PRPF19 promotes the proliferation, migration, and inhibits autophagy in prostate cancer by suppressing SLC40A1

Guofei Zhang; Wansong Zhang; Mingjiang Dan; Feng Zou; Chunming Qiu; Canbiao Sun

doi:10.4103/cjop.CJOP-D-22-00152

ORIGINAL ARTICLE

Year : 2023 | Volume : 66 | Issue : 5 | Page : 379-387

PRPF19 promotes the proliferation, migration, and inhibits autophagy in prostate cancer by suppressing SLC40A1

Guofei Zhang¹, Wansong Zhang¹, Mingjiang Dan², Feng Zou¹, Chunming Qiu¹, Canbiao Sun¹
¹ Department of Urology, The Seventh Affiliated Hospital, Southern Medical University, Foshan, Guangdong, China
² Department of Urology, Hui Ya Hospital of The First Affiliated Hospital, Sun Yat-sen University Huizhou, Guangdong, China

Date of Submission	19-Dec-2022
Date of Decision	05-May-2023
Date of Acceptance	19-May-2023
Date of Web Publication	18-Aug-2023

Correspondence Address:
Dr. Guofei Zhang
Department of Urology, The Seventh Affiliated Hospital, Southern Medical University, No. 28, Liguan Road, Lishui Town, Nanhai District, Foshan, Guangdong
China

Source of Support: None, Conflict of Interest: None

DOI: 10.4103/cjop.CJOP-D-22-00152

Abstract

Prostate cancer (PCa) is a common cancer and the leading cause of cancer-related death in men. To investigate the role of pre-mRNA processing factor 19 (PRPF19) in proliferation, migration of PCa, and evaluate the potential ability of PRPF19 as a therapeutic target. PRPF19 expression was analyzed from The Cancer Genome Atlas and GEPIA databank. Quantitative real-time polymerase chain reaction (qRT-PCR) was performed to evaluate the transcription of PRPF9 and solute carrier family 40 member 1 (SLC40A1). Immunohistochemistry (IHC) was used to test PRPF9 expression in PCa tissues. The cell viability and 5-ethynyl-2′-deoxyuridine incorporation analysis were performed to assess cell proliferation. Transwell assay was performed to investigate the migration and invasion of cancer cells. Western blot was used to measure the expression level of PRPF9, E-cadherin, Vimentin and α-smooth muscle actin (α-SMA), SLC40A1, LC3, Beclin-1 and ATG7. Immunofluorescence assay was performed to measure LC3 expression in PCa cells. The bioinformatic analysis revealed PRPF19 was highly expressed in PCa which was certified by qRT-PCR, western blot and IHC detection in PCa tissues. The proliferation of PCa cells could be promoted by PRPF19 overexpression and suppressed by PRPF19 knockdown. Moreover, the migration and invasion of PCa cells could be positively regulated by PRPF19 which promoted the expression of E-cadherin, Vimentin, and α-SMA. Furthermore, the expression of LC3, Beclin-1, and ATG7 was negatively regulated by PRPF19, indicating that PRPF19 inhibited autophagy in PCa cells. In the double knockdown of PRPF19 and SLC40A1, PRPF19 repressed the mRNA and reduced protein level of SLC40A1, and SLC40A1 antagonized effects of PRPF19 on proliferation, migration and autophagy of PCa cells. PRPF19 promoted proliferation and migration, and inhibited autophagy in PCa by attenuating SLC40A1 expression, indicating PRPF19 was a potential therapeutic target for PCa treatment.

Keywords: Autophagy, migration, PRPF19, proliferation, prostate cancer, SLC40A1

How to cite this article:
Zhang G, Zhang W, Dan M, Zou F, Qiu C, Sun C. PRPF19 promotes the proliferation, migration, and inhibits autophagy in prostate cancer by suppressing SLC40A1. Chin J Physiol 2023;66:379-87

How to cite this URL:
Zhang G, Zhang W, Dan M, Zou F, Qiu C, Sun C. PRPF19 promotes the proliferation, migration, and inhibits autophagy in prostate cancer by suppressing SLC40A1. Chin J Physiol [serial online] 2023 [cited 2023 Dec 4];66:379-87. Available from: https://www.cjphysiology.org/text.asp?2023/66/5/379/383913

Introduction

Prostate cancer (PCa) is a common cancer and the leading cause of cancer-related death in men. For early stage PCa, androgen ablation is a successful treatment to inhibit tumor progression. However, in the later stages, androgen receptor is often persistently activated in the absence of androgen.^[1],[2] It is necessary to discover tumor suppressor genes and proto-oncogenes for clinical progression and treatment strategies to avoid and treat advanced and metastatic tumors.^[3]

Pre-mRNA processing factor 19 (PRPF19) is a highly conserved splicing factor in eukaryotes, consisting of an N-terminal U-box domain, a coiled-coil domain, and a C-terminal WD40 repeat.^[4] PRPF19 is associated with DNA damage response, pre-mRNA processing and genome maintenance.^[5],[6] Studies have shown that the expression of PRPF19 is reduced during replicative senescence, and knockdown of PRPF19 induces cell cycle arrest by inhibiting MDM4-mediated inactivation of p53, which finally leads to cellular senescence.^[7] PRPF19 is highly expressed in the liver cancer tissues, which is related to poor prognosis.^[8] In addition, PRPF19 is also aberrantly expressed in ovarian tumor cells.^[9]

PRPF19 overexpression facilitates cell proliferation and migration of tongue cancer through inhibiting solute carrier family 40 member 1 (SLC40A1) and single ADP-ribose hydrolase 2 (MACROD2) expression.^[10] Previous research showed that SLC40A1 was lowly expressed in PCa, which promoted proliferation and migration of PCa cells.^[11] Overexpressed SLC40A1 inhibits tumor progression of hepatocellular carcinoma cells by stimulating autophagy.^[12]

However, the role of PRPF19 in PCa remains unclear. In this study, PRPF19 expression in PCa was first analyzed using the Cancer Genome Atlas (TCGA) and GEPIA database, and its expression was measured in PCa cell lines and tissues. Further studies were performed to evaluate the role of PRPF19 in proliferation, migration and autophagy in PCa cells. Moreover, the effect of SLC40A1 on tumor activity promoted by PRPF9 was investigated as well.

Materials and Methods

Cell culture

The prostate cell line RWPE2 and PCa cell line LNCaP were cultivated in RPMI-1640 medium plus 5% fetal bovine serum (FBS) (Gibco). Another two PCa cell lines including PC-3 and DU145 were cultured in F-12K Medium (ATCC 30-2004)/Eagle's Minimum Essential Medium (ATCC 30-2003) containing 10% FBS, respectively. All these cells were housed in a 37°C incubator supplemented with 5% CO₂.

Overexpression or knockdown

PRPF19 coding sequence was amplified with primers of PRPF19 overexpression [Table 1] and inserted into lentivirus control vector (LVCV-19) (Sino Biological, Beijing, China). shRNA-coding sequences of PRPF19 or SLC40A1 were amplified with corresponding primers [Table 1] and inserted into lentivirus vector pLKO.1. The overexpression plasmid of PRPF19 or the shRNA lentiviral plasmids were transfected into 293T cells with pMD2G and psPAX2 which are necessary for packaging recombinant lentivirus. The lentiviral particles in supernatant were harvested after 48 h. PC-3 or DU145 cells were infected with these recombinant lentiviruses and then treated with puromycin. Single colonies were picked, and cultured in new individual plates. Western blot was used to screen the target colonies with high expression and knockdown efficiency. The certified recombinant lentiviruses were used to infect PC-3 or DU145 cells for overexpression or knockdown.

Table 1: Primers list

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The Cancer Genome Atlas data analysis

The RNA-seq data were derived from TCGA and were used to analyze the gene expression on UALCAN web portal which is available at http://ualcan.path.uab.edu. Then we performed analysis of PRPF19 expression in PCa tissues via online tool (http://gepia.cancer-pku.cn/).

Quantitative real-time polymerase chain reaction

Total RNA was isolated using TRIzol reagent (15596026, Invitrogen, USA) following the protocol. The reverse transcription was conducted with a total of 500 ng RNA by Prime Script RT Reagent Kit (Takara, Dalian, China). Bio-Rad real-time polymerase chain reaction (RT-PCR) system was used to do qRT-PCR with iTaq universal SYBR Green Supermix (1725121, Bio-Rad Laboratories Inc., Hercules, CA, USA). The transcription of target genes was determined by 2^−ΔΔCT, and the relative transcription was normalized to the β-actin level. The PCR primer sequences were listed in [Table 1].

Western blot

The cellular protein was extracted by radioimmunoprecipitation assay lysis buffer (89901, Thermo Scientific, Carlsbad, CA, USA). The lysates were processed for immunoblot with the primary antibodies listed in [Table 2]. Horseradish peroxidase-conjugated goat anti-rabbit immunoglobulin G (IgG) was used as the secondary antibodies (B900210, ProteinTech Group; 1:5000). Finally, the target bands were visualized with enhanced chemiluminescence Western Blotting Detection Kit (Solarbio Life Sciences, Beijing, China). For quantification of western blot signal, the relative intensity of each band was measured by ImageJ software, and the relative expression levels were normalized to the relative β-actin levels.

Table 2: Antibodies information

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Immunohistochemistry analysis

The clinical tumor tissues and their para-carcinoma tissues from 30 patients with PCa were collected. All procedures performed in studies involving human participants were in accordance with the standards upheld by the Ethics Committee of The Seventh Affiliated Hospital (Approval No. 2016(06) 0254), Southern Medical University, and with those of the 1964 Helsinki Declaration and its later amendments for ethical research involving human subjects. The prostate tissues were fixed by formalin, embedded with paraffin, and then cut into 5-μm sections. 0.01 M citrate sodium buffer (pH = 6.0) was used in antigen retrieval at 100°C for 10 min. Then, sections were blocked with 0.3% H₂O₂ and 10% bovine serum albumin, respectively. Then, the sections were incubated with primary antibody against PRPF19. After being washed for three times, the sections were incubated with anti-rabbit IgG, developed in DAB substrate and counterstained with hematoxylin. Written informed consent was obtained from a legally authorized representative(s) for anonymized patient information to be published in this article.

Cell proliferation assay

PC-3 or DU145 cells were seeded into 96-well plates and infected with lentiviruses of PRPF19 overexpression or shRNAs. The supernatant was replaced with new medium supplemented with 10 μL CCK-8 (Dojindo Molecular Technologies, Japan) in each well and the cells were cultured in incubator with 5% CO₂ at 37°C for 0, 1, 2 and 3 days. Absorbance was detected at 450 nm wavelength, and the cell viability ratio was calculated.

5-ethynyl-2'-deoxyuridine assay

PC-3 or DU145 cells in 96-well plates were separately infected with lentiviruses of PRPF19 overexpression or shRNAs, and cultured for another 2 days. Then, cells were incubated with 50 μmol/L 5-ethynyl-2′-deoxyuridine (EdU) for 2 h, fixed with 4% paraformaldehyde, treated with 2 mg/mL glycine, and permeabilized with 0.5% Triton X-100. Cells were further incubated with Apollo staining reaction solution for 30 min followed by DAPI staining (28718-90-3, Sigma-Aldrich, Shanghai, China) for 5 min in a dark room. The washed cells were observed under a fluorescence microscope, and cells in captured pictures were counted for calculating EdU/DAPI ratio.

Transwell assay

PC-3 or DU145 cells were infected with lentiviruses of PRPF19 overexpression or shRNAs, and seeded in the upper chamber of transwell plates (PI8P01250, Merck KGaA, Darmstadt, Germany) with membrane inserts. For migration assay, there is no Matrigel (E1270, Sigma Aldrich, Shanghai, China) coated in these member inserts, but the Matrigel is necessary to be added into the upper compartment of the inserts for invasion assay. In transwell plates, the lower chamber was supplemented with media containing 10% FBS but the upper one was filled with serum-free medium. Then, the plates were incubated at 37°C for 48 h. The cells which passed through the polycarbonate membrane were fixed with 5% glutaraldehyde and stained with 1% crystal violet. For each sample, more than 10 fields were selected randomly and the mean number of stained cells was counted.

Indirect immunofluorescent assay

PC-3 or DU145 cells were infected with lentiviruses for PRPF19 overexpression or knockdown, then fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100. The cells were incubated with anti-LC3 antibody, washed for three times, and stained with fluorescein isothiocyanate conjugated goat anti-rabbit IgG. Finally, the cells were washed again, stained with DAPI, and examined under the fluorescence microscope (Nikon, TE2000U, Melville, NY, USA).

Quantification and statistical analysis

Statistical analysis was done by GraphPad Prism 8.0 (GraphPad Software, Boston, MA, USA). Data were presented as mean ± standard deviation (SD) from three biological replicates, and the differences between any two groups were compared by unpaired t-tests. Multiple group comparisons were analyzed with ANOVA. *(^) P < 0.05, **(^^) P < 0.01, ***(^^^) P < 0.001 were considered statistically significant.

Results

PRPF19 was highly expressed in prostate cancer

To evaluate the PRPF19 expression level in malignant tumors, especially in PCa, an analysis of RNA-seq data derived from TCGA on UALCAN web portal was first performed, which revealed that PRPF19 was upregulated in 497 samples of primary tumors [Figure 1]a. Then, we performed another analysis from GEPIA databank, and the results showed that PRPF19 expression in PCa tissues was elevated as well [Figure 1]b. To verify that PRPF19 expression was upregulated in PCa tissues, the clinical tumor tissues and their para-carcinoma tissues from 30 patients with PCa were collected. Their RNA transcriptions were assessed by qRT-PCR, and the data suggested that the relative transcription level of PRPF19 to β-actin in these PCa tissues was significantly higher than that in the para-carcinoma tissues [Figure 1]c. Furthermore, a Western blot was performed to measure the PRPF19 protein level, and the results indicated that the protein expression of PRPF19 in these PCa tissues was elevated compared to that in para-carcinoma tissues [Figure 1]d. Further, IHC was performed to detect PRPF19 in PCa tissues and their corresponding para-carcinoma tissues, and the results showed that the staining for PRPF19 in PCa tissues was significantly stronger than that in para-carcinoma tissues [Figure 1]e. Moreover, PRPF19 expression in cell lines was also assessed by Western blot, and the data revealed that its protein levels in PCa cell lines including PC-3, DU145, and LNCaP were much higher than that in RWPE2, which is a normal cell line isolated from prostate [Figure 1]f. To sum up, PRPF19 was highly expressed in PCa.

Figure 1: PRPF19 was highly expressed in PCa. (a) Analysis of RNA-seq data derived from TCGA on UALCAN web portal, PRPF19 was upregulated in 497 samples of primary tumors. (b) Analysis from GEPIA databank, PRPF19 expression in PCa tissues was elevated as well. (c) qRT-qPCR was performed to assess the PRPF9 transcription in clinical tumor tissues from 30 patients with PCa. (d) Western blot was performed to measure the PRPF19 protein level in PCa tissues of four patients. (e) IHC was performed to detect PRPF19 expression in PCa tissues. (f) Western blot was used to evaluate PRPF19 expression in normal prostate cell line RWPE2 and three PCa cell lines including PC-3, DU145, and LNCaP. Three replicate experiments were conducted for statistical analysis. ***Means the comparison to PWPE2. *P < 0.05, ***P < 0.001. PRPF19: Pre-mRNA processing factor 19, PCa: Prostate cancer, TCGA: The Cancer Genome Atlas, qRT-qPCR: Quantitative real-time polymerase chain reaction, IHC: Immunohistochemistry.

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PRPF19 promoted proliferation in prostate cancer cells

Given that increased expression of PRPF19 in PCa, the proliferation of PCa cells with PFPR19 overexpression or knockdown was studied. PC-3 and DU145 cells were selected to be infected with lentivirus of PRPF19 overexpression (marked as PRPF19) or shRNA of PRPF19 including shPRPF19-1# and shPRPF19-2# or with shNC as the negative control. First, a Western blot was performed to test the PRPF19 expression, and the results showed that PRPF19 was elevated in cells infected with lentivirus of PRPF19 overexpression compared to the control group. However, shPRPF19-2# or shPRPF19-1# could significantly reduce PRPF19 expression compared to shNC group in PC-3 or DU145 cells [Figure 2]a. Furthermore, the data on cell viability illustrated that PRPF19 overexpression in PRPF19 group significantly promoted cell proliferation, but which was reduced significantly in shPRPF19-2# group compared to sh-NC [Figure 2]b. Moreover, EdU incorporation analysis was performed to test cell proliferation. As shown in PC-3 or DU145 cells, PRPF19 overexpression increased EdU incorporation (EdU/DAPI) compared to the control group, while shPRPF19 potently reduced EdU incorporation in contrast to sh-NC [Figure 2]c. Therefore, PRPF19 played a positive role in regulating the proliferation of PCa cells.

Figure 2: PRPF19 promoted proliferation in PCa cells. (a) Western blot was performed to test the PRPF19 expression in PC-3 and DU145 cells infected with lentivirus for PRPF19 overexpression (marked as PRPF19) or shRNA of PRPF19 including shPRPF19-1# and shPRPF19-2# or the control sh-NC, respectively. (b) The cell viability of PC-3 and DU145 was studied to assess the effect of PRPF19 overexpression or knockdown on cell proliferation. (c) EdU incorporation analysis was performed to test cell proliferation. The t-tests were analyzed on three biological replicates. *P < 0.05, **P < 0.01, ***P < 0.001, PRPF19 versus Control. ^^P < 0.01, ^^^P < 0.001, shPRPF19-2# versus shNC. PRPF19: Pre-mRNA processing factor 19, EdU: 5-ethynyl-2'-deoxyuridine.

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PRPF19 promoted migration of prostate cancer cells

Cell migration occurs during cancer development and is especially important during invasion, which is the initial step of metastasis. It is necessary to investigate the effect of PRPF19 on migration here. The data from the transwell assay revealed that PRPF19 facilitated the migration of PCa cells. However, downregulation of PRPF19 by shPRPF19-2# significantly weakened the migration and invasion of PC-3 or DU145 cells compared to shNC group [Figure 3]a.

Figure 3: PRPF19 promoted migration of PCa cells. (a) Transwell assay was performed to investigate the effect of PRPF19 expression on the migration and invasion of PC-3 and DU145 cells. (b) Western blot was performed to evaluate the expression of migration-related regulatory proteins including E-cadherin, Vimentin and α-SMA. At least three repeats were used for statistical analysis. *P < 0.05, **P < 0.01, ***P < 0.001, PRPF19 versus Control. ^^P < 0.01, ^^^P < 0.001, shPRPF19-2# versus shNC. PRPF19: Pre-mRNA processing factor 19, α-SMA: α-Smooth muscle actin.

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Further, a Western blot was performed to evaluate the expression of migration-related regulatory proteins including E-cadherin, Vimentin, and α-smooth muscle actin (α-SMA). The results demonstrated that these proteins were regulated by PRPF19 expression in PC-3 or DU145 cells. For E-cadherin, its expression was reduced in PRPF19 overexpression group but increased in shPRPF19-2# group. For Vimentin and α-SMA, their expressions were elevated in PRPF19 overexpression group, but decreased in shPRPF19-2# treated cells. When PRPF19 was overexpressed, low level of E-cadherin, or high expression of Vimentin and α-SMA facilitated the cell migration [Figure 3]b. Thus, PRPF19 played a positive role in regulating migration of PCa cells.

PRPF19 inhibited autophagy in prostate cancer cells

To further elucidate the positive effect of PRPF19 on the proliferation of PCa cells, the autophagy in PC-3 or DU145 cells was evaluated as well. First, a Western blot was performed to test the expression of LC3, Beclin-1, and ATG7. As LC3-II was regarded as the typical marker of autophagy, its expression was decreased in PRPF19 overexpression compared to the control group. While more LC3-II was detected when PRPF19 expression was reduced in shPRPF19-2# group. Similarly, the expression of Beclin-1 and ATG7 in PRPF19 overexpression group was lower than that in the control group, while their expression in shPRPF19-2# group was higher than that in shNC group. These data showed that PRPF19 suppressed autophagy in PC-3 or DU145 cells [Figure 4]a and [Figure 4]b.

Figure 4: PRPF19 inhibited autophagy in PCa cells. (a) Western blot was used to test the effect of PRPF19 on the expression of autophagy related proteins including LC3, Beclin-1 and ATG7. (b) The quantitative analysis of a. (c) IFA was performed to measure the quantity of LC3 in PC-3 and DU145 cells. Three replicate experiments were conducted for statistical analysis. *P < 0.05, **P < 0.01, ***P < 0.001, PRPF19 versus Control. ^P < 0.05, ^^P < 0.01, ^^^P < 0.001, shPRPF19-2# versus shNC. PRPF19: Pre-mRNA processing factor 19, PCa: Prostate cancer, IFA: Immunofluorescence assay.

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Meanwhile, immunofluorescence assay was conducted to measure the quantity of LC3 in PC-3 and DU145 cells. The green fluorescent signal strength indicating LC3 expression was negatively associated with PRPF19. In brief, the LC3 signal was weakened significantly in PRPF19 overexpressed cells. Once PRPF19 expression was attenuated by shPRPF19-2#, LC3 signal was elevated compared to shNC [Figure 4]c. All the data above elucidated that PRPF19 inhibited autophagy in PCa cells.

PRPF19 attenuated the expression of SLC40A1

It has been reported that low expression of SLC40A1 promoted proliferation and migration of PCa cells,^[11] indicating SCL40A1 antagonized the effect of PRPF19 on PCa. Hence, it is necessary to clarify the relationship of these proteins. First, TCGA analysis showed that SLC40A1 was downregulated in PCa tissues [Figure 5]a. Moreover, qRT-PCR was performed to evaluate mRNA level of SLC40A1, and the data unveiled that high expression of PRPF19 repressed the transcription of SLC40A1 in both PC-3 and DU145 cells [Figure 5]b. Moreover, the protein level of SLC40A1 was measured by Western blot. Consistent with the above, PRPF19 overexpression decreased SLC40A1 expression, while PRPF19 downregulation in shPRPF19-2# increased SLC40A1 expression in PC-3 and DU145 cells [Figure 5]c. In sum, PRPF19 attenuated the expression of SLC40A1 in PCa cells.

Figure 5: PRPF19 attenuated the expression of SLC40A1. (a) SLC40A1 was downregulated in PCa from TCGA analysis. (b) qRT-PCR was performed to evaluate the effect of PRPF19 on SLC40A1 transcription in both PC-3 and DU145 cells. (c) Western blot was used to measure the effect of PRPF19 on SLC40A1 expression in PC-3 and DU145 cells. (d) Western blot was performed to measure the protein level of PRPF19 and SLC40A1 in a double knockdown model infected with lentiviruses of shPRPF19 and shSLC40A1. (e) The cell viability was analyzed to elucidate the effect of SLC40A1 knockdown on cell viability in PC-3 with PRPF19 silence. (f) Transwell assay was performed to investigate the effect of SLC40A1 knockdown on migration and invasion of PC-3 with PRPF19 silence. (g) Western blot analysis of E-cadherin, Vimentin and α-SMA expression in PC-3 cells. (h) Western blot was conducted to evaluate the expression of LC3, Beclin-1, and ATG7 in a double knockdown of PRPF19 plus SLC40A1. The statistical analysis contains at least three replicate data. *P < 0.05, **P < 0.01, ***P < 0.001, shPRPF19-2# versus shNC. ^P < 0.05, ^^P < 0.01, ^^^P < 0.001, shPRPF19-2# + sh SLC40A1 versus shPRPF19-2#. ^#P < 0.05, ^##P < 0.01, ^###P< 0.001, shPRPF19-2# + shSLC40A1 versus shSLC40A1. PRPF19: Pre-mRNA processing factor 19, SLC40A1: Solute carrier family 40 member 1, PCa: Prostate cancer, qRT-qPCR: Quantitative real-time polymerase chain reaction.

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Furthermore, PC-3 cells were used to be infected with lentiviruses of shPRPF19 and shSLC40A1, to establish a double knockdown model. Western blot results showed that shPRPF19 reduced PRPF19 expression but promoted SLC40A1 protein expression. In the double knockdown model, both PRPF19 and SLC40A1 protein level were decreased to a considerably low level [Figure 5]d. Then, the cell viability data revealed that PRPF19 knockdown drastically reduced cell proliferation compared to the control (shNC), but low level of SLC40A1 in double knockdown model significantly elevated cell proliferation compared with that in shPRPF19 group, in which the cell proliferation almost recovered to the level of shNC group [Figure 5]e. Moreover, the migration and invasion of cells in shPRPF19 + shSLC40A1 group were at the similar level as that in shNC group, indicating that SLC40A1 knockdown reversed the inhibition of shPRPF19 on migration and invasion [Figure 5]f. SLC40A1 knockdown significantly declined E-cadherin expression, while elevated Vimentin and α-SMA expression compared to shPRPF19 group [Figure 5]g. Finally, the expression of LC3, Beclin-1, and ATG7 was increased in shPRPF19 group. However, their expression level in double knockdown of PRPF19 plus SLC40A1 was decreased significantly compared with that in shPRPF19 group, meaning that SLC40A1 downregulation reversed the effect of PRPF19 down regulation on PCa cell autophagy [Figure 5]h. In a word, downregulation of SLC40A1 reversed the anticancer activity of PRPF19 knockdown.

PRPF19 promotes migration and invasion, inhibits autophagy by suppressing SLC40A1

PRPF19 is highly expressed in PCa, PRPF19 down regulates the transcription level and protein expression of SLC40A1 in PCa cells. Through its inhibitory effect on SLC40A1, PRPF19 attenuates the expression of Beclin-1, LC3, and ATG7, repressing autophagy production; PRPF19 promotes the expression of α-SMA and Vimentin, while reducing E-cadherin level, which enhances migration and invasion of PCa cells [Figure 6]. In sum, PRPF19 plays a vital role in promoting cell proliferation and migration, while inhibiting autophagy in PCa by decreasing SLC40A1 expression.

Figure 6: Model for PRPF19 regulating migration, invasion, and autophagy in PCa cells. Through inhibiting SLC40A1, PRPF19 attenuates the expression of Beclin-1, LC3, and ATG7, repressing autophagy production. PRPF19 promotes the expression of α-SMA and Vimentin, reduces E-cadherin level, and enhances migration and invasion of PCa cells. PRPF19: Pre-mRNA processing factor 19, PCa: Prostate cancer, SLC40A1: Solute carrier family 40 member 1, α-SMA: α-Smooth muscle actin.

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Discussion

PCa is a common cancer and the leading cause of cancer-related death in men. It is necessary to discover tumor suppressor genes and proto-oncogenes for clinical progression and treatment strategies, so as to avoid or treat advanced and metastatic tumors.^[3] PRPF19 is highly expressed in the liver cancer tissues and ovarian tumor cells, which is associated with poor prognosis.^[8],[9] Consistent with these reported studies, the bioinformatics analysis showed PRPF19 was upregulated in PCa. Moreover, qRT-PCR, western blot and IHC detection on clinical PCa tissues confirmed the transcription and expression levels of PRPF19 were significantly elevated in PCa. Thus, it can be concluded that PRPF19 was highly expressed in PCa.

Previous studies reported that PRPF19 overexpression promoted tongue cancer cell proliferation.^[10] A similar study was performed in this work, and the cell viability and EdU incorporation analysis illustrated that cell proliferation could be promoted by PRPF19 overexpression and suppressed by PRPF19 knockdown. As is known that PRPF19 was reduced during replicative senescence, inducing cell cycle arrest by inhibiting MDM4-mediated inactivation of p53, resulting in cellular senescence.^[7] Hence, it makes sense that PRPF19 promoted proliferation in PCa cells.

The migration was also investigated in this work. Transwell assay revealed that PRPF19 played a positive role in regulating migration and invasion of PC-3 or DU145 cells. Moreover, the expression of E-cadherin, Vimentin, and α-SMA was also positively regulated by PRPF19. It has been reported that PRPF19 overexpression promoted tongue cancer cell migration and tumor development.^[10] E-cadherin, Vimentin, and α-SMA are the markers of epithelial-mesenchymal transition which is the key factor for cancer cells migration and invasion.^[13] Their expression is also related to poor prognosis of cancer patients.^[14] It is easy to conclude that PRPF19 promoted the migration of PCa cell through regulating expression of E-cadherin, Vimentin and α-SMA.

Autophagy plays dual roles in tumor-promoting or suppressing cancer-cell development and proliferation.^[15] Hence, it is necessary to evaluate the function of PRPF19 in regulating autophagy. This work confirmed the autophagy-related proteins including LC3, Beclin-1, and ATG7 were negatively regulated by PRPF19, indicating that PRPF19 inhibited autophagy in PCa cells. PRPF19 significantly increased LC3-II expression, which is one of the typical markers for autophagosome production.^[16] Beclin-1 plays a key role in regulating autophagy and cell death.^[17] During the initiation of autophagy, ATG7 is an essential regulator of autophagosome assembly.^[15] Therefore, low expression of these molecules in PRPF19-overexpressed cells indicated that PRPF19 suppressed autophagy.

Previous research showed that SLC40A1 is lowly expressed in PCa, which promoted the proliferation and migration of PCa cells.^[11] Overexpressed SLC40A1 inhibits tumor progression of hepatocellular carcinoma cells by stimulating autophagy.^[12] It is meaningful to clarify the relationship between PRPF19 and SLC40A1 in PCa cells. Hence, the double knockdown of PRPF19 and SLC40A1 was established, and cell proliferation, migration, and autophagy were investigated in this double knockdown model. The data elucidated that PRPF19 repressed the mRNA and protein level of SLC40A1, and SLC40A1 antagonized PRPF19's effects on proliferation, migration, and autophagy. It is easy to conclude that PRPF19 promoted cell proliferation and migration through decreasing SLC40A1 expression.

Conclusion

In summary, this study demonstrated PRPF19 played a vital role in promoting cell proliferation and migration and inhibiting autophagy in PCa by decreasing SLC40A1 expression. Therefore, PRPF19 is expected to be considered a novel therapeutic target for PCa treatment.

Data availability statement

All data generated or analyzed during this study are included in this published article. The datasets used and/or analyzed during the present study are available from the corresponding author on reasonable request.

Ethics approval

Ethical approval was obtained from the Ethics Committee of the Seventh Affiliated Hospital, Southern Medical University (Approval No. 2016(06)0254).

Statement of informed consent

Written informed consent was obtained from a legally authorized representative(s) for anonymized patient information to be published in this article.

Author contributions

Conceptualization, methodology, and original draft writing: Guofei Zhang. Formal analysis, resources, and investigation: Wansong Zhang. Formal analysis, visualization and data curation: Mingjiang Dan. Project administration, supervision, and validation: Feng Zou. Validation, supervision, and writing (review and editing): Chunming Qiu and Canbiao Sun. All authors read and approved the final manuscript.

Financial support and sponsorship

Foshan Science and Technology Bureau, self-funded science and technology innovation project (Project No: 2220001005685).

Conflicts of interest

There are no conflicts of interest.

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