A Pilot Study-Comparison between miRNA-200 c and miR-205 in Breast Cancer Cells

Autophagy appears to have differing roles in varied types of breast cancer cells. Autophagy plays a complex role in tumor initiation and progression. MicroRNAs (miRNAs) are non-coding small RNAs that play an important role in the post-transcriptional regulation of gene expression. Recent studies indicated that miRNAs are also mediators in breast cancer progression. A thorough understanding of the molecular mechanisms controlling autophagy in homeostasis may eventually allow for the manipulation of the process specific target of breast cancer migration and invasion in therapeutic applications. Results demonstrate how a breast cancer cell model can be used to study the relationship between miRNA-200c and miRNA-205, and autophagy. Confocal microscopy was utilized to examine changes in autophagic flux. This study revealed that miRNA-200c and miRNA-205 were altered significantly in autophagic flux in comparison with the FDA-approved drug (chloroquine-CQ) in the BT549 and MCF7 breast cancer cell types.

Autophagy is derived from the Greek words auto, which means "self", and phagy, which means "to eat". The autophagic process breaks down cellular components and plays a particularly important role during periods of starvation, or when organelles are damaged [7]. This allows cells to balance synthesis, degradation, and recycling of cellular structures. Autophagosomes are double-membrane cytosolic vesicles that encapsulate superfluous or damaged cellular components and break them down to either supply nutrients to the cell or fine-tune organelle content [8][9][10]. Not surprisingly, then, during development autophagy plays a significant role in cell growth, and in homeostasis. Macroautophagy, often referred to simply as autophagy, involves the formation of double membrane vesicles around organelles and other cytoplasmic components that then fuse with lysosomes [11].
Different miRNAs have been found to maintain protein translator and homeostasis in enriched breast-cancer subsets. Furthermore, miRNA biogenesis and regulation are involved in selective autophagy. MicroRNAs (miRNAs) are short non-coding RNAs that regulate the function of target genes at the post-transcriptional phase. miRNAs are known involved in disease processes. miRNAs regulate gene expression post-transcriptional by binding to the 3'-Untranslated Regions (3'UTR) of a target mRNA and inhibited translation [12]. Different miRNAs have been found to enriched in breast cancer subsets and maintain in protein translation homeostasis [13][14][15][16]. Furthermore, miRNA biogenesis and regulation are involved with selective autophagy [17].
To antagonize the function of miRNAs, several antagomirs have been engineered and are commercially available. On the contrary, miRNAs effect can be enhanced with miRNA mimics, which are chemically modified short double-stranded RNA sequences. Recent studies have suggested a role of miRNA in modulating breast cancer cells. The two-major miRNA are miRNA-200c and miRNA-205 which were thoroughly studied, and data suggested that miRNA-mediated control of EMT Non-coding miRNAs that selectively bind mRNAs, thus inhibiting their translation or promoting their degradation, also regulate the epithelial phenotype and EMT [18]. Members of the miR-200 family and miR-205 repress the translation of ZEB1 and ZEB2 mRNAs, and double-negative feedback controls ZEB and miR-200 expression, with ZEB proteins repressing the expression of miR-200 miRNAs, and miR-200 suppressing ZEB expression [19]. During EMT, decreased miR-200 expression results in increased ZEB1 and ZEB2 levels and EMT progression [20]. Additionally, p53 represses liver carcinoma cell EMT by increasing the expression of miR-200 and miR-192 (a miR-215 homologue), which target and reduce ZEB1 and ZEB2 expression [21,22].

Transfection
200 pM miRNA (Ambion) was transfected into cells using Trans-Bra transfection reagent (Invitrogen) compared to a scrambled control miRNA. Cells were incubated at 37ºC, 5% CO 2 for 24 or 48 hours respectively, and harvested with RIPA buffer for immunoblotting. Unmodified miRNA mimics were obtained from Ambion and proprietary modified miRNAs (designed for selective incorporation of the guide strand into RISC) are from Ambion.

Immunoblotting (Western Blotting)
Cells were seeded at 1x10 6 per 6 cm culture dish and treated under conditions equal to those chosen for the immunocytochemistry. Cells were lysed in ice-cold RIPA buffer supplemented with protease inhibitor (Roche) for protein extraction. The total protein concentration was determined using a spectrometer. Equally loaded proteins were separated in 8% and 20% SDS-PAGE gels and then transferred to nitrocellulose membranes. The membranes were probed with the following: (1) Rabbit monoclonal anti-LC3 (1:1000, Cell Signaling Technology), (2) Mouse monoclonal anti-ZEB1 antibody (1:500, Santa Cruz, USA), and (3) Mouse monoclonal anti-actin antibody (1:5000, Sigma) at 4ºC overnight ( Table 3). The primary antibodies were detected using a horseradish peroxidase-conjugated anti-rabbit (1:5000 or anti-mouse (1:5000) goat secondary, respectively. Blotting was visualized with a Chemiluminescence (ECL) kit; then, the membrane was exposed to chemiluminescence imaging system.

Phases
Cell Type Biomarkers Mirnas

Immuno Fluorescence (IFC) analysis
Cells were fixed and incubated with primary antibodies at a dilution of 1:100, fluorescence dye-conjugated secondary antibodies, and Hochester 33342, according to standard protocols. Cells were examined using a confocal microscope (Olympus) with a 60x oil immersion objective.

Image analysis and quantification and statistical analysis
Total numbers of LC3B-GFP puncta per cell in a frame was quantified by using ImageJ, and the Analyze Particles Plug in (a constant threshold for all the images within each experiment was applied). One hundred cells per condition were used for quantification. To quantitate the autophagosome per sample, open the digital image in the image analysis software and measure the entire cross-section area of the frame. This can be done by setting the threshold compared with the original image to ensure there has little information lost during threshold set. Second, identify and manually measure by analysis particles. Then exported the excel for statistical analysis. Data is represented as mean ± S.E.M. N values indicate the number of images. Images were taken in a blinded fashion. Significant differences between experimental groups were measured by the GraphPad Prism with statistically significance (0.001, ***).      In MCF7, miRNA antagonists have successfully stimulated ZEB1 expression, whereas chloroquine 50 uM did not significantly increase statistically based upon analysis ( Figure 3B). Interestingly, both miRNA antagonists did not stimulate LC3 expression compared with chloroquine 50 uM, which stimulated more significantly in LC-II than LC3-I in MCF7.

Discussion
This study revealed that miRNA-200c and miRNA-205 did not significantly alter autophagic flux compared to a FDA-approved drug (Chloroquine-CQ) in the BT549 and MCF7 breast cancer cell types. The process of Epithelial to Mesenchymal Transition (EMT) is believed the driving force to promote early invasive cancer growth by changing the cell morphology. ZEB can be regulating by miRNA 200 and miR205 family members, respectively [23,24]. MicroRNAs (miRNAs) are short non-coding RNAs that regulate the function of target genes at the post-transcriptional phases. They it has been suggested a therapeutic toward to cancer research. However, there has little information correlate between EMT, miRNAs with autophagy activities. Dysregulation of miRNAs contributes to tumor progression and therapeutic resistance in cancer cells [15,20,25]. Studies have shown that luminal phenotype breast cancer cells (MCF7 and MCF-12A) have higher miR-200c expression compared to basal phenotype breast cancer cells (MDA-MB-231 and BT549) [26,27].
We have used immunoblotting to evaluate the overall autophagic flux in differing treatments within 24 hours. Different breast cancer cells with different genetic composition exhibited different responses to miRNA treatment. In BT549, miR-200c over expression treatment resulted in decreased lipidation of LC3, whereas the MDA-MB-231 exhibited a reduction in LC-II expression by miR-205, and MCF7 exhibited no autophagic flux changes by miRNA (200c and 205) inhibition treatment. We found that the miR-200c over expression in MDA-MB-231 resulted in decreased lipidation of LC3. These results are similar to those of other studies [27].
To further investigate changes in autophagic flux in the heterogeneity of breast cancer cells, we use confocal imaging to observe. Our results demonstrated the changes in autophagic flux in real time in a time-dependent manner. miRNAs (200c and 205) were shown to decrease the autophagic flux in a 2-day treatment in MDA-MB-231, but not in BT549. It suggested that different behaviors depend on individual genetic composition in luminal phenotype breast cancer. Moreover, the study has shown that breast cancer cell lines possess heterogeneity which varies by cancer stem cell potential and different drug resistance populations [19]. Furthermore, the study also demonstrated that over expression of miR-200c reduced the survival fraction of the MDA-MB-231 and BT549 to radiation, whereas inhibition miR-200c expression resulted in increased survival fraction in MCF7 cells after radiation [27]. It indicates that miR-200c and 205 can serve in different breast cancer cells based on sensitivity in a manner associated with the miRNAs treatment.
Autophagy is a canonical pathway in which autophagosomes are fused with lysosomes and degraded within vesicles, and the nutrients are recycled back to cells. Autophagy can serve as a two sided function: it can suppress tumorigenesis [28] or it can serve as a pro-survival mechanism when cancer cells are subjected to damage by chemical or physical treatments [29,30]. Chloroquine (CQ) has been widely applied and is an FDA-approved drug. CQ is known as lysosome inhibitor which inhibits lysosomes from fusing with autophagosomes. Often, the side effect is cell apoptosis which is not specifically targeted to cancer cells. It has also been shown in our study that chloroquine significantly induces autophagic flux in immunoblotting in 1-day treatments, and confocal images in 1-day and 2-days treatments, respectively. Autophagic flux was also demonstrated in chloroquine-treated samples. This manuscript consists of a preliminary inspection of the results, which indicated additive effects and further investigations are required.
Our preliminary results suggested a potential sensitivity to autophagy in certain cancer cells than others, probably due to the heterogeneity between cells. Future experiments to be conducted will determine the cytotoxicity of miRNA treatments to specific cell populations or in a general population. Furthermore, the nature of the combination of chloroquine and miRNAs on apoptosis rate induced by miRNAs treatment will be investigated. Overall, our findings provide induction in autophagy flux is associated with cell death in normal cells and in some of breast cancer cells. It is crucial to understand the role of autophagy in the treatment of breast cancer.  In conclusion, our results indicate that the autophagy flux changed in response to miRNA treatment, at least in the first day of treatment. In addition, chloroquine has significantly stimulated the autophagy flux in breast cancer cell lines. These findings are promising, but in consideration of the limits of our investigation, further research is needed before suggesting its use in mechanistic and clinical applications.