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Up-Regulation of SNHG15 Facilitates Cell Proliferation, Migration, Invasion and Suppresses Cell Apoptosis in Breast Cancer by Regulating Mir-411-5P/VASP Axis

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Up-Regulation of SNHG15 Facilitates Cell Proliferation, Migration, Invasion and Suppresses Cell Apoptosis in Breast Cancer by Regulating Mir-411-5P/VASP Axis European Review for Medical and Pharmacological Sciences 2020; 24: 1899-1912 Up-regulation of SNHG15 facilitates cell proliferation, migration, invasion and suppresses cell apoptosis in breast cancer by regulating miR-411-5p/VASP axis L.-B. LIU1, Z.-J. JIANG2, X.-L. JIANG3, S. WANG2 1Department of Colorectal Surgery, 2Department of Thyroid Breast Surgery, 3Department of Radiation; Affiliated Nanhua Hospital, University of South China, Hengyang, China Libing Liu and Zhongjun Jiang contributed equally to the writing of this article Abstract. – OBJECTIVE: Breast cancer (BC) larly, VASP attenuated the regulatory effects of is an intractable cancer with a rising incidence. SNHG15 silencing on BC cell progression. Fur- Small nucleolar RNA host gene 15 (SNHG15) is thermore, SNHG15 elimination hindered tumor a novel biomarker of multiple cancers. However, growth in vivo. the molecular mechanism of SNHG15 during on- CONCLUSIONS: SNHG15 contributes to BC cogenesis of BC is still poorly understood. cell progression by sponging miR-411-5p and MATERIALS AND METHODS: Expression enhancing VASP expression, providing essen- of SNHG15, microRNA (miR)-411-5p and vaso- tial biomarkers for BC therapy. dilator stimulated phosphoprotein (VASP) was measured by quantitative real-time polymerase Key Words: chain reaction (qRT-PCR). Cell proliferation was SNHG15, MiR-411-5p, VASP, Progression, BC. evaluated by colony formation and 3-(4,5-di- methyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) assay. Cell apoptosis was deter- mined by flow cytometry and caspase-3 activi- Introduction ty assay. Cell migration and invasion were exam- ined by transwell assay. The interaction between Breast cancer (BC) is a malignant tumor which miR-411-5p and SNHG15 or VASP was validated threatens the health of millions of people, in par- by dual-luciferase reporter assay. Protein ex- ticular women1. The pathogenesis of BC is com- pression of VASP, B cell lymphoma (Bcl-2), Bcl-2 associated X (Bax), vascular endothelial growth plicated, including hereditary, smoking, alcohol factor (VEGF), and matrix metalloproteinas- consumption, late age of pregnancy, no breastfeed- es (MMP-9, MMP-14) was measured by Western ing and delayed menopause2-4. Despite earlier diag- blot. Xenograft mice were established by sub- nosis and advanced treatment methods, the 5-year cutaneously injecting SKBR-3 cells transfected survival rate is still unpleasant5,6. Therefore, it is of with sh-SNHG15 and sh-NC. great importance to disclose the molecular mecha- RESULTS: SNHG15 and VASP were over-ex- pressed whereas miR-411-5p was low-ex- nism implicated in BC cell progression. pressed in BC tumors and cells compared with Long non-coding RNAs (lncRNAs) are funda- the normal counterparts. Next, SNHG15 knock- mental modulators of many pathological process- down attenuated cell proliferation, migration, in- es, such as cell survival, metabolism, differenti- vasion and stimulated cell apoptosis in BC. In ation, migration, inflammation and apoptosis7-9. addition, SNHG15 acted as a sponge while VASP Small nucleolar RNA host gene 15 (SNHG15), a acted as a target of miR-411-5p. Rescue experi- long transcript mapped on human chromosome ment revealed that miR-411-5p inhibitor could al- 7p13, was involved in the oncogenesis of various leviate SNHG15 silencing-induced inhibitive ef- 10 fects on cell proliferation, migration, invasion cancers . However, its role was controversial in and promotive effects on cell apoptosis. Simi- different cancers. SNHG15 acted as competing Corresponding Author: Zhongjun Jiang, MM; e-mail: [email protected] 1899 L.-B. Liu, Z.-J. Jiang, X.-L. Jiang, S. Wang endogenous RNA (ceRNA) to expedite osteo- ticipants by surgery. All the patients have signed sarcoma cell progression by interacting with informed consents and the protocols have been miR-14111. In addition, SNHG15 improved the approved by Ethics Committee of Affiliated Nan- aggressiveness of prostate cancer by accelerating hua Hospital, University of South China. cell proliferation and epithelial to mesenchymal transition (EMT) via sponging miR-338-3p and Cell Transfection increasing FKBP1A expression12. On the con- BT-474, MDA-MB-468, SKBR-3, MCF-7 cells trary, SNHG15 exerted its suppressive function in and human breast epithelial cells MCF-10A were thyroid cancer to restrain cell growth, migration purchased from American Type Culture Collec- and invasion13. Therefore, the exact role of SN- tion (ATCC, Manassas, VA, USA) and cultured HG15 during the initiation and progression of BC in complete Dulbecco’s Modified Eagle’s Medi- requires in-depth exploration. um (DMEM) medium (Gibco, Rockville, MD, MicroRNAs (miRNAs) are signal-stranded USA). Small harboring RNA targeting SNHG15 transcripts with 17-26 nucleotides in length14. MiR- (sh-SNHG15), negative control (sh-NC), pcDNA, NAs could negatively regulate gene expression by SNHG15 and VASP overexpression vectors were interacting with the messenger RNA (mRNA) and synthesized by Genepharma (Shanghai, China). leading to mRNA degradation and protein trans- MiR-411-5p, miR-411-5p inhibitor (anti-miR- lation repression15,16. They were closely linked 411-5p), negative control (miR-NC) and negative with carcinogenesis by affecting cell cycle, EMT, control inhibitor (anti-miR-NC) were purchased migration, autophagy, apoptosis and death17-19. For from RIBOBIO (Guangzhou, China). The vec- instance, miR-889-3p served as tumor promoter to tors were transfected into SKBR-3 and MCF-7 accelerate cell growth in osteosarcoma by interact- cells using Lipofectamine 2000 (Invitrogen, ing with MNDA20. By contrast, miR-202-3p acted Carlsbad, CA, USA). as tumor suppressor to repress colorectal carcino- ma cell growth by targeting ARL5A21. In addition, Quantitative Real-Time Polymerase Chain miR-411 could alleviate the malignancy of gastric Reaction (qRT-PCR) cancer by restricting cell viability and migration The tissues and the cells were re-suspended in via regulating SETD622. However, the regulatory TRIzol reagent (Invitrogen, Carlsbad, CA, USA) to effects of miR-411-5p on BC cell development re- extract total RNA. The cDNA for SNHG15, miR-411- main unclear. 5p and VASP was synthesized by All-in-One™ Kit Owing to the controversial role of SNHG15 (FulenGen, Guangzhou, China). Next, SYBR green in cancer cells, we aimed to disclose the exact (Applied Biosystems, Foster City, CA, USA) was ex- regulatory role and molecular mechanism of SN- ploited for qRT-PCR. Glyceraldehyde-3-phosphate HG15 in BC progression. The expression of SN- dehydrogenase (GAPDH) and U6 were employed HG15, miR-411-5p and vasodilator stimulated as internal references. The primers for SNHG15, phosphoprotein (VASP) was measured to reveal miR-411-5p, VASP, GAPDH and U6 were listed: their potential role. Rescue experiment was per- SNHG15, (Forward, 5’-TTGTGAAGCCCAGT- formed to clarify the regulatory effects of SN- GAAAGTACTGC-3’; Reverse, 5’-TTCACTGT- HG15, miR-411-5p and VASP on NC cell pro- GGAGACTGTCGTTGGT-3’); miR-411-5p, (For- gression. To the best of our knowledge, this is the ward, 5’-CCGGAACCCCCTCCTTACTC-3’; Re- first study to explore the influence of SNHG15/ verse, 5’-AATGGGATGTGTCCGAAGGA-3’); miR-411-5p/VASP feedback loop on BC cell de- VASP (Forward, 5’-CTGGGAGAAGAACAGCA- velopment. CAACC-3’; Reverse, 5’-CTGGGAGAAGAACAG- CACAACC-3’); GAPDH, (Forward, 5’-AGGTC- GGTGTGAACGGATTTG-3’; Reverse, 5’-GG- Materials and Methods GGTCGTTGATGGCAACA-3’); U6, (Forward, 5’-ACCCTGAGAAATACCCTCACAT-3’; Reverse, Patient Samples 5’-GACGACTGAGCCCCTGATG-3’). BC patients (n=35) were recruited from the Affiliated Nanhua Hospital, University of South Colony Formation China. Those patients have not received pre-op- Transfected BC cells were mixed with top agar erative therapies, including chemotherapy, radio- and seeded on the base agar in 6-well plates. After therapy and immunotherapy. Fresh BC tumors 2 weeks’ incubation, the cells were stained with and normal tissues were collected from the par- 0.1% crystal violet (Sigma-Aldrich, St. Louis, 1900 SNHG15 up-regulation and BC cells tumorigenesis by miR-411-5p/VASP axis MO, USA). Finally, the colonies ≥ 0.5 mm were upper chamber pre-treated with Matrigel (Sig- analyzed by a microscope. ma-Aldrich). After migrating or invading for 48 h, the cells at the lower chamber of transwell were 3-(4,5-Dimethyl-2-Thiazolyl)-2,5-Diphenyl- stained with 0.1% crystal violet (Sigma-Aldrich). 2-H-Tetrazolium Bromide (MTT) Assay Lastly, the migration and invasion rate were ana- Transfected BC cells were inoculated in lyzed by a microscope. 96-well plates. Then, the cells were incubated for 24 h, 48 h and 72 h and reacted with 10 μL MTT Dual-Luciferase Reporter Assay (Beyotime, Shanghai, China) for 4 h. Next, 100 The interaction between miR-411-5p and SN- μL dimethyl sulfoxide (DMSO) (Sigma-Aldrich) HG15 or VASP was certified by dual-lucifer- was added in the cells for 2 h. Finally, the optical ase reporter assay. In brief, wild type SNHG15 density (OD) value at 490 nm was detected by a (SNHG15-WT) and mutant type SNHG15 (SN- spectrophotometer. HG15-MUT) luciferase vectors were constructed. Meanwhile, wild type VASP (VASP 3’UTR-WT) Flow Cytometry and mutant type VASP (VASP 3’UTR-MUT) Transfected BC cells were inoculated in luciferase vectors were constructed. Next, they 24-well plates. After incubating for 48 h, the cells were co-transfected in BC cells with miR-411-5p were collected and stained by fluorescein isothio- or miR-NC to construct dual-luciferase system. cyanate tagged Annexin V (Annexin
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