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 , 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 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 /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-”, “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 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 genomic analysis, each cancer contains characteristic , suggesting molecular biological differences [1–3]. ICC is the second most common cancer of the liver after (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 ), 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 Project has revealed the presence of a vast number of non- coding RNA molecules (ncRNAs) in the . 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 [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 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 Omnibus (GEO) database (GSE: 53870). These datasets tained 63 ICC tissues and 9 normal intrahepatic bile ducts tissues. Downregulation of miR- contained 63 ICC tissues and 9 normal intrahepatic bile ducts tissues. Downregulation 29a-3p, miR-29b-3p and miR-29c-3p was confirmed in ICC tissues compared with normal of miR-29a-3p, miR-29b-3p and miR-29c-3p was confirmed in ICC tissues compared with tissues (p = 0.0003, p < 0.001 and p = 0.0210, respectively; Figure 1A). These miRNAs were normal tissues (p = 0.0003, p < 0.001 and p = 0.0210, respectively; Figure1A). These miRNAs clustered at two different human loci: miR-29b-1 and miR-29a were at 7q32.3 and miR-29b- were clustered at two different human loci: miR-29b-1 and miR-29a were at 7q32.3 and 2, and miR-29c was at 1q32.2. Mature sequences of miR-29a-3p and miR-29c-3p are identi- miR-29b-2, and miR-29c was at 1q32.2. Mature sequences of miR-29a-3p and miR-29c-3p are cal. On the other hand, the mature sequence of miR-29b-3p differs at two bases on the identical. On the other hand, the mature sequence of miR-29b-3p differs at two bases on the 3′end. The seed sequences of the three miRNAs are identical (red letters; Figure 1B). 30end. The seed sequences of the three miRNAs are identical (red letters; Figure1B).

FigureFigure 1. 1. InIn silico silico expression expression analysis analysis of ofmiRmiR-29a-3p-29a-3p, miR, miR-29b-3p-29b-3p andand miRmiR-29c-3p-29c-3p. (A).( ExpressionA) Expression levelslevels of of miRmiR-29a-3p-29a-3p, ,miRmiR-29b-3p-29b-3p andand miRmiR-29c-3p-29c-3p inin ICC ICC tissues. tissues. A A total total of of 63 63 ICC ICC cancer cancer tissues tissues and and 99 normal normal intrahepatic intrahepatic bile bile ducts ducts (NIBDs) (NIBDs) were were analyzed. analyzed. ( (BB)) Mature Mature sequences sequences of of the the miR miR-29-family-29-fam- ilyare are shown. shown. Mature Mature sequences sequences ofof miR-29a-3pmiR-29a-3p and miRmiR-29c-3p-29c-3p areare identical. identical. The The mature mature sequence sequence ofof miRmiR-29b-3p-29b-3p differsdiffers in in 2 bases 2 bases on on the the 3′ end. 30 end. The The seed seed sequences sequences of the of 3 the miRNAs 3 miRNAs are identical are identical (red letters). (red letters).

22.2..2. Effects Effects of of E Ectopicctopic E Expressionxpression of of miR miR-29a-3p,-29a-3p, miR miR-29b-3p-29b-3p and and miR miR-29c-3p-29c-3p on on ICC ICC C Cellell PProliferation,roliferation, M Migrationigration and and Invasion Invasion TheThe tumor tumor-suppressive-suppressive roles roles of of miRmiR-29a-3p-29a-3p, ,miRmiR-29b-3p-29b-3p andand miRmiR-29c-3p-29c-3p werewere assessed assessed byby using using ectopic ectopic expression expression assays assays in in HuCCT1 HuCCT1 and and RBE RBE cells. cells. Cell Cell proliferation proliferation assays assays showedshowed no no significant significant effects effects of of these these miRNAs miRNAs transfected transfected in in two two cell cell lines lines (Figure (Figure 2A).2A). In In contrast,contrast, cell cell migration migration and and invasive invasive abilities abilities were were significantly significantly inhibited inhibited in in miRmiR-29a-3p-29a-3p, , miRmiR-29b-3p-29b-3p andand miRmiR-29c-3p-29c-3p-transfected-transfected cells cells (HuCCT1 (HuCCT1 and and RBE) RBE) compared compared with with mock mock-- or or miRmiR-control-transfected-control-transfected cells cells (Fig (Figureure 2B,2B,C).C).

Cancers 2021, 13, 2804 4 of 21 Cancers 2021, 13, x 4 of 21

Figure 2. EffectsFigure of miR 2.-29aEffects-3p, ofmiRmiR-29a-3p-29b-3p and, miR-29b-3p miR-29c-3pand on functionsmiR-29c-3p (cellon prolife functionsration, (cell migra- proliferation, migra- tion and invasion)tion andof intrahepatic invasion) of cholangiocarcinoma intrahepatic cholangiocarcinoma (ICC) cell lines (ICC) (HuCCT1 cell lines and (HuCCT1RBE). (A) andCell RBE). (A) Cell proliferation wasproliferation assessed using was assessedXTT assays. using Data XTT were assays. collected Data 72 were h after collected miicro 72RNA h after transfec- miicroRNA transfec- tion. Ectopic expressiontion. Ectopic of miR expression-29a-3p, miR of miR-29a-3p-29b-3p and, miR-29b-3p miR-29c-3pand did miR-29c-3pnot affect celldid proliferation. not affect cell proliferation. (B,C) Cell migration(B,C) Cell and migrationinvasive abilities and invasive were significantly abilities were blocked significantly by ectopic blocked expression by ectopic of miR expression- of miR- 29a-3p, miR-29b29a-3p-3p and, miR-29b-3p miR-29c-3pand in ICCmiR-29c-3p cells. Errorin ICC bars cells. represent Error bars mean represent ± standard mean error± standard(SE). error (SE).

2.3. Identification2.3. of Identification Putative Pathways of Putative and PathwaysTargets of and the TargetsmiR-29 of-3p the-Family miR-29-3p-Family (miR-29a/b/c- (miR-29a/b/c-3p)3p) Regulation in RegulationICC Cells in ICC Cells The seed sequencesThe seed of sequencesthe miR-29 of-3p the-familymiR-29-3p- are identicalfamily are(Figure identical 1B). Thus, (Figure we1B). pre- Thus, we pre- dicted that thedicted pathways that theand pathways targets controlled and targets by these controlled miRNAs by w theseould miRNAsbe the same. would Our be the same. strategy for theOur selection strategy for of themiR selection-29-3p-fam ofilymiR-29-3p controlled-family pathways controlled and pathways target genes and istarget genes is shown in Figureshown 3. in Figure3.

Cancers 2021, 13, x 5 of 21 Cancers 2021, 13, 2804 5 of 21

Figure 3. Flowchart summarizing the search for oncogenic targets of miR-29-3p regulation in ICC Figure 3. Flowchart summarizing the search for oncogenic targets of miR-29-3p regulation in ICC cells. To identify miR- cells. To identify miR-29-3p target genes, we screened putative targets using the TargetScan database 29-3p target genes, we screened putative targets using the TargetScan database and TCGA database. A total of 888 targets were upregulated in cholangiocarcinoma tissues.and TCGAThese genes database. were A then total categorized of 888 targets into were KEGG upregulated (Kyoto Encyclopedia in cholangiocarcinoma of tissues. These Genes and ) pathways using the GeneCodisgenes were database. then categorized Finally, 20 into pathways KEGG (Kyotothat were Encyclopedia regulated by of miR Genes-29- and3p Genomes) pathways were identified. using the GeneCodis database. Finally, 20 pathways that were regulated by miR-29-3p were identified.

First, based on theFirst, TargetScan based on database the TargetScan (release database 7.2), we (releasesearched 7.2), for weputative searched genes for putative genes that have targetthat sites have for the target miR sites-29-3p for-family the miR-29-3p- (AGCACCA).family A (AGCACCA). total of 3543 genes A total were of 3543 genes were identified. Second,identified. we selected Second, the upregulated we selected genes the in upregulated cholangiocarcinoma genes in cholangiocarcinomatissues com- tissues pared with normalcompared tissues with by using normal TCGA tissues database by using (36 TCGA cancer database and 9 (36 normal cancer tissues, and 9 normal tissues, TCGA-CHOL) and Subio platform. A total of 4999 upregulated genes (log2 ratio > 1) were TCGA-CHOL) and Subio platform. A total of 4999 upregulated genes (log2 ratio > 1) were identified. Next, the two datasets were merged, and a total of 888 genes were selected as identified. Next, the two datasets were merged, and a total of 888 genes were selected as putative targets of miR-29-3p (i.e., upregulated genes in cholangiocarcinoma tissues that putative targets of miR-29-3p (i.e., upregulated genes in cholangiocarcinoma tissues that have miR-29-3p binding sites). Finally, we classified 888 genes (Table S1) based on their have miR-29-3p binding sites). Finally, we classified 888 genes (Table S1) based on their molecular functions using KEGG pathways (GeneCodis 4.0). molecular functions using KEGG pathways (GeneCodis 4.0). A total 20 pathways were identified as the miR-29-3p-family controlled oncogenic A total 20 pathways were identified as the miR-29-3p-family controlled oncogenic pathways in ICC cells (Table1; Tables S2–S7). In this study, we focused on ITGA6 and ITGB1, pathways in ICC cells (Table 1; Tables S2–S7). In this study, we focused on ITGA6 and both of which involve multiple pathways, indicating these two genes closely contributed ITGB1, both of which involve multiple pathways, indicating these two genes closely con- to ICC oncogenesis. tributed to ICC oncogenesis. Table 1. Significantly enriched pathways regulated by the miR-29-3p-family in ICC cells. Table 1. Significantly enriched pathways regulated by the miR-29-3p-family in ICC cells. p Number of Number of Genes -Value Annotations p-Value − Annotations Genes 30 1.07 × 10 6 (KEGG) 04510: Focal adhesion 19 2.04 × 10−6 (KEGG) 05222: Small cell 30 1.07 × 10−6 (KEGG) 04510: Focal adhesion 18 4.72 × 10−5 (KEGG) 04974: digestion and absorption −6 19 2.04 × 1048 6.98 ×(KEGG)10−5 05222: Small(KEGG) cell lung 05200: cancer Pathways in cancer 18 4.72 × 1035−5 (KEGG)7.63 × 10 04974:−5 Protein(KEGG) digestion 05165: Humanand absorption papillomavirus infection 48 6.98 × 1016−5 8.83 ×(KEGG)10−5 05200: Pathways(KEGG) 04512: in cancer ECM-receptor interaction 35 7.63 × 1028−5 (KEGG)0.000184427 05165: Human papillomavirus(KEGG) 04144: infe Endocytosisction 34 0.000560778 (KEGG) 04151: PI3K-Akt signaling pathway 16 8.83 × 10−5 (KEGG) 04512: ECM-receptor interaction 20 0.000585526 (KEGG) 04390: 28 0.00018442713 0.00911125(KEGG) 04144: Endocytosis (KEGG) 05215: Prostate cancer 34 0.000560778 (KEGG) 04151:(KEGG) PI3K- 04933:Akt signaling AGE-RAGE pathway signaling pathway in diabetic 13 0.0112006 20 0.000585526 (KEGG) 04390: Hippo signalingcomplications pathway 13 0.0091112512 0.0247066(KEGG) 05215: (KEGG) Prostate 05231: ca Cholinencer metabolism in cancer 18 0.0252777 (KEGG) 04360: Axon guidance (KEGG) 04933: AGE-RAGE signaling pathway in dia- 13 0.011200610 0.0253268 (KEGG) 05218: Melanoma 8 0.026522betic complications (KEGG) 04340: Hedgehog signaling pathway 12 0.0247066 (KEGG) 05231: Choline metabolism in cancer 18 0.0252777 (KEGG) 04360: Axon guidance

Cancers 2021, 13, x 6 of 21

Cancers 2021, 13, 2804 6 of 21

10 0.0253268 (KEGG) 05218: Melanoma 8 Table 1.0.026522Cont. (KEGG) 04340: Hedgehog signaling pathway (KEGG) 04540: Signaling pathways regulating pluripo- 15 Number0.0283549 of Genes p-Value Annotations tency of stem cells (KEGG) 04540: Signaling pathways regulating pluripotency 15 0.0283549 19 0.0302379 (KEGG) 05205: Proteoglycansof stem cellsin cancer 15 0.035434519 0.0302379(KEGG) 04072: Phospholipase (KEGG) 05205: D Proteoglycans signaling pathway in cancer 11 0.041227915 0.0354345(KEGG) 04350: (KEGG) TGF 04072:-beta Phospholipase signaling Dpathway signaling pathway 11 0.0412279 (KEGG) 04350: TGF-beta signaling pathway 20 0.0463112 (KEGG) 04014: Ras signaling pathway 20 0.0463112 (KEGG) 04014: Ras signaling pathway

2.4. Expression of ITGA6/ITGA6 and ITGB1/ITGB1 Genes and in ICC Clinical 2.4. Expression of ITGA6/ITGA6 and ITGB1/ITGB1 Genes and Proteins in ICC Specimens Clinical Specimens We analyzedWe expression analyzed expression levels of levelsITGA6 of andITGA6 ITGB1and (RNAITGB1-Sequence(RNA-Sequence data in data 36 incases 36 cases of of TCGA-CHOL)TCGA-CHOL) using the using GEPIA2 the GEPIA2 database database platform. platform. Expression Expression of ofboth both ITGA6ITGA6 andand ITGB1 ITGB1 was wassignificantly significantly upregulated upregulated (p (

Figure 4. ExpressionFigure 4. ofExpression ITGA6 and of ITGB1ITGA6 inand ICC.ITGB1 (A) inExpression ICC. (A) Expression levels of ITGA6 levels and of ITGA6 ITGB1and in ITGB1ICC tissuesin ICC and tissues normal and normal tissues obtainedtissues from obtained TCGA- fromCHOL TCGA-CHOL based on the based GEPIA2 on the platform. GEPIA2 platform.(B) H&E- (stainedB) H&E-stained sections sections of human of humanICC tissue. ICC tissue. Areas Areas in in the boxed regionthe boxed at t regionhe left at are the shown left are magnified shown magnified at right. at right.(C,D) (CRepresentative,D) Representative images images of tissues of tissues immunostained immunostained for for ITGA6 ITGA6 and ITGB1.and ITGB1. ITGA6 ITGA6 stained stained positive positive in the in membranes the membranes of cancer of cancer cells cells in a inclinical a clinical sample sample (original (original magnification magnification × × 100). 100). ITGB1 stainedITGB1 stainedpositive positive in the inmembr the membranes,anes, cytoplasm and anda part a part of the of the nuclei nuclei of of cancer cancer cells cells in in a clinicalclinical samplesample (original (original magnificationmagnification × 100).× 100).

2.5. Direct R2.5.egulation Direct of Regulation ITGA6 and of ITGA6 ITGB1 and by ITGB1miR-29a by-3p, miR-29a-3p, miR-29b- miR-29b-3p3p and miR and-29c miR-29c-3p-3p in in ICC Cells ICC Cells ITGA6 miR-29-3p We investigatedWe investigated direct regulation direct regulationof ITGA6 by of the miR-by29- the3p-family in ICC-family cells. in Ex- ICC cells. Expression levels of both mRNA and protein were significantly reduced by ectopic expres- pression levels of both mRNA and protein were significantly reduced by ectopic expres- sion of miR-29a-3p, miR-29b-3p and miR-29c-3p in ICC cells (HuCCT1 and RBE, Figure5 , sion of miR-29a-3p, miR-29b-3p and miR-29c-3p in ICC cells (HuCCT1 and RBE, Figure 5, Figure S2A). Similarly, ectopic expression of miR-29a-3p, miR-29b-3p and miR-29c-3p re- Figure S2Aduced). Similarly, expression ectopic levels expression of ITGB1 /ITGB1of miR- (Figure29a-3p,6 miR, Figure-29b S2B).-3p and miR-29c-3p re- duced expression levels of ITGB1/ITGB1 (Figure 6, Figure S2B).

CancersCancers 2021 2021,Cancers ,13 13, ,x x 2021, 13, 2804 77 of of 21 217 of 21

Figure 5. Regulation of ITGA6/ITGA6 expression by ectopic expression of miR-29a-3p, miR-29b-3p FigureFigure 5. 5. Regulation Regulation of of ITGA6ITGA6/ITGA6/ITGA6 expression expression by by ectopic ectopic expression expression of of miRmiR--29a29a--3p3p, ,miR miR--29b29b--3p3p miR-29c-3p A ITGA6 andand miR miR--29c29cand--3p3p in in intrahepatic intrahepaticin intrahepatic cholangiocarcinoma cholangiocarcinoma cholangiocarcinoma ( (ICCICC)) cells. cells. (ICC) ( (AA cells.)) Expression Expression ( ) Expression levels levels levelsof of ITGA6ITGA6 of were werewere significantly significantlysignificantly reduced reduced reduced by by miR bymiRmiR-29-3p--2929--3p3p transfection transfectiontransfection into into into ICC ICC ICC cells. cells. cells. GUSB GUSBGUSB was was used usedused asas anan internalinternal internal control. control.control. ( (BB()B) Protein )Protein Protein expression expression expression levels levels ofof ITGA6ITGA6 werewere were significantly significantly significantly reduced reduced reduced by by bymiR-29- miRmiR--29family29--familyfamily transfection trans- trans- into fectionfection into intoICC ICC ICC cells cells cells (72 (72 (72 h afterh h afterafter transfection). transfection). transfection). GAPDH GAPDHGAPDH was was was used used used as an as as internal an an internal internal control. control.control.

Figure 6. Regulation of ITGB1/ITGB1 expression by ectopic expression of miR-29a-3p , miR-29b-3p FigureFigure 6. 6. Regulation Regulationand miR-29c-3p of of ITGB1ITGB1in intrahepatic/ITGB1/ITGB1 expressionexpression cholangiocarcinoma by by ectopic ectopic (ICC) expressionexpression cells. ( Aof of) miR ExpressionmiR--29a29a--3p3p, levels ,miR miR--29b of29bITGB1--3p3p were and miR-29c-3p in intrahepatic cholangiocarcinoma (ICC) cells. (A) Expression levels of ITGB1 and miR-29csignificantly-3p in intrahepatic reduced by cholangiocarcinomamiR-29-3p transfection (ICC into) cells. ICC cells.(A) ExpressionGUSB was usedlevels as of an ITGB1 internal control. were significantly reduced by miR-29-3p transfection into ICC cells. GUSB was used as an internal were significantly(B) Protein reduced expression by miR levels-29 of-3p ITGB1 transfection were significantly into ICC cells. reduced GUSB by miR-29-3pwas used transfectionas an internal into ICC control. (B) Protein expression levels of ITGB1 were significantly reduced by miR-29-3p transfec- control. (Bcells) Protein (72 h afterexpression transfection). levels of GAPDH ITGB1 waswere used significantly as an internal reduced control. by miR-29-3p transfec- tiontion into into ICC ICC cells cells (72 (72 h h after after transfection). transfection). GAPDH GAPDH was was usedused asas an an internal internal control. control.

Cancers 2021, 13, 2804 8 of 21 Cancers 2021, 13, x 8 of 21

Next, to assessNext, whether to assess the whethermiR-29-family the miR-29- directlyfamily binds directlyto the ITGA6 binds target to the siteITGA6 in target site ICC cells, wein ICCperformed cells, we dual performed-luciferase dual-luciferase reporter assays. reporter Luciferase assays. activity Luciferase was signifi- activity was sig- cantly decreasednificantly following decreased co-transfection following of co-transfection miR-29c-3p and of a miR-29c-3pvector carryingand the a vector wild- carrying the type miR-29cwild-type-3p targetmiR-29c-3p site. In contrast,target site.luciferase In contrast, activity luciferase was not changed activity wasfollowing not changed co- following transfectionco-transfection of miR-29c-3p and of miR-29c-3p a vector carryingand a the vector deletion carrying-type of the the deletion-type miR-29c-3p target of the miR-29c-3p site (Figuretarget 7A). These site (Figure results7 indicatedA). These that results ITGA6 indicated was directly that ITGA6regulatedwas by directly miR-29a regulated-3p, by miR- miR-29b-3p29a-3p and miR, miR-29b-3p-29c-3p in ICCand cells.miR-29c-3p Similar resultsin ICC were cells. confirmed Similar results in the dual were-lucif- confirmed in the erase reporterdual-luciferase assays of ITGB1 reporter and assaysmiR-29c of-3pITGB1, indicatingand miR-29c-3p that ITGB1, indicating was directly that regu-ITGB1 was directly lated by miRregulated-29a-3p, miR by miR-29a-3p-29b-3p and, miRmiR-29b-3p-29c-3p inand ICCmiR-29c-3p cells (Figurein ICC7B). cells (Figure7B).

Figure 7. DirectFigure regulation 7. Direct of regulationITGA6 and ofITGB1ITGA6 expressionand ITGB1 by miRexpression-29-3p in ICC by miR-29-3p cells. (A) Dualin ICC- cells. (A) Dual- luciferase reporterluciferase assays reporter showed assays that luminescence showed that luminescenceactivity was reduced activity by was co-transfection reduced by with co-transfection with wild-type vectorwild-type (containing vector the (containing miR-29-3p thebindingmiR-29-3p site of ITGA6binding) and site m ofiR-ITGA629c-3p )precursor and miR-29c-3p in precursor in HuCCT1 andHuCCT1 RBE cells. and In RBE contrast, cells. no In change contrast, of no luminescence change of luminescence activity occurred activity after occurred co-transfec- after co-transfection tion with deletion-type vector luciferase activity with miR-29-3p transfection into ICC cells. Nor- with deletion-type vector luciferase activity with miR-29-3p transfection into ICC cells. Normalized malized data were calculated as the ratios of Renilla/firefly. (B) The luminescence activity was re- duced by codata-transfection were calculated with wild as-type the vector ratios of(containingRenilla/firefly. miR-29 (-B3p) Thebinding luminescence site of ITGB1 activity) and was reduced by miR-29c-3p precursorco-transfection in HuCCT1 with wild-typeand RBE cells. vector In (containingcontrast, no miR-29-3pchange of luminescencebinding site ofactivityITGB1 ) and miR-29c-3p occurred afterprecursor co-transfection in HuCCT1 with anddeletion RBE-type cells. vector In contrast, luciferase no activity. change of luminescence activity occurred after co-transfection with deletion-type vector luciferase activity.

Cancers 2021, 13, x 9 of 21

2.6. Effects of Knockdown of ITGA6 and ITGB1 on Cell Proliferation, Migration, and Invasion in ICC Cells To assess the oncogenic functions of ITGA6/ITGB1 in ICC cells, we conducted knock- down assays of corresponding genes using small interfering (siRNAs). First, we

Cancersevaluated2021, 13, 2804 the mRNA and protein knockdown efficiencies of siRNAs9 of that 21 targeted ITGA6 and ITGB1 in HuCCT1 and RBE cells. The expression levels of ITGA6/ITGA6 and

ITGB1/ITGB1 were2.6. Effects significantly of Knockdown of ITGA6reduced and ITGB1 by on all Cell siRNA Proliferation,-transfected Migration, and Invasion cells, in HuCCT1 and RBE (Figures 8 andICC 9, Cells Figure S3A,B). Functional assays using siRNAs demonstrated that To assess the oncogenic functions of ITGA6/ITGB1 in ICC cells, we conducted knock- knockdown of ITGA6down assays and of correspondingITGB1 suppressed genes using small cancer interfering cell RNAsmali (siRNAs).gnant phenotypes, First, especially cell migration we and evaluated invasive the mRNA abilities and protein (Figure knockdowns 10 efficiencies and of11). siRNAs Furthermore, that targeted both integrins ITGA6 and ITGB1 in HuCCT1 and RBE cells. The expression levels of ITGA6/ITGA6 and (ITGA6 and ITGB1ITGB1)/ITGB1 were were knocked significantly down reduced at by the all siRNA-transfected same time to cells, examine HuCCT1 and the tumor suppres- RBE (Figures8 and9 , Figure S3A,B). Functional assays using siRNAs demonstrated that sor effects, e.g., knockdowncell proliferation of ITGA6 and ITGB1 and suppressed migration, cancer celland malignant invasive phenotypes, abilities. especially By knocking down both integrins, cellthe migration malignant and invasive phenotypes abilities (Figures of 10cancer and 11). cells Furthermore, were both further integrins controlled (Figure (ITGA6 and ITGB1) were knocked down at the same time to examine the tumor suppressor S4). effects, e.g., cell proliferation and migration, and invasive abilities. By knocking down both integrins, the malignant phenotypes of cancer cells were further controlled (Figure S4).

Figure 8. Knockdown efficiency of siITGA6 in HuCCt1 and RBE cells. (A) Expression of ITGA6 in ICC cell lines was evaluated 72 h after transfection with si-ITGA6-1 or si-ITGA6-2. GUSB was used as Figure 8. Knockdownan internal efficiency control. (B) Expression of siITGA6 of ITGA6 in in ICCHuCCt1 cell lines was and evaluated RBE by cells. Western (A blot) analysisExpression of ITGA6 in ICC cell lines was72 evaluated h after transfection 72 with h aftersi-ITGA6-1 transfectionor si-ITGA6-2. GAPDH with wassi-ITGA6 used as internal-1 or control. si-ITGA6-2. GUSB was used as an internal control. (B) Expression of ITGA6 in ICC cell lines was evaluated by Western blot analysis 72 h after transfection with si-ITGA6-1 or si-ITGA6-2. GAPDH was used as internal con- trol.

Cancers 2021, 13, x 10 of 21 Cancers 2021, 13, 2804 10 of 21

Figure 9. Knockdown efficiency of siITGB1 in HuCCT1 and RBE cells. (A) Expression of ITGB1 in ICC cell lines was evaluated 72 h after transfection with si-ITGB1-1 or si-ITGB1-2. GUSB was used as Figure 9. .Knockdownan internal control.efficiency (B) Expression of si ofITGB1 ITGB1 in in intrahepatic HuCCT1 cholangiocarcinoma and RBE (ICC)cells. cell ( linesA) wasExpression of ITGB1 in evaluated by Western blot analysis 72 h after transfection with si-ITGB1-1 and si-ITGB1-2. GAPDH ICC cell lines waswas usedevaluated as internal control.72 h after transfection with si-ITGB1-1 or si-ITGB1-2. GUSB was used as an internal control. (B) Expression of ITGB1 in intrahepatic cholangiocarcinoma (ICC) cell lines was evaluated by Western blot analysis 72 h after transfection with si-ITGB1-1 and si-ITGB1-2. GAPDH was used as internal control.

Cancers 2021, 13, x 11 of 21

Cancers 2021, 13, 2804 11 of 21

Figure 10. Effects of knockdown of ITGA6 on cell proliferation, migration and invasion in HuCCT1 Figure 10. Effects of knockdown of ITGA6 on cell proliferation, migration and invasion in and RBE cell lines. (A) Cell proliferation was assessed using XTT assays. Data were collected 72 h HuCCT1 and RBEafter cell miRNA lines. transfection. (A) Cell proliferation (B) Cell migration was was assessed assessed using using wound XTT healing assays. assays. Data (C were) Cell col- lected 72 h after invasionmiRNA was transfection. determined 72 h(B after) Cell seeding migration microRNA-transfected was assessed cells using into chambers wound using healing Matrigel assays. (C) Cell invasioninvasion was determined assays. Error bars72 h are after represented seeding as mean micro± standardRNA-transfected error (SE). cells into chambers using Matrigel invasion assays. Error bars are represented as mean ± standard error (SE).

Cancers 2021, 13, x 12 of 21 Cancers 2021, 13, 2804 12 of 21

Figure 11. Effects of knockdown of ITGB1 on cell proliferation, migration and invasion in HuCCT1 Figure 11. Effects ofand knockdown RBE. (A) Cell of proliferation ITGB1 on was cell assessed proliferation, using XTT assays. migration Data were and collected invasion 72 h after in HuCCT1 miRNA and RBE. (A) Cell prolifetransfection.ration (B was) Cell assessed migration wasusing assessed XTT usingassays. wound Data healing were assays. collected (C) Cell 72 invasion h after was miRNA transfection.determined (B) Cell 72migration h after seeding was miRNA-transfected assessed using cells wound into chambers healing using assays. Matrigel (C invasion) Cell assays.inva- Error bars are represented as mean ± standard error (SE). sion was determined 72 h after seeding miRNA-transfected cells into chambers using Matrigel invasion assays. Error bars are represented as mean ± standard error (SE).

2.7. Identification of Transcriptional Regulators of ITGA6 and ITGB1 in ICC Cells We further investigated the involvement of transcription factors (TFs) that positively regulated ITGA6 and ITGB1 expression in ICC cells based on in silico database analysis. GeneCodis database analysis and previous studies identified a total of 18 TFs (Table 2) [25–27].

Cancers 2021, 13, 2804 13 of 21

2.7. Identification of Transcriptional Regulators of ITGA6 and ITGB1 in ICC Cells We further investigated the involvement of transcription factors (TFs) that posi- tively regulated ITGA6 and ITGB1 expression in ICC cells based on in silico database analysis. GeneCodis database analysis and previous studies identified a total of 18 TFs (Table2)[25–27].

Table 2. Transcription factors for ITGA6 or ITGB1.

Expression in Correlation Correlation Gene miR-29-3p Gene Name ICC 1 Cancer with ITGA6 with ITGB1 Reference Symbol Binding Site Tissues (p-Value) (p-Value) (p-Value) SP1 <0.01 <0.01 <0.01 + [24,25] cAMP responsive element CREB1 <0.01 <0.01 <0.01 − [25] binding protein 1 MAX MYC associated factor X <0.01 <0.01 0.011 − [25] four and a half LIM FHL2 <0.01 <0.01 <0.01 − [25] domains 2 regulatory factor X, 1 RFX1 (influences HLA class II <0.01 <0.01 <0.01 − [25] expression) hypoxia inducible factor 1, alpha subunit (basic HIF1A <0.01 <0.01 0.019 − [25] helix-loop-helix transcription factor) transcription factor AP-2 TFAP2A alpha (activating enhancer <0.01 N.S <0.01 − [25] binding protein 2 alpha) CUX1 cut-like 1 <0.01 <0.01 N.S − [25] upstream transcription USF1 <0.01 N.S <0.01 − [25] factor 1 transcription factor AP-2 gamma (activating TFAP2C <0.01 N.S N.S + [25] enhancer binding protein 2 gamma) FOXM1 forkhead box M1 <0.01 N.S N.S − [26] SPI1 Spi-1 proto-oncogene <0.05 N.S N.S − [25] MAX interactor 1, MXI1 N.S 2 0.011 N.S + [25] dimerization protein PAX6 paired box 6 N.S <0.01 <0.01 − [25] ESR1 estrogen receptor 1 N.S 0.015 <0.01 − [25] MEF2A myocyte enhancer factor 2A N.S <0.01 0.038 − [25] HOXD3 homeobox D3 N.S 0.012 N.S − [25] proto-, MYC N.S N.S N.S − [25] bHLH transcription factor 1 Intrahepatic cholangiocarcinoma, 2 No significant difference.

Among these TFs, expression of 12 TFs was upregulated in cholangiocarcinoma tissues (Figure S5). Moreover, expressions of six TF genes (SP1, CREB1, MAX, FHL2, RFX1, and HIF1A) was positively correlated with the expression of ITGA6 and ITGB1 in cholangiocarcinoma tissues (Figure S6). These TFs might be involved with the enhanced expression of ITGA6 and ITGB1 in ICC cells. Thus, further investigation was conducted.

2.8. Regulation of SP1 by miR-29a-3p, miR-29b-3p and miR-29c-3p in ICC Cells Expression levels of SP1/SP1 in clinical specimens and positive correlations of SP1/ITGA6 and SP1/ITGB1 are shown in Figure 12 and Figure S7. Cancers 2021, 13, x 14 of 21 Cancers 2021, 13, 2804 14 of 21

Figure 12. Expression of SP1 in intrahepatic cholangiocarcinoma (ICC) clinical specimens. (A) Ex- Figure 12. Expressionpression levels of of SP1SP1 in in ICC intrahepatic tissues, normal cholangiocarcinoma tissues obtained from TCGA-CHOL (ICC) clinical through specimens. the GEPIA2 (A) Ex- pression levelsplatform. of SP1 (B )in Immunostaining ICC tissues, ofnormal SP1 in ICC tissues clinical obtained specimen. from Areas TCGA in the boxed-CHOL region through above the GE- PIA2 platform.are shown(B) Immunostaining magnified below (original of SP1 magnification in ICC clinical above: specimen.×40, below: ×Areas200). ( Cin) Correlationthe boxed of region above are shownexpression magnified of SP1/ITGA6 belowand (originalSP11/ITGB1 magnificationin cholangiocarcinoma above: tissues ×40, through below: GEPIA2 ×200). platform. (C) Correla- tion of expressionPearson’s of rank SP1 tests/ITGA6 between and the S expressionP11/ITGB1 levels in of cholangiocarcinomaSP1/ITGA6 and SP1/ITGB1 tissues. through GEPIA2 platform. PearsonInterestingly,’s rank testsmiR-29-3p betweenbinding the expression sites were detected levels in of the SP1/ITGA6 30-UTR region and of SP1 SP1/ITGB1mRNA. . To evaluate whether direct regulation of SP1 by miR-29-3p-family in ICC cells occurred, we Interestingly,performed miR dual-luciferase-29-3p binding reporter assays. sites Luciferase were detected activity was in significantly the 3′-UTR decreased region of SP1 following co-transfection of miR-29c-3p and a vector carrying the wild-type miR-29c-3p mRNA. Totarget evaluate site. In whether contrast, luciferase direct activityregulation was not of changed SP1 by following miR-29 co-transfection-3p-family in of miR-ICC cells oc- curred, we 29c-3pperformedand a vector dual carrying-luciferase the deletion-type reporter assays. of the miR-29c-3p Luciferasetar-get activity site (Figure was 13 significantlyA). decreased These following results coindicated-transfection that SP1 was of directlymiR-29c regulated-3p and by a the vectormiR-29-3p- carryingfamily in the ICC wild-type cells. Furthermore, both mRNA and protein expression levels of SP1/SP1 were reduced miR-29c-3pby target ectopic site. expression In contrast, of miR-29a-3p luciferase, miR-29b-3p activityand miR-29c-3p was notin changed HuCCT1 and following RBE cells co-trans- fection of miR(Figure-29c 13-B,C,3p and Figure a S8).vector These carry data indicateding the thatdeletion downregulation-type of of themiR-29a-3p miR-29c, miR-29b--3p tar-get site (Figure 13A).3p and ThesemiR-29c-3p resultsenhanced indicatedSP1 expression, that SP1 and was this eventdirectly accelerated regulated the overexpression by the miR-29-3p- of ITGA6 and ITGB1 in ICC cells. family in ICC cells. Furthermore, both mRNA and protein expression levels of SP1/SP1 were reduced by ectopic expression of miR-29a-3p, miR-29b-3p and miR-29c-3p in HuCCT1 and RBE cells (Figure 13B,C, Figure S8). These data indicated that downregulation of miR- 29a-3p, miR-29b-3p and miR-29c-3p enhanced SP1 expression, and this event accelerated the overexpression of ITGA6 and ITGB1 in ICC cells.

Cancers 2021Cancers, 13, x 2021, 13, 2804 15 of 21 15 of 21

Figure 13.Figure Direct 13.regulationDirect regulation of SP1 byof miRSP1-29by-familymiR-29 in-family intrahepatic in intrahepatic cholangiocarcinoma cholangiocarcinoma (ICC) (ICC) cells. cells. (A) The(A) Theluminescence luminescence activity activity was reduced was reduced by co by-transfection co-transfection with wild with-type wild-type vector vector(con- (containing taining miRmiR-29-3p-29-3p bindingbinding site site of ofSP1SP1) a)nd and m miRiR-29c-3p-29c-3p precursorprecursor in inHuCCT1 HuCCT1 and and RBE RBE cells. cells. In Incon- contrast, no trast, no change of luminescence activity occurred after co-transfection with deletion-type vector change of luminescence activity occurred after co-transfection with deletion-type vector luciferase luciferase activity. (B) Expression levels of SP1 were significantly reduced by miR-29a-3p, miR-29b- activity. (B) Expression levels of SP1 were significantly reduced by miR-29a-3p, miR-29b-3p and 3p and miR-29c-3p transfection into ICC cells. GUSB was used as an internal control. (C) Protein expressionmiR-29c-3p levels of SP1transfection were significantly into ICC cells. reducedGUSB bywas miR used-29a-3p as, anmiR internal-29b-3p, control. and miR (C-29c) Protein-3p expression transfectionlevels into of ICC SP1 cells were (72 significantly h after transfection). reduced by GAPDHmiR-29a-3p was, miR-29b-3pused as an ,internal and miR-29c-3p control. transfection into ICC cells (72 h after transfection). GAPDH was used as an internal control. 3. Discussion 3. Discussion Among primary liver cancers, the incidence of ICC (15% of liver cancers) is second Among primary liver cancers, the incidence of ICC (15% of liver cancers) is second only to hepatocellular carcinoma (HCC), and the global incidence of ICC has increased only to hepatocellular carcinoma (HCC), and the global incidence of ICC has increased over the overpast thethree past decades three decades[4,8]. Patients [4,8]. Patientswith ICC with are ICCadvanced are advanced at the time at theof initial time of initial diagnosisdiagnosis because becausethere are there no effective are no effectiveearly diagnostic early diagnostic markers markers[10,11]. Unfortunately, [10,11]. Unfortunately, there is no effective treatment for inoperable cases [1,2]. Many studies have attempted to

Cancers 2021, 13, 2804 16 of 21

there is no effective treatment for inoperable cases [1,2]. Many studies have attempted to elucidate the molecular mechanisms underlying ICC [28,29]. Recently, genome-based analyses, including ncRNAs and miRNAs, have been vigorously conducted [12,15]. Recent study demonstrated that lncRNA-PAICC was overexpressed in ICC tissues, and expression of PAICC enhanced proliferation and invasion of ICC cells [30]. Importantly, oncogenic PAICC functioned as competitive endogenous RNA (ceRNA) that adsorbed tumor-suppressive miRNAs, miR-141-3p and miR-27a-3p, in cancer cells. Silencing of miR-141-3p and miR-27a-3p induced activation of the Hippo pathway in ICC cells [30]. Similarly, overexpression of lncRNA-UCA1 sponged miR-122, and this event enhanced proliferation and invasion of ICC cells [31]. Aberrant expression of lncRNAs and silencing tumor-suppressive miRNAs was closely associated with ICC oncogenesis [31]. In this study, we focused on the miR-29-3p-family because previous ICC signature and our miRNA signatures in several cancers revealed that the miR-29-3p-family was signifi- cantly downregulated in cancer tissues, suggesting that its members acted as key regulators, negatively controlling pivotal oncogenic targets and pathways [22,23,32]. Downregulation and tumor-suppressive functions of the miR-29-3p-family have been reported in several types of cancers [33,34]. Thus far, no detailed functional and targeted analysis of the miR- 29-3p-family in ICC cells has been performed. In HCC cells, several studies showed that the miR-29-3p-family acted as tumor-suppressive miRNAs via negative control of several oncogenic targets, e.g., IGF2BP1, RPS15A and LOXL2 [35–37]. In cholangiocarcinoma, the serum concentration of miR-29b-3p was elevated compared to healthy controls and patients with primary sclerosing cholangitis [38]. The molecular mechanism increasing the level of miR-29b-3p in serum is not understood. It may be released from cancerous tissue. Our next interest was the search for oncogenic molecular networks in ICC cells that were controlled by the tumor-suppressive miR-29-3p-family. Our in silico analysis identi- fied 20 pathways, including “focal adhesion”, “ECM-receptor”, “endocytosis”, “PI3K-Akt signaling” and “Hippo signaling”. Analysis of the genes contained in these molecular pathways revealed that ITGA6 and ITGB1 were involved in multiple pathways. Integrins are cell surface proteins that interact with the extracellular matrix (ECM), modulating cell characteristics, such as cell shape, proliferation, and motility [39]. We also analyzed expres- sion levels of other ITGs that form heterodimers with ITGB1 in ICC clinical specimens using GEPIA2 database (Figure S9). The expression of some ITGs, e.g., ITGAV, ITGA2, ITGA3, ITGA5, and ITGA7 were upregulated in ICC tissues. Additionally, upregulation of ITGB4 (form heterodimers with ITGA6) was detected in ICC tissues. It was suggested that not only the ITGA6/ITGB1 dimer but also the ITGs that form dimers with each of these may play an important role in ICC. Additionally, liver fibrosis is characterized by an abnormal accumu- lation of extracellular matrix (ECM) and is a common feature of chronic liver damage. The hepatic stellate cell (HSC) is involved in liver fibrosis, which is the formation of scar tissue in response to liver damage. Previous study showed that miR-29b was downregulated during HSC activation [40]. Ectopic expression of miR-29b inhibited several genes involved in HSC activation, e.g., COL1A1, COL1A2, DDR2, FN1, and ITGB1 [41]. A vast number of studies have shown that aberrant expression of ECM/integrin-mediated oncogenic signaling enhanced cancer cell malignant transformation, e.g., invasion, migration, the epithelial mesenchymal transition, metastasis and drug resistance [17,32]. Previous studies showed that several miRNAs (e.g., miR-29, miR-124-3p, miR-150, miR-218 and miR-199) directly controlled expression of ITGA6 and ITGB1 in several cancers. Ectopic expression of these miRNAs attenuated cancer cell migration, invasion and metas- tasis [17,32,42,43]. Our previous study of pancreatic ductal adenocarcinoma (PDAC) cells showed that aberrant expression of ITGA3 and ITGB1 (genes for two integrins that form homodimers) was significantly associated with poor prognosis of patients with PDAC [17]. Expression of ITGA3 and ITGB1 was directly regulated by tumor-suppressive miR-124- 3p. Moreover, overexpression of miR-124-3p inhibited ITGA3/ITGB1-mediated oncogenic signaling, and attenuated cancer cell migration and invasive abilities [17]. In head and neck squamous cell carcinoma cells, ectopic expression of the miR-29-3p-family inhib- Cancers 2021, 13, 2804 17 of 21

ited ITGB1-mediated oncogenic signaling [22]. At this time, no molecular-targeted drugs targeting the integrin-family are used for ICC. Searching for specific integrin-mediated molecular pathways in ICC will help develop new therapeutic agents. Integrin inhibitors have been clinically applied in autoimmune diseases, thrombosis, and several cancers, but with poor results, especially for malignant tumors [44–46]. This may be due to two aspects of their function: (1) integrins require structural changes due to their inside-out signals, and (2) multiple integrin units’ function in cancer cells [47]. Therefore, future development of therapy must control both integrin-related cascades as well as the presence of currently expressed ligands. In this study, we also investigated the involvement of transcription factors (TFs) that positively regulated ITGA6 and ITGB1 expression in ICC cells. Our in silico analysis revealed that a total of six TFs (SP1, CREB1, MAX, FHL2, RFX1 and HIF1A) were upregulated in cholangiocarcinoma tissues. We also found a positive correlation between ITGA6 and ITGB1 expression. Notably, among these TFs, SP1 has a miR-29-family binding site in the 30-UTR region. Several studies showed that miR-29b and miR-29c directly regulated SP1 [48,49]. Our present data also showed inhibited expression of SP1 by ectopic expression of the miR-29-3p-family in ICC cells.

4. Conclusions These findings indicated that downregulation of the miR-29-3p-family caused upregu- lation of ITGA6/ITGB1, and their transcriptional modulator SP1 in ICC cells. Overexpres- sion of SP1 induces the expression of several genes involved in malignant transformation of cancer cells [50]. Therefore, reducing the expression of SP1 should permit control of can- cers. A vast number of studies indicated that small-molecule drugs and natural products (bortezomib, retinoids, aspirin, metformin, and curcumin) downregulated SP1 expression in cancer cells [51,52]. It may be possible to control ICC by combining it with existing anti-cancer drugs.

5. Materials and Methods 5.1. ICC Cell Lines and Cell Culture We used 2 human ICC cell lines: HuCCT1 and RBE, both purchased from the RIKEN Cell Bank (Tsukuba, Ibaraki, Japan). The cell lines were maintained in RPMI 1640 medium supplemented with 10% fetal bovine serum in a humidified atmosphere of 5% CO2 and 95% air at 37 ◦C. These cell lines were transferred from the RIKEN Bank in 2020 and were used in the present analysis.

5.2. RNA Extraction and Quantitative Real-Time Reverse Transcription Polymerase Chain Reaction (qRT-PCR) The methods for total RNA extraction from clinical specimens and cell lines and for qRT-PCR have been described previously [16,17]. GUSB was used as the normalized control. The reagents used in the analysis are listed in Table S8.

5.3. Transfection of miRNAs, siRNAs, and Vectors into ICC Cells The transfection procedures of miRNAs, siRNAs, and plasmid vectors into ICC cells were described previously [16,17]. The reagents used in this study are listed in Table S8.

5.4. Functional Assays in ICC Cells (Cell Proliferation, Migration and Invasion) The procedures for conducting the functional assays in cancer cells (e.g., proliferation, migration and invasion) have been described previously [16,17]. In brief, for proliferation assays, cells were transferred to 96-well plates. HuCCT1 and RBE cells were plated at 2.4 × 103 cells per well. Cell proliferation was evaluated using the XTT assays 72 h after the transfection procedure. For migration and invasion assays, HuCCT1 cells or RBE cells at 1.2 × 105 were transfected in 6-well plates. After 72 h, HuCCT1 or RBE cells were added into each chamber at 2.5 × 105 per well. After 48 h, the cells on the lower surface were counted for analysis. All experiments were performed in triplicate. Cancers 2021, 13, 2804 18 of 21

5.5. Expression Analysis of miRNA, Target Genes and Transcriptional Factors Using Public Databases To search for downregulated miRNAs in ICC clinical specimens, we obtained expres- sion data from the GEO database (GSE53870). The expression data of 63 patients with ICC and 9 normal intrahepatic bile ducts are stored in the dataset. We imported TCGA- CHOL RNA seq data into Subio platform (https://www.subioplatform.com/ (accessed on 29 October 2020)) and analyzed gene expression by using the “compare 2 groups” tool of Subio platform. The expression level of each gene was extracted and analyzed from the TCGA-CHOL database through the GEPIA2 platform (http://gepia2.cancer-pku.cn/ #index (accessed on 10 February 2021)).

5.6. Plasmid Construction and Dual-Luciferase Reporter Assays Plasmid vectors containing wild type and deletion type sequences of miR-29-3p bind- ing site in 30-UTR of ITGA6 or ITGB1 or SP1 were prepared. The predicted binding site se- quences were obtained from the TargetScanHuman database (https://www.targetscan.org/, release 7.2 (accessed on 1 December 2020)). Cells were co-transfected with miR-29c-3p and the plasmid vectors for 36 h. The detailed procedures for dual-luciferase reporter assay were described in our previous studies [16,17].

5.7. Western Blotting and Immunohistochemistry Cell lysates were prepared in RIPA buffer 72 h after transfection. Polyacrylamide gel electrophoresis was performed using 30 µg of protein lysate, and the protein lysates were transferred onto PVDF membranes (Thermo Fisher Scientific, Waltham, MA, USA). Membranes were blocked with skim milk and incubated with the indicated primary antibodies overnight at 4 ◦C. The antibodies used in this study are shown in Table S8. GAPDH was used as the internal control. The quantitation of the protein band intensity was analyzed by ImageJ software (NIH, Bethesda, MD, USA). We assessed expression of ITGA6, ITGB1 and SP1 proteins by immunohistochemistry. The procedure for immunostaining was described previously [16,17]. Clinical sample were obtained from a patient following resection at Kagoshima University Hospital at March 2017. The patient was diagnosed with mass-forming type ICC, T3N0M0 stage IIIA according to the 8th edition of the Union for International Cancer Control (UICC).

5.8. Statistical Analyses All statistical analyses were performed using JMP Pro 15 (SAS Institute Inc., Cary, NC, USA). Differences between 2 groups were evaluated using Mann–Whitney U tests. For multiple groups, one-way analysis of variance and Dunnett’s test were applied. Correlation coefficients were evaluated using Pearson’s test. All data are presented as the mean ± SD. p-values less than 0.05 were considered significant.

Supplementary Materials: The following are available online at https://www.mdpi.com/article/ 10.3390/cancers13112804/s1, Figure S1: Immunostaining of ITGA6 and ITGB1 stained sections in normal liver tissues of 2 ICC clinical specimens. Arrowheads point to normal cholangiocytes, Figure S2A: Downregulation of ITGA6 by miR-29-3p-family transfection in ICC cells, Figure S2B: Downregulation of ITGB1 by miR-29-3p-family transfection in ICC cells, Figure S3A: Downregulation of ITGA6 by siRNA transfection in ICC cells, Figure S3B: Downregulation of ITGB1 by siRNA trans- fection in ICC cells, Figure S4: Effects of knockdown of both ITGA6 and ITGB1 on cell proliferation, migration and invasion in HuCCT1 and RBE, Figure S5: Expression of 11 upregulated transcrip- tion factors in cholangiocarcinoma tissues except SP1, Figure S6A: Correlation between ITGA6 and transcription factors in cholangiocarcinoma tissues. Figure S6B: Correlation between ITGB1 and transcription factors in cholangiocarcinoma tissues, Figure S7 (Related to Figure 12): Immunos- taining of SP1 and H&E stained sections in 2 ICC clinical specimens Arrow heads point to normal cholangiocytes, Figure S8: Downregulation of SP1 by miR-29-3p-family transfection in ICC cells, Figure S9: Expression levels of ITGs forming dimer with ITGA6 and ITGB1 in ICC tissues and normal Cancers 2021, 13, 2804 19 of 21

tissues obtained from TCGA-CHOL based on the GEPIA2 platform, Table S1: Candidate target genes regulated by miR-29-3p-family, Table S2: Putative target genes of intrahepatic cholangiocarcinoma for miR-29-3p-family in focal adhesion pathway, Table S3: Putative target genes of intrahepatic cholangiocarcinoma for miR-29-3p-family in small cell lung cancer, Table S4: Putative target genes of intrahepatic cholangiocarcinoma for miR-29-3p-family in pathways in cancer, Table S5: Putative target genes of intrahepatic cholangiocarcinoma for miR-29-3p-family in Human papillomavirus infection, Table S6: Putative target genes of intrahepatic cholangiocarcinoma for miR-29-3p-family in ECM-receptor interaction, Table S7: Putative target genes of intrahepatic cholangiocarcinoma for miR-29-3p-family in PI3K-Akt signaling pathway, Table S8: Reagents used in this study. Author Contributions: Conceptualization, Y.H. and N.S.; data curation, Y.H., T.T. and S.M.; formal analysis, Y.H. and T.T.; funding acquisition, N.S., T.I., M.W., K.T. and T.O.; investigation, Y.H., Y.K. and Y.M.; methodology, N.S. and T.O.; project administration, N.S.; resources, T.I., H.K. and T.O.; supervision, T.O.; validation, T.T. and S.M.; visualization, Y.H. and N.S.; writing—original draft preparation, Y.H.; writing—review and editing, N.S., S.A. and H.K.; All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by JSPS KAKENHI grant (No. 18K08687, No.18K16422, No.19K03085, No.19K09077, No.20H03753). Institutional Review Board Statement: The study was conducted according to the guidelines of the Declaration of Helsinki and approved by Ethics Committee of Kagoshima University (approval no. 160038 28-65, date of approval: 4 September 2016). Informed Consent Statement: Written prior informed consent and approval were obtained from all patients who were diagnosed with ICC and resected at Kagoshima University Hospital. Data Availability Statement: The data presented in this study are available on request from the corresponding author. Conflicts of Interest: The authors declare no conflict of interest.

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