Pitx controls amphioxus asymmetric morphogenesis by promoting left-side development and repressing right-side formation.

BACKGROUND: Left-right (LR) asymmetry is an essential feature of bilateral animals. Studies in vertebrates show that LR asymmetry formation comprises three major steps: symmetry breaking, asymmetric gene expression, and LR morphogenesis. Although much progress has been made in the first two events, mechanisms underlying asymmetric morphogenesis remain largely unknown due to the complex developmental processes deployed by vertebrate organs. RESULTS: We here addressed this question by studying Pitx gene function in the basal chordate amphioxus whose asymmetric organogenesis, unlike that in vertebrates, occurs essentially in situ and does not rely on cell migration. Pitx null mutation in amphioxus causes loss of all left-sided organs and incomplete ectopic formation of all right-sided organs on the left side, whereas Pitx partial loss-of-function leads to milder phenotypes with only some LR organs lost or ectopically formed. At the N1 to N3 stages, Pitx expression is gradually expanded from the dorsal anterior domain to surrounding regions. This leads to activation of genes like Lhx3 and/or Prop1 and Pit, which are essential for left-side organs, and downregulation of genes like Hex and/or Nkx2.1 and FoxE4, which are required for right-side organs to form ectopically on the left side. In Pitx mutants, the left-side expressed genes are not activated, while the right-side genes fail to decrease expression on the left side. In contrast, in embryos overexpressing Pitx genes, the left-side genes are induced ectopically on the right side, and the right-side genes are inhibited. Several Pitx binding sites are identified in the upstream sequences of the left-side and right-side genes which are essential for activation of the former and repression of the latter by Pitx. CONCLUSIONS: Our results demonstrate that (1) Pitx is a major (although not the only) determinant of asymmetric morphogenesis in amphioxus, (2) the development of different LR organs have distinct requirements for Pitx activity, and (3) Pitx controls amphioxus LR morphogenesis probably through inducing left-side organs and inhibiting right-side organs directly. These findings show much more dependence of LR organogenesis on Pitx in amphioxus than in vertebrates. They also provide insight into the molecular developmental mechanism of some vertebrate LR organs like the lungs and atria, since they show a right-isomerism phenotype in Pitx2 knockout mice like right-sided organs in Pitx mutant amphioxus. Our results also explain why some organs like the adenohypophysis are asymmetrically located in amphioxus but symmetrically positioned in vertebrates.

Circular RNA ITCH Suppresses Cell Proliferation but Induces Apoptosis in Oral Squamous Cell Carcinoma by Regulating miR-421/PDCD4 Axis.

BACKGROUND: Circular RNAs (circRNAs), a group of covalently closed non-coding RNAs, serve critical regulatory roles in many human cancers, including oral squamous cell carcinoma (OSCC). The purpose of this study was to investigate the functional role of circular RNA ITCH (circ-ITCH) in OSCC and the underlying mechanisms. METHODS: RT-qPCR analysis was applied to detect the expression levels of circ-ITCH in OSCC tissues and cell lines. MTT assay and flow cytometer analysis were used to evaluate the effects of circ-ITCH overexpression on the proliferation and apoptosis of OSCC cells. Bioinformatics analysis and dual-luciferase reporter assay were applied to determine the binding relation between circ-ITCH and miR-421 as well as PDCD4 mRNA and miR-421. RESULTS: Our results showed that circ-ITCH expression was remarkably decreased in OSCC tissues and cell lines. Low circ-ITCH expression was strongly associated with adverse clinicopathological characteristics of OSCC patients. Moreover, functional assays demonstrated that circ-ITCH overexpression significantly inhibited OSCC cell proliferation and induced cell apoptosis. Our data further uncovered that circ-ITCH could bind directly to miR-421 and block its repression on PDCD4 in OSCC. MiR-421 expression was significantly increased in OSCC tissues and was inversely correlated with circ-ITCH expression. Notably, miR-421 restoration blocked the tumor-suppressive role of circ-ITCH in OSCC cells. CONCLUSION: In conclusion, our study reveals that circ-ITCH serves as a tumor suppressor in OSCC partly by regulating miR-421/PDCD4 axis.

Molecular cloning and expression analysis of inhibitor of growth protein 3 (ING3) in the Manila clam, Ruditapes philippinarum.

Inhibitor of growth protein 3 (ING3), a new member of ING family, is involved in the regulation of various processes. In this study, a full-length cDNA of ING3 (named as RpING3) was cloned from the gill of Ruditapes philippinarum by rapid amplification of cDNA ends method for the first time. The cDNA obtained was 1442 bp exclusive of poly (A) residues with a 1248 bp open reading frame encoding 415 amino acids. The RpING3 protein has a calculated molecular weight of 46.59 kDa and isoelectric point of 6.62. Two conserved motif and some functional sites were found. Tissue distribution analysis of the RpING3 mRNA revealed that the RpING3 expression level was much higher in gill and digestive gland while lower in mantle, foot and adductor muscle. The temporal expression of RpING3 in digestive gland after lead exposure was recorded by quantitative real-time PCR. The result showed that RpING3 was rapidly up-regulated at 6 h post-exposure and reached tenfold of the control group. These results suggest that RpING3 dependent signaling pathway is present in Manila clam and RpING3 may play important roles in protecting cells from heavy metal damage in R. philippinarum.

[Comparison of acellular bovine pericardium material with collagen membrane in guiding bone regeneration].

OBJECTIVE: To compare the effect of guiding bone regeneration between 1-ethyl-3(3-diaminopropyol)-carbodiimide (EDAC)-crosslinked acellular bovine pericardium (ABP) and medical collagen membrane (CM). METHODS: Defects of 7 mm X 7 mm x 5 mm were created in both mandibles of 24 rabbits, which weighted 2.6-3.5 kg. One side defect was covered with EDAC-crosslinked ABP(EDAC-crosslinked ABP group), the other side defect with medical CM as control(CM group). The ability of bone defect repair and change of both membrane materials were evaluated by gross observation, histological study and computer graphic analysis in the 4th, 8th, 16th and 24th weeks after operation. RESULTS: The surface of bone defects was even, consistent with adjacent normal bone in EDAC-crosslinked ABP group, while that of bone defects was of no evenness in CM group in the 16th and the 24th weeks. The histological observation showed that bone trabecula formed in the EDAC-crosslinked ABP group and fibrous connective tissue was seen in CM group in the 16th and the 24th weeks. There were no significant differences in new bone percentage of bone defects between 2 groups in the 4th and the 8th weeks (P > 0.05). In the 16th week new bone percentage of bone defects was 81.99% +/- 3.92% in EDAC-crosslinked ABP group and 76.35% +/- 4.29% in CM group, showing significant difference (P < 0.05). The average percentage of absorption in EDAC-crosslinked ABP group was 16.57%, 27.94%, 65.61 and 85.72% in the 4th, 8th, 16th and 24th weeks respectively, while that in CM group was more than 50% in the 4th week and completely degraded at the end of 8 weeks. CONCLUSION: EDAC-crosslinked ABP has a better effect on guiding bone regeneration than CM in the repair of bone defects.

[Experimental study on acellular bovine pericardium guided bone regeneration material].

OBJECTIVE: To choose the best procedure on preparation of acellular bovine pericardium (ABP) guided bone regeneration (GBR) material. METHODS: The BP was decellularized with 0. 25% Trypsin + 0.5% Triton X-100. The acellular bovine pericardiums (ABPs) were treated with phosphate-buffered saline(PBS) (group A), 95% glycerol (group B), EDAC (group C), and EDAC and 95% glycerol (group D) respectively. The treated ABPs were implanted subcutaneously in the back of SD rats respectively at random and no material was implanted as control. Seven rats were sacrificed at 2 weeks, twelve at 4 weeks, twelve at 8 weeks, seven at 16 weeks. Local reaction was studied grossly. The amount of antigen presenting cell (APC) and the percentage of ABP degeneration were reckoned by images analysis system. RESULTS: The ABPs were replaced by fibroblasts completely in group A at 8 weeks, in group C at 16 weeks, but only less than 50% till 16 weeks in groups B and D. In all groups, the depth of surrounding fibres attenuated time-dependently. The APC amount of the groups B and D was higher than that of the control group, and the ABP of the groups B and D degraded partly at 16 weeks. CONCLUSION: The ABP treated with EDAC can be replaced by the surrounding tissues and has good biocompatibility.