RNA-seq has become an essential tool in molecular research. Nevertheless, application of RNA-seq was limited by cost and technical difficulties. Illumina has introduced the cost effective and ease to handle Truseq Targeted RNA Sequencing. In this study, we present the requirements and the optimization procedure for this Truseq Targeted RNA sequencing on cell line. Total RNA was recommended as starting materials but it required optimization including additional purification step and adjusting the AMPure beads ratio to eliminate unwanted contaminants. This can be resolved by using PolyA-enriched mRNA as starting material. TREx is a useful assay to evaluate gene expression. Quality library of TREx can be prepared by adding multiple washing steps or changing input sample to mRNA.
Two-third of breast cancer patients expressed estrogen receptors (ER)s and received endocrine treatment with established anti-estrogens such as tamoxifen. But the action and acquired resistance during treatment are largely unknown. In contrary, phytochemicals are more selective and less cytotoxic to normal cells. Accordingly, we found aloe emodin, an anthraquinone to inhibit the proliferation of ER+-breast cancer cells, MCF-7 with IC50 of 80 µM, but not affecting control breast cells, MCF-10A. Tamoxifen was non-selective to both cells with IC50 of 27 and 38 μM, respectively. Thus, we aimed to investigate the anti-proliferative mechanism of aloe emodin on MCF-7 and its underlying signalling compared to tamoxifen. Cells were treated separately with aloe emodin and tamoxifen at respective IC50 for 72 h. Apoptosis was determined using Annexin V-FITC/PI staining. The expression of insulin-like growth factor-1 receptor (IGF-1R), insulin like growth factor binding protein (IGFBP)-2 and B-raf gene was investigated using QuantiGene 2.0 Plex assay. Pairedstudent t-test and ANOVA test were used to compare between untreated and treated cells on the measured parameters. Each treatment was conducted in triplicate and repeated three times. Significance was set at p<0.05. The presences of early and late apoptosis in MCF-7 were seen in both treatments. All target genes were down regulated. The anti-proliferation effect of aloe emodin on MCF-7 is similar with tamoxifen which mediates inhibition of IGF-1R signalling pathway. This suggests aloe emodin as a potential anti-cancer agent to be used in combined anti-estrogen therapy to enhance its efficacy in ER+-breast cancer treatment.
Conjugated linoleic acids (CLA) act as an important ligand for nuclear receptors in adipogenesis and fat deposition in mammals and avian species. This study aimed to determine whether similar effects are plausible on avian abdominal fat adipocyte size, as well as abdominal adipogenic transcriptional level. CLA was supplemented at different levels, namely, (i) basal diet without CLA (5% palm oil) (CON), (ii) basal diet with 2.5% CLA and 2.5% palm oil (LCLA), and (iii) basal diet with 5% CLA (HCLA).The content of cis-9, trans-11 CLA was between 1.69- and 2.3-fold greater (P < 0.05) than that of trans-10, cis-12 CLA in the abdominal fat of the LCLA and HCLA group. The adipogenic capacity of the abdominal fat depot in LCLA and HCLA fed chicken is associated with a decreased proportion of adipose cells and monounsaturated fatty acids (MUFA). The transcriptional level of adipocyte protein (aP2) and peroxisome proliferator-activated receptor gamma (PPARγ) was downregulated by 1.08- to 2.5-fold in CLA supplemented diets, respectively. It was speculated that feeding CLA to broiler chickens reduced adipocyte size and downregulated PPARγ and aP2 that control adipocyte cellularity. Elevation of CLA isomers into their adipose tissue provides a potential CLA-rich source for human consumption.
Monobenzyltin Schiff base complex, [N-(3,5-dichloro-2-oxidobenzylidene)-4-chlorobenzyhydrazidato](o-methylbenzyl)aquatin(IV) chloride, C1, is an organotin non-platinum metal-based agent. The present study was conducted to investigate its effects on MCF-7 cells with respect to the induction of apoptosis and its inhibitory effect against MCF-7 breast cancer stem cells. As determined in a previous study, compound C1 revealed strong antiproliferative activity on MCF-7 cells with an IC50 value of 2.5 μg/mL. Annexin V/propidium iodide staining coupled with flow cytometry indicated the induction of apoptosis in treated cells. Compound C1 induced apoptosis in MCF-7 cells and was mediated through the intrinsic pathway with a reduction in mitochondrial membrane potential and mitochondrial cytochrome c release to cytosol. Complex C1 activated caspase 9 as a result of cytochrome c release. Subsequently, western blot and real time PCR revealed a significant increase in Bax and Bad expression and a significant decrease in the expression levels of Bcl2 and HSP70. Furthermore, a flow cytometric analysis showed that treatment with compound C1 caused a significant arrest of MCF-7 cells in G0/G1 phase. The inhibitory analysis of compound C1 against derived MCF-7 stem cells showed a significant reduction in the aldehyde dehydrogenase-positive cell population and a significant reduction in the population of MCF-7 cancer stem cells in primary, secondary, and tertiary mammospheres. Moreover, treatment with C1 down-regulated the Wnt/β-catenin self-renewal pathway. These findings indicate that complex C1 is a suppressive agent of MCF-7 cells that functions through the induction of apoptosis, cell cycle arrest, and the targeting of MCF-7-derived cancer stem cells. This work may lead to a better treatment strategy for the reduction of breast cancer recurrence.