Patterns and functional implications of rare germline variants across 12 cancer types

Large-scale cancer sequencing data enable discovery of rare germline cancer susceptibility variants. Here we systematically analyse 4,034 cases from The Cancer Genome Atlas cancer cases representing 12 cancer types. We find that the frequency of rare germline truncations in 114 cancer-susceptibility-associated genes varies widely, from 4% (acute myeloid leukaemia (AML)) to 19% (ovarian cancer), with a notably high frequency of 11% in stomach cancer. Burden testing identifies 13 cancer genes with significant enrichment of rare truncations, some associated with specific cancers (for example, RAD51C, PALB2 and MSH6 in AML, stomach and endometrial cancers, respectively). Significant, tumour-specific loss of heterozygosity occurs in nine genes (ATM, BAP1, BRCA1/2, BRIP1, FANCM, PALB2 and RAD51C/D). Moreover, our homology-directed repair assay of 68 BRCA1 rare missense variants supports the utility of allelic enrichment analysis for characterizing variants of unknown significance. The scale of this analysis and the somatic-germline integration enable the detection of rare variants that may affect individual susceptibility to tumour development, a critical step toward precision medicine

MiR-21 Simultaneously Regulates ERK1 Signaling in HSC Activation and Hepatocyte EMT in Hepatic Fibrosis

<div><p>Background</p><p>MicroRNA-21 (miR-21) plays an important role in the pathogenesis and progression of liver fibrosis. Here, we determined the serum and hepatic content of miR-21 in patients with liver cirrhosis and rats with dimethylnitrosamine-induced hepatic cirrhosis and examined the effects of miR-21 on <i>SPRY2</i> and <i>HNF4α</i> in modulating ERK1 signaling in hepatic stellate cells (HSCs) and epithelial-mesenchymal transition (EMT) of hepatocytes.</p><p>Methods</p><p>Quantitative RT-PCR was used to determine miR-21 and the expression of <i>SPRY2, HNF4α</i> and other genes. Immunoblotting assay was carried out to examine the expression of relevant proteins. Luciferase reporter assay was performed to assess the effects of miR-21 on its predicted target genes <i>SPRY2</i> and <i>HNF4α</i>. Primary HSCs and hepatocytes were treated with miR-21 mimics/inhibitors or appropriate adenoviral vectors to examine the relation between miR-21 and <i>SPRY2</i> or <i>HNF4α.</i></p><p>Results</p><p>The serum and hepatic content of miR-21 was significantly higher in cirrhotic patients and rats. <i>SPRY2</i> and <i>HNF4α</i> mRNA levels were markedly lower in the cirrhotic liver. MiR-21 overexpression was associated with enhanced ERK1 signaling and EMT in liver fibrosis. Luciferase assay revealed suppressed <i>SPRY2</i> and <i>HNF4α</i> expression by miR-21. Ectopic miR-21 stimulated ERK1 signaling in HSCs and induced hepatocyte EMT by targeting <i>SPRY2</i> or <i>HNF4α</i>. Downregulating miR-21 suppressed ERK1 signaling, inhibited HSC activation, and blocked EMT in TGFβ1-treated hepatocytes.</p><p>Conclusions</p><p>MiR-21 modulates ERK1 signaling and EMT in liver fibrosis by regulating <i>SPRY2</i> and <i>HNF4α</i> expression. MiR-21 may serve as a potentially biomarker as well as intervention target for hepatic cirrhosis.</p></div

Clinical features of the patients providing cirrhotic liver tissues.

<p>Clinical features of the patients providing cirrhotic liver tissues.</p

Liver fibrosis is associated with broad changes in the expression of miR-21 and its targeted genes, ERK1 signaling and EMT-associated genes.

<p>Serum miR-21 contents in cirrhotic patients (n = 20) and normal subjects (n = 20) (A) and in rats with dimethylnitrosamine-induced liver cirrhosis (B). MiR-21 expression was normalized against miR-238 in serum. **<i>P</i><0.01 vs. normal controls. Quantitative RT-PCR examination of the expression of miR-21 and its targeted genes <i>SPRY2</i> and <i>HNF4α</i>, <i>ERK1</i> and its downstream target <i>RSK2</i>, and epithelial-mesenchymal transition (EMT)-associated genes <i>E-cadherin</i> and <i>vimentin</i> in the liver tissues of cirrhotic patients (n = 7) (C) and rats (n = 10) (D). *<i>P</i><0.05 and **<i>P</i><0.01 vs. normal subjects or rats. Primary HSCs were treated with TGFβ1 for 7 days (E) and primary hepatocytes for 48 h (F) as detailed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0108005#s2" target="_blank">methods</a>. Quantitative RT-PCR examination of the expression of miR-21 and its targeted genes <i>SPRY2</i> and <i>HNF4α, ERK1</i> and <i>RSK2</i>, <i>E-cadherin</i>, <i>vimentin</i> and <i>MMP-9</i>, liver fibrogenesis-associated genes α<i>-SMA</i> and <i>TIMP1</i>, and liver specific genes <i>ALB</i> and <i>CYP1a2</i>. MiR-21 expression was normalized against <i>Rnu6-2</i> and mRNA expression was normalized against <i>β-actin</i>. *<i>P</i><0.05 and **<i>P</i><0.01 vs. non-stimulated HSCs or hepatocytes. Each value represents the mean with the SD (error bars) for triplicate samples.</p

MiR-21 and <i>HNF4α</i> mutually modulate the expression of each other in regulating EMT of primary hepatocytes.

<p>Primary rat hepatocytes were treated with miR-21 mimics for 48 h. MiR-21 and the mRNA transcript levels of <i>HNF4α</i>, EMT associated genes <i>E-cadherin</i>, <i>vimentin</i> and <i>slug</i> (A), <i>MMP-2</i> and <i>MMP-9</i>, fibrogenesis-associated genes α<i>-SMA</i> and <i>TIMP1</i> and liver specific genes <i>ALB</i>, <i>CYP1a2</i> and <i>AFP</i> (B) were examined by quantitative RT-PCR. <i>HNF4α</i>, <i>E-cadherin</i> and <i>vimentin</i>, ALB, <i>MMP-2</i> and <i>TIMP1</i> were examined by Western blotting assays (C). GADPH was used as a loading control. Blots representative of at least three independent experiments are shown. Primary hepatocytes were treated with TGFβ1 for 48 h followed by AdHNF4α infection 48 h later (D). Primary hepatocytes were transfected with miR-21 mimics followed by AdHNF4α (E) or AdshHNF4α (F) infection 48 h later. MiR-21 and the mRNA transcript levels of <i>HNF4α</i>, <i>E-cadherin</i> and <i>vimentin</i> were then examined by quantitative RT-PCR. Each value in (A), (B), (D), (E) and (F) represents the mean with the SD (error bars) for triplicate samples of at least three independent experiments and miR-21 expression was normalized against <i>Rnu6-2</i> and mRNA expression was normalized against <i>β-actin</i>. *<i>P</i><0.05 or **<i>P</i><0.01 vs. controls.</p

MiR-21 inhibitors target ERK1 signaling and <i>SPRY2</i> in HSC activation and block EMT of TGFβ1-treated hepatocytes by targeting <i>HNF4α</i>.

<p>Primary HSCs were treated with TGFβ1 for 5 d followed by transfection with miR-21 inhibitors. MiR-21 and the mRNA transcript levels of <i>SRPY2</i>, <i>ERK1</i> and <i>RSK2</i> (A), <i>α-SMA</i>, <i>TIMP1</i>, <i>MMP-2</i> and <i>MMP-13</i> (B) were examined by quantitative RT-PCR. Immunoblotting assays were done to detect the expression of <i>SPRY2</i>, <i>phospho-ERK1</i>, <i>phospho-ERK2</i> and <i>RSK2</i> (C). HSC-T6 cells were transfected with miR-21 inhibitors. MiR-21 and the mRNA transcript levels of <i>SRPY2</i>, <i>ERK1</i> and <i>RSK2</i>, <i>MMP-9</i>, <i>MMP-13</i>, fibrogenesis-associated genes <i>TGFβ1</i>, α<i>-SMA</i>, <i>Col I</i>, <i>Col III</i> and <i>TIMP1</i> were examined by quantitative RT-PCR (D, E). Immunoblotting assays were done to detect the expression of <i>SPRY2</i>, <i>phospho-ERK1</i>, <i>phospho-ERK2</i> and <i>RSK2</i>, <i>Col I</i> and <i>TIMP1</i> (F). HSC-T6 cells were transfected with miR-21 inhibitors and transwell cell migration assays were performed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0108005#s2" target="_blank">methods</a> (G, H). Hepatocytes were treated with TGFβ1 for 48 h followed by transfection with miR-21 inhibitors. MiR-21 and the mRNA transcript levels of <i>HNF4α</i>, <i>E-cadherin</i> and <i>vimentin</i>, <i>MMP-2</i>, <i>MMP-9</i> and α<i>-SMA</i>, liver specific genes <i>CYP1a2</i> and <i>AFP</i> were examined by quantitative RT-PCR (I, J). Immunoblotting assays were done to detect the expression of <i>HNF4α</i>, <i>E-cadherin</i> and <i>vimentin</i>, <i>MMP2</i>, <i>α-SMA</i> and <i>TIMP1</i> (K). GADPH was used as a loading control. Blots representative of at least three independent experiments are shown. MiR-21 expression was normalized against <i>Rnu6-2</i> and mRNA expression was normalized against <i>β-actin</i>. Each value in (A), (B), (D), (E), (H), (I) and (J) represents the mean with the SD (error bars) for triplicate samples of at least three independent experiments. *<i>P</i><0.05 or **<i>P</i><0.01 vs. controls.</p