Long Noncoding RNA PVT1 Promotes Melanoma Progression via Endogenous Sponging miR-26b.

Melanoma is an extremely aggressive malignant skin tumor with a high mortality. Various long noncoding RNAs (lncRNAs) have been reported to be associated with the oncogenesis of melanoma. The purposes of this study were to investigate the potential role of lncRNA PVT1 in melanoma progression and to explore its possible mechanisms. A total of 35 patients who were diagnosed with malignant melanoma were enrolled in this study. Expression of PVT1 was significantly upregulated in melanoma tissue and was associated with a poor prognosis. Loss-of-function experiments showed that PVT1 knockdown markedly suppressed the proliferation activity, induced cell cycle arrest at the G0/G1 phase, and enhanced the apoptosis of melanoma cell lines. Bioinformatics analysis and dual-luciferase reporter assay revealed that PVT1 directly bound to miR-26b, which had been verified to be a tumor suppressor in melanoma. Moreover, further functional rescue experiments revealed that PVT1 knockdown could observably reverse the tumor-promoting role of the miR-26b inhibitor. Overall, our study demonstrates the oncogenic role of PVT1 as a miR-26b sponge, possibly providing a novel therapeutic target for melanoma.

INTRODUCTION

Melanoma is an aggressive malignant skin tumor with a high mortality rate and a high potential for metastasis1. With great development and evolution in cancer diagnostic and therapeutic methods, the occurrence rate of various tumors is declining, except in melanoma2. Although primary melanoma is curable by surgical resection, the metastatic rate is extremely high and is accompanied by a high mortality rate and an insufficiency of therapy. If early stage surgical excision is missed, the 5-year survival rate is as low as 16% because of the high probability for metastasis3. Because of high mortality and a poor prognosis, an understanding of in-depth regulating mechanisms and the development of specific treatments are urgently required for melanoma.

Epigenetics is a burgeoning research field in pathophysiology. Changes that do not affect DNA sequence, including DNA methylation and histone modifications, can cause inheritable phenotypic variation4–6. An increasing volume of literature has paid close attention to the physiological function of long noncoding RNAs (lncRNAs). Dysregulation of lncRNAs has recently been shown to be involved in various tumors, as well as melanoma. For instance, HOTAIR acts as a competing endogenous RNA (ceRNA) to mediate cell proliferation, migration, and invasion in various tumors, such as chondrosarcoma7, glioma8, and osteosarcoma9.

Similarly, lncRNAs have been verified to participate in the occurrence and progression of melanoma. In general, lncRNAs may act as oncogenes or tumor suppressors to regulate tumorigenesis, proliferation, invasion, and metastasis10. lncRNA SPRY4-IT1 could suppress growth and differentiation and induce a higher rate of apoptosis in melanoma cell lines11. lncRNA BANCR is involved in the proliferation of melanoma cells and activates the MAPK pathway, especially ERK1/2 and JNK, to mediate its tumorigenesis12. Furthermore, the level of serum PVT1 has been verified to be in accord with melanoma cell proliferation, progression, and migration, suggesting a potential diagnostic biomarker and therapeutic target for melanoma13.

MicroRNAs (miRNAs) are a type of noncoding RNA that negatively regulate the translation of mRNAs via binding the complementary sites in the 3′-UTR14. Various miRNAs have been proven to participate in the oncogenesis of melanoma. For instance, miR-26b inhibits the proliferation of melanoma cells and promotes apoptosis via inhibiting MAPK activation. With the popularization of high-throughput sequencing and bioinformatics analysis, more lncRNAs are being found that act as ceRNAs to directly mediate miRNAs and indirectly mediate the corresponding target genes15.

Emerging evidence has demonstrated that lncRNAs exert extensive regulating roles in melanoma; however, the potential role of lncRNA PVT1 has not yet been reported. In this study, we investigate the potential regulatory mechanism of lncRNA PVT1 in the tumor characteristics and further discover novel therapeutic targets for melanoma.

MATERIALS AND METHODS

Patients and Clinical Samples

Thirty-five malignant melanoma patients were enrolled in our study, and none of them received any chemotherapy or radiotherapy before surgery. All tumor samples were confirmed by histological diagnosis. All enrolled patients signed written informed consent. The study was approved by the review board of the ethics committee of The Second Hospital of Hengshui City and Harrison International Heping Hospital.

Cell Lines and Culture

Human melanoma cell lines (M21, B16F10, MM200, MEL-RM, A375, and A2058) were purchased from the American Type Culture Collection (ATCC; Manassas, VA, USA) and cultured in Dulbecco’s modified Eagle’s medium (DMEM; Gibco, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS; Gibco) and 100 U/ml penicillin/streptomycin. Human epidermal melanocytes neonatal (HEMn) cells were purchased from the Type Culture Collection of the Chinese Academy of Sciences (Shanghai, P.R. China) and cultured in melanocyte growth media (PromoCell, Shanghai, P.R. China). Cells were cultured in a suitable environment at 37°C with 5% CO2.

siRNA Transfection

Multiple synthetic interfering RNAs (siRNA) and mimic oligonucleotide sequences that target PVT1 and miR-26b were synthesized by GenePharma Co. Ltd. (Shanghai, P.R. China). The transfection was performed using Lipofectamine 2000 transfection reagent according to the manufacturer’s instructions. The oligonucleotide sequences were provided by GenePharma.

Quantitative Real-Time PCR (qRT-PCR)

Total RNA was isolated from melanoma tissues, adjacent nontumor tissues, and cell lines using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s protocol. The cDNA was synthesized from primers and corresponding total RNA using RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher, Waltham, MA, USA). qRT-PCR was then performed using a SYBR-Green PCR Master Mix Kit (Takara, Dalian, P.R. China). The following primers were used: PVT1, 5′-ATTGATTTTGTTTGGATGGA-3′ (forward) and 5′-TGAGAAGTATGCTGAATGGC-3′ (reverse); GAPDH, 5′-GCACCGTCAAGGCTGAGAAC-3′ (forward) and 5′-TGGTGAAGACGCCAGTGGA-3′ (reverse). Values obtained for target gene expression were normalized to GAPDH and quantified relative to their expression in the control samples.

Cell Proliferation and Colony Formation Assay

Cell viability was assessed by the cell counting kit-8 (CCK-8; Dojindo, Japan). A total of 5 × 103 cells/well were plated in 96-well plates. After being cultured for 0, 1, 2, 3, and 4 days, the optical absorbance of samples was detected using a microplate reader (Bio-Rad, Hertfordshire, UK) at 450 nm. Melanoma cells (M21 and A375) were cultured in six-well plates and incubated for 2 weeks. The cell colonies were then fixed and stained with 10% crystal violet and counted under a microscope. All assays were performed in triplicate.

Dual-Luciferase Reporter Assay

The 3′-UTR sequence of PVT1 was amplified from normal human genomic DNA and subcloned into the pRL-CMV luciferase reporter vector (Ambion, Austin, TX, USA). HEK293T cells were seeded at a density of 5 × 103 cells/well in 96-well plates and cotransfected with firefly luciferase target reporter (50 ng) and pRL-CMV Renilla luciferase control reporter (5 ng) and miRNA mimics or negative control using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions. After incubation for 48 h, the firefly and Renilla luciferase vitality were assayed using the Dual Luciferase System (E1910; Promega, Madison, WI, USA).

Cell Cycle Analysis

After transfection, cell cycle analysis was performed using a cell cycle analysis kit (Lianke, Shanghai, P.R. China). Briefly, cells (4 × 105 per well) were seeded onto six-well plates and starved in FBS-free medium for 12 h. Cells were then washed with PBS, digested by trypsin, and collected by centrifugation at 1,000 rpm for 5 min. After centrifugation, DNA staining was performed with 10 mg PI/ml PBS and 2.5 mg of Ag DNase-free RNase/ml PBS for 30 min. Cell cycle profiles were generated with Modifit software (BD Biosciences, Franklin Lakes, NJ, USA).

Cell Apoptosis

Apoptotic cells of the M21 and A375 cell lines were identified by Annexin-V/Dead Cell Apoptosis Kit (Invitrogen). Briefly, cells were seeded onto six-well plates at a density of 4 × 105 per well. Before treatment with the indicated agents, cells were starved in FBS-free medium for 12 h. After washing with ice-cold PBS twice, cells were harvested and resuspended in annexin-binding buffer, and then 5 μl of annexin V–FITC and 1 μl of PI were appended. Cells were then incubated at room temperature for 15 min in the dark. The apoptosis cells were analyzed by flow cytometry.

Statistical Analysis

Data are shown as the mean ± standard deviation (SD). Student’s t-test and one-way ANOVA were performed to calculate the differences. A value of p < 0.05 was considered to be a significant difference.

RESULTS

PVT1 Was Upregulated in Human Melanoma Tissue and Related to Poor Prognosis

Our study performed qRT-PCR to measure the expression level of PVT1 in human melanoma tissue. Results showed that the expression levels of PVT1 in 35 pairs of melanoma tissues were observably higher than those of adjacent noncancerous tissues (Fig. 1A). PVT1 expression was upregulated in 88% (31/35) of the melanoma tissues compared with their adjacent nontumor tissues (Fig. 1B). Moreover, Kaplan–Meier analysis and log-rank test were performed to assess the prognosis of melanoma patients. The differences in high- and low-expression PVT1 revealed that patients with a higher PVT1 expression level had lower overall survival and a poorer prognosis than those with a lower PVT1 expression (Fig. 1C). The above results indicate that the aberrantly overexpressed PVT1 may play an oncogenic role in the progression of melanoma.

Figure 1

Expression of PVT1 was upregulated in the human melanoma tissue. (A) Expression levels of PVT1 were detected in the melanoma tissues and adjacent nontumor tissues using quantitative real-time PCR (qRT-PCR). (B) Expression of PVT1 was upregulated in 88% (31/35) of melanoma tissues compared with their pair-matched adjacent nontumor tissues. (C) Comparison of overall survival between high and low PVT1 expression was demonstrated by Kaplan–Meier analysis. **p < 0.01 compared to the noncancerous group.

Downregulated PVT1 Suppressed Melanoma Cell Proliferation and Promoted Apoptosis

In melanoma tissues, PVT1 was found to be overexpressed and associated with a poor prognosis and overall survival. In subsequent experiments, we assessed the expression of PVT1 in melanoma cell lines, suggesting a similar overexpression of PVT1 as in melanoma tissues. Our research team transfected three siRNAs into M21 and A375 cells to knock down the expression of PVT1, displaying disparate interference efficiency (Fig. 2B). CCK-8 and colony formation assays showed that PVT1 silencing inhibited proliferation activity and colony number (Fig. 2C and D). Furthermore, PVT1 knockdown could significantly induce cell cycle arrest in the G0/G1 phase and enhance the apoptosis of M21 and A375 cells (Fig. 2E and F). In conclusion, the above loss-of-function experiments demonstrated that PVT1 took part in the regulation of melanoma tumorigenesis.

Figure 2

Downregulated PVT1 suppressed melanoma cell proliferation and promoted apoptosis. (A) Overexpression of PVT1 in melanoma cell lines (M21, B16F10, MEL-RM, MM200, A375, and A2058) compared to normal HEMn. (B) Silencing of PVT1 inferenced with three biosynthetic synthetic interfering RNAs (siRNAs) in M21 and A375 cell lines. (C) Cell counting kit-8 (CCK-8) assay showed cell viability at indicated time points after seeding into 96-well plates. (D) Colony formation assay showed the proliferation ability of M21 and A375 cell lines. (E) FCM showed cell cycle arrest in the G0/G1 phase. (F) FCM showed the apoptosis of melanoma cells. Data are presented as the mean ± standard deviation (SD). *p < 0.05, **p < 0.01 compared to the HEMn or si-NC groups.

PVT1 Targeted miR-26b and Inhibited its Expression

Increasing evidence has proven that lncRNAs might function as an endogenous miRNA “sponge” to exert regulation on tumorigenesis. Our study verified that PVT1 plays an oncogenic role in the occurrence of melanoma; thus, we hypothesized that PVT1 could also act as an miRNA “sponge” to induce the tumor progression of melanoma. We performed bioinformatics analysis (starBase, http://starbase.sysu.edu.cn) to predict the potential target miRNAs, and we focused on miR-26b, which had been validated as a tumor suppressor in melanoma oncogenesis. The predicted complementary binding sites at the 3′-UTR are shown in Figure 3A. Dual-luciferase reporter assay verified the paired binding (Fig. 3B). Moreover, miR-26b was downregulated in melanoma tissue samples compared to noncancerous tissue (Fig. 3C). In addition, miR-26b expression was downregulated in 91% (32/35) of OS tissues compared to the adjacent nontumor tissues (Fig. 3D). Pearson’s correlation showed that PVT1 was negatively correlated to miR-26b expression in human melanoma samples (Fig. 3E). These data powerfully suggest that PVT1 inhibits miR-26b expression in melanoma tissue by targeting 3′-UTR.

Figure 3

PVT1 inhibited miR-26b expression in melanoma tissues. (A) Bioinformatics analysis predicted the putative complementary sites at the 3′-UTR, including PVT1 wild and mutant types. (B) Luciferase activity of wild or mutant types of PVT1 3′-UTR reporter gene in M21 and A375 cells. (C) qRT-PCR analysis of the expression of miR-26b in 35 melanoma patients. (D) Expression of miR-26b was downregulated in 91% (32/35) of the melanoma tumor tissues compared with their adjacent nontumor tissues. (E) Pearson’s correlation analysis showed the correlation between PVT1 and miR-26b expression in 35 cases of human melanoma tissue. Data were calculated and presented as the mean ± SD. **p < 0.01 compared with the control group.

PVT1 Mediated Melanoma Proliferation and Apoptosis via Targeting miR-26b

To investigate the regulation of PVT1 on the tumorigenesis of melanoma targeting miR-26b, we performed functional experiments to validate the ceRNA mechanism in M21 cell lines. The expression of miR-26b was downregulated in various melanoma cell lines (M21, B16F10, MEL-RM, MM200, A375, and A2058) compared to normal HEMn (Fig. 4A). After cotransfection of miR-26b mimics, inhibitor, and si-PVT1-3, the expression of miR-26b was observably upregulated and downregulated, respectively (Fig. 4B). CCK-8 and colony formation assays showed that miR-26b mimics suppressed the tumorigenesis of melanoma, and si-PVT1 could reverse the tumor-promoting effect of the miR-26b inhibitor (Fig. 4C and D). Furthermore, for the FCM analysis of cell cycle and apoptosis, PVT1 knockdown could significantly inhibit cell cycle arrest in the G0/G1 phase and apoptosis by targeting miR-26b (Fig. 4E and F).

Figure 4

PVT1 mediated melanoma proliferation and apoptosis via targeting miR-26b in M21 cell lines. (A) Relative expression of miR-26b in melanoma cell lines (M21, B16F10, MEL-RM, MM200, A375, and A2058) compared to normal HEMn. (B) Expression of miR-26b was measured in various groups with transfected miR-26b mimics, inhibitor, or si-PVT1. (C) Cell proliferation viability of M21 cells was detected by CCK-8 assay. (D) Cell colony formation assay. (E) Cell cycle arrest detected by flow cytometry. (F) Apoptosis was detected by flow cytometry. Data are presented as the mean ± SD. *p < 0.05, **p < 0.05 compared to the control.

In conclusion, our results indicate that PVT1 acts as an oncogene in the progression of melanoma through targeting miR-26b.

DISCUSSION

Emerging evidence has demonstrated that lncRNAs exert enormous crucial functions in the physiological/pathological process, especially in oncogenesis involving multiple tumors16,17. These findings guided us to further investigate the underlying mechanism of lncRNAs in melanoma.

Our study revealed that lncRNA PVT1 was overexpressed in 35 pairs of melanoma tissues compared to their paired adjacent noncancerous tissues, being upregulated in 88% (31/35) of the samples, indicating the poor prognosis of melanoma patients. Therefore, our results indicated that PVT1 may play an oncogenic role in the progression of melanoma. Chen et al. reported that the serum PVT1 level was significantly upregulated in melanoma patients compared to the matched nonmelanoma controls, and PVT1 depletion significantly inhibited cell progression, indicating a potential diagnostic biomarker and therapeutic target for melanoma13. In cervical cancer, Zhang et al. reported that lncRNA PVT1 promotes the progression through epigenetically silencing miR-200b18. In osteosarcoma cells, PVT1 silencing by siRNA suppressed proliferation, migration, and invasion and promoted cell cycle arrest and apoptosis via sponging miR-19519. Hence, we concluded that it acts as an oncogene in the tumorigenesis of melanoma.

Emerging evidence has indicated that lncRNAs serve as ceRNAs in the regulation of oncogenesis through binding miRNAs20. Because we have affirmed that PVT1 participates in the oncogenesis of melanoma, we predicted and screened the probable target miRNAs using bioinformatics analysis. Finally, with comprehensive comparison, we focused on miR-26b, which has been certified to be a tumor suppressor in the melanoma tumorous progression. Dual-luciferase reporter assay verified the paired binding. Moreover, miR-26b expression was downregulated in 91% (32/35) of melanoma samples compared to noncancerous tissues. To verify the ceRNA role of PVT1 on miR-26b, functional experiments were performed. The results showed that PVT1 knockdown could significantly inhibit proliferation activity, induce cell cycle arrest at the G0/G1 phase, and facilitate apoptosis by targeting miR-26b. In conclusion, our results indicated that PVT1 acted as an oncogene in the progression of melanoma through targeting miR-26b.

lncRNAs, including cis- and trans-acting, could participate in tumorigenesis by modulating cell growth, migration, invasion, cycle, or apoptosis in multiple cancers21. Owing to the undefined pathogeny, melanoma has a lack of effective treatment methods. Recently, the extensive regulatory functions of lncRNAs have been increasingly discovered and have attracted the attention of researchers. In the tumorigenesis of melanoma, a series of lncRNAs have been identified. For instance, UCA1 or MALAT-1 could attenuate the migrational ability of melanoma cells in in vitro studies22. Overexpression of GAS5 reduces the invasion of melanoma cells by mediating MMP2 expression23. The memorable regulatory mechanism of lncRNA in melanoma is ceRNA. For example, MALAT1 acts as a ceRNA to promote malignant melanoma growth and metastasis by sponging miR-2224.

In conclusion, our study demonstrates that PVT1 modulates melanoma tumorigenesis by acting as an endogenous sponge of miR-26b. These results suggest a novel insight for melanoma and provide a novel therapeutic target.