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Uc.77- Downregulation Promotes Colorectal Cancer Cell Proliferation by Inhibiting FBXW8-Mediated CDK4 Protein Degradation

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Uc.77- Downregulation Promotes Colorectal Cancer Cell Proliferation by Inhibiting FBXW8-Mediated CDK4 Protein Degradation ORIGINAL RESEARCH published: 19 May 2021 doi: 10.3389/fonc.2021.673223 uc.77- Downregulation Promotes Colorectal Cancer Cell Proliferation by Inhibiting FBXW8-Mediated CDK4 Protein Degradation † † † Zhijian Zheng 1 , Dan Hong 1 , Xiaodong Zhang 2 , Yixin Chang 1, Ning Sun 1, Zhenni Lin 1, Hongyan Li 1, Shirui Huang 3, Ruirui Zhang 1, Qipeng Xie 3, Haishan Huang 1* and Honglei Jin 1* 1 Zhejiang Provincial Key Laboratory of Medical Genetics, Key Laboratory of Laboratory Medicine, Ministry of Education, School of Laboratory Medicine and Life Sciences, Wenzhou Medical University, Wenzhou, China, 2 Department of Colorectal Anal Surgery, The First Affiliated Hospital of Wenzhou Medical University, Wenzhou, China, 3 Department of Clinical Edited by: Laboratory, The Second Affiliated Hospital & Yuying Children’s Hospital of Wenzhou Medical University, Wenzhou, China Ruowen Zhang, Stony Brook University, Transcribed ultraconserved regions (T-UCRs) are a new type of long non-coding RNA, United States Reviewed by: and the UCR has 481 segments longer than 200 base pairs that are 100% conserved Yohei Sekino, between humans, rats, and mice. T-UCRs involved in colorectal cancer (CRC) have not Hiroshima University, Japan been studied in detail. We performed T-UCR microarray analysis and found that uc.77- Yulei Wang, fi Guangdong Provincial was signi cantly downregulated in CRC tissues and cell lines. Ectopic expression of People’s Hospital, China uc.77- significantly inhibited the proliferation of CRC cells in vitro and the growth of *Correspondence: xenograft tumors in nude mice in vivo. Mechanistic studies showed that uc.77- competed Honglei Jin with FBXW8 mRNA for binding to microRNA (miR)-4676-5p through a competing [email protected] Haishan Huang endogenous RNA mechanism and inhibited the proliferation of CRC cells by negatively [email protected] regulating CDK4. The present findings highlight the role of the uc.77-/miR-4676-5p/ † These authors have contributed FBXW8 axis in CRC and identify uc.77- as a potential novel target for the treatment equally to this work of CRC. Specialty section: Keywords: uc.77-, miR-4676-5p, FBXW8, CDK4, colorectal cancer This article was submitted to Molecular and Cellular Oncology, a section of the journal Frontiers in Oncology INTRODUCTION Received: 27 February 2021 Accepted: 06 April 2021 Colorectal cancer (CRC) is the third most common cancer worldwide. Approximately 1.93 million Published: 19 May 2021 new cases of CRC were reported in 2020, with an incidence rate of 10.0% and a mortality rate of Citation: 9.4% (1). In China, CRC is the third most common malignant tumor and the fourth leading cause of Zheng Z, Hong D, Zhang X, Chang Y, cancer-related death (2). Elucidating the pathogenesis of CRC and identifying diagnostic markers Sun N, Lin Z, Li H, Huang S, Zhang R, are urgent needs. Xie Q, Huang H and Jin H (2021) Ultraconserved regions (UCRs) are non-coding gene sequences composed of 481 segments that uc.77- Downregulation Promotes are 100% conserved among mammals such as humans, rats, and mice. These segments can Colorectal Cancer Cell Proliferation by be located on exons or introns of the coding gene. RNAs transcribed from the UCR region are Inhibiting FBXW8-Mediated CDK4 Protein Degradation. called T-UCRs or ultra-conserved (uc) RNAs. Non-coding RNAs transcribed from UCRs may Front. Oncol. 11:673223. regulate gene expression (3).T-UCRs are a new type of long non-coding RNAs (lncRNAs) that can doi: 10.3389/fonc.2021.673223 function as oncogenes or tumor suppressors in different tumor types (4). uc.338 is highly expressed Frontiers in Oncology | www.frontiersin.org 1 May 2021 | Volume 11 | Article 673223 Zheng et al. uc.77- Inhibits CRC Cell Growth in liver cancer cells, and it promotes cell proliferation by affecting sites at uc.77- and at the FBXW8 3′ untranslated region cell cycle progression (5). uc.338 is also expressed at high levels in (3′-UTR) were purchased from TsingKe Biological Technology. CRC, and it promotes metastasis and invasion of CRC (6). Short hairpin RNAs (shRNAs) against FBXW8 and the On the other hand, uc.160+ is expressed at low levels in control plasmid were purchased from Open Biosystems gastric cancer, where it plays a tumor suppressor role (7). The (Thermo Fisher Scientific, NY, USA). Antibodies against role of T-UCRs in tumor development is not well understood. CDK2 (sc-6248), CDK4 (sc-260), CDK6 (sc-177), cyclin D1 Cyclin-dependent kinase 4 (CDK4) is an important regulator (sc-20044), cyclin E2 (sc-481), and FBXW8 (sc-514385) were of the cell cycle that forms a complex with CDK6 and cyclin D1, purchased from Santa Cruz Biotechnology (Santa Cruz, CA, and phosphorylates retinoblastoma (Rb) protein. Inactivated Rb USA). Antibodies against cyclin E2 (sc-481), FBXW11 (13149-1- releases E2F, thereby promoting the transition from G0/G1 AP), and HECTD3 (11487-1-AP) were purchased from phase to S phase of the cell cycle (8, 9). CDK4 is frequently Proteintech (Chicago, IL, USA). Antibody against a-tubulin dysregulated in cancer, and the development of CDK4-targeted (ab7291) was purchased from Abcam (Cambridge, UK). therapies is important. However, the development of CDK Cycloheximide (CHX) and MG132 were purchased from inhibitors is challenging, and several clinical trials have shown Calbiochem (San Diego, CA, USA). disappointing results (10, 11). uc.77-, a new lncRNA that was identified as a potential target for the treatment of CRC, is Cell Culture and Transfection involved in the regulation of CDK4. In this study, we show that The human CRC cell lines HCT116, HT-29, LoVo, and SW620 uc.77- competes with FBXW8 mRNA for binding to microRNA were purchased from the Shanghai Chinese Academy of (miR)-4676-5p, thereby promoting the expression of FBXW8 Sciences. SW480 and CCD-18Co cells were purchased from and the ubiquitination of CDK4, which inhibits the proliferation American Type Culture Collection. HCT116 cells were of CRC cells. cultured in M5A medium (PM150710; Procell) supplemented with 10% fetal bovine serum (FBS; 42Q8982K; Gibco), and SW620 cells were cultured in RPMI 1640 (11875–093, Gibco) supplemented with 10% FBS. The Polyjet Transfection Reagent MATERIALS AND METHODS (SL100688, SignaGen Laboratories) was used for stable transfection. According to the different antibiotic resistance T-UCR Microarray plasmids transfected, selection was performed using puromycin This study was previously detected by our team at Kangcheng Bio (4–6 mg/mL; J593; Amresco Inc.) or G418 (1000–1500 mg/mL; (Shanghai, China). The total RNA of each sample was quantified sc-29065;Santa Cruz, Dallas, TX, USA) for 3–4 weeks. with NanoDrop ND-1000 (Thermo Fisher Scientific), and RNA integrity was evaluated by standard denaturing agarose gel Lentiviral Infection electrophoresis. For microarray analysis, the Agilent array The 293T cells were inoculated into 6-well plates in 2 mL DMEM platform (Agilent Technologies, Santa Clara, California) was containing 10% FBS and grown until reaching a density of 60– employed. Sample preparation and microarray hybridization 70%. A combination of 1.2 mg pMD2.G (12259, Addgene), 1.2 mg were performed according to the manufacturer’sstandard psPAX.2 (12260, Addgene), and 2 mg plasmid was mixed with protocol. In short, the purified mRNA and LncRNA were 50 mL serum-free DMEM containing 6 mL Polyjet. The mixture amplified by random primer method (Arraystar Flash RNA was incubated at room temperature for 15 min and added to fl Labeling Kit, Arraystar) and transcribed into uorescent cRNA 293T cells in 6-well plates, which were placed in a 5% CO2 along the full length of the transcript. The labeled cRNA was incubator at 37°C for 6–8 h. After replacing the medium with hybridized to human T-UCR array (8×60K, Arraystar). After fresh DMEM containing 10% FBS, the plates were incubated for cleaning the slides, the array was scanned by the Agilent scanner 48 h. Then, 2 mL of supernatant containing virus was collected, G2505C (Agilent Technologies). The extracted data was centrifuged at 3000 rpm for 30 min, and passed through a 0.45 normalized using Agilent GeneSpring GX v12.1 software. Use mm filter with a syringe. The virus supernatant was added to the GeneSpring GX v12.1 and a signal processing algorithm called cell culture medium at a ratio of 1:1, and stable cell lines were Otsu method for further data analysis. Finally, the differentially selected with puromycin. expressed T-UCRs between the samples were identified by fold change filtering. Anchorage-Independent Growth To examine the anchorage-independent growth of CRC cells, a Plasmids, Antibodies, and Reagents suspension culture containing 104 stably transfected HCT116 The plasmid overexpressing uc.77- and the corresponding (uc.77-) or SW620 (uc.77-) cells and their control carrier cells control plasmid were purchased from TsingKe Biological was placed in a 6-well plate containing 0.33% agar and 10% FBS Technology (Beijing, China). The CDK4 plasmid was basal medium. The plates were incubated at 37°C and 5% CO2 constructed and used in a previous study (12). Mimics for for 2–3 weeks, and the number of colonies was counted under a miR-4676-5p and miR-3605-5p were purchased from Ruibo DMi1 microscope (Leica Microsystems, Buffalo Grove, IL, USA). Biotechnology (Guangzhou, China). The wild-type and mutant The result is presented as the mean ± SD of colonies/10,000 luciferase reporter genes for the predicted miR-4676-5p binding inoculated cells (13). Frontiers in Oncology | www.frontiersin.org 2 May 2021 | Volume 11 | Article 673223 Zheng et al. uc.77- Inhibits CRC Cell Growth Cell Proliferation were processed with ECF (RPN5787; GE Healthcare, PA, USA) for Approximately 1500 HCT116 (vector), HCT116 (uc.77-), signal detection, and images were captured on the Typhoon FLA SW620 (vector), or SW620 (uc.77-) cells were seeded into 96- 7000 scanner (GE Healthcare) (16).
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