CircRNA-UCK2 increased TET1 inhibits proliferation and invasion of prostate cancer cells via sponge miRNA-767-5p

2.1 Cell culture

The human PCa cell line C4-2 was obtained from the American Type Culture Collection (ATCC, Rockville, MD, USA) and cultured in RPMI-1640 supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin, in a 5% (v/v) CO2 humidified incubator at 37°C. EnzR-C4-2 cells were generated by culturing the EnzS-C4-2 cells in the presence of increasing Enz concentrations from 10–40 μM (every 20 days) for 3 months. All EnzR cells were maintained with 10 µM Enz.

2.2 MTT assay

Cell proliferation was evaluated by MTT assay as reported work [14]. The cells were seeded in 96-well plate at a density of 1000 cells/well, adhered overnight, and cultured at 37°C, 5% CO2 for days 2, 4, and 6. Then, the cells were harvested and the cell number was calculated using an MTT reagent. DMSO was used as the control.

2.3 Tests of RNase R resistance

The total RNAs were isolated from cells using TRIzol (Invitrogen), followed by PureLink purification of the aqueous phase (Life Technologies). An equivalent of 1 µg of total RNA was treated with 0 (mock treatment) or 20 U of RNase R (Epicenter) in 1X RNase R buffer in a 10 µL reaction containing 1U/µL murine ribonuclease inhibitor (New England Biolabs). The reaction was incubated at 37°C for 1 h, followed by the addition of 1 µL of 10 mM dNTP, 1 µL of 1 mM EDTA, and 1 µL of 100 µM random hexamers. The RNA was denatured at 65°C for 5 min and placed on ice. Then, 4 µL of buffer (250 mM Tris-HCl pH 8, 125 mM KCl, 15 mM MgCl2), 1 µL murine ribonuclease inhibitor (40 U/ µL), and 1 µL Superscript III (Life Technologies) constituted the reaction for the synthesis of cDNA at 25°C for 10 min, 50°C 50 min, 55°C 10 min, 85°C 5 min, then held at 4°C. Subsequently, 1 µL cDNA reaction was adopted as the template for qPCR.

2.4 Lentiviral expression plasmids and virus production

The plasmids pLCDH-circUCK2, Plv-miR-767-5p, pLKO.1-TET1, the psPAX2 packaging plasmid, and pMD2.G envelope plasmid, were transfected into HEK-293T cells using the standard calcium chloride transfection method for 48 h to obtain the lentivirus soup. These soups were collected, concentrated by density gradient centrifugation, and used immediately or preserved at -80°C for later use.

2.5 RNA extraction and quantitative real-time PCR (qRT-PCR) analysis

The total RNA was extracted using TRIzol reagent. An equivalent of 1 µg of total RNA was subjected to reversed transcription into cDNA using PrimeScript™ RT reagent Kit(Takara) and Mir-X™ miRNA qRT-PCR SYBR® Kit (Takara). The mRNA and miRNA expression of the target gene was determined by qRT-PCR conducted on ABI-7900 system with SYBR Green (Takara). The expression of a target gene was normalized to that of GAPDH or U6.

2.6 Western Blot Analysis

We follow the method with reported work[15]. Cells were lysed in RIPA buffer, and an equivalent of 40 µg total protein was separated on 10% SDS-PAGE and transferred onto 0.45 μm polyvinylidene fluoride (PVDF) membranes (Millipore). The membranes were blocked and probed with specific primary antibodies, followed by incubation with HRP-conjugated secondary antibodies. The immunoreactive bands were visualized using the ECL system (Thermo Fisher Scientific).

2.7 Transwell invasion assay

The cell invasion capacity was assessed by a Matrigel invasion assay by coating the Matrigel on 8.0-μm filter membranes. An equivalent of 1 × 105 cells in 150 μL serumfree medium was plated onto each filter, with 750 μL of 10% fetal bovine serum-containing medium placed in the lower chamber. The system was incubated for 24 h in a humidified tissue culture incubator at 37°C in a 5% CO2 atmosphere. After 24 h, the membranes were fixed with 4% paraformaldehyde on both sides, washed with phosphate buffer saline (PBS), and stained with crystal violet. The cells on the upper surface of the filters were removed with cotton swabs, and those that invaded the lower surface of the filter were counted under the microscope. The relative invasion was determined by setting the number of invading cells in the ethanol-treated group as one.

2.8 Pull-down assay

The cells were harvested and lysed in RIPA lysis buffer. The supernatant was incubated with 500 pM of anti-sense oligos supplemented with RNase inhibitor overnight at 4°C. Subsequently, 10u µL Streptavidin Agarose beads were added and to the cells, and the mixture homogenized for 2 h at 4°C. Then, the streptavidin Agarose beads were incubated with the supernatant for 2 h. The complex was centrifuged at a speed of 3000 rpm for 10 mins, following which, the beads were washed five times with RIPA lysis buffer. The RNA was extracted using TRIzol according to the manufacturer’s protocol and subjected to RT-PCR analysis.

2.9 In vivo studies

7-week-old NOD/SCID mice were purchased from Slac Laboratory Animal and divided into two groups (9 mice/ group) for injection of 1×106 EnzR-C4-2 cells and pre-cultured as follows: 1: pLCDH; 2: oe-circUCK2. A total of 1x106 EnzR-C4-2 cells were mixed with Matrigel (1:1) and transplanted into the prostate of the male NOD/SCID mice. Next, the mice were injected with 20mg/kg enzalutamide intravenously every 2 days. Tumour development was monitored by non-invasive In Vivo Fluorescent Imager (IVIS Spectrum, Caliper Life Sciences, Cleveland, USA) once per week. The mice were sacrificed after 8 weeks, and any metastatic tumours were removed for subsequent analysis. This study revealed in specific pathogen-free (SPF) conditions in strict accordance with the Ethics Committee of Tongji Hospital.

2.10 Immunohistochemistry (IHC) staining

Mouse tissues were fixed in 10% (v/v) formaldehyde in PBS and embedded in paraffin. The samples were sliced into 5-μm-thick sections, dehydrated with xylene, and used for IHC staining with a specific primary antibody against TET1. To enhance antigen exposure, the slides were treated with 1X EDTA at 98°C for 10 min for antigen retrieval. The slices were incubated with endogenous peroxidase blocking solution, followed by incubation with the primary antibody TET1 overnight at 4 ºC. After rinsing with the phosphate buffer saline, the slides were incubated with biotinylated secondary antibody for 1 h at room temperature, washed, and incubated with enzyme conjugated horseradish peroxidase (HRP)-streptavidin. Freshly prepared DAB (Zymed, San Francisco, CA, USA) was used as a substrate to detect the HRP. Finally, slides were counterstained with hematoxylin and mounted with aqueous mounting media. The number of positive cells was calculated as the number of immune-positive cells × 100% / the total number of cells/field in 10 random fields at 400X magnification.

2.11 Statistical analysis

All statistical analyses were carried out using SPSS 19.0 (SPSS Inc, Chicago, IL, USA). Data were presented as the mean ± SD or SEM. The differences in the mean values between two groups were analyzed by two-tailed Student’s t-test, and the mean values of more than two groups were compared using one-way ANOVA. p ≤ 0.05 was considered statistically significant.

3.1 CircUCK2 decrease EnzR cell proliferation and invasion

PCa eventually progressed to castration-resistant PCa after antiandrogen therapy[16], and enzalutamide only extended the patient’s survival an extra 4.8 months[17]. The Transwell invasion assay showed that EnzR-C4-2 cells were more invasive than EnzS-C4-2 cells (Fig. 1A). In our previous study, circRNAs were found to play a role in the development of PCa. Based on the previous results, ten genes related to tumour growth and invasion were identified as candidates through prediction literature research and database. Hsa_circ-001128 and hsa_circ_001357 (circUCK2) were found to be significantly decreased in EnzR-C4-2 cells compared to EnzS-C4-2 cells (Fig. 1B). CircUCK2 was insensitive to RNase-R, which was confirmed by RNase-R assay. (Fig. 1C). We found that circUCK2 can change the proliferation and invasion abilities of PCa cells, while hsa_circ-001128 cannot. EnzS-C4-2 and circUCK2 knockdown can accelerate cell proliferation and invasion (Fig. 1D-E). After the overexpression of circUCK2, cell proliferation slowed down, and invasion decreased (Fig. 1F-G).

Figure 1

CircUCK2 decrease EnzR cell proliferation and invasion. (A) Transwell invasion assay was performed to show the different invasion capacity in Enzalutamide-sensitive C4-2 cell line (EnzS-C4-2) and Enzalutamide-resistance C4-2 cell line (EnzR-C4-2). (B) Ten circRNAs related to cell proliferation and invasion were screened from the literature and expressed differently in the comparator between EnzS cells and EnzR cells. (C) The RNase-R assay was used to determine the sensitivity of circUCK2 to RNase digestion. (D-E) Knocking down circUCK2 in EnzS-C4-2 cells leads to increase cell proliferation and invasion. (F-G) The overexpression of circUCK2 in EnzR-C4-2 cells leads to decreased cell proliferation and invasion. The data shown represent the mean of three independent experiments. *p < 0.05 by Student’s t-test for two groups or ANOVA for more than two groups.

Taken together, the results from Fig. 1A–G suggested that circUCK2 can suppress the growth of EnzR PCa cells.

3.2 Mechanism dissection of how circUCK2 can suppress the PCa cell growth: via sponge miR-767-5p

To detect whether circUCK2 can regulate the expression of miRNAs, we screened 72 circUCK2-related miRNAs and did not find a significant increase in miRNAs when we knocked down circUCK2 in EnzS-C4-2 cells (Fig. 2A). Moreover, predictive analysis showed that circUCK2 has binding sites of miR-767-5p (Fig. 2B), suggesting a vital role of circUCK2 through the miRNA sponge. Then, the pull-down assay using the biotinylated oligo was employed to examine the interaction of circUCK2 with miR-767-5p in EnzR-C4-2 cells, and the results revealed that circUCK2 could interact with miR-767-5p (Fig. 2C). To verify that circUCK2 inhibits EnzR-C4-2 cells by lowering the level of miR-767-5p, we co-overexpressed circUCK2 and miR-767-5p. The MTT assay showed that miR-767-5p could reverse the inhibitory effect of circUCK2 on cell proliferation (Fig. 2D) and cell invasion (Fig. 2E).

Figure 2

Mechanism dissection of how circUCK2 can suppress PCa cell growth via sponge the miR-767-5p. (A) The expression of miRNAs related to circUCK2 was determined by RT-PCR after knocking down circUCK2 in EnzS-C4-2 cells. (B) According to the prediction of Bioinformatics tools (miRBase, circRBase, and RNA22), circUCK2 has binding sites with miR-767-5p. (C) The miR-767-5p can physically interact with circUCK2. The biotinylated oligo that is complementary to the junction region of circUCK2 is mixed with the EnzR-C4-2 cell lysate, followed by purification with streptavidin beads. miR-767-5p was quantified in the pull-down complex as well as the control complex without the biotinylated oligo. (D-E) MTT assay shows that cell proliferation (D) and invasion (E) inhibition effect by overexpressing circUCK2 can be reversed by overexpressing miR-767-5p in EnzR-C4-2 cells. The data shown represent the mean of three independent experiments. *p < 0.05 by Student’s t-test for two groups or ANOVA for more than two groups.

Together, these results suggested that circUCK2 can sponge the miR-767-5p to reduce the proliferation and invasion ability of EnzR-C4-2 cells.

3.3 Mechanism dissection of how circUCK2 can suppress the PCa cell growth by increasing the TET1 expression

To understand the mechanism underlying the miR-767-5p-regulated cell proliferation and invasion, we examined the literature and bioinformatics tools (miRbase, Target-scan, and RNA22) and found that miR-767-5p can interact with the 3’-UTR of TET1 (Fig. 3A). Moreover, the expression of TET1 was decreased in EnzR-C4-2 cells (Fig. 3B). TET1 was also increased when circUCK2 was overexpressed and could be reversed by overexpressing miR-767-5p in EnzR-C4-2 cells (Fig. 3C). Furthermore, we knocked down TET1 in EnzR cells overexpressing circUCK2 and found that the cell proliferation was increased (Fig. 3D). In addition, the cell invasion could be reversed by knocking down TET1 in EnzR-C4-2 cells (Fig. 3E).

Figure 3

Mechanism dissection of how circUCK2 can suppress the PCa cell growth: via increase TET1 expression. (A) After searching the literature and database, we found that miR-767-5p can interact with 3’-UTR of TET1. (B) West blotting shows that the expression of TET1 was decreased in EnzR-C4-2 cells. (C) West blotting shows that the level of TET1 was increased by overexpressing circUCK2, which can be reversed by overexpressing miR-767-5p in EnzR-C4-2 cells. (D-E) MTT assay shows that cell proliferation (D) and invasion (E) inhibition by overexpressing circUCK2 can be reversed by knocking down TET1 in EnzR-C4-2 cells. The data shown represent the mean of three independent experiments. *p < 0.05 by Student’s t-test for two groups or ANOVA for more than two groups.

Together, these results Fig. 3A–E suggested that circUCK2 inhibits cell proliferation and invasion by increasing the level of TET1.

3.4 Preclinical study using the mouse model to prove that circUCK2 can inhibit EnzR cell growth

To elucidate whether the high expression of circUCK2 gene can slow down the cell proliferation in vivo experiments, NOD/SCID mice were transplanted with EnzR-C4-2 pLCDH cells and EnzR-C4-2 oe-circUCK2 cells in situ. After 8 weeks, the EnzR-C4-2 circUCK2 group was found to have a smaller tumour size than the control group (Fig. 4A). IHC showed an increased expression of tumour TET1 protein in the EnzR-C4-2 circUCK2 group (Fig. 4B).

Figure 4

Preclinical study using the mouse model to prove that circUCK2 can inhibit the EnzR cell growth. (A) EnzR-C4-2 cells were transfected with pLCDH and circUCK2 and implanted into NDO/SCID mice. After 8 weeks, the tumour weights of the xenografts were tabulated and shown in dot plot. (B) The expression of TET1 in the two groups of mice. (C) Schematic of the circUCK2 pathway. The data shown represent the mean of 9 NDO/SCID mice in each group. *p < 0.05 by Student’s t-test for two groups.

Together, the in vivo results showed that high circUCK2 expression inhibits the EnzR-C4-2 cell growth.