Integrated High-Throughput Bioinformatics (Microarray, RNA-Seq, and RNA Interaction) and qRT-PCR Investigation of BMPR1B Axis as a Potential Diagnostic Biomarker of Isfahan Breast Cancer.

Background: According to the bioinformatics analyses and previous studies, bone morphogenetic protein receptor type 1B (BMPR1B) dysregulation could remarkably affect breast cancer (BC) status as a potential biomarker and tumor suppressor. Therefore, the analysis of the expression level of BMPR1B and other relevant biological factors such as microRNAs, long non-coding RNAs, downstream proteins in the relevant signaling pathways, and finding the accurate biological mechanism of BMPR1B could be helpful for a better understanding of BC pathogenicity and discovering the new treatment methods and drugs. Materials and Methods: R Studio software (4.0.2) was used for microarray data analyses. GSE31448 dataset was downloaded by GEOquery package and analyzed by limma package. STRING and miRWalk online databases and Cytoscape software were used for interaction analyses. Quantitative measurement of BMPR1B expression level was performed by qRT-PCR experiment. Result: Microarray and real-time PCR analysis revealed that BMPR1B has a significant downregulation in the transforming growth factor (TGF)-beta and bone morphogenic protein (BMP) signaling pathways in BC samples. BMPR1B is a potential diagnostic biomarker, regulated by hsa-miR-181a-5p. Also, BMPR1B regulates the function of BMP2, BMP6, SMAD4, SMAD5, and SMAD6 proteins. Discussion: BMPR1B have a significant role in the development of BC by regulating the potential proteins' function, playing the diagnostic biomarker role, and regulation of TGF-beta and BMP signaling pathways. The high amount of BMPR1B protein helps in increasing the survival rate of the patients.

NEAT1 can be a diagnostic biomarker in the breast cancer and gastric cancer patients by targeting XIST, hsa-miR-612, and MTRNR2L8: integrated RNA targetome interaction and experimental expression analysis.

BACKGROUND: The most frequent malignancy in women is breast cancer (BC). Gastric cancer (GC) is also the leading cause of cancer-related mortality. Long non-coding RNAs (lncRNAs) are thought to be important neurotic regulators in malignant tumors. In this study, we aimed to evaluate the expression level of NEAT1 and the interaction of this non-coding RNA with correlated microRNAs, lncRNAs, and mRNAs or protein coding genes, experimentally and bioinformatically. METHODS: For the bioinformatics analyses, we performed RNA-RNA and protein-protein interaction analyses, using ENCORI and STRING. The expression analyses were performed by five tools: Microarray data analysis, TCGA data analysis (RNA-seq, R Studio), GEPIA2, ENCORI, and real-time PCR experiment. qRT-PCR experiment was performed on 50 GC samples and 50 BC samples, compared to adjacent control tissue. RESULTS: Based on bioinformatics and experimental analyses, lncRNA NEAT1 have a significant down-regulation in the breast cancer samples with tumor size lower than 2 cm. Also, it has a significant high expression in the gastric cancer patients. Furthermore, NEAT1 have a significant interaction with XIST, hsa-miR-612 and MTRNR2L8. High expression of NEAT1 have a correlation with the lower survival rate of breast cancer samples and higher survival rate of gastric cancer patients. CONCLUSION: This integrated computational and experimental investigation revealed some new aspects of the lncRNA NEAT1 as a potential prognostic biomarker for the breast cancer and gastric cancer samples. Further investigations about NEA1 and correlated mRNAs, lncRNAs, and microRNAs - specially the mentioned RNAs in this study - can lead the researchers to more clear information about the role of NEAT1 in the breast cancer and gastric cancer.

Development and evaluation of bioactive 3D zein and zein/nano-hydroxyapatite scaffolds for bone tissue engineering application.

The aim of this study is to generate and investigate biodegradable and biocompatible zein and zein/nano-hydroxyapatite composite scaffolds for bone defect healing. 3D zein scaffold was successfully fabricated using the salt-leaching method and incorporated with 12.5 wt% nHA for osteogenic differentiation of murine myoblast cell line (C2C12 cells). The scaffolds were subjected to physicochemical and biomechanical characterizations using the scanning electron microscopy (SEM), Fourier-transform infrared spectroscopy (FTIR), X-ray diffraction (XRD), biodegradation, porosity, mechanical tests. C2C12 cells were cultured on scaffolds and incubated for 21 days. Cell proliferation was detected by the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. Quantitative real-time PCR was used to test the expression of osteoblastic-related genes including Runx2, ALP, and Col1A1. The scaffolds had an adequate mean pore size and a total porosity of 61.1%-70.6%. The addition of 12.5 wt% nHA to the zein scaffold increased the compressive modulus to 79.1 MPa and the ultimate strength to 2.7 MPa. The qRT-PCR analysis confirmed that mRNA transcript levels were significantly higher (p < 0.05) on the zein/nHA than on the pure zein scaffold. The results suggested that the developed scaffolds could be a potential candidate for bone tissue engineering due to their promising osteoinductivity, surface topography, mechanical behavior, biodegradability.

The miR526b-5p-Related Single Nucleotide Polymorphisms, rs72618599, Located in 3'-UTR of TCF3 Gene, is Associated with the Risk of Breast and Gastric Cancers

Background: Single nucleotide polymorphisms result in dysregulation of the proto-oncogene TCF3 gene, which is associated with the development, metastasis, and chemoresistance of different malignancies. Methods: GSE10810 microarray dataset and GEPIA2 online software were used to find differentially expressed genes and the TCF3 status in breast cancer (BC) and gastric cancer (GC), respectively. Plots and figures of microarray analysis were prepared by ggplot2 and pheatmap packages. Differentially expressed genes were obtained by the Bioconductor limma package. In silico analysis was used to predict the functions of rs72618599. BC (n = 123), GC (n = 132) and healthy age and gender matched controls (n = 184) were genotyped, using the high-resolution melting technique. Results: Based on the allelic comparison study, C allele of rs72618599 was associated with the BC tumor stage IV (66.1%, 78/120, p < 0.0001) and grade III (52.4%, 55/72, p < 0.0001), while the T allele was associated with metastasis (84.2%, 10/162, p < 0.0001). However, in GC patients, the C allele was significantly correlated with H. pylori infection (51.7%, 30/58, p = 0.008), stage III of primary tumors (47.7%, 62/88, p = 0.017), stage II of lymph node status (35.5%, 44/74, p = 0.017), and metastasis (52.9%, 90/132, p = 0.044). In silico analysis predicted that rs72618599 leads to the creation of a binding site for hsa-miR526b-5p in the 3'-UTR of TCF3 transcript. Conclusion: Regarding the rs72618599 SNP, the C allele, is associated with poor prognosis of BC and GC. Furthermore, rs72618599 may be associated with cancer progression by altering the regulatory affinity of hsa-miR526b-5p to 3'-UTR of TCF3.

rs6426881 in the 3'-UTR of PBX1 is involved in breast and gastric cancers via altering the binding potential of miR-522-3p.

BACKGROUND: Breast and gastric cancers are the most important diseases that lead to cancer death and social healthcare challenge. Overexpression of PBX1, a proto-oncogene, is correlated with the progression and metastasis of various cancers. For the first time, in this study the researchers evaluated the relationship between rs6426881, affecting miR-522-3p binding to the PBX1, with breast and gastric cancers. METHODS AND RESULTS: The Microarray analysis was performed for finding the relative expression level of PBX1 and hsa-miR-522-3p, based on high throughput experiments. The GSE54397, GSE112369, GSE10810, GSE241585.ER, GSE24185.PR, GSE68373, and GSE38167 datasets were analyzed. A case-control study was carried out in 123 Iranian suffering from breast cancer and 132 participants as control samples as well as 130 people suffering from gastric cancer and 54 people as control group members. SNP rs6426881 in the 3'-UTR of PBX1 was genotyped by the High-Resolution Melting (HRM) method. Association analysis revealed that rs6426881 is correlated with Estrogen and Progesterone receptors, grade, and stage of breast cancer. Furthermore, a significant relationship was observed between the genotypes and blood groups in gastric cancer, while the distribution of alleles was significantly related to smoking, status of the primary tumor, and metastasis (Chi-Square P < 0.05). Finally, Bioinformatics analyses suggested that rs6426881 contains binding sites for miR-522-3p in the 3'-UTR of PBX1 transcript. The finding suggested that TT genotype is associated with poor prognosis in breast and gastric cancer. CONCLUSIONS: The rs6426881 T allele at PBX1 3'-UT is significantly related to breast and gastric cancers by altering the regulatory affinity of miR-522-3p to PBX1 3'-UTR and may be suggested as a novel prognostic biomarker for the diseases.

Influence of a 5-bp Indel Polymorphism at Promoter of the GAS5 lncRNA and Risk of Breast Cancer.

Long non-coding RNAs (lncRNAs) are RNA molecules (>200 nucleotides in length) with no protein-coding capacity. Recent studies have demonstrated that lncRNAs involve in the regulation of their target genes at transcriptional, post-transcriptional and epigenetic levels. The aim of this case-control study was to explore whether growth arrest-specific 5 (GAS5) lncRNA 5-bp Ins/Del (rs145204276) polymorphism is involved in the breast cancer susceptibility. A total of 170 cases and 220 age matched controls were recruited in this study. GAS5 lncRNA polymorphism was genotyped using tetra primers amplification refractory mutation system polymerase chain reaction (T-ARMS-PCR) method. Statistical analysis was performed using SPSS. The distribution of the genotype ins/ins, ins/del and del/del were %75.29, 21.76% and 2.94% and 52.27%, 39.55% and 8.81% in the cases and controls, respectively. The ins/del or del/del genotype had a significantly decreased risk of breast cancer as compared with the ins/ins genotype under a codominant model (OR=0.38, 95%CI 0.24-0.60, p=0.0001; OR= 0.25, 95%CI 0.09-0.69, p=0.008, respectively). Moreover, the deletion allele of this polymorphic site is associated with a protective effect (OR=0.41, 95%CI 0.28-0.60, p=0.0001). Our study provided the first evidence that the deletion allele of GAS5 rs145204276 may have a protective role in mediating individual susceptibility to breast cancer. However, further comprehensive studies are warranted in a larger sample.
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Expression Alteration of Candidate Rice MiRNAs in Response to Sheath Blight Disease.

BACKGROUND: MicroRNAs, as small non-coding RNAs, are recently reported to be involved in plant defense system against pathogens including fungi. OBJECTIVE: In this research, it was intended to investigate candidate susceptible rice (Oryza Sativa) Osa-miRNA expression alteration following the infection by Rhizoctonia solani. MATERIALS AND METHODS: To this aim, literature review suggested eight conserved plant miRNAs that are involved in other plant-pathogen interactions. Then, sixty days old rice plants (Hashemi, susceptible cultivar) were inoculated with R. solani and candidate miRNA expression alterations were investigated 2 hpi (hours post inoculation), 2 dpi (days post inoculation) and 6 dpi. RESULTS: RT-qPCR analysis suggested four subgroups of candidate miRNAs based on the time of their responses to the pathogenesis of R. solani. While Osa-miR-156 was early-responsive, Osa-miR159 was the last-responsive and Osa-miR167, Osa-miR171, Osa-miR408, and Osa-miR444 were late responsive to R. solani infection. Osa-miR166 and Osa-miR393 were non-responsive to this infection, compared to the mock-inoculated control group. Consistently, Os-SPL3 and Os-MADS known target genes were expressed in reverse correlation to Osa-miR156 and Osa-miR444, respectively. CONCLUSIONS: From these data, it is suggested that both early (Osa-miR-156) and late (Osa-miR167, Osa-miR171, Osa- miR408, Osa-miR444) responsive miRNAs might be involved in R. solani infection in rice plants.