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Original Article Mir-876-3P Targets KIF20A to Block JAK2/STAT3 Pathway in Glioma

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Original Article Mir-876-3P Targets KIF20A to Block JAK2/STAT3 Pathway in Glioma Am J Transl Res 2019;11(8):4957-4966 www.ajtr.org /ISSN:1943-8141/AJTR0098906 Original Article MiR-876-3p targets KIF20A to block JAK2/STAT3 pathway in glioma Jiao Tang2*, Jie Xu3*, Zhongwen Zhi1, Xiang Wang1, Yu Wang4, Yong Zhou1, Rui Chen1 1Department of Neurology, The Second People’s Hospital of Huai’an and The Affiliated Huai’an Hospital of Xuzhou Medical University, Huai’an, Jiangsu Province, China; 2Department of Neurology, Yan Cheng City No. 1 People’s Hospital, Yancheng City, Jiangsu Province, China; 3Department of General Surgery, Huai’an First People’s Hospital and The Affiliated Huai’an No. 1 People’s Hospital of Nanjing Medical University, Huai’an, Jiangsu Province, China; 4Department of Neurology, The Fourth Affiliated Hospital of Nanjing Medical University, Nanjing Pukou Hospital, Nanjing 210031, Jiangsu Province, China. *Equal contributors. Received February 15, 2019; Accepted June 25, 2019; Epub August 15, 2019; Published August 30, 2019 Abstract: Aberrant expression of miRNAs has been reported to be involved in the development and progression of glioma. But the function of miR-876-3p in glioma is unknown. We found that miR-876-3p is significantly downregu- lated in glioma tissues and cell lines. Overexpression of miR-876-3p suppressed glioma cell proliferation, epithe- lial-mesenchymal transition, migration, and invasion. By prediction combining with luciferase reporter assay, we identified that miR-876-3p could decrease the expression of KIF20A by directly targeting the region of its 3’UTR. Furthermore, we observed that overexpression of miR-876-3p inhibited the expression of KIF20A, thus blocking the protein kinase JAK2/STAT3 pathway. Overexpressed KIF20A reversed miR-876-3p-induced suppression of glio- ma cell proliferation, migration, and invasion. We also demonstrated the inhibitory effect of miR-876-3p on tumor growth in glioma using an in vivo model. The miR-876-3p/KIF20A-axis mediated JAK2/STAT3 pathway have thera- peutic potential in glioma treatment. Keywords: Glioma, miR-876-3p, proliferation, EMT, KIF20A Introduction Here, we determine whether miR-876-3p direct- ly represses KIF20A expression, which leads to MiRNAs are a class of short single-strand non- the subsequent inhibition of glioma cell prolif- coding RNAs, which length are about 19-25 eration, epithelial-mesenchymal transition (EMT) nucleotides [1-3]. MiRNAs negatively regulate and in vivo tumor growth. In an exploration of gene expression through gene translational the mechanism of miR-876-3p action, we dem- repression by binding to the 3’ untranslated onstrate that dysregulation of miR-876-3p/ region (UTR) [4, 5]. The aberrantly expression of KIF20A signalling activates the JAK2/STAT3 miRNAs occur in various types of human can- pathway. cers, which can act as tumor suppressors or promoters [6, 7]. Materials and methods Kinesin family member 20A (KIF20A) is a mem- Cell lines and human tissue samples ber of the kinesin super family-6 [8, 9]. Previous studies showed that KIF20A could play a impor- Human glioma cell lines (U251, T98, U87, and tant role in the development and progression of LN229) were obtained from the Cell Bank of human cancers, including gastric [10], pancre- the Chinese Academy of Sciences (Shanghai, atic cancer [11, 12], breast cancer [13] and China). Normal human astrocytes (NHAs) were hepatocellular carcinoma [8]. In addition, KIF- obtained from Lonza (Walkersville, MD, USA). 20A has been reported to be involved in prolif- Glioma cells were cultured in Dulbecco’s modi- eration, migration, invasion and angiogenesis fied Eagle’s medium (DMEM) supplemented [11, 14]. Furthermore, KIF20A is a tumor-asso- with 10% of fetal bovine serum (FBS) in a hu- ciated antigen involved in the glioma cell growth midified incubator at 37°C and 5% CO2. NHAs and cell survival [15, 16]. were grown in the astrocyte growth media sup- miR-876-3p inhibits glioma by target KIF20A plemented with rhEGF, insulin, L-glutamine, with ethanol and stained with crystal violet. GA-1000, ascorbic acid and 5% FBS. MiRNA Colonies with ≥ 50 cells were calculated as one expression data was obtained from Chinese colony. Glioma Genome Atlas (CGGA) database (http:// www.cgga.org.cn/portal.php). Gene expression Wound-healing assay data was obtained from Cancer Genome Atlas (TCGA) database (http://tcga-data.nci.nih.gov). Cells transfected with the miR-876-3p or its negative control were seeded in 6-well plates. A Nontumorous brain tissues (NBTs) and glioma linear wound was created with a plastic pipette tissues were obtained from the Department of tip. Cells were then cultured in serum-free me- Neurosurgery, Huai’an Hospital Affiliated to dium and photographed under a microscope at Xuzhou Medical University, Second People’s oh and 48 h. Hospital of Huai’an City. Of these 30 samples, 6 were NBTs, 12 were low-grade glioma sam- Transwell invasion assay ples (grade II, LGGs) and 12 were high-grade glioma samples (grade III and IV, HGGs). Written 2 × 105 cells were seeded in the upper cham- informed consent was obtained from all the bers of 24-well Transwell plates precoated with patients. 10 μg of Matrigel (BD Biosciences, Franklin, NJ, USA). DMEM containing 10% FBS was added to RNA isolation and quantitative real-time PCR the lower chamber. After incubation for 36 h, (qRT-PCR) the cells on the upper chambers were removed. Invading cells on the bottom were fixed and qRT-PCR were performed using Mir-XTM miRNA ® stained with 1% crystal violet and photogra- qRT-PCR SYBR Kit (Takara) for miR-876-3p. phed with microscope. The expression of U6 was used as an endoge- nous control. qRT-PCR was performed using TB Three-dimensional spheroid assay GreenTM Fast qPCR Mix (Takara) for KIF20A. The expression of GAPDH was used as an inter- Cells were seeded at a 3 × 103 cells/ml density nal control. in 96-well ultra-low adherence plates. After 48 hours, these cells were induced to aggregate Cell transfection into a multicellular spheroid, and then matrigel was added into wells. After 48 h, motion of cells Lentiviruses carrying hsa-miR-876-3p or its was confirmed as fully formed with micro- negative control were purchased from Gene- scopy. pharma (Shanghai, China). For over-expression of KIF20A, cells were transfected with KIF20A Dual luciferase reporter assay over-expressing recombinant vector (GenePh- arma, Shanghai, China). Stable cell lines were The wild-type (wt) 3’-UTR of the KIF20A se- established by infecting U251 and T98 cells, quence containing a binding site for miR-876- which were collected for the experiments after 3p was amplified from a human cDNA library 48 h of transfection. and cloned into the downstream region of the luciferase gene. The mutant (mt) 3’-UTR of Cell Counting Kit-8 assay KIF20A was created by mutating the seed region of the miR-876-3p-binding site. For re- Cells (2 × 103) were seeded to 96-well plate. porter assay, cells were co-transfected with After incubation for 0, 1, 2, 3 and 4 days, 10 μL miR-876-3p and wild-type or mutant 3’UTR of of CCK8 reagent was added to each well and KIF20A by Lipofectamine 3000. Renilla lucifer- cells were cultured for 4 h at 37°C. Then, cell ase served for normalization. proliferation was quantified by measuring the absorbance at 450 nm. Western blotting assay Colony information assay Western blot assay was performed as describ- ed previously [17]. Antibodies against KIF- Cells transfected with miR-876-3p or its nega- 20A (ab104118), Cyclin D1 (ab16663), CDK2 tive control (500/well) were planted in a 6-well (ab32147), CDK4 (ab108357), Vimentin (ab- plate for about 2 weeks. Then, cells were fixed 195877), Fibronectin (ab2413), E-cadherin 4958 Am J Transl Res 2019;11(8):4957-4966 miR-876-3p inhibits glioma by target KIF20A the GraphPad Prism 5 soft- ware. The significance of the differences in variables were calculated using Student’s t test. P values < 0.05 were considered significant. Results Expression levels of miR-876- 3p in human glioma tissues and cell lines To investigate miR-876-3p ex- pression in glioma, we initially analyzed its expression by database of CGGA (Grade II: 63; Grade III: 44 and Grade IV: 91). The expression of miR- 876-3p was higher in low gra- de glioma tissues (grade II, LGGs) than that in high grade Figure 1. miR-876-3p is downregulated in glioma. A. The expression of miR- glioma tissues (grades III and 876-3p in LGGs and HGGs. *P < 0.05. B. The expression of miR-876-3p in IV, HGGs) (Figure 1A). Se- clinical samples (NBTs: 6, LGGs: 12, and HGGs: 12). Data were expressed as the mean ± SEM from three independent experiments. *P < 0.05. C. qRT- condly, we detected miR-876- PCR analysis of miR-876-3p expression in NHAs and glioma cell lines U251, 3p expression in 30 samples, T98, U87, and LN229. Transcript levels were normalized by U6 expression. including 6 NBTs, 12 LGG Data were expressed as the mean ± SEM from three independent experi- cases and 12 HGG cases by ments. *P < 0.05. D. The expression of miR-876-3p in NBTs and glioma qRT-PCR. The expression of specimens was assessed by fluorescence in situ hybridization. miR-876-3p was decreased observed in LGGs and HGGs (ab40772), N-cadherin (ab18203), STAT3 (ab- compared with nontumorous brain tissues 68153), p-STAT3 (ab76315), Actin (ab8227, (NBTs) (Figure 1B). Thirdly, we investigated the Abcam); JAK2 (D2E12), and p-JAK2 (D15E2, expression of miR-876-3p in glioma cell lines Cell Signaling Technology) were used for west- (U251, T98, U87, and LN229) and normal hu- ern blotting assay. man astrocytes (NHAs). Compared with NHAs, miR-876-3p was downregulated in glioma cells Xenograft assay (Figure 1C).
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