Bioinformatics analysis of the potentially functional circRNA-miRNA-mRNA network in breast cancer.

Breast cancer (BC) is the most common cancer among women with high morbidity and mortality. Therefore, new research is still needed for biomarker detection. GSE101124 and GSE182471 datasets were obtained from the Gene Expression Omnibus (GEO) database to evaluate differentially expressed circular RNAs (circRNAs). The Cancer Genome Atlas (TCGA) and Molecular Taxonomy of Breast Cancer International Consortium (METABRIC) databases were used to identify the significantly dysregulated microRNAs (miRNAs) and genes considering the Prediction Analysis of Microarray classification (PAM50). The circRNA-miRNA-mRNA relationship was investigated using the Cancer-Specific CircRNA, miRDB, miRTarBase, and miRWalk databases. The circRNA-miRNA-mRNA regulatory network was annotated using Gene Ontology (GO) analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway database. The protein-protein interaction network was constructed by the STRING database and visualized by the Cytoscape tool. Then, raw miRNA data and genes were filtered using some selection criteria according to a specific expression level in PAM50 subgroups. A bottleneck method was utilized to obtain highly interacted hub genes using cytoHubba Cytoscape plugin. The Disease-Free Survival and Overall Survival analysis were performed for these hub genes, which are detected within the miRNA and circRNA axis in our study. We identified three circRNAs, three miRNAs, and eighteen candidate target genes that may play an important role in BC. In addition, it has been determined that these molecules can be useful in the classification of BC, especially in determining the basal-like breast cancer (BLBC) subtype. We conclude that hsa_circ_0000515/miR-486-5p/SDC1 axis may be an important biomarker candidate in distinguishing patients in the BLBC subgroup of BC.

Turmeric Inhibits MDA-MB-231 Cancer Cell Proliferation, Altering miR-638-5p and Its Potential Targets.

Objective: Recent research suggests curcumin extracted from the turmeric plant may inhibit the proliferation of cancer cells by controlling the expression of microRNAs (miRNAs). The effect of phenolic curcumin on miR-638-5p and potential target gene expressions in the triple negative breast cancer (TNBC) cell line MDA-MB-231 was investigated in this study. Materials and Methods: GSE154255 and GSE40525 datasets were downloaded and analyzed using GEO2R to identify dysregulated miRNAs in TNBC. To find differently expressed genes in breast cancer (BRCA), The Cancer Genome Atlas Program data was examined. Utilizing in silico tools, KEGG, GO, and other enrichment analyses were performed. The databases miRNet, miRTarBase v8.0, and TarBase v.8 were used for miRNA and mRNA matching. Real-time quantitative reverse transcription polymerase chain reaction was used to examine the levels of miRNA and its targets in miRNA mimic transfected/curcumin-treated MDA-MB-231 cultures and controls. The cell viability detection kit-8 method was used to assess cell viability, and the scratch assay was used to conduct migration assessment. Results: Bioinformatics analysis showed that miR-638-5p was significantly reduced in TNBC patients. Experimental results showed that miR-638-5p was upregulated in MDA-MB-231 treated with curcumin, while the potential target genes of miR-638-5p, CFL1, SIX4, MAZ, and CDH1 were downregulated. Mimic miR-638-5p transfection inhibited MDA-MB-231 cell proliferation and reduced migration and expression of CFL1, SIX4, and MAZ genes was decreased in mimic miR-638-5p transfected cells. Conclusion: These findings suggest that curcumin exerts its anticancer effects on MDA-MB-231 cells by modulating the expression of miR-638-5p and its possible target genes.

Functional characterization of a novel TP53RK mutation identified in a family with Galloway-Mowat syndrome.

Galloway-Mowat syndrome (GAMOS) is a very rare condition characterized by early-onset nephrotic syndrome and microcephaly with variable neurologic features. While considerable genetic heterogeneity of GAMOS has been identified, the majority of cases are caused by pathogenic variants in genes encoding the four components of the Kinase, endopeptidase, and other proteins of small size (KEOPS) complex, one of which is TP53RK. Here we describe a 3-year-old male with progressive microcephaly, neurodevelopmental deficits, and glomerular proteinuria. He was found to carry a novel homozygous TP53RK missense variant, c.163C>G (p.Arg55Gly), which was considered as potentially disease-causing. We generated a morpholino tp53rk knockdown model in Xenopus laevis showing that the depletion of endogenous Tp53rk caused abnormal eye and head development. This phenotype could be rescued by the expression of human wildtype TP53RK but not by the c.163C>G mutant nor by another previously described GAMOS-associated mutant c.125G>A (p.Gly42Asp). These findings support the pathogenic role of the novel TP53RK variant.

Cytogenetic and molecular characterization of a patient having infertility and mild intellectual disability with a very rare unstable ring chromosome 13.

INTRODUCTION: Ring chromosomes arise from breakage and fusion at distal regions of short and long arms of the chromosomes. The effect of the ring chromosome on the phenotype may vary widely depending on the amount of the deletion in the chromosomal areas and genes implicated in these regions. CASE PRESENTATION: We present a 35-year-old male patient with infertility and mild intellectual disability (MID) who has de novo ring 13 (r(13)) chromosomes. To determine chromosomal abnormality, we performed karyotype analysis, Y chromosome microdeletion analysis, FISH, and aCGH techniques. CONCLUSION: The patient's karyotype analysis result was mos46,XY,r(13)(p13q34)[75]/45,XY,-13[14]/46,XY,dic (13;13)[8]/47,XY,r(13), + r(13)[2]/46,XY,tetrac r(13;13;13;13)[1]. FISH analysis supported the findings of the cytogenetic analysis. Y microdeletion analysis showed that the AZF region was intact. On aCGH analysis, we detected a 1.5 megabase deletion at the end of chromosome 13, including the CHAMP1 gene. The loss of the CHAMP1 gene, in particular, may explain our patient's MID, and the other deleted genes at 13q34 may explain our patient's infertility.

The effect of Anzer honey on X-ray induced genotoxicity in human lymphocytes: An in vitro study.

Anzer honey is well known in Turkey and used for its medicinal properties, especially for pharyngitis, tonsillitis, ulcers and cancer. In this study, we investigated whether Anzer honey, which is shown to have antioxidant, anti-tumoral, and anti-inflammatory properties, has a protective effect against X-ray induced genotoxic damage by cytogenetic methods. Peripheral blood lymphocytes isolated from 20 healthy volunteers were divided into two groups and cultivated by conventional methods. Study group lymphocytes were treated with 10% diluted honey while those in the control group were not. Both groups were exposed to a high dose (2 Gy) X-ray at the 48th hour of culture. Conventional cytogenetic staining and Giemsa banding methods were applied to evaluate chromosomal breakage and ring formation. Micronucleus frequencies were determined by the cytokinesis-block micronucleus (CBMN) assay. Paired sample t test was used to compare groups. Anzer honey, which was analyzed melissopalynologically, was used. Micronucleus frequency was significantly decreased in the study group (CI = 348.75 ± 31, median 326, min. 98, max. 704) compared to the control group (CI = 489.10 ± 27, median 500, min. 216, max. 645) (p = .001). Chromosomal breakage was also significantly decreased in the study group (CI = 118.70 ± 16, median 109, min. 12, max. 316) compared to the control group (CI = 233.60 ± 25, median 225, min. 65, max. 492) (p < .0001). This is the first study indicating that genotoxic damage in the peripheral blood lymphocytes of healthy volunteers induced by X-radiation may be prevented or alleviated by adding Anzer honey in vitro. These results encourage further research about the protective effects of honey. RESEARCH HIGHLIGHTS: Anzer honey has a genoprotective effect against radiation-induced genotoxicity, probably by preventing oxidation damage.

CASC11 promotes aggressiveness of prostate cancer cells through miR-145/IGF1R axis.

BACKGROUND: Prostate cancer (PCa) is the most common malignancy diagnosed among men after lung cancer in developed countries. Investigation of the underlying molecular mechanisms of PCa is urgently needed in order to develop better therapeutic strategies and to reveal more effective therapeutic targets. In this study, we aimed at exploring the potential functions of CASC11 in association with miR-145 and IGF1R during the malignant progression of PCa cells. METHODS: We initially investigated the oncogenic potential of noncoding members of CASC gene family and analyzed the effects of CASC11 overexpression on proliferation, migration, and colony formation ability of DU145, LNCaP, and PC3 PCa cells. We, then, exprlored the association of CASC11, miR-145, and IGF1R expression and their impacts on PI3K/AKT/mTOR signaling pathway in in vitro models. RESULTS: In silico analysis revealed that of the CASC family only CASC11 showed consistent results considering its differential expression as well as its association with the overall survival of patients. We demonstrated that ectopic overexpression of CASC11 significantly increased the proliferation, colony formation, and migration capacity in all three cell lines. CASC11 overexpression caused suppression of miR-145 and overexpression of IGF1R, leading to activation of PI3K/AKT/mTOR signaling pathway. CONCLUSION: In summary, we found that CASC11 is upregulated in PCa cells and clinical tumor samples in comparison to corresponding controls and revealed that ectopic CASC11 overexpression promotes cellular phenotypes associated with PCa progression through CASC11/miR-145/IGF1R axis.

Case Report: a novel chromosomal insertion, 46, XY, inv ins(18;2)(q11.2;q13q22), in a patient with infertility and mild intellectual disability.

Infertility is an important health problem affecting 15% of couples worldwide. Intellectual disability (ID) is characterized with significant impairment of intellectual function, adaptive daily life skills and social skills. Insertion is a rare chromosomal rearrangement causing infertility and ID. Here, we report a 39-year-old man presenting with primary infertility and mild ID. The patient's spermiogram was consistent with azoospermia. Conventional cytogenetic analysis showed a novel inversion/insertion type of chromosomal aberration involving chromosomes 18 and 2: 46, XY, inv ins(18;2)(q11.2;q13q22). We carried out the array comparative genomic hybridization analysis to confirm the cytogenetic findings. Y micro-deletion analysis demonstrated that the AZF region as intact. We suggest that the novel insertion found in this case [46, XY, inv ins(18;2)(q11.2;q13q22)] may have caused infertility and mild ID in our patient. To the best of our knowledge, this chromosomal insertion has not previously been reported.

Revealing the functions of novel mutations in RAB3GAP1 in Martsolf and Warburg micro syndromes.

PURPOSE: Martsolf (MS) and Warburg micro syndromes (WARBM) are rare autosomal recessive inherited allelic disorders, which share similar clinical features including microcephaly, intellectual disability, brain malformations, ocular abnormalities, and spasticity. Here, we revealed the functions of novel mutations in RAB3GAP1 in a Turkish female patient with MS and two siblings with WARBM. We also present a review of MS patients as well as all reported RAB3GAP1 pathogenic mutations in the literature. METHODS: We present a female with MS phenotype and two siblings with WARBM having more severe phenotypes. We utilized whole-exome sequencing to identify the molecular basis of these syndromes and confirmed suspected variants by Sanger sequencing. Quantitative (q) RT-PCR analysis was carried out to reveal the functions of novel splice site mutation detected in MS patient. RESULTS: We found a novel homozygous c.2607-1G>C splice site mutation in intron 22 of RAB3GAP1 in MS patient and a novel homozygous c.2187_2188delinsCT, p.(Met729_Lys730delinsIleTer) mutation in exon 19 of RAB3GAP1 in the WARBM patients. We showed exon skipping in MS patient by Sanger sequencing and gel electrophoresis. qRT-PCR analysis demonstrated the reduced expression of RAB3GAP1 in the patient with the c.2607-1G>C splice site mutation compared to a healthy control individual. CONCLUSION: Here, we have studied two novel RAB3GAP1 mutations in two different phenotypes; a MS associated novel splice site mutation, and a WARBM1 associated novel deletion-insertion mutation. Our findings suggest that this splice site mutation is responsible for milder phenotype and the deletion-insertion mutation presented here is associated with severe phenotype.

MicroRNAs as prognostic markers in prostate cancer.

BACKGROUND: Prostate cancer (PCa) is the most commonly diagnosed malignancy in men who are especially over the age of 50 years in the western countries. Currently used therapeutic modalities mostly fail to give positive clinical outcomes and nearly 30% of the PCa patients eventually develop clinical recurrence. Therefore, understanding the underlying mechanisms of PCa progression is of paramount importance to help determining the course of disease. In this study, we aimed at profiling the differentially expressed microRNAs in recurrent PCa samples. METHODS: We profiled the microRNA expression of 20 recurrent and 20 non-recurrent PCa patients with microRNA microarray, and validated the differential expression of significantly deregulated microRNAs in 40 recurrent and 39 non-recurrent PCa specimens using quantitative reverse-transcription PCR (qRT-PCR). Data were statistically analyzed using two-sided Student's t-test, Pearson Correlation test, Receiver operating characteristic (ROC) analysis. RESULTS: Our results demonstrated that a total of 682 probes were significantly deregulated in recurrent versus non-recurrent PCa specimen comparison. Among those, we confirmed the significant downregulation of miR-424 and upregulation of miR-572 with further qRT-PCR analysis in a larger sample set. Further ROC analysis showed that these microRNAs have enough power to distinguish recurrent specimens from non-recurrent ones on their own. CONCLUSIONS: Here, we report that differential expression of miR-424 and miR-572 in recurrent PCa specimens can serve as novel biomarkers for prediction of PCa progression.

Identification of microRNA profile specific to cancer stem-like cells directly isolated from human larynx cancer specimens.

BACKGROUND: Emerging evidences proposed that microRNAs are associated with regulation of distinct physio-pathological processes including development of normal stem cells and carcinogenesis. In this study we aimed to investigate microRNA profile of cancer stem-like cells (CSLCs) isolated form freshly resected larynx cancer (LCa) tissue samples. METHODS: CD133 positive (CD133+) stem-like cells were isolated from freshly resected LCa tumor specimens. MicroRNA profile of 12 pair of CD133+ and CD133- cells was determined using microRNA microarray and differential expressions of selvected microRNAs were validated by quantitative real time PCR (qRT-PCR). RESULTS: MicroRNA profiling of CD133+ and CD133- LCa samples with microarray revealed that miR-26b, miR-203, miR-200c, and miR-363-3p were significantly downregulated and miR-1825 was upregulated in CD133+ larynx CSLCs. qRT-PCR analysis in a total of 25 CD133+/CD133- sample pairs confirmed the altered expressions of these five microRNAs. Expressions of miR-26b, miR-200c, and miR-203 were significantly correlated with miR-363-3p, miR-203, and miR-363-3p expressions, respectively. Furthermore, in silico analysis revealed that these microRNAs target both cancer and stem-cell associated signaling pathways. CONCLUSIONS: Our results showed that certain microRNAs in CD133+ cells could be used as cancer stem cell markers. Based on these results, we propose that this panel of microRNAs might carry crucial roles in LCa pathogenesis through regulating stem cell properties of tumor cells.

The role of miR-145 in stem cell characteristics of human laryngeal squamous cell carcinoma Hep-2 cells.

The cancer stem-like cells (CSLCs) are tumorigenic cells promoting initiation, progression, and spread of the tumor. Accumulating evidences suggested the presence of CSLCs in distinct tumors including laryngeal squamous cell carcinoma (LSCC). MicroRNAs have been proposed as significant regulators of carcinogenesis, and several of them have been demonstrated to have direct roles in survival of CSLCs. In this study, we aimed to explore the role of miR-145, which is downregulated in LSCC, on cancer stem cell potency of laryngeal cancer cells. We initially showed the downregulation of miR-145 expression in tumor tissue samples and in CD133-enriched CSLCs. Quantitative reverse-transcription PCR (qRT-PCR) analysis of miR-145-transfected Hep-2 cells demonstrated the inhibitory role of miR-145 on stem cell markers like SOX2, OCT4, KLF4, and ABCG2. We, then, investigated the stem cell features of miR-145-overexpressing Hep-2 cells by sphere formation assay, single-cell cloning assay, and aldehyde dehydrogenase (ALDH) assay, which all demonstrated the inhibition of stem cell potency upon miR-145 overexpression. Further qRT-PCR analysis demonstrated altered expression of epithelial to mesenchymal transition markers in miR-145-overexpressing Hep-2 cells. In conclusion, we demonstrated the regulatory role of miR-145 in stem cell characteristics of Hep-2 cells. Based on these results, we propose that miR-145 might carry crucial roles in LSCC tumorigenesis, prognosis, metastasis, chemoresistance, and recurrence through regulating stem cell properties of tumor cells.

Role of miR-145 in human laryngeal squamous cell carcinoma.

BACKGROUND: Laryngeal squamous cell carcinoma (SCC), being an aggressive malignancy, is one of the most commonly diagnosed malignant types of head and neck SCC worldwide. Incidences of laryngeal SCC have been reported to increase recently. In this study, we aimed to explore the biological effects of miR-145 on laryngeal cancer cells. METHODS: The relative miR-145 level in laryngeal SCC tumor tissues and normal samples was investigated. Then, Hep-2 cells were utilized for functional analysis of miR-145. The proliferation abilities of transfected cells were measured using MTS assay. Scratch assay and single colony migration assay were performed to observe the alterations in migration behavior of transfected cells. Caspase assay and cell cycle analysis were used to investigate the underlying reasons of proliferative inhibition in cells in which miR-145 is overexpressed. Moreover, expression of SOX2 was analyzed using quantitative reverse transcription polymerase chain reaction (qRT-PCR) and Western blot analysis in Hep-2 cells upon miR-145 transfection and its expression was evaluated in tumor and normal tissue sample of the larynx. RESULTS: The miR-145 expression in laryngeal SCC tumor samples has been shown to be downregulated. The miR-145 overexpression caused inhibition of proliferation and migration in Hep-2 cells through induction of apoptosis and cell cycle arrest. The SOX2 level was demonstrated to be overexpressed in tumor samples and its expression was significantly decreased in miR-145 overexpressed Hep-2 cells. CONCLUSION: We have demonstrated the deregulation of miR-145 and SOX2 in laryngeal SCC. Based on these results, we propose that miR-145, as an important regulator of SOX2, carries crucial roles in laryngeal SCC tumorigenesis.

miR-1 and miR-133b are differentially expressed in patients with recurrent prostate cancer.

Prostate cancer (PCa) is currently the most frequently diagnosed malignancy in the western countries. It is more prevalent in older men with 75% of the incident cases above 65 years old. After radical prostatectomy, approximately 30% of men develop clinical recurrence with elevated serum prostate-specific antigen levels. Therefore, it is important to unravel the molecular mechanisms underlying PCa progression to develop novel diagnostic/therapeutic approaches. In this study, it is aimed to compare the microRNA (miRNA) profile of recurrent and non-recurrent prostate tumor tissues to explore the possible involvement of miRNAs in PCa progression. Total RNA from 41 recurrent and 41 non-recurrent PCa tissue samples were used to investigate the miRNA signature in PCa specimens. First of all, 20 recurrent and 20 non-recurrent PCa samples were profiled using miRNA microarray chips. Of the differentially expressed miRNAs, miR-1, miR-133b and miR-145* were selected for further validation with qRT-PCR in a different set of 21 recurrent and 21 non-recurrent PCa samples. Data were statistically analyzed using two-sided Student's t-test, Pearson Correlation test, Receiver operating characteristic analysis. Our results demonstrated that miR-1 and mir-133b have been significantly downregulated in recurrent PCa specimens in comparison to non-recurrent PCa samples and have sufficient power to distinguish recurrent specimens from non-recurrent ones on their own. Here, we report that the relative expression of miR-1 and mir-133b have been significantly reduced in recurrent PCa specimens in comparison to non-recurrent PCa samples, which can serve as novel biomarkers for prediction of PCa progression.

Characterization of stem-like cells directly isolated from freshly resected laryngeal squamous cell carcinoma specimens.

Larynx cancer (LCa) is an aggressive malignancy, which is the second most common malignant neoplasm of head and neck squamous cell carcinoma. Its incidences have been reported to increase and therapeutic options mostly fail to give positive clinical response especially for the advanced LCa cases. In this study we aimed to isolate stem-like cells from freshly resected LCa tumor specimens and characterize them by quantitative real time PCR (qRT-PCR) for expression of cancer stem cell markers including SOX2, OCT4, KLF4, ABCG2, CXCR4 and CD44. Our results showed that CD133(high) cells directly isolated from freshly resected tumor specimens exhibit elevated levels of SOX2, OCT4 and KLF4, and have increased expression levels of ABCG2 and CXCR4, which were associated with resistance of tumors to regular chemotherapeutic reagents. In conclusion, this study offers a useful approach utilizing CD133 to isolate stem cells directly from fresh tissues, which gives the opportunity to develop novel therapeutic tools specifically targeting these cells through their further characterization.