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A KLF4/Pihl/EZH2/HMGA2 Regulatory Axis and Its Function in Promoting Deng et al. Cell Death and Disease (2021) 12:485 https://doi.org/10.1038/s41419-021-03753-1 Cell Death & Disease ARTICLE Open Access A KLF4/PiHL/EZH2/HMGA2 regulatory axis and its function in promoting oxaliplatin-resistance of colorectal cancer Xuan Deng1, Fanyang Kong2,SiLi3,HaoqinJiang1,LiuDong1,XiaoXu1, Xinju Zhang1,HongYuan3,YingXu4, Yimin Chu4,HaixiaPeng4 andMingGuan 1 Abstract Long noncoding RNAs (lncRNAs) have emerged as a new class of regulatory molecules implicated in therapeutic resistance, yet the mechanisms underlying lncRNA-mediated oxaliplatin resistance in colorectal cancer (CRC) are poorly understood. In this study, lncRNA P53 inHibiting LncRNA (PiHL) was shown to be highly induced in oxaliplatin- resistant CRC cells and tumor tissues. In vitro and in vivo models clarified PiHL’s role in conferring resistance to oxaliplatin-induced apoptosis. PiHL antagonized chemosensitivity through binding with EZH2, repressing location of EZH2 to HMGA2 promoter, and downregulating methylation of histone H3 lysine 27 (H3K27me3) level in HMGA2 promoter, thus activating HMGA2 expression. Furthermore, HMGA2 upregulation induced by PiHL promotes PI3K/Akt phosphorylation, which resulted in increased oxaliplatin resistance. We also found that transcription factor KLF4 was downregulated in oxaliplatin-resistant cells, and KLF4 negatively regulated PiHL expression by binding to PiHL promoter. In vivo models further demonstrated that treatment of oxaliplatin-resistant CRC with locked nucleic acids targeting PiHL restored oxaliplatin response. Collectively, this study established lncRNA PiHL as a chemoresistance 1234567890():,; 1234567890():,; 1234567890():,; 1234567890():,; promoter in CRC, and targeting PiHL/EZH2/HMGA2/PI3K/Akt signaling axis represents a novel choice in the investigation of drug resistance. Introduction eventually cause cell cycle arrest and apoptosis2. The Poor response to chemotherapy is one of the top chal- third-generation platinum analog, oxaliplatin, is with lenges patients are facing in advanced cancer care. How- promising activity in treating colon and rectal cancer3. ever, the molecular mechanism underlying this complex The combination of oxaliplatin plus 5-fluorouracil (5- event has not been fully elucidated. Platinum-based FU)/leucovorin (FOLFOX) has improved response rates antineoplastic drugs are among the widely used first-line to more than 50% and median survival time approaching agents for treating solid tumors1. By forming drug-DNA 2 years for patients with metastatic colorectal cancer adducts, platinum is able to produce DNA lesions that (CRC)4. Unfortunately, 40–50% of stage II and stage III impair DNA replication and transcription, which would CRC patients have shown chemo-resistance to oxaliplatin-based adjuvant therapy and have recurrent disease over the course of treatment5. Thus, there is an Correspondence: Haixia Peng ([email protected])or unmet need for a deep understanding of the molecular Ming Guan ([email protected]) mechanisms that contribute to chemotherapy resistance. 1Department of Laboratory Medicine, Huashan Hospital, Shanghai Medical College, Fudan University, Shanghai 200040, China Long non-coding RNAs (lncRNAs) are transcripts from 2Department of Gastroenterology, Changhai Hospital, Second Military Medical non-protein-coding regions of the genome with more University, Shanghai 222300, China than 200 nucleotides (nt) in length6. They have been Full list of author information is available at the end of the article These authors contributed equally: Xuan Deng, Fanyang Kong shown to modulate transcription, translation, and post- Edited by G. Blandino © The Author(s) 2021 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a linktotheCreativeCommons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. Official journal of the Cell Death Differentiation Association Deng et al. Cell Death and Disease (2021) 12:485 Page 2 of 13 transcriptional modification of mRNAs and regulate the tong University School. Briefly, tumor resections were cellular process involved in cancer pathogenesis7,8. P53 manually minced and digested for 1 h with 0.1% trypsin inHibiting LncRNA (PiHL) was a newly identified lncRNA (Invitrogen, Carlsbad, CA, USA) and 10 U/ml of DNase I located on human chromosome 8. Our previous study (Promega, Madison, WA, USA) in a 37 °C Innova 40 revealed that PiHL had three exons with a full length of temperature incubator (New Brunswick Scientific, Edison, 599 nt9. We clarified that PiHL upregulation is an inde- NJ, USA), followed by a red blood cell lysis using ACK pendent predictor for poor prognosis of CRC patients in lysis buffer (Beyotime, Shanghai, China). Tissues then two independent cohorts, and PiHL was able to promote were washed and triturated bypassed through a 100 μm proliferation and inhibit apoptosis of CRC in vitro and cell strainer. Oxa-resistant CRC C4R, R21R, 480R, and in vivo9. It is generally acknowledged that “futile” 116R cells were induced from C4, R21, SW480, and attempts to repair DNA damage generated by cytotoxic HCT116 cells, respectively. Cells were cultured in DMEM drugs usually finish in cell death activation. Therefore, medium (Gibco, Grand Island, NY, USA), supplemented changes in key regulators of cell death (which might be a with 10% fetal bovine serum (Gibco) at 37 °C in an major culprit for drug resistance) often compromise the atmosphere with 5% CO2. Culture environments were efficacy of chemotherapy10. Indeed, gene expression tested and certified as mycoplasma-free. affected by PiHL silencing was significantly enriched in the cell apoptotic process in our mRNA profile of CRC RNA isolation and quantitative RT-PCR analyses cells (GSE124526), prompting us to further explore the RNA isolation was performed as previously described9. role and mechanism of PiHL in drug resistance. Briefly, total RNA from the CRC tissue specimens and cell In this study, we demonstrated a novel KLF4/lncRNA- lines was extracted using TRIzol reagent (Invitrogen, PiHL/EZH2/HMGA2 signaling axis that mediated the Carlsbad, CA, USA), and reverse transcribed using Pri- response of CRC cells to oxaliplatin treatment. We fur- meScript™ RT reagent Kit with gDNA Eraser (Takara, thermore provided strategies to reverse such resistance Beijing, China). SYBR® Premix Ex Taq™ GC (Takara) was in CRC. used for qPCR with primers listed in Supplementary Table 1. Expression levels were calculated relative to Materials and methods β-actin and normalized to control samples. Patient sample In this study, fresh CRC tissues were collected from 100 Western blot CRC patients by surgical resection (Shanghai Jiao-tong Western blot analysis was performed as previously University School of Medicine affiliated Tongren Hospi- described9. Cells were suspended in RIPA lysis buffer tal). All patients were selected based on a clear patholo- (Beyotime, Shanghai, China) containing a protease inhi- gical and imaging diagnosis with stage II and stage III bitor cocktail (Sigma-Aldrich, St. Louis, MO, USA). Cell CRC. None of the patients had received any prior antic- lysates or retrieved proteins were analyzed by immuno- ancer treatment but underwent oxaliplatin-based adju- blot with primary antibodies and HRP-conjugated sec- vant chemotherapy after surgery. Treatment response was ondary antibodies. assessed with morphologic imaging (CT) and classified according to the response evaluation criteria in solid Plasmid construction and cell transfection tumors guideline into complete response, partial Full-length PiHL cDNA was cloned into pCDH-CMV- response, stable disease, and progressive disease. Written puro (short for pCDH) vector (System Biosciences, Palo informed consent for using tumor tissues and clinical data Alto, CA, USA) as previously described9. PiHL promoter in this study was obtained from each patient. This was amplified from 1000 bp to 1 bp upstream of PiHL’s research was approved by the Ethics Committee of TSS. These constructs of truncated PiHL promoter were Shanghai Jiao-tong University. subsequently cloned into the pGL3 vector. Cells seeded on the plate overnight were transfected Cell lines and cell culture with plasmids as indicated in figure legends using Lipo- Human CRC Caco-2, RKO, and DLD-1 cells were fectamine® 3000 (Thermo Fisher Scientific, Waltham, purchased from the Chinese Academy of Sciences Cell MA, USA) transfection reagent following the manu- Bank or ATCC. Human CRC SW480, HCT116, and HT- facturer’s protocol. Cells were harvested at 48–72 h post- 29 were gifts from Dr.Fanyang Kong’s lab. All cells were transfection for future experiments. authenticated by STR profiling (Genetic Testing Bio- technology, Suzhou, China). Patient-derived cancer cells RNA interference C4 and R21 were isolated from discarded CRC specimens, SiRNA oligonucleotides targeting PiHL, HMGA2, and with patients’ written
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