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Aberrantly expressed microRNAs (miRNAs) disrupt intracellular RNA networks and contribute to malignant transformation of cancer cells. Utilizing the latest RNA sequencing technology, we newly created the miRNA expression signature of esophageal squamous cell carcinoma (ESCC). A total of 47 miRNAs were downregulated in ESCC tissues, and these miRNAs were candidates for antitumor miRNAs in ESCC cells. Analysis of the signature revealed that several passenger strands of miRNAs were significantly downregulated in ESCC, e.g., miR-28-3p, miR-30a-3p, miR-30c-3p, miR-133a-3p, miR-139-3p, miR-143-5p, and miR-145-3p. Recent studies indicate that some passenger strands of miRNAs closely involved in cancer pathogenesis. In this study, we focused on both strands of pre-miR-143, and investigated their antitumor roles and target oncogenes in ESCC. Ectopic expression of miR-143-5p and miR-143-3p significantly attenuated malignant phenotypes (e.g., proliferation, migration, and invasive abilities) in ESCC cell lines. We revealed that six genes (HN1, HMGA2, NETO2, STMN1, TCF3, and MET) were putative targets of miR-143-5p regulation, and one gene (KRT80) was a putative target of miR-143-3p regulation in ESCC cells. Our ESCC miRNA signature and analysis strategy provided important insights into the molecular pathogenesis of ESCC.

Simple Summary Even today, there are no effective targeted therapies for intrahepatic cholangiocarcinoma (ICC) patients. Clarifying the molecular pathogenesis of ICC will contribute to the development of treatment strategies for this disease. In this study, we searched for the role of the miR-29-3p-family and its association with oncogenic pathway. Interestingly, aberrant expression of ITGA6 and ITGB1 was directly regulated by the miR-29-3p-family which are involved in multiple oncogenic pathways in ICC, and enhanced malignant transformation of ICC cells. Furthermore, SP1 which is a transcriptional activator of ITGA6/ITGB1, is regulated by the miR-29-3p-family. These molecules may be novel therapeutic targets for ICC. Abstract The aggressive nature of intrahepatic cholangiocarcinoma (ICC) renders it a particularly lethal solid tumor. Searching for therapeutic targets for ICC is an essential challenge in the development of an effective treatment strategy. Our previous studies showed that the miR-29-3p-family members (miR-29a-3p, miR-29b-3p and miR-29c-3p) are key tumor-suppressive microRNAs that control many oncogenic genes/pathways in several cancers. In this study, we searched for therapeutic targets for ICC using the miR-29-3p-family as a starting point. Our functional studies of cell proliferation, migration and invasion confirmed that the miR-29-3p-family act as tumor-suppressors in ICC 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 analysis focused on the genes for integrin-α6 (ITGA6) and integrin-β1 (ITGB1), which are involved in multiple pathways. Overexpression of ITGA6 and ITGB1 enhanced malignant transformation of ICC cells. Both ITGA6 and ITGB1 were directly regulated by the miR-29-3p-family in ICC cells. 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.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive types of cancer, and its prognosis is abysmal, only 25% of patients survive one year, and 5% live for five years. MicroRNA (miRNA) signature analysis of PDAC revealed that both strands of pre-miR-30c (miR-30c-5p, guide strand, miR-30c-2-3p, passenger strand) were significantly downregulated, suggesting they function as tumor-suppressors in PDAC cells. Ectopic expression assays demonstrated that these miRNAs attenuated the aggressiveness of PDAC cells, e.g., cell proliferation, migration, and invasiveness. Through a combination of in silico analyses and gene expression data, we identified 216 genes as putative oncogenic targets of miR-30c-5p and miR-30c-2-3p regulation in PDAC cells. Among these, the expression of 18 genes significantly predicted the 5-year survival rates of PDAC patients (p &lt, 0.01). Importantly, the expression levels of 10 genes (YWHAZ, F3, TMOD3, NFE2L3, ENDOD1, ITGA3, RRAS, PRSS23, TOP2A, and LRRFIP1) were found to be independent prognostic factors for patient survival (p &lt, 0.01). We focused on TOP2A (DNA Topoisomerase II Alpha) and investigated its potential as a therapeutic target for PDAC. The overexpression of TOP2A and its transcriptional activators (SP1 and HMGB2) was detected in PDAC clinical specimens. Moreover, the knockdown of TOP2A enhanced the sensitivity of PDAC cells to anticancer drugs. Our analyses of the PDAC miRNA signature and tumor-suppressive miRNAs provide important insights into the molecular pathogenesis of PDAC.

RNA-sequencing-based microRNA (miRNA) expression signatures have revealed that miR-148a-5p (the passenger strand of the miR-148a-duplex) is downregulated in various kinds of cancer tissues. Analysis of The Cancer Genome Atlas (TCGA) database showed that low expression of miR-148a-5p was predictive of a lower survival rate (p = 0.041) in patients with gastric cancer (GC). Downregulation of miR-148a-5p was confirmed in GC clinical specimens, and its ectopic expression attenuated GC cell proliferation. Our search for miRNA target genes identified a total of 18 oncogenic targets of miR-148a-5p in GC cells. Among these targets, high expression levels of six genes (THBS2, P4HA3, SERPINH1, CDH11, BCAT1, and KCNG3) were closely associated with a poor prognosis (10-year survival rates) in GC patients (p

Triple-negative breast cancer (TNBC) is an aggressive type of cancer associated with a poor prognosis. Identification of novel therapeutic targets in TNBC is urgently needed. Here, we investigated the microRNA (miRNA) expression signature of TNBC using clinical specimens. In total, 104 miRNAs (56 upregulated and 48 downregulated) were significantly dysregulated in TNBC tissues; miR-204-5p showed the most dramatic downregulation. We then examined the antitumor roles of miR-204-5p in breast cancer (BC) cells. Notably, cancer cell migration and invasion were significantly reduced by ectopic expression of miR-204-5p in BC cells. Genome-wide gene expression analysis and in silico database search revealed that 32 genes were putative miR-204-5p targets. High expression of AP1S3, RACGAP1, ELOVL6, and LRRC59 was significantly associated with poor prognosis in patients with BC, and adaptor-related protein complex 1 sigma 3 subunit (AP1S3) was directly regulated by miR-204-5p, as demonstrated by luciferase reporter assays. AP1S3 overexpression was detected in TNBC clinical specimens and enhanced cancer cell aggressiveness. We further analyzed downstream RNA networks regulated by AP1S3 in BC cells. Overall, this miRNA signature is expected to be an effective tool for identification of miRNA-mediated molecular mechanisms of TNBC pathogenesis.

MicroRNAs (miRNAs) are unique in that a single miRNA molecule regulates a vast number of RNA transcripts. Thus, aberrantly expressed miRNAs disrupt tightly controlled RNA networks in cancer cells. Our functional screening showed that expression of miR-124-3p was downregulated in pancreatic ductal adenocarcinoma (PDAC) tissues. Here, we aimed to investigate the anti-tumor roles of miR-124-3p in PDAC cells and to identify miR-124-3p-mediated oncogenic signaling in this disease. Ectopic expression of miR-124-3p inhibited cancer cell migration and invasion in PDAC cells. Moreover, restoration of miR-124-3p suppressed oncogenic signaling, as demonstrated by reduced phosphorylation of focal adhesion kinase, AKT, and extracellular signal-regulated kinase, in PDAC cells. Our in silico database analyses and luciferase reporter assays showed that two cell-surface matrix receptors, integrin α3 (ITGA3) and integrin β1 (ITGB1), were directly regulated by miR-124-3p in PDAC cells. Overexpression of ITGA3 and ITGB1 was confirmed in PDAC clinical specimens. Interestingly, a large number of cohort analyses from TCGA database showed that high expressions of ITGA3 and ITGB1 were significantly associated with poor prognosis of patients with PDAC. Knockdown of ITGA3 and ITGB1 by siRNAs markedly suppressed the migration and invasion abilities of PDAC cells. Moreover, downstream oncogenic signaling was inhibited by ectopic expression of miR-124-3p or knockdown of the two integrins. The discovery of anti-tumor miRNAs and miRNA-mediated oncogenic signaling may provide novel therapeutic targets for the treatment of PDAC.

Analysis of our original microRNA (miRNA) expression signature of patients with advanced renal cell carcinoma (RCC) showed that microRNA-10a-5p (miR-10a-5p) was significantly downregulated in RCC specimens. The aims of the present study were to investigate the antitumor roles of miR-10a-5p and the novel cancer networks regulated by this miRNA in RCC cells. Downregulation of miR-10a-5p was confirmed in RCC tissues and RCC tissues from patients treated with tyrosine kinase inhibitors (TKI). Ectopic expression of miR-10a-5p in RCC cell lines (786-O and A498 cells) inhibited cancer cell migration and invasion. Spindle and kinetochore-associated protein 1 (SKA1) was identified as an antitumor miR-10a-5p target by genome-based approaches, and direct regulation was validated by luciferase reporter assays. Knockdown of SKA1 inhibited cancer cell migration and invasion in RCC cells. Overexpression of SKA1 was observed in RCC tissues and TKI-treated RCC tissues. Moreover, analysis of The Cancer Genome Atlas database demonstrated that low expression of miR-10a-5p and high expression of SKA1 were significantly associated with overall survival in patients with RCC. These findings showed that downregulation of miR-10a-5p and overexpression of the SKA1 axis were highly involved in RCC pathogenesis and resistance to TKI treatment in RCC.

// Keiichi Yonemori 1 , Naohiko Seki 2 , Tetsuya Idichi 1 , Hiroshi Kurahara 1 , Yusaku Osako 1 , Keiichi Koshizuka 2 , Takayuki Arai 2 , Atsushi Okato 2 , Yoshiaki Kita 1 , Takaaki Arigami 1 , Yuko Mataki 1 , Yuko Kijima 1 , Kosei Maemura 1 and Shoji Natsugoe 1 1 Department of Digestive Surgery, Breast and Thyroid Surgery, Graduate School of Medical Sciences, Kagoshima University, Sakuragaoka, Kagoshima 890-8520, Japan 2 Department of Functional Genomics, Chiba University Graduate School of Medicine, Chuo-ku, Chiba 260-8670, Japan Correspondence to: Naohiko Seki, email: naoseki@faculty.chiba-u.jp Keywords: pancreatic ductal adenocarcinoma, microRNA, expression signature, miR-216b-3p , FOXQ1 Received: February 07, 2017 Accepted: June 26, 2017 Published: July 26, 2017 ABSTRACT We analysed the RNA sequence-based microRNA (miRNA) signature of pancreatic ductal adenocarcinoma (PDAC). Aberrantly expressed miRNAs were successfully identified in this signature. Using the PDAC signature, we focused on 4 clustered miRNAs, miR-216a-5p , miR-216a-3p , miR-216b-5p and miR-216b-3p on human chromosome 2p16.1. All members of the miR-216 cluster were significantly reduced in PDAC specimens. Ectopic expression of these miRNAs suppressed cancer cell aggressiveness, suggesting miR-216 cluster as anti-tumour miRNAs in PDAC cells. The impact of miR-216b-3p (passenger strand of pre- miR-216b ) on cancer cells is still ambiguous. Forkhead box Q1 ( FOXQ1 ) was directly regulated by miR-216b-3p and overexpression of FOXQ1 was confirmed in clinical specimens. High expression of FOXQ1 predicted a shorter survival of patients with PDAC by Kaplan–Meier analysis. Loss-of-function assays showed that cancer cell migration and invasion activities were significantly reduced by si FOXQ1 transfectants. We investigated pathways downstream from FOXQ1 by using genome-wide gene expression analysis. Identification of the miR-216-3p/FOXQ1 -mediated network in PDAC should enhance understanding of PDAC aggressiveness at the molecular level.

// Tetsuya Idichi 1 , Naohiko Seki 2 , Hiroshi Kurahara 1 , Keiichi Yonemori 1 , Yusaku Osako 1 , Takayuki Arai 2 , Atsushi Okato 2 , Yoshiaki Kita 1 , Takaaki Arigami 1 , Yuko Mataki 1 , Yuko Kijima 1 , Kosei Maemura 1 and Shoji Natsugoe 1 1 Department of Digestive Surgery, Breast and Thyroid Surgery, Graduate School of Medical Sciences, Kagoshima University, Kagoshima, Japan 2 Department of Functional Genomics, Chiba University Graduate School of Medicine, Chiba, Japan Correspondence to: Naohiko Seki, email: naoseki@faculty.chiba-u.jp Keywords: microRNA, miR-217 , pancreatic ductal adenocarcinoma, ANLN , tumor-suppressor Received: January 11, 2017 Accepted: May 08, 2017 Published: May 29, 2017 ABSTRACT Analysis of our microRNA (miRNA) expression signature of pancreatic ductal adenocarcinoma (PDAC) revealed that microRNA-217 ( miR-217 ) was significantly reduced in cancer tissues. The aim of this study was to investigate the antitumor roles of miR-217 in PDAC cells and to identify miR-217 -mediated molecular pathways involved in PDAC aggressiveness. The expression levels of miR-217 were significantly reduced in PDAC clinical specimens. Ectopic expression of miR-217 significantly suppressed cancer cell migration and invasion. Transcription of actin-binding protein Anillin (coded by ANLN ) was detected by our in silico and gene expression analyses. Moreover, luciferase reporter assays showed that ANLN was a direct target of miR-217 in PDAC cells. Overexpression of ANLN was detected in PDAC clinical specimens by real-time PCR methods and immunohistochemistry. Interestingly, Kaplan–Meier survival curves showed that high expression of ANLN predicted shorter survival in patients with PDAC by TCGA database analysis. Silencing ANLN expression markedly inhibited cancer cell migration and invasion capabilities of PDAC cell lines. We further investigated ANLN -mediated downstream pathways in PDAC cells. “Focal adhesion” and “Regulation of actin binding protein” were identified as ANLN -modulated downstream pathways in PDAC cells. Identification of antitumor miR-217 / ANLN -mediated PDAC pathways will provide new insights into the potential mechanisms underlying the aggressive course of PDAC.

Aberrantly expressed microRNA (miRNA) are known to disrupt intracellular RNA networks in cancer cells. Exploring miRNA‐dependent molecular networks is a major challenge in cancer research. In this study, we performed RNA‐sequencing of breast cancer (BrCa) clinical specimens to identify tumor‐suppressive miRNA in BrCa. In total, 64 miRNA were identified as candidate tumor‐suppressive miRNA in BrCa cells. Analysis of our BrCa signature revealed that several miRNA duplexes (guide strand/passenger strand) derived from pre‐miRNA were downregulated in BrCa tissues (e.g. miR‐99a‐5p/‐3p, miR‐101‐5p/‐3p, miR‐126‐5p/‐3p, miR‐143‐5p/‐3p, and miR‐144‐5p/‐3p). Among these miRNA, we focused on miR‐101‐5p, the passenger strand of pre‐miR‐101, and investigated its tumor‐suppressive roles and oncogenic targets in BrCa cells. Low expression of miR‐101‐5p predicted poor prognosis in patients with BrCa (overall survival rate: P = 0.0316). Ectopic expression of miR‐101‐5p attenuated aggressive phenotypes, e.g. proliferation, migration, and invasion, in BrCa cells. Finally, we identified seven putative oncogenic genes (i.e. High Mobility Group Box 3, Epithelial splicing regulatory protein 1, GINS complex subunit 1 (GINS1), Tumor Protein D52, Serine/Arginine‐Rich Splicing Factor Kinase 1, Vang‐like protein 1, and Mago Homolog B) regulated by miR‐101‐5p in BrCa cells. The expression of these target genes was associated with the molecular pathogenesis of BrCa. Furthermore, we explored the oncogenic roles of GINS1, whose function had not been previously elucidated, in BrCa cells. Aberrant expression of GINS1 mRNA and protein was observed in BrCa clinical specimens, and high GINS1 expression significantly predicted poor prognosis in patients with BrCa (overall survival rate: P = 0.0126). Knockdown of GINS1 inhibited the malignant features of BrCa cells. Thus, identification of tumor‐suppressive miRNA and molecular networks controlled by these miRNA in BrCa cells may be an effective strategy for elucidation of the molecular pathogenesis of this disease., We showed that miR‐101‐5p acted as an anti‐tumor miRNA in breast cancer (BrCa) cells through targeting several oncogenic genes (i.e. High Mobility Group Box 3, Epithelial splicing regulatory protein 1, GINS complex subunit 1 (GINS1), Tumor Protein D52, Serine/Arginine‐Rich Splicing Factor Kinase 1, Vang‐like protein 1, and Mago Homolog B). Aberrant expression of GINS1 mRNA and protein was observed in BrCa clinical specimens, and high GINS1 expression significantly predicted poor prognosis in patients with BrCa (overall survival rate: P = 0.0126).

Our ongoing analyses identifying dysregulated microRNAs (miRNAs) and their controlled target RNAs have shed light on novel oncogenic pathways in pancreatic ductal adenocarcinoma (PDAC). The PDAC miRNA signature obtained by RNA sequencing showed that both strands of pre-miR-130b (miR-130b-5p, the passenger strand and miR-130b-3p, the guide strand) were significantly downregulated in cancer tissues. Our functional assays revealed that miR-130b-5p significantly blocked the malignant abilities of PDAC cell lines (PANC-1 and SW1990), e.g., cancer cell proliferation, migration, and invasion. A total of 103 genes were identified as possible oncogenic targets by miR-130b-5p regulation in PDAC cells based on genome-wide gene expression analysis and in silico database search. Among the possible targets, high expression of 9 genes (EPS8, ZWINT, SMC4, LDHA, GJB2, ZCCHC24, TOP2A, ANLN, and ADCY3) predicted a significantly poorer prognosis of PDAC patients (5-year overall survival, p

Lymphoepithelioma-like carcinoma (LELC) of the liver is extremely rare, and lymphoepithelioma-like hepatocellular carcinoma (LEL-HCC) is an uncommon variant form of HCC, exhibiting relatively good prognosis compared to conventional HCC. LELC is defined as a tumour composed of undifferentiated epithelial cells with densely infiltrating lymphoid stroma. LEL-HCC represents a unique immune response against tumour cells, which may contribute to the superior clinical outcomes. However, the exact aetiology remains unknown. We report a case of a 76-year-old man with a prior hepatitis B virus infection. A 20 mm tumour was detected in the liver. Microscopically, the tumour displayed characteristics of poorly differentiated HCC with a dense lymphocytic and plasma cell infiltration and was diagnosed as LEL-HCC. We describe a unique pathological finding with immunohistochemical data demonstrating T-cell dominant lymphocytic and polyclonal plasma cell infiltration, along with programmed death ligand 1 expression in the tumour cells and lymphocytes.

Although miR‑145‑5p (the guide strand of the miR‑145 duplex) is established as a tumor suppressive microRNA (miRNA or miR), the functional significance of miR‑145‑3p (the passenger strand of the miR‑145 duplex) in cancer cells and its targets remains obscure. In our continuing analysis of esophageal squamous cell carcinoma (ESCC) pathogenesis, the aim of the present study was to identify important oncogenes and proteins that are controlled by miR‑145‑3p. Overexpression of miR‑145‑3p significantly reduced cancer cell proliferation, migration and invasive abilities, and further increased apoptotic abilities. In ESCC cells, 30 possible oncogenic targets were identified that might be regulated by miR‑145‑3p. Among these targets, dehydrogenase/reductase member 2 (DHRS2) and myosin IB (MYO1B) were focused on to investigate their functional roles in ESCC cells. DHRS2 and MYO1B were directly regulated by miR‑145‑3p in ESCC cells by dual luciferase reporter assays. Aberrantly expressed DHRS2 and MYOIB were detected in ESCC clinical specimens, and their overexpression enhanced cancer cell aggressiveness. Genes regulated by antitumor miR‑145‑3p were closely associated with the molecular pathogenesis of ESCC. The approach based on antitumor miRNAs may contribute to the understanding of ESCC molecular pathogenesis.

Analyses of microRNA (miRNA) expression signatures obtained by RNA sequencing revealed that some passenger miRNAs (miR-144-5p, miR-145-3p, miR-149-3p, miR-150-3p, and miR-199a-3p) acted as anti-tumor miRNAs in several types of cancer cells. The involvement of passenger strands in the pathogenesis of human cancer is a novel concept. Based on the miRNA signature of bladder cancer (BC) obtained by RNA sequencing, we focused on both strands of the miR-223-duplex (miR-223-5p and miR-223-3p) and investigated their functional significance in BC cells. Ectopic expression of these miRNAs showed that both miR-223-3p (the guide strand) and miR-223-5p (the passenger strand) inhibited cancer cell migration and invasion of BC cells. The role of miR-223-5p (the passenger strand) has not been well studied. Combining gene expression studies and in silico database analyses, we demonstrated the presence of 20 putative target genes that could be regulated by miR-223-5p in BC cells. Among these targets, high expression of five genes (ANLN, INHBA, OIP5, CCNB1, and CDCA2) was significantly associated with poor prognosis of BC patients based on The Cancer Genome Atlas (TCGA) database. Moreover, we showed that a gene (ANLN) encoding a multifunctional actin-binding protein was directly regulated by miR-223-5p in BC cells. Overexpression of ANLN was observed in BC clinical specimens and high expression of ANLN was significantly associated with poor prognosis of BC patients. We suggest that studies of regulatory cancer networks, including the passenger strands of miRNAs, may provide new insights into the pathogenic mechanisms of BC.

We previously used RNA sequencing to establish the microRNA (miRNA) expression signature of pancreatic ductal adenocarcinoma (PDAC). We found that both strands of pre-miR-148a (miR-148a-5p: the passenger strand and miR-148a-3p: the guide strand) were downregulated in cancer tissues. Ectopic expression of miR-148a-5p and miR-148a-3p significantly inhibited cancer cell migration and invasion, indicating that both strands of pre-miR-148a had tumor-suppressive roles in PDAC cells. In silico database and genome-wide gene expression analyses identified a total of 15 genes that were putative targets regulated by these miRNAs. High expression of miR-148a-5p targets (PHLDA2, LPCAT2 and AP1S3) and miR-148a-3p targets (SMA, ENDOD1 and UHMK1) was associated with poor prognosis of patients with PDAC. Moreover, knockdown of PHLDA2 expression inhibited cancer cell aggressiveness, suggesting PHLDA2 acted as an oncogene in PDAC cells. Involvement of the passenger strand of pre-miR-148a (miR-148-5p) is a new concept in cancer research. Novel approaches that identify tumor-suppressive miRNA regulatory networks in lethal PDAC might provide new prognostic markers and therapeutic targets for this disease.

Analysis of the microRNA (miRNA) expression signature of head and neck squamous cell carcinoma (HNSCC) based on RNA sequencing showed that dual strands of pre‑miR‑145 (miR‑145‑5p, guide strand; and miR‑145‑3p, passenger strand) were significantly reduced in cancer tissues. In miRNA biogenesis, passenger strands of miRNAs are degraded and have no biological activities in cells. The aims of this study were to investigate the functional significance of the passenger strand of miR‑145 and to identify miR‑145‑3p‑regulated oncogenic genes in HNSCC cells. Expression levels of miR‑145‑5p and miR‑145‑3p were significantly downregulated in HNSCC tissues and cell lines (SAS and HSC3 cells). Ectopic expression of miR‑145‑3p inhibited cancer cell proliferation, migration and invasion, similar to miR‑145‑5p, in HNSCC cells. Myosin 1B (MYO1B) was directly regulated by miR‑145‑3p, and knockdown of MYO1B by siRNA inhibited cancer cell aggressiveness. Overexpression of MYO1B was confirmed in HNSCC clinical specimens by analysis of protein and mRNA levels. Interestingly, high expression of MYO1B was associated with poor prognosis in patients with HNSCC by analysis of The Cancer Genome Atlas database (p=0.00452). Our data demonstrated that the passenger strand of miR‑145 acted as an antitumor miRNA through targeting MYO1B in HNSCC cells. The involvement of dual strands of pre‑miR‑145 (miR‑145‑5p and miR‑145‑3p) in the regulation of HNSCC pathogenesis is a novel concept in present RNA research.

Objective Our recent studies have revealed that both strands of pre-miRNAs, the guide strand and the passenger strand, are involved in cancer pathogenesis. Analyses of miRNA expression signatures by RNA sequencing in head and neck squamous cell carcinoma (HNSCC) showed that both of the strands of pre-miR-150 (miR-150-5p and miR-150-3p) were significantly downregulated, and that these miRNAs acted as antitumor miRNAs in HNSCC cells. The aim of this study was to identify oncogenic genes in HNSCC cells that were regulated by miR-150-5p and miR-150-3p. Methods Genome-wide gene expression studies, in silico analyses and dual-luciferase reporter assays were carried out to predict miR-150-5p and miR-150-3p regulation in HNSCC cells. Knockdown assay was applied to investigate the functional significance of the target gene. Overall patient survival as a function of target gene expression was estimated by The Cancer Genome Atlas (TCGA) database. Results A total of 19 genes were putative targets of both miR-150-5p and miR-150-3p regulation. Among them, SPOCK1 (SPARC/osteonectin, cwcv and kazal-like domains proteoglycan 1) was directly regulated by both miRNAs in HNSCC cells. Knockdown studies using si-SPOCK1 showed that expression of SPOCK1 enhanced HNSCC cell aggressiveness. Overexpression of SPOCK1/SPOCK1 was confirmed in HNSCC clinical specimens. Interestingly, analysis of a large number of patients in the TCGA database (n = 248) demonstrated that patients with high SPOCK1 expression had significantly shorter survival than did those with low SPOCK1 expression (P = 0.0003). Moreover, 15 pathways were identified as SPOCK1-mediated downstream pathways. Conclusion Downregulation of both strands of pre-miR-150 (miR-150-5p and miR-150-3p) and overexpression of SPOCK1 contribute to the aggressive nature of HNSCC. The involvement of passenger strand miRNA in the regulation of HNSCC pathogenesis is a novel concept in RNA research.

Analysis of our microRNA (miRNA) expression signatures of human cancers based on RNA sequencing have shown that both strands of pre-miR-150, miR-150-5p (the guide strand) and miR-150-3p (the passenger strand), are significantly reduced in cancer tissues. We have investigated the functional significance of both strands of pre-miR-150 in cancer cells. The aim of this study was to investigate the antitumor function of these miRNAs and how these miRNAs regulated oncogenic targets in esophageal squamous cell carcinoma (ESCC). Ectopic expression studies demonstrated that both strands of pre-miR-150 miRNA inhibited ESCC cancer cell migration and invasion, indicating that both miR-150-5p and miR-150-3p acted as antitumor miRNAs. A combination of genome-wide gene expression analyses and in silico database searches showed that SPOCK1 (SPARC/osteonectin, cwcv and kazal-like domains proteoglycan 1) was a candidate target of miR-150-5p and miR-150-3p in ESCC cells. Luciferase reporter assays showed that SPOCK1 was directly regulated by these miRNAs. Silencing of SPOCK1 by small interfering RNA inhibited cancer cell migration and invasion. Overexpression of SPOCK1/SPOCK1 was confirmed by real-time PCR methods and immunohistochemistry. Taken together, downregulation of both strands of pre-miR-150 and overexpression of SPOCK1 are involved in ESCC pathogenesis. The involvement of passenger strand miRNAs in the regulation of cancer cell aggressiveness is a novel concept in RNA research.

Analysis of our microRNA (miRNA) expression signature in human cancers has shown that guide and passenger strands of pre-miR‑150, i.e., miR‑150‑5p and miR‑150‑3p, are significantly downregulated in cancer tissues. In miRNA biogenesis, the passenger strand of miRNA is degraded and is thought to have no functions. Thus, the aim of this study was to investigate the functional significance of miR‑150‑5p and miR‑150‑3p in naive prostate cancer (PCa) and castration-resistant prostate cancer (CRPC). Ectopic expression assays showed that both strands of miRNAs significantly suppressed cancer cell migration and invasion. Our strategies of miRNA target searching demonstrated that SPOCK1 (SPARC/osteonectin, cwcv and kazal like domains proteoglycan 1) was directly regulated by miR‑150‑5p and miR‑150‑3p. Knockdown of SPOCK1 by siRNA inhibited cancer cell aggressiveness. Moreover, overexpression of SPOCK1 was observed in naive PCa and CRPC tissues. Taken together, dual strands of pre-miR‑150 (miR‑150‑5p and miR‑150‑3p) acted as antitumor miRNAs in naive PCa and CRPC cells. Expression of oncogenic SPOCK1 was involved in naive PCa and CRPC pathogenesis. Novel approaches to analysis of antitumor miRNA-regulated RNA networks in cancer cells may provide new insights into the pathogenic mechanisms of naive PCa and CRPC.

Previously, we established a microRNA (miRNA) expression signature in pancreatic ductal adenocarcinoma (PDAC) tissues using RNA sequencing and found significantly reduced expression of miR-204-5p. Here, we aimed to investigate the functional significance of miR-204-5p and to identify miR-204-5p target genes involved in PDAC pathogenesis. Cancer cell migration and invasion were significantly inhibited by ectopic expression of miR-204-5p in PDAC cells. Comprehensive gene expression analyses and in silico database searches revealed 25 putative targets regulated by miR-204-5p in PDAC cells. Among these target genes, high expression levels of RACGAP1, DHRS9, AP1S3, FOXC1, PRP11, RHBDL2 and MUC4 were significant predictors of a poor prognosis of patients with PDAC. In this study, we focused on RACGAP1 (Rac guanosine triphosphatase-activating protein 1) because its expression was most significantly predictive of PDAC pathogenesis (overall survival rate: p = 0.0000548, disease-free survival rate: p = 0.0014). Overexpression of RACGAP1 was detected in PDAC clinical specimens, and its expression enhanced the migration and invasion of PDAC cells. Moreover, downstream genes affected by RACGAP1 (e.g., MMP28, CEP55, CDK1, ANLN and S100A14) are involved in PDAC pathogenesis. Our strategy to identify antitumor miRNAs and their target genes will help elucidate the molecular pathogenesis of PDAC.

Due to its aggressive nature, pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal and hard-to-treat malignancies. Recently developed targeted molecular strategies have contributed to remarkable improvements in the treatment of several cancers. However, such therapies have not been applied to PDAC. Therefore, new treatment options are needed for PDAC based on current genomic approaches. Expression of microRNA-375 (miR-375) was significantly reduced in miRNA expression signatures of several types of cancers, including PDAC. The aim of the present study was to investigate the functional roles of miR-375 in PDAC cells and to identify miR-375-regulated molecular networks involved in PDAC aggressiveness. The expression levels of miR-375 were markedly downregulated in PDAC clinical specimens and cell lines (PANC-1 and SW1990). Ectopic expression of miR-375 significantly suppressed cancer cell proliferation, migration and invasion. Our in silico and gene expression analyses and luciferase reporter assay showed that zinc finger protein 36 ring finger protein-like 2 (ZFP36L2) was a direct target of miR-375 in PDAC cells. Silencing ZFP36L2 inhibited cancer cell aggressiveness in PDAC cell lines, and overexpression of ZFP36L2 was confirmed in PDAC clinical specimens. Interestingly, Kaplan-Meier survival curves showed that high expression of ZFP36L2 predicted shorter survival in patients with PDAC. Moreover, we investigated the downstream molecular networks of the miR-375/ZFP36L2 axis in PDAC cells. Elucidation of tumor-suppressive miR-375-mediated PDAC molecular networks may provide new insights into the potential mechanisms of PDAC pathogenesis.

Background MicroRNAs (miRNAs) belong to a group of small non-coding RNA molecules that act as pivotal agents responsible for fine-tuning RNA expression in a sequence-dependent manner. A large number of studies showed that dysregulated miRNAs are deeply involved in the development of cancer cells, as well as their metastasis and drug resistance. Based on our original miRNA expression signatures by RNA-sequencing revealed that both strands of miR-150–5p (the guide strand) and miR-150–3p (the passenger strand) was downregulated in several cancers. The general concept of miRNA biogenesis posits that the passenger strand of miRNA (the minor strand or miRNA*) derived from duplex miRNA is degraded and does not regulate gene expression. Here, we aimed that to investigate functional significance of these miRNAs in esophageal squamous cell carcinoma (ESCC). Methods Cancer cell proliferation, migration and invasion abilities were performed by using mature miRNAs or siRNAs. Genome-wide gene expression analyses and in silico analyses were applied to identify miRNA target genes in ESCC cells. Results Expression levels of miR-150–5p and miR-150–3p were significantly reduced in ESCC clinical specimens and cell lines. Cancer cell aggressiveness was inhibited by ectopic expression of these miRNAs. A total of 12 genes were identified as oncogenic targets by both miR-150–5p and miR-150–3p in ESCC cells. SPOCK1 (SPARC/osteonectin, cwcv and kazal-like domains proteoglycan 1) was directly regulated by both miR-150–5p and miR-150–3p by luciferase reporter assay. Overexpression of SPOCK1 was detected in ESCC specimens and knockdown of SPOCK1 by siRNA significantly inhibited cancer cell migration and invasion abilities. Conclusion Both strands of miR-150-duplex (miR-150–5p and miR-150–3p) acted as anti-tumor miRNAs in ESCC. Overexpression of SPOCK1 was enhanced cancer cell aggressiveness. Involvement of passenger strand of miRNA in cancer pathogenesis is novel concept in cancer research. We suggest that identification of novel function of passenger strands of miRNAs and the RNA networks they regulate might enhance our understanding of the molecular pathogenesis of ESCC. Disclosure All authors have declared no conflicts of interest.