Comparative genome anatomy reveals evolutionary insights into a unique amphitriploid fish.

Triploids are rare in nature because of difficulties in meiotic and gametogenic processes, especially in vertebrates. The Carassius complex of cyprinid teleosts contains sexual tetraploid crucian carp/goldfish (C. auratus) and unisexual hexaploid gibel carp/Prussian carp (C. gibelio) lineages, providing a valuable model for studying the evolution and maintenance mechanism of unisexual polyploids in vertebrates. Here we sequence the genomes of the two species and assemble their haplotypes, which contain two subgenomes (A and B), to the chromosome level. Sequencing coverage analysis reveals that C. gibelio is an amphitriploid (AAABBB) with two triploid sets of chromosomes; each set is derived from a different ancestor. Resequencing data from different strains of C. gibelio show that unisexual reproduction has been maintained for over 0.82 million years. Comparative genomics show intensive expansion and alterations of meiotic cell cycle-related genes and an oocyte-specific histone variant. Cytological assays indicate that C. gibelio produces unreduced oocytes by an alternative ameiotic pathway; however, sporadic homologous recombination and a high rate of gene conversion also exist in C. gibelio. These genomic changes might have facilitated purging deleterious mutations and maintaining genome stability in this unisexual amphitriploid fish. Overall, the current results provide novel insights into the evolutionary mechanisms of the reproductive success in unisexual polyploid vertebrates.

Comparative mitogenome analyses uncover mitogenome features and phylogenetic implications of the subfamily Cobitinae.

BACKGROUND: Loaches of Cobitinae, widely distributed in Eurasian continent, have high economic, ornamental and scientific value. However, the phylogeny of Cobitinae fishes within genera or family level remains complex and controversial. Up to now, about 60 Cobitinae mitogenomes had been deposited in GenBank, but their integrated characteristics were not elaborated. RESULTS: In this study, we sequenced and analyzed the complete mitogenomes of a female Cobits macrostigma. Then we conducted a comparative mitogenome analysis and revealed the conserved and unique characteristics of 58 Cobitinae mitogenomes, including C. macrostigma. Cobitinae mitogenomes display highly conserved tRNA secondary structure, overlaps and non-coding intergenic spacers. In addition, distinct base compositions were observed among different genus and significantly negative linear correlation between AT% and AT-skew were found among Cobitinae, genus Cobitis and Pangio mitogenomes, respectively. A specific 3 bp insertion (GCA) in the atp8-atp6 overlap was identified as a unique feature of loaches, compared to other Cypriniformes fish. Additionally, all protein coding genes underwent a strong purifying selection. Phylogenetic analysis strongly supported the paraphyly of Cobitis and polyphyly of Misgurnus. The strict molecular clock predicted that Cobitinae might have split into northern and southern lineages in the late Eocene (42.11 Ma), furthermore, mtDNA introgression might occur (14.40 Ma) between ancestral species of Cobitis and ancestral species of Misgurnus. CONCLUSIONS: The current study represents the first comparative mitogenomic and phylogenetic analyses within Cobitinae and provides new insights into the mitogenome features and evolution of fishes belonging to the cobitinae family.

Molecular characterization and functional analysis of the vitellogenin receptor in the rice stem borer, Chilo suppressalis.

As a member of the low-density lipoprotein receptor (LDLR) superfamily, vitellogenin (Vg) receptor (VgR) is responsible for the uptake of Vg into developing oocytes and is a potential target for pest control. Here, a full-length VgR complementary DNA (named as CsVgR) was isolated and characterized in the rice stem borer, Chilo suppressalis. The composite CsVgR gene contained an open reading frame of 5,484 bp encoding a protein of 1,827 amino acid residues. Structural analysis revealed that CsVgR contained two ligand-binding domains (LBDs) with four Class A (LDLRA ) repeats in LBD1 and seven in LBD2, which was structurally different from most non-Lepidopteran insect VgRs having five repeats in LBD1 and eight in LBD2. The developmental expression analysis showed that CsVgR messenger RNA expression was first detectable in 3-day-old pupae, sharply increased in newly emerged female adults, and reached a peak in 2-day-old female adults. Consistent with most other insects VgRs, CsVgR was exclusively expressed in the ovary. Notably, injection of dsCsVgR into late pupae resulted in fewer follicles in the ovarioles as well as reduced fecundity, suggesting a critical role of CsVgR in female reproduction. These results may contribute to the development of RNA interference-mediated disruption of reproduction as a control strategy of C. suppressalis.

LncRNA BLACAT1 is involved in chemoresistance of non­small cell lung cancer cells by regulating autophagy.

The aim of the present study was to determine the effect of the long non­coding RNA (lncRNA) bladder cancer­associated transcript 1 (BLACAT1) in chemoresistance of non­small cell lung cancer (NSCLC) cells. Expression of lncRNA BLACAT1, microRNA (miR)­17, autophagy­related protein 7 (ATG7), multidrug­resistance protein 1 (MRP1), and the autophagy­associated proteins light chain 3 (LC3)­II/LC3­I and Beclin 1 were detected using the reverse transcription­quantitative polymerase chain reaction and western blot analysis. Cell viability was determined using an MTT assay. The interaction between BLACAT1 and miR­17 was determined using RNA immunoprecipitation and RNA pull­down assays. A cisplatin (DDP)­resistant NSCLC cell A549/DDP xenograft model in nude mice was established to investigate the effect of BLACAT1 on the chemoresistance of NSCLC cells. Compared with in DDP­sensitive NSCLC cells, expression of BLACAT1, ATG7, MRP1, LC3­II/LC3­I and Beclin 1 was significantly upregulated in DDP­resistant NSCLC cells, whereas miR­17 was downregulated in DDP­resistant NSCLC cells. Short interfering RNA against BLACAT1 decreased the viability of DDP­resistant NSCLC cells. In addition, BLACAT1 interacted with miR­17, and negatively regulated miR­17. BLACAT1 promoted ATG7 expression through miR­17, and facilitated autophagy and promoted chemoresistance of NSCLC cells through miR­17/ATG7. Finally, in vivo experiments indicated that inhibition of BLACAT1 ameliorated the chemoresistance of NSCLC. BLACAT1 was upregulated in DDP­resistant NSCLC cells, and promoted autophagy and chemoresistance of NSCLC cells through the miR­17/ATG7 signaling pathway.

Effects of mechanical ventilation with different tidal volume on oxidative stress and antioxidant in lung.

PURPOSE: The aim of this study was to investigate the changes in oxidative stress and antioxidants in lung tissue under different tidal volume ventilation conditions. METHODS: Forty-eight male Wistar rats were randomized into four groups, namely, group C, the control group, which was not ventilated, and groups C1, C2 and C3, the treatment groups, which were ventilated for 2 h with tidal volumes of 8, 30 and 42 ml/kg, respectively. The right middle lobe was assayed for malondialdehyde (MDA), the right posterior lobe was assayed using Western blotting for Nrf2, GCLm and SrX1 and the left lobe was assayed for Nrf2, GCLm and SrX1 mRNA. RESULTS: The MDA levels were increased in the three treatment groups, with MDA levels highest in group C3 and lowest in group C1 (C3 > C2 > C1) (all P < 0.05). The mRNA expression of Nrf2, GCLm and SrX1 was highest in group C3 and lowest in group 1 (C3 > C2 > C1) (all P < 0.05). No significant difference was observed between group C1 and group C (P > 0.05). A Western blot analysis showed that Nrf2, GCLm and SrX1 expression was highest in group C3 and lowest in group C1 (C3 > C2 > C1) (all P < 0.05). No significant difference was observed between group C1 and group C (P > 0.05). CONCLUSIONS: Oxidative stress and antioxidant enzyme levels in the lungs of rats were positively associated with the tidal volumes of mechanical ventilation, suggesting that higher tidal volumes cause more severe oxidative stress and increased antioxidant responses.

Stat3 inhibits Beclin 1 expression through recruitment of HDAC3 in nonsmall cell lung cancer cells.

Recent studies have shown that Beclin 1, a key regulator of autophagic process, is frequently downregulated and may serve as an independent prognostic biomarker for nonsmall cell lung cancer. However, the molecular mechanisms underlying its downregulation remain poorly understood. The signal transducer and activator of transcription 3 (Stat3) is a transcription factor which plays a crucial role for multiple tumor growth and progression. Here, we demonstrate that Beclin 1 is a direct transcriptional target of Stat3 in lung cancer cells. Interleukin-6 (IL-6) treatment or transfection of a constitutively activated Stat3 in AGS and NCI-H1650 cells inhibited Beclin 1 expression. At the molecular level, we further revealed that Stat3 could directly bind to the promoter region of Beclin 1 and repressed its transcription through recruiting histone deacetylase 3 (HDAC3). Collectively, our results suggest that the activated Stat3 may represent an important mechanism for Beclin 1 downregulation in nonsmall cell lung cancer development.

MiR-449c targets c-Myc and inhibits NSCLC cell progression.

MicroRNAs (miRNA) play an important role in tumorigenesis, proliferation, and differentiation. Altered miRNA expression in cancer indicates that miRNAs can function as tumor suppressors or oncogenes. MiR-449c downregulation in non-small cell lung cancer (NSCLC) compared with normal lung tissues was investigated in this study. NSCLC cell proliferation and invasion assays indicate that transfection of miR-449c expression plasmid inhibits the proliferation and invasion ability of NCI-H23 and NCI-H838 cells. In addition, miR-449c overexpression could suppress tumor growth in vivo. Morever, c-Myc was identified as a direct target gene of miR-449c. These findings clearly suggest that miR-449c downregulation and c-Myc amplification may be involved in the development of NSCLC.

[Correlation of P27 expression and localization to phosphorylated AKT in non-small cell lung cancer].

BACKGROUND & OBJECTIVE: Inactivation of tumor suppressor gene plays an important role in tumorigenesis and tumor development. Functional inactivation of the tumor suppressor gene p27 in human cancer occurs either through the loss of expression or through phosphorylation-dependent cytoplasmic mislocalization. AKT, known as protein kinase B, represents a subfamily of serine/threonine protein kinases. Activated AKT (phosphorylated AKT, p-AKT) could promote cell cycle progression by modulating many effectors in cell cycle, including the expression and localization of P27. This study was to investigate the expression and localization of P27 protein in human non-small cell lung cancer (NSCLC), and further to explore its correlation to p-AKT. METHODS: The expression and subcellular localization of P27 and the expression of p-AKT in 80 cases of NSCLC and 35 cases of non-cancerous lung disease were detected by immunohistochemistry, and their correlations to clinicopathologic factors were statistically analyzed. RESULTS: The total and nuclear positive rates of p27 were significantly lower in NSCLC than in non-cancerous lung disease (72.5% vs. 94.3%, 46.3% vs. 94.3%, P<0.01). There were 21 cases of NSCLC with only cytoplasmic expression of P27, while no cytoplasmic expression alone was found in 35 cases of non-cancerous lung disease (P<0.01). The nuclear positive rate of P27 was significantly higher in NSCLC without lymph node metastasis than in NSCLC with lymph node metastasis (P<0.05), and higher in well-and moderately-differentiated NSCLC than in poorly-differentiated NSCLC (P<0.05). The nuclear and cytoplasmic expression of P27 has no correlation to clinicopathologic factors of NSCLC (P>0.05). The positive rate of p-AKT was significantly higher in NSCLC than in non-cancerous lung disease (78.8% vs. 0, P<0.05); it was significantly higher in well-and moderately-differentiated adenocarcinoma than in poorly-differentiated adenocarcinoma (95.0% vs. 50.0%, P<0.05). In NSCLC, the expression of p-AKT was positively correlated to the cytoplasmic expression of P27 (r=0.437, P<0.01), but had no correlation to the nuclear expression of P27 (r=0.175, P>0.05). CONCLUSIONS: Reduced nuclear expression or loss of expression of P27 may be associated to the development of NSCLC. The localization of P27 only in cytoplasm may be one of the characteristics of lung cancer cells. P-AKT might play a role in the cytoplasmic localization of P27, but has no correlation to its nuclear expression.

[Expression of AKT2, cyclin D1, and MMP-9 and their correlations to clinicopathologic features of non-small cell lung cancer].

BACKGROUND & OBJECTIVE: AKT2, a serine/threonine kinase, is confirmed to be an oncogene. Abnormal expression and activation of AKT2 is observed in many kinds of tumor tissues. AKT2 is associated with the proliferation and invasion of cancers. This study was designed to investigate the expression of AKT2, Cyclin D1, and matrix metalloproteinase-9 (MMP-9) in human non-small cell lung cancer (NSCLC) tissue and their correlations to clinicopathologic features of NSCLC. METHODS: The expression of AKT2, Cyclin D1, and MMP-9 in 68 specimens of NSCLC, 38 specimens of corresponding adjacent tissues, and 14 specimens of no-cancerous lung tissue were assessed by immunohistochemistry; their correlations to clinicopathologic factors were analyzed using Chi-square test. RESULTS: The positive rates of AKT2, Cyclin D1, and MMP-9 were significantly higher in NSCLC than in adjacent tissues and no-cancerous lung tissues (91.2% vs. 3.8%, 76.5% vs. 0%, 72.1% vs. 13.5%, P<0.05). The expression of AKT2 wasn't correlated to age, sex, histologic subtype and differentiation, and TNM stage of NSCLC patients (P>0.05), but was correlated to lymph node metastasis (P<0.05). The expression of Cyclin D1 and MMP-9 was correlated to lymph node metastasis and differentiation of squamous cell carcinoma (P<0.05); the expression of MMP-9 was correlated to TNM stage. The expression of AKT2 was positively correlated to the expression of Cyclin D1 and MMP-9 (P<0.05). CONCLUSIONS: AKT2, Cyclin D1, and MMP-9 is related to development of lung cancer. The overexpression of Cyclin D1 and MMP-9 may relate to AKT2 regulation.