The Role of Differentially Expressed miRNAs and Potential miRNA-mRNA Regulatory Network in Prostate Cancer Progression and Metastasis

The Role of Differentially Expressed and Potential Regulatory in Abstract Purpose: Aberrant expression of microRNAs (miRNAs) has been discovered in prostate cancer progression however their function is not well understood, thereby further investigation is required to understand the importance of underlying mechanisms and their involvement in multiple signaling pathways, as well as their potential as therapeutic targets. In this study the role and expression levels of three miRNAs were evaluated: miR-21, miR-221 and miR-200c in different prostate cancer cell lines. In addition, based on the latest studies on miRNAs function, their association with other target genes and molecules were analyzed using bioinformatic tools. Methods: Three PCa cell lines PC3, LNCaP and VCaP and normal prostate epithelial cell line PNT1A were screened for miRNA expression levels using qPCR. miRNA target genes and their association with signaling pathways were analyzed through several Network and pathway analysis online tools. Findings: Upregulation of miR-21 and miR-221 was observed in PC3 and VCaP prostate cancer cells, respectively. According to KEGG analysis, we found that Hippo signaling pathway and cytokine-cytokine receptor interactions were affected by miR-21 while miR-221 would interfere with ECM-receptor interaction, Fatty acid elongation and Huntington disease molecular networks. Exposure of PC3 cells to TGF-β (10 µM) caused upregulation of miR-21 with the evidence with increased invasion potential. Discussion and Conclusion: miRNAs could regulate several genes in multiple signaling pathways. Here, we demonstrated that in a panel of PCa cell lines, both mir-21 and miR-221 expressions were upregulated. miR-21 may be a diagnostic and prognostic biomarker for PCa.


Introduction
Prostate cancer (PCa) is the most common cancer in men and a major cause of cancer-related deaths worldwide [1] PCa often progresses from hormone sensitive to castrate resistance [2]. The rates of PCa mortalities increase each year due to recurrence and metastasis, where developing new biomarkers could lead to early diagnosis, prognosis and effective treatment. microRNAs (miRs) are endogenous small non-coding RNA molecules with around 22 nucleotides and are regulators of gene expression at the post transcriptional level that can affect mRNA translation [3]. Highly conserved and stable miRNAs are expressed uniquely across different types of human cancers including PCa [4]. miR-21 is amongst the most commonly studied miRNAs, acts as oncogene being called oncomiR, it is thought to be highly expressed in both androgen dependent and independent PCa [5,6]. miR-221 however, is expressed together with miR-222 and its function can be both oncogenic and tumour suppressor depending on the tissue type [7]. It is unclear whether miR-221 regulates PCa progression from androgen dependence to castration resistance while acting as oncomiR. On the other hand, a member of the miR-200 family, miR-200c, is reported to be involved in the progression of various cancers as a tumour suppressor and inhibits PI3K-Akt pathway [8].
Transforming growth factor beta (TGF-β) activator of SMADs and superfamily of cytokines is a signaling process involved in cancer progression. TGF-B is known to be a target for one or several miRNAs, leading to changes in the cell behaviour and tumor microenvironment [9]. Both miR-21 and miR-200 are identified to be targeted by the components of TGF-β pathway, and this association could lead to potential therapeutic targets for PCa [10]. Epithelial-mesenchymal transition (EMT), a process that changes epithelial cells into fibroblast looking cell types known as mesenchymal cells, accordingly, known to play an important role in morphological changes during embryonic development [11]. This is highly regulated process is identified to play a role in tumour metastasis; thereby EMT markers could be potential targets for cancer diagnosis and prognosis. E-cadherin, an epithelial marker from a family of cell adhesion molecules (CAMs) is one of the most important molecules encoded by CDH1 gene. It plays an important role as a tumour suppressor and found to be suppressed in many cancers such as PCa suggesting that it can be an important marker in cancer progression and metastasis [12].
Though epithelial markers are important biomarkers, mesenchymal markers such as N-cadherin and Vimentin also play a crucial role in migration and metastasis and are often overexpressed in cancers. Other EMT markers such as matrix metalloproteinases (e.g. MMP2 and MMP9) are significant as they have the ability to breakdown collagen, an important structural component of basement membrane, in other words it is an essential factor in extracellular matrix (ECM) breakdown. MMP is also known to be highly associated with disease development and metastasis [13,14] and miR-21 might alter MMP signalling [15]. EMT genes could be targeted by several miRNAs, which subsequently deregulate their function and expression levels. Epidermal growth factor (EGF) could induce RAS and mitogen-activated protein kinases (MAPK) signalling through receptor tyrosine kinase (RTK) activation, where downstream components of this pathway were studied in PCa. miRs could also alter PI3K/AKT/mTOR signalling axis, which is one of the central signalling pathways that is involved in cell proliferation, differentiation and cell survival [16].
An important regulator in the PI3K pathway known as phosphatase and tensing homolog (PTEN) is a tumour suppressor, targeted and regulated by miR-21, leading to its suppression in tumour cells. Many miRNAs are known to regulate signalling pathways and be regulated through several mRNAs within these cascades, either enhancing or suppressing tumorigenesis. Having all this info together, this study evaluates the influence of cytokine activity within several signalling cascades and how cross-talks between these pathways could lead to initiation of EMT and metastasis in PCa. were used as described before [17]. 2ΔΔCT method [18] was used for calculating relative Mir expression levels and for normalization.

Cell Lines and Cell Culture
Each experiment was repeated three times.

Cell Viability and Invasion Assays
Cell invasiveness by assessed as described before [19].

Data Analysis
The graphs and histograms were prepared, and statistical analysis was performed using GraphPad Prism version 6 (GraphPad Software, San Diego, U.S.A.). One-way ANOVA was performed followed by Tukey's post-hoc analysis; significant differences were considered as p ≤ 0.05.

MicroRNA Expression of PCa Cell Lines
The

Molecular Signalling Pathways Affected By miR-200c, MIR-221 AND MIR-21
We then run an in-silico experiment to identify molecular networks orchestrated by these miRs. According to KEGG analysis, we found that Hippo signalling pathway and cytokinecytokine receptor interactions were affected by miR-21 while miR-221 would interfere with ECM-receptor interaction, Fatty acid elongation and Huntington disease molecular networks. We did not detect any overlapping network hierarchy for any Mir targets (Figure 2A). Gene Ontology (GO) analysis showed that only organelle functions could be a similar target for miR-21 and miR-221. In turn, GO analysis also showed that miR-21 expression levels significantly affect mechanisms as gene expression, nucleic acid binding transcription factor activity, neurotrophin TRK receptor signalling pathway, cellular nitrogen metabolic compound process and biosynthetic process ( Figure 2B). Here, we concluded that  A.
KEGG analysis shows Hippo signalling pathway and cytokine-cytokine receptor interactions were affected by miR-21 and ECM-receptor interaction, Fatty acid elongation and Huntington disease molecular networks were controlled by miR-221.

B.
GO analysis demonstrated miR-21 regulates gene expression, nucleic acid binding transcription factor activity, neurotrophin TRK receptor signalling pathway, cellular nitrogen metabolic compound process and biosynthetic process. B. TGF-β promoted a slight but insignificant increase in the proliferation ability of PC3 cells (n=3; p=0.079).

C.
A significant increase of PC3 cells invasion capacity was triggered by TGF-β (n=3; p= 0.001), crystal violet staining was performed to confirmed both cell proliferation and invasion after 16h. The pictures on the left represent, cell proliferation for control (upper left) and cellular invasion (lower left), the right side of the pictures represent cell proliferation of PC3 cells treated with TGF-β (upper right) and cellular invasion (lower right). Scale bar, 20 µm (applicable to both panels).

Discussion
Increasing clinical and biological evidence showed that a number of miRNAs have potential to monitor disease progression and clarify the molecular mechanism of diseases. miR-21 is an oncogene and found upregulated in six different types of cancer.
The upregulated expression level of miR-21 is associated with poor therapeutic response and poor prognosis of disease. Its molecular function is to target several tumour suppressor genes, including programmed cell death 4 (PDCD4) and TGFβR2, which are linked to stemness properties of cancer cells [20]. It was shown that miR- miR-21 was suggested with a mechanistic role in stemness properties of cells via affecting TGFβR2 to modulate Wnt/β-catenin signalling [12]. One of the crucial targets of miR-21 is PTEN that often found either deleted or mutated to suppress its function in PCa [6]. Here, we found that PC3 is a PTEN deleted cell line, that We concluded that miR-21 significant upregulation in PC3 cells might be a reason for PTEN deletion and increased the invasion potential of cells through modulating TGF-β-TIMP signalling axis to regulate MMPs [3]. We found that TGF-β upregulated miR-21 expression in PC3 cells. However, another study showed that upregulation of miR-21 due to TGF-β treatment led to growth arrest related to TGFBR2 and PTEN regulation in HaCaT keratinocytes [12]. Thus, the mechanistic role of miR-21 might have potential to regulate the ability of epithelial cells to respond to TGF-β, with potential impact on epithelium homeostasis, wound-healing and tumorigenesis [12]. According to previous studies, miR-21 downregulated Hippo signalling via targeting KIBRA, which promoted the progression of lung adenocarcinoma 28. Hippo signalling axis proteins regulates the miRs biogenesis by activating p72 with YAP to decrease expression levels of miRs in tumour tissues in a cell density dependent manner.

Conclusions
The role of microRNAs in PCa is increasing its importance. Here, in a panel of PCa cell lines, both mir-21 and miR-221 were seen to be increased agreeing with other studies. We have also looked at potential signalling pathways that miR-21 can regulate. miR-21 may be a diagnostic and prognostic biomarker for PCa. Based on this concept, further research on miRNA signalling pathways are needed.