Identification of miR-660-5p targets involved in breast cancer progression

Background: Breast cancer (BC) is the most diagnosed cancer in women globally. MicroRNAs (miRNAs) participate in different processes of BC; their deregulation can make them act as oncogenes or tumor suppressors, participating in cancer progression. Using the TCGA (The Cancer Genome Atlas) database, we found miR-660-5p significantly overexpressed and associated with poor survival in patients with this pathology. It is reported that miR-660-5p induces proliferation, migration, and invasion in BC. However, the specific targets of this miRNA that induce each of these processes are unknown. In this project we propose to identify the targets of miR-660-5p involved in proliferation, migration, invasion, and angiogenesis in BC cells. Methods: The basal levels of miR-660-5p were determined by RT-qPCR. The effect of miR-660-5p was evaluated on proliferation, invasion, and migration processes in MDA-MB-231 and MCF-7 cells, and angiogenesis in HUVEC cells transfected with the miR-660-5p inhibitor. We identified targets of miR-660-5p using different databases, and we evaluated their expression by RT-qPCR in plate. Results: In this study, we found that miR-660-5p is significantly upregulated in BC cells MDA-MB-231 and MCF-7, compared to normal breast cells MCF-10A. In addition, we observed a significantly decrease in the processes of proliferation, migration, and invasion in BC cells, compared to untreated cells and negative control group. Similarly, we observed a significantly decrease in the angiogenesis process in HUVEC cells, compared to untreated cells and negative control group. Likewise, by analyzing the different databases and the literature, we found a total of 28 miR-660-5p targets involved in oncological processes. Conclusions: miR-660-5p is overexpressed in BC cells compared to healthy breast cells. Furthermore, miR-660-5p induces the processes of proliferation, migration and invasion in BC cells, and angiogenesis in HUVEC cells

Upregulation of miR-21 in Cisplatin Resistant Ovarian Cancer via JNK-1/c-Jun Pathway

<div><p>Cisplatin has been the most accepted drug for the treatment of ovarian cancer for almost 40 years. Although the majority of patients with ovarian cancer respond to front-line platinum combination chemotherapy, many patients will develop cisplatin-resistance disease, which is extremely rapid and fatal. Although various mechanisms of cisplatin resistance have been postulated, the key molecules involved in such resistance have not been identified. MiRNAs are endogenously expressed small non-coding RNAs, which are evolutionarily conserved and function as post-transcriptional regulators of gene expression. Dysregulation of miRNAs have been associated with cancer initiation, progression and drug resistance. The oncogenic miRNA-21, one of the best-studied miRNAs, is upregulated in almost all human cancers. However, the regulation of miR-21 in cisplatin resistant ovarian cancer cells has not been assessed. In this study, we measured the miR-21 expression by real-time PCR and found upregulation of miR-21 in cisplatin resistant compared with cisplatin sensitive ovarian cancer cells. Chromatin immunoprecipitation studies demonstrated the association of the c-Jun transcription factor to the pri-mir-21 DNA promoter regions. Blocking the JNK-1, the major activator of c-Jun phosphorylation, reduced the expression of pre-mir-21 and increased the expression of its well-known target gene, PDCD4. Overexpression of miR-21 in cisplatin sensitive cells decreased PDCD4 levels and increased cell proliferation. Finally, targeting miR-21 reduced cell growth, proliferation and invasion of cisplatin resistant ovarian cancer cells. These results suggest that the JNK-1/c-Jun/miR-21 pathway contributes to the cisplatin resistance of ovarian cancer cells and demonstrated that miR-21 is a plausible target to overcome cisplatin resistance.</p></div

Effect of miR-21 inhibition in cell growth, proliferation and invasion.

<p>(<b>A</b>) A2780CP20 cells were transiently transfected with a miR-21 antagomir or with a negative antagomir (-) as described in the “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0097094#s2" target="_blank">Materials and Methods</a>” section. Eight and 24 hours after transfection cells were collected and RNA (including miRNAs) was isolated as described in the “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0097094#s2" target="_blank">Materials and Methods</a>” section. (<b>B</b>) MiR-21 inhibition reduced cell growth compared to untreated cells (NT) or with the negative control inhibitor (NC-Inh). (<b>C</b>) A2780CP20 cells were transfected as in 5A. A thousand cells were seeded in Petri dishes. Ten days later, the colonies were stained and counted. Inhibition of miR-21 decreased the ability of cells to undergo unlimited division compared with the NC-Inh. (<b>D</b>) Cell invasion was carried out as described in the “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0097094#s2" target="_blank">Materials and Methods</a>” section. The number of invaded cells was expressed in percentages, taken the NC-Inh as 100%. *p<0.05, **p<0.01 compared to NC-Inh. Columns represent the means of triplicates ± S.E.M.</p

Chromatin immunoprecipitation assay (ChIP).

<p>ChIP assay was performed as described in the “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0097094#s2" target="_blank">Materials and Methods</a>” section. (<b>A</b>) SYBR-I-based real-time PCR amplification of the region containing the c-Jun recognition sequence in the pri-miR-21 DNA. The phospho-c-Jun levels bound to the pri-miR-21 promoter was higher in A2780CP20 cells compared with A2780 cells. (<b>B</b>) SYBR-I-based real-time PCR amplification of a DNA region far of the pre-mir-21 promoter was performed as a control. *p<0.05 compared to control. Columns represent the means of triplicates ± S.E.M.</p

RT-PCR and western blot analysis of miRNA-21-related molecules.

<p>(<b>A</b>) Validation of microarrays by RT-PCR. (<b>B</b>) MiR-21 levels in a panel of ovarian cancer cells. MiR-21 levels were expressed relative to the A2780 cells miR-21 levels. IC50s were calculated after 72-hr treatment of cells with different concentrations of cisplatin as described in the “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0097094#s2" target="_blank">Material and Methods</a>” section. (<b>C</b>) Evaluation of c-Jun and p-c-Jun protein expression in A2780 and A2780CP20 cells. (<b>D</b>) Protein expression analysis of MAPKs in total and nuclear fractions of A2780 and A2780CP20 cells. Expression level in Figures A, C and D are without cisplatin treatment. *p<0.05, **p<0.01, ***p<0.001 compared to control. Columns represent the means of triplicates ± S.E.M.</p

Effect of pre-mir-21 overexpression in A2780 cells.

<p>(<b>A</b>) A2780 cells were stably transfected with pCMV-miR21 or empty pCMV-EV vectors. The miR-21 expression was quantified by qRT-PCR. (<b>B</b>) Western blot analysis shows a decreased expression of PDCD4 levels in A2780-miR-21 compared with A2780-EV cells. (<b>C</b>) Overexpression of miR21 increased cell proliferation (13%, ***p<0.001) in A2780-miR-21 compared with A2780-EV cells. (<b>D</b>) A2780-miR-21 overexpressed clones were more resistant to cisplatin-induced cell death compared with untransfected A2780 cells or with the A2780-EV cells. **p<0.01. Columns represent the means of triplicates ± S.E.M.</p

Effect of JNK-1 inhibition in miR-21 and PDCD4 expression.

<p>A2780CP20 cells were treated with 10 µM SP600125. (<b>A</b>) Western blot shown the inhibition of p-c-Jun after treatment with SP600125 in A2780CP20 cells compared to control (DMSO). (<b>B</b>) SYBR-I-based real-time PCR was performed to calculate the relative pre-mir-21 expression in A2780CP20 cells after treatment with SP600125 inhibitor. (<b>C</b>) Western blot and densitometric analysis of PDCD4 protein expression levels after treatment of A2780CP20 cells with SP600125. (<b>D</b>) PDCD4 protein expression levels after transfection of A2780CP20 with miR-21 oligonucleotide inhibitor. (<b>E</b>) A2780 CP20 cells were transiently transfected with two c-Jun-targeted siRNAs as described in the “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0097094#s2" target="_blank">Materials and Methods</a>” section. Western blot analysis shows that both c-Jun-siRNAs decreased the c-Jun levels. SYBR-I-based real-time PCR was performed (see “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0097094#s2" target="_blank">Materials and Methods</a>” section) to calculate the relative pre-mir-21 expression levels in A2780CP20 cells after siRNA-mediated c-Jun silencing. *p<0.05, **p<0.01, ***p<0.001 compared to control. Columns represent the means of triplicates ± S.E.M.</p

Biological Functions and Therapeutic Potential of Lipocalin 2 in Cancer

Lipocalin-2 (LCN2) is a secreted glycoprotein linked to several physiological roles, including transporting hydrophobic ligands across cell membranes, modulating immune responses, maintaining iron homeostasis, and promoting epithelial cell differentiation. Although LNC2 is expressed at low levels in most human tissues, it is abundant in aggressive subtypes of cancer, including breast, pancreas, thyroid, ovarian, colon, and bile duct cancers. High levels of LCN2 have been associated with increased cell proliferation, angiogenesis, cell invasion, and metastasis. Moreover, LCN2 modulates the degradation, allosteric events, and enzymatic activity of matrix metalloprotease-9, a metalloprotease that promotes tumor cell invasion and metastasis. Hence, LCN2 has emerged as a potential therapeutic target against many cancer types. This review summarizes the most relevant findings regarding the expression, biological roles, and regulation of LCN2, as well as the proteins LCN2 interacts with in cancer. We also discuss the approaches to targeting LCN2 for cancer treatment that are currently under investigation, including the use of interference RNAs, antibodies, and gene editing

Upregulation of the Long Noncoding RNA CASC10 Promotes Cisplatin Resistance in High-Grade Serous Ovarian Cancer

Despite initial responses to first-line treatment with platinum and taxane-based combination chemotherapy, most high-grade serous ovarian carcinoma (HGSOC) patients will relapse and eventually develop a cisplatin-resistant fatal disease. Due to the lethality of this disease, there is an urgent need to develop improved targeted therapies against HGSOC. Herein, we identified CASC10, a long noncoding RNA upregulated in cisplatin-resistant ovarian cancer cells and ovarian cancer patients. We performed RNA sequencing (RNA-seq) in total RNA isolated from the HGSOC cell lines OVCAR3 and OV-90 and their cisplatin-resistant counterparts. Thousands of RNA transcripts were differentially abundant in cisplatin-sensitive vs. cisplatin-resistant HGSOC cells. Further data filtering unveiled CASC10 as one of the top RNA transcripts significantly increased in cisplatin-resistant compared with cisplatin-sensitive cells. Thus, we focused our studies on CASC10, a gene not previously studied in ovarian cancer. SiRNA-mediated CASC10 knockdown significantly reduced cell proliferation and invasion; and sensitized cells to cisplatin treatment. SiRNA-mediated CASC10 knockdown also induced apoptosis, cell cycle arrest, and altered the expression of several CASC10 downstream effectors. Multiple injections of liposomal CASC10-siRNA reduced tumor growth and metastasis in an ovarian cancer mouse model. Our results demonstrated that CASC10 levels mediate the susceptibility of HGSOC cells to cisplatin treatment. Thus, combining siRNA-mediated CASC10 knockdown with cisplatin may represent a plausible therapeutic strategy against HGSOC