were treated with dexamethasone and miR-21-5p inhibitor. In human brown cells, inhibition of miR-21-5p increased brown adipocyte differentiation and prevented GC-induced glucose uptake, resulting in a lower glycolysis rate. In conclusion, high-dose GC therapy significantly impacts brown adipose tissue function, with a notable association between glucose uptake and miR-21-5p.

Tumor-exosomal miR-205-5p as a diagnostic biomarker for colorectal cancer.

BACKGROUND: Tumor-derived exosomal miRNAs play crucial roles in cancer diagnosis. Current studies aim to identify exosomal miRNAs associated with colorectal cancer (CRC) that are noninvasive, sensitive, and specific. PATIENTS AND METHODS: Exosomes were extracted from CRC patients and healthy donors via ultracentrifugation, followed by verification via transmission electron microscopy (TEM), qNano, and Western blot analysis. The differential expression levels and clinical characteristics of miR-205-5p were analyzed in CRC via data from The Cancer Genome Atlas (TCGA). Real-time quantitative PCR was used to assess the expression levels of exosomal miRNAs in 157 primary CRC patients, 20 patients with benign diseases, and 135 healthy donors. Predictions regarding target genes were made to guide further exploration of the disease's etiopathogenesis through bioinformatics. RESULTS: Compared with that in healthy donors, the expression of miR-205-5p in colorectal cancer (CRC) patients was significantly lower, as determined through analysis of the TCGA database. We conducted a prediction and analysis of the functional enrichment of downstream target genes regulated by miR-205-5p. A lower level of exosomal miR-205-5p in the serum of CRC patients than in that of healthy controls (p < 0.0001) and patients with benign disease (p < 0.0001) was observed. Furthermore, the expression levels of exosomal miR-205-5p were significantly lower in early-stage CRC patients than in the comparison groups (p<0.001 and p < 0.0001). Notably, the expression levels of exosomal miR-205-5p significantly increased postoperatively (p = 0.0053). CONCLUSIONS: The present study demonstrated that serum exosomal miR-205-5p may be a diagnostic biomarker for CRC.

LncRNA PTENP1/miR-21/PTEN Axis Modulates EMT and Drug Resistance in Cancer: Dynamic Boolean Modeling for Cell Fates in DNA Damage Response.

It is well established that microRNA-21 (miR-21) targets phosphatase and tensin homolog (PTEN), facilitating epithelial-to-mesenchymal transition (EMT) and drug resistance in cancer. Recent evidence indicates that PTEN activates its pseudogene-derived long non-coding RNA, PTENP1, which in turn inhibits miR-21. However, the dynamics of PTEN, miR-21, and PTENP1 in the DNA damage response (DDR) remain unclear. Thus, we propose a dynamic Boolean network model by integrating the published literature from various cancers. Our model shows good agreement with the experimental findings from breast cancer, hepatocellular carcinoma (HCC), and oral squamous cell carcinoma (OSCC), elucidating how DDR activation transitions from the intra-S phase to the G2 checkpoint, leading to a cascade of cellular responses such as cell cycle arrest, senescence, autophagy, apoptosis, drug resistance, and EMT. Model validation underscores the roles of PTENP1, miR-21, and PTEN in modulating EMT and drug resistance. Furthermore, our analysis reveals nine novel feedback loops, eight positive and one negative, mediated by PTEN and implicated in DDR cell fate determination, including pathways related to drug resistance and EMT. Our work presents a comprehensive framework for investigating cellular responses following DDR, underscoring the therapeutic potential of targeting PTEN, miR-21, and PTENP1 in cancer treatment.

The diagnostic and prognostic role of miR-146b-5p in differentiated thyroid carcinomas.

Introduction: Samples classified as indeterminate correspond to 10-20% of cytologies obtained by fine needle biopsy of thyroid nodules, preventing an adequate distinction between benign and malignant lesions and leading to diagnostic thyroidectomies that often prove unnecessary, as most cases are benign. Furthermore, although the vast majority of patients with differentiated thyroid cancer (DTC) have such a good prognosis that active surveillance is permitted as an initial therapeutic option, relapses are not rare, and a non-negligible number of patients experience poor outcomes. MicroRNAs (miR) emerge as potential biomarkers capable of helping to define more precise management of patients in all these situations. Methods: Aiming to investigate the clinical utility of miR-146b-5p in the diagnostic of thyroid nodules and evaluating its prognostic potential in a realworld setting, we studied 89 thyroid nodule samples, correlating miR-146b-5p expression with clinical tools such as the 8th edition from the American Joint Committee on Cancer (AJCC/UICC) and the American Thyroid Association Guideline Stratification Systems for the rate of recurrence (RR). Results: miR-146b-5p expression levels distinguished benign from malignant thyroid FNA samples (p< 0.0001). For indeterminate nodules, overexpression of miR-146b-5p with a cut-off of 0.497 was able to diagnose malignancy with a 90% accuracy; specificity=87.5%; sensitivity=100%. An increased expression of miR-146b-5p was associated with greater RR (p=0.015). A cut-off of 2.21 identified cases with more vascular involvement (p=0.013) and a cut-off of 2.420 was associated with a more advanced TNM stage (p-value=0.047). Discussion: We demonstrated that miR-146b5p expression in FNA samples is able to differentiate benign from malignant indeterminate nodules and is associated with an increased risk of recurrence and mortality, suggesting that this single miRNA may be a useful diagnostic and prognostic marker in the personalized management of DTC patients.

microRNA-181a-5p promotes fibroblast differentiation of mesenchymal stem cells in rats with pelvic floor dysfunction.

The use of stem cells capable of multilineage differentiation in treating Pelvic Floor Dysfunction (PFD) holds great promise since they are susceptible to entering connective tissue of various cell types and repairing damaged tissues. This research investigated the effect of microRNA-181a-5p (miR-181a-5p) on Bone Marrow Mesenchymal Stem Cells (BMSCs) in rats with PFD. BMSCs were transfected and analyzed for their fibroblast differentiation ability. miR-181a-5p, MFN1, and fibroblast-related genes were quantitatively analyzed. Whether MFN1 is a target gene of miR-181a-5p was predicted and confirmed. The efficacy of BMSCs in vivo rats with PFD was evaluated by measuring Leak Point Pressure (LPP), Conscious Cystometry (CMG), hematoxylin and eosin staining, and Masson staining. The present results discovered that miR-181a-5p was up-regulated and MFN1 was down-regulated during the differentiation of BMSCs into fibroblasts. Fibroblast differentiation of BMSCs was promoted after miR-181a-5p was induced or MFN1 was suppressed, but it was suppressed after miR-181a-5p was silenced. miR-181a-5p improved LPP and conscious CMG outcomes in PDF rats by targeting MFN1 expression, thereby accelerating fibroblast differentiation of BMSCs. In brief, miR-181a-5p induces fibroblast differentiation of BMSCs in PDF rats by MFN1, potentially targeting PDF therapeutics.

Next-generation sequencing profiling of miRNAs in individuals with 22q11.2 deletion syndrome revealed altered expression of miR-185-5p.

BACKGROUND: The 22q11.2 deletion syndrome (22q11.2DS) is a microdeletion syndrome with highly variable phenotypic manifestations, even though most patients present the typical 3 Mb microdeletion, usually affecting the same ~ 106 genes. One of the genes affected by this deletion is DGCR8, which plays a crucial role in miRNA biogenesis. Therefore, the haploinsufficiency of DGCR8 due to this microdeletion can alter the modulation of the expression of several miRNAs involved in a range of biological processes. RESULTS: In this study, we used next-generation sequencing to evaluate the miRNAs profiles in the peripheral blood of 12 individuals with typical 22q11DS compared to 12 healthy matched controls. We used the DESeq2 package for differential gene expression analysis and the DIANA-miTED dataset to verify the expression of differentially expressed miRNAs in other tissues. We used miRWalk to predict the target genes of differentially expressed miRNAs. Here, we described two differentially expressed miRNAs in patients compared to controls: hsa-miR-1304-3p, located outside the 22q11.2 region, upregulated in patients, and hsa-miR-185-5p, located in the 22q11.2 region, which showed downregulation. Expression of miR-185-5p is observed in tissues frequently affected in patients with 22q11DS, and previous studies have reported its downregulation in individuals with 22q11DS. hsa-miR-1304-3p has low expression in blood and, thus, needs more validation, though using a sensitive technology allowed us to identify differences in expression between patients and controls. CONCLUSIONS: Thus, lower expression of miR-185-5p can be related to the 22q11.2 deletion and DGCR8 haploinsufficiency, leading to phenotypic consequences in 22q11.2DS patients, while higher expression of hsa-miR-1304-3p might be related to individual genomic variances due to the heterogeneous background of the Brazilian population.

DLK1-DIO3 region as a source of tumor suppressor miRNAs in papillary thyroid carcinoma.

BACKGROUND: In previous studies, we demonstrated the downregulation of several miRNAs from the DLK1-DIO3 genomic region in papillary thyroid carcinoma (PTC). Due to the large number of miRNAs within this region, the individual contribution of these molecules to PTC development and progression remains unclear. OBJECTIVE: In this study, we aimed to clarify the contribution of DLK1-DIO3-derived miRNAs to PTC. METHODS: We used different computational approaches and in vitro resources to assess the biological processes and signaling pathways potentially modulated by these miRNAs. RESULTS: Our analysis suggests that, out of more than 100 mature miRNAs originated from the DLK1-DIO3 region, a set of 12 miRNAs accounts for most of the impact on PTC development and progression, cooperating to modulate distinct cancer-relevant biological processes, such as cell migration, extracellular matrix remodeling, and signal transduction. The restoration of the expression of one of these miRNAs (miR-485-5p) in a BRAFT199A-positive PTC cell line impaired proliferation and migration, suppressing the expression of GAB2 and RAC1, validated miR-485-5p targets. CONCLUSIONS: Overall, our results shed light on the role of the DLK1-DIO3 region, which harbors promising tumor suppressor miRNAs in thyroid cancer, and open prospects for the functional exploration of these miRNAs as therapeutic targets for PTC.

Downregulation of hsa-miR-100-5p May Be a Protective Factor in the Early Stages of Nephropathy in Type 1 Diabetes Mellitus.

Type 1 Diabetes Mellitus (T1DM) can generate severe complications, such as Diabetic Kidney Disease (DKD) or Diabetic Nephropathy (DN), with it emerging as the leading cause of terminal (end-stage) renal disease all over the world. For T1DM, the clinical evaluation of DKD uses markers like the Glomerular Filtration Rate (GFR) and the Urinary Albumin Excretion (UAE). However, early diagnosis of DKD is still a challenge. For this reason, investigating molecular markers, such as microRNAs (miRNAs), offers a promising perspective to an early diagnosis, highlighting the stability and the ability to reflect incipient molecular manifestations. Thus, here we investigated four miRNAs (hsa-let-7i-5p, hsa-miR-143-3p, hsa-miR-501-3p, and hsa-miR-100-5p) regarding nephropathy in patients with T1DM, considering the albuminuria (micro and macro) as a standard to evaluate the groups. As a result, we found a reduced expression of miR-100-5p in patients with MIC, indicating a protective role in nephropathy. Beyond that, expression levels between the groups (Non vs. UAE) were not significant when comparing the miRNAs miR-501-3p and miR-143-3p. Finally, miR-143-3p and miR-100-5p were linked to some target genes such as AKT1, MMP13, and IGF1R, that are connected to signal pathways and cellular metabolism.

miR-137 confers robustness to the territorial restriction of the neural plate border.

The neural plate border (NPB) of vertebrate embryos is segregated from the neural plate (NP) and epidermal regions, and comprises an intermingled group of progenitors with multiple fate potential. Recent studies have shown that, during the gastrula stage, TFAP2A acts as a pioneer factor in remodeling the epigenetic landscape required to activate components of the NPB induction program. Here, we show that chick Tfap2a has two highly conserved binding sites for miR-137, and both display a reciprocal expression pattern at the NPB and NP, respectively. In addition, ectopic miR-137 expression reduced TFAP2A, whereas its functional inhibition expanded their territorial distribution overlapping with PAX7. Furthermore, we demonstrate that loss of the de novo DNA methyltransferase DNMT3A expanded miR-137 expression to the NPB. Bisulfite sequencing revealed a markedly elevated presence of non-canonical CpH methylation within the miR-137 promoter region when comparing NPB and NP samples. Our findings show that miR-137 contributes to the robustness of NPB territorial restriction in vertebrate development.

Single-Base Gene Variants in MIR-146A and SCN1A Genes Related to the Epileptogenic Process in Drug-Responsive and Drug-Resistant Temporal Lobe Epilepsy-A Preliminary Study in a Brazilian Cohort Sample.

The drug-resistant temporal lobe epilepsy (TLE) has recently been associated with single nucleotide variants (SNVs) in microRNA(miR)-146a (MIR-146A) (rs2910164) and Sodium Voltage-Gated Channel Alpha Subunit 1 (SCN1A) (rs2298771 and rs3812718) genes. Moreover, no studies have shown an association between these SNVs and susceptibility to drug-resistant and drug-responsive TLE in Brazil. Thus, deoxyribonucleic acid (DNA) samples from 120 patients with TLE (55 drug-responsive and 65 drug-resistant) were evaluated by real-time polymerase chain reaction (RT-PCR). A total of 1171 healthy blood donor individuals from the Online Archive of Brazilian Mutations (ABraOM, from Portuguese Arquivo Brasileiro On-line de Mutações), a repository containing genomic variants of the Brazilian population, were added as a control population for the studied SNVs. MIR-146A and SCN1A relative expression was performed by quantitative RT-PCR (qRT-PCR). The statistical analysis protocol was performed using an alpha error of 0.05. TLE patient samples and ABraOM control samples were in Hardy-Weinberg equilibrium for all studied SNVs. For rs2910164, the frequencies of the homozygous genotype (CC) (15.00% vs. 9.65%) and C allele (37.80% vs. 29.97%) were superior in patients with TLE compared to controls with a higher risk for TLE disease [odds ratio (OR) = 1.89 (95% confidence interval (95%CI) = 1.06-3.37); OR = 1.38 (95%CI = 1.04-1.82), respectively]. Drug-responsive patients also presented higher frequencies of the CC genotype [21.81% vs. 9.65%; OR = 2.58 (95%CI = 1.25-5.30)] and C allele [39.09% vs. 29.97%; OR = 1.50 (95%CI = 1.01-2.22)] compared to controls. For rs2298771, the frequency of the heterozygous genotype (AG) (51.67% vs. 40.40%) was superior in patients with TLE compared to controls with a higher risk for TLE disease [OR = 2.42 (95%CI = 1.08-5.41)]. Drug-resistant patients presented a higher AG frequency [56.92% vs. 40.40%; OR = 3.36 (95%CI = 1.04-17.30)] compared to the control group. For rs3812718, the prevalence of genotypes and alleles were similar in both studied groups. The MIR-146A relative expression level was lower in drug-resistant compared to drug-responsive patients for GC (1.6 vs. 0.1, p-value = 0.049) and CC (1.8 vs. 0.6, p-value = 0.039). Also, the SCN1A relative expression levels in samples from TLE patients were significantly higher in AG [2.09 vs. 1.10, p-value = 0.038] and GG (3.19 vs. 1.10, p-value < 0.001) compared to the AA genotype. In conclusion, the rs2910164-CC and rs2298771-AG genotypes are exerting significant risk influence, respectively, on responsive disease and resistant disease, probably due to an upregulated nuclear factor kappa B (NF-kB) and SCN1A loss of function.

CircUBE2D2 regulates HMGB1 through miR-885-5p to promote ovarian cancer malignancy.

BACKGROUND: The newly discovered CircUBE2D2 has been shown to abnormally upregulate and promote cancer progression in a variety of cancers. The present study explored circUBE2D2 (hsa_circ_0005728) in Ovarian Cancer (OC) progression. METHODS: CircUBE2D2, miR-885-5p, and HMGB1 were examined by RT-qPCR or WB. SKOV-3 cell functions (including cell viability, apoptosis, migration, and invasion) were validated using the CCK-8, flow cytometry, scratch assay, and transwell assay, respectively. The direct relationship between miR-885-5p and circUBE2D2 or HMGB1 was confirmed by a dual-luciferase reporter and RNA pull-down analysis. circUBE2D2's role in vivo tumor xenograft experiment was further probed. RESULTS: OC tissue and cell lines had higher circUBE2D2 and HMGB1 and lower miR-885-5p. Mechanically, CircUBE2D2 shared a binding relation with miR-885-5p, while miR-885-5p can directly target HMGB1. Eliminating circUBE2D2 or miR-885-5p induction inhibited OC cell activities. However, these functions were relieved by down-regulating miR-885-5p or HMGB1 induction. Furthermore, circUBE2D2 knockout reduced tumor growth. CONCLUSION: CircUBE2D2 regulates the expression of HMGB1 by acting as a sponge of ceRNA as miR-885-5p, thereby promoting the control of OC cell proliferation and migration and inhibiting cell apoptosis. Targeting CircUBE2D2 could serve as a new potential treatment strategy for OC.

Circ-USP9X accelerates deep vein thrombosis after fracture by acting as a miR-148b-3p sponge and upregulates SRC kinase signaling inhibitor 1.

OBJECTIVES: This study aims to elucidate the role of circUSP9X (Circular RNA Ubiquitin Specific Peptidase 9 X-Linked) in the development of venous thrombosis in the lower extremities. METHODS: An animal model of Deep Vein Thrombosis (DVT) and a hypoxic model of Human Umbilical Vein Endothelial Cells (HUVECs) treated with Cobalt (II) Chloride (CoCl2) were developed. The expression levels of circUSP9X, microRNA-148b-3p (miR-148b-3p), and SRC Kinase Signaling Inhibitor 1 (SRCIN1) were quantified using quantitative reverse transcription Polymerase Chain Reaction and Western blot analysis. Cell cytotoxicity, viability, apoptosis, and inflammation in HUVECs were assessed via Lactate Dehydrogenase (LDH) assay, MTT assay, flow cytometry, Enzyme-Linked Immunosorbent Assay, and Western blot, respectively. Hematoxylin and Eosin staining were employed for histopathological examination of the venous tissues in the animal model. The interaction between circUSP9X, miR-148b-3p, and SRCIN1 was further explored through dual-luciferase reporter assays and RNA Immunoprecipitation experiments. RESULTS: The present findings reveal a significant upregulation of circUSP9X and SRCIN1 and a concurrent downregulation of miR-148b-3p in DVT cases. Knockdown of circUSP9X or overexpression of miR-148b-3p ameliorated CoCl2-induced apoptosis in HUVECs, reduced LDH release, enhanced cellular viability, and mitigated inflammation. Conversely, overexpression of circUSP9X intensified CoCl2's cytotoxic effects. The effects of manipulating circUSP9X expression were counteracted by the corresponding modulation of miR-148b-3p and SRCIN1 levels. Additionally, circUSP9X knockdown effectively inhibited the formation of DVT in the mouse model. A competitive binding mechanism of circUSP9X for miR-148b-3p, modulating SRCIN1 expression, was identified. CONCLUSION: circUSP9X promotes the formation of DVT through the regulation of the miR-148b-3p/SRCIN1 axis.

LINC00662 Promotes Aggressive Traits by Modulating OCT4 Expression through miR-335-5p in Gallbladder Cancer Cells.

Long non-coding RNAs (lncRNAs) are nucleotide sequences that participate in different biological processes and are associated with different pathologies, including cancer. Long intergenic non-protein-coding RNA 662 (LINC00662) has been reported to be involved in different cancers, including colorectal, prostate, and breast cancer. However, its role in gallbladder cancer has not yet been described. In this article, we hypothesize that LINC00662 has an important role in the acquisition of aggressiveness traits such as a stem-like phenotype, invasion, and chemoresistance in gallbladder cancer. Here, we show that LINC00662 is associated with larger tumor size and lymph node metastasis in patients with gallbladder cancer. Furthermore, we show that the overexpression of LINC00662 promotes an increase in CD133+/CD44+ cell populations and the expression of stemness-associated genes. LINC00662 promotes greater invasive capacity and the expression of genes associated with epithelial-mesenchymal transition. In addition, the expression of LINC00662 promotes resistance to cisplatin and 5-fluorouracil, associated with increased expression of chemoresistance-related ATP-binding cassette (ABC) transporters in gallbladder cancer (GBC) cell lines. Finally, we show that the mechanism by which LINC00662 exerts its function is through a decrease in microRNA 335-5p (miR-335-5p) and an increase in octamer-binding transcription factor 4 (OCT4) in GBC cells. Thus, our data allow us to propose LINC00662 as a biomarker of poor prognosis and a potential therapeutic target for patients with GBC.

Therapeutic potential of oleic acid supplementation in myotonic dystrophy muscle cell models.

BACKGROUND: We recently reported that upregulation of Musashi 2 (MSI2) protein in the rare neuromuscular disease myotonic dystrophy type 1 contributes to the hyperactivation of the muscle catabolic processes autophagy and UPS through a reduction in miR-7 levels. Because oleic acid (OA) is a known allosteric regulator of MSI2 activity in the biogenesis of miR-7, here we sought to evaluate endogenous levels of this fatty acid and its therapeutic potential in rescuing cell differentiation phenotypes in vitro. In this work, four muscle cell lines derived from DM1 patients were treated with OA for 24 h, and autophagy and muscle differentiation parameters were analyzed. RESULTS: We demonstrate a reduction of OA levels in different cell models of the disease. OA supplementation rescued disease-related phenotypes such as fusion index, myotube diameter, and repressed autophagy. This involved inhibiting MSI2 regulation of direct molecular target miR-7 since OA isoschizomer, elaidic acid (EA) could not cause the same rescues. Reduction of OA levels seems to stem from impaired biogenesis since levels of the enzyme stearoyl-CoA desaturase 1 (SCD1), responsible for converting stearic acid to oleic acid, are decreased in DM1 and correlate with OA amounts. CONCLUSIONS: For the first time in DM1, we describe a fatty acid metabolism impairment that originated, at least in part, from a decrease in SCD1. Because OA allosterically inhibits MSI2 binding to molecular targets, reduced OA levels synergize with the overexpression of MSI2 and contribute to the MSI2 > miR-7 > autophagy axis that we proposed to explain the muscle atrophy phenotype.

LncRNA XIST promotes neovascularization in diabetic retinopathy by regulating miR-101-3p/VEGFA.

Objective: This study sought to investigate the regulation of long noncoding RNA (lncRNA) XIST on the microRNA (miR)-101-3p/vascular endothelial growth factor A (VEGFA) axis in neovascularization in diabetic retinopathy (DR). Materials and methods: Serum of patients with DR was extracted for the analysis of XIST, miR-101-3p, and VEGFA expression levels. High glucose (HG)-insulted HRMECs and DR model rats were treated with lentiviral vectors. MTT, transwell, and tube formation assays were performed to evaluate cell viability, migration, and angiogenesis, and ELISA was conducted to detect the levels of inflammatory cytokines. Dual-luciferase reporter, RIP, and RNA pull-down experiments were used to validate the relationships among XIST, miR-101-3p, and VEGFA. Results: XIST and VEGFA were upregulated and miR-101-3p was downregulated in serum from patients with DR. XIST knockdown inhibited proliferation, migration, vessel tube formation, and inflammatory responsein HG-treated HRMECs, whereas the above effects were nullified by miR-101-3p inhibition or VEGFA overexpression. miR-101-3p could bind to XIST and VEGFA. XIST promoted DR development in rats by regulating the miR-101-3p/VEGFA axis. Conclusion: LncRNA XIST promotes VEGFA expression by downregulating miR-101-3p, thereby stimulating angiogenesis and inflammatory response in DR.

PITPNA-AS1 Inhibits Cell Proliferation and Migration in Ovarian Cancer by Regulating the MIR-223-3p/RHOB Axis.

Background: Ovarian cancer is a fatal gynecologic malignancy. Long non-coding RNA (lncRNA) has been verified to serve as key regulator in ovarian cancer tumorigenesis. Objective: The aim of the study was to study the functions and mechanism of lncRNA PITPNA-AS1 in ovarian cancer cellular process. Methods: Clinical ovarian cancer samples were collected and stored at an academic medical center. Cellular fractionation assays and fluorescence in situ hybridization were conducted to locate PITPNA-AS1 in OC cells. TUNEL staining, colony-forming assays, and Transwell assays were performed for evaluating cell apoptosis as well as proliferative and migratory abilities. Western blot was conducted for quantifying protein levels of epithelialmesenchymal transition markers. The binding relation between genes was verified by RNA pulldown, RNA immunoprecipitation, and luciferase reporter assays. Gene expression levels in ovarian cancer tissues and cells were subjected to RT-qPCR. Results: PITPNA-AS1 level was downregulated in ovarian cancer samples and cells. PITPNA-AS1 overexpression contributed to the accelerated ovarian cancer cell apoptosis and inhibited cell migration, proliferation, and epithelial-mesenchymal transition process. In addition, PITPNA-AS1 interacted with miR-223-3p to regulate RHOB. RHOB knockdown partially counteracted the repressive impact of PITPNA-AS1 on ovarian cancer cell activities. Conclusion: PITPNA-AS1 inhibited ovarian cancer cellular behaviors by targeting miR-223-3p and regulating RHOB.

IDH1 mutation produces R-2-hydroxyglutarate (R-2HG) and induces mir-182-5p expression to regulate cell cycle and tumor formation in glioma.

BACKGROUND: Mutations in isocitrate dehydrogenase 1 and 2 (IDH1 and IDH2), are present in most gliomas. IDH1 mutation is an important prognostic marker in glioma. However, its regulatory mechanism in glioma remains incompletely understood. RESULTS: miR-182-5p expression was increased within IDH1-mutant glioma specimens according to TCGA, CGGA, and online dataset GSE119740, as well as collected clinical samples. (R)-2-hydroxyglutarate ((R)-2HG) treatment up-regulated the expression of miR-182-5p, enhanced glioma cell proliferation, and suppressed apoptosis; miR-182-5p inhibition partially eliminated the oncogenic effects of R-2HG upon glioma cells. By direct binding to Cyclin Dependent Kinase Inhibitor 2 C (CDKN2C) 3'UTR, miR-182-5p inhibited CDKN2C expression. Regarding cellular functions, CDKN2C knockdown promoted R-2HG-treated glioma cell viability, suppressed apoptosis, and relieved cell cycle arrest. Furthermore, CDKN2C knockdown partially attenuated the effects of miR-182-5p inhibition on cell phenotypes. Moreover, CDKN2C knockdown exerted opposite effects on cell cycle check point and apoptosis markers to those of miR-182-5p inhibition; also, CDKN2C knockdown partially attenuated the functions of miR-182-5p inhibition in cell cycle check point and apoptosis markers. The engineered CS-NPs (antagomir-182-5p) effectively encapsulated and delivered antagomir-182-5p, enhancing anti-tumor efficacy in vivo, indicating the therapeutic potential of CS-NPs(antagomir-182-5p) in targeting the miR-182-5p/CDKN2C axis against R-2HG-driven oncogenesis in mice models. CONCLUSIONS: These insights highlight the potential of CS-NPs(antagomir-182-5p) to target the miR-182-5p/CDKN2C axis, offering a promising therapeutic avenue against R-2HG's oncogenic influence to glioma.