miR-301a expression: Diagnostic and prognostic marker for prostate cancer

Highlights

• miR-301a expression is higher in serum and tissue and correlates with increased Gleason score.

• RUNX3 has been identified as a target for miR-301a and activates Rho signaling in prostate cancer.

• Combining miR-301a with PSA values may help to stratify risk in patients and improve biopsy candidacy

Abstract

Background

Prostate-specific antigen screening for prostate cancer (CaP) remains controversial. This study establishes the role of microRNA 301a (miR-301a) as a supplemental biomarker that can distinguish between patients with benign prostate hyperplasia and clinically significant CaP. We evaluate the ability of miR-301a to predict the adverse pathology of CaP.

Methods

In the first cohort, serum and prostate tumor samples were obtained from thirteen patients with Benign prostate hyperplasia (BPH), twelve patients with Gleason 6, and sixteen patients with Gleason 7 prostate adenocarcinoma. In the second cohort, 40 prostatectomy cases were selected (BPH:12, Gleason 6:12 and Gleason 7:16). MiRNA was extracted from serum and tumor samples. Quantitative reverse transcription-polymerase chain reaction was performed for detection of miR-301a. To understand the molecular role of miR-301a, we performed cell viability, Western blots, promoter analysis, overexpression, and silencing studies in BPH and DU-145 cell lines.

Results

MiR-301a demonstrated a significantly higher expression in both serum and tumor tissue in patients with CaP when compared to patients with BPH (P = 0.011 and 0.013 for serum and tissue expression, respectively). Expression of miR-301a in prostatectomy specimens correlated with increased Gleason score. We demonstrated that miR-301a inhibited the pro-apoptotic function of RUNX3, and activated ROCK1-mediated pro-survival signal in CaP. Silencing miR-301a initiated the pro-apoptotic function of RUNX3 by inhibiting ROCK1 expression in CaP cells.

Conclusions

Expression of miR-301a could be a valuable adjunct tool for stratifying patients with elevated prostate-specific antigen, as well as those diagnosed with CaP. Including the miR-301a as an additional variable in MSKCC post-prostatectomy nomogram improved its ability in facilitating clinical decision-making.

Introduction

Prostate cancer (CaP) is the most common malignancy in males in the United States with an estimated 164,690 new cases in 2018 by American Cancer Society. Prostate-specific antigen (PSA) screening has led to an increase in the detection of early stage CaP. As a diagnostic test (PSA is a screening test), PSA has poor specificity, which leads to the over-diagnosis and over-treatment of CaP. Patients with low-risk or indolent tumors are often subjected to treatments that are not without complications. Over diagnosis and over-treatment of CaP is a major concern, and there is a critical need for improved diagnostic tools to identify patients with high-risk CaP, who benefit from radical therapy. As such, the goal of this study was to discover a novel biomarker, which would differentiate between indolent vs. aggressive disease, and will facilitate better clinical decision-making.

MicroRNAs (miRNA) are short non-coding single-stranded RNA molecules (approximately 18–22 nucleotides) that are evolutionarily conserved post-transcriptional gene regulators [1], which may repress gene expression or activate a specific gene or transcription factors [2]. miRNAs are involved in numerous cellular processes such as proliferation, differentiation, migration, invasion, apoptosis, and promoter methylation [3]. Several studies have demonstrated miRNAs as potential biomarkers for cancer detection, prognosis, and treatment [4]. Several miRNAs have been identified, and their expression levels were linked to the clinical outcome of CaP [5], [6]. miR-301a has gained attention in cancer research due to its significant role in differentiation, inflammation, apoptosis, and pathogenesis of cancer [7]. Previous studies have demonstrated higher expression of miR-301a in different cancer types [8], [9], including CaP [10]. It has been suggested that miR-301a could be a predictive marker for metastasis [[11], [12]]. Nam et al., have identified miR-301a as a prognostic marker for CaP metastasis [12]. It has been established that miR-301a functions as an oncogene in CaP by directly targeting p63, leading to loss of E-cadherin, thereby resulting in epithelial to mesenchymal transition [11].

Runt-related transcription factor 3 (RUNX3) is a master regulator of genes responsible for cell proliferation and differentiation [13], [14]. The RUNX3 family proteins, RUNX-1 and RUNX-2, may play important roles in normal developmental processes and tumorigenesis [15], [16]. Overexpression of RUNX3 suppresses tumor growth, cellular proliferation, migration, and invasion potential in breast cancer, colon cancer, renal cell carcinoma, and glioma [17], [18]. In metastatic gastric cancers, a significant downregulation of RUNX3 expression was seen compared to healthy counterparts [19]. Similarly, downregulation of RUNX3 expression was correlated with the TNM stage in patients with CaP [20], although the molecular mechanism by which RUNX3 function is attenuated in CaP, and remains unclear.

Rho GTPase family members, including rho-associated coiled-coil-containing protein kinase 1 (ROCK1), regulate many functions, such as controlling microtubule dynamics by reorganizing actin cytoskeleton structure, which in turn influences adhesions, motility, and cellular polarity [21]. Higher expression of Rho kinases is positively correlated with aggressiveness of many cancer types (breast, colon, and lung) [22], [23] vs. CaP [24], compared to normal or benign CaP. Inhibition of ROCK activity in tumor cells decreases migration and invasive capacity in pancreatic cancer [25], [26]. Notably, an overexpression of Rho kinase signaling is correlated with metastasis and poor prognosis of the disease [27], [28]; this occurs by activating the downstream events of Rho-associated kinases 1 and 2 (ROCK1 and 2) [29], [30]. Activation of ROCK1 enhances the formation of stress fibers and cellular contraction, an essential part of cancer cell migration and invasion.

To our knowledge, this is the first study to have investigated the levels of miR-301a in paired serum and tumor tissues in CaP patients, and correlated miR-301a expression with Gleason score and tumor progression. Previously, we reported that miR-301a expression in prostate tumor tissues was higher compared to prostate tissue in patients with benign prostatic hypertrophy (BPH) [31]. In this study, we demonstrate that miR-301a expression in serum and tumor tissues correlates with the Gleason score and aggressiveness of the disease. We also identified that miR-301a inhibits the tumor suppressor function of RUNX3 and concomitantly activates Rho signaling in CaP.