Bioinformatics-integrated screening of systemic sclerosis-specific expressed markers to identify therapeutic targets

BackgroundSystemic sclerosis (SSc) is a rare autoimmune disease characterized by extensive skin fibrosis. There are no effective treatments due to the severity, multiorgan presentation, and variable outcomes of the disease. Here, integrated bioinformatics was employed to discover tissue-specific expressed hub genes associated with SSc, determine potential competing endogenous RNAs (ceRNA) regulatory networks, and identify potential targeted drugs.MethodsIn this study, four datasets of SSc were acquired. To identify the genes specific to tissues or organs, the BioGPS web database was used. For differentially expressed genes (DEGs), functional and enrichment analyses were carried out, and hub genes were screened and shown in a network of protein-protein interactions (PPI). The potential lncRNA–miRNA–mRNA ceRNA network was constructed using the online databases. The specifically expressed hub genes and ceRNA network were validated in the SSc mouse and in normal mice. We also used the receiver operating characteristic (ROC) curve to determine the diagnostic values of effective biomarkers in SSc. Finally, the Drug-Gene Interaction Database (DGIdb) identified specific medicines linked to hub genes.ResultsThe pooled datasets identified a total of 254 DEGs. The tissue/organ-specifically expressed genes involved in this analysis are commonly found in the hematologic/immune system and bone/muscle tissue. The enrichment analysis of DEGs revealed the significant terms such as regulation of actin cytoskeleton, immune-related processes, the VEGF signaling pathway, and metabolism. Cytoscape identified six gene cluster modules and 23 hub genes. And 4 hub genes were identified, including Serpine1, CCL2, IL6, and ISG15. Consistently, the expression of Serpine1, CCL2, IL6, and ISG15 was significantly higher in the SSc mouse model than in normal mice. Eventually, we found that MALAT1-miR-206-CCL2, let-7a-5p-IL6, and miR-196a-5p-SERPINE1 may be promising RNA regulatory pathways in SSc. Besides, ten potential therapeutic drugs associated with the hub gene were identified.ConclusionsThis study revealed tissue-specific expressed genes, SERPINE1, CCL2, IL6, and ISG15, as effective biomarkers and provided new insight into the mechanisms of SSc. Potential RNA regulatory pathways, including MALAT1-miR-206-CCL2, let-7a-5p-IL6, and miR-196a-5p-SERPINE1, contribute to our knowledge of SSc. Furthermore, the analysis of drug-hub gene interactions predicted TIPLASININ, CARLUMAB and BINDARIT as candidate drugs for SSc

Genomic Analyses Reveal Mutational Signatures and Frequently Altered Genes in Esophageal Squamous Cell Carcinoma

Esophageal squamous cell carcinoma (ESCC) is one of the most common cancers worldwide and the fourth most lethal cancer in China. However, although genomic studies have identified some mutations associated with ESCC, we know little of the mutational processes responsible. To identify genome-wide mutational signatures, we performed either whole-genome sequencing (WGS) or whole-exome sequencing (WES) on 104 ESCC individuals and combined our data with those of 88 previously reported samples. An APOBEC-mediated mutational signature in 47% of 192 tumors suggests that APOBEC-catalyzed deamination provides a source of DNA damage in ESCC. Moreover, PIK3CA hotspot mutations (c.1624G>A [p.Glu542Lys] and c.1633G>A [p.Glu545Lys]) were enriched in APOBEC-signature tumors, and no smoking-associated signature was observed in ESCC. In the samples analyzed by WGS, we identified focal (<100 kb) amplifications of CBX4 and CBX8. In our combined cohort, we identified frequent inactivating mutations in AJUBA, ZNF750, and PTCH1 and the chromatin-remodeling genes CREBBP and BAP1, in addition to known mutations. Functional analyses suggest roles for several genes (CBX4, CBX8, AJUBA, and ZNF750) in ESCC. Notably, high activity of hedgehog signaling and the PI3K pathway in approximately 60% of 104 ESCC tumors indicates that therapies targeting these pathways might be particularly promising strategies for ESCC. Collectively, our data provide comprehensive insights into the mutational signatures of ESCC and identify markers for early diagnosis and potential therapeutic targets

TGF-β1 Reduces miR-29a Expression to Promote Tumorigenicity and Metastasis of Cholangiocarcinoma by Targeting HDAC4

<div><p>Transforming growth factor β1 (TGF-β1) and miRNAs play important roles in cholangiocarcinoma progression. In this study, miR-29a level was found significantly decreased in both cholangiocarcinoma tissues and tumor cell lines. TGF-β1 reduced miR-29a expression in tumor cell lines. Furthermore, anti-miR-29a reduced the proliferation and metastasis capacity of cholangiocarcinoma cell lines in vitro, overexpression of miR-29a counteracted TGF-β1-mediated cell growth and metastasis. Subsequent investigation identified HDAC4 is a direct target of miR-29a. In addition, restoration of HDAC4 attenuated miR-29a-mediated inhibition of cell proliferation and metastasis. Conclusions: TGF-β1/miR-29a/HDAC4 pathway contributes to the pathogenesis of cholangiocarcinoma and our data provide new therapeutic targets for cholangiocarcinoma.</p></div

Transwell cell invasion assay.

<p>(A and B) Transwell assay was administrated in the two tumor cell lines, 48h after treatment with anti-miR-29a. When the two tumor cell lines were treated with miR-29a mimic, the migrating cells were counted after additional incubation with 5ng ml^-1TGF-β1 for 48h. Data are shown as mean±SD; **P<0.01;*** P<0.001.</p

Up-regulation of miR-29a attenuates TGF-β1-mediated cholangiocarcin- oma cell growth.

<p>Cell proliferation assay was performed in FRH–0201 (A) and CCLP–1 (B) cells, 48h after transfection with anti-miR-29a. When the two tumor cell lines were treated with miR-29a mimic, cell proliferation assay was performed after additional treatment with 5ng/ml TGF-β1 for 48h. Data are shown as mean±SD; *p<0.05;**P<0.01.</p

HDAC4 overexpression induces an EMT phenotype.

<p>(A) Enhanced HDAC4 expression led to increased MMP2 and Vimentin, decreased E-cadherin expression in FRH–0201 cells and CCLP-1cells (B).</p

Enhanced expression of miR-29a attenuates TGF-β1-mediated cholangio- carcinoma cell metastasis.

<p>Wound healing assay was performed in FRH–0201 and CCLP–1 cells (A-C), 48h after transfection with anti-miR-29a. When the two tumor cell lines were treated with miR-29a mimic, wound healing assay was performed after additional treatment with 5ng ml^-1TGF-β1 for 48h. Data are shown as mean±SD; *p<0.05;**P<0.01;*** P<0.001.</p

The expression of miR-29a was decreased in cholangiocarcinoma samples and cell lines.

<p>(A) The average expression level of miR-29a was measured in forty human cholangiocarcinoma tissues and matched cancer-adjacent (normal) tissues. (B) Expression of miR-29a in the human intrahepatic bile duct epithelial cell line HIBEC and two cholangiocarcinoma cell lines. Data are shown as mean±SD; *P<0.05.</p

TGF-β1 reduced the expression of miR-29a in the two cholangiocarcinoma cell lines.

<p>(A and B) qRT-PCR was performed respectively in FRH–0201 and CCLP–1 cell lines after treatment with 5ng/ml TGF-β1 for 48h. Data are shown as mean±SD; *p<0.05;**P<0.01.</p

HDAC4 is a target of miR-29a.

<p>(A) Predicted putative (upper) and mutated (lower, shown in red) binding sequence in the 3’UTR of HDAC4 mRNA, displayed from 5’ to 3’. (B) The relative luciferase activity. miR-29a mimic (50nM) or NC (50nM) were co-transfected with wt or mut–3’UTR luciferase reporter. (C) Western blot analysis was performed in FRH–0201 and CCLP–1 cells. HDAC4 was reduced by miR-29a mimic (50nM), compared with NC. (D) The relative mRNA expression of HDAC4. Data are shown as mean±SD; **P<0.01.</p