|Year : 2023 | Volume
| Issue : 5 | Page : 295-305
LncRNA VPS9D1-AS1 regulates miR-187-3p/fibroblast growth factor receptor-like 1 axis to promote proliferation, migration, and invasion of prostate cancer cells
Chenguang Wu, Jian Chen, Dong Wang
Department of Urology Surgery, 900 Hospital of the Joint Logistics Team, Fuzhou, Fujian, China
|Date of Submission||23-Apr-2023|
|Date of Decision||30-Jun-2023|
|Date of Acceptance||16-Jul-2023|
|Date of Web Publication||26-Oct-2023|
Dr. Dong Wang
Department of Urology Surgery, 900 Hospital of the Joint Logistics Team, 156 West Erhuan Rd., Fuzhou 350025
Source of Support: None, Conflict of Interest: None
The morbidity and mortality of prostate cancer are increasing year by year, and the survival rate of prostate cancer patients after treatment is low. Therefore, investigating the molecular mechanism underlying prostate cancer is crucial for developing effective treatments. Recent studies have shown the important role of long-chain non-coding RNAs (lncRNAs) in tumorigenesis. VPS9D1-AS1 can modulate the progression of multiple cancers, but its molecular action mechanism in prostate cancer remains unknown. This study, therefore, intended to investigate the regulatory mechanism of VPS9D1-AS1 in prostate cancer. First, differentially expressed lncRNAs in prostate cancer were identified through bioinformatics approaches. The target lncRNA for the study was determined by reviewing the relevant literature and its downstream miRNA/mRNA axis was uncovered. Then, quantitative reverse transcription polymerase chain reaction was introduced to assess the expression of VPS9D1-AS1, miR-187-3p, and fibroblast growth factor receptor-like 1 (FGFRL1) at a cellular level, and Western blot was conducted to assess the protein level of FGFRL1 in cells. The results indicated that VPS9D1-AS1 and FGFRL1 were highly expressed in prostate cancer while miR-187-3p was less expressed. Besides, MTT, colony formation, wound healing, and cell invasion assays showed that silencing VPS9D1-AS1 inhibited the viability, migration ability, and invasion ability of prostate cancer cells. Dual-luciferase assay and RNA binding protein immunoprecipitation assay were performed to explore the interplay of miR-187-3p and VPS9D1-AS1 or FGFRL1. The results showed that VPS9D1-AS1 could sponge miR-187-3p, and FGFRL1 could serve as a direct target of miR-187-3p. Moreover, combined with the results of the rescue experiment, VPS9D1-AS1 was found to upregulate FGFRL1 by competitively sponging miR-187-3p to accelerate the malignant behaviors of prostate cancer cells. In conclusion, VPS9D1-AS1 could promote the phenotype progression of prostate cancer cells through targeting the miR-187-3p/FGFRL1 axis, and it has the potential to be a target for prostate cancer patients.
Keywords: Fibroblast growth factor receptor-like 1, invasion, migration, miR-187-3p, proliferation, prostate cancer, VPS9D1-AS1
|How to cite this article:|
Wu C, Chen J, Wang D. LncRNA VPS9D1-AS1 regulates miR-187-3p/fibroblast growth factor receptor-like 1 axis to promote proliferation, migration, and invasion of prostate cancer cells. Chin J Physiol 2023;66:295-305
|How to cite this URL:|
Wu C, Chen J, Wang D. LncRNA VPS9D1-AS1 regulates miR-187-3p/fibroblast growth factor receptor-like 1 axis to promote proliferation, migration, and invasion of prostate cancer cells. Chin J Physiol [serial online] 2023 [cited 2023 Dec 9];66:295-305. Available from: https://www.cjphysiology.org/text.asp?2023/66/5/295/388468
| Introduction|| |
Prostate cancer occurs in the prostatic epithelium. The latest data from the Global Cancer Survey 2020 exhibited that there were about 1.4 million new cases and 375,000 deaths in 2020 throughout the world. Furthermore, morbidity and mortality of prostate cancer are rising year by year, which severely restricts life quality. Thus far, treatments for prostate cancer mainly include drug therapy and excision. In recent years, a breakthrough has been achieved in therapeutic approaches. Nevertheless, poor prognosis of prostate cancer patients is unavoidable and tumor often reoccurs and metastasizes. Once those happened, the patients' five-survival rate will be <30%.,, Hence, it is of great importance to probe into prostate cancer molecular mechanisms and provide treatment targets for prostate cancer patients to improve their survival rate.
Long non-coding RNA (lncRNA), with a length of more than 200 nucleotides, has been regarded as “transcriptional noise” previously without functions in the human body., VPS9D1-AS1 is an antisense RNA, which is highly expressed in colorectal cancer, esophageal squamous cell carcinoma, and liver cancer, exerting an oncogenic effect. Despite existing reports on the effects of VPS9D1-AS1 on prostate cancer,, the specific molecular modulatory mechanism of VPS9D1-AS1 in this disease remains uncertain and needs to be explored.
LncRNA can serve as a sponge of miRNA to modulate levels of downstream genes, thus influencing tumor progression. MiR-187-3p exhibits low expression in multiple tumors. For example, it is decreased in infant hemangioma tissue and accelerates the sensitivity of hemangioma stem cells to propranolol. MiR-187-3p decreases breast cancer cell sensitivity to gemcitabine by targeting fibroblast growth factor 9 (FGF9). Besides, ATF2-induced lncRNA GAS8-AS1 hastens autophagy in thyroid cancer cells through miR-187-3p/ATG5 axis. However, the function of miR-187-3p in prostate cancer is rarely studied.
FGF receptor-like 1 (FGFRL1) modulates multiple cellular processes. FGFRL1 facilitates the progression of ovarian cancer through crosstalk with Hedgehog signaling. Besides, FGFRL1 is modulated by miRNA and participates in tumor occurrence and progression. For instance, lung cancer-derived exosomal miR-210-3p facilitates lung cancer cell metastasis by down-regulation of FGFRL1 level. FGD5-AS1 increases FGFRL1 level through sponging miR-107 to foster the proliferation of non-small cell lung cancer (NSCLC) cells. But no studies have been done about the molecular mechanism of FGFRL1 in prostate cancer.
Bioinformatics revealed highly-expressed lncRNA VPS9D1-AS1 and FGFRL1 and lowly-expressed miR-187-3p in prostate cancer. We then speculated that lncRNA VPS9D1-AS1/miR-187-3p/FGFRL1 could influence prostate cancer progression. Afterward, we manifested through molecular and cellular experiments that lncRNA VPS9D1-AS1 increased FGFRL1 level through competitively sponging miR-187-3p, thus facilitating the malignant phenotype of prostate cancer cells. These efforts will provide a theoretical basis for VPS9D1-AS1 as a possible treatment target for prostate cancer patients.
| Materials and Methods|| |
Expression data of lncRNA (normal: 52, tumor: 499), miRNA (normal: 52, tumor: 499), and mRNA (normal: 52, tumor: 499) as well as clinical data were got from The Cancer Genome Atlas (TCGA) database. Next, differential expression between normal and tumor groups of lncRNA was assessed by utilizing the “edgeR” package (|logFC| >1.5, FDR < 0.05). Afterward, the target lncRNA of the study was determined through literature citation, and then subcellular localization analysis was conducted on this gene by using the lncATLAS database to confirm its expression location in cells. Furthermore, R package “edgeR” was utilized again to measure differences between normal and tumor groups of miRNA and mRNA expression data (miRNA: |log FC| >1.5, FDR < 0.05; mRNA: |log FC| >1.5, FDR < 0.05). Moreover, the starBase database was introduced to predict miRNAs that interact with the target lncRNA. Then the predicted miRNAs were overlapped with differentially downregulated miRNAs in prostate cancer, and the miRNA showing the highest negative correlation was chosen as the study of interest. Similarly, mirDIP and starBase were applied to predict regulatory target genes downstream of miRNA. The candidate target genes were got from overlapping of differentially upregulated mRNAs in prostate cancer and predicted target genes. Finally, mRNA with the highest inverse correlation was chosen as the study of interest.
Prostate epithelial cell line RWPE-1 (No. 3131C0001000200037) and prostate cancer cell line VCaP (No. 3131C0001000700220) were accessed from Cell Bank, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences. Prostate cancer cell lines PC-3 (No. 3111C0001CCC000115) and DU-145 (No. 3111C0001CCC000006) were bought from Cell Resource Center, Institute of Basic Medicine, Chinese Academy of Medical Sciences. PC-3 cells and DU-145 cells were cultured in the F12K medium (Thermo Fisher Scientific, USA) and serum-free cell RPMI-1640 medium (Thermo Fisher Scientific, USA), respectively. Other cell lines were prepared in DMEM (Thermo Fisher Scientific, USA). All of the mediums were added with 10% fetal bovine serum (FBS) (Life Technologies, Inc., USA).
Sh-VPS9D1-AS1, miR-187-3p-inhibitor, miR-187-3p-mimic, oe-FGFRL1, and negative controls (NC) were provided by RiboBio Company (China). According to the instructions of the manufacturer, all of them were transfected into PC-3 cells with Lipofectamine 2000 kit (Invitrogen, USA), and transfected efficiency was tested at 48 h after transfection.
Quantitative reverse transcription polymerase chain reaction
Total RNA was extracted from cells with TRIzol kit (Thermo Fisher Scientific, USA), and was reversely transcribed into cDNA with reverse transcription kit (Invitrogen, USA), followed by qRT-PCR on Applied Biosystems 7500 (Applied Biosystems, USA) detection system with SYBR Green kit (Takara, Japan). GAPDH acted as the endogenous reference for VPS9D1-AS1 and FGFRL1 while U6 for miR-187-3p. Relative expression levels of VPS9D1-AS1, miR-187-3p, and FGFRL1 were calculated by the 2−ΔΔCT method. qRT-PCR primers were presented in [Table 1].
|Table 1: Quantitative reverse transcription polymerase chain reaction primer sequences|
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Cytoplasm and nucleus in PC-3 cells were fractionated with PARIS kit (Thermo Fisher Scientific, USA). RNA was extracted from them. The location of VPS9D1-AS1 in the cells was analyzed using qRT-PCR, with GAPDH and U6 serving as cytoplasm and nucleus controls, respectively.
Total proteins were isolated utilizing RIPA kit (Nanjing KeyGen Biotech Co., Ltd., China) and then the proteins underwent SDS-PAGE. Thereafter, proteins were transferred onto a nitrocellulose membrane (Millipore, USA) and supplemented with primary antibodies rabbit anti-FGFRL1 (ab95940, 1:1000, Abcam, UK) or GAPDH (ab181602, 1:10000, Abcam, UK) for overnight culture at 4°C. Upon finish, the proteins were probed with secondary antibody goat anti-rabbit IgG H & L coupled with horse radish peroxidase at room temperature for 1 h. Finally, the expression levels of proteins were observed using an ECL kit (Sigma-Aldrich, USA).
The viability of PC-3 cells was assessed using MTT. In brief, 2 × 103 PC-3 cells were seeded into a 96-well plate. Cells were cultivated for 0, 24, 48, and 72 h before being treated with 10 μL MTT kit (Solarbio, China) for 4 h of incubation. Thereafter, the residue was removed. Then, 110 μL Formazan was added into the wells. A microplate reader (SpectraMax M2, Molecular Devices, USA) was implemented for the assessment of optical density at 490 nm.
Colony formation assay
1 × 103 PC-3 cells were seeded into 6-well plates. The culture was stopped when visible spots appeared in the culture holes. After discarding the culture solution, the cells were fixed with 4% paraformaldehyde (PA) and dyed with 0.1% crystal violet for 10 min. In the end, the colony in each well was counted.
Wound healing assay
4 × 105 PC-3 cells were plated into 6-well plates. After cell confluence reached 90%, cells were scraped with a 200 μL pipette tip and cultured with DMEM without FBS. After 0 h and 48 h of culture, the observed areas were photographed with a microscope and the wound healing rate of each group was computed. Computational formulas: cell migration rate = (0 h wound area – 48 h wound area)/0 h wound area.
Cell invasion assay
2 × 104 PC-3 cells were inoculated into a transwell room laid with Matrigel (BD Biosciences, USA), and mitomycin C was added. The lower room was supplemented with 600 μL DMEM + 20% FBS. The uninvading cells were swabbed with a cotton swab 24 h later. Invading cells underwent fixation with 4% PA for 15 min, followed by staining with 0.1% crystal violet for 10 min. Finally, cell number within five random fields was calculated under a microscope.
The 3' untranslated region of wild type (wt) VPS9D1-AS1/FGFRL1 having binding sites with miR-187-3p was cloned to multi colony sites in the downstream of pmirGLO (Promega, USA) luciferase gene (VPS9D1-AS1-Wt/FGFRL1-Wt). At the same time, VPS9D1-AS1/FGFRL1 mutant (mut) vector was constructed (VPS9D1-AS1-Mut/FGFRL1-Mut). Afterward, VPS9D1-AS1-Wt/FGFRL1-Wt or VPS9D1-AS1-Mut/FGFRL1-Mut and miR-187-3p were co-transfected into PC-3 cells using Lipofectamine 2000. Forty-eight hour later, activities of firefly and renilla luciferase were assessed on the luciferase reporting analysis system (Promega, USA), and the renilla luciferase viability was utilized for standard processing on data.
RNA binding protein immunoprecipitation
Magna RIP™ RIP Kit was applied to conduct RIP assay. PC-3 cells were lysed in RIP lysis buffer and cultivated with a magnetic bead coupled with anti-Ago2 or anti-IgG antibody. Finally, levels of VPS9D1-AS1 and miR-187-3p in antibody precipitation complexes were evaluated by qRT-PCR.
All cell assays in the study were repeated alone three times. All of the data were processed via GraphPad Prism 6 (GraphPad Software Inc., San Diego, CA, USA) and presented in the form of mean ± standard deviation. Differential expression analysis between the two groups was compared through t-test and P < 0.05 demonstrated the statistical significance.
| Results|| |
VPS9D1-AS1 level is upregulated in prostate cancer
As described earlier, lncRNA expression levels of the prostate cancer group and normal group in TCGA were analyzed using edgeR. Altogether, 790 substantially dysregulated lncRNAs in prostate cancer were obtained [Figure 1]a. Previous studies showed that VPS9D1-AS1 level was elevated in NSCLC and hastened cancer progression. In addition, by analyzing 790 lncRNAs, we found that VPS9D1-AS1 was also substantially increased in prostate cancer [Figure 1]b. To validate the result of bioinformatics analysis, we utilized qRT-PCR to detect VPS9D1-AS1 expression in RWPE-1, VCaP, PC-3, and DU-145 cells. Compared with RWPE-1 cells, the VPS9D1-AS1 level in VCaP, PC-3, and DU-145 cells was substantially upregulated [Figure 1]c. As a result, we validated that VPS9D1-AS1 was highly expressed in prostate cancer. Besides, since VPS9D1-AS1 was most substantially upregulated in PC-3 cells, we chose PC-3 cells for study in subsequent assays.
|Figure 1: VPS9D1-AS1 level is elevated in prostate cancer. (a) Volcano plot of differentially expressed lncRNAs in prostate cancer in The Cancer Genome Atlas (TCGA) database, in which upregulated and downregulated lncRNAs in prostate cancer were represented by red dots and green dots, respectively. (b) Specific expression of VPS9D1-AS1 in prostate cancer and normal groups in TCGA database, with normal tissues shown with green and prostate cancer tissues shown with red. (c) In quantitative real-time reverse transcription polymerase chain reaction assay, VPS9D1-AS1 expression of RWPE-1, VCaP, PC-3, and DU-145 cells. *P < 0.05 relative to RWPE-1 group, n = 3.|
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Silencing VPS9D1-AS1 represses malignant phenotypes of prostate cancer cells
To assess whether VPS9D1-AS1 can modulate prostate cancer cells, we transfected sh-VPS9D1-AS1 into PC-3 cells. Afterward, qRT-PCR was carried out to confirm that transfection of sh-VPS9D1-AS1 contributed to a significant decrease of VPS9D1-AS1 in PC-3 cells [Figure 2]a. Afterward, we performed cell biological functional assays to examine the impacts of silencing VPS9D1-AS1 on the phenotype progression of PC-3 cells. We noticed that silencing VPS9D1-AS1 led to significant reductions in the proliferation and colony formation of PC-3 cells through MTT and colony formation assays [Figure 2]b and [Figure 2]c. Moreover, silencing VPS9D1-AS1 notably lessened migratory and invasive properties of PC-3 cells through wound healing and invasion assays [Figure 2]d and [Figure 2]e. Based on the assays above, we identified the promotion effect of VPS9D1-AS1 on the phenotype progression of prostate cancer cells.
|Figure 2: Silencing VPS9D1-AS1 represses malignant phenotypes of prostate cells. (a) Impact of transfecting sh-VPS9D1-AS1 on VPS9D1-AS1 level in PC-3 cells was tested in quantitative real-time reverse transcription polymerase chain reaction assay. (b) Impact of silencing VPS9D1-AS1 on the proliferation of PC-3 cells was detected in MTT assay. (c) Impact of silencing VPS9D1-AS1 on the colony formation of PC-3 cells was assayed in a colony formation assay. (d) Impact of silencing VPS9D1-AS1 on the migration of PC-3 cells was evaluated in a wound healing assay (magnification: ×40). (e) Impact of silencing VPS9D1-AS1 on the invasion of PC-3 cells was estimated in cell invasion assay (magnification: ×100). *P < 0.05 relative to sh-NC group, n = 3.|
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VPS9D1-AS1 sponges miR-187-3p in prostate cancer
Subcellular fractionation was utilized to perform the expressed location of VPS9D1-AS1 in PC-3 cells and revealed that VPS9D1-AS1 was distributed in the cell nucleus and cytoplasm. This exhibited the capability of VPS9D1-AS1 to be a ceRNA [Figure 3]a. Thereby, miRNAs were differentially analyzed. Totally, 51 differentially expressed miRNAs (DEmiRNAs) were obtained (15 downregulated miRNAs and 36 upregulated miRNAs) [Figure 3]b. In addition, we used the starBase database to predict miRNAs that bind to VPS9D1-AS1. The predicted miRNAs were then intersected with downregulated DEmiRNAs, and 2 miRNAs were obtained (miR-187-3p and miR-23c) [Figure 3]c. Through correlation analysis, the inverse correlation of miR-187-3p and VPS9D1-AS1 was found to be the highest [Figure 3]d. MiR-187-3p level was markedly lower in prostate cancer tissues than in normal tissues as revealed by bioinformatics analysis [Figure 3]e. To verify the bioinformatics prediction above, qRT-PCR was carried out to investigate the expression of miR-187-3p in RWPE-1, VCaP, PC-3, and DU-145 cells. The outcome demonstrated a low miR-187-3p level in prostate cancer cells [Figure 3]f. Afterward, we designed a mutant sequence of VPS9D1-AS1 according to the binding site of VPS9D1-AS1 and miR-187-3p in the starBase database [Figure 3]g. The binding site was then observed in a dual-luciferase assay. The result indicated that miR-187-3p notably hampered the luciferase viability of the VPS9D1-AS1-Wt reporter gene, while it had no impact on VPS9D1-AS1-Mut reporter genes [Figure 3]h. In addition, there was an interplay between VPS9D1-AS1 and miR-187-3p by RIP assay [Figure 3]i. Finally, we examined miR-187-3p level in PC-3 cells that transfected with sh-VPS9D1-AS1 via qRT-PCR, and the outcome revealed that miR-187-3p expression could be markedly upregulated by transfecting sh-VPS9D1-AS1 [Figure 3]j. These assays validated that VPS9D1-AS1 could sponge miR-187-3p in prostate cancer.
|Figure 3: VPS9D1-AS1 sponges miR-187-3p in prostate cancer. (a) Locations of VPS9D1-AS1 in the nucleus and cytoplasm were observed in the subcellular fractionation assay. (b) Volcano plot of differentially expressed miRNAs in prostate cancer in The Cancer Genome Atlas (TCGA) database, in which upregulated and downregulated miRNAs in prostate cancer are expressed as red dots and green dots respectively. (c) Venn diagram of intersecting miRNAs that share binding site with VPS9D1-AS1 in starBase database/differentially downregulated miRNAs in prostate cancer in TCGA database. (d) Heat map of correlative analysis on the expressions of VPS9D1-AS1, miR-187-3p, and miR-23c in TCGA database. (e) MiR-187-3p level in prostate cancer tissues (red) and normal tissues (green). (f) MiR-187-3p expression in RWPE-1, VCaP, PC-3, and DU-145 cells was examined via quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) *P < 0.05 relative to the RWPE-1 group, n = 3. (g) Binding site of VPS9D1-AS1-Wt and miR-187-3p in starBase database. (h) The binding relationship between VPS9D1-AS1 and miR-187-3p was verified in dual-luciferase assy. *P < 0.05 relative to VPS9D1-AS1-Wt + negative controls (NC)-mimic group, n = 3. (i) The interplay between VPS9D1-AS1 and miR-187-3p was confirmed in the RIP assay. *P < 0.05 relative to IgG group, n = 3. (j) The impact of silencing VPS9D1-AS1 on miR-187-3p expression in PC-3 cells was assessed utilizing qRT-PCR. *P < 0.05 relative to sh-NC group, n = 3.|
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Downregulation of miR-187-3p reverses the repressive impact of sh-VPS9D1-AS1 on the progression of prostate cancer cells
To evaluate whether VPS9D1-AS1 can hasten the phenotype progression of prostate cancer cells through sponging miR-187-3p, the following groupings were set up: sh-NC + NC-inhibitor, sh-VPS9D1-AS1 + NC-inhibitor, and sh-VPS9D1-AS1 + miR-187-3p-inhibitor, which were transfected into PC-3 cells. qRT-PCR assay was done to assess miR-187-3p level in PC-3 cells in three groups. The result displayed that the accelerated effect of sh-VPS9D1-AS1 on miR-187-3p level could be rescued by miR-187-3p-inhibitor [Figure 4]a. Next, we observed the malignant phenotype of PC-3 cells in three groups. The outcome denoted that, after miR-187-3p-inhibitor was transfected, the suppressive impact of sh-VPS9D1-AS1 on cancer cell malignant phenotypes was notably reversed [Figure 4]b, [Figure 4]c, [Figure 4]d, [Figure 4]e. As a result, miR-187-3p was ascertained to be a downstream target of VPS9D1-AS1 in modulating the progression of prostate cancer.
|Figure 4: Repressive impact of sh-VPS9D1-AS1 on phenotype progression is rescued by downregulating miR-187-3p. (a) qRT-PCR was done to test miR-187-3p level in PC-3 cells in 3 groups (sh-NC + NC-inhibitor, sh-VPS9D1-AS1 + NC-inhibitor, and sh-VPS9D1-AS1 + miR-187-3p-inhibitor). (b) MTT assay detected the proliferative capacity of PC-3 cells in 3 groups. (c) Colony formation assay was carried out to examine the colony formation ability of PC-3 cells in 3 groups. (d) Wound healing assay was launched to assess the migratory property of PC-3 cells in three groups (magnification: ×40). (e) Cell invasion assay estimated the invasive property of PC-3 cells in 3 groups (magnification: ×100). *P < 0.05 relative to sh-NC + NC-inhibitor group, #P < 0.05 relative to sh-VPS9D1-AS1 + NC-inhibitor group, n = 3. NC: Negative controls.|
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FGFRL1 is a downstream target of miR-187-3p in prostate cancer
We mined mRNAs downstream of miR-187-3p to explore the molecular mechanism of VPS9D1-AS1. A total of 1402 DEmiRNAs (743 downregulated mRNAs and 659 upregulated mRNAs) in prostate cancer were obtained through TCGA database [Figure 5]a. Next, target mRNAs of miR-187-3p were predicted utilizing mirDIP and starBase databases. Then, mRNAs were overlapped with upregulated mRNAs in prostate cancer in TCGA database and 7 mRNAs were obtained (HOXC9, GATA4, ERG, GBX2, CAMK2N2, LRFN1, and FGFRL1) [Figure 5]b. By analyzing their correlations, FGFRL1 was found to have the highest negative correlation with miR-187-3p [Figure 5]c. In TCGA database, the FGFRL1 level was pronouncedly higher in prostate cancer tissues than in normal tissues [Figure 5]d. FGFRL1 mRNA and protein levels in prostate cancer cells were conspicuously elevated through qRT-PCR and western blot analyses [Figure 5]e and [Figure 5]f. Next, we utilized the binding site of miR-187-3p and FGFRL1 in the starBase database to design the mutant sequences of FGFRL1 [Figure 5]g and then performed a dual-luciferase assay. The result manifested that miR-187-3p reduced the luciferase viability of FGFRL1-Wt, but it had no impact on that of FGFRL1-Mut [Figure 5]h. Finally, we performed qRT-PCR and western blot assays to reveal the effect of overexpression of miR-187-3p on the expression of VPS9D1-AS1 and FGFRL1 in PC-3 cells. As a result, FGFRL1 mRNA and protein expression levels in PC-3 cells were significantly inhibited by overexpression of miR-187-3p but increased by inhibition of miR-187-3p. However, the increase or decrease of miR-187-3p expression had no significant effect on the expression of its upstream regulatory gene VPS9D1-AS1 [Figure 5]i and [Figure 5]j. FGFRL1 was identified as a downstream target of miR-187-3p.
|Figure 5: FGFRL1 is a downstream target of miR-187-3p in prostate cancer. (a) Volcano plot of differentially expressed mRNAs in prostate cancer in The Cancer Genome Atlas (TCGA) database, in which red dots and green dots indicate upregulated and downregulated mRNAs in prostate cancer, respectively. (b) Venn diagram of the intersection of mRNAs sharing the binding site with miR-187-3p in starBase and mirDIP databases and DEmiRNAs in prostate cancer in TCGA database. (c) Heat plot about the correlation analysis on expressions of miR-187-3p and 7 mRNAs (HOXC9, GATA4, ERG, GBX2, CAMK2N2, LRFN1, and FGFRL1) in TCGA database. (d) FGFRL1 level in prostate cancer tissues (red) and normal tissues (green) in TCGA database. (e) Gene expression of FGFRL1 in RWPE-1, VCaP, PC-3, and DU-145 cells was assessed through quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR). *P < 0.05 relative to RWPE-1 group, n = 3. (f) Western blot was done to test the protein level of FGFRL1 in cells. (g) Binding site of miR-187-3p and FGFRL1-WT in starBase database. (h) Binding relationship of miR-187-3p and FGFRL1 was identified through conducting a dual-luciferase assay. *P < 0.05 relative to FGFRL1-Wt + NC-mimic group, n = 3. (i) The effects of overexpression or inhibition of miR-187-3p on the expression of VPS9D1-AS1 and FGFRL1 in PC-3 cells were tested by qRT-PCR assay. *P < 0.05 relative to NC-mimic group, n = 3. (j) The effect of overexpression or inhibition of miR-187-3p on FGFRL1 protein level in PC-3 cells was evaluated by Western blot.|
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Upregulation of FGFRL1 rescues the repressive effect of miR-187-3p mimic on proliferation, migration, and invasion of prostate cancer cells
We carried out a rescue assay to research the regulatory impact of FGFRL1 on prostate cancer cells. Transfection treatment was performed in PC-3 cells and cell groups were set as NC-mimic + oe-NC, miR-187-3p-mimic + oe-NC, NC-mimic + oe-FGFRL1, and miR-187-3p-mimic + oe-FGFRL1. We noticed that transfection of miR-187-3p mimic could downregulate the mRNA and protein expression of FGFRL1 in PC-3 cells through qRT-PCR and Western blot, while transfecting oe-FGFRL1 could reverse such inhibitory effect [Figure 6]a and [Figure 6]b. Besides, oe-FGFRL1 greatly reversed the repressive influence of miR-187-3p mimic on malignant phenotypes of PC-3 cells by cell biological functional assays [Figure 6]c, [Figure 6]d, [Figure 6]e, [Figure 6]f. Based on the above results, overexpression of miR-187-3p could inhibit FGFRL1 expression and promote the malignant phenotypes of prostate cancer cells.
|Figure 6: Repressive effect of miR-187-3p mimic on malignant phenotypes of prostate cancer cells was rescued through upregulating FGFRL1. (a) Quantitative real-time reverse transcription polymerase chain reaction was utilized to examine FGFRL1 level in PC-3 cells of 4 groups (NC-mimic + oe-NC, miR-187-3p-mimic + oe-NC, NC-mimic + oe-FGFRL1, and miR-187-3p-mimic + oe-FGFRL1). (b) Western blot was employed for measuring FGFRL1 protein level in PC-3 cells of 4 groups. (c) MTT was done to test the proliferative ability of PC-3 cells in 4 groups. (d) Colony formation assay was conducted for evaluating the colony formation property of PC-3 cells in 4 groups. (e) A wound healing assay was introduced for examination of the migratory capacity of PC-3 cells in 4 groups (magnification: ×40). (f) Cell invasion assay was launched for testing the invasive capacity of PC-3 cells in 4 groups (magnification: ×100). *P < 0.05 relative to NC-mimic + oe-NC group, #P < 0.05 relative to NC-mimic + oe-FGFRL1 group, n = 3.|
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Upregulation of FGFRL1 rescues the repressive effect of sh-VPS9D1-AS1 on proliferation, migration, and invasion of prostate cancer cells
We carried out a rescue assay to research the regulatory impact of FGFRL1 on prostate cancer cells. Transfection treatment groups were set as sh-NC + oe-NC, sh-VPS9D1-AS1 + oe-NC, sh-NC + oe-FGFRL1, and sh-VPS9D1-AS1 + oe-FGFRL1 based on PC-3 cells. Through qRT-PCR and western blot assays, we noticed that transfection of sh-VPS9D1-AS1 could downregulate the mRNA and protein expressions of FGFRL1 in PC-3 cells while transfecting oe-FGFRL1 could reverse such inhibitory effect [Figure 7]a and [Figure 7]b. Besides, the outcome of the cell biological functional assay revealed that oe-FGFRL1 greatly reversed the repressive influence of sh-VPS9D1-AS1 on the malignant phenotype of PC-3 cells [Figure 7]c, [Figure 7]d, [Figure 7]e, [Figure 7]f. In combination with the previous studies, it could be concluded that VPS9D1-AS1 indirectly upregulated FGFRL1 by competitively binding to miR-187-3p, thereby suppressing the malignant phenotypes of prostate cancer cells.
|Figure 7: Repressive effect of sh-VPS9D1-AS1 on malignant phenotypes of prostate cancer cells was rescued through upregulating fibroblast growth factor-like-1 (FGFRL1). (a) Quantitative real-time reverse transcription polymerase chain reaction was utilized to examine FGFRL1 level in PC-3 cells of 4 groups (sh-NC + oe-NC, sh-VPS9D1-AS1 + oe-NC, sh-NC + oe-FGFRL1, and sh-VPS9D1-AS1 + oe-FGFRL1). (b) Western blot was employed for measuring FGFRL1 protein level in PC-3 cells of 4 groups. (c) MTT was done to test the proliferative ability of PC-3 cells in 4 groups. (d) Colony formation assay was conducted for evaluating the colony formation property of PC-3 cells in 4 groups. (e) A wound healing assay was introduced for examination of the migratory capacity of PC-3 cells in 4 groups (magnification: ×40). (f) Cell invasion assay was launched for testing the invasive capacity of PC-3 cells in 4 groups (magnification: ×100). *P < 0.05 relative to sh-NC + oe-NC group, #P < 0.05 relative to sh-NC + oe-FGFRL1 group, n = 3.|
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| Discussion|| |
LncRNA VPS9D1-AS1 has been demonstrated to be highly expressed in prostate cancer and to stimulate prostate cancer cells to proliferate and migrate, which is consistent with the results of this study. In addition, lncRNA VPS9D1-AS1 is highly expressed in various cancers., These studies suggest that lncRNA VPS9D1-AS1 has the potential to become a key node in regulating tumor development, but more research data are needed to support this hypothesis. LncRNAs function as “sponges” or “ceRNAs” in the modulatory network including lncRNAs, miRNAs, and target genes, affecting the interaction between miRNAs and mRNAs, and thereby influencing the treatment and prognosis of tumors.,, Recent literature has shown that VPS9D1-AS1 can act as a ceRNA for miR-184 and upregulate c-Myc expression in prostate cancer. In this study, bioinformatics techniques were used to predict that VPS9D1-AS1 contained complementary binding sites for miR-187-3p. The binding association between miR-187-3p and VPS9D1-AS1 was validated using luciferase reporter gene and RIP assays. MiR-187-3p was expressed at a low level in prostate cancer tissues and negatively linked with the expression of VPS9D1-AS1 according to bioinformatics analysis. VPS9D1-AS1 knockdown increased miR-187-3p expression in prostate cancer cells, reduced the proliferation and migration of prostate cancer cells, and this suppressive effect was counteracted by suppressing the expression of miR-187-3p. In combination with previous research, it has been found that VPS9D1-AS1 has multiple downstream regulatory target genes in prostate cancer, such as miR-187-3p, miR-184, and miR-4739.
MiR-187-3p is downregulated in NSCLC, pituitary adenoma, breast cancer, thyroid cancer, liver cancer, and colon cancer, but upregulated in ovarian cancer. MiR-187-3p has been reported as a candidate diagnostic screening miRNA for prostate cancer by multiple scholars.,, However, in this study, two mRNAs, LRFN1 and FGFRL1, were found to have the same negative correlation score with miR-187-3. The correlation between miR-187-3p and LRFN1 has been investigated in renal cancer but has not yet been discovered in prostate cancer. In this work, we found the link between miR-187-3p and FGFRL1 for the first time. In addition, we found that ERG fusion status in 7 candidate genes played an important role in the development of prostate cancer., Subsequently, we found that FGFRL1 was significantly down-regulated in the ERG fusion state through bioinformatics analysis [Supplementary Figure 1], which is a direction worthy of further study. Herein, FGFRL1 mRNA is the downstream target of miR-187-3p in prostate cancer cells, and overexpression of miR-187-3p can inhibit the expression of downstream FGFRL1 at mRNA and protein levels, thereby suppressing the malignant progression of prostate cancer cells.
FGFRL1 is a type of FGF receptor in which the intracellular tyrosine kinase domain is replaced by a short histidine-rich C-terminal tail that is incapable of transmitting canonical signal transduction via receptor auto-phosphorylation. The association between elevated expression of FGFRL1 and progression of prostate cancer has been shown by previous research., We demonstrated that overexpression of FGFRL1 promoted the clonogenic and invasive abilities of prostate cancer cells, suggesting that FGFRL1 played an oncogenic role in prostate cancer. FGFRL1 overexpression could restore the suppressive effect of knocking down VPS9D1-AS1 and miR-187-3p mimic on prostate cancer, and FGFRL1 was directly regulated by miR-187-3p. Finally, this work found a novel signaling axis, VPS9D1-AS1-miR-187-3p-FGFRL1, which was associated with the malignant progression of prostate cancer.
| Conclusion|| |
Herein, VPS9D1-AS1 and FGFRL1 were found to be highly expressed in prostate cancer tissues, while miR-187-3p was expressed at a low level. Knockdown of VPS9D1-AS1 could suppress the proliferation and migration of PC-3 cells. Silencing VPS9D1-AS1 could upregulate its downstream target gene miR-187-3p, and overexpression of miR-187-3p could downregulate its downstream target gene FGFRL1, demonstrating the molecular mechanism of the VPS9D1-AS1/miR-187-3p/FGFRL1 axis in affecting proliferation and migration of PC-3 cells. However, our research still has some limitations, and we plan to validate the results at the animal and clinical levels. VPS9D1-AS1 is expected to be a molecular target for patients with prostate cancer, but extensive research work is still needed to achieve this goal.
Data availability statement
The data and materials in the current study are available from the corresponding author on reasonable request.
Chenguang Wu participated in the design and interpretation of the data and drafted/revised the manuscript. Dong Wang and Jian Chen conceived of the study, and participated in its design and interpretation and helped to draft the manuscript. Chenguang Wu performed the statistical analysis and revised the manuscript critically. All the authors read and approved the final manuscript.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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