Guo et al. Cell Death and Disease (2020) 11:869 https://doi.org/10.1038/s41419-020-03026-3 Cell Death & Disease ARTICLE Open Access LINC01106 drives colorectal cancer growth and stemness through a positive feedback loop to regulate the Gli family factors Kun Guo1, Wenbin Gong2,QinWang3,GuoshengGu4,TaoZheng4,YingLi5, Weijie Li4,MiaoFang4, Haohao Xie4, Chao Yue6,JianboYang1 and Zhiqiang Zhu1 Abstract Long non-coding RNAs (lncRNAs) are essential contributors to the progression of various human cancers. Long intergenic non-protein coding RNA 1106 is a member of lncRNAs family. Until now, the specific role of LINC01106 in CRC remains undefined. The aim the current study was to unveil the functions of LINC01106 and explore its potential molecular mechanism in CRC. Based on the data of online database GEPIA, we determined that LINC01106 was expressed at a high level in colon adenocarcinoma (COAD) tissues compared to normal colon tissues. More importantly, high level of LINC01106 had negative correlation with the overall survival of COAD patients. Additionally, we also determined the low level of LINC01106 in normal colon tissues based on UCSC database. Through qRT-PCR, we identified that LINC01106 was highly expressed in CRC tissues compared to adjacent normal ones. Similarly, we detected the expression of LINC01106 and confirmed that LINC01106 was expressed higher in CRC cells than that in normal cells. Subsequently, LINC01106 was mainly distributed in the cytoplasm. LINC01106 induced the proliferation, migration, and stem-like phenotype of CRC cells. Mechanistically, cytoplasmic LINC01106 positively modulated Gli4 in CRC cells by serving as a miR-449b-5p sponge. 1234567890():,; 1234567890():,; 1234567890():,; 1234567890():,; Furthermore, nuclear LINC01106 could activate the transcription of Gli1 and Gli2 through recruiting FUS to Gli1 and Gli2 promoters. Mechanism of investigation unveiled that Gli2 was a transcription activator of LINC01106. In conclusion, Gli2-induced upregulation of LINC01106 aggravates CRC progression through upregulating Gli2, Gli2, and Gli4. Introduction with CRC deterioration4,5. Furthermore, it is predicted Colorectal cancer (CRC) results in 600,000 fatalities that the mortality of CRC will be climbing until 20356. each year worldwide. It ranks third among all usually Increasing comprehension on the molecular mechanism diagnosed human cancers1,2. Due to the improvement of underneath CRC progression will help to find novel anti-tumor therapeutics, annual fatalities are gradually methods for the treatment. decreasing in recent decades3. Genetic and epigenetic Long non-coding RNAs (lncRNAs) are RNA molecules alterations are recognized as factors closely associated without protein-coding capacity. Dysregulation of lncRNAs is illuminated to be associated with the biological functions, including cell growth, mobility, and stemness. For exam- Correspondence: Chao Yue ([email protected])or Jianbo Yang ([email protected]) or Zhiqiang Zhu (zhuzhiqiang711010@163. ples, lncRNA UICLM induces CRC metastasis via com) increasing ZEB2 expression7. LncRNA AB073614 accel- 1 fi Department of General Surgery, the First Af liated Hospital of USTC, Division erates in CRC cell proliferation and invasiveness8. LncRNA of Life Sciences and Medicine, University of Science and Technology of China, 230001 Hefei, Anhui Province, P. R. China BCAR4 maintains stemness and facilitates tumorigenesis in 2Department of General Surgery, Jinling Hospital, School of Medicine, CRC via targeting to miR-665/STAT3 signaling9.Micro- Southeast University, 210009 Nanjing, Jiangsu Province, P. R. China RNAs (miRNAs) are small ncRNAs, which function in Full list of author information is available at the end of the article These authors contributed equally: Kun Guo, Wenbin Gong, Qin Wang human cancers through post-transcriptionally modulating Edited by G. Blandino © The Author(s) 2020 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 Guo et al. Cell Death and Disease (2020) 11:869 Page 2 of 15 gene expression10. Studies suggest miR-495 suppresses RPMI-1640 (Gibco, Grand Island, NY, USA) with 10% of CRC cell proliferation and migration by targeting fetal bovine serum (FBS; Gibco) at 37 °C in 5% CO2. FAM83D11. miR-144 plays essential role in suppressing CRC cell proliferation and migration through modulation Cell transfection of GSPT112. Mechanistically, cytoplasmic lncRNAs are Specific shRNAs against LINC01106 (sh/LINC01106#1/ capable of arresting miRNA function via sequestering 2), FUS (sh/FUS#1/2), Gli1 (sh/Gli1#1/2), Gli2 (sh/ miRNAs to upregulate mRNAs13. Moreover, nuclear Gli2#1/2), and their relative negative control (sh/ctrl), lncRNAs have been proven to be the scaffold for proteins were obtained from Genechem (Shanghai, China), toge- to regulate downstream targets and thus affect tumor cell ther with pcDNA3.1/Gli4, pcDNA3.1/FUS, pcDNA3.1/ – growth, stem-like potency, and mobility14 16. Based on the Gli1, pcDNA3.1/Gli2, and the pcDNA3.1 empty vector data of online databases, LINC01106 was dysregulated in (negative control). MiRNA mimics and NC mimics, miR- CRC samples and associated with patients’ poor prognosis. 449b-5p inhibitor and NC inhibitor were all synthesized LINC01106 has been reported to be participant in by GenePharma (Shanghai, China). All above plasmids lncRNA–miRNA–mRNA axis17. However, the specific were constructed into the pLKO.1 vector (Addgene functions of LINC01106 in CRC progression and its #10878), followed by lentiviral particle preparation as upstream or downstream molecular mechanism remain previously described26. Thereafter, lentiviral particles unclear. Thus, the aim of this study was to unmask the containing above plasmids were separately transfected mechanism by which LINC01106 functions in CRC into LoVo or SW1116 cells for 48 h by Lipofectamine progression. 3000 (Invitrogen, Carlsbad, CA, USA). Finally, stably Mechanistically, lncRNAs can regulate the expression of transfected cells were screened out via 2 μg/ml of pur- their downstream genes at transcriptional or post- omycin treatment for additional 48 h. transcriptional level. LncRNAs can act as positive reg- – ulators for mRNAs by acting as sponges for miRNAs18 21. qRT-PCR In the current study, the possible downstream miRNAs of Utilizing TRIzol (Invitrogen), total RNA was isolated cytoplasmic LINC01106 were also detected. In addition, from LoVo and SW1116 cells and then reverse- nuclear lncRNAs can act as scaffold and recruit tran- transcribed into cDNA. qRT-PCR was conducted by scriptional factors to the promoter region of their down- using SYBR-green IMaster Mix (Roche, Mannheim, −ΔΔ stream targets22. Here, we also detected whether nuclear Germany). Gene expression was calculated using 2 Ct LINC01106 exert functions in CRC in the same way. and quantified by normalizing to GAPDH or U6. Transcriptional regulation is an important factor lead- – ing to the upregulation of lncRNAs23 25. In our current Northern blot study, we also investigated the mechanism which induced Northern blot assay was conducted in accordance with a the upregulation of LINC01106 in CRC. previous protocol27. To sum up, our work was aimed at unveiling the asso- ciation of LINC01106 with CRC progression. Fluorescence in situ hybridization (FISH) LoVo or SW1116 cells were loaded on glass coverslips, Materials and methods followed by being washed with PBS (Sigma-Aldrich, St. Tissue collection Louis, MO, USA). Fixation for 30 min in 4% formaldehyde CRC tissues (n = 68) and adjacent non-tumor tissues (n (Sigma-Aldrich) was later conducted. After being per- = 68) were collected via surgical operation from the First meabilized using 70% ethanol (Sigma-Aldrich) for one Affiliated Hospital of USTC. All patients did not receive night, cells were rinsed twice by PBS. Cells were incubated any therapy before surgery. The participants were blinded with LINC01106 probe (GenePharma) at 37 °C overnight. to the group allocation during the experiment. Fresh Cells were dyed for 10 min with DAPI (Sigma-Aldrich), samples were snap frozen in the liquid nitrogen and washed in saline-sodium citrate (SSC; Sigma-Aldrich) and preserved at −80 °C. Informed consents were signed by photographed by fluorescence microscope (Olympus, each participant, with the approval obtained from the Tokyo, Japan). Ethics Committee of the First Affiliated Hospital of USTC (Registration number: 2018NZGKJ-016). Subcellular fractionation The cytoplasmic and nuclear RNA purification kit Cell culture and treatment acquired from Norgen (Thorold, ON, Canada) was Normal colon epithelial cell line (FHC) and CRC cell applied to isolate the nuclear and cytoplasmic fractions. lines (HT29, HCT116, SW480, LoVo and SW1116)