LncRNA LINC00662 promotes colon cancer tumor growth and metastasis by competitively binding with miR-340-5p to regulate CLDN8/IL22 co-expression and activating ERK signaling pathway

Cheng et al. Journal of Experimental & Clinical Cancer Research https://doi.org/10.1186/s13046-019-1510-7 (2020) 39:5 RESEARCH Open Access LncRNA LINC00662 promotes colon cancer tumor growth and metastasis by competitively binding with miR-340-5p to regulate CLDN8/IL22 co-expression and activating ERK signaling pathway Bo Cheng1*, Aimei Rong2, Quanbo Zhou3 and Wenlu Li4 Abstract Background: LncRNA LINC00662 is closely related to the occurrence and development of cancer. This study aims to explore the effect of LINC00662 on colon cancer tumor growth and metastasis and its molecular mechanism. Methods: CCK8, colony formation, transwell, scratch wound, TUNEL, flow cytometry, RT-PCR, western blotting and immunohistochemistry assays were used to detect the proliferation, apoptosis, invasion and migration of colon cancer cell and mRNA and protein expressions. Luciferase reporter and RNA pull down assays were used to detect the combination of LINC00662 and miR-340-5p or IL22 and the combination of miR-340-5p and CLDN8/IL22. Coimmunoprecipitation were used to detect the co-expression of CLDN8 and IL22 in colon cell lines. The targets of LINC00662 were predicated by Starbase v2.0. The target genes of miR-340-5p were predicated by miRDB and TargetScan. GO and KEGG enrichment analysis were performed by DAVID website. Results: LINC00662 was up-regulation in colon cancer tissues and cell lines. Univariate Cox regression analysis showed that the LINC00662 expression level was related to the poor prognosis. LINC00662-WT and miR-340-5p mimics co-transfection depressed luciferase activity and IL22/CLDN8-WT and miR-340-5p inhibitors co-transfection memorably motivated luciferase activity. LINC00662 overexpression promoted cell proliferation, invasion and migration, and inhibited cell apoptosis in colon cancer. In vivo xenograft studies in nude mice manifested that LINC00662 overexpression prominently accelerate tumor growth. There was an opposite reaction in the biological functions of colon cells and tumor growth between LINC00662 overexpression and LINC00662 inhibition in vitro and in vivo. The functions of miR-340-5p mimics regulating the biological functions of colon cells and tumor growth were consistent with those of LINC00662 inhibition. CLDN8 and IL22, as target genes of miR-340-5p, reversed the functions of LINC00662 affecting the biological functions of colon cells and the protein levels of Bax, Bcl-2, XIAP, VEGF, MMP-2, E-cadherin and N-cadherin. Co-immunoprecipitation experiments indicated that CLDN8 directly interact with IL22 in colon cell lines. LINC00662 regulated CLDN8 and IL22 expressions and the activation of ERK signaling pathway via targeting miR-340-5p. Conclusion: LINC00662 overexpression promoted the occurrence and development of colon cancer by competitively binding with miR-340-5p to regulate CLDN8/IL22 co-expression and activating ERK signaling pathway. Keywords: LncRNA LINC00662, Colon cancer, miR-340-5p, CLDN8, IL22, Growth, Metastasis * Correspondence: 1 Department of Emergency Surgery, The First Affiliated Hospital of Zhengzhou University, No. 1, Jianshe East Road, Zhengzhou City 410008, Henan Province, China Full list of author information is available at the end of the article © The Author(s). 2020 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Cheng et al. Journal of Experimental & Clinical Cancer Research (2020) 39:5 Background Colon cancer is a common malignant tumor of digestive tract in clinic, and its incidence and mortality are high [1]. With the adjustment of lifestyle and diet, the incidence of colon cancer is increasing year by year and becoming younger in China [2]. Like most malignant tumors, the pathogenesis of colon cancer is not entirely clear. At present, colon cancer is considered to be the combined effect of environmental factors and genetic factors. Studies have shown that the main factors affecting the incidence of colon cancer include environment, intestinal homeostasis, diet, alcohol and tobacco addiction and physical exercise [3]. Colon cancer treatment still is primarily surgical, chemotherapy and radiotherapy are supplementary. For therapeutic effect, there are significant individual differences among patients with colon cancer. In the patients with advanced colon cancer, the defects of the above therapy are obvious resulting in a poor prognosis. Postoperative metastasis for colon cancer chiefly includes hematological metastasis, peritoneal metastasis and distant lymph node metastasis, which are frequently accompanied by local recurrence [4]. Hematogenous metastasis is the dominating cause of failure in the treatment of colon cancer. The survival rate of colon cancer is overtly relevant to clinical stage, and the 5-year survival rates of patients with no metastasis, local metastasis and distant metastasis are 90, 70 and 10%, respectively [5]. Therefore, to find the markers of early diagnosis and explore the key molecules involved in the growth and metastasis of colon cancer is the focus of current research. Long-stranded non-coding RNA is a class of RNA molecules whose transcriptional length exceeds that of 200 nt and can’t carry out coding proteins [6]. LncRNA usually is located in cytoplasm or nucleus. The number of lncRNA in the human genome is astonishingly large [7]. LncRNA participates in the regulatory processes of chromatin modification, transcriptional interference, transcriptional activation, nuclear transport, selective splicing and regulation of proto-oncogene activation, so as to regulate gene expression at epigenetic, transcriptional or posttranscriptional levels [8, 9]. Abnormal expression and functions of lncRNA are involved in the occurrence and development of many diseases, especially malignant tumors. It is reported in colon cancer that LINC01082 and lncRNA THOR can regulate cell proliferation, migration and invasion [10, 11] . LncRNA can not only directly participate in the posttranscriptional regulation of mRNA, including variable splicing, RNA editing, protein translation and transport, but also affect the expression of target genes by controlling microRNA [12]. In some tumor cells, Page 2 of 21 lncRNA carries the seed sequence of miRNA to prevent miRNA from binding to its target mRNA. The functions of lncRNA AWPPH in proliferation of colon cancer cells were regulated by targeting GLUT1 [13]; LncRNA CCAT1 promotes autophagy of liver cancer cells via regulating ATG7 (...truncated)