THE JOURNAL OF EXPERIMENTAL BIOLOGY 1266 growth performance (El-Sayed and Kawanna, 2008;Yan and Wang, 2010; Chen et al., 2011): (1) initial mass (g), wet mass of fish at the beginning of culture; (2) final mass (g), wet mass of fish at the final of culture; (3) daily mass gain (DMG), calculated as (final mass-initial mass)/culture days; (4) coefficient of variation (CV) for final mass, calculated as mean standard deviation of the final mass/mean final mass; (5) feed conversion ratio, calculated as feed consumption/mass gain; and (6) feed intake, calculated as 100×feed consumption/[days×(final mass+initial mass)/2)]. Quantitative PCRTotal RNAs were extracted using Trizol reagent (Invitrogen, Carlsbad, CA, USA), and miRNAs were extracted using the miRNeasy kit (Qiagen, Gaithersburg, MD, USA). Real-time PCR for IGF-1 was performed using SYBR Green PCR mixture (Takara Bio, Otsu, Shiga, Japan) in a MyiQ5 Real-time PCR Detection System (Bio-Rad Laboratories, Hercules, CA, USA). Gene expression was normalized relative to the housekeeping gene (β-actin). The primer sequences are as follows: IGF-1, 5′-GTCTGTGGAGAGCGAGGCTTT-3′ (forward), 5′-CACGTGAC -CGCCTTGCA-3′ (reverse); β-actin, 5′-GTCCCTGTACGC-CTCTGGTCG-3′ (forward), 5′-GCCGGACTCATCGTACTCCTG-3′ (reverse). The relative amount of miRNA was detected using stemloop PCR, and U6 RNA was detected as the internal control. Relative gene or miRNA expression was determined using the comparative C t method, which is also referred to the 2 -ΔΔCt method (Chen et al., 2005;Schmittgen and Livak, 2008). Silencing of miR-206 in vivo using the antagomir methodThe antagomirs used in the study are single-stranded RNAs, which consist of 21-23 nucleotides modified as follows: antagomir-133a, CsAsGCUGGUUGAAGGGGACCsAs As As-Chol-3′; antagomir-206, CsCsACACACACUUCCUUACAUUs CsC sAs-Chol-3′; and mismatch antagomir, CsAsCGGUUCCAGGCACUGUsG sUs AsChol-3′. All nucleotides are 2′-OMe-modified; the 's' represents a phosphorothioate linkage; 'Chol' represents cholesterol linked through a hydroxyprolinol linkage. They were deprotected, desalted and purified by high-performance liquid chromatography. Antagomir was dissolved in phosphate-buffered saline before injection. Tilapia weighing approximately 1g received a 0.02ml tail-vein injection of saline or antagomir at a dose of 60mgkg -1 body mass twice a week. Tissues were harvested, snap-frozen and stored at -80°C.Cell culture and 3′-UTR luciferase reporter assay HEK 293T cells were obtained from the American Type Culture Collection (Rockville, MD, USA). They were maintained in Dulbecco's modified Eagle's medium with 10% fetal bovine serum, 100Uml -1 penicillin, 100μgml -1 streptomycin and 250ngml -1 amphotericin B, and maintained at 37°C in a humidified 5% CO 2 incubator.To generate the 3′ UTR luciferase reporter construct, the full length of the 3′ UTR from IGF-1 was cloned into the downstream of the firefly luciferase gene in pGL3-control vector (Promega, Madison, WI, USA). The QuikChange site-directed mutagenesis kit (Stratagene, La Jolla...

No abstract

Long noncoding RNAs (lncRNAs) represent a new frontier in molecular genetics and molecular biology. They have a tremendous potential for advancing our comprehensive understanding of biological processes in huma n health and disease. The transcripts of lncRNAs are easy to find, but sorting out what they do remains the biggest challenge in lncRNAs' research field. In the paper, we highlight recent progress regarding the methods to explore the roles of lncRNAs.

SUMMARY

No abstract

No abstract

BackgroundKRAS-mutant lung adenocarcinomas (LUADs) are heterogeneous and frequently occur in smokers. The heterogeneity of KRAS-mutant LUAD has been an obstacle for the drug discovery.MethodsWe integrated multiplatform datatypes and identified two corresponding subtypes in the patients and cell lines. We further characterized the features of these two subtypes and performed drug screening to identify subtype-specific drugs. Finally, we used the defining features of the KRAS subtypes for drug sensitivity prediction.FindingsPatient-Subtype 1 (PS1) was characterized by increased smoking-related mutational signature activity, a low tumor-infiltrating lymphocyte (TIL)-associating score and STK11/KEAP1 co-mutations. Patient-Subtype 2 (PS2) was characterized by an increased smoking-related methylation signature activity, a high TIL-associating score and increased KRAS dependency. The cell line subtypes faithfully recapitulated all the patients' features. Drug screening of the two cell line subtypes yielded several potential candidates, such as cytarabine and enzastaurin for Cell-line-Subtype 1 (CS1) and a BTK inhibitor QL-XII-61 for Cell-line-Subtype 2 (CS2). The defining features, such as smoking-related methylation signature, were significantly associated with the sensitivity to several drugs.InterpretationThe heterogeneity of KRAS-mutant LUAD is associated with smoking-related genomic and epigenomic aberration along with other features such as immunogenicity, KRAS dependency and STK11/KEAP1 co-mutations. These features might be used as biomarkers for drug sensitivity prediction.FundThis research was funded by the Young Scientists Fund of the National Natural Science Foundation of China, the Natural Science Foundation of Fujian Province, China and the Education and Research Foundation for Young Scholars of Education Department of Fujian Province, China.

BackgroundEsophageal squamous cell carcinomas (ESCC) is the fourth most lethal cancer in China. Previous studies reveal several highly conserved mutational processes in ESCC. However, it remains unclear what are the true regulators of the mutational processes.ResultsWe analyzed the somatic mutational signatures in 302 paired whole-exome sequencing data of ESCC in a Chinese population for potential regulators of the mutational processes. We identified three conserved subtypes based on the mutational signatures with significantly different clinical outcomes. Our results show that patients of different subpopulations of Chinese differ significantly in the activity of the “NpCpG” signature (FDR = 0.00188). In addition, we report ZNF750 and CDC27, of which the somatic statuses and the genetic burdens consistently influence the activities of specific mutational signatures in ESCC: the somatic ZNF750 status is associated with the AID/APOBEC-related mutational process (FDR = 0.0637); the somatic CDC27 copy-number is associated with the “NpCpG” (FDR = 0.00615) and the AID/APOBEC-related mutational processes (FDR = 8.69 × 10− 4). The burdens of germline variants in the two genes also significantly influence the activities of the same somatic mutational signatures (FDR < 0.1).ConclusionsWe report multiple factors that influence the mutational processes in ESCC including: the subpopulations of Chinese; the germline and somatic statuses of ZNF750 and CDC27 and exposure to alcohol and tobacco. Our findings based on the evidences from both germline and somatic levels reveal potential genetic regulators of the somatic mutational processes and provide insights into the biology of esophageal carcinogenesis.Electronic supplementary materialThe online version of this article (10.1186/s12864-018-4906-4) contains supplementary material, which is available to authorized users.