Methods: The mechanism involved in the cytotoxic activities of F5 against MCF7 cells was elucidated by flow cytometry-based apoptosis detection, caspases activity measurement, and expression profiling of apoptosis markers by western blotting. Molecular attributes of F5 were further mined from L. rhinocerus's published genome and transcriptome for future exploration.
Results and Discussion: Apoptosis induction in MCF7 cells by F5 may involve a cross-talk between the extrinsic and intrinsic apoptotic pathways with upregulation of caspase-8 and -9 activities and a marked decrease of Bcl-2. On the other hand, the levels of pro-apoptotic Bax, BID, and cleaved BID were increased accompanied by observable actin cleavage. At gene level, F5 composed of three predicted non-synonymous single nucleotide polymorphisms (T > C) and an alternative 5' splice site.
Conclusions: Findings from this study provide an advanced framework for further investigations on cancer therapeutics development from L. rhinocerus.
RESULTS: Several ascending and descending monotonic key genes were identified by Monotonic Feature Selector. The identified descending monotonic key genes are related to stemness or regulation of cell cycle while ascending monotonic key genes are associated with the functions of mesangial cells. The TFs were arranged in a co-expression network in order of time by Time-Ordered Gene Co-expression Network (TO-GCN) analysis. TO-GCN analysis can classify the differentiation process into three stages: differentiation preparation, differentiation initiation and maturation. Furthermore, it can also explore TF-TF-key genes regulatory relationships in the muscle contraction process.
CONCLUSIONS: A systematic analysis for transcriptomic profiling of MSC differentiation into mesangial cells has been established. Key genes or biomarkers, TFs and pathways involved in differentiation of MSC-mesangial cells have been identified and the related biological implications have been discussed. Finally, we further elucidated for the first time the three main stages of mesangial cell differentiation, and the regulatory relationships between TF-TF-key genes involved in the muscle contraction process. Through this study, we have increased fundamental understanding of the gene transcripts during the differentiation of MSC into mesangial cells.
Methods: Human ALDH1+ BCSCs were grown in serum-free Dulbecco's Modified Eagle Medium (DMEM)/F12, while MCF-7 and MDA-MB-231 were cultured in DMEM supplemented with 10% foetal bovine serum under standard conditions. Total RNA was extracted using the Tripure Isolation Reagent. The relative mRNA expressions of OCT4, ALDH1A1 and CD44 associated with stemness as well as TGF-β1, TβR1, ERα1 and MnSOD associated with aggressiveness in BCSCs and MCF-7 cells were determined using the quantitative real-time PCR (qRT-PCR).
Results: The mRNA expressions of OCT4 (5.19-fold ± 0.338; P = 0.001), ALDH1A1 (3.67-fold ± 0.523; P = 0.006), CD44 (2.65-fold ± 0.307; P = 0.006), TGF-β1 (22.89-fold ± 6.840; P = 0.015), TβR1 (3.74-fold ± 1.446; P = 0.045) and MnSOD (4.6-fold ± 1.096; P = 0.014) were higher in BCSCs than in MCF-7 but were almost similar to MDA-MB-231 cells. In contrast, the ERα1 expression of BCSCs (0.97-fold ± 0.080; P = 0.392) was similar to MCF-7 cells, indicating that BSCSs are oestrogen-dependent breast cancer cells.
Conclusion: The oestrogen-dependent BCSCs express stemness and aggressiveness genes at a higher level compared to oestrogen-dependent MCF-7 but are almost similar to oestrogen-independent MDA-MB-231 cells.