Investigation of the effect of Prunus Amygdalus Amara on the expression of some genes of apoptosis and immortality in breast cancer cells (MCF-7)

Background: Anti-cancer effects of almond nuts or oil have been approved, but there are a few pieces of research that have evaluated, in detail, almond and other seeds' effects on cancer. Therefore, in the present project, the aim was to explore the regulatory effect of the bitter almond extract (Prunus amygdalus Batsch) on the apoptotic and anti-cancer potency of MCF-7 cells. Objectives: In the current experimental research, the Almond effect on MCF7 cells was evaluated by investigating the expression and the balance between Bcl-2, Bax genes to unmark the potential molecular mechanism. Methods: For 24 and 48h, the MCF7 cells were treated with the bitter almond extract (187.5-3000 µg/mL). MTT assay was used to assess the viability, and Real-time-PCR was applied to determine the expression of Bax and Bcl-2, facing β-actin. Results: Our results revealed a significant difference between different extract concentrations on the viability of MCF7 cell lines in 24 and 48 h; cell viability decreased time-dependently (P < 0.05). After 24 and 48h of extract facing MCF7 cells, the evaluated IC50 value was 3000 and 1500 µg/mL, respectively. Based on Real Time-PCR analysis, after 24 and 48 h, the mRNA levels of BCL-2 decreased by the extract, whereas BAX was in the MCF-7 cell line. Conclusion: From the results, it can be concluded that bitter almond extract has anti-cancer properties that may influence the apoptotic pathways by regulating relative gene expression.

Download Full-text

Genome-wide screening of m6A-modified transcript in the Wilms’ tumor

10.21203/rs.3.rs-197376/v1 ◽

2021 ◽

Author(s):

Zhuo Liu ◽

Feng He ◽

Jing Liu ◽

Shengrong OuYang ◽

Zexi Li ◽

...

Keyword(s):

Gene Ontology ◽

Real Time ◽

Real Time Pcr ◽

Wilms Tumor ◽

Expression Patterns ◽

Mrna Levels ◽

Gene Ontology Analysis ◽

Quantitative Real Time Pcr ◽

Related Factors ◽

Significant Difference

Abstract Background Wilms’ tumor, also called nephroblastoma, is the most common pediatric renal malignancy. The pathogenesis of Wilms’ tumor has been attributed to several genetic and epigenetic factors. However, the most pervasive internal mRNA modification that affects almost every process of RNA metabolism, RNA N6-Methyladenosine (m6A) methylation, has not been characterized in Wilms’ tumor. Methods Wilms’ tumor (WT) and adjacent non-cancerous (NC) tissue samples were obtained from 23 children with nephroblastoma, and the global m6A levels were measured by mass spectrometry. Analyses by m6A-mRNA epitranscriptomic microarray and mRNA microarray were performed, and m6A-related mRNAs were validated by quantitative real-time PCR for input and m6A-immunoprecipitated RNA samples from WT and NC tissues. Gene ontology analysis and KEGG pathway analysis were performed for differentially expressed genes, and expression of RNA methylation-related factors was measured by quantitative real-time PCR. Results The total m6A methylation levels in total RNA of WT samples and NC samples were (0.21 ± 0.01)% and (0.22 ± 0.01)%, respectively, with no statistically significant difference. Fifty-nine transcripts were differentially m6A-methylated between the WT and NC groups, which showed distinct m6A modification patterns. Gene ontology analysis indicated that m6A-modified genes were enriched in cancer-associated pathways, including the mTOR pathway, and conjoint analysis of the unique methylation and gene expression patterns in WT samples suggested an association with metabolic pathways.The mRNA levels of the m6A-related “reader” genes, YTHDF1, YTHDF2 and IGF2BP3, were statistically higher in WT samples than in NC samples. Conclusion This is the first study to determine the m6A modification profiles in Wilms’ tumor. Our data provide novel information regarding patterns of m6A modification that correlate with carcinogenesis in Wilms’ tumor.

Download Full-text

Effect of dietary α-lipoic acid on the mRNA expression of genes involved in drug metabolism and antioxidation system in rat liver

British Journal Of Nutrition ◽

10.1017/s0007114514000841 ◽

2014 ◽

Vol 112 (3) ◽

pp. 295-308 ◽

Cited By ~ 10

Author(s):

Takashi Ide

Keyword(s):

Drug Metabolism ◽

Mrna Expression ◽

Real Time ◽

Dna Microarray ◽

Lipoic Acid ◽

Real Time Pcr ◽

Glutathione Transferase ◽

Redox System ◽

Pcr Analysis ◽

Mrna Levels

In the present study, the mRNA levels of hepatic proteins involved in the drug metabolism of rats fed α-lipoic acid were evaluated by DNA microarray and real-time PCR analyses. Experimental diets containing 0, 0·1, 0·25 and 0·5 % (w/w) α-lipoic acid were fed to four groups of rats consisting of seven animals each for 21 d. DNA microarray analysis revealed that the diet containing 0·5 % α-lipoic acid significantly (P< 0·05) increased the mRNA levels of various phase I drug-metabolising enzymes up to 15-fold and phase II enzymes up to 52-fold in an isoenzyme-specific manner. α-Lipoic acid also up-regulated the mRNA levels of some members of the ATP-binding cassette transporter superfamily, presumed to be involved in the exportation of xenobiotics, up to 6·6-fold. In addition, we observed that α-lipoic acid increased the mRNA levels of many proteins involved in antioxidation, such as members of the thiol redox system (up to 5·5-fold), metallothioneins (up to 12-fold) and haeme oxygenase 1 (1·5-fold). These results were confirmed using real-time PCR analysis, and α-lipoic acid dose dependently increased the mRNA levels of various proteins involved in drug metabolism and antioxidation. Consistent with these observations, α-lipoic acid dose dependently increased the hepatic concentration of glutathione and the activities of glutathione reductase and glutathione transferase measured using 1-chloro-2,4-dinitrobenzene and 1,2-dichloro-4-nitrobenzene as substrates, but decreased the hepatic and serum concentrations of malondialdehyde. In conclusion, the present study unequivocally demonstrated that α-lipoic acid increases the mRNA expression of proteins involved in drug metabolism and antioxidation in the liver.

Download Full-text

Real-Time PCR and Immunocytochemical Study of Chondroitin Sulfate Proteoglycans after Scratch Wounding in Cultured Astrocytes / PCR I IMUNOCITOHEMIJSKA STUDIJA EKSPRESIJE HONDROITIN-SULFATNIH PROTEOGLIKANA NAKON POVREDE ASTROCITA U KULTURI

Journal of Medical Biochemistry ◽

10.2478/jomb-2013-0036 ◽

2013 ◽

Vol 32 (4) ◽

pp. 398-405

Author(s):

Ana Parabucki ◽

Anja Santrač ◽

Danijela Savić ◽

Sanja Dacić ◽

Ivana Bjelobaba ◽

...

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Complex Disease ◽

In Vitro Models ◽

Pcr Analysis ◽

Mrna Levels ◽

Cultured Astrocytes ◽

Scratch Wound

Summary Background: Various in vivo and in vitro models have been described in order to elucidate the pathobiology underlying the traumatic brain injury (TBI) and test potentially suitable treatments. Since TBI is a complex disease, models differ in regard to the aspect of TBI that is being investigated. One of the used in vitro models is the scratch wound assay, first established as a reproducible, low-cost assay for the analysis of cell migration in vitro. The aim of the present study was to further investigate the relevancy of this model as a counter- part of in vivo TBI models. Methods: We have examined the astrocytic response to a mechanical injury in terms of expression of chondroitin sul- fate proteoglycans (CSPGs) - phosphacan, neurocan and brevican, using real-time PCR and immunocytochemistry. Results: Our results indicate that in vitro scratch wounding alters the expression profile of examined CSPGs. Four hours after the scratch injury of the astrocytic monolayer, real-time PCR analysis revealed upregulation of mRNA levels for phos- phacan (3-fold) and neurocan (2-fold), whereas brevican mRNA was downregulated (2-fold). Immunofluorescent sig- nal for phosphacan and neurocan was more intense in astro- cytes close to the injury site, while brevican was scarcely present in cultured astrocytes. Conclusions: Obtained results indicate that CSPGs are differ- entially expressed by astrocytes after scratch wounding, demonstrating that the scratch wound model might be suit- able for investigation of astrocyte-derived response to injury.

Download Full-text

Cloning and Expression of kiss2 in the Zebrafish and Medaka

Endocrinology ◽

10.1210/en.2008-0940 ◽

2009 ◽

Vol 150 (2) ◽

pp. 821-831 ◽

Cited By ~ 207

Author(s):

Takashi Kitahashi ◽

Satoshi Ogawa ◽

Ishwar S. Parhar

Keyword(s):

Mrna Expression ◽

Real Time ◽

Real Time Pcr ◽

Mature Female ◽

Cloning And Expression ◽

Hypothalamic Nucleus ◽

Pcr Analysis ◽

Mrna Levels ◽

The Novel ◽

Female Zebrafish

Newly discovered kisspeptin (metastin), encoded by the Kiss1/KISS1 gene, is considered as a major gatekeeper of puberty through the regulation of GnRH. In the present study, we cloned a novel kisspeptin gene (kiss2) in the zebrafish Danio rerio and the medaka Oryzias latipes, which encodes a sequence of 125 and 115 amino acids, respectively, and its core sequence (FNLNPFGLRF, F-F form) is different from the previously characterized kiss1 (YNLNSFGLRY, Y-Y form). Our in silico data mining shows kiss1 and kiss2 are highly conserved across nonmammalian vertebrate species, and we have identified two putative kisspeptins in the platypus and three forms in Xenopus. In the brain of zebrafish and medaka, in situ hybridization and laser capture microdissection coupled with real-time PCR showed kiss1 mRNA expression in the ventromedial habenula and the periventricular hypothalamic nucleus. The kiss2 mRNA expression was observed in the posterior tuberal nucleus and the periventricular hypothalamic nucleus. Quantitative real-time PCR analysis during zebrafish development showed a significant increase in zebrafish kiss1, kiss2 (P < 0.002), gnrh2, and gnrh3 (P < 0.001) mRNA levels at the start of the pubertal phase and remained high in adulthood. In sexually mature female zebrafish, Kiss2 but not Kiss1 administration significantly increased FSH-β (2.7-fold, P < 0.05) and LH-β (8-fold, P < 0.01) mRNA levels in the pituitary. These results suggest that the habenular Kiss1 and the hypothalamic Kiss2 are potential regulators of reproduction including puberty and that Kiss2 is the predominant regulator of gonadotropin synthesis in fish. Habenular kisspeptin-1 (Kiss1) and the novel hypothalamic Kiss2 are potential regulators of puberty. Kiss2 is the predominant regulator of gonadotropin synthesis in fish.

Download Full-text

Decreased Platelet Expression of MYL9 (Myosin Regulatory Light Chain Polypeptide) and Other Genes with Platelets Dysfunction and CBFA2 Mutation: Insights from Platelet Expression Profiling.

Blood ◽

10.1182/blood.v104.11.737.737 ◽

2004 ◽

Vol 104 (11) ◽

pp. 737-737

Author(s):

Liansheng Sun ◽

J. Rafael Gorospe ◽

Eric P. Hoffman ◽

A. Koneti Rao

Keyword(s):

Real Time ◽

Light Chain ◽

Real Time Pcr ◽

Expression Profiling ◽

Regulatory Light Chain ◽

Mrna Levels ◽

Myosin Regulatory Light Chain ◽

Platelet Factor ◽

Significant Difference ◽

12 Lipoxygenase

Abstract We have previously reported (Blood, 87:1368, 1996) a patient with a mild thrombocytopenia, and markedly abnormal aggregation, secretion, phosphorylation of pleckstrin and myosin light chain (MLC), and activation of GPIIb-IIIa complex. We recently showed (Blood 2004, 103: 948–954) in this patient a heterozygous mutation in hematopoietic transcription factor CBFA2 (Core Binding factor A2, also called AML1) and decreased platelet PKC-theta. We postulated that platelet expression profiling would provide insights into the pathophysiological mechanisms and gene expression changes resulting from a CBFA2 mutation. We performed platelet expression profiling in 4 control subjects (6 profiles, 2 individuals profiled twice) and the patient (profiled twice, 10 months apart) using the Affymetrix microarray Human U133A and B GeneChips (total ~44000 probe sets). ~10,000 probe sets (genes and ESTs) are expressed in human platelets. Iterative comparisons of data from each individual platelet profiles had correlation coefficients (r) > 0.85. Using GeneSpring® analytical software we selected genes that showed at least 1 present call in the 8 profiles, and p<0.05 showing a significant difference between the 2 groups. Relative expression values <0.5 and >2 were considered as significant changes. In the patient, 298 probe sets were significantly down regulated and 29 significantly upregulated. We focused on genes showing a > 5-fold decrease (fold change, fc < = 0.20). There was a striking ~75 fold decrease (fc = 0.013; p=0.00022) in myosin regulatory light chain polypeptide (MYL9 gene). Other down-regulated genes included calcium binding protein 5 (CABP5, fc = 0.02; a protein with similarity to calmodulin), Na/K/Ca exchanger member 3 (SLC24A3, fc = 0.11), b3-endonexin (ITGB3BP, fc = 0.01), platelet factor 4 variant 1 (PF4V1, fc = 0.03), chemokine CXCL5 (ENA-78) (fc = 0.20), tropomyosin I (alpha) (TPM1, fc = 0.13) and arachidonate 12-lipoxygenase (ALOX-12, fc = 0.22). The expression array findings were validated by decreased platelet mRNA levels on real time PCR or by immunoblotting (platelet PF4). Several down-regulated genes are directly relevant to the patient’s platelet defect. Agonist-stimulated MLC phosphorylation is impaired in patient platelets. The strikingly decreased expression of MYL9 with normal expression of MLC kinase (MLCK, by array and real time PCR) suggests that the blunted myosin phosphorylation arises from decreased MYL9 than due to decreased MLCK. Myosin light chains play a role in diverse processes including in cell contractility, mobility and cytokinesis, and are relevant to impaired platelet function and production. The decreased expression of the Na/K/Ca exchanger and b3-endonexin 3 (both implicated in activation of GPIIb-IIIa) provide mechanisms for the diminished GPIIb-IIIa activation. Chemokine CXCL5 and PF4V1 are expressed in megakaryocytes, and the genes are colocalized to “chemokine cluster” on chromosome 4. 12-lipoxygenase, a major platelet enzyme, regulates arachidonic acid metabolism and production of the 12-hydroxy eicosanoids. This is the first proof of concept that platelet expression profiling can be applied to obtain new insights into the molecular basis of inherited platelet dysfunction. Further studies will provide new information on the genes regulated by CBFA2, and on mechanisms regulating platelet production and activation.

Download Full-text

Low expression of Talin1 is associated with advanced pathological features in colorectal cancer patients

Scientific Reports ◽

10.1038/s41598-020-74810-6 ◽

2020 ◽

Vol 10 (1) ◽

Author(s):

Somayeh Vafaei ◽

Leili Saeednejad Zanjani ◽

Zohreh Habibi Shams ◽

Marzieh Naseri ◽

Fahimeh Fattahi ◽

...

Keyword(s):

Protein Expression ◽

Real Time ◽

Real Time Pcr ◽

Mrna Level ◽

Univariate Analysis ◽

Survival Rates ◽

Tnm Stage ◽

Mrna Levels ◽

Significant Difference ◽

Low Expression

Abstract To explore the proper prognostic markers for the likelihood of metastasis in CRC patients. Seventy-seven fresh CRC samples were collected to evaluate the mRNA level of the selected marker using Real-time PCR. Moreover, 648 formalin-fixed paraffin-embedded CRC tissues were gathered to evaluate protein expression by immunohistochemistry (IHC) on tissue microarrays. The results of Real-Time PCR showed that low expression of Talin1 was significantly associated with advanced TNM stage (p = 0.034) as well as gender (p = 0.029) in mRNA levels. Similarly, IHC results indicated that a low level of cytoplasmic expression of Talin1 was significantly associated with advanced TNM stage (p = 0.028) as well as gender (p = 0.009) in CRC patients. Moreover, decreased expression of cytoplasmic Talin1 protein was found to be a significant predictor of worse disease-specific survival (DSS) (p = 0.011) in the univariate analysis. In addition, a significant difference was achieved (p = 0.039) in 5-year survival rates of DSS: 65% for low, 72% for moderate, and 88% for high Talin1 protein expression. Observations showed that lower expression of Talin1 at both the gene and protein level may drive the disparity of CRC patients’ outcomes via worse DSS and provide new insights into the development of progression indicators because of its correlation with increased tumor aggressiveness.

Download Full-text