Higher level of miR-423-3p in CRPC exosomes suggested that miR-423-3p could potentially promote tumour progression. To evaluate the function of miR-423-3p prostate cancer cells in relation to CRPC development, I overexpressed miR-423- 3p in hormone-dependent PCa cell line, LNCaP, and evaluated the cell proliferation, migration and invasion under the hormone-depleted environment. The increased migration and invasion ability of LNCaP cells overexpressing miR- 423-3p indicated that miR-423-3p could induce hormone-independent migration and invasion of LNCaP cells. The higher expression of miR-423-3p in C4-2 cells and higher migration and invasion ability of C4-2 cells compared to LNCaP cells also supported CRPC development is associated with increased cancer cell migration/invasion ability and miR-423-3p could promote CRPC via promoting hormone-independent migration and invasion.
Figure 33. Bioanalyzer evaluation of RNA extracted from PC3 cell exosomes. MiRNAs are < 40 nt and the peak at 60 nt represents tRNAs.
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MiR-423-3p has been reported to act as an oncogene via promoting cell proliferation, cell migration and invasion [402, 405, 406]. However, I did not observe changes in cell proliferation when I overexpressed miR-423-3p in LNCaP cells in hormone-depleted medium. My data indicated that miR-423-3p is mainly associated with cell migration and invasion in castration environment. Some studies have revealed that miR-423-3p is involved in cancer metastasis. In lung adenocarcinoma, miR-423-3p was found highly expressed in patients with brain metastasis [406] and overexpression of miR-423-3p promoted lung cancer cell migration [407]. CRPC is highly related to metastasis, as only small proportion of CRPC patients have non-metastatic disease when diagnosed with CRPC and about half of the non-metastatic CRPC patients would develop metastasis within 3 years of diagnosis [408]. Thus, it is potential that miR-423-3p promoted CRPC development by inducing cell migration and invasion. Further study is warranted to understand the role of miR-423-3p in CRPC and the association of cancer cell invasion with CRPC development.
MiRNAs downregulate gene expression through incorporation into the RISC, which then binds to partially complementary sites mainly in the 3′UTR (also can be in 5’ UTR) of their mRNA targets. According to TargetScan 7.1, there are in total 18 predicted target genes of miR-423-3p with conserved sites and MEIS1 is the top third target. Although it is not predicted by other prediction tool, this lower expression of this gene has shown positive association with PCa progression, while there is limited information of other predicted targets based on literatures. MEIS1 is a homeodomain transcription factor. In PCa, MEIS1 level exhibits a stepwise decrease in from benign epithelia, to primary tumour, and then to metastatic tissues and low expression of MEIS1 is associated with poor prognosis [409, 410]. MEIS1 has been identified as an AR negative regulator, which could inhibit the AR transcriptional activity and reduced the expression of AR target gene as well as disrupt the cytoplasm to nucleus translocation of AR in response to androgen [411]. Thus, I decided to explore if miR-423-3p could function as a regulator of MEIS1 in PCa cells. My WB results showed that over-expression of miR-423-3p decreased the protein level of MEIS1, but the effect was not big. This result cannot rule out the potential that MEIS1 is a miR-423-3p target. Further studies will be done to investigate if MEIS1 is a direct target of miR-423-3p.
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Other genes may also be involved in the development of CRPC via regulation by miR-423-3p. In hepatocellular carcinoma [412], lung adenocarcinoma[406], and colorectal cancer [405], it has been demonstrated that miR-423-3p induces cell proliferation and invasion by directly targeting p21 (WAF1/CIP1), a cyclin dependent kinase inhibitor, although p21 (WAF1/CIP1) is not predicted as target of miR-423-3p either by Targetscan or miRDB. Previous reports of p21 (WAF1/CIP1) function in PCa progression are controversial. Early studies found p21 (WAF1/CIP1) is highly expressed in androgen-independent tumours and correlates with poor prognosis [413, 414], while other studies showed increased p21(WAF1/CIP1) production directly suppresses proliferation of PCa cells regardless of androgen sensitivity [415] and downregulation of both p21(WAF1/CIP1) and p27/Kip1 could produces a more aggressive prostate cancer phenotype[416]. Further studies should be done to determine the interaction of miR-423-3p and p21(WAF1/CIP1) and their role in PCa progression. As I found different expression levels of miR-423-3p in plasma exosomes of CRPC and non-CRPC patients, it would be interesting to explore the potential role of exosomal miR-423-3p. The exosomes in plasma may come from various cell types. Currently we do not have evidence of the source of the exosomes. However, we observed higher levels of miR-423-3p in C4-2 cells compared to LNCaP cells, indicating that plasma exosomes miR-423-3p expression potentially resembles that in the tumour tissue. It has been demonstrated that exosomes from malignant cells could deliver miRNAs to recipient cells and induce their change from non-malignant to malignant [197, 417]. Due to short of time, the role of exosome-derived miRNAs in CRPC development have not been elucidated in the present study. However, I have tested method of ultracentrifugation to enrich exosomes from cell culture medium, which may facilitate further studies exploring the mechanism of miR-423-3p packaging in exosomes, and investigation of whether exosomes can deliver exogenous miR-423-3p to LNCaP cells and mediate its hormone-sensitivity. Exosomal miRNAs have also been demonstrated to playing important roles in regulating cancer microenvironment and promoting metastasis [203, 418, 419]. The effect of exosomal miRNAs on other cells, for example cancer-associated fibroblast and immune cells may also be explored.
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