Bio-Prospecting of Crude Leaf Extracts from Thirteen Plants of Brazilian Cerrado Biome on Human Glioma Cell Lines.

(1) Background: Malignant gliomas are aggressive tumors characterized by fast cellular growth and highly invasive properties. Despite all biological and clinical advances in therapy, the standard treatment remains essentially palliative. Therefore, searching for alternative therapies that minimize adverse symptoms and improve glioblastoma patients' outcomes is imperative. Natural products represent an essential source in the discovery of such new drugs. Plants from the cerrado biome have been receiving increased attention due to the presence of secondary metabolites with significant therapeutic potential. (2) Aim: This study provides data on the cytotoxic potential of 13 leaf extracts obtained from plants of 5 families (Anacardiaceae, Annonaceae, Fabaceae, Melastomataceae e Siparunaceae) found in the Brazilian cerrado biome on a panel of 5 glioma cell lines and one normal astrocyte. (3) Methods: The effect of crude extracts on cell viability was evaluated by MTS assay. Mass spectrometry (ESI FT-ICR MS) was performed to identify the secondary metabolites classes presented in the crude extracts and partitions. (4) Results: Our results revealed the cytotoxic potential of Melastomataceae species Miconia cuspidata, Miconia albicans, and Miconia chamissois. Additionally, comparing the four partitions obtained from M. chamissois crude extract indicates that the chloroform partition had the greatest cytotoxic activity against the glioma cell lines. The partitions also showed a mean IC50 close to chemotherapy, temozolomide; nevertheless, lower toxicity against normal astrocytes. Analysis of secondary metabolites classes presented in these crude extracts and partitions indicates the presence of phenolic compounds. (5) Conclusions: These findings highlight M. chamissois chloroform partition as a promising component and may guide the search for the development of additional new anticancer therapies.

Transcript Expression Profiles and MicroRNA Regulation Indicate an Upregulation of Processes Linked to Oxidative Stress, DNA Repair, Cell Death, and Inflammation in Type 1 Diabetes Mellitus Patients.

Type 1 diabetes (T1D) arises from autoimmune-mediated destruction of insulin-producing ß-cells leading to impaired insulin secretion and hyperglycemia. T1D is accompanied by DNA damage, oxidative stress, and inflammation, although there is still scarce information about the oxidative stress response and DNA repair in T1D pathogenesis. We used the microarray method to assess mRNA expression profiles in peripheral blood mononuclear cells (PBMCs) of 19 T1D patients compared to 11 controls and identify mRNA targets of microRNAs that were previously reported for T1D patients. We found 277 differentially expressed genes (220 upregulated and 57 downregulated) in T1D patients compared to controls. Analysis by gene sets (GSA and GSEA) showed an upregulation of processes linked to ROS generation, oxidative stress, inflammation, cell death, ER stress, and DNA repair in T1D patients. Besides, genes related to oxidative stress responses and DNA repair (PTGS2, ATF3, FOSB, DUSP1, and TNFAIP3) were found to be targets of four microRNAs (hsa-miR-101, hsa-miR148a, hsa-miR-27b, and hsa-miR-424). The expression levels of these mRNAs and microRNAs were confirmed by qRT-PCR. Therefore, the present study on differential expression profiles indicates relevant biological functions related to oxidative stress response, DNA repair, inflammation, and apoptosis in PBMCs of T1D patients relative to controls. We also report new insights regarding microRNA-mRNA interactions, which may play important roles in the T1D pathogenesis.

Comprehensive Molecular Landscape of Cetuximab Resistance in Head and Neck Cancer Cell Lines.

Cetuximab is the sole anti-EGFR monoclonal antibody that is FDA approved to treat head and neck squamous cell carcinoma (HNSCC). However, no predictive biomarkers of cetuximab response are known for HNSCC. Herein, we address the molecular mechanisms underlying cetuximab resistance in an in vitro model. We established a cetuximab resistant model (FaDu), using increased cetuximab concentrations for more than eight months. The resistance and parental cells were evaluated for cell viability and functional assays. Protein expression was analyzed by Western blot and human cell surface panel by lyoplate. The mutational profile and copy number alterations (CNA) were analyzed using whole-exome sequencing (WES) and the NanoString platform. FaDu resistant clones exhibited at least two-fold higher IC50 compared to the parental cell line. WES showed relevant mutations in several cancer-related genes, and the comparative mRNA expression analysis showed 36 differentially expressed genes associated with EGFR tyrosine kinase inhibitors resistance, RAS, MAPK, and mTOR signaling. Importantly, we observed that overexpression of KRAS, RhoA, and CD44 was associated with cetuximab resistance. Protein analysis revealed EGFR phosphorylation inhibition and mTOR increase in resistant cells. Moreover, the resistant cell line demonstrated an aggressive phenotype with a significant increase in adhesion, the number of colonies, and migration rates. Overall, we identified several molecular alterations in the cetuximab resistant cell line that may constitute novel biomarkers of cetuximab response such as mTOR and RhoA overexpression. These findings indicate new strategies to overcome anti-EGFR resistance in HNSCC.

MicroRNA Biomarkers of High-Grade Cervical Intraepithelial Neoplasia in Liquid Biopsy.

New prevention strategies are needed to detect cervical intraepithelial neoplasia (CIN). The microRNA expression analysis has already been reported as molecular biomarkers in the early detection of cervical cancer (CC) through minimally invasive samples, such as liquid biopsy, obtained through collection using liquid-based cytology (LBC). In this study, we aimed to identify molecular signatures of microRNAs in cervical precursor lesions from LBC cervical and the molecular pathways potentially associated with the CC progression. We analyzed 31 LBC cervical samples from women who underwent colposcopy. These samples were divided into two groups: the first group was composed of samples without precursor lesions of CC, considering the control group, referred to as healthy female subjects (HFS; n = 11). The second group corresponded to women diagnosed with cervical interepithelial neoplasia grade 3 (CIN 3; n = 20). We performed microRNA and gene expression profiling using the nCounter® miRNA Expression Assays (NanoString Technology) and PanCancer Pathways (NanoString Technology), respectively. A microRNA target prediction was performed by mirDIP, and molecular pathway interaction was constructed using Cytoscape. Bidirectional in silico analyses and Pearson's correlation were performed for associated the relation between genes, and miRNAs differentially expressed related cervical cancer progression were performed. We found that the expression of nine microRNAs was significantly higher, two were downregulated (miR-381-3p and miR-4531), and seven miRNAs were upregulated (miR-205-5p, miR-130a-3p, miR-3136-3p, miR-128-2-5p, let-7f-5p, miR-202-3p, and miR-323a-5p) in CIN 3 (fold change ≥ 2 and p ≤ 0.05). The miRNA expression patterns were independent of hr-HPV infection. We identified four miRNAs (miR-205-5p, miR-130a-3p, miR-4531, and miR-381-3p) that could be used as biomarkers for CIN 3 in LBC samples through multiple logistic regression analyses. We found 16 genes differentially expressed between CIN 3 and HSF samples (fold change ≥ 2 and p ≤ 0.05). We found the correlation between miR-130a-3p and CCND1(R = -0.52; p = 0.0029), miR-205-5p and EGFR (R = 0.53; p = 0.0021), and miR-4531 and SMAD2 (R = -0.54; p = 0.0016). In addition, we demonstrated the most significant pathways of the targets associated with cervical cancer progression (FDR-corrected p < 0.001). This study demonstrated that miRNA biomarkers may distinguish healthy cervix and CIN 3 and regulate important molecular pathways of carcinogenesis.

Bioprospecting of Natural Compounds from Brazilian Cerrado Biome Plants in Human Cervical Cancer Cell Lines.

Cervical cancer is the third most common in Brazilian women. The chemotherapy used for the treatment of this disease can cause many side effects; then, to overcome this problem, new treatment options are necessary. Natural compounds represent one of the most promising sources for the development of new drugs. In this study, 13 different species of 6 families from the Brazilian Cerrado vegetation biome were screened against human cervical cancer cell lines (CCC). Some of these species were also evaluated in one normal keratinocyte cell line (HaCaT). The effect of crude extracts on cell viability was evaluated by a colorimetric method (MTS assay). Extracts from Annona crassiflora, Miconia albicans, Miconia chamissois, Stryphnodendron adstringens, Tapirira guianensis, Xylopia aromatica, and Achyrocline alata showed half-maximal inhibitory concentration (IC50) values < 30 µg/mL for at least one CCC. A. crassiflora and S. adstringens extracts were selective for CCC. Mass spectrometry (Electrospray Ionization Fourier Transform Ion Cyclotron Resonance Mass Spectrometer (ESI FT-ICR MS)) of A. crassiflora identified fatty acids and flavonols as secondary compounds. One of the A. crassiflora fractions, 7C24 (from chloroform partition), increased H2AX phosphorylation (suggesting DNA damage), PARP cleavage, and cell cycle arrest in CCC. Kaempferol-3-O-rhamnoside and oleic acid were bioactive molecules identified in 7C24 fraction. These findings emphasize the importance of investigating bioactive molecules from natural sources for developing new anti-cancer drugs.

Role of glioblastoma stem cells in cancer therapeutic resistance: a perspective on antineoplastic agents from natural sources and chemical derivatives.

Glioblastoma (GBM) is the highest-grade form of glioma, as well as one of the most aggressive types of cancer, exhibiting rapid cellular growth and highly invasive behavior. Despite significant advances in diagnosis and therapy in recent decades, the outcomes for high-grade gliomas (WHO grades III-IV) remain unfavorable, with a median overall survival time of 15-18 months. The concept of cancer stem cells (CSCs) has emerged and provided new insight into GBM resistance and management. CSCs can self-renew and initiate tumor growth and are also responsible for tumor cell heterogeneity and the induction of systemic immunosuppression. The idea that GBM resistance could be dependent on innate differences in the sensitivity of clonogenic glial stem cells (GSCs) to chemotherapeutic drugs/radiation prompted the scientific community to rethink the understanding of GBM growth and therapies directed at eliminating these cells or modulating their stemness. This review aims to describe major intrinsic and extrinsic mechanisms that mediate chemoradioresistant GSCs and therapies based on antineoplastic agents from natural sources, derivatives, and synthetics used alone or in synergistic combination with conventional treatment. We will also address ongoing clinical trials focused on these promising targets. Although the development of effective therapy for GBM remains a major challenge in molecular oncology, GSC knowledge can offer new directions for a promising future.

Integrated analysis of mRNA and miRNA profiles revealed the role of miR-193 and miR-210 as potential regulatory biomarkers in different molecular subtypes of breast cancer.

BACKGROUND: Breast cancer is the most frequently diagnosed malignancy among women. However, the role of microRNA (miRNA) expression in breast cancer progression is not fully understood. In this study we examined predictive interactions between differentially expressed miRNAs and mRNAs in breast cancer cell lines representative of the common molecular subtypes. Integrative bioinformatics analysis identified miR-193 and miR-210 as potential regulatory biomarkers of mRNA in breast cancer. Several recent studies have investigated these miRNAs in a broad range of tumors, but the mechanism of their involvement in cancer progression has not previously been investigated. METHODS: The miRNA-mRNA interactions in breast cancer cell lines were identified by parallel expression analysis and miRNA target prediction programs. The expression profiles of mRNA and miRNAs from luminal (MCF-7, MCF-7/AZ and T47D), HER2 (BT20 and SK-BR3) and triple negative subtypes (Hs578T e MDA-MB-231) could be clearly separated by unsupervised analysis using HB4A cell line as a control. Breast cancer miRNA data from TCGA patients were grouped according to molecular subtypes and then used to validate these findings. Expression of miR-193 and miR-210 was investigated by miRNA transient silencing assays using the MCF7, BT20 and MDA-MB-231 cell lines. Functional studies included, xCELLigence system, ApoTox-Glo triplex assay, flow cytometry and transwell inserts were performed to determine cell proliferation, cytotoxicity, apoptosis, migration and invasion, respectively. RESULTS: The most evident effects were associated with cell proliferation after miR-210 silencing in triple negative subtype cell line MDA-MB-231. Using in silico prediction algorithms, TNFRSF10 was identified as one of the potential regulated downstream targets for both miRNAs. The TNFRSF10C and TNFRSF10D mRNA expression inversely correlated with the expression levels of miR-193 and miR210 in breast cell lines and breast cancer patients, respectively. Other potential regulated genes whose expression also inversely correlated with both miRNAs were CCND1, a known mediator on invasion and metastasis, and the tumor suppressor gene RUNX3. CONCLUSIONS: In summary, our findings identify miR-193 and miR-210 as potential regulatory miRNA in different molecular subtypes of breast cancer and suggest that miR-210 may have a specific role in MDA-MB-231 proliferation. Our results highlight important new downstream regulated targets that may serve as promising therapeutic pathways for aggressive breast cancers.

Deregulated microRNAs Are Associated with Patient Survival and Predicted to Target Genes That Modulate Lung Cancer Signaling Pathways.

(1) Background: Although the advances in diagnostic and treatment strategies, lung cancer remains the leading cause of cancer-related deaths, worldwide, with survival rates as low as 16% in developed countries. Low survival rates are mainly due to late diagnosis and the lack of effective treatment. Therefore, the identification of novel, clinically useful biomarkers is still needed for patients with advanced disease stage and poor survival. Micro(mi)RNAs are non-coding RNAs and potent regulators of gene expression with a possible role as diagnostic, prognostic and predictive biomarkers in cancer. (2) Methods: We applied global miRNA expression profiling analysis using TaqMan® arrays in paired tumor and normal lung tissues (n = 38) from treatment-naïve patients with lung adenocarcinoma (AD; n = 23) and lung squamous cell carcinoma (SCC; n = 15). miRNA target genes were validated using The Cancer Genome Atlas (TCGA) lung AD (n = 561) and lung SCC (n = 523) RNA-Seq datasets. (3) Results: We identified 33 significantly deregulated miRNAs (fold change, FC ≥ 2.0 and p < 0.05) in tumors relative to normal lung tissues, regardless of tumor histology. Enrichment analysis confirmed that genes targeted by the 33 miRNAs are aberrantly expressed in lung AD and SCC, and modulate known pathways in lung cancer. Additionally, high expression of miR-25-3p was significantly associated (p < 0.05) with poor patient survival, when considering both tumor histologies. (4) Conclusions: miR-25-3p may be a potential prognostic biomarker in non-small cell lung cancer. Genes targeted by miRNAs regulate EGFR and TGFß signaling, among other known pathways relevant to lung tumorigenesis.

Expression of GNAS, TP53, and PTEN Improves the Patient Prognostication in Sonic Hedgehog (SHH) Medulloblastoma Subgroup.

Medulloblastoma (MB) is the most common malignant brain tumor in children. It is currently classified in four main molecular subgroups with different clinical outcomes: sonic hedgehog, wingless, group 3, and group 4 (MBSHH, MBWNT, MBGRP3, or MBGRP4). Presently, a 22-gene expression panel has been efficiently applied for molecular subgrouping using nCounter technology. In this study, formalin-fixed, paraffin-embedded samples from 164 Brazilian medulloblastomas were evaluated, applying the 22-gene panel, and subclassified into the low and high expression of nine key medulloblastoma-related genes. In addition, TP53 mutation status was assessed using TruSight Tumor 15 Panel, and its correlation with expression and prognostic impact was evaluated. Samples from 149 of 164 patients (90%) were classified into MBSHH (47.7%), MBWNT (16.1%), MBGRP3 (15.4%), and MBGRP4 (20.8%). GNAS presented the highest expression levels, with higher expression in MBSHH. TP53, MYCN, SOX2, and MET were also up-regulated in MBSHH, whereas PTEN was up-regulated in MBGRP4. GNAS, TP53, and PTEN low expression was associated with the unfavorable patient outcome only for MBSHH (P = 0.04, P = 0.01, and P = 0.02, respectively). TP53 mutations were detected in 28.57% of MBSHH cases and exhibited association with lower expression and worse clinical outcome, although not statistically significant. The 22-gene panel for molecular classification of medulloblastoma associated with the expression of GNAS, TP53, and PTEN improves the patient prognostication in MBSHH subgroup and can be easily incorporated in the 22-gene panel without any additional costs.

Identification of Cell-Free Circulating MicroRNAs for the Detection of Early Breast Cancer and Molecular Subtyping.

Early detection is crucial for achieving a reduction in breast cancer mortality. Analysis of circulating cell-free microRNAs present in the serum of cancer patients has emerged as a promising new noninvasive biomarker for early detection of tumors and for predicting their molecular classifications. The rationale for this study was to identify subtype-specific molecular profiles of cell-free microRNAs for early detection of breast cancer in serum. Fifty-four early-stage breast cancers with 27 age-matched controls were selected for circulating microRNAs evaluation in the serum. The 54 cases were molecularly classified (luminal A, luminal B, luminal B Her2 positive, Her-2, triple negative). NanoString platform was used for digital detection and quantitation of 800 tagged microRNA probes and comparing the overall differences in serum microRNA expression from breast cancer cases with controls. We identified the 42 most significant (P ≤ 0.05, 1.5-fold) differentially expressed circulating microRNAs in each molecular subtype for further study. Of these microRNAs, 19 were significantly differentially expressed in patients presenting with luminal A, eight in the luminal B, ten in luminal B HER 2 positive, and four in the HER2 enriched subtype. AUC is high with suitable sensitivity and specificity. For the triple negative subtype miR-25-3p had the best accuracy. Predictive analysis of the mRNA targets suggests they encode proteins involved in molecular pathways such as cell adhesion, migration, and proliferation. This study identified subtype-specific molecular profiles of cell-free microRNAs suitable for early detection of breast cancer selected by comparison to the microRNA profile in serum for female controls without apparent risk of breast cancer. This molecular profile should be validated using larger cohort studies to confirm the potential of these miRNA for future use as early detection biomarkers that could avoid unnecessary biopsy in patients with a suspicion of breast cancer.

Euphol, a tetracyclic triterpene, from Euphorbia tirucalli induces autophagy and sensitizes temozolomide cytotoxicity on glioblastoma cells.

Glioblastoma (GBM) is the most frequent and aggressive type of brain tumor. There are limited therapeutic options for GBM so that new and effective agents are urgently needed. Euphol is a tetracyclic triterpene alcohol, and it is the main constituent of the sap of the medicinal plant Euphorbia tirucalli. We previously identified anti-cancer activity in euphol based on the cytotoxicity screening of 73 human cancer cells. We now expand the toxicological screening of the inhibitory effect and bioactivity of euphol using two additional glioma primary cultures. Euphol exposure showed similar cytotoxicity against primary glioma cultures compared to commercial glioma cells. Euphol has concentration-dependent cytotoxic effects on cancer cell lines, with more than a five-fold difference in the IC50 values in some cell lines. Euphol treatment had a higher selective cytotoxicity index (0.64-3.36) than temozolomide (0.11-1.13) and reduced both proliferation and cell motility. However, no effect was found on cell cycle distribution, invasion and colony formation. Importantly, the expression of the autophagy-associated protein LC3-II and acidic vesicular organelle formation were markedly increased, with Bafilomycin A1 potentiating cytotoxicity. Finally, euphol also exhibited antitumoral and antiangiogenic activity in vivo, using the chicken chorioallantoic membrane assay, with synergistic temozolomide interactions in most cell lines. In conclusion, euphol exerted in vitro and in vivo cytotoxicity against glioma cells, through several cancer pathways, including the activation of autophagy-associated cell death. These findings provide experimental support for further development of euphol as a novel therapeutic agent for GBM, either alone or in combination chemotherapy.

Reproducibility of the NanoString 22-gene molecular subgroup assay for improved prognostic prediction of medulloblastoma.

Medulloblastoma is the most frequent malignant brain tumor in children. Four medulloblastoma molecular subgroups, MBSHH , MBWNT , MBGRP3 and MBGRP4 , have been identified by integrated high-throughput platforms. Recently, a 22-gene panel NanoString-based assay was developed for medulloblastoma molecular subgrouping, but the robustness of this assay has not been widely evaluated. Mutations in the gene for human telomerase reverse transcriptase (hTERT) have been found in medulloblastomas and are associated with distinct molecular subtypes. This study aimed to implement the 22-gene panel in a Brazilian context, and to associate the molecular profile with patients' clinical-pathological features. Formalin-fixed, paraffin-embedded (FFPE) medulloblastoma samples (n = 104) from three Brazilian centers were evaluated. Expression profiling of the 22-gene panel was performed by NanoString and a Canadian series (n = 240) was applied for training phase. hTERT mutations were analyzed by PCR followed by direct Sanger sequencing and the molecular profile was associated with patients' clinicopathological features. Overall, 65% of the patients were male, average age at diagnosis was 18 years and 7% of the patients presented metastasis at diagnosis. The molecular classification was attained in 100% of the cases, with the following frequencies: MBSHH (n = 51), MBWNT (n = 19), MBGRP4 (n = 19) and MBGRP3 (n = 15). The MBSHH and MBGRP3 subgroups were associated with older and younger patients, respectively. The MBGRP4 subgroup exhibited the lowest 5-year cancer-specific overall survival (OS), yet in the multivariate analysis, only metastasis at diagnosis and surgical resection were associated with OS. hTERT mutations were detected in 29% of the cases and were associated with older patients, increased hTERT expression and MBSHH subgroup. The 22-gene panel provides a reproducible assay for molecular subgrouping of medulloblastoma FFPE samples in a routine setting and is well-suited for future clinical trials.

Expression of tumor suppressors miR-195 and let-7a as potential biomarkers of invasive breast cancer.

OBJECTIVE: MicroRNAs (miRNAs) are small non-coding RNAs that regulate gene expression at the posttranscriptional level. Some miRNAs, including let-7a and miR-195, have been described as tumor suppressors. However, the roles of these microRNAs in breast cancer progression remain controversial. The aim of this study is to evaluate miR-195 and let-7a expression as potential biomarkers of invasive breast cancer. METHODS: In the present study, 200 individuals were separated into three groups: (i) 72 women constituting the control group who were selected according to rigorous and well-established criteria; (ii) 56 patients with benign breast tumors; and (iii) 72 patients with malignant breast cancers of different clinical stages. The miR-195 and let-7a expression levels in serum were evaluated by real-time PCR. The results were assessed alone and in combination, and the analysis included an estimation of sensitivity and specificity in ROC curves. RESULTS: Compared with the benign and control groups, both microRNAs were downregulated in the malignant breast cancer patient group. Compared with the malignant group, the combination of both biomarkers in the control and benign groups showed good sensitivity and specificity in the serum with AUCs of 0.75 and 0.72, respectively. The biomarker combination for the control group versus the malignant group exhibited a better sensitivity and specificity than for the benign group versus the malignant group. CONCLUSION: These findings support the evidence that the analysis of miR-195 and let-7a can be used as a non-invasive biomarker for breast cancer detection.

Expression of tumor suppressors miR-195 and let-7a as potential biomarkers of invasive breast cancer

OBJECTIVE: MicroRNAs (miRNAs) are small non-coding RNAs that regulate gene expression at the posttranscriptional level. Some miRNAs, including let-7a and miR-195, have been described as tumor suppressors. However, the roles of these microRNAs in breast cancer progression remain controversial. The aim of this study is to evaluate miR-195 and let-7a expression as potential biomarkers of invasive breast cancer. METHODS: In the present study, 200 individuals were separated into three groups: (i) 72 women constituting the control group who were selected according to rigorous and well-established criteria; (ii) 56 patients with benign breast tumors; and (iii) 72 patients with malignant breast cancers of different clinical stages. The miR-195 and let-7a expression levels in serum were evaluated by real-time PCR. The results were assessed alone and in combination, and the analysis included an estimation of sensitivity and specificity in ROC curves. RESULTS: Compared with the benign and control groups, both microRNAs were downregulated in the malignant breast cancer patient group. Compared with the malignant group, the combination of both biomarkers in the control and benign groups showed good sensitivity and specificity in the serum with AUCs of 0.75 and 0.72, respectively. The biomarker combination for the control group versus the malignant group exhibited a better sensitivity and specificity than for the benign group versus the malignant group. CONCLUSION: These findings support the evidence that the analysis of miR-195 and let-7a can be used as a non-invasive biomarker for breast cancer detection.

Overexpression of mir-183 and mir-494 promotes proliferation and migration in human breast cancer cell lines.

Breast cancer (BC) is a leading cause of cancer-associated mortality in females worldwide. MicroRNAs (miRNAs or miRs), a type of non-coding RNA, have been reported to be important in the regulation of BC onset and progression. Several studies have implicated the role of miR-183 and miR-494 in different types of cancer. However, the biological functions of these miRNAs in BC remain largely unknown. In the present study, the expression of both miRNAs was assessed in the MDA-MB-231 and MDA-MB-468 BC cell lines. It was hypothesized that miR-183 and miR-494 serve an important role in regulating the expression of key genes associated with the metastatic phenotype of BC cells. To further understand their role, the expression of these miRNAs was restored in selected BC cell lines. Functional assays revealed that overexpression of miR-183 or miR-494 modulated the proliferation and migration of MDA-MB-231 and MDA-MB-468 cells in vitro. Additionally, retinoblastoma 1 (RB1) was identified to be a downstream target of both miRNAs by in silico analysis. Western blotting revealed that upregulation of miR-183 was associated with downregulation of RB1 protein in MDA-MB-231 cells. In conclusion, the present results support the hypothesis that miR-183 and miR-494 serve a pivotal role in BC metastasis, and that miR-183 may act as an oncogene by targeting RB1 protein in MDA-MB-231 cells.

Detection of ALK fusion transcripts in FFPE lung cancer samples by NanoString technology.

BACKGROUND: ALK-rearranged lung cancers exhibit specific pathologic and clinical features and are responsive to anti-ALK therapies. Therefore, the detection of ALK-rearrangement is fundamental for personalized lung cancer therapy. Recently, new molecular techniques, such as NanoString nCounter, have been developed to detect ALK fusions with more accuracy and sensitivity. METHODS: In the present study, we intended to validate a NanoString nCounter ALK-fusion panel in routine biopsies of FFPE lung cancer patients. A total of 43 samples were analyzed, 13 ALK-positive and 30 ALK-negative, as previously detected by FISH and/or immunohistochemistry. RESULTS: The NanoString panel detected the presence of the EML4-ALK, KIF5B-ALK and TFG-ALK fusion variants. We observed that all the 13 ALK-positive cases exhibited genetic aberrations by the NanoString methodology. Namely, six cases (46.15%) presented EML-ALK variant 1, two (15.38%) presented EML-ALK variant 2, two (15.38%) presented EML-ALK variant 3a, and three (23.07%) exhibited no variant but presented unbalanced expression between 5'/3' exons, similar to other positive samples. Importantly, for all these analyses, the initial input of RNA was 100 ng, and some cases displayed poor RNA quality measurements. CONCLUSIONS: In this study, we reported the great utility of NanoString technology in the assessment of ALK fusions in routine lung biopsies of FFPE specimens.

Assessment of DNA damage and mRNA/miRNA transcriptional expression profiles in hyperglycemic versus non-hyperglycemic patients with type 2 diabetes mellitus.

The development of type 2 diabetes mellitus (T2D) is associated with a number of genetic and environmental factors. Hyperglycemia, a T2D hallmark, is related to several metabolic complications, comorbidities and increased DNA damage. However, the molecular alterations of a proper glucose control are still unclarified. In this study, we aimed to evaluate DNA damage (comet assay), as well as to compare the transcriptional expression (mRNA and miRNA analyzed by the microarray technique) displayed by peripheral blood mononuclear cells (PBMCs) from three distinct groups: hyperglycemic T2D patients (T2D-H, n=14), non-hyperglycemic T2D patients (T2D-N, n=15), and healthy non-diabetic individuals (n=16). The comet assay revealed significantly (p<0.05) higher levels of DNA damage in T2D-H group compared to both T2D-N and control groups, while a significant difference was not observed between the control and T2D-N groups. After bioinformatics analysis, the differentially expressed mRNAs were subjected to functional enrichment analysis (DAVID) and inflammatory response was among the enriched terms found when comparing T2D-N with controls and T2D-H with T2D-N. Concerning the gene set enrichment and gene set analyses, among the differentially expressed gene sets, three were of interest: regulation of DNA repair (T2D-H versus T2D-N), superoxide response (T2D-H versus control group), and response to endoplasmic reticulum stress (T2D-H versus control group). We also identified miRNAs related with T2D and hyperglycemia not yet associated with these conditions in the literature. Some of the differentially expressed mRNAs were among the predicted targets of the differentially expressed miRNAs. Our results showed the association of hyperglycemia with increased DNA damage and aberrant expression of miRNAs and genes related to several biological processes, such as inflammation, DNA repair, ROS production and antioxidant defense, highlighting the importance of proper glycemic control. Moreover, the transcriptional expression of miRNAs provided novel information for understanding the regulatory mechanisms involved in the T2D progression.

MicroRNA expression profiling and functional annotation analysis of their targets in patients with type 1 diabetes mellitus.

Type 1 diabetes mellitus (T1DM) results from an autoimmune attack against the insulin-producing pancreatic ß-cells, leading to elimination of insulin production. The exact cause of this disorder is still unclear. Although the differential expression of microRNAs (miRNAs), small non-coding RNAs that control gene expression in a post-transcriptional manner, has been identified in many diseases, including T1DM, only scarce information exists concerning miRNA expression profile in T1DM. Thus, we employed the microarray technology to examine the miRNA expression profiles displayed by peripheral blood mononuclear cells (PBMCs) from T1DM patients compared with healthy subjects. Total RNA extracted from PBMCs from 11 T1DM patients and nine healthy subjects was hybridized onto Agilent human miRNA microarray slides (V3), 8x15K, and expression data were analyzed on R statistical environment. After applying the rank products statistical test, the receiver-operating characteristic (ROC) curves were generated and the areas under the ROC curves (AUC) were calculated. To examine the functions of the differentially expressed (p-value<0.01, percentage of false-positives <0.05) miRNAs that passed the AUC cutoff value ≥ 0.90, the database miRWalk was used to predict their potential targets, which were afterwards submitted to the functional annotation tool provided by the Database for Annotation, Visualization, and Integrated Discovery (DAVID), version 6.7, using annotations from the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis. We found 57 probes, corresponding to 44 different miRNAs (35 up-regulated and 9 down-regulated), that were differentially expressed in T1DM and passed the AUC threshold of 0.90. The hierarchical clustering analysis indicated the discriminatory power of those miRNAs, since they were able to clearly distinguish T1DM patients from healthy individuals. Target prediction indicated that 47 candidate genes for T1DM are potentially regulated by the differentially expressed miRNAs. After performing functional annotation analysis of the predicted targets, we observed 22 and 12 annotated KEGG pathways for the induced and repressed miRNAs, respectively. Interestingly, many pathways were enriched for the targets of both up- and down-regulated miRNAs and the majority of those pathways have been previously associated with T1DM, including many cancer-related pathways. In conclusion, our study indicated miRNAs that may be potential biomarkers of T1DM as well as provided new insights into the molecular mechanisms involved in this disorder.

Identifying common and specific microRNAs expressed in peripheral blood mononuclear cell of type 1, type 2, and gestational diabetes mellitus patients.

BACKGROUND: Regardless the regulatory function of microRNAs (miRNA), their differential expression pattern has been used to define miRNA signatures and to disclose disease biomarkers. To address the question of whether patients presenting the different types of diabetes mellitus could be distinguished on the basis of their miRNA and mRNA expression profiling, we obtained peripheral blood mononuclear cell (PBMC) RNAs from 7 type 1 (T1D), 7 type 2 (T2D), and 6 gestational diabetes (GDM) patients, which were hybridized to Agilent miRNA and mRNA microarrays. Data quantification and quality control were obtained using the Feature Extraction software, and data distribution was normalized using quantile function implemented in the Aroma light package. Differentially expressed miRNAs/mRNAs were identified using Rank products, comparing T1DxGDM, T2DxGDM and T1DxT2D. Hierarchical clustering was performed using the average linkage criterion with Pearson uncentered distance as metrics. RESULTS: The use of the same microarrays platform permitted the identification of sets of shared or specific miRNAs/mRNA interaction for each type of diabetes. Nine miRNAs (hsa-miR-126, hsa-miR-1307, hsa-miR-142-3p, hsa-miR-142-5p, hsa-miR-144, hsa-miR-199a-5p, hsa-miR-27a, hsa-miR-29b, and hsa-miR-342-3p) were shared among T1D, T2D and GDM, and additional specific miRNAs were identified for T1D (20 miRNAs), T2D (14) and GDM (19) patients. ROC curves allowed the identification of specific and relevant (greater AUC values) miRNAs for each type of diabetes, including: i) hsa-miR-1274a, hsa-miR-1274b and hsa-let-7f for T1D; ii) hsa-miR-222, hsa-miR-30e and hsa-miR-140-3p for T2D, and iii) hsa-miR-181a and hsa-miR-1268 for GDM. Many of these miRNAs targeted mRNAs associated with diabetes pathogenesis. CONCLUSIONS: These results indicate that PBMC can be used as reporter cells to characterize the miRNA expression profiling disclosed by the different diabetes mellitus manifestations. Shared miRNAs may characterize diabetes as a metabolic and inflammatory disorder, whereas specific miRNAs may represent biological markers for each type of diabetes, deserving further attention.