LncRNA PROX1 antisense RNA 1 promotes PD-L1-mediated proliferation, metastasis, and immune escape in colorectal cancer by interacting with miR-520d.

It was recently found that lncRNA PROX1 antisense RNA 1 (PROX1-AS1) manifested oncogenicity in a variety of malignancies. This work intended to investigate the molecular mechanisms of PROX1-AS1 in colorectal cancer (CRC) development and immune evasion. In this study, both PROX1-AS1 and PD-L1 expressions were lifted in CRC tissues and cells. PROX1-AS1 interference restrained CRC cell proliferation, migration, invasion, as well as CD8 + T-lymphocyte apoptosis, but increased the cytotoxicity and percentage of CD8 + T lymphocytes. The inhibitory effects of PROX1-AS1 inhibition on CRC progression and immune escape were positively related to PD-L1 suppression. PROX1-AS1 absorbed miR-520d to upregulate PD-L1 expression. PROX1-AS1 facilitated CRC progression and immune escape by targeting miR-520d. Furthermore, PROX1-AS1 deletion impaired CRC tumor growth in vivo . To sum up, this study affirmed that PROX1-AS1 could absorb miR-520d to upregulate PD-L1 in CRC, thereby promoting tumor progression and immune escape.

Duodenal-jejunal bypass reduces serum ceramides via inhibiting intestinal bile acid-farnesoid X receptor pathway.

BACKGROUND: Bile acids play an important role in the amelioration of type 2 diabetes following duodenal-jejunal bypass (DJB). Serum bile acids are elevated postoperatively. However, the clinical relevance is not known. Bile acids in the peripheral circulation reflect the amount of bile acids in the gut. Therefore, a further investigation of luminal bile acids following DJB is of great significance. AIM: To investigate changes of luminal bile acids following DJB. METHODS: Salicylhydroxamic acid (SHAM), DJB, and DJB with oral chenodeoxycholic acid (CDCA) supplementation were performed in a high-fat-diet/streptozotocin-induced diabetic rat model. Body weight, energy intake, oral glucose tolerance test, luminal bile acids, serum ceramides and intestinal ceramide synthesis were analyzed at week 12 postoperatively. RESULTS: Compared to SHAM, DJB achieved rapid and durable improvement in glucose tolerance and led to increased total luminal bile acid concentrations with preferentially increased proportion of farnesoid X receptor (FXR) - inhibitory bile acids within the common limb. Intestinal ceramide synthesis was repressed with decreased serum ceramides, and this phenomenon could be partially antagonized by luminal supplementation of FXR activating bile acid CDCA. CONCLUSION: DJB significantly changes luminal bile acid composition with increased proportion FXR-inhibitory bile acids and reduces serum ceramide levels. There observations suggest a novel mechanism of bile acids in metabolic regulation after DJB.

ISLR affects colon cancer progression by regulating the epithelial-mesenchymal transition signaling pathway.

This study aims to determine the mechanism of ISLR on the progression of colon cancer. TCGA database was used to analyze ISLR expression in colon cancer tumor tissues. QRT-PCR and western blotting were used to detect ISLR expression in colon cancer cells. CCK-8, colony formation, EDU, wound healing and transwell assays were used to measure cell viability, proliferation, migration and invasion of colon cancer cells, respectively. The signaling pathway enrichment analysis of ISLR was analyzed on the basis of the KEGG database. The protein expression of genes related to signaling pathway was measured by western blotting. Results of TCGA analysis, qRT-PC and western blotting showed that ISLR was upregulated in colon cancer tumor tissues and cells. High level of ISLR was related to low overall survival of patients with colon cancer. ISLR silence significantly inhibited cell viability, proliferation, migration and invasion of colon cancer cells. ISLR overexpression markedly enhanced the cell viability, proliferation, migration and invasion of colon cancer cells. KEGG database analyzed showed that ISLR can activate the EMT signaling pathway. Inhibition of the EMT signaling pathway can suppress the growth, migration, and invasion of colon cancer cells and eliminate the promoted effect of ISLR overexpression on colon cancer progression. ISLR promotes the progression of colon cancer by activating the EMT signaling pathway.

Circular RNA SMARCA5 functions as an anti-tumor candidate in colon cancer by sponging microRNA-552.

It was reported that circular RNA (circRNA) circSMARCA5, as a tumor-related molecule, could modulate development of cancers, including prostatic cancer and cervical cancer. Nevertheless, the essential function of circSMARCA5 in colon cancer has not yet been confirmed. We aimed to investigate the role of circSMARCA5 in colon cancer. CircSMARCA5 expression in tumor cells was detected using RT-qPCR. CCK-8, colony formation, flow cytometry and Transwell assays evaluated the influences of circSMARCA5 in colon cancer cells. RT-qPCR, prediction database and luciferase report assay were accomplished for revealing the correlation between circSMARCA5 and miR-552. After transfection with miR-552 mimic, colon cancer cell behaviors were re-evaluated. Wnt and YAP1 pathways were explored by western blot. Our data presented that circSMARCA5 was under-expressed in colon cancer tissues. Transfection with overexpressing circSMARCA5 plasmid restrained growth, migration and invasion of colon cancer cells. Besides, circSMARCA5 directly sponged to miR-552 and miR-552 up-regulation offset the effects of circSMARCA5 on SW480 and SW620 cells. Furthermore, circSMARCA5 inactivated Wnt and YAP1 pathways by inhibiting miR-552. Anti-tumor role of sircSMARCA5 was showed in colon cancer cells as sponging miR-552 and blocking Wnt and YAP1 pathways.

Application of mesenchymal stem cells derived from human pluripotent stem cells in regenerative medicine.

Mesenchymal stem cells (MSCs) represent the most clinically used stem cells in regenerative medicine. However, due to the disadvantages with primary MSCs, such as limited cell proliferative capacity and rarity in the tissues leading to limited MSCs, gradual loss of differentiation during in vitro expansion reducing the efficacy of MSC application, and variation among donors increasing the uncertainty of MSC efficacy, the clinical application of MSCs has been greatly hampered. MSCs derived from human pluripotent stem cells (hPSC-MSCs) can circumvent these problems associated with primary MSCs. Due to the infinite self-renewal of hPSCs and their differentiation potential towards MSCs, hPSC-MSCs are emerging as an attractive alternative for regenerative medicine. This review summarizes the progress on derivation of MSCs from human pluripotent stem cells, disease modelling and drug screening using hPSC-MSCs, and various applications of hPSC-MSCs in regenerative medicine. In the end, the challenges and concerns with hPSC-MSC applications are also discussed.

Downregulated let-7d positively stimulates rectum adenocarcinoma cell malignant biological behaviors by upregulating ATP binding cassette subfamily C member 2.

BACKGROUND: Let-7d has been reported to serve as a tumor suppressor in numerous cancers, however, the function in rectum adenocarcinoma has not been illuminated. In this study, we aimed to explore whether let-7d functions in rectum adenocarcinoma and its functional significance links to ATP binding cassette subfamily C member 2 (ABCC2). METHODS: The expression patterns of let-7d and ABCC2 were gained from TCGA database. Then, cell proliferation, invasion and migration assays were conducted to detect the influence on rectum adenocarcinoma cells behaviors after over-expression of let-7d. Subsequently, the potential target gene of let-7d was predicted and identified through bioinformatics prediction analysis and luciferase reporter assay. Analyses against prognostic value and independent predictor were acquired from Kaplan-Meier, Univariate and Multivariate analysis of Cox regression. Finally, con-transfection experiments were performed to investigate let-7d/ABCC2 pairs function on rectum adenocarcinoma cells after co-transfected with let-7d mimic and si-ABCC2. mRNA and protein levels were assessed by reverse transcription quantitative polymerase chain reaction (qRT-PCR) and western blot. RESULTS: The data from TCGA indicated that let-7d was down-regulated in rectum adenocarcinoma samples, whilst ABCC2 was showed a trend of high expression and its overexpression hinted to worse overall survival of rectum adenocarcinoma patients. Cells proliferation, invasion and migration properties were restrained after over-expression of let-7d in SW837 cells. Further investigations showed that over-expression of let-7d induced the inhibitory effect on SW837 cells proliferative, migrant and invasive capacities was augmented by silencing ABCC2. CONCLUSIONS: All results in this study indicated that up-regulation of let-7d could suppress SW837 cells growth, invasion and migration abilities by reducing ABCC2 expression, providing a new insight into molecular mechanism of let-7d/ABCC2 as a significant mediator for tumor progression and development of rectum adenocarcinoma.

Sperm nuclear transfer and transgenic production in the fish medaka.

Sperm nuclear transfer or intracytoplasmic sperm injection (ICSI) is a powerful assisted reproductive technology (ART) for treating human male infertility. Controversial reports of increased birth defects have raised concerns about the ART's safety. The cause for birth defects, however, has remained elusive for analysis in human because of the sample size, male infertility genetics, physiological heterogeneity and associated procedures such as embryo manipulations. Animal models are required to evaluate factors leading to the increased birth defects. Here we report the establishment of medakafish model for ICSI and transgenic production. This small laboratory fish has high fecundity and easy embryology. We show that ICSI produced a 5% high percentage of fertile animals that exhibited both paternal and maternal contribution as evidenced by the pigmentation marker. Furthermore, when sperm were pre-incubated with a plasmid ubiquitously expressing RFP and subjected to ICSI, 50% of sperm nuclear transplants showed germline transmission. We conclude that medaka is an excellent model for ICSI to evaluate birth defects and that sperm nuclear transfer can mediate stable gene transfer at high efficiency. Although more demanding for experimentation, sperm-mediated transgenesis should be particularly applicable for aquaculture species with a lengthy generation time and/or a large adult body size.

Sequence context- and temperature-dependent nucleotide excision repair of a benzo[a]pyrene diol epoxide-guanine DNA adduct catalyzed by thermophilic UvrABC proteins.

The influence of DNA base sequence context on the removal of a bulky benzo[a]pyrene diol epoxide-guanine adduct, (+)-trans-B[a]P-N2-dG (G*), by UvrABC nuclease from the thermophilic organism Bacillus caldotenax was investigated. The lesion was flanked by either T or C in otherwise identical complementary 43-mer duplexes (TG*T or CG*C, respectively). It was reported earlier that in the CG*C context, a dominant minor groove adduct structure was observed by NMR methods with all Watson-Crick base pairs intact, and the duplex exhibited a rigid bend. In contrast, in the TG*T context, a highly flexible bend was observed, base pairing at G*, and two 5'-base pairs flanking the adduct were impaired, and multiple solvent-accessible adduct conformations were observed. The TG*T-43-mer duplexes are incised with consistently greater efficiency by UvrABC proteins from B. caldotenax by a factor of 2.3 +/- 0.3. The rates of incisions increase with increasing temperature and are characterized by linear Arrhenius plots with activation energies of 27.0 +/- 1.5 and 23.4 +/- 1.0 kcal/mol for CG*C and TG*T duplexes, respectively. These values reflect the thermophilic characteristics of the UVrABC nuclease complex and the contributions of the different DNA substrates to the overall activation energies. These effects are consistent with base sequence context-dependent differences in structural disorder engendered by a loss of local base stacking interactions and Watson-Crick base pairing in the immediate vicinity of the lesions in the TG*T duplexes. The local weakening of base pairing interactions constitutes a recognition element of the UvrABC nucleotide excision repair apparatus.

Activation of the mouse Oct4 promoter in medaka embryonic stem cells and its use for ablation of spontaneous differentiation.

The determination and maintenance of the cell fate is ultimately due to differential gene activity. In the mouse, expression of the transcription factor Oct4 is high in totipotent inner cell mass, germ cells and undifferentiated embryonic stem (ES) cells, but dramatically reduced or extinct upon differentiation. Here, we show that medaka blastula embryos and cells of the ES cell line MES1 are able to activate the Oct4 promoter. Ectopic expression of a fusion gene for beta-galactosidase and neomycin resistance from the Oct4 promoter conferred resistance to G418. G418 selection led to a homogeneous population of undifferentiated ES cells which were able to undergo induced or directed differentiation into various cell types including neuron-like cells and melanocytes. Furthermore, GFP-labeled GOF18geo-MES1 cells after differentiation ablation were able to contribute to a wide variety of organ systems derived from all the three germ layers. Most importantly, we show that drug ablation of differentiation on the basis of Oct4 promoter is a useful tool to improve ES cell cultivation and chimera formation: MES1 cells after differentiation ablation appeared to be better donors than the parental MES1 line, as the permissive number of input donor cells increases from 100 to 200, resulting in an enhanced degree of chimerism. Taken together, some transcription factors and cis-acting regulatory sequences controlling totipotency-specific gene expression appear to be conserved between mammals and fish, and medaka ES cells offer an in vitro system for characterizing the expression of totipotency-specific genes such as putative Oct4 homologs from fish.

Establishment of a normal medakafish spermatogonial cell line capable of sperm production in vitro.

Spermatogonia are the male germ stem cells that continuously produce sperm for the next generation. Spermatogenesis is a complicated process that proceeds through mitotic phase of stem cell renewal and differentiation, meiotic phase, and postmeiotic phase of spermiogenesis. Full recapitulation of spermatogenesis in vitro has been impossible, as generation of normal spermatogonial stem cell lines without immortalization and production of motile sperm from these cells after long-term culture have not been achieved. Here we report the derivation of a normal spermatogonial cell line from a mature medakafish testis without immortalization. After 140 passages during 2 years of culture, this cell line retains stable but growth factor-dependent proliferation, a diploid karyotype, and the phenotype and gene expression pattern of spermatogonial stem cells. Furthermore, we show that this cell line can undergo meiosis and spermiogenesis to generate motile sperm. Therefore, the ability of continuous proliferation and sperm production in culture is an intrinsic property of medaka spermatogonial stem cells, and immortalization apparently is not necessary to derive male germ cell cultures. Our findings and cell line will offer a unique opportunity to study and recapitulate spermatogenesis in vitro and to develop approaches for germ-line transmission.

The toxic effects of microcystin-LR on embryo-larval and juvenile development of loach, Misguruns mizolepis Gunthe.

Microcystin-LR, a specific and potent hepatotoxin, was tested for its effects on loach embryo-larval and juvenile development. The results of this study showed that loach embryos were more sensitive when exposed to microcystin-LR at a later than at an earlier stage of development. Juveniles were far less sensitive to MC-LR than were embryos and larvae. Mortality and developmental abnormality were proven to be dose-dependent and to be stage-specific sensitive. Among the abnormal changes noted were: pericardial edema and tubular heart, bradycardia, homeostasis, poor yolk resumption, small head, curved body and tail, and abnormal hatching. Liver and heart were the main targets of microcystin-LR toxicity. Ultrastructural analysis documented a complex set of sublethal effects of microcystin-LR on loach hepatocytes, chiefly including morphological alteration in nuclear and RER of loach liver cells. In addition, microcystin-LR was lethal to loach juvenile in the subacute (7 days) exposure (LC(50)=593.3 microg/l).