Circular RNAs （circRNAs） are recently identified as a naturally occurring family of widespread and diverse endogenous noncoding RNAs that may regulate gene expression in mammals . They are unusually ...sta- ble RNA molecules with cell type- or developmental stage-specific expression patterns . Exosomes are small membrane vesicles of endocytic origin secreted by most cell types. They contain a specific cargo of protein, mRNA and microRNA species, which can modulate recipient cell behaviors and may be used as biomarkers for diagnosis of human diseases .
Circular RNAs (circRNAs) represent a class of widespread and diverse endogenous RNAs that may regulate gene expression in eukaryotes. However, the regulation and function of human circRNAs remain ...largely unknown. Here we generate ribosomal-depleted RNA sequencing data from six normal tissues and seven cancers, and detect at least 27,000 circRNA candidates. Many of these circRNAs are differently expressed between the normal and cancerous tissues. We further characterize one abundant circRNA derived from Exon2 of the HIPK3 gene, termed circHIPK3. The silencing of circHIPK3 but not HIPK3 mRNA significantly inhibits human cell growth. Via a luciferase screening assay, circHIPK3 is observed to sponge to 9 miRNAs with 18 potential binding sites. Specifically, we show that circHIPK3 directly binds to miR-124 and inhibits miR-124 activity. Our results provide evidence that circular RNA produced from precursor mRNA may have a regulatory role in human cells.
Summary Background Hepatocellular carcinoma (HCC) is prevalent worldwide and improvements in timely and effective diagnosis are needed. We assessed whether measurement of Dickkopf-1 (DKK1) in serum ...could improve diagnostic accuracy for HCC. Methods We analysed data for patients with HCC, chronic hepatitis B virus (HBV) infection, liver cirrhosis, and healthy controls, recruited from two Chinese centres between December, 2008, and July, 2009. A validation cohort matched for age and sex was recruited from another centre in China between February, 2009, and June, 2011. DKK1 was measured in serum by ELISA by independent researchers who had no access to patients' clinical information. We used receiver operating characteristics (ROC) to calculate diagnostic accuracy. Findings We assessed serum DKK1 in 831 participants: 424 with HCC, 98 with chronic HBV infection, 96 with cirrhosis, and 213 healthy controls. The validation cohort comprised 453 participants: 209 with HCC, 73 with chronic HBV infection, 72 with cirrhosis, and 99 healthy controls. Levels of DKK1 in serum were significantly higher in patients with HCC than in all controls. ROC curves showed the optimum diagnostic cutoff was 2·153 ng/mL (area under curve AUC 0·848 95% CI 0·820–0·875, sensitivity 69·1%, and specificity 90·6% in the test cohort; 0·862 0·825–0·899, 71·3%, and 87·2% in the validation cohort). Similar results were noted for early-stage HCC (0·865 0·835–0·895, 70·9%, and 90·5% in the test cohort; 0·896 0·846–0·947, 73·8%, and 87·2% in the validation cohort). Furthermore, DKK1 maintained diagnostic accuracy for patients with HCC who were α-fetoprotein (AFP) negative (0·841 0·801–0·882, 70·4%, and 90·0% in the test cohort; 0·869 0·815–0·923, 66·7%, and 87·2% in the validation cohort), including for patients with early-stage HCC (0·870 0·829–0·911, 73·1%, and 90·0% in the test cohort; 0·893 0·804–0·983, 72·2%, and 87·2% in the validation cohort), compared with all controls. Raised concentrations of DKK1 in serum could differentiate HCC from chronic HBV infection and cirrhosis (0·834 0·798–0·871, 69·1%, and 84·7% in the test cohort; 0·873 0·832–0·913, 71·3%, and 90·6% in the validation cohort). Moreover, measurement of DKK1 and AFP together improved diagnostic accuracy for HCC versus all controls compared with either test alone (0·889 0·866–0·913, 73·3%, and 93·4% in the test cohort; 0·888 0·856–0·920, 78·5%, and 87·2% in the validation cohort). Interpretation DKK1 could complement measurement of AFP in the diagnosis of HCC and improve identification of patients with AFP-negative HCC and distinguish HCC from non-malignant chronic liver diseases. Funding National Key Basic Research Programme of China, National Key Sci-Tech Special Projects of Infectious Diseases, National Natural Science Foundation of China, Research Fund for the Doctoral Programme of Higher Education of China.
Mice housed in an enriched environment display a tumor-resistant phenotype due to eustress stimulation. However, the mechanisms underlying enriched environment-induced protection against cancers ...remain largely unexplained. In this study, we observed a significant antitumor effect induced by enriched environment in murine pancreatic cancer and lung cancer models. This effect remained intact in T/B lymphocyte-deficient Rag1
mice, but was nearly eliminated in natural killer (NK) cell-deficient Beige mice or in antibody-mediated NK-cell-depleted mice, suggesting a predominant role of NK cells in enriched environment-induced tumor inhibition. Exposure to enriched environment enhanced NK-cell activity against tumors and promoted tumoral infiltration of NK cells. Enriched environment increased the expression levels of CCR5 and NKG2D (KLRK1) in NK cells; blocking their function effectively blunted the enriched environment-induced enhancement of tumoral infiltration and cytotoxic activity of NK cells. Moreover, blockade of β-adrenergic signaling or chemical sympathectomy abolished the effects of enriched environment on NK cells and attenuated the antitumor effect of enriched environment. Taken together, our results provide new insight into the mechanism by which eustress exerts a beneficial effect against cancer.
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Collagens are the most abundant proteins in extra cellular matrix and important components of tumor microenvironment. Recent studies have showed that aberrant expression of collagens can influence ...tumor cell behaviors. However, their roles in hepatocellular carcinoma (HCC) are poorly understood.
In this study, we screened all 44 collagen members in HCC using whole transcriptome sequencing data from the public datasets, and collagen type IV alpha1 chain (COL4A1) was identified as most significantly differential expressed gene. Expression of COL4A1 was detected in HCC samples by quantitative real-time polymerase chain reaction (qRT-PCR), western blot and immunohistochemistry (IHC). Finally, functions and potential mechanisms of COL4A1 were explored in HCC progression.
COL4A1 is the most significantly overexpressed collagen gene in HCC. Upregulation of COL4A1 facilitates the proliferation, migration and invasion of HCC cells through FAK-Src signaling. Expression of COL4A1 is upregulated by RUNX1 in HCC. HCC cells with high COL4A1 expression are sensitive to the treatment with FAK or Src inhibitor.
COL4A1 facilitates growth and metastasis in HCC via activation of FAK-Src signaling. High level of COL4A1 may be a potential biomarker for diagnosis and treatment with FAK or Src inhibitor for HCC.
Small cell lung cancer (SCLC) is an aggressive disease with poor survival. A few sequencing studies performed on limited number of samples have revealed potential disease-driving genes in SCLC, ...however, much still remains unknown, particularly in the Asian patient population. Here we conducted whole exome sequencing (WES) and transcriptomic sequencing of primary tumors from 99 Chinese SCLC patients. Dysregulation of tumor suppressor genes TP53 and RB1 was observed in 82% and 62% of SCLC patients, respectively, and more than half of the SCLC patients (62%) harbored TP53 and RB1 mutation and/or copy number loss. Additionally, Serine/Arginine Splicing Factor 1 (SRSF1) DNA copy number gain and mRNA over-expression was strongly associated with poor survival using both discovery and validation patient cohorts. Functional studies in vitro and in vivo demonstrate that SRSF1 is important for tumorigenicity of SCLC and may play a key role in DNA repair and chemo-sensitivity. These results strongly support SRSF1 as a prognostic biomarker in SCLC and provide a rationale for personalized therapy in SCLC.
Individuals with Down syndrome have a low risk for many solid tumors, prompting the search for tumor suppressor genes on human chromosome 21 (HSA21). We aimed to identify and explore potential ...mechanisms of tumor suppressors on HSA21 in hepatocellular carcinoma (HCC).
We compared expression of HSA21 genes in 14 pairs of primary HCC and adjacent noncancer liver tissues using the Affymetrix HG-U133 Plus 2.0 array (Affymetrix, Santa Clara, CA). HCC tissues and adjacent normal liver tissues were collected from 108 patients at a hospital in China for real-time polymerase chain reaction and immunohistochemical analyses; expression levels of regulator of calcineurin 1 (RCAN1) isoform 4 (RCAN1.4) were associated with clinical features. We overexpressed RCAN1.4 from lentiviral vectors in MHCC97H and HCCLM3 cells and knocked expression down using small interfering RNAs in SMMC7721 and Huh7 cells. Cells were analyzed in proliferation, migration, and invasion assays. HCC cells that overexpressed RCAN1.4 or with RCAN1.4 knockdown were injected into livers or tail veins of nude mice; tumor growth and numbers of lung metastases were quantified. We performed bisulfite pyrosequencing and methylation-specific polymerase chain reaction analyses to analyze CpG island methylation. We measured phosphatase activity of calcineurin in HCC cells.
RCAN1.4 mRNA and protein levels were significantly decreased in primary HCC compared with adjacent noncancer liver tissues. Reduced levels of RCAN1.4 mRNA were significantly associated with advanced tumor stages, poor differentiation, larger tumor size, and vascular invasion. Kaplan-Meier survival analysis showed that patients with HCCs with lower levels of RCAN1.4 mRNA had shorter time of overall survival and time to recurrence than patients whose tumors had high levels of RCAN1.4 mRNA. In HCC cell lines, expression of RCAN1.4 significantly reduced proliferation, migration, and invasive activity. HCC cells that overexpressed RCAN1.4 formed smaller xenograft tumors, with fewer metastases and blood vessels, than control HCC cells. In HCC cells, RCAN1.4 inhibited expression of insulin-like growth factor 1 and vascular endothelial growth factor A by reducing calcineurin activity and blocking nuclear translocation of nuclear factor of activated T cells (NFAT1). HCC cells incubated with the calcineurin inhibitor cyclosporin A had decreased nuclear level of NFAT1. HCC cells had hypermethylation of a CpG island in the 5’ regulatory region of RCAN1.4, which reduced its expression.
RCAN1.4 is down-regulated in HCC tissues, compared with non-tumor liver tissues. RCAN1.4 prevents cell proliferation, migration, and invasion in vitro; overexpressed RCAN1.4 in HCC cells prevents growth, angiogenesis, and metastases of xenograft tumors by inhibiting calcineurin activity and nuclear translocation of NFAT1.
Hormones and their corresponding receptors are vital in controlling metabolism under normal physiologic and pathologic conditions, but less is known about their roles in the metabolism of cancer. ...Using a small interfering RNA screening approach, we examined the effects of silencing 20 well‐known hormone receptors on the Warburg effect, specifically by measuring the production of lactate in four established hepatocellular carcinoma (HCC) cell lines. We found that silencing a variety of hormone receptors had effects on the production of this metabolite. Unexpectedly silencing of mineralocorticoid receptor (MR) significantly increased lactate production in all these HCC cell lines. Subsequent in vitro and in vivo studies showed that gain‐ and loss‐of‐function of MR significantly influenced HCC cellular proliferation, cell cycle distribution, and apoptosis. Furthermore, mechanistic studies revealed that MR as a transcriptional factor directly regulated the expression of miR‐338‐3p, suppressing the Warburg effects of HCC cells by targeting a key enzyme of glycolysis: pyruvate kinase, liver and red blood cells. Moreover, MR expression was significantly down‐regulated in 81% of HCC patient tissues, caused by both chromosome deletion and histone deacetylation. Low expression of MR in tumor tissues was associated with poor patient prognosis. The expression level of miR‐338‐3p was found to positively correlate with the expression of MR in HCC tissues and to inversely correlate with expression of the enzyme pyruvate kinase, liver and red blood cells. Conclusion: MR affects HCC development by modulating the miR‐338‐3p/pyruvate kinase, liver and red blood cells axis with an ability to suppress the Warburg effect. (Hepatology 2015;62:1145‐1159)
The pathological relevance and significance of microRNAs (miRNAs) in hepatocarcinogenesis have attracted much attention in recent years; however, little is known about the underlying molecular ...mechanisms through which miRNAs are involved in the development and progression of hepatocellular carcinoma (HCC). In this study, we demonstrate that miR‐30d is frequently up‐regulated in HCC and that its expression is highly associated with the intrahepatic metastasis of HCC. Furthermore, the enhanced expression of miR‐30d could promote HCC cell migration and invasion in vitro and intrahepatic and distal pulmonary metastasis in vivo, while silencing its expression resulted in a reduced migration and invasion. Galphai2 (GNAI2) was identified as the direct and functional target of miR‐30d with integrated bioinformatics analysis and messenger RNA array assay. This regulation was further confirmed by luciferase reporter assays. In addition, our results, for the first time, showed that GNAI2 was frequently suppressed in HCC by way of quantitative reverse‐transcription polymerase chain reaction and immunohistochemical staining assays. The increase of the GNAI2 expression significantly inhibits, whereas knockdown of the GNAI2 expression remarkably enhances HCC cell migration and invasion, indicating that GNAI2 functions as a metastasis suppressor in HCC. The restoration of GNAI2 can inhibit miR‐30d–induced HCC cell invasion and metastasis. Conclusion: The newly identified miR‐30d/GNAI2 axis elucidates the molecular mechanism of HCC cell invasion and metastasis and represents a new potential therapeutic target for HCC treatment. (HEPATOLOGY 2010.)
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