Original Article Knockdown of Lncrna XIST Suppresses Osteosarcoma Progression by Inactivating AKT/Mtor Signaling Pathway by Sponging Mir-375-3P
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Int J Clin Exp Pathol 2019;12(5):1507-1517 www.ijcep.com /ISSN:1936-2625/IJCEP0091590 Original Article Knockdown of lncRNA XIST suppresses osteosarcoma progression by inactivating AKT/mTOR signaling pathway by sponging miR-375-3p Xin Sun, Bo Wei, Zhi-Heng Peng, Qing-Long Fu, Chao-Jun Wang, Jin-Chang Zheng, Jie-Cong Sun Orthopaedic Center, Affiliated Hospital of Guangdong Medical University, Xiashan District, Zhanjiang, Guangdong Province, China Received January 21, 2019; Accepted February 22, 2019; Epub May 1, 2019; Published May 15, 2019 Abstract: Background: Osteosarcoma (OS) is one of the most common bone tumors in adolescents and young adults. Emerging evidence suggested ncRNA (lncRNA and miRNA) are closely associated with cell progression, apoptosis and autophagy. However, the role of regulatory network between ncRNA and mRNA in OS has not been fully verified. Methods: lncRNA XIST, miRNA expression were detected by qRT-PCR. The protein expression of LC3, p62, AKT, p-AKT, mTOR and p-mTOR was measured by western blot. MTT assay and flow cytometry were applied to measure cell proliferation and apoptosis. Luciferase assay was used to ensure the relationship between lncRNA, miRNA and mRNA. GFP-LC3 cells were observed using fluorescence microscope. Results: XIST expression was up- regulated but miR-375-3p was down-regulated in OS tissues and cells. Luciferase assay results demonstrated that miR-375-3p was a target of XIST and mTOR was a target mRNA of miR-375-3p. In addition, knockdown of XIST and mTOR inhibited OS cell proliferation and autophagy, but induced apoptosis. Knockdown of XIST could reverse the effect of miR-375-3p inhibitor on OS cells. The effects of si-mTOR of OS cells could be reversed by silencing miR- 375-3p. Moreover, knockdown of XIST inhibited AKT/mTOR signaling pathway via sponging miR-375-3p. Conclusion: Knockdown of XIST inhibited cell growth and autophagy but induced cell apoptosis by suppressing the AKT/mTOR signaling pathway by sponging miR-375-3p. Keywords: Osteosarcoma, XIST, miR-375-3p, AKT/mTOR Introduction MicroRNAs (miRNAs) are ~22 nucleotides small non-coding RNAs that repress gene expression Osteosarcoma (OS) is the most common pri- at post-transcription by binding to mRNAs [11]. mary bone tumor in adolescents and young Additionally, miRNAs, as oncogenes or tumor adults, exhibiting osteogenic differentiation suppressors, play an important role in cell pro- and producing malignant bones [1, 2]. How- liferation, migration, apoptosis, and drug resis- ever, research on the underlying mechanisms tance of many cancers [12-15]. For example, of OS is still lacking. miR-155, as an oncogene, is up-regulated in liposarcoma and promotes tumor cell growth Long non-coding RNAs (lncRNAs) are a class through targeting casein kinase-1α [16]. miR- of RNAs, which are ~200 nucleotides in length. 145, as a tumor suppressor, was down-regulat- Evidence determined that lncRNAs are widely ed in colon cancer and inhibits cell growth by involved in multiple biologic processes, includ- regulating HDAC4 [17]. ing cell progression, inflammation and tumori- genesis [3-5]. LncRNA-XIST (X inactive-specific Recently, some studies reported that lncRNA transcript) is an oncogenic lncRNA in various act as competing endogenous RNA (ceRNA) cancers, including pancreatic cancer, non- with miRNAs by binding miRNAs contained small cell lung cancer, hepatocellular carcino- common sites [18]. Until now, many regulatory ma, pancreatic cancer and osteosarcoma [6-9]. mechanisms of cell growth have been investi- Some studies showed that LncRNA-XIST was gated in OS [19-22], but the role of regulatory up-regulated in OS and associated with cellular network lncRNA, miRNA, and mRNA has not processes [9, 10]. been fully elucidated. In our study, we found lncRNA XIST/miR-375-3p promotes OS progression that XIST was up-regulated and miR-375-3p manufacturer’s instructions. RNA concentra- was down-regulated in OS, and bioinformatic tion was detected using the NanoDropND- analysis showed that miR-375-3p was a poten- 1000 spectrophotometer (NanoDrop Techno- tial target miRNA of XIST and mTOR was a logies, Wilmintgon, DE, USA). cDNA were re- potential target mRNA of miR-375-3p. In addi- verse transcribed using TaqMan MicroRNA tion, autophagy plays an important role in regu- Reverse Transcription Kit for microRNA (Applied lating cellular progression and tumor formation Biosystems, Foster City, CA, USA) or M-MLV [23]. Therefore, we hypothesized XIST can regu- Reverse Transcriptase (Invitrogen) for mRNA. late mTOR expression by sponging miR-375-3p Then the SYBR® Green (Promega, Madison, and that the regulatory network was associated WI, USA) was applied to detect the expression with OS cell growth and autophagy. of XIST, miR-375-3p and mTOR, according to the manufacturer’s instruction. GAPDH and Materials and methods U6 were used as reference gene for lncRNA, Patients and tissues specimens mRNA and miRNA. Quantitative real-time PCR was performed by using an iQTM5 Multicolor 20 pairs of tissues and adjacent tissues speci- Real-Time PCR Detection System (Bio-Rad, mens were obtained from 20 OS patients with Hercules, CA, USA). The primer: U6: forward no chemotherapy or radiation therapy at Affi- 5’-CTCGCTTCGGCAGCACA-3’ and reverse 5’- liated Hospital of Guangdong Medical Univer- AACGCTTCACGAATTTGCGT-3’; miR-375-3p: for- sity. Written informed consent was obtained ward 5’-ACACTCCAGCTGGGTTTGTTCGTTCGGC- from patients and their guardians. This study TC-3’ and reverse 5’-CTCAACTGGTGTCGTGGA- has been approved by Experiments committee GTCGGCAATTCAGTTGAGTCACGCGA-3’ GAPDH: of Affiliated Hospital of Guangdong Medical forward 5’-GCACCGTCAAGGCTGAGAAC-3’ and University. reverse 5’-ATGGTGGTGAAGACGCCAGT-3’; XIST: forward 5’-ACGCTGCATGTGTCCTTAG-3’ and re- Cell culture verse 5’-GAGCCTCTTATAAGCTGTTTG-3’; mTOR: The human OS cell lines MG-63, HOS, U2-OS forward 5’-ACATGCAGCTGTCCTGGTTC-3’ and and Saos-2, and the human normal cell line reverse 5’-TGAGGCTTCTGCATCTCCTT-3’. hFOB1.19 were purchased from the American Type Cultured Collection (Manassas, VA, USA). Western blot The cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM) with 10% fetal bovine Total proteins were extracted from tissues and serum (FBS, Gemini Bio-Products, West Sacra- cells with RIPA buffer and separated from using mento, CA), penicillin and streptomycin at 37°C sodium dodecyl sulphate-polyacrylamide gel with 5% CO . electrophoresis (SDS-PAGE), and then trans- 2 ferred onto a polyvinylidene fluoride (PVDF) Cell transfection membrane (Millipore, Bedford, MA, USA). We blocked the membrane with 5% dried skim milk miR-375-3p mimic, miR-375-3p inhibitor, sh- in TBS and the membrane was incubated with XIST, pc-XIST and their respective negative the rabbit anti-LC3, anti-p62, anti-AKT, anti- controls were obtained from GenePharma p-AKT, anti-mTOR and anti-p-mTOR (1:2000 (Shanghai, China). si-mTOR, pc-mTOR and their dilution, Santa Cruz Biotechnology Inc., Santa respective negative controls were purchased Cruz, CA, USA) and mouse anti-GAPDH anti- form from Ribobio (Guangzhou, Guangdong, body (1:2000 dilution, Santa Cruz Biotechno- China). All the vectors and fragments were logy Inc.) at 4°C overnight. After washing in transfected into OS cell lines using Lipofecta- TBST, the membrane was incubated with HRP- mine 2000 (Invitrogen, Carlsbad, CA) according conjugated anti-rabbit IgG secondary antibody to the manufacturer’s instruction. (1:2000 dilution, Santa Cruz Biotechnology Reverse transcription and quantitative real- Inc.) and anti-mouse IgG secondary antibody time PCR (1:2000 dilution, Santa Cruz Biotechnology Inc.). The blot was measured using PierceTM Total RNA was extracted from tissues and cells ECL western blotting substrate (Thermo Fisher by TRIzol reagent (Invitrogen) according to the Scientific). 1508 Int J Clin Exp Pathol 2019;12(5):1507-1517 lncRNA XIST/miR-375-3p promotes OS progression gen). Cell collected at 48 h after transfection, and the luciferase activity was detect- ed using the Dual luciferase reporter assay system (Pro- mega). Cell apoptosis Cell apoptosis was detect- ed using flow cytometry and Annexin-V fluorescein isothio- cyanate and propidium iodide (FITC-Annexin V/PI) apoptosis detection kit (Life Technolo- gy, Waltham, MA, USA). Cells were stained by Annexin-V FITC/PI. Then each sample Figure 1. XIST expression was up- regulated and miR-375-3p was down- was measured using flow cy- regulated in OS tissues and cells. (A tometry. and B) The expression of XIST (A) and miR-375-3p (B) were detected in tu- Microcopy mor tissues and adjacent tissues by qRT-PCR. (C and D) The expression of Cells stably expression GFP- XIST (C) and miR-375-3p (D) were de- LC3 were cultured. Then tected in hFOB1.19, MG-63, HOS, U2- the GFP-LC3 cells expressed OS and Saos-2 cell lines by qRT-PCR. green fluorescence were ob- (E) XIST and miR-375-3p correlation relationship was analyzed by Pear- served using Fluorescence son’s correlation analysis. *P < 0.05. microscope (Nikon, Tokyo, Japan). Cell proliferation Statistical analysis 2 × 103 cells were seeded into the 96-well cell All statistical analyses were performed using culture plates (Corning Inc., Corning, NY, USA). GraphPad Prism (GraphPad Software, San Cell proliferation was measured using 3-(4,5- Diego, CA, USA). All results were showed as Dimethyl-2-thiazolyl)-2,5-diphenyl-2Htetrazoli- mean ± SD (standard deviation) at least three um bromide (MTT) assay kit (Sigma-Aldrich, St. repeated individual experiments. All compari- Louis, MO, USA) according to the manufactur- sons groups were analyzed using Student t- er’s protocol. 30 µL serum-free media with MTT test. P-value < 0.5 was considered significant. solution were added into each well and incu- Results bated for 4 h at 37°C. Then 150 ul MTT solvent were added and incubated for 3 h at 37°C. The XIST expression was up-regulated and miR- absorbance at 450 nm was measured using a 375-3p was down-regulated in OS tissues and spectrophotometric microplate reader (Beyo- cells time Institute of Biotechnology, Haimen, China).