LMX1B Activated Circular RNA GFRA1 Modulates the Tumorigenic Properties and Immune Escape of Prostate Cancer

Prostate cancer (PCa) is the most common cancer affecting men, with increasing global mortality and morbidity rates. Despite the progress in the diagnosis and treatment of PCa, patient outcomes remain poor, and novel therapeutic targets for PCa are urgently needed. Recently, circular RNAs (circRNAs) have been studied in-depth as potential biomarkers for many diseases. In this study, circRNA microarrays using four pairs of PCa tissues were utilized to show that circGFRA1 was upregulated in PCa tumor tissues. CircGFRA1 is suggested to play an oncogene role in PCa progression as the silencing of circGFRA1 inhibited the proliferation, migration, and immune escape activity of PCa cells. Furthermore, by utilizing bioinformatics analysis, RIP, RNA pull-down, and luciferase reporter assays, our results showed that LMX1B could bind to the GFRA1 promoter and regulate circGFRA1 expression in PCa cells and circGFRA1 upregulated HECTD1 expression through sponging miR-3064-5p. This novel LMX1B/circGFRA1/miR-3064-5p/HECTD1 axis identified in PCa provides new insights for developing novel therapeutic strategies for PCa.


Introduction
Prostate cancer (PCa) is ranked the second major cause of global human malignancy [1], with increasing mortality and morbidity rates in multiple countries in the past two decades [2]. In 2020, PCa contributed to 10% of cancerrelated deaths in men [3] and is considerable economic burden for health-care systems. Multiple molecular mechanisms in the initiation or progression of PCa have been explored, including aberrant RNA splicing, irregular ubiquitination and methylation, functional gene dysregulation, and DNA mutations [4,5], but the specific mechanism underlying PCa initiation and progression remains unclear.
This study aimed to identify a novel circRNA utilizing circRNA microarray analysis using four pairs of PCa tumor tissues and normal adjacent tissues. The analysis revealed that circGFRA1 was markedly upregulated in PCa tumor tissues and is induced by LMX1B, playing a promotive effect in PCa progression via the miR-3064-5p/HECTD1 axis.    2.2. Cell Culture and Treatment. Prostate cancer cell lines (DU145, PC-3, LNCap, and 22Rv1) and normal prostatic epithelial cells (RWPE-1) were purchased from the American Type Culture Collection and cultured in DMEM medium (Invitrogen, Thermo Fisher, USA) supplemented with 10% fetal bovine serum (Gibco) and 100 units/ml penicillin and 100 g/ml streptomycin in a 37°C environment with 5% CO 2 . The siRNA targets circGFRA1, LMX1B, and HECTD1, pcDNA3.1-LMX1B vector, and miR-3064-5p mimic were synthesized and obtained from Genepharm (Shanghai, China). All transfections were performed using a Lipofectamine® 3000 kit (Invitrogen; Thermo Fisher, USA) following the manufacturer's instructions.

Western
Blotting. RIPA lysis buffer (Beyotime, Shanghai, China) was used to extract proteins from cells and tissues. The protein samples were separated on a 10% SDS-PAGE gel and transferred to PVDF membranes (Millipore). The membranes were blocked with 5% nonfat milk before incubation with primary antibodies (LMX1B: CST, 1 : 1000, 13457S; HECTD1: Abcam, 1 : 5000, ab101992, GAPDH: Abcam, 1 μg/ml, ab37168) overnight at 4°C and then incubated with secondary antibodies for 2 h at room temperature. An enhanced chemiluminescent (ECL) system (Beyotime) was used to visualize the protein bands with GAPDH used as the internal control.
2.5. Cell Proliferation Detection. The Cell Counting Kit-8 (CCK-8) and 5-ethynyl-2′-deoxyuridine (EdU) assay were used to detect cell proliferation levels. For the CCK-8 assay, cells were seeded in a 96-well plate and cultured at 37°C with 5% CO 2 for three days before the addition of 10 μl of CCK-8 solution at each time point and cultured for 4 h. The optical density was measured at 450 nm. For the EdU assay, the cells   Journal of Immunology Research       Relative expression of LMX1B   2.9. Cytotoxicity Activity Analysis. To validate the cytokineinduced killer (CIK) cell-induced cytotoxicity towards PCa cells, the cells were co-cultured at 10: 1, 20 : 1, and 40 : 1 ratios, respectively, in a 37°C environment with 5% CO 2 for one day. Subsequently, 20 μl of CCK-8 solution was added to 100 μl of cell medium for 4 h before the optical density was measured at 450 nm. The survival rate was calculated as follows: survival ð%Þ = ðfunctional&target cell mixture − functional cellÞ/target cell * 100%.
2.11. RNA Pull-down. CircGFRA1 or miR-3064-5p was biotinylated to construct bio-circGFRA1 or bio-miR-3064-5p probes. Subsequently, 2 μg cell lysate was incubated with 100 pmol biotinylated probes before the addition of streptavidin agarose beads for 1 h at room temperature. The beads were boiled in SDS and analyzed by RT-qPCR.

Statistical
Analysis. The statistical analyses were performed using SPSS 19.0 software (IBM Corporation, USA). The data were presented as mean ± SD and subjected to one-way ANOVA and Student t-tests. All experiments were performed in triplicate, and a P value <0.05 was considered statistically different.

Expression of circGFRA1 in PCa.
The expression profile of circRNAs in four pairs of PCa tissues was examined by microarray analysis (Figure 1(a)      Journal of Immunology Research is derived from gene GFRA1 (GDNF family receptor alpha 1), was abundantly expressed in all four PCa tumor tissues compared to normal tissues. CircGFRA1 was also upregulated in PCa cell lines, especially in PC-3 and LNCap cells (Figure 1(b)). The schematic diagram of circGFRA1 is shown in Figure 1(c), and to confirm that circGFRA1 was indeed circular in PCa cells, cells are treated with the transcription inhibitor actinomycin D (ActD) showing that the half-life of circGFRA1 was significantly longer than GFRA1 mRNA (Figures 1(d) and 1(e)). Moreover, circGFRA1 was more resistant to RNase R digestion than its linear form (Figures 1(f) and 1(g)). Next, the use of random and oligo(dT)18 primers in reverse transcription to deplete cir-cRNAs in the 3 ′ pA tail, as expected, reduced circGFRA1 level in PC-3 and LNCap cells compared to the linear form (Figures 1(h) and 1(i)). In addition, the subcellular location assay showed that circGFRA1 was mainly distributed in the cell cytoplasm (Figures 1(j) and 1(k)), indicating that cir-cGFRA1 might participate in the cellular behaviors of PCa.

Silencing circGFRA1 Attenuates the Tumorigenic
Properties and Immune Escape of PCa. To study the function of circGFRA1, circGFRA1 but not GFRA1 was silenced in PC-3 and LNCap cells (Figures 2(a) and 2(b)), resulting in reduced cell proliferation (Figures 2(c)-2(f)) and cell migration (Figures 2(g) and 2(h)). It has been demonstrated that immune escape is essential for the development of PCa [18], and we determined the expression of immunosuppressive factors VEGF, IL-10, and TGF-β1 in the supernatant of circGFRA1 silenced PC-3 and LNCap cells. As shown in Figures 2(i)-2(k), VEGF, IL-10, and TGF-β1 are markedly decreased, and CIK cell-induced cytotoxic activity against circGFRA1 silenced cells was higher compared to normal control cells in the same conditions (Figures 2(l) and 2(m)), suggesting that silencing circGFRA1 inhibits the immune escape of PCa.

LMX1B Binds to GFRA1 and Upregulates circGFRA1
Expression in PCa Cells. Interestingly, putative LMX1B binding sites were identified in promoter regions of GFRA1, so the effects of LMX1B on circGFRA1 expression in PC-3 and LNCap cells were quantified by qRT-PCR, showing that circGFRA1 expression was upregulated by LMX1B overexpression in a dose-dependent manner (Figure 3(a)) and downregulated by LMX1B knockdown (Figure 3(b)). The schematic diagram of the putative binding sites of LMX1B (Figure 3(c)) or GFRA1 promoter regions is shown in Figure 3(d). The ChIP assay revealed that the P2 and P4 regions on the GFRA1 promoter were markedly enriched by anti-LMX1B compared to anti-IgG in PC-3 and LNCap cells (Figures 3(e) and 3(f)). Furthermore, the luciferase activity increased when the P2 or P4 regions on the GFRA1 promoter were mutated (Figure 3(g)) and was unchanged when both P2 and P4 regions on the GFRA1 promoter were mutated (Figure 3(h)). Taken together, these results suggest that LMX1B regulates circGFRA1 expression in PCa cells.

LMX1B Modulates the Tumorigenic Properties and
Immune Escape of PCa. To demonstrate the role of LMX1B in PCa, LMX1B expression was analyzed in thirty pairs of PCa tissues by qRT-PCR and then validated in five pairs of PCa tissues by western blotting. As shown in Figures 4(a) and 4(b), the expression of LMX1B in PCa tumors is markedly higher than in normal tissues. Furthermore, LMX1B was upregulated in PCa cell lines (Figure 4(c)). Loss-offunction studies by stably knocking down LMX1B expression in PC-3 and LNCap cells were conducted (Figures 4(d) and    Journal of Immunology Research 4(e)) showing that LMX1B knockdown significantly inhibited cell proliferation (Figures 4(f) and 4(i)) and markedly suppressed cell migration level (Figures 4(j) and 4(k)). Moreover, LMX1B knockdown significantly decreased VEGF, IL-10, and TGF-β1 in the supernatant of PCa cells (Figures 4(l)-4(n)) and markedly increased the CIK cellinduced cytotoxic activity towards PCa cells (Figures 4(l) and 4(m)).

CircGFRA1
Functions as a Molecular Sponge for miR-3064-5p. Since circRNA can function as a molecular sponge for microRNAs (miRNA), we analyzed whether circGFRA1 binds to miRNA through a competitive endogenous RNA (ceRNA) mechanism. It is well known that AGO2 is essential for the biogenesis and mature of miRNAs, and our results showed that circGFRA1 can bind to AGO2 suggesting that circGFRA1 may bind to miRNA in PCa cells ( Figure 5(a)). Subsequent bioinformatics analysis identified four miRNAs with relatively high scores, and the qRT-PCR assay results showed that only miR-3064-5p was abundantly pulled down by the circGFRA1 probe in PC-3 and LNCap cells ( Figure 5(b)), and miR-3064-5p expression was upregulated by circGFRA1 knockdown in PC-3 and LNCap cells ( Figure 5(c)). The predicted binding sites between cir-cGFRA1 and miR-3064-5p are shown in Figure 5(d), and the dual-luciferase reporter assay results suggested that cir-cGFRA1 can directly bind to miR-3064-5p in PCa cells.

Discussion
PCa is the most common cancer in men and a great threat to the genitourinary health of men [3]. In recent years, great progress has been made in the diagnosis or treatment of PCa with the identification of multiple diagnostic targets such as the main biomarker prostate-specific antigen (PSA), PCa antigen 3 (PCA3), the gene fusion test of TMPRSS2-ERG, circulating tumor cells, lncRNA biomarkers, and microRNA biomarkers [19][20][21][22]. PCa therapy involves radical prostatectomy or radical radiotherapy, endocrine therapy [23], and enzalutamide treatment when the PCa progresses to castration-resistant PCa [24]. However, despite the wide application of these new diagnostic biomarkers or treatment strategies, patients remain poor, so novel therapeutic targets for PCa are urgently needed. There is emerging evidence of the role of circRNA in multiple cancers, including PCa [25,26]. The present study showed that circGFRA1 was upregulated in PCa tumor tissues compared to normal tissues. CircGFRA1 has been investigated in several cancers, such as nonsmall cell lung cancer, ovarian cancer, breast cancer, and hepatocellular cancer [27][28][29][30], but its role in PCa has not been fully elucidated. Our results suggest that circGFRA1 plays an onco-gene role in PCa progression, which renewed the profile of circGFRA1 in tumorigenesis progression.
Recently, the interaction between circRNA and transcription factors has been demonstrated to be important for circRNA maintenance and function [31,32]. For instance, circRNA ARF1 expression in glioma stem cells is regulated by U2AF2 [33]. circRNA circHipk2 expression in C2C12 myoblasts is mediated by Sp1 [34], and circ-FOXP1 in hepatocellular carcinoma cells is regulated by SOX9 [9][10][11]. Therefore, we investigated the upstream regulator of circGFRA1 in PCa cells showing that circGFRA1 is regulated by LMX1B, which has previously been shown to be involved in many cancers, including ovarian, esophageal, and glioma [35][36][37].
Furthermore, circGFRA1 upregulated HECTD1 expression to promote PCa progression by sponging miR-3064-5p. Interestingly, despite the investigations of miR-3064-5p or HECTD1 in various cancers [38][39][40][41][42], no study has been conducted in PCa. The present study is the first to explore the biological or mechanical role of miR-3064-5p or HECTD1 in PCa, which might be useful for the basic research conducted in PCa.
Although this study has partially demonstrated the functional role of circGFRA1 in PCa, further investigation of more PCa tissues and the underlying molecular pathway of HECTD1 in PCa cellular behaviors are required to confirm the clinical significance of circGFRA1. In conclusion, the present study has partially revealed the involvement of the LMX1B/circGFRA1/miR-3064-5p/HECTD1 axis in PCa progression, providing new insights for developing novel diagnostic or therapeutic targets for PCa.

Data Availability
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.