Zhao et al. Cell Death Discovery (2020) 6:99 https://doi.org/10.1038/s41420-020-00332-9 Cell Death Discovery ARTICLE Open Access LINC00355 induces gastric cancer proliferation and invasion through promoting ubiquitination of P53 Wenjing Zhao1,YanJin1,PengWu1,JianYang1, Yuanyuan Chen2,QianluYang2,XinyingHuo1, Juxue Li2,WeiDe2, Jinfei Chen3,4 and Fen Yang2 Abstract Long noncoding RNAs (LncRNAs) have been reported to play critical roles in gastric cancer, but true biomarkers remain unknown. In this study, we found a new lncRNA LINC00355 that was involved in malignant progression of gastric cancer (GC) and further revealed its role and mechanism. Differentially expressed lncRNAs were identified through bioinformatics, and qRT-PCR was used to validate the expression of LINC00355 in gastric cancer tissues and cells. The biological role of LINC00355 in GC was detected by gene overexpression and knockdown experiments. Subcellular fractionation, qRT-PCR, and FISH were performed to detect the subcellular localization. Co-IP and western blotting were used to study the ubiquitination-mediated regulation of P53 and the expression of the E3 ligases RAD18 and UBE3C. The results showed that LINC00355 was significantly increased in gastric cancer cell lines and patient tissues and closely correlated with late stages, distant metastasis, and poor prognosis of patients. High expression of LINC00355 promoted the proliferation and invasion of gastric cancer cells in vivo and in vitro. Mechanistic studies found that LINC00355 that mainly located in the nucleus, acting as a transcriptional activator, promoted transcription of RAD18 and UBE3C, which both bind to P53 and mediate the ubiquitination and degradation of P53. Furthermore, LINC00355 overexpression enhanced the ubiquitination process, and LINC00355 knockdown alleviated it. These results indicated that LINC00355 induces gastric cancer cell proliferation and invasion by promoting transcription of RAD18 and UBE3C, which mediates 1234567890():,; 1234567890():,; 1234567890():,; 1234567890():,; ubiquitination of P53 and thereby plays a critical role in survival and tumorigenicity of gastric cancer cells. LINC00355 may represent a new mechanism for GC progression and provide a potential marker for GC diagnosis and treatment. Introduction main methods for treating gastric cancer, but patients Gastric cancer (GC) is the fifth most frequently diag- with advanced gastric cancer do not have the option of nosed cancer and the third leading cause of cancer-related surgery, and the prognosis is very poor4,5. Therefore, death worldwide1. Although there are many available elucidating the molecular biological mechanism of gastric methods for screening gastric cancer, many patients are cancer is indispensable for finding a sensitive diagnostic still diagnosed at advanced stages, and some patients are method for early gastric cancer and improving prognosis. even diagnosed with metastasis in lymph nodes or other Long noncoding RNA (lncRNA) is a class of molecules organs2,3. Surgery resection and chemotherapy are the that have transcripts longer than 200 nt, lack an open- reading frame, and have limited protein-coding function6. In the human genome, as common epigenetic regulatory Correspondence: Jinfei Chen ([email protected])or molecules, lncRNAs play an important role in epigenetics Fen Yang ([email protected]) and are involved in transcriptional regulation, RNA 1Department of Oncology, Nanjing First Hospital, Nanjing Medical University, 210006 Nanjing, People’s Republic of China shearing and modification, mRNA stabilization, transla- 2Department of Biochemistry and Molecular Biology, School of Basic Medical tional regulation, protein stabilization and transport, ’ Sciences, Nanjing Medical University, 211166 Nanjing, People s Republic of chromosome formation, and structural stability7,8. They China Full list of author information is available at the end of the article participate in regulating embryo development, tissue Edited by Ivano Amelio © 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 Zhao et al. Cell Death Discovery (2020) 6:99 Page 2 of 12 differentiation, organ formation, and the occurrence and GSE27342 from GEO datasets. Bioinformatics analysis development of some diseases9,10. The dysregulation of showed that there were 353, 37, and 69 upregulated lncRNAs can promote tumorigenesis and enhance the lncRNAs in the TCGA, GSE27342, and GSE58828 data- development of cancer by regulating proliferation, inva- sets, respectively (Fig. 1a). FEZF1-AS1 and LINC00355 sion, and metastasis11,12. were only two overlapping lncRNAs (Fig. 1b, left panel). A Many lncRNAs have been found to act as oncogenes previous study reported that FEZF1-AS1 promoted gastric and tumor suppressors, and many lncRNAs act by inter- cancer cell proliferation and that high expression of – acting with familiar oncogenes or tumor suppressors13 15. FEZF1-AS1 predicted poor prognosis in gastric cancer P53, encoded by the TP53 gene, is an important tumor patients24. We also found that 167 lncRNAs in GSE27342, suppressor that has the nickname of “gene guardian,” and 18 lncRNAs in GSE58828, and 310 lncRNAs in TCGA its transcriptional activity is crucial for cell-cycle pro- were downregulated, and that LINC00982 was the only – gression, apoptosis, and DNA repair16 18. Although TP53 overlapping lncRNA (Fig. 1b, right panel). It was reported is largely accepted as a tumor-suppressor gene, oncogenic that LINC00982 was expressed at low levels in gastric effects of mutant P53 proteins, such as deregulated cancer tissues, and that decreased LINC00982 expression metabolic pathways, increased tumor invasion, and was negatively correlated with patient prognosis25. enhanced chemotherapy resistance, have also been Therefore, we presumed that LINC00355, a novel reported, indicating a gain-of-function role for mutant lncRNA, might have an important role in the develop- P53, including in gastric cancer18,19. Post-translational ment and prognosis of gastric cancer. modification of P53, mainly including ubiquitination, phosphorylation, and acetylation, is one of the important LINC00355 is upregulated in gastric cancer and predicts mechanisms of regulating P53 activity20. The poor prognosis in patients ubiquitin–proteasome pathway plays a crucial role in To investigate the relevance between LINC00355 and regulating P53 protein levels21. It is well known that gastric cancer development, we examined LINC00355 MDM2, functioning as an E3 ubiquitin ligase, promotes expression in gastric cancer tissue by qRT-PCR from 72 the degradation of P53, and there are also a number of clinical gastric cancer patients and the corresponding other E3 ubiquitin ligases that bind to P53 to facilitate paracarcinoma tissue. The results showed that the proteasome-mediated ubiquitination of P5322,23. expression of LINC00355 in gastric cancer tissues was In this study, we screened lncRNA profiles using significantly higher than that in adjacent normal tissues microarray analysis and identified a novel lncRNA, (Fig. 2a), and its level was positively associated with depth LINC00355, in gastric cancer tissue from patients. It was of invasion and TNM stage (Fig. 2b, c). The survival highly expressed in gastric cancer tissues compared with analysis based on patients’ clinical data showed that high adjacent normal gastric mucosal epithelial tissues, and expression of LINC00355 significantly shortened the was associated with poor prognosis in patients. High survival time of patients (Fig. 2d), which suggested that expression of LINC00355 promoted gastric cancer cell high expression of LINC00355 predicted poor prognosis proliferation and tumorigenicity in immunodeficient of gastric cancer. mice. High-throughput RNA sequencing upon The correlational analysis between LINC00355 expres- LINC00355 knockdown revealed that the expression of sion and clinicopathological factors of gastric cancer RAD18 and UBE3C is positively correlated with patients determined that higher LINC00355 expression LINC00355 expression. We predicted substrates of the E3 levels were significantly correlated with advanced patho- ubiquitin ligases RAD18 and UBE3C using UbiBrowser logic stage (P = 0.000), invasion depth (P = 0.000), and (http://ubibrowser.ncpsb.org) and found that P53 was distant metastasis (P = 0.029) in gastric cancer patients, their only “highly probable” substrate. Our data showed but it was not correlated with other factors, including that LINC00355 enhanced the interaction between P53 histologic differentiation, lymphatic metastasis, sex, and and the E3 ubiquitin ligases RAD18 and UBE3C, thereby age (Table 1). facilitating ubiquitination and