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Ribosomal Protein L15 Is Involved in Colon Carcinogenesis Zhixiong Dong1,2,3, Hongyu Jiang2,3, Shuangshuang Liang2,5, Yajie Wang2,3, Wei Jiang3,4, Changjun Zhu2,3

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Ribosomal Protein L15 Is Involved in Colon Carcinogenesis Zhixiong Dong1,2,3, Hongyu Jiang2,3, Shuangshuang Liang2,5, Yajie Wang2,3, Wei Jiang3,4, Changjun Zhu2,3 Int. J. Med. Sci. 2019, Vol. 16 1132 Ivyspring International Publisher International Journal of Medical Sciences 2019; 16(8): 1132-1141. doi: 10.7150/ijms.34386 Research Paper Ribosomal Protein L15 is involved in Colon Carcinogenesis Zhixiong Dong1,2,3, Hongyu Jiang2,3, Shuangshuang Liang2,5, Yajie Wang2,3, Wei Jiang3,4, Changjun Zhu2,3 1. Key Laboratory of Laboratory Medicine, School of Laboratory Medicine and Life Science, Wenzhou Medical University, Wenzhou, Zhejiang 325035, China; 2. Tianjin Key Laboratory of Animal and Plant Resistance, College of Life Sciences, Tianjin Normal University, Tianjin 300387, China; 3. Key Laboratory of Molecular and Cellular Systems Biology, College of Life Sciences, Tianjin Normal University, Tianjin 300387, China; 4. State Key Laboratory of Molecular Oncology, Cancer Institute and Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100021, China; 5. AstraZeneca Pharmaceutical Co Ltd, Xi'an, 710100, China. Corresponding author: Changjun Zhu, Email: [email protected]; We Jiang, Email: [email protected]; Zhixiong Dong, Email: [email protected]. © The author(s). This is an open access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/). See http://ivyspring.com/terms for full terms and conditions. Received: 2019.02.24; Accepted: 2019.05.03; Published: 2019.08.06 Abstract Ribosomal biogenesis is responsible for protein synthesis in all eukaryotic cells. Perturbation of ribosomal biogenesis processes can cause dysfunctions of protein synthesis and varieties of human diseases. In this study, we examine the role of RPL15, a large ribosomal subunit protein, in human colon carcinogenesis. Our results reveal that RPL15 is remarkably upregulated in human primary colon cancer tissues and cultured cell lines when compared with paired non-cancerous tissues and non-transformed epithelium cells. Elevated expression of RPL15 in colon cancer tissues is closely correlated with clinicopathological characteristics in patients. We determine the effects of RPL15 on nucleolar maintenance, ribosomal biogenesis and cell proliferation in human cells. We show that RPL15 is required for maintenance of nucleolar structure and formation of pre-60S subunits in the nucleoli. Depletion of RPL15 causes ribosomal stress, resulting in a G1-G1/S cell cycle arrest in non-transformed human epithelium cells, but apoptosis in colon cancer cells. Together, these results indicate that RPL15 is involved in human colon carcinogenesis and might be a potential clinical biomarker and/or target for colon cancer therapy. Key words: RPL RPL15; nucleolus; ribosome biogenesis; colon cancer; apoptosis 15; nucleolus; ribosome biogenesis; colon cancer; apoptosis Introduction Ribosome is an intracellular organelle that is apoptosis[2, 3]. responsible for translating genetic information Given the important function of ribosome and encoded in messenger RNAs (mRNAs) into functional ribosomal proteins (RPs) in cells, ribosomal proteins to sustain cell growth, proliferation, dysfunction would result in a variety of diseases [4-7]. differentiation and metabolism, etc. Eukaryotes have It has been shown that plenty of birth defects and 80S ribosome consisting of a large (60S) and a small anemia were closely associated with mutations or (40S) subunit. The 60S subunit consists of a 5S rRNA, deletions of ribosomal proteins. There are abundant a 5.8S rRNA, a 28S rRNA and about 49 proteins genetic and experimental evidences demonstrate that (RPLs). The 40S subunit is composed of an 18S rRNA Diamond-Blackfan anemia (DBA), a dominant and approximately 33 proteins (RPSs)[1]. In addition autosomal bone marrow failure syndrome, is due to to protein synthesis, ribosomal proteins have been mutations in ribosomal genes including RPS19, demonstrated to take part in several other RPS24, RPL5, RPL11, and RPS29, etc[8-12]. In extraribosomal functions. For example, several addition, it was also reported that ribosomal protein ribosomal proteins have been found to play a role in disorders associated with malignancies. DBA patients DNA repair, transcription, RNA processing and have higher risk of cancer than the general http://www.medsci.org Int. J. Med. Sci. 2019, Vol. 16 1133 population, specifically a higher risk of developing and/or RNAiMAX Reagent (Life Technologies Inc) acute myelocytic leukemia (AML), osteosarcoma, or according to manufacturer's protocol. In brief, HeLa, colon cancer[13]. Changes in the expression levels of HCT116 and RPE1 cells were plated in 96-well plates RPs in cancer are common[14]. For example, RPS2 is (3000 cells/well), 24-well plates (2×104 cells/well) or found to be overexpressed in liver cancer[15], and 6-well plates (1×105 cells/well) for 16 h at 37°C before RPL7A, RPL19, RPL37 are overexpressed in prostate transfection. Then, cells were incubated with cancer[16, 17]. In addition, response to ribosome indicated plasmids/siRNAs plus transfection reagent stress, RPL5, RPL11 and 5S rRNA have been mixture in medium for 24 h and then changed into demonstrated to interact with Mdm2 and inhibit fresh medium. Mdm2 E3 ligase activity, thereby increase p53 protein Plasmids, siRNAs and antibodies. The stability and transcriptional activity, thus activating full-length coding region of RPL15 cDNA was p53-dependent cell cycle checkpoints[18-20]. RPL11 generated by PCR and subcloned into EcoR I–Sal I and RPL5 have been referred as tumor suppressors, sites of the mammalian expression vector pEGFP-C2. and their deletion or mutation were found in several The construct was fully sequenced. siRNAs specific cancers[21-23]. targeting to RPL15 (1#: 5’-UGGUGUUA Previous studies reported that Ribosomal ACCAGCUAAAGdTdT-3’; 2#: 5'-UCCAGGAGC protein L15 (RPL15), a component of 60S subunit, not UAUGGAGAAAdTdT-3') were synthesized by only participates in ribosomal assembly but also Genepharma (Shanghai, China). Polyclonal rabbit regulates pre-rRNA processing[12, 24]. RPL15 was α-RPL15 were generated against peptide of found to be dysregulated in many types of diseases. CSRRAAWRRRNTLQLHRYR. Mouse α-UBF For instance, RPL15 was detected existing depletion in (#sc-13125), mouse α-p53 (#sc-126), rabbit α-p21 a DBA patient[12]. However, the correlation profiles (#sc-397) and mouse α-nucleolin (#sc-8031) were of RPL15 with cancer are different depending on the purchased from Santa Cruz Biotechnology. Mouse type of cancer. Wang et al. found that RPL15 is α-fibrillarin (#ab4566) was purchased from Abcam. overexpressed in esophageal cancer[25]. Additionally, Mouse α-BrdU (#5292) and mouse α-RPS6 (#2317) it was reported that upregulation of RPL15 is were purchased from Cell Signaling Technology. associated with cell proliferation in gastric cancer[23]. Mouse α-RPL11 (#37-3000) was purchased from Life However, other studies revealed that RPL15 is Technologies Inc. Rabbit α-H2AX (#3522-1) was downregulated in cutaneous squamous cell purchased from Epitomics. Mouse α-Bip (#27033) was carcinoma and pancreatic ductal adenocarcinoma[26, purchased from signalway antibody. Mouse 27], which were contrast to the former two studies. To anti-α-tubulin antibody (T5168) was purchased from date, little is known about the role of RPL15 in colon Sigma-Aldrich. All secondary antibodies were cancer. obtained from Life Technologies Inc. In this study, we investigate the role of RPL15 in Tissue samples. Colon cancer specimens and colon carcinogenesis. We find that RPL15 is adjacent histologic normal tissues were obtained from overexpressed in colon cancer cells and tissues, and 25 patients who underwent surgery in Department of the expression of RPL15 is closely associated with Cancer Research Institute, Cancer Affiliated Hospital colon cancer carcinogenesis. Furthermore, we of Xinjiang Medical University, Urumqi, China. All demonstrate that depletion of RPL15 induces specimens were immediately frozen in liquid nitrogen apoptosis in colon cancer cells, but cell cycle arrest in and stored at -80°C until use. The patients’ medical non-transformed RPE1 cells. These results support the data and lifestyle for cancer risk factors (e.g. family potential value of RPL15 as a therapeutic target in history of cancer) were documented with informed colon cancer treatment. consent. Preribosome preparation. Nuclear extracts were Materials and Methods fractionated and preribosome preparation was Cell culture, transfection and drug treatment. performed as described previouslywith minor Human cervical carcinomas HeLa cells, human colon modifications[28]. In brief, HeLa cells were swollen in carcinoma HCT116 cells and RPE1 (hTERT-RPE1) ice-cold hypotonic lysis buffer (10 mM Tris [pH 7.4], cells were purchased from ATCC. HeLa and HCT116 10 mM KCl, 2 mM MgCl2, 0.05% Triton X-100, 1 mM cells were cultured in DMEM (HyClone) EGTA, 1 mM DTT, 40 mg/ml of supplemented with 10% fetal bovine serum (FBS) phenylmethylsulfonyl fluoride, and 10 mg/ml of (HyClone). RPE1 cells were cultured in DMEM: F-12 protease inhibitor cocktail). The nuclei pellet was (1:1) (HyClone) containing 10% FBS. All cells were collected by centrifugation at 500×g for 5 min. The cultured at 37°C in 5% CO2. Plasmid/siRNA nuclear lysate was extracted in extraction buffer (25 transfection was conducted with Lipofectamine 3000 mM Tris (pH 7.5), 100 mM KCl, 1 mM DTT, 2 mM http://www.medsci.org Int. J. Med. Sci. 2019, Vol. 16 1134 EDTA, 0.1% NP-40, 1 mM NaF, 40 mg/ml of calculated using Image-Pro Plus 7.0[32]. phenylmethylsulfonyl fluoride, 10 mg/ml of protease RNA isolation and Real-time RT-PCR. Total inhibitor cocktail and 0.1U/ml of RNasin (Promega) RNA was isolated using the Trizol reagent and sonicated. The nuclear lysate was overlaid on 10 (Invitrogen) following manufacturer’s instructions. to 30% (wt/wt) sucrose gradients in preribosome One microgram RNA was used for cDNA synthesis buffer (25 mM Tris (pH 7.5), 100 mM KCl, 1 mM DTT using a reverse transcriptase reaction kit (Promega) and 2 mM EDTA) and centrifuged at 36,000 rpm for 3 and quantitative real-time PCR was performed on an h at 4°C in a Beckman SW41Ti rotor.
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