European Review for Medical and Pharmacological Sciences 2020; 24: 8429-8438 LINC00662 promotes cell proliferation, migration and invasion of melanoma by sponging miR-890 to upregulate ELK3 X.-Q. XIA1,2, W.-L. LU1,2, Y.-Y. YE3, J. CHEN1,2 1Department of Dermatology, Shanghai Ninth People’s Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, China 2Department of Laser Cosmetology, Shanghai Ninth People’s Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, China 3Department of Dermatology, Hwa Mei Hospital, University of Chinese Academy of Sciences (Ningbo No. 2 Hospital), Ningbo, China Abstract. – OBJECTIVE: Melanoma is one of Key Words: the most malignant types of skin tumors and LINC00662, MiR-890, ELK3, Melanoma. accounts for the majority of skin cancer-related deaths. LINC00662 is a tumor promoter in multi- ple types of cancer, but the role of LINC00662 in melanoma has not been fully elucidated. MATERIALS AND METHODS: The expres- Introduction sion levels of LINC00662, miR-890, and ELK3 were detected by Real Time-quantitative Poly- Melanoma is a malignant tumor which origi- merase Chain Reaction (RT-qPCR). MTT assay 1,2 was performed to measure the cell proliferation nated from melanocytes . Despite recent major ability in A375 and SK-MEL-1 cells. Cell mi- advances in tumor therapy, including surgery, gration and invasion abilities were measured radiotherapy and chemotherapy, the survival rate by wound healing assay and transwell assay, for patients with melanoma is still unsatisfacto- respectively. Besides, Luciferase reporter as- ry3,4. Accordingly, the study of novel molecular say was employed to examine the interaction targets in melanoma is an important challenge. between miR-890 and LINC00662 or ELK3. Long non-coding RNAs (LncRNAs) are a RESULTS: In the present study, it was demon- class of transcribed RNAs with a length of more strated that melanoma patients with high ex- 5-7 pression levels of LINC00662 had a shorter than 200 nucleotides and they participate in survival time than those with low expression lev- the regulation of various types of cancer by act- els of LINC00662. LINC00662 exhibited higher ing as a tumor promoter or suppressor8,9. In fact, expression levels in melanoma tissues and cell lncRNA PVT1 accelerates cell proliferation and lines. Additionally, suppression of LINC00662 invasion in gliomas by sponging miR-200a10. The impaired cell proliferation, migration, and inva- upregulation of lncRNA LINC00163 inhibits the sion. Furthermore, animal experiments demon- 11 strated that LINC00662 facilitated tumor growth development of lung cancer . LncRNA PICART1 in vivo. LINC00662 was confirmed to bind with represses cell proliferation and invasion in non- miR-890, and ELK3 was identified as a down- small cell lung cancer through affecting the stream target gene of miR-890. Furthermore, AKT1 signaling pathway12. Many lncRNAs are miR-890 was found to negatively regulate ELK3 also involved in the progression of melanomas. expression. Through rescue assays, overex- LncRNA CPS1-IT1 restrains melanoma cell me- pression of ELK3 reversed the inhibitive effects tastasis by competitively binding to BRG1 to sup- of LINC00662 knockdown or miR-890 mimics 13 on the cell proliferative, migratory, and invasive press Cyr61 . LncRNA RMEL3 enhances cell abilities. proliferation and tumor growth in melanoma14. CONCLUSIONS: Our results demonstrated LINC00662 is a novel lncRNA and acts as an on- that LINC00662 facilitated the occurrence and cogene in human cancers. For example, lncRNA development of melanoma by sponging miR- LINC00662 modulates the Hippo-YAP1 pathway 890 to upregulate ELK3. This discovery implied 15 that LINC00662 may be a promising prognos- to stimulate the progression of gastric cancer . tic and therapeutic biomarker for patients with LINC00662 acts as a competitive endogenous melanoma. RNA (ceRNA) to promote prostate cancer tum- Corresponding Author: Jun Chen, MD; e-mail: [email protected] 8429 X.-Q. Xia, W.-L. Lu, Y.-Y. Ye, J. Chen origenesis by regulating miR-34a expression16. NY, USA) and penicillin-streptomycin (penicil- However, the underlying molecular mechanisms lin: 100 UI/mL and streptomycin: 100 µg/mL; of LINC00662 in melanoma are unknown. Thermo Fisher Scientific, Waltham, MA, USA). Another important set of non-coding RNAs HEMa-LP cells were cultured in medium 254 are microRNAs (miRNAs), whose length is typ- (Thermo Fisher Scientific, Waltham, MA, USA) ically 18-24 nucleotides17,18. MiRNAs play im- and Human Melanocyte Growth Supplement-2 portant roles in the occurrence and development (HMGS-2; Thermo Fisher Scientific, Waltham, of various types of cancer, including melanoma. MA, USA). The cells were cultured at 37°C in an MiR-375 attenuates cell proliferation in esoph- atmosphere with 5% CO2. ageal squamous cell carcinoma by directly tar- The short-hairpin RNA against LINC00662 geting SP119. miR-485-5p suppresses cell prolif- (sh-LINC00662; 5’-GCUGCUGCCACU- eration in gliomas by directly targeting paired GUAAUAAUU-3’) with negative control (sh- box 320. MiR-338-5p targets CD82 to enhance NC; 5’-AAUUCUCGGAACGUCUGACGU-3’), the growth and metastasis of malignant melano- miR-890 mimics (5’- UACUUGGAAAGG- mas21. The miR-425/IGF-1 axis weakens mela- CAUCAGUUGtt-3’), miR-890 inhibitor (5’- noma metastasis by blocking the PI3K-Akt path- CAACUGAUGCCUUUCCAAGUAtt-3’) and way22. Recently, lncRNAs have been reported negative control (NC, 5’- AGACGUGUAUC- to act as ‘sponges’ to bind with miRNAs, which GUAACUGAUGtt- 3’) were synthesized by Ge- can regulate cancer. LncRNA small nucleolar nePharma (Shanghai, China). The full length of RNA host gene 1 (SNHG1) accelerates osteo- ELK3 was subcloned into pcDNA3.1 to overex- sarcoma progression by sequestrating miR-577 press ELK3 levels with empty pcDNA3.1 serving and activating the WNT2B/Wnt/β-catenin sig- as the control. The pcDNA3.1 vector was bought naling pathway23. LncRNA LINC00511 aggra- from GenePharma (Shanghai, China). The afore- vates breast cancer tumorigenesis by targeting mentioned plasmids were transfected into A375 the miR-185-3p/E2F1/Nanog axis24. LncRNA or SK-MEL-1 cells using Lipofectamine 2000 Re- DSCAM-AS1 contributes to the development of agent (Invitrogen, Carlsbad, CA, USA). melanomas by sponging miR-13625. In the pres- ent study, bioinformatics tools predicted that Real Time-Quantitative Polymerase Chain LINC00662 had the binding sequences for miR- Reaction (RT-qPCR) 890. miR-890 has been reported to attenuate TRIzol reagent (Invitrogen, Carlsbad, CA, triple-negative breast cancer progression26, but USA) was used according to the manufacturer’s its role in melanomas has not been investigated. protocol to isolate total RNA from melanoma As such, the present investigation further inves- cells. Total RNA was reverse transcribed into tigated the interaction between LINC00662 and cDNA using a PrimeScript RT reagent kit (Ta- miR-890 in melanoma. kara, Dalian, China). PCR amplification reaction In the present study, the role of LINC00662 was prepared using SYBR Green Real-Time PCR in melanoma was explored. It was confirmed that Master Mix (Thermo Fisher Scientific, Waltham, LINC00662 accelerated the progression of mela- MA, USA). The relative expression levels were noma by targeting the miR-890/ELK3 axis. calculated using the 2-ΔΔCq method. The expres- sion levels of LINC00662 or ELK3 were normal- ized to GAPDH. The expression levels of miR- Materials and Methods 890 were normalized to U6. The sequences of the primers were: LINC00662 forward, 5’-CAC- Cell Culture and Transfection GCTTCTGAAACTGGTGT-3’ and reverse, The normal human epidermal melanin cell 5’-TGTACAGCCTGGTGACAGAG-3’; miR-890 line, HEMa-LP, was acquired from Invitrogen forward, 5’-GCTTGGAAAGGCATCAGTTG-3’ (Carlsbad, CA, USA), and melanoma cell lines and reverse, 5’-CTCAACTGGTGTCGTG- including A375, SK-MEL-1, and SK-MEL-2 GAGTC-3’; ELK3 forward, 5’-ACCCAAAG- cells were purchased from the American Type GCTTGGAAATCT-3’ and reverse, 5’-TGTAT- Culture Collection (ATCC; Manassas, VA, GCTGGAGAGCAGTGG-3′; GAPDH forward, USA). Melanoma cells were incubated in Dul- 5’-ACCACAGTCCAT GCCATCAC-3’ and re- becco’s Modified Eagle’s Medium (DMEM; verse 5’-TCCACCACCCTGTTGCTGTA-3’; U6 Corning Life Sciences, MA, USA) with 10% forward, 5’-CTCGCTTCGGCAGCACAT-3’ and fetal bovine serum (FBS; Gibco, Grand Island, reverse, 5’-AACGCTTCACGAATTTGCGT-3’. 8430 LINC00662 promotes cell proliferation, migration and invasion of melanoma MTT Assay Animal Experiments Cell proliferation was measured using MTT Animal experiments were performed to deter- assays. Melanoma cells, at a density of 1x104 cells/ mine the impact of LINC00662 on tumor growth well, were cultured on 96-well plates. 10 μL MTT in vivo. The experiments were carried out on male (5 mg/ml; Bioswamp, Wuhan, China) was added BALB/C nude mice. Briefly, cells transfected into each well at the time of determination (0, 24, with sh-NC or sh-LINC00662 were injected into 48, and 72 h). Subsequently, dimethyl sulfoxide nude mice. Every 7 days, the volume and weight (~100 μl; Bio-Swamp, Wuhan, China) was then of tumors were calculated. 4 weeks later, mice added after incubation for 4 h. The optical density were sacrificed. The tumors were dissected and was measured at 450 nm using a microplate read- detected. The animal experiments were approved er (Bio-Rad, Hercules, CA, USA). by the Ethics Committee of Shanghai Ninth Peo- ple’s Hospital. Wound Healing Assay The transfected melanoma cells were seeded Statistical Analysis onto 6-well plates and cultured in standard con- Data are expressed as the mean ± SD and ditions until 80-90% confluent. A straight wound all experiments were repeated 3 times. Statis- was made using a sterile pipette tip. The size of tical analysis was conducted using the SPSS the wound was captured at 0 and 24 h using an 22.0 software (IBM Inc., Armonk, NY, USA). optical microscope (Leica DMI4000B, Milton Comparisons among the groups were tested us- Keynes, Bucks, UK) and the migration ratio was ing Student’s t-tests (for two groups) or one-way calculated. ANOVAs (for more than two groups) followed by Tukey’s test.