RESUMO
BACKGROUND: Carcinoma of the breast, a prevailing factor in female mortality worldwide, involves dysregulation of lncRNAs and microRNAs. AIM: The main goal of this research was to predict and experimentally examine the LINC01405 expression status in breast cancer subtypes, along with investigation of its interaction with miR-29b and miR-497-5p that results in regulating PI3-Kinase, WNT, and TGF-beta signaling pathways. METHODS AND RESULTS: We performed a meta-analysis of five GEO datasets, encompassing microarray and RNA-seq data, to identify differentially expressed genes. The Cancer Genome Atlas transcriptome dataset was also analyzed to determine essential gene modules, associated with different stages of breast cancer by weighted gene co-expression networks. In addition, networks of drug-gene interactions were constructed to explore potential treatment options. LINC01405 as a microRNA sponge was chosen and examined. furthermore, downstream target genes were discovered. Experimental validation consisted of plasmid constructs used in cell culture experiments, RT-qPCR for expression analysis, and cell cycle assays. Our bioinformatics findings showed higher LINC01405 expression in Basal-like triple-negative breast carcinoma. In contrast, lower expression in Luminal samples was observed compared with normal samples, which was consistently observed in both breast cancer tissues and cell lines. LINC01405 expression level was correlated with miR-29b and miR-497 levels. The MDA-MB-231 cell line demonstrated higher LINC01405 expression and lower miR-29b and miR-497 expression levels. However, SKBR3 and MCF7 cells had lower LINC01405 expression and higher miR-29b and miR-497 levels, suggesting a regulatory role for LINC01405 as a competing endogenous RNA. This was experimentally confirmed when LINC01405 was overexpressed in SKBR3 cells, and the common target genes of miR-29b and miR-497 were upregulated. Additionally, LINC01405 upregulation led to the increased cell populations, proliferation, and upregulation of critical cancer-related genes, including AKT1, AKT3, mTOR, WNT3A, SMAD3, CYCLIN D1, CYCLIN D2, BCL2, and GSK3B. CONCLUSION: We revealed the differential expression of LINC01405 in several types of breast cancer and its role in regulating signaling pathways, potentially via scavenging miRNAs. These findings clarified the role of LINC01405 in breast cancer development and identified potential therapeutic targets.
Assuntos
Neoplasias da Mama , MicroRNAs , RNA Longo não Codificante , Feminino , Humanos , Neoplasias da Mama/genética , Linhagem Celular Tumoral , MicroRNAs/genética , MicroRNAs/metabolismo , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , Transdução de Sinais , RNA Longo não Codificante/metabolismoRESUMO
HER2 signaling upregulation is a hallmark of breast cancer which is the second cause of cancer-related death in women. Here, we were looking for the candidate microRNAs which is capable of regulating the HER2 receptor and the genes of its downstream. To this aim, preliminary bioinformatics analysis introduced hsa-miR-1254 (miR-1254) as a potential common regulator of HER2, HER3, PIK3R2, and AKT1 genes. Then, reverse-transcription quantitative polymerase chain reaction (RT-qPCR) analysis indicated a lower expression level of miR-1254 in breast cancer specimens, compared to their normal pairs. Furthermore, overexpression of miR-1254 resulted in HER2, HER3, PIK3R2, and AKT1 genes downregulation, detected by RT-qPCR and confirmed by western blot analysis and enzyme-linked immunosorbent assay test against AKT1, BAX, FADD, and HER2 protein levels in SKBR3 cells. Dual-luciferase assay also supported direct interaction of miR-1254 with MREs within 3' untranslated region sequences of HER2, HER3, PIK3R2, and AKT1 target genes. Overexpression of miR-1254 in SKBR3 cells was followed by increased BAX/BCL2 expression ratio, detected by RT-qPCR, and increased proportion of G1 cell population, detected by flow cytometry. Corroborated by cell cycle analysis, MTT, Annexin V-FITC, and Live-Dead cell staining assays, overexpression of miR-1254 in MDA-MB-231 cells showed opposing results following the overexpression of miR-1254. Taken together, results indicated that miR-1254 acts as cell-type-specific tumor suppressor that targets HER2, HER3, PIK3R2, and AKT1 transcripts. These results suggest miR-1254 as a potential therapeutic candidate for breast cancer subtypes.
Assuntos
Neoplasias da Mama , MicroRNAs , Regiões 3' não Traduzidas , Neoplasias da Mama/metabolismo , Feminino , Humanos , MicroRNAs/genética , Receptor ErbB-2 , Transdução de Sinais/genética , Proteína X Associada a bcl-2/genéticaRESUMO
PI3K/AKT signaling has a crucial role in breast cancer incidence and finding the miRNAs that regulate this pathway enhances our understanding of breast cancer regulation. Firstly, our bioinformatics analysis suggested miR-1226-3p as a bona fide regulator of HER2, PIK3R2, and AKT1 putative target genes. Secondly, RT-qPCR, ELISA test and western blotting showed that overexpression of miR-1226 was followed by reduced expression of HER2, PIK3R2, and AKT1 putative targets genes in SKBR3 cells. Third, dual luciferase assay verified direct interaction of miR-1226-3p with 3'UTR sequences of these target genes. Then, overexpression of miR-1226 in SKBR3 cells brought about increased population of sub-G1 and decreased populations of G1 cells, measured by flow cytometry. This was consistent to the reduction of p-AKT protein and increased BAX protein levels, detected by western analysis and consistent to decreased CCND1 genes expression, detected by RT-qPCR. The reduced survival and increased apoptosis rate of these cells was also verified through MTT, Annexin V-FITC and Live-Dead cell staining assays. Our results suggest that miR-1226-3p is a tumor suppressor in SKBR3 cells. However, following the overexpression of miR-1226 in MDA-MB-231 cells, Bax/Bcl2 ratio and CCND1 genes expression levels were not significantly changed, sub-G1 and G1 cell cycle population were reduced while, S and G2/M cell populations were increased, consistent to the results acquired from the apoptosis and staining assays. Finally, TCGA data analysis and RT-qPCR against 20 pairs of Normal/Tumor breast tissues indicated that miR-1226-3p has been downregulated in breast cancer. Overall, the present study gathered shreds of evidence that suggest miR-1226-3p as a tumor suppressor that exerts its inhibitory effect on SKBR3 cells through targeting of HER2, PIK3R2, and AKT1 genes and downregulates PI3K/AKT pathway.
Assuntos
MicroRNAs/genética , Fosfatidilinositol 3-Quinases/química , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Receptor ErbB-2/antagonistas & inibidores , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Regulação para Baixo , Feminino , Regulação Neoplásica da Expressão Gênica , Genes Supressores de Tumor , Humanos , MicroRNAs/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptor ErbB-2/metabolismo , Transdução de SinaisRESUMO
PURPOSE: Dysregulation of HER2 signaling pathway in breast cancer is well documented. Our bioinformatics analysis predicted hsa-miR-512-3p (miR-512-3p) as a bona fide regulator of HER2 as well as HER3, PIK3R2, and AKT1 genes. Then, we intended to examine the effect of miR-512-3p on the predicted target genes that are involved in HER2 signaling pathway. METHODS AND RESULTS: RT-qPCR results indicated lower expression of miR-512-3p in breast cancer specimens, compared to their normal pairs. Overexpression of miR-512-3p resulted in HER2, HER3, PIK3R2, and AKT1 gene downregulation, detected by RT-qPCR and the result was confirmed by western analysis and ELIZA test against p-AKT, BAX, FADD, and HER2 proteins in SKBR3 cells, respectively. Then, dual-luciferase assay supported the direct interaction of miR-512-3p with 3'UTR sequences of HER2, HER3, PIK3R2, and AKT1 target genes. When miR-512-3p was overexpressed, BAX/BCL2 expression ratio and proportion of sub-G1 cell population were increased in transfected SKBR3 cells, detected by RT-qPCR and flow cytometry, respectively. These results were consistent with the decreased viability of transfected cells, documented by MTT assay. In addition, results were consistent with the upregulation of BAX, BAK, BOK, PTEN, P53, and P21 genes and downregulation of CCND1 gene in SKBR3 cells. Although the overexpression of miR-512 resulted in cell cycle arrest at Sub-G1 stage in MDA-MB-231 cells, this effect seemed independent of targeting HER2, HER3, PIK3R2, and AKT1 target genes. CONCLUSION: Overall, results indicated that miR-512-3p acts as a cell-type-specific tumor suppressor, through targeting HER2, HER3, PIK3R2, and AKT1 transcripts. These results suggest miR-512-3p as a potential candidate marker for breast cancer diagnosis.
Assuntos
Neoplasias da Mama , MicroRNAs , Neoplasias da Mama/genética , Feminino , Humanos , MicroRNAs/genética , Transdução de Sinais , Regulação para CimaRESUMO
OBJECTIVE: Wharton's jelly (WJ) is the gelatinous connective tissue from the umbilical cord. It is composed of mesenchymal stem cells, collagen fibers, and proteoglycans. The stem cells in WJ have properties that are interesting for research. For example, they are simple to harvest by noninvasive methods, provide large numbers of cells without risk to the donor, the stem cell population may be expanded in vitro, cryogenically stored, thawed, genetically manipulated, and differentiated in vitro. In our study, we investigated the effect of human cerebrospinal fluid (CSF) on neural differentiation of human WJ stem cells. MATERIAL & METHODS: The cells in passage 2 were induced into neural differentiation with different concentrations of human cerebrospinal fluid. Differentiation along with neural lineage was documented by expression of three neural markers: Nestin, Microtubule-Associated Protein 2 (MAP2), and Glial Fibrillary Astrocytic Protein (GFAP) for 21 days. The expression of the identified genes was confirmed by Reverse Transcriptase PCR (RT-PCR). RESULTS: Treatment with 100 and 200µg/ml CSF resulted in the expression of GFAP and glial cells marker on days 14 and 21. The expression of neural-specific genes following CSF treatment was dose-dependent and time-dependent. Treatment of the cells with a twofold concentration of CSF, led to the expression of MAP2 on day 14 of induction. No expression of GFAP was detected before day 14 or MAP2 before day 21, which shows the importance of the treatment period. In the present study, expression analysis for the known neural markers: Nestin, GFAP, and MAP2 using RT-PCR were performed. The data demonstrated that CSF could play a role as a strong inducer. CONCLUSION: RT-PCR showed that cerebrospinal fluid promotes the expression of Nestin, MAP2, and GFAP mRNA in a dose-dependent manner, especially at a concentration of 200 µl/ml. In summary, CSF induces neurogenesis of WJ stem cells that encourages tissue engineering applications with these cells for treatments of neurodegenerative defects and traumatic brain injury.