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Molecular Pathogenesis and Regulation of the Mir-29-3P-Family: Involvement of ITGA6 and ITGB1 in Intra-Hepatic Cholangiocarcinoma

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cancers Article Molecular Pathogenesis and Regulation of the miR-29-3p-Family: Involvement of ITGA6 and ITGB1 in Intra-Hepatic Cholangiocarcinoma Yuto Hozaka 1 , Naohiko Seki 2,* , Takako Tanaka 1, Shunichi Asai 2, Shogo Moriya 3, Tetsuya Idichi 1, Masumi Wada 1, Kiyonori Tanoue 1, Yota Kawasaki 1, Yuko Mataki 1 , Hiroshi Kurahara 1 and Takao Ohtsuka 1 1 Department of Digestive Surgery, Breast and Thyroid Surgery, Graduate School of Medical and Dental Sciences, Kagoshima University, Kagoshima 890-8520, Japan; [email protected] (Y.H.); [email protected] (T.T.); [email protected] (T.I.); [email protected] (M.W.); [email protected] (K.T.); [email protected] (Y.K.); [email protected] (Y.M.); [email protected] (H.K.); [email protected] (T.O.) 2 Department of Functional Genomics, Graduate School of Medicine, Chiba University, Chiba 260-8670, Japan; [email protected] 3 Department of Biochemistry and Genetics, Graduate School of Medicine, Chiba University, Chiba 260-8670, Japan; [email protected] * Correspondence: [email protected]; Tel.: +81-43-226-2971 Simple Summary: Even today, there are no effective targeted therapies for intrahepatic cholan- giocarcinoma (ICC) patients. Clarifying the molecular pathogenesis of ICC will contribute to the Citation: Hozaka, Y.; Seki, N.; development of treatment strategies for this disease. In this study, we searched for the role of the Tanaka, T.; Asai, S.; Moriya, S.; Idichi, miR-29-3p-family and its association with oncogenic pathway. Interestingly, aberrant expression of T.; Wada, M.; Tanoue, K.; Kawasaki, ITGA6 and ITGB1 was directly regulated by the miR-29-3p-family which are involved in multiple Y.; Mataki, Y.; et al. Molecular oncogenic pathways in ICC, and enhanced malignant transformation of ICC cells. Furthermore, SP1 Pathogenesis and Regulation of the which is a transcriptional activator of ITGA6/ITGB1, is regulated by the miR-29-3p-family. These miR-29-3p-Family: Involvement of molecules may be novel therapeutic targets for ICC. ITGA6 and ITGB1 in Intra-Hepatic Cholangiocarcinoma. Cancers 2021, 13, 2804. https://doi.org/10.3390/ Abstract: The aggressive nature of intrahepatic cholangiocarcinoma (ICC) renders it a particularly cancers13112804 lethal solid tumor. Searching for therapeutic targets for ICC is an essential challenge in the devel- opment of an effective treatment strategy. Our previous studies showed that the miR-29-3p-family Academic Editor: Takahiro Kodama members (miR-29a-3p, miR-29b-3p and miR-29c-3p) are key tumor-suppressive microRNAs that con- trol many oncogenic genes/pathways in several cancers. In this study, we searched for therapeutic Received: 31 March 2021 targets for ICC using the miR-29-3p-family as a starting point. Our functional studies of cell prolifera- Accepted: 31 May 2021 tion, migration and invasion confirmed that the miR-29-3p-family act as tumor-suppressors in ICC Published: 4 June 2021 cells. Moreover, in silico analysis revealed that “focal adhesion”, “ECM-receptor”, “endocytosis”, “PI3K-Akt signaling” and “Hippo signaling” were involved in oncogenic pathways in ICC cells. Our Publisher’s Note: MDPI stays neutral analysis focused on the genes for integrin-α6 (ITGA6) and integrin-β1 (ITGB1), which are involved with regard to jurisdictional claims in in multiple pathways. Overexpression of ITGA6 and ITGB1 enhanced malignant transformation published maps and institutional affil- of ICC cells. Both ITGA6 and ITGB1 were directly regulated by the miR-29-3p-family in ICC cells. iations. Interestingly, expression of ITGA6/ITGB1 was positively controlled by the transcription factor SP1, and SP1 was negatively controlled by the miR-29-3p-family. Downregulation of the miR-29-3p-family enhanced SP1-mediated ITGA6/ITGB1 expression in ICC cells. MicroRNA-based exploration is an attractive strategy for identifying therapeutic targets for ICC. Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. Keywords: intrahepatic cholangiocarcinoma; miR-29a-3p; miR-29b-3p; miR-29c-3p; tumor-suppressor; This article is an open access article ITGA6; ITGB1; SP1 distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). Cancers 2021, 13, 2804. https://doi.org/10.3390/cancers13112804 https://www.mdpi.com/journal/cancers Cancers 2021, 13, 2804 2 of 21 1. Introduction Cholangiocarcinoma is anatomically divided into intrahepatic cholangiocarcinoma (ICC) and extrahepatic cholangiocarcinoma. From cancer genomic analysis, each cancer contains characteristic gene mutations, suggesting molecular biological differences [1–3]. ICC is the second most common cancer of the liver after hepatocellular carcinoma (HCC). ICC constitutes about 5% of primary liver cancers, and global morbidity and mortality of the disease have increased significantly in recent years [4–7]. Due to the aggressive features of ICC (the high rate of metastasis), the prognosis of patients is very poor (5-year survival rate is less than 10%) [8]. Although surgical resection is the only strategy aiming at a complete cure, only about 30% of patients are operable [9]. Even today, there are no effective anti-cancer drugs or molecularly targeted therapies for ICC patients in the advanced stage of disease [1,2]. The lack of effective diagnostic markers for ICC is a major factor that delays the detection of this disease [10,11]. Clarifying the molecular pathogenesis of ICC based on the latest genomic analyses will contribute to the development of treatment strategies for this disease. The Human Genome Project has revealed the presence of a vast number of non- coding RNA molecules (ncRNAs) in the human genome. These ncRNAs have important function within cells [12]. MicroRNAs (miRNAs) constitute a set of small ncRNAs (19 to 23 bases) that negatively regulate the expression of RNA transcripts in a sequence- dependent manner [13]. Over the last decade, accumulating evidence has shown that aberrantly expressed miRNAs are closely involved in cancer cell development, metastasis and drug-resistance [12,14,15]. For example, dysregulated miRNAs can disrupt tightly controlled RNA networks, triggering the transition of normal cells to diseased cells. Identification of aberrantly expressed miRNAs in each type of cancer is the first step towards elucidating the roles of miRNA in the molecular pathogenesis of cancers. Based on miRNA expression signatures, we have revealed some of the roles of tumor-suppressive miRNAs and their controlled oncogenic targets and pathways in gastrointestinal cancers, e.g., esophageal cancer, gastric cancer and pancreatic ductal adenocarcinoma [16–18]. Previous study of ICC signatures showed that a total of 30 miRNAs (10 of which were upregulated and 20 were downregulated) were dysregulated in ICC tissues [19]. Moreover, the expression of three miRNAs (miR-675-5p, miR-652-3p and miR-338-3p) was characteristic of ICC. Those miRNA signatures were independent prognostic indicators based on multivariate analysis [19]. Other signatures showed that 38 miRNAs were differ- entially expressed in tumor and normal tissues [20]. Among these miRNAs, exogenous expression of miR-320 and miR-204 negatively controlled Mcl-1 and BCl-2 expression, respectively. Expression of those miRNAs facilitates chemotherapeutic drug-triggered apoptosis [20]. Upregulation of miR-191 in ICC tissues was detected by next-generation sequencing technology [21]. Overexpression of miR-191 enhanced ICC aggressive pheno- types, e.g., proliferation, invasion and migration abilities in vitro and in vivo [21]. Analysis of our RNA-sequence-based signatures revealed that the miR-29-3p-family (miR-29a-3p, miR-29b-3p and miR-29c-3p) was frequently downregulated in several cancers, suggesting these miRNAs acted as pivotal tumor-suppressors in cancer cells [22,23]. Impor- tantly the miR-29-3p-family is downregulated in ICC tissues [24]. The aim of the study was to investigate the functional significance of the miR-29-3p-family and to identify oncogenic targets/pathways in ICC cells. Our ectopic expression assays showed that all members of the miR-29-3p-family acted as tumor-suppressors in ICC cells. Several oncogenic pathways (e.g., focal adhesion, ECM-receptor, endocytosis, PI3K–Akt signaling and Hippo signaling) were subject to control by the miR-29-3p-family. Furthermore, we revealed that the genes for integrin alfa-6 (ITGA6) and beta-1(ITGB1) were directly regulated by the miR-29-3p-family, and their overexpression enhanced migration and invasive abilities in ICC cells. In addition, we showed that transcription factor SP1 is involved in ITGA6 and ITGB1 expression, and that SP1 expression was negatively controlled by the miR-29-family in ICC cells. Downregulation of the miR-29-3p-family activated several oncogenic pathways, and these events were closely involved in ICC oncogenesis. Oncogenic signaling pathways Cancers 2021, 13, x 3 of 21 Cancers 2021, 13, 2804 3 of 21 Downregulation of the miR-29-3p-family activated several oncogenic pathways, and these events were closely involved in ICC oncogenesis. Oncogenic signaling pathways mediated by ITGA6/ITGB1 are likely therapeutic targets for this disease. Our miRNA- basedmediated approach by ITGA6 will accelerate/ITGB1 are the likely understanding therapeutic of targetsthe molecular for this pathogenesis disease. Our of miRNA- ICC. based approach will accelerate the understanding of the molecular pathogenesis of ICC. 2. Results 2. Results 2.1. Downregulation of miR-29a-3p, miR-29b-3p and miR-29c-3p in ICC Clinical Specimens 2.1. Downregulation of miR-29a-3p, miR-29b-3p and miR-29c-3p in ICC Clinical Specimens The expression levels of miR-29a-3p, miR-29b-3p and miR-29c-3p were investigated to utilizeThe the expressionGene Expression levels ofOmnibusmiR-29a-3p (GEO), miR-29b-3p database (GSE:and miR-29c-3p 53870). Thesewere datasets investigated con- to utilize the Gene Expression Omnibus (GEO) database (GSE: 53870). These datasets tained 63 ICC tissues and 9 normal intrahepatic bile ducts tissues.
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