Induction of promyelocytic leukemia zinc finger protein by miR-200c-3p restores sensitivity to anti-androgen therapy in androgen-refractory prostate cancer and inhibits the cancer progression via down-regulation of integrin α3β4

Androgen-refractory prostate cancer (ARPC) is one of the aggressive human cancers with metastatic capacity and resistance to androgen deprivation therapy (ADT). The present study investigated the genes responsible for ARPC progression and ADT resistance, and their regulatory mechanisms. Transcriptome analysis, co-immunoprecipitation, confocal microscopy, and FACS analysis were performed to determine differentially-expressed genes, integrin α3β4 heterodimer, and cancer stem cell (CSC) population. miRNA array, 3′-UTR reporter assay, ChIP assay, qPCR, and immunoblotting were used to determine differentially-expressed microRNAs, their binding to integrin transcripts, and gene expressions. A xenograft tumor model was used to assess tumor growth and metastasis. Metastatic ARPC cell lines (PC-3 and DU145) exhibiting significant downregulation of ZBTB16 and AR showed significantly upregulated ITGA3 and ITGB4. Silencing either one of the integrin α3β4 heterodimer significantly suppressed ARPC survival and CSC population. miRNA array and 3′-UTR reporter assay revealed that miR-200c-3p, the most strongly downregulated miRNA in ARPCs, directly bound to 3′-UTR of ITGA3 and ITGB4 to inhibit the gene expression. Concurrently, miR-200c-3p also increased PLZF expression, which, in turn, inhibited integrin α3β4 expression. Combination treatment with miR-200c-3p mimic and AR inhibitor enzalutamide showed synergistic inhibitory effects on ARPC cell survival in vitro and tumour growth and metastasis of ARPC xenografts in vivo, and the combination effect was greater than the mimic alone. This study demonstrated that miR-200c-3p treatment of ARPC is a promising therapeutic approach to restore the sensitivity to anti-androgen therapy and inhibit tumor growth and metastasis.


Introduction
Prostate cancer (PCa) is the second leading annual cause of cancer mortality in men worldwide [1], and the 5-year survival rate of the patients with distant metastasis remains dismal [2]. Initially, most prostate cancers respond to androgen deprivation therapy (ADT). However, ADT fails and the cancers progress to androgen-refractory state [3]. Recurrent and metastasised prostate cancer is considered castrationresistant or androgen-refractory prostate cancer (ARPC). ARPC also develops resistance to other treatment options including bone metastasis-targeted radiopharmaceuticals [4,5].
The promyelocytic leukaemia zinc finger (PLZF), also known as ZBTB16, is a zinc finger transcription factor. PLZF is widely distributed in many cell types including stem cells and progenitor cells, but its expression is higher in some tissues such as prostate than in other tissues [6]. PLZF knockout in mouse induced several defects in musculoskeletal development and germ cell production [7,8]. PLZF is also known to maintain self-renewal of stem cells, but the action is cell-type specific [9]. In cancers, PLZF expression level varies depending on cancer type; decrease in liver and pancreatic cancer but increase in colorectal cancer compared to normal tissue [10]. In prostate cancer, PLZF expression is inversely correlated with high grade [11]. PLZF expression is reported to be under control of AR [12], but the opposite seems to be possible: PLZF knock-down in androgen responsive prostate cancer cells (e.g., LNCaP) induces resistance to ADT and enhances androgen-independent growth of the cells [13]. Despite the various studies on the role of PLZF in cancer progression, it is still uncovered the exact regulatory mechanism on PLZF expression and its specific action in ARPC progression.
MicroRNAs (miRNAs), a class of small single-stranded non-coding RNA molecules targeting about 60% of the human genes [29,30], regulate expression of genes related to proliferation, differentiation, and apoptosis [31][32][33][34]. miRNAs in recurrent prostate cancer cells are differentially expressed compared to those in non-recurrent prostate cancer cells [35]. Differentially expressed miRNAs regulate CSC maintenance directly-by targeting CSC markers-as well as indirectly by targeting signalling molecules involved in EMT, metastasis, and drug resistance [36,37].
In the present study, we investigated how ARPCs acquire ADT resistance and metastatic phenotype through comprehensive analysis of genes and miRNAs differentially expressed between hormone-sensitive and ARPC cells.

Cell culture
Human prostate cancer cell lines (LNCaP, PC-3, and DU145) and human embryonic kidney cell line (HEK-293) purchased from the Korean Cell Line Bank (Seoul, South Korea) were cultured in DMEM/High glucose (DU145) or RPMI1640 (PC-3 and LNCaP) or MEM (HEK-293) supplemented with 10% fetal bovine serum, and maintained in a humidified incubator at 37 °C in a 5% CO 2 atmosphere.

Transcriptome analysis
mRNAs were extracted and analysed using the Nanostring nCounter PanCancer Progression and Cancer Pathway array kits and systems by PhileKorea (Seoul, South Korea). Genes with ≥ 2-fold change in expression (p < 0.05) were considered differentially expressed. The functional network of differentially expressed genes (DEGs) was analysed using STRING version 11.0 [38] or Panther classification system 16.0 [39]. In Gene Ontology (GO) enrichment analysis, pathways with false discovery rate (FDR) < 0.05 for the DEGs were considered the most significantly and differentially regulated. membranes (Amersham Life Science, Buckinghamshire, UK). After non-specific binding was blocked using 5% bovine serum albumin in TBST for 1 h, membranes were incubated overnight with anti-integrin β4 antibody (Cell Signalling Technology Inc. Danvers, MA, USA), or other antibodies (Abcam, Waltham, MA, USA), at 4 °C. Then, the membranes were washed and incubated with horseradish peroxidase-conjugated secondary antibody. β-Actin was used as loading control. Chemiluminescence was detected using ECL reagent on a luminescent image analyser, LAS-4000 mini.
For co-IP, total proteins were lysed in IP lysis buffer (Thermo Fisher Scientific; Waltham, MA, USA).

Sphere forming assay
Prostate cancer sphere formation assay was performed as described previously [40]. Briefly, cells (3 × 10 3 ) in prEGM media (Lonza, Basel, Switzerland) were seeded in the bottom of an ultra-low attachment 24 well plate after treatment with or without siRNA or miRNA for 72 h using Lipofectamine 2000. Images of spheres were captured and only those with a diameter > 50 μm were counted.

Cell viability and proliferation assay
For the measurement of cell viability, non-target siRNA (siNT)-or siRNA-transfected cells were seeded in a 96-well plate in a serum-starved (1%) medium. For the measurement of cell proliferation, the siRNA-transfected cells were seeded in a 96-well plate in a complete medium containing 10% FBS. After 48 h, the number of viable cells was measured by MTT assay as described earlier.

miRNA array and target gene prediction
miRNAs were extracted and analyzed for differential expression using Affymetrix miRNA kit and systems (Affymetix; Santa Clara, CA, USA) by Macrogen (Seoul, South Korea). miRNAs with ≥ 2-fold change were considered differentially expressed. False discovery rate (FDR) was controlled by adjusting the p-value using Benjamini-Hochberg algorithm. All Statistical tests and DEG visualisation were conducted using R 3.3.2.

Confocal laser-scanning microscopy
Briefly, cells (1 × 10 4 ) cultured on a 35-millimetre glass bottom dish were fixed with 4% paraformaldehyde prepared in PBS (pH 7.4), washed, and then incubated with 1% bovine serum albumin prepared in PBST for 1 h to prevent nonspecific binding. Then, the cells were incubated overnight with integrin α3 or β4 antibody solution at 4 °C, and probed with fluorescence-conjugated secondary antibodies in the dark for 1 h at 25 °C. Stained cells were washed and counterstained with DAPI. Images were captured at 200× magnification using a Nikon A1Si confocal microscope (Nikon Instruments Inc., Tokyo, Japan).

Statistical analysis
One-way ANOVA followed by Student-Newman-Keuls comparison (GraphPad Prism 8.0 software; San Diego, CA, USA) was used for statistical analysis. Data are presented as means ± SEMs, and p < 0.05 was considered significant.

Survival and growth of ARPC cells deficient AR and PLZF is maintained by overexpressed integrin α3 and β4
We investigated the molecules responsible for the growth of ARPC cells by comparing the gene expression levels of hormone-sensitive LNCaP and ARPC cell lines, PC-3 and DU145 [42]. Transcriptome analysis of the total 1,333 genes revealed significantly up-and down-regulated in ARPC cell lines (compared to LNCaP cells) ( Supplementary Fig. S1A). Functionally enriched pathway analysis of 147 genes that were significantly up-regulated in both PC-3 and DU145 ( Supplementary Fig. S1B) showed that the genes were associated with focal adhesion, signalling receptor binding, and integrin complex/signalling pathway as well as gonadotropin releasing hormone receptor (GnRHR) (Supplementary Fig. S1C and S1D). On the other hand, the most strongly down-regulated gene in both PC-3 and DU-145 cells was ZBTB16 ( Supplementary Fig. S1A and Supplementary Table S1). The 55 genes that were commonly down-regulated in the ARPC cell lines ( Supplementary Fig. S1E) were mostly associated with the GnRHR pathway and gene expression regulatory process (Supplementary Fig. S1G and S1F). The transcriptome analysis result indicates that in addition to the genes of GnRHR pathway, genes associated with integrin signalling pathway play a critical role in the ARPC cell survival and growth. Indeed, most of the integrin genes were up-regulated in ARPC cells, among which ITGA3 and ITGB4 were the most strongly and significantly upregulated ( Table 1). Similar to the mRNA levels, the protein levels of AR and PLZF in PC-3 and DU145 cells were almost not detectable (Fig. 1A). On the other hand, integrin α3 and β4 protein expressions were strongly increased in PC-3 and DU-145 cells which also showed E-cadherin down-regulation and Snail up-regulation, corresponding to the metastatic nature of the cells (Fig. 1A). The expression level of integrin α3 and β4 in PC-3 cells was stronger than DU145 cells. We, then, examined whether integrin α3 and β4 are involved in PLZF down-regulation as well as CSC population responsible for survival and growth of ARPC cells, by silencing ITGA3 and ITGB4. In silencing ITGA3 or ITGB4 with two different siRNA sequences in ARPC cells, For RT-PCR, mRNAs were extracted from cancer cells using TRIzol reagent, and cDNA was synthesised using GoScript Reverse Transcriptase kit (Promega Corporation, WI, USA). qPCR was performed using primers described above.

Anti-tumour and anti-metastatic activity measurements using a xenograft tumour model
The chick embryo experiments were approved beforehand by the Institutional Animal Care and Use Committee of Yeungnam University and were performed according to the guidelines issued by the Institute of Laboratory Animal Resources (1996) and Yeungnam University (The care and use of animals 2009).
On the 9th day of fertilised chicken egg incubation (37℃, 55% relative humidity), false air sac was generated on the relatively flat side of the eggs using a negative pressure technique. A small window (1 cm 2 ) was created on the false air sac surface, through which cancer cells (1.5 × 10 6 cells/CAM) labelled with cell-tracking red-florescent dye and mixed in 50% Matrigel were implanted on the exposed CAM. On the 5th day of implantation, tumour weight and vessel branch points within the tumour were analysed. Protein extracts from the tumor tissues were analyzed for integrin α3 and β4 expressions. For metastasis experiments, the lower CAM and liver of developing chicken embryo were collected to evaluate metastatic cells using fluorescence-aided Leica L2 microscope (Leica, Tokyo, Japan). The lower CAM and liver tissues were further analysed to detect human DNA hypoxanthine phosphoribosyltransferase (HPRT), ITGA3, and ITGB4 using PCR. PCSC population in ARPC cells was similar to that of CD44 + CD133 + α3 + or CD44 + CD133 + β4 + , indicating that PCSCs expressed integrin α3 and β4 (Fig. 2B). Consistent to the sphere forming ability, the PCSC CD44 + CD133 + α3 + or CD44 + CD133 + β4 + population counts were higher in PC-3 than in DU145 (Fig. 2B). To investigate whether integrin α3 and β4 were expressed specifically in PCSCs, the expression of α3 and β4 in non-stem cells (CD44 − CD133 − ) was also examined. The integrin α3 expression population in non-stem cells identified as CD44 − CD133 − α3 + was 4.23% in PC-3 and 3.20% in DU145, much lower than that in PCSCs, while CD44 − CD133 − β4 + population was 0.03% in PC-3 and 0.03% in DU145 (Fig. 2C). The results showed that integrin α3, which was expressed in non-stem cells, was further increased in PCSCs, while integrin β4 was only expressed in PCSCs. Nonetheless, silencing either ITGA3 or ITGB4 completely abolished CD44 + CD133 + PCSC in both PC-3 and DU-145 cells (Fig. 2D), suggesting that α3β4 heterodimer was critical for the PCSC maintenance.
siRNA sequence-2 (siRNA-2) was used throughout the experiments, as siRNA-2 of ITGA3 and ITGB4 was more selective (Fig. 1B). Knock-down of ITGA3 and ITGB4 did not change the expression level of PLZF, while AR expression levels were slightly increased by the siRNAs, but not significantly. However, ITGA3 and ITGB4 silencing significantly suppressed the protein expression of CD44 and stemness-associated TFs, Sox2, Oct4 and Nanog (Fig. 1B).
Consistently, ITGA3 or ITGB4 knockdown significantly inhibited sphere formation to the same level, regardless of differences in sphere-forming ability between ARPC and LNCaP cells (Fig. 1C). In addition, although ITGA3 or ITGB4 knock-down suppressed survival (Fig. 1D) and proliferation ( Supplementary Fig. S1H) of PC-3 cells by more than 50%, enzalutamide did not alter the ITGA3 or ITGB4-silenced cell viability, unlike the cells treated with non-target siRNA (siNT). Because integrins normally function as a αβ heterodimer, we investigated whether integrins α3 and β4 form a heterodimer. The co-IP result revealed that integrin α3 bound to β4, forming a heterodimer in PC-3 cells (Fig. 2A). Co-IP with α3 antibody resulted in a higher protein yield compared to Co-IP with β4 antibody, but Co-IP results in both cases were the same. In addition, the population of CD44 + CD133 + were predicted using TargetScan [44,45]. miR-130a-3p and miR-29b-1-5p showed negative correlation with ZBTB16 and AR transcripts ( Table 2). In contrast, miR-200c-3p and miR-148a-3p showed a positive correlation with ZBTB16, whereas miR-99a-5p showed no correlation with either ZBTB16 or AR as target genes in ARPC cells (Table 2). Accordingly, we focused on four miRNAs and further examined their interaction with highly up-and downregulated genes. Bioinformatic analysis revealed that the 3′-UTR of ITGA3 and ITGB4 contained putative binding sites for the four miRNAs ( Supplementary Fig. S2). However, the 3′-UTR reporter gene assay revealed that only the miR-200c-3p mimic significantly repressed the normalised luciferase activity of ITGA3 and ITGB4 reporter genes (Fig. 3C). In case of miR-148 mimic treatment, inhibition of the reporter gene activity was weak, and the mix of the four different miRNA mimics exhibited similar effect on the 3′-UTR of ITGA3 as the miR-200c-3p mimic. Likewise, the mix of the four different miRNA inhibitors did not alter the 3′-UTR reporter luciferase activity. Such regulatory activity of miR-200c-3p was also confirmed by mRNA (Fig. 3D) and protein (Fig. 3E, Supplementary Fig. S3A) expressions of integrin α3β4 in both LNCaP and PC-3 cells. The changes in α3β4 expression levels by miR-200c-3p mimic and inhibitors were also confirmed by fluorescence microscopy (Fig. 3F). Importantly, PLZF and AR were up-and downregulated by the miR-200c-3p mimic and inhibitor, respectively ( Fig. 3D and E). We also searched for relationships

Differentially expressed miRNAs regulate androgen-refractory genes (AR and PLZF) and integrin α3β4 in ARPC cell lines
Next, we investigated that the most significantly down-regulated AR and PLZF are involved in the up-regulation of ITGA3 and ITGB4. Along with differentially up-regulated ITGA3 and ITGB4, the top three down-regulated genes were ZBTB16, KLK3, and TMPRSS2 (Supplementary Table S1). Although these genes are under direct transcriptional regulation of AR [10,43], the relative expression of the three genes in ARPC cells was lower than that of AR (Supplementary Table S1), implicating molecules other than AR may be involved in down-regulation of the genes. To elucidate the involvement of miRNAs as master regulatory molecules associated with these gene expressions, miRNA array was performed. Among the total 2,578 mature miR-NAs, 184 and 314 miRNAs were differentially expressed in PC-3 and DU145 cells, respectively (compared to LNCaP cells) (Fig. 3A). The top two upregulated miRNAs in PC-3 and DU145 cells were miR-130a-3p and miR-29b-1-5p, while the top two downregulated miRNAs in PC-3 and DU145 cells were miR-200c-3p and miR-99a-5p in PC-3 versus miR-99a-5p and miR-148a-3p in DU145 (Fig. 3B,  Supplementary Table S2). In case of miR-148a-3p, relative expression in PC-3 and DU145 was similar (Supplementary Table S2). For the five miRNAs, putative target genes

Both miR-200c-3p and miR-200c-3p-induced PLZF suppress integrin α3β4 expression
As upregulation of integrin α3 and β4 by miR-200c-3p inhibitor was accompanied by downregulation of AR and PLZF, we examined whether miR-200c-3p-regulated PLZF was also involved in the expression of α3 and β4. In case of ChIP-qPCR with anti-AR antibody, there was no binding of AR to the integrin genes, whereas with anti-PLZF antibody it showed that PLZF directly bound to the ITGA3 and ITGB4 promoter in both LNCaP and PC-3 cells, with greater binding affinity to ITGA3 than to ITGB4 (Fig. 4A). Notably, PLZF binding to the genes was significantly increased by miR-200c-3p mimic (Fig. 4A). We then examined the enhanced binding of PLZF to ITGA3 and ITGB4 promoter induce down regulation of integrin α3β4 by comparing the effect of PLZF siRNA, miR-200c-3p, and combination of the two. Treatment of cells with miR-200c-3p mimic strongly enhanced PLZF expression, but suppressed between miR-200c-3p, PLZF, and AR expression in clinical samples using public databases. A previous human prostate cancer cohort study [46] has reported miR-200c-3p expression is significantly lower in metastatic ARPC than primary PCa. PLZF data mining of the public prostate cancer database PCaDB [47] using GSE2443 [48] confirmed lower PLZF expression in human ARPC samples than in androgen-dependent PCa samples (Supplementary Fig. S3B). The data mining results from clinical samples correlate to our present findings that miR-200c-3p and PLZF levels were lower in ARPC cells. Corresponding to those gene expression changes, spheroid formation was significantly inhibited by the miR-200c-3p mimic, but significantly increased by miR-200c-3p inhibitor in both LNCaP and ARPC cell lines (Fig. 3G). and PLZF expression changes, the population counts of PCSCs that also express α3 (Fig. 5A) and β4 (Fig. 5B) were completely suppressed by miR-200c-3p mimic. In contrast, 200c-3p inhibitor that induced PLZF down-regulation increased synergistically enhanced the PCSC population upon co-treatment with PLZF siRNA.
gene, HPRT level (Fig. 6F). Similarly, significant level of ITGA3 and ITGB4 genes in the metastases to liver and bottom side CAM was detected, which was almost fully inhibited by miR-200c-3p mimic, but not by enzalutamide alone (Fig. 6G).

Discussion
Here, we demonstrated the mechanism by which prostate cancer acquires resistance and metastatic potentials. Integrin α3β4 overexpressed in ARPC cells were functional as a heterodimer for PCSC maintenance, and the gene expression was suppressed by both transcription factor PLZF and miR-200c-3p. Notably, PLZF itself expression was induced by miR-200c-3p. By restoring miR-200c-3p level, which was the most strongly decreased in ARPCs, sensitivity to antiandrogen therapy was recovered in ARPCs, and the growth and metastasis of ARPC tumors were greatly suppressed. Transcriptome analysis revealed that highly metastatic ARPC cells expressed significantly higher level of integrins than LNCaP; integrin α3β4 expression being the highest.
of LNCaP xenograft, but not PC-3 xenograft (Fig. 6B C). However, when enzalutamide was co-administered with miR-200c-3p mimic, the growth of PC-3 xenografts was synergistically inhibited, and the effect was greater than that of miR-200c-3p mimic alone (Fig. 6B C). In tumor masses isolated from CAM tumours, integrin α3 and β4 expressions were not detected in LNCaP-tumors, but detected at significant levels in PC-3-tumours ( Fig. 6D and Supplementary  Fig. S4). The integrin α3 and β4 expressions were significantly suppressed by treatment with miR-200c-3p mimic, but not by enzalutamide. Co-administration with miR-200c-3p mimic and enzalutamide synergistically inhibited the α3 and β4 expressions, and the effect was greater than that of miR-200c-3p mimic alone ( Fig. 6D and Supplementary Fig. S4). Moreover, unlike LNCaP implants with non-detectable metastasis, PC-3 implants significantly metastasised to the developing chick liver and to the bottom side CAM (Fig. 6E). Importantly, PC-3 xenograft metastasis to the liver and lower CAM was completely blocked by miR-200c-3p mimic alone as well as by the combination treatment with miR-200c-3p mimic and enzalutamide, as revealed by determining human specific house-keeping , and for co-localisation of integrin α3 and β4 by confocal microscopy (F). *p < 0.05, compared to Mock, scrambled (SC) miRNA, or NT siRNA. # p < 0.05, compared to PLZF siRNA-treated group. $ p < 0.05, compared to miR-200c-3p mimic or inhibitor alone-treated group. (G) The viability of cells transfected with miR-200c-3p mimic was measured in the absence and presence of enzalutamide. *p < 0.05, compared to miR-SC-treated control. # p < 0.05, compared to vehicle-treated group by each other, and that α3β4 complex was the major player in maintaining prostate CSCs.
Although a cross-talk existed between integrin α3 and β4, the major regulator of α3β4 expression was miR-200c-3p. miR-200c-3p binds directly to the 3′-UTR of integrin α3β4, resulting in post-transcriptional repression, in spite of the fact that no canonical binding site was predicted in the 3′-UTR of integrin α3 or β4 that matched the miR-200c-3p seed region. Rather, compensatory pairing sites were centered at 13-17th miRNA nucleotides at the 5′ end and extended more than five contiguous Watson-Crick pairs [56]. Regardless of canonical or compensatory sites, the pairing and binding of miRNA to target mRNA causes posttranscriptional repression [30], which was further proved in the present study through down-and up-regulation of the genes by miR-200c-3p mimic and inhibitor, respectively.
Another way via which miR-200c-3p regulated α3β4 expression involved PLZF, which binds to α3β4 DNA and represses transcription of these genes. As a transcription repressor, PLZF exhibited greater capacity to bind α3 than to β4. 200c-3p mimic up-regulated both AR and PLZF, but only PLZF was involved in α3β4 gene expression. Interestingly, stringent seed pairing predicted PLZF as one of the target genes of both upregulated miRNAs (miR-130a-3p Studies have shown that prostate primary tumour CSCs expressing integrin α2β1 high are more invasive than the cells not expressing α2β1 [49,50]. Metastatic ARPCs with upregulation of α2β1, and much higher expression of α3β4 levels than α2β1, correlated with the degree of CSC enrichment in ARPCs. As integrins regulate maintenance of CSCs contributing to cancer progression and heterogeneity [51,52], α3β4 silencing in the present study resulted in significant decrease in CSC counts, consequently, tumour growth and metastasis of ARPCs. Consistent with previous reports [28,53,54], our results also indicate that prostate cancer aggressiveness was dependent on the expression of integrin β4. Additionally, our results revealed integrin α3 upregulation in ARPC, highlighting its important role in prostate cancer aggressiveness and transformation into ARPC. Notably, unlike normal prostate stem cells in which integrin β4 is tightly associated with α6 [55], our study revealed that integrin β4 also work together with α3 in metastatic ARPCs. Integrin α6 expression was much less than β4, whereas α3 expression was more than enough to form dimer with β4 in PC and DU145, as demonstrated by co-IP. More importantly, silencing either α3 or β4 inhibited expression of both α3 and β4 along with complete suppression of CSC formation, indicating that α3 and β4 were mutually inter-regulated α3-APC, and integrin β4-PE-CY7, before analysing by FACS. Bar graph indicates relative number of triple-positive (CD44 + CD133 + α3 + or CD44 + CD133 + β4 + ) CSC population (n = 3). *p < 0.05, compared to Mock, scrambled (SC) miRNA, or NT siRNA. # p < 0.05, compared to PLZF siRNA-treated group. $ p < 0.05, compared to miR-200c-3p mimic or inhibitor alone-treated group LNCaP xenografts. Despite the anti-tumour activity of miR-200c-3p being proven in prostate cancer [62], our study provides more detailed mechanism by which reduced miR-200c-3p levels induce anti-androgen resistance and metastasis in vivo. In both LNCaP and PC-3 xenografts, the effect of combination treatment with miR-200c-3p mimic and enzalutamide on the tumour growth was much greater than that of miR-200c-3p mimic or enzalutamide alone. More importantly, miR-200c-3p mimic almost completely inhibited metastasis of PC-3 xenografts, indicating the excellency in the therapeutic efficacy of miR-200c-3p in ARPC.
In this study, we demonstrated that the relatively lower expression of ZBTB16 (PLZF) than AR in ARPC cells, as determined by transcriptome analysis, was due to the fact that besides AR, other molecule such as miR-200c-3p, was involved in the regulation of PLZF expression. In addition, through ChIP-qPCR analysis, we also demonstrated that PLZF binds to the ITGA3 and ITGB4 gene promoters, while AR does not. Taken together, the results suggest and miR-29b-1-5p) and downregulated miRNAs (miR-200c-3p and miR-148a-3p), which did not follow classical miRNA actions, i.e., translational repression or mRNA degradation. Rather, regulation of PLZF expression by miR-200c-3p seemed to involve recently discovered miRNA functions, such as post-transcriptional upregulation of target genes through direct and indirect mechanisms, ligand for cell surface receptors, or nuclear transcription factor activator [57][58][59]. 3′-UTR reporter assay and ChIP-qPCR confirmed that miR-200c-3p performed a dual regulatory action in α3β4 expression, i.e., PLZF-mediated and direct binding to the genes.
Concurrent with the correlation between PLZF levels and inhibition of prostate cancer proliferation [12,60,61], our in vivo results confirmed that tumour growth of PC-3 xenografts with non-detectable PLZF was greater than that of LNCaP xenografts with more PLZF. Further, the miR-200c-3p mimic inhibited the growth of enzalutamideinsensitive PC-3 xenografts to the same extent as that of The D and V represent dorsal and ventral side of cancer-inoculated membrane, respectively. Tumour masses harvested (B) were further analyzed for their weight (C), and the number of new vessel branches formed on CAM were counted using ImageJ (C). *p < 0.05, compared to LNCaP cells. # p < 0.05, compared to Mock-treated group. $ p < 0.05, compared to miR-200c-3p mimic or enzalutamide alone-treated group. Integrin α3 and β4 expression in tumour tissues were analyzed by immunoblotting (D). (E-G) LNCaP and PC-3 cells labelled with CMTPX, a cell-tracker emitting red fluorescence, were implanted on the CAM tissue. For detection of metastasis of xenografts, lower CAM, the opposite site of tumour implants, and liver were collected at day 14, and photographed using stereo and fluorescence microscopy (E). The human cancer cells that had metastasised into the bottom side CAM and liver tissues were quantified based on qPCR for human HPRT (F). ITGA3 and ITGB4 gene expressions in metastases of liver and bottom side CAM were analyzed by qPCR (G). *p < 0.05, compared to LNCaP cells. # p < 0.05 compared to the vehicle or miR-SC-treated group that miR-200c-3p regulates the expression of both AR and PLZF, but unlike PLZF, AR is not involved in the expression of integrins α3 and β4. That is, the expression of integrins α3 and β4 is regulated through the miR-200c-3p-PLZF axis but not the 200c-3p-AR-PLZF axis.