MiR-141-3p promotes malignant progression in prostate cancer through AlkB homolog 5-mediated m6A modification of protein arginine methyltransferase 6

Xun Li; Bide Liu; Shuheng Wang; Jiuzhi Li; Xiaohu Ge

doi:10.4103/cjop.CJOP-D-22-00071

ORIGINAL ARTICLE

Year : 2023 | Volume : 66 | Issue : 1 | Page : 43-51

MiR-141-3p promotes malignant progression in prostate cancer through AlkB homolog 5-mediated m⁶A modification of protein arginine methyltransferase 6

Xun Li¹, Bide Liu², Shuheng Wang², Jiuzhi Li², Xiaohu Ge³
¹ Xinjiang Medical University, Urumqi, China
² Department of Urology, People's Hospital of Xinjiang Uygur Autonomous Region, Urumqi, China
³ Department of Vascular Surgery, People's Hospital of Xinjiang Uygur Autonomous Region, Urumqi, China

Date of Submission	08-Aug-2022
Date of Decision	21-Oct-2022
Date of Acceptance	27-Oct-2022
Date of Web Publication	20-Jan-2023

Correspondence Address:
Dr. Xiaohu Ge
Department of Vascular Surgery, People's Hospital of Xinjiang Uygur Autonomous Region, No. 91, Tianchi Road, Urumqi, Xinjiang Uygur Autonomous Region
China

Source of Support: None, Conflict of Interest: None

DOI: 10.4103/cjop.CJOP-D-22-00071

Abstract

Prostate cancer (PCa) is one of the leading causes of cancer-related death in males worldwide and exploring more reliable biomarkers for PCa is essential for the diagnosis and therapeutics for the disease. Although the functions of miR-141-3p and AlkB homolog 5 (ALKBH5) were identified in some cancers, whether they were involved in the development of PCa remains unclear. In this study, reverse transcription-quantitative polymerase chain reaction unveiled that the expression of ALKBH5 was reduced in PCa tissues and was negatively correlated with miR-141-3p. ALKBH5 attenuated the malignant development of PCa through suppressing the growth, migration, invasion, and sphere formation abilities of PCa cells. In addition, the luciferase activity assay identified that ALKBH5 was corroborated as a downstream target of miR-141-3p. Moreover, miR-141-3p expression was boosted in PCa tissues and cells and inhibition of miR-141-3p suppressed the tumor growth of PCa in vivo. Moreover, ALKBH5 was confirmed to suppress protein arginine methyltransferase 6 (PRMT6) expression through N6-methyladenosine (m⁶A) modification. We further identified that miR-141-3p-modulated PRMT6 level through mediating ALKBH5. Furthermore, PRMT6 level was positively correlated with miR-141-3p level and negatively associated with ALKBH5 level. Finally, rescue assays also uncovered that miR-141-3p aggravated PCa development by regulating PRMT6. In conclusion, miR-141-3p accelerated the malignant progression of PCa through ALKBH5-mediated m6A modification of PRMT6, which might offer a novel insight into the role of miR-141-3p and ALKBH5 in the treatments of PCa patients.

Keywords: miR-141-3p, prostate cancer, AlkB homolog 5, m⁶A, protein arginine methyltransferase 6

How to cite this article:
Li X, Liu B, Wang S, Li J, Ge X. MiR-141-3p promotes malignant progression in prostate cancer through AlkB homolog 5-mediated m⁶A modification of protein arginine methyltransferase 6. Chin J Physiol 2023;66:43-51

How to cite this URL:
Li X, Liu B, Wang S, Li J, Ge X. MiR-141-3p promotes malignant progression in prostate cancer through AlkB homolog 5-mediated m⁶A modification of protein arginine methyltransferase 6. Chin J Physiol [serial online] 2023 [cited 2023 Sep 28];66:43-51. Available from: https://www.cjphysiology.org/text.asp?2023/66/1/43/370012

Introduction

Prostate cancer (PCa) is a malignant cancer occurring in the epithelial cells of the prostate, and is one of the major causes of cancer-related death in males worldwide.^[1] It has been reported that metastasis occurred in approximately 10% of newly diagnosed PCa patients and 5% of patients eventually developed metastasis postsurgery.^[2],[3] At present, the most common treatments for PCa patients were active surveillance, radical prostatectomy, brachytherapy, and external beam radiotherapy.^[4],[5] Despite increasing efforts by experts were made to improve the treatments of PCa, the 5-year overall survival of PCa patients was still unfavorable because of the lack of reliable biomarkers for the detection and treatments.^[6] Thus, exploring more reliable biomarkers for PCa is crucial for the diagnosis and therapeutics for the diseases.

In recent years, growing studies have identified that the abnormal expression of microRNAs (miRNAs) might affect the development of a variety of diseases.^[7],[8],[9] As an endogenous, and highly conserved single-stranded non-coding small RNAs, miRNAs are found to negatively modulate the expressions of target genes through binding to their 3'-UTR.^[10] miRNAs were previously observed to be implicated in the development of various diseases including cancers.^{[11],[12],[13]} As a member of miRNAs, miR-141-3p has been previously studied and was shown to participate in the growth of several diseases. For instance, miR-141-3p plays a role in the cell growth, migration, invasion, and apoptosis in colon cancer through binding with Bcl2.^[14] The proliferation, migration, and invasion of colorectal cancer cells are reported to be suppressed by miR-141-3p/TRAF5 axis.^[15] Another comprehensive study revealed that miR-141-3p enhancement might be a prospective treatment target for endometrial carcinoma.^[16] In addition, Zhou et al. unveiled that miR-141-3p modulates the glutaminase-mediated glutamine metabolism to increase the cisplatin sensitivity in osteosarcoma cells.^[17] MiR-141-3p is implicated in nasopharyngeal carcinoma cell proliferation and metastasis by the modulation of NME1.^[18] Although the functions of miR-141-3p were identified in some cancers, whether it is engaged in the development of PCa is unclear.

Based on the data from Starbase (https://starbase.sysu.edu.cn/), miR-141-3p had potential binding sites for AlkB homolog 5 (ALKBH5), implied that ALKBH5 was a downstream target of miR-141-3p in PCa. However, the roles of miR-141-3p/ALKBH5 axis in PCa remain unclear. The current study aimed to explore the role and mechanism of miR-141-3p in PCa. The results demonstrated that miR-141-3p accelerated malignant progression of PCa through ALKBH5-mediated N6-methyladenosine (m⁶A) modification of PRMT6, which might highlight the role of miR-141-3p in the treatment of PCa.

Materials and Methods

Tissue samples

PCa tissues (n = 30) and benign prostatic hypertrophy tissues (n = 30) were obtained from patients at People's Hospital of Xinjiang Uygur Autonomous Region. All patients have signed the informed consents, and they received no treatment before. This work was approved by the Ethics Committee of People's Hospital of Xinjiang Uygur Autonomous Region. The tissues for further experiments were maintained in liquid nitrogen at once.

Real-time quantitative reverse transcription-polymerase chain reaction assay

Trizol reagent (Invitrogen, Carlsbad, CA, USA) was applied for total RNA extraction from PCa tissues and cell lines. Then cDNA was reverse transcribed from RNAs by use of PrimeScript RT Reagent (Takara, Dalian, China). The reverse transcription-quantitative polymerase chain reaction (RT-qPCR) was executed using the SYBR Premix Ex Taq II Kit (TaKaRa) on Applied Biosystems 7500 Sequence Detection through the 2^−ΔΔct method. U6 and GAPDH were employed as respective controls.

The primer sequences were: miR-141-3p (F: 5'-CTTCC AGTACAGTGTTGG-3'; R: 5'-GAACATGTCTGCGTATCTC-3'), ALKBH5 (F: 5'-CGGCGAAGGCTACACTTACG-3'; R: 5'-CCACCAGCTTTTGGATCACCA-3'), PRMT6 (F: 5'-CAAGACACGGACGTTTCAG-3'; R: 5'-CCTGGTCTCCCACTTTGTA-3'), U6 (F: 5'-GCTTCGGCAGCACATATACTAAAAT-3'; R: 5'-CGCTTCACGAATTTGCGTGTCAT-3'), GAPDH (F: 5'-CACTCCTCCACCTTTGACGC-3'; R: 5'-CTGTTGCTGTAGCCAAATTCGT-3').

Cell culture and transfection

The human prostatic epithelial cell-1 and PCa cells (LNCaP, PC3, DU145 and 22RV1) were obtained from Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences. Cells were cultured in RPMI-1640 medium (Thermo Fisher Scientific, Waltham, MA, USA) containing fetal bovine serum (FBS) (10%), penicillin (100 U/mL), and streptomycin (100 μg/mL, Thermo Fisher Scientific) with 5% CO₂ at 37°C.

The miR-141-3p mimic, negative control (NC) mimic, miR-141-3p inhibitor, NC inhibitor, small interfering RNA for ALKBH5 (si-ALKBH5), and PRMT6 (si-PRMT6) were synthesized in RiboBio Company (Guangzhou, China). ALKBH5 overexpression plasmid (OE-ALKBH5) was generated by cloning the full-length ORF of human ALKBH5 gene (NM_017758.3) into pcDNA3.1 vector. PC3 cells were grown in 6-well plates and when cells reached 70%–80% confluence, transfection was conducted. The transfection of vectors into cells was performed through Lipofectamine 2000.

miR-141-3p mimic for overexpressing miR-141-3p;
miR-141-3p inhibitor for inhibiting miR-141-3p;
si-ALKBH5 for inhibiting ALKBH5;
si-PRMT6 for inhibiting PRMT6;
OE-ALKBH5 for overexpressing ALKBH5.

Western blot analysis

The radioimmunoprecipitation assay lysis buffer was utilized for the lysis of PCa tissues or cells. After collecting the supernatant of the lysate, the samples were subjected to sodium dodecyl sulfate-polyacrylamide gel (10%), and then the proteins were transferred onto a polyvinylidene difluoride membrane. Primary antibodies were ALKBH5 (1:1000, ab195377, Abcam, Shanghai, China), PRMT6 (1:1000, ab271091, Abcam) and GAPDH (1:2500, ab9485, Abcam). Further, the secondary antibodies (horseradish peroxidase-labeled immunoglobulin G (IgG), 1: 5000, Beijing CoWin Biosciences, China) were supplemented into the membranes. An ECL western blotting kit (Thermo Fisher Scientific) was applied for the visualization of the protein bands.

Cell proliferation detection

For Cell Counting Kit-8 (CCK-8) assay, the transfected PC3 cells were plated in 96-well plates for 1 h at 37°C. Ten microliter of the CCK-8 solution was then added to the plates at 0 day, 1 day, 2 days, and 3 days. The absorbance at 450 nm was assessed through a microplate reader (BioTek Instruments, Winooski, VT, USA).

For colony formation assay, the transfected PC3 cells were grown in 12-well plates and incubated for a week at 37°C. Every 4 days, the culture medium was refreshed. After washed by phosphate buffered saline (PBS), the cells were fixed with 4% paraformaldehyde followed by staining with crystal violet (0.5%, Sigma-Aldrich; Merck KGaA) at room temperature for 10 min. A microscope (Olympus, Tokyo, Japan) was finally applied for observing the number of colonies (containing more than 50 cells).

Luciferase activity assay

The Promega company provided us with the oligonucleotides containing wild-type (WT) or mutant (MUT) binding sequences of ALKBH5 for miR-141-3p and subcloned in the pmirGLO plasmids (Madison, WI, USA) (ALKBH5-WT and ALKBH5-MUT). Posttransfected with these vectors into PC3 cells with miR-141-3p mimic and NC mimic, the relative luciferase activity was tested through a Dual-Luciferase Reporter Assay System (Promega).

Transwell assay

The invasion of PCa cells was tested through transwell assay in transwell chambers precoated with reconstituted basement membrane matrix (50 μg, BD Biosciences, MD, USA). In the upper chamber, serum-free RPMI-1640 medium was added while RPMI-1640 medium with FBS (20%) was filled in the lower chamber. The crystal violet was employed for the staining of cells invaded the lower chambers for 15 min. A light microscope was finally used to observe and count the number of invaded cells.

RNA immunoprecipitation assay

The protein-RNA interaction was measured through RNA immunoprecipitation (RIP) assay. Total Polyadenylated RNA was grown with anti-IgG, anti-m⁶A (202003, sysy), and anti-ALKBH5 (ABE547, Merck Millipore) or rabbit IgG-binding protein A/G magnetic beads in 500 μl IP buffer. The RNA of PRMT6 was immunoprecipitated with the beads and tested through RT-qPCR.

Methylated RIP qPCR was performed using Magna meRIP^™ M6A kit to confirm that M6A-specific antibody can enrich PRMT6.

RIP qPCR was performed using Magna RIP kit to confirm that ALKBH5 antibody can enrich PRMT6.

Immunohistochemistry staining

The tissues from PCa mice model were dissociated using xylene followed by dehydrating with gradient alcohol. Postantigen retrieving in sodium citrate buffer in a microwave oven (100°C) for 10 min, the tissues were incubated with hydrogen peroxide (3%) to inactive the endogenous peroxidase. Afterward, the primary antibodies of ALKBH5 (1:1000, ab195377, Abcam) and PRMT6 (1:1000, ab271091, Abcam) were incubated with the tissue slides overnight at 4°C. Next, the biotinylated secondary antibodies were added and Diaminobenzidine (Zytomed Systems, Berlin, Germany) was applied to dying the tissues. Subsequently, the tissues were counterstained with hematoxylin (Servicebio, Wuhan, China) after washing. Finally, running tap water was used to rinse the slides for 5 min and an Olympus multifunction microscope (Olympus, Tokyo, Japan) was used for capture the images.

Wound healing assay

PC3 cells were grown on 6-well plates to 80%–90% confluence. A sterile pipette tip (10 μL) was applied for scratching the cell layer. PBS was applied for cleaning the damaged cells. Further, the RPMI-1640 medium was employed for the growth of the cells for 0 h and 24 h. The pictures were obtained and analyzed to evaluate the migration of PCa cells.

Sphere formation assay

The transfected PC3 cells were plated on a 6-well ultra-low attachment cell culture plate (Corning, USA). The bFGF (20 ng/mL, R and D Systems, USA), 1×B27 supplement (Invitrogen, USA), and EGF (20 ng/mL, Sigma, USA) were added in stem cell medium DMEM/F12 and incubated with the cells. After 10 days, the number and sizes of the sphere were observed.

Animals

The animal experiments applied the approval from the Institutional Animal Care and Use Committee of People's Hospital of Xinjiang Uygur Autonomous Region (Approval No. 2021-028). The Model Animal Research Center of Nanjing University provided us 4-week-old male nude mice (n = 10). The mice were caged in a house (temperature of 22°C ± 1°C and humidity of 55% ± 5%) with adequate food and water. Each mouse was injected with PC3 cells transfected miR-141-3p inhibitor or NC inhibitor to the left and right flanks. After 28 days, the mice were sacrificed and the tumor size, weight, and volume were evaluated.

Statistical analysis

All data were analyzed through GraphPad Prism 8 software (GraphPad Prism, San Diego, CA, USA) and displayed as the mean ± standard deviation. Student's t-test was applied for difference comparison between two groups and analysis of variance, followed by Tukey post hoc test was applied for comparing differences among multiple groups. Spearman correlation analysis was employed for exploring the correlation between genes. The assays were repeated for three times. P < 0.05 was deemed as a significant difference.

Results

AlkB homolog 5 was down-regulated in prostate cancer tissues and negatively associated with miR-141-3p

To identify the role of miR-141-3p and ALKBH5 in PCa, the levels of them were evaluated in PCa tissues and normal tissues. The data depicted that miR-141-3p was elevated in PCa tissues [Figure 1]a. Moreover, the mRNA of ALKBH5 was decreased in tumor tissues relative to normal tissues [Figure 1]b. Spearman correlation analysis indicated that ALKBH5 was negatively correlated with miR-141-3p [Figure 1]c. Through TCGA database, ALKBH5 expression was also discovered to be down-regulated [Figure 1]d. In addition, ALKBH5 protein level was found to be lowered in the tumor tissues (n = 5 pairs) [Figure 1]e. In a word, ALKBH5 was down-regulated in PCa and negatively correlated with miR-141-3p.

Figure 1: ALKBH5 was down-regulated in PCa and negatively correlated with miR-141-3p. (a and b) RT-qPCR was applied to evaluate the levels of miR-141-3p and ALKBH5 in PCa and normal tissues. (c) Spearman correlation analysis revealed the correlation of miR-141-3p and ALKBH5. (d) Through TCGA database, ALKBH5 expression was verified in PCa and normal tissues. (e) The protein level of ALKBH5 in PCa and normal tissues (n = 5 pairs) was explored through western blot analysis. **P < 0.01, ***P < 0.001 relative to normal group. ALKBH5: AlkB homolog 5, PCa: Prostate cancer, RT-qPCR: Reverse transcription-quantitative polymerase chain reaction.

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AlkB homolog 5 inhibited the malignant development of prostate cancer

Next, the functions of ALKBH5 in PCa cells were measured. ALKBH5 was found to be reduced in PCa cells compared with REPE-1 cells and had the lowest level in PC3 cells [Figure 2]a. Thus, the following experiments were performed in PC3 cells. Firstly, ALKBH5 was knocked down and overexpressed in PC3 cells. The level of ALKBH5 in PC3 cells was evidently downregulated through transfecting si-ALKBH5 and upregulated through transfecting OE-ALKBH5, implying the favorable knockdown or overexpression efficiency of si-ALKBH5 or OE-ALKBH5 vectors [Figure 2]b. Further, the proliferation of PCa cells was detected. The results unveiled that the viability of PC3 cells was elevated by the inhibition of ALKBH5 but repressed by overexpression of ALKBH5 [Figure 2]c. Similarly, upregulation of ALKBH5 restrained and suppression of ALKBH5 increased the proliferation of PC3 cells [Figure 2]d. The invasion of PC3 cells was inhibited by ALKBH5 enhancement and aggravated by ALKBH5 reduction [Figure 2]e. Alleviation of ALKBH5 elevated and upregulation of ALKBH5 decreased the migration of PC3 cells [Figure 2]f. Besides, the number of spheres was increased by downregulation of ALKBH5 and decreased by overexpression of ALKBH5 [Figure 2]g. To sum up, ALKBH5 inhibited the malignant development of PCa.

Figure 2: ALKBH5 inhibited the malignant development of PCa. (a) Western blot analysis manifested the protein level of ALKBH5. *P < 0.05, **P < 0.01, ***P < 0.001 relative to normal group. (b) Western blot analysis manifested the protein level of ALKBH5. **P < 0.01, ***P < 0.001 relative to si-NC group. (c and d) The proliferation of PC3 cells was measured through CCK-8 and colony formation assays. (e) The invasion of PC3 cells was evaluated through transwell assay. (f) The migration of PC3 cells was tested by wound healing assay. (g) Sphere formation assay was applied for measuring the number and size of spheres. **P < 0.01, ***P < 0.001 relative to si-NC group. ^#P < 0.05, ^##P < 0.01, ^###P < 0.001 relative to vector group. ALKBH5: AlkB homolog 5, PCa: Prostate cancer, CCK-8: Cell counting kit-8.

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AlkB homolog 5 was a target of miR-141-3p

Afterward, the regulatory mechanism of ALKBH5 and miR-141-3p in PCa cells was probed. According to the data from Starbase (https://starbase.sysu.edu.cn/), ALKBH5 and miR141-3p had potential binding sites [Figure 3]a. The binding affinity of ALKBH5 and miR141-3p was verified through luciferase activity assay. The data showed that the enhancement of miR-141-3p decreased the luciferase activity of ALKBH5-WT rather than the ALKBH5-MUT, suggesting that miR-141-3p could bind with ALKBH5 [Figure 3]b. Furthermore, we found that ALKBH5 protein level was reduced by upregulation of miR-141-3p but aggrandized by downregulation of miR-141-3p [Figure 3]c. Taken together, ALKBH5 was a target of miR-141-3p.

Figure 3: ALKBH5 was a target of miR-141-3p. (a) The potential binding sites for ALKBH5 and miR-141-3p. (b) The binding affinity of ALKBH5 and miR141-3p was verified via luciferase activity assay. ***P < 0.001 relative to NC mimic group. (c) Western blot analysis was performed to assess the protein level of ALKBH5. **P < 0.01 relative to NC mimic group, ^##P < 0.01 relative to NC inhibitor group. ALKBH5: AlkB homolog 5, NC: Negative control.

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AlkB homolog 5 suppressed protein arginine methyltransferase 6 through m⁶A modification

Thereafter, the mechanisms of ALKBH5 in PCa were further explored. RIP assay uncovered a large amount of PRMT6 expression in the ALKBH5 antibody precipitate instead of the IgG group [Figure 4]a. The meRIP assay revealed that ALKBH5 suppression enhanced the enrichment m6A of PRMT6 [Figure 4]b. Furthermore, the protein level of PRMT6 was elevated in the si-ALKBH5 group and suppressed in OE-ALKBH5 group [Figure 4]c. All in all, ALKBH5 suppressed PRMT6 expression through m⁶A modification.

Figure 4: ALKBH5 suppressed protein arginine methyltransferase 6 expression via m⁶A modification. (a) RIP assay was employed for detecting the binding ability of ALKBH5 and PRMT6. **P < 0.01 relative to IgG group. (b) meRIP was applied to evaluate whether ALKBH5 mediating PRMT6 in a m⁶A-dependent manner. ***P < 0.001 relative to si-NC-IgG group, ^###P < 0.001 relative to si-ALKBH5-IgG group. ^{^^^}P < 0.001 relative to si-NC-m⁶A group. (c) The protein level of PRMT6 was examined via western blot analysis. *P < 0.05, relative to si-NC group. ^##P < 0.01 relative to vector group. ALKBH 5: AlkB homolog 5, RIP: RNA immunoprecipitation, PRMT: Protein arginine methyltransferase, IgG: Immunoglobulin G, NC: Negative control.

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MiR-141-3p modulated protein arginine methyltransferase 6 level through mediating AlkB homolog 5

Subsequently, whether miR-141-3p modulated PRMT6 level through mediating ALKBH5 was investigated. Western blot analysis delineated that miR-141-3p enhancement elevated and miR-141-3p downregulation inhibited the protein level of PRMT6 [Figure 5]a. In addition, the downregulated expression of PRMT6 induced by miR-141-3p inhibition was reversed by ALKBH5 suppression [Figure 5]b. Besides, the elevated mRNA and protein levels of PRMT6 were observed in the PCa tumor tissues [Figure 5]c and [Figure 5]d. Moreover, results showed that the PRMT6 level was positively correlated with miR-141-3p level and negatively correlated with ALKBH5 level [Figure 5]e and [Figure 5]f. To conclude, miR-141-3p modulated PRMT6 level through mediating ALKBH5.

Figure 5: MiR-141-3p-modulated protein arginine methyltransferase 6 level through mediating ALKBH5. (a) The protein level of PRMT6 was evaluated through western blot analysis. **P < 0.01 relative to NC mimic group, ^#P < 0.05 relative to NC inhibitor group. (b) Western blot analysis showed PRMT6 protein level. **P < 0.01 relative to NC inhibitor + si-NC group, ^###P < 0.001 relative to miR-141-3p inhibitor + si-NC group. (c and d) RT-qPCR and western blot analysis was executed to examine the level of PRMT6 in PCa tissues. ***P < 0.001 relative to the normal group. (e and f) Spearman correlation analysis uncovered the association between miR-141-3p and PRMT6 as well as ALKBH5 and PRMT6. ALKBH5: AlkB homolog 5, PRMT: Protein arginine methyltransferase, NC: Negative control, RT-qPCR: Reverse transcription-quantitative polymerase chain reaction, PCa: Prostate cancer.

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MiR-141-3p aggravated prostate cancer development by regulating protein arginine methyltransferase 6

Next, rescue assays were conducted to further investigate the miR-141-3p/PRMT6 axis in PCa cells. CCK-8 and colony formation assays manifested that the increased proliferation ability of PC3 cells resulted from miR-141-3p upregulation was offset by PRMT6 silencing [Figure 6]a and [Figure 6]b. The increased migration ability of PC3 cells in miR-141-3p mimic group was inversely altered by PRMT6 downregulation [Figure 6]c. Moreover, downregulation of miR-141-3p enhanced the number of spheres, but this effect was attenuated by the knockdown of PRMT6 [Figure 6]d. Altogether, these results suggested that miR-141-3p aggravated PCa development by regulating PRMT6.

Figure 6: MiR-141-3p aggravated PCa development by regulating protein arginine methyltransferase 6. (a) The proliferation of PC3 cells was measured via CCK-8 assay. (b) The invasion of PC3 cells was evaluated through transwell assay. (c) The migration of PC3 cells was tested by wound healing assay. (d) Sphere formation assay was applied for measuring the number and size of spheres. **P < 0.01, ***P < 0.001 relative to NC mimic group. ^##P < 0.01, ^###P < 0.001 relative to miR-141-3p mimic group. PCa: Prostate cancer, CCK-8: Cell counting kit-8, NC: Negative control.

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MiR-141-3p facilitated prostate cancer tumor growth in vivo

At last, the function of miR-141-3p in PCa was explored in vivo. As depicted in [Figure 7]a, the tumor sizes were smaller by the suppression of miR-141-3p. In addition, miR-141-3p silencing also alleviated the tumor volume and weight [Figure 7]b. MiR-141-3p level in vivo was decreased in the miR-141-3p inhibitor group [Figure 7]c. Immunohistochemistry staining revealed that the expression of ALKBH5 was enhanced and PRMT6 was suppressed in vivo by miR-141-3p downregulation [Figure 7]d. In summary, miR-141-3p facilitated PCa tumor growth in vivo.

Figure 7: MiR-141-3p facilitated PCa tumor growth in vivo. (a and b) The tumor size, volume, and weight were evaluated in vivo. (c) The expression of miR-141-3p in mice model was measured through RT-qPCR. (d) IHC staining revealed the expression of ALKBH5 and PRMT6 in vivo. ***P < 0.001 relative to NC inhibitor group. PCa: Prostate cancer, RT-qPCR: Reverse transcription-quantitative polymerase chain reaction, ALKBH5: AlkB homolog 5, PRMT: Protein arginine methyltransferase, NC: Negative control.

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Discussion

PCa is a prevalent cancer occurring in males, which threatens the health of men with high mortality and causes a substantial burden.^[19] PCa is a slow-growing malignancy, indicating that patients have an opportunity to receive timely interventions to delay tumor growth and increase life expectancy.^[20] Currently, despite the improvements in care and treatments of the diseases, the outcomes of patients with PCa were still not optimistic. It is essential to find more reliable biomarkers for the early diagnosis and interventions of PCa, thereby improving the prognosis of these patients. Nowadays, the researchers have confirmed that various proteins were involved in the development of PCa. For instance, tribbles 2 pseudokinase is corroborated to participate in the enzalutamide resistance in PCa through enhancing lineage plasticity.^[21] FOXA1 is involved in the angiogenesis of PCa via upregulating the level of pro-angiogenic factors.^[22] Interacting with Wig1, EIF5B modulates the development of PCa through mediating PD-L1 level.^[23] Although the roles of various proteins in PCa were probed, more effective biomarkers are still needed.

As a methylation of N6 on adenosine, m⁶A is one of the common modifications of mRNAs.^[24] ALKBH5 is reported to be a m⁶A eraser catalyzing the demethylation of m⁶A.^[25] ALKBH5 that belongs to the dioxygenase AlkB family, has been widely shown to be implicated with the development of tumors. For example, ALKBH5 mediates casein kinase 2α-associated glycolysis to modulate bladder cancer progression in an m⁶A-dependent manner.^[26] ALKBH5 influences the development of osteosarcoma in m⁶A/YTHDF2-dependent manner to modulate the activity of STAT3.^[27] In addition, ALKBH5 expression is decreased in PCa.^[28],[29] Nevertheless, whether ALKBH5 exerted a role in PCa still requires investigation. Here, we found that ALKBH5 was decreased in PCa tissues and was negatively correlated with miR-141-3p. ALKBH5 attenuated the malignant development of PCa through suppressing the proliferation, migration, invasion, and sphere formation abilities of PCa cells. Besides, ALKBH5 was confirmed to be a downstream target of miR-141-3p. MiR-141-3p expression was boosted in PCa tissues and cells. Inhibition of miR-141-3p suppressed the tumor growth of PCa in vivo. In conclusion, miR-141-3p accelerated PCa development through targeting ALKBH5.

PRMT6 is a type I PRMT that participates in epigenetic modulation of the expression of genes through the methylation of histone or nonhistone proteins.^[30] Its role and function have been extensively reported in PCa. For instance, PRMT6 suppression is involved in the inhibition of cell growth and migration of PCa.^[31] Almeida-Rios et al. revealed that PRMT6 aggravates the progression of PCa.^[32] In addition, ALKBH5 has been reported to downregulate PRMT6 expression through m⁶A modification.^[33] Nonetheless, whether ALKBH5 participated in PCa through modulating PRMT6 in one m⁶A-dependent manner was still unclear. Herein, ALKBH5 was confirmed to suppress PRMT6 expression via m⁶A modification. Further, we identified that miR-141-3p-modulated PRMT6 level through mediating ALKBH5. Moreover, PRMT6 was positively correlated with miR-141-3p and is negatively associated with ALKBH5. Finally, rescue assays uncovered that miR-141-3p aggravated PCa development by regulating PRMT6.

Conclusion

In summary, this study indicated that miR-141-3p accelerated the malignant progression of PCa through ALKBH5-mediated m⁶A modification of PRMT6. These findings might offer a novel insight into the roles of miR-141-3p and ALKBH5 in the treatments of PCa patients.

Ethics approval

Ethical approval was obtained from the Ethics Committee of People's Hospital of Xinjiang Uygur Autonomous Region (Approval No.2021-028).

Financial support and sponsorship

This work was supported by the Natural Science Foundation of Xinjiang Uygur Autonomous Region Youth Fund Project (Grant No. 2019D01C159).

Conflicts of interest

There are no conflicts of interest.

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