Background & Aims Small non-coding RNAs (ncRNA) are increasingly recognized to play important roles in tumorigenesis. With the advent of deep sequencing, efforts have been put forth to profile the ...miRNome in a number of human malignancies. However, information on ncRNA in hepatocellular carcinoma (HCC), especially the non-microRNA transcripts, is still lacking. Methods Small RNA transcriptomes of two HCC cell lines (HKCI-4 and HKCI-8) and an immortalized hepatocyte line (MIHA) were examined using Illumina massively parallel sequencing. Dysregulated ncRNAs were verified in paired HCC tumors and non-tumoral livers (n = 73) by quantitative reverse transcription-polymerase chain reaction. Clinicopathologic correlations and in vitro functional investigations were further carried out. Results The combined bioinformatic and biological analyses showed the presence of ncRNAs and the involvement of a new PIWI-interacting RNA (piRNA), piR-Hep1, in liver tumorigenesis. piR-Hep1 was found to be upregulated in 46.6% of HCC tumors compared to the corresponding adjacent non-tumoral liver. Silencing of piR-Hep1 inhibited cell viability, motility, and invasiveness, with a concomitant reduction in the level of active AKT phosphorylation. In the analysis of miRNA, we showed for the first time, the abundant expression of miR-1323 in HCC and its distinct association in tumors arising from a cirrhotic background. Furthermore, miR-1323 overexpression in cirrhotic HCC correlated with poorer disease-free and overall survivals of patients ( p <0.009). Conclusions Our study demonstrated the value of next-generation sequencing in dissecting the ncRNome in cancer. The comprehensive definition of transcriptome unveils virtually all types of ncRNAs and provides new insight into liver carcinogenetic events.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
Background & Aims: Recent studies have emphasized causative links between microRNA (miRNA) deregulations and cancer development. In hepatocellular carcinoma (HCC), information on differentially ...expressed miRNA remained largely undefined. Methods: Array-based miRNA profiling was performed on HCC cells that were derived from chronic carriers of hepatitis B virus (HBV) and hepatitis C virus (HCV), and nonviral-associated patients. Specific microRNA (miR)-223 and miR-222 deregulations were verified in an independent series of tumors. The functional effect of miR-223 was examined further. An integrative analysis of messenger RNA (mRNA) array with in silico predictions defined potential downstream targets of miR-223. A luciferase reporter assay was conducted to confirm target association. Results: Distinct up-regulations of miR-222, miR-221, and miR-31, and down-regulations of miR-223, miR-126, and miR-122a were identified. Further investigations suggested the highly deregulated miR-223 and miR-222 could unequivocally distinguish HCC from adjacent nontumoral liver, irrespective of viral associations ( P ≤ .0002). Re-expression of miR-223 in HBV, HCV, and non-HBV non-HCV–related HCC cell lines revealed a consistent inhibitory effect on cell viability ( P < .01). Integrative analysis further implicated Stathmin 1 ( STMN1 ) as a downstream target of miR-223. A strong inverse relationship between STMN1 mRNA and miR-223 expressions was shown ( P = .006). A substantial reduction in STMN1 protein was further demonstrated upon restoration of miR-223 expression in HCC cell lines. We further showed that miR-223 readily could suppress the luciferase activity in reporter construct containing the STMN1 3' untranslated region ( P = .02). Conclusions: Our study revealed specific miRNA differential expressions in HCC and underscores the potential importance of miR-223 down-regulations in the development of HCC.
Stathmin1 (STMN1) is a candidate oncoprotein and prognosis marker in several kinds of cancers. This study was aimed to analyze its expression and biological functions in gastric cancer. The ...expression of STMN1 was evaluated by qRT-PCR, western blot and immunohistochemistry. The biological function of STMN1 was determined by MTT proliferation assays, monolayer colony formation and cell invasion assays using small interference RNA technique in gastric cancer cell lines. We also explored the regulation of STMN1 expression by microRNA-223. STMN1 was upregulated in gastric cancer cell lines and primary gastric adenocarcinomas. STMN1-positive tumors were more likely to be found in old age group and associated with p53 nuclear expression. In diffuse type gastric adenocarcinomas, STMN1 expression was correlated with age (p = 0.043), T stage (p = 0.004) and lymph node metastasis (p = 0.046). Expression of STMN1 in diffuse type gastric adenocarcinoma was associated with poor disease specific survival by univariate analysis (p = 0.01). STMN1 knockdown in AGS and MKN7 cell lines suppressed proliferation (p<0.001), reduced monolayer colony formation (p<0.001), inhibited cell invasion and migration ability (p<0.001) and induced G1 phase arrest. siSTMN1 could also suppress cell growth in vivo (p<0. 01). We finally confirmed that STMN1 is a putative downstream target of miR-223 in gastric cancer. Our findings supported an oncogenic role of STMN1 in gastric cancer. STMN1 might serve as a prognostic marker and a potential therapeutic target for gastric cancer.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Yes-associated protein 1 (YAP1) is a multifunctional protein that can interact with different transcription factors to activate gene expression. The role of YAP1 in tumorigenesis is unclear. We aimed ...to investigate the functional role of YAP1 in tumorigenesis of gastric cancer.
YAP1 expression in gastric adenocarcinoma was evaluated. The biological function was determined by proliferation assay, colony formation, cell invasion, and flow cytometric analysis through knocking down or ectopic expressing YAP1 in gastric cancer cell lines coupled with in vivo study. The possible downstream effectors of YAP1 were investigated by expression microarray.
YAP1 protein expression was upregulated in gastric cancer. Nuclear accumulation of YAP1 was associated with poor disease-specific survival (P = 0.021), especially in patients with early-stage diseases (P < 0.001). Knockdown YAP1 resulted in a significant reduction in proliferation, anchorage-dependent colony formation, cell invasion, and cell motility. Ectopic YAP1 expression promoted anchorage-independent colony formation, induced a more invasive phenotype, and accelerated cell growth both in vitro and in vivo. Microarray analysis highlighted the alteration of MAPK (mitogen-activated protein kinase) pathway by YAP1. We confirmed a constitutive activation of RAF/MEK/ERK (extracellular signal-regulated kinase) in YAP1-expressing MKN45 cells and further showed that YAP1 enhanced serum/epidermal growth factor-induced c-Fos expression in gastric cancer cells.
Our findings supported that YAP1 exhibits oncogenic property in gastric cancer. We provided the first evidence that YAP1 exerted the oncogenic function by enhancing the capacity to activate the early-response gene pathway. YAP1 could be a prognostic biomarker and potential therapeutic target for gastric cancer.
The mutagenic effect of hepatitis B (HBV) integration in predisposing risk to hepatocellular carcinoma (HCC) remains elusive. In this study, we performed transcriptome sequencing of HBV-positive HCC ...cell lines and showed transcription of viral-human gene fusions from the site of genome integrations. We discovered tumor-promoting properties of a chimeric HBx-LINE1 that, intriguingly, functions as a hybrid RNA. HBx-LINE1 can be detected in 23.3% of HBV-associated HCC tumors and correlates with poorer patient survival. HBx-LINE1 transgenic mice showed heightened susceptibility to diethylnitrosamine-induced tumor formation. We further show that HBx-LINE1 expression affects β-catenin transactivity, which underlines a role in activating Wnt signaling. Thus, this study identifies a viral-human chimeric fusion transcript that functions like a long noncoding RNA to promote HCC.
•HBV integration into the host genome can cause transcription of viral-human chimeras•HBx-LINE1 exerts oncogenic functions as a long noncoding RNA-like transcript•HBx-LINE1 promotes tumorigenicity via activation of Wnt/β-catenin signaling•HBx-LINE1 increases risk of HCC development
Lau et al. discover a viral-human fusion transcript that is detectable in a quarter of hepatitis B virus-associated hepatocellular carcinomas (HCCs). This chimeric transcript promotes HCC by functioning like a long noncoding RNA to activate the Wnt pathway.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
MicroRNAs (miRNAs) have been reported to play an important role in tumorigenesis. In this study, the role of miR-15a and miR-16-1 in gastric adenocarcinoma (GAC) was investigated.
The expression of ...miR-15a and miR-16-1 in cell lines and primary tumors was examined by miRNA qRT-PCR. Proliferative assays, colony formation, cell invasion and migration, flow cytometry analysis and in vivo study were performed by ectopic expression of miR-15a and miR-16-1. The putative target genes of miR-15a and miR-16-1 were explored by TargetScan and further validated.
We found that miR-15a and miR-16-1 were down-regulated in GAC cell lines and primary tumor samples compared with normal gastric epithelium. Functional study demonstrated that ectopic expression of miR-15a and miR-16-1 suppressed cell proliferation, monolayer colony formation, invasion and migration, and xenograft formation in vivo. In addition, miR-15a and miR-16-1 induced G0/G1 cell cycle arrest which was further confirmed by Western blot and qRT-PCR of related cell cycle regulators. YAP1 was confirmed to be a functional target of miR-15a and miR-16-1 in GAC. YAP1 re-expression partly abrogated the inhibitory effect of miR-15a and miR-16-1 in GAC cells. In clinical samples, YAP1 protein expression shows negative correlation with miR-15a and miR-16-1 expression.
In conclusion, targeting YAP1 by tumor suppressor miRNA miR-15a and miR-16-1 plays inhibitory effect and this might have a therapeutic potential in GAC.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
miR-375 is a tumor-suppressive microRNA (miRNA) in gastric cancer (GC). However, its molecular mechanism remains unclear. The aim of this study is to comprehensively investigate how miR-375 is ...involved in Hippo pathway by targeting multiple oncogenes. miR-375 expression in gastric cancer cell lines and primary GC was investigated by qRT-PCR. The regulation of YAP1, TEAD4, and CTGF expression by miR-375 was evaluated by qRT-PCR, western blot, and luciferase reporter assays, respectively. The functional roles of the related genes were examined by siRNA-mediated knockdown or ectopic expression assays. The clinical significance and expression correlation analysis of miR-375, YAP1, and CTGF were performed in primary GCs. TCGA cohort was also used to analyze the expression correlation of YAP1, TEAD4, CTGF, and miR-375 in primary GCs. miR-375 was down-regulated in GC due to promoter methylation and histone deacetylation. miR-375 downregulation was associated with unfavorable outcome and lymph node metastasis. Ectopic expression of miR-375 inhibited tumor growth in vitro and in vivo. Three components of Hippo pathway, YAP1, TEAD4 and CTGF, were revealed to be direct targets of miR-375. The expression of three genes showed a negative correlation with miR-375 expression and YAP1 re-expression partly abolished the tumor-suppressive effect of miR-375. Furthermore, CTGF was confirmed to be the key downstream of Hippo-YAP1 cascade and its knockdown phenocopied siYAP1 or miR-375 overexpression. YAP1 nuclear accumulation was positively correlated with CTGF cytoplasmic expression in primary GC tissues. Verteporfin exerted an anti-oncogenic effect in GC cell lines by quenching CTGF expression through YAP1 degradation. In short, miR-375 was involved in the Hippo pathway by targeting YAP1-TEAD4-CTGF axis and enriched our knowledge on the miRNA dysregulation in gastric tumorigenesis.
The echinoderm microtubule-associated protein-like 4 anaplastic lymphoma kinase (EML4-ALK) fusion gene has been identified as a potent oncogenic driver in non–small-cell lung cancer, in particular ...adenocarcinoma (ADC). It defines a unique subgroup of lung ADC, which may be responsive to ALK inhibitors. Detection of ALK rearrangement by fluorescence in situ hybridization (FISH) or reverse transcriptase polymerase chain reaction (RT-PCR) is considered to be the standard procedure, but each with its own limitation. We evaluated the practical usefulness of immunohistochemistry (IHC) to detect ALK expression as a reliable detection method of ALK rearrangement in lung ADC.
We tested 373 lung ADCs for ALK rearrangement by IHC and FISH. Multiplex RT-PCR was performed to confirm the fusion variants.
Twenty-two of 373 lung ACs (5.9%) were positive for ALK immunoreactivity. ALK-positive tumor cells demonstrated strong and diffused granular staining in the cytoplasm. All the ALK IHC-positive cases were confirmed to harbor ALK rearrangement, either by FISH, or RT-PCR. Two cases with positive ALK protein expression, but negative for breakapart FISH signal were shown to harbor EML4-ALK variant 1 by RT-PCR. None of the ALK IHC-negative cases were FISH-positive. In addition, we identified a novel EML4-ALK fusion variant (E3:ins53A20), and its potent transformation potential has been confirmed by in vivo tumorigenicity assay.
IHC can effectively detect ALK rearrangement in lung cancer. It might provide a reliable and cost-effective diagnostic approach in routine pathologic laboratories for the identification of suitable candidates for ALK-targeted therapy.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
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