MARCH5 promotes aerobic glycolysis to facilitate ovarian cancer progression via ubiquitinating MPC1.

MARCH5 is a ring-finger E3 ubiquitin ligase located in the outer membrane of mitochondria. A previous study has reported that MARCH5 was up-regulated and contributed to the migration and invasion of OC cells by serving as a competing endogenous RNA. However, as a mitochondrial localized E3 ubiquitin ligase, the function of MARCH5 in mitochondrial-associated metabolism reprogramming in human cancers remains largely unexplored, including OC. We first assessed the glycolysis effect of MARCH5 in OC both in vitro and in vivo. Then we analyzed the effect of MARCH5 knockdown or overexpression on respiratory activity by evaluating oxygen consumption rate, activities of OXPHOS complexes and production of ATP in OC cells with MARCH5. Co-immunoprecipitation, western-blot, and in vitro and vivo experiments were performed to investigate the molecular mechanisms underlying MARCH5-enhanced aerobic glycolysis s in OC. In this study, we demonstrate that the abnormal upregulation of MARCH5 is accompanied by significantly increased aerobic glycolysis in OC. Mechanistically, MARCH5 promotes aerobic glycolysis via ubiquitinating and degrading mitochondrial pyruvate carrier 1 (MPC1), which mediates the transport of cytosolic pyruvate into mitochondria by localizing on mitochondria outer membrane. In line with this, MPC1 expression is significantly decreased and its downregulation is closely correlated with unfavorable survival. Furthermore, in vitro and in vivo assays revealed that MARCH5 upregulation-enhanced aerobic glycolysis played a critical role in the proliferation and metastasis of OC cells. Taken together, we identify a MARCH5-regulated aerobic glycolysis mechanism by degradation of MPC1, and provide a rationale for therapeutic targeting of aerobic glycolysis via MARCH5-MPC1 axis inhibition.

Rs6757 in microRNA-3976 binding site of CD147 confers risk of hepatocellular carcinoma in South Chinese population.

BACKGROUND: Cluster of differentiation 147 (CD147) overexpression plays a key role in the proliferation, differentiation, invasion, metastasis, and prognosis of hepatocellular carcinoma (HCC). The aim of this study was to explore the relationship between rs6757 and the HCC risk in the South Chinese population, and the functional significance of rs6757 by affecting the efficacy of microRNA-3976 (miR-3976) binding to the CD147 3'-UTR. METHODS: We performed a retrospective case-control study to analyze the association between rs6757 and the risk of HCC. We chose candidate microRNAs with the potential of interacting with rs6757 through a series of silico analyses. A luciferase reporter gene assay was implemented to detect the binding extent of microRNAs to each polymorphic allele of rs6757. RESULTS: An obvious association between rs6757 and the risk of HCC was detected in C vs. T (OR = 1.826, 95% CI [1.263-2.642]), CC vs. TT (OR = 4.513, 95% CI [1.510-13.489]), dominant genetic model (OR = 1.824, 95% CI [1.120-2.965]), and recessive genetic model (OR = 3.765, 95% CI [1.286-11.020]). Bioinformatics analysis indicated that miR-3976 binding sites containing the rs6757-T allele had lower free energies than those with the C allele, the lower free energies, the higher affinities. Luciferase activity was remarkably decreased by miR-3976 binding to the CD147 3'-UTR bearing rs6757 T allele, which could be reversed by miR-3976 inhibitors. Furthermore, miR-3976 reduced the luciferase expression in a manner of dose-dependent when cotransfected with constructs with the CD147-TT-pSICHECK2. CONCLUSIONS: The research we have done suggests that rs6757 confers the CD147 allele-specific translational suppression by miR-3976, which provides a theoretical basis for antineoplastic therapy targeting CD147.

Identification of two novel insertion abnormal transcripts in two Chinese families affected with Dystrophinopathy.

BACKGROUND: Duchenne muscular dystrophy (DMD) is an X-linked recessive inheritance muscle dystrophy disease, associated with pathogenic variants in the DMD gene. MLPA, DHPLC and DMD sequence studies fail to found the causative alteration in two cases. This study intends to evaluate the disease-causing mutations and explains the correlation genotype-phenotype. METHODS: The mRNA analysis and Long-range PCR with sequencing were used for molecular diagnosis. RESULTS: In case one, an insertion of 78 nucleotides between exons 40 and 41 (r.5739_5740insMN602429:r415_492) was identified in case one. The insertion sequences were highly homologous to the intron 40 (NG_012232.1:g.1001760_g.1001837). Long-range PCR with sequencing analysis showed that a novel deep intronic DMD mutation (NG_012232.1:g.1001838A>G) was identified, generating a premature stop codon and terminating protein translation. The likely pathogenic mutation was detected in fetal sample. In case two, an insertion of 74 nucleotides which located inside the consensus sequence AG/GT was detected between exons 2 and 3 (r.93_94insMN584887:r61_134), which resulted in a premature stop codon. The insertion sequences were traceable in the intron 2 of DMD gene (NG_012232.1:g.415926_g.415999). We did not perform prenatal DMD gene diagnosis for case two due to lack of sufficient genetic information. CONCLUSION: These findings clarify importance of proceeding to the mRNA analysis when no causative mutations were found neither by MLPA/DHPLC nor gene sequencing so as to reach the molecular confirmation of DMD and carry out an accurate genetic assessment/ carrier status testing.

Composition of Bacterial and Archaeal Communities in an Alkali-Surfactant-Polyacrylamide-Flooded Oil Reservoir and the Responses of Microcosms to Nutrients.

The microbial communities in alkali-surfactant-polyacrylamide-flooded (ASP-flooded) oil reservoirs have rarely been investigated compared to those in water-flooded oil reservoirs. Here, the bacterial and archaeal communities in an ASP-flooded reservoir and the adjacent water-flooded block, and responses of the microbial communities in microcosms to nutrients were investigated by 16S rRNA gene sequencing and cultivation. Compared with the water-flooded block, both the bacterial and archaeal communities inhabiting the ASP-flooded block had lower Sobs indices (91:232 and 34:55, respectively), lower Shannon indices (1.296:2.256 and 0.845:1.627, respectively) and higher Simpson indices (0.391:0.248 and 0.678:0.315, respectively). Halomonas (58.4-82.1%) and Anoxynatronum (14.5-18.2%) predominated in the ASP-flooded production wells, and were less than 0.05% in the bacterial communities of the adjacent water-flooded production wells, which were dominated by Pseudomonas and Thauera. Methanobacterium accounted for 65.0-94.5% of the archaeal communities inhabiting the ASP-flooded production wells, and Methanosaeta (36.7-94.5%) dominated the adjacent water-flooded production wells. After nutrients stimulation, the quantity of cultivable microorganisms increased from 103/mL to 107/mL. Community analysis indicated that the relative abundances of some species that belonged to Halomonas and Pseudomonas obviously increased, yet there were no oil emulsification or dispersion and changes of surface tension of the water-oil mixture. In addition, 6 alkali-tolerating strains showing 98% similarity of 16S rRNA genes with those of Halomonas alkalicola and Halomonas desiderata and 2 strains with 99% similarity with Pseudomonas stutzeri gene were isolated from the nutrients stimulated brines. In summary, this study indicated that Halomonas, Anoxynatronum, and Methanobacterium were dominant populations in the ASP-flooded reservoir, the extreme environment decreased microbial diversity, and restricted microbial growth and metabolisms.

Aloperine Activates the PI3K/Akt Pathway and Protects Against Coronary Microembolisation-Induced Myocardial Injury in Rats.

BACKGROUND: Coronary microembolisation (CME)-induced myocardial apoptosis is a key factor in progressive cardiac dysfunction. Aloperine (ALO) plays a protective role in the cardiovascular system, but its role and the mechanism -underlying its protection against CME are unclear. Therefore, we aimed to verify whether ALO has a protective effect against CME-induced myocardial injury, as well as whether this effect has a relationship with regulation of the PI3K/Akt pathway for rats. METHODS: Forty Sprague-Dawley rats were randomised into 4 equal groups: CME, CME + ALO, CME + ALO + LY294002 (LY) and a Sham group. Twelve hours after surgery, the rats' cardiac function, apoptosis index, microinfarct and serum cardiac-troponin I (cTnI) level were measured. Levels of p-Akt, total Akt, Bcl-2, Bax and cleaved caspase-3 were detected. RESULTS: ALO improved cardiac dysfunction induced by CME, while also decreasing serum levels of cTnI and microinfarct areas. In addition, ALO inhibited myocardial apoptosis, which may have been partially as a result of downregulated cleaved caspase-3 and Bax, upregulated Bcl-2 and increased protein levels in phosphorylated Akt. However, these ALO effects were blocked if ALO was administered along with LY. CONCLUSIONS: ALO can inhibit cardiomyocyte apoptosis and consequently attenuate CME-induced myocardial injury. These functions are realised by activating PI3K/Akt signalling pathway.

Role of near-infrared heptamethine cyanine dye IR-783 in diagnosis of cervical cancer and its mechanism.

Near-IR fluorescence imaging is a novel modality and has great potential for detection of tumors. The goal of this study was to evaluate the value of the IR-783 dye, a near-infrared heptamethine carbocyanine, as an imaging agent for rapid detection of human cervical cancer and the underlying mechanism of IR-783 mediated imaging. The imaging was investigated in cervical cancer cells for detecting the uptake, accumulation and subcellular localization of IR-783 dye. Whole-body imaging of mice bearing human cervical cancer xenografts and freshly harvested clinical cervical cancer specimens were used for assessment of specific dye uptake and retention. Frozen tissue section was used for confirming the dye accumulation at the tissue and cellular levels. The detection of circulating tumor cells in peripheral blood spiked with cervical cancer cells was made. The results revealed that IR-783 dye could be specifically uptaken by cultured cervical cancer cells, human cervical cancer cell-spiked whole blood, human cervical cancer xenografts and freshly harvested human cervical cancer tissue, but not by normal cell tissues. In the present study, we proved that IR-783 has potential for detecting cervical cancer cells in clinical specimens and in circulating blood. It can be further developed as modality agent for deep tissue imaging of cervical cancer in clinic.

Platelet Distribution Width in Hepatocellular Carcinoma.

BACKGROUND Platelets and platelet activation are involved in the critical steps of tumor cell growth, metastasis, and angiogenesis. In this study, we analyzed the correlation between platelet distribution width (PDW) and hepatocellular carcinoma (HCC). MATERIAL AND METHODS HCC patients at the First Affiliated Hospital of Guangxi Medical University, China, from January 2017 to October 2017, were analyzed retrospectively. The control group comprised healthy persons at the hospital for medical check-ups during the same period. The Mann-Whitney U test was performed to compare the differences in relevant laboratory parameters between the HCC and control groups. Differences in PDW at different stages of HCC were calculated by using one-way analysis of variance. The Spearman correlation analysis was used to analyze the correlations between the PDW and relevant experimental parameters in HCC patients. RESULTS The platelet distribution width (PDW), mean platelet volume (MPV), and neutrophil-to-lymphocyte ratio (NLR) were higher in HCC patients than in the control group. The platelet count (P), absolute lymphocyte count (L), absolute neutrophil count (N), and platelet-to-lymphocyte ratio (PLR) were distinctly lower in the HCC patients than in the control group. There were significant differences in the PDW between the 4 stages of HCC. The Spearman correlation analysis demonstrated that the PDW was positively correlated with the cancer stage, alpha fetoprotein (AFP), and MPV, and negatively correlated with the platelet-to-lymphocyte ratio (PLR) and platelet count (P). The area under the receiver-operating characteristic curve of the platelet distribution width-to-platelet count ratio was 0.727 (95% confidence interval 0.691-0.762). CONCLUSIONS PDW is associated with HCC and may be a potential marker for its progression.

Increased serum concentrations of asymmetric dimethylarginine (ADMA) in patients with early-onset coronary artery disease.

BACKGROUND: Asymmetric dimethylarginine (ADMA) has been associated with an increased risk of cardiovascular disease. We investigated the role of serum ADMA concentrations in early-onset coronary artery disease (EOCAD). METHODS: Candidates for coronary artery angiography (age<50y for men and <55y for women) who met the inclusion criteria were enrolled in this study. Serum concentrations of ADMA were determined using ELISA. Severity of coronary atherosclerosis was estimated by number of diseased vessels. RESULTS: A total of 601 subjects (286 with EOCAD patients and 315 controls) were included in the study. ADMA concentrations were found to be significantly higher in the EOCAD group (0.480±0.110µmol/l) than in the control group (0.457±0.091, P=0.007). ADMA concentrations significantly increased with the number of diseased vessels (P<0.001). In addition, serum ADMA concentrations were affected by diabetes mellitus and smoking status, and were positively correlated with serum creatinine and body mass index (BMI). CONCLUSIONS: Our results show that serum ADMA concentrations were associated with the presence and severity of EOCAD, suggesting that ADMA may be involved in the progression of EOCAD.

[C1q and tumor necrosis factor related protein 4 (CTRP4) suppresses caspase-1/IL-1ß inflammatory pathway in trophoblasts of rat models with preeclampsia].

Objective To explore the effect of C1q/tumor necrosis factor related protein 4 (CTRP4) on the placental trophoblasts of preeclampsia model rats. Methods Placental trophoblastic tissues were respectively collected from normal pregnant rats and model rats with preeclampsia, and then mRNA and protein expression levels of CTRP4, interleukin 1ß (IL-1ß), and caspase-1 were detected with quantitative real-time PCR (qRT-PCR) and Western blotting. Primary placental trophoblasts were isolated from normal pregnant rats and model rats; at different time points, flow cytometry was used to detect the number of PI+caspase-1+ pyroptotic cells; and qRT-PCR and Western blotting were used to detect expression levels of IL-1ß and caspase-1. Finally, recombinant CTRP4 protein (at the doses of 0.5, 5, 15, 25 or 50 ng/mL) or neutralizing CTRP4 antibody (at the doses of 10 or 20 ng/mL) were added into the medium of trophoblasts from model rats; after incubation for 72 h, the number of pyroptotic cells and the expression levels of IL-1ß and caspase-1 were detected. ResultsCaspase-1/IL-1ß inflammatory pathway was activated and CTRP4 expression was downregulated in placenta trophoblastic tissue from rats with preeclampsia. CTRP4 recombinant protein treatment significantly inhibited pyroptosis and the caspase-1/IL-1ß pathway in trophoblasts derived from rats with preeclampsia, while CTRP4 neutralizing antibody treatment had an opposite effect on pyroptosis and inflammation. Conclusion CTRP4 can significantly inhibit the activation of caspase-1/IL-1ß inflammatory pathway, and suppress the pyroptosis of trophoblasts derived from rats with preeclampsia.

Mitochondrial membrane potential and reactive oxygen species in cancer stem cells.

Cancer stem cells (CSCs) are believed as the initiators of the occurrence, development and recurrence of malignant tumors. Targeting this unique cell population would provide a less toxic approach than regular chemotherapeutic agents that kill bulk rapid proliferating tumor cells and also normal cells which divide rapidly. To date, major research effort has been aimed at identifying and eradicating CSC population. The metabolism heterogeneity of mitochondria in CSCs shows a big promise for cancer research. Of them, mitochondrial membrane potential (Δψm), reflecting the functional status of the mitochondrion is proved to be highly related to cancer malignancy. Reactive oxygen species, mainly produced from mitochondria, are also increased in many types of cancer cells. However, their statuses in CSCs remain poorly understood. Here we shall review the mitochondrial membrane potential and reactive oxygen species of CSCs and propose the novel potential targets for cancer therapy.

[A pilot study on transdifferentiation of skin stem cell in reconstructing corneal epithelium].

OBJECTIVE: To observe the differentiation and development of skin stem cells on corneal stroma and to discuss the possibility of reconstructing corneal epithelium with skin stem cells. METHODS: Pieces of human and rabbit skin were obtained during operation. Rabbit eye balls were taken, and pieces of corneal stroma without epithelium were prepared. Skin stem cells from the rabbit skin and human skin were cultured. The human skin stem cells of the first generation to 4th generation were implanted on the rabbit corneal stroma and cultured. Three rabbits underwent autotransplantation of the rabbit skin stem cells of the first generation to 4th generation on the pieces of corneal stroma with the superficial lamina removed and then fed for 100 approximately 114 days. Another 3 rabbits underwent allotransplantation of the rabbit skin stem cells of first to 4th generation on the pieces of corneal stroma with the superficial lamina removed and then fed for 100 days. Then the rabbits were killed and their eye balls taken out. The rabbit corneas implanted with human or rabbit epithelial cells and the rabbit corneas with the autogeneous or heterogeneous epithelial cells were sliced and underwent immunohistochemistry with human AE5 antibody corresponding to the specific surface marker keratin K3/K12 common to humankind and rabbit, and human epithelial cell keratin K-19 monoclonal antibody. RESULTS: Since the 3(rd) day of transplantation the transplanted human epithelial cells formed multiplayer and were human AE5 antibody and human K19 monoclonal antibody positive. The autotransplanted corneas remained basically transparent without obvious vascular hyperplasia till the cornea specimens were taken. Histological examination showed intact multiplayer epithelium and immunohistochemistry showed human AE5 positive. The allotransplanted\rabbit corneas showed congestion since the 9(th) day. Histological examination showed that the corneas were nor so transparent as the autotransplanted ones and the epithelium was nor intact with a lot of lymphocyte infiltration. CONCLUSION: Corneal epithelium can be reconstructed from skin stem cell, which may be an alternative for constructing autogeneous bioengineered corneas.

[Experimental study on promoting the engraftment of hematopoietic stem/progenitor cell by all-trans retinoic acid].

OBJECTIVE: To explore the effect of all-trans retinoic acid (RA) on the engraftment of unrelated umbilical cord blood stem/progenitor cell transplantation (UCBT) in murine model. METHODS: 1 x 10(6) and 0.5 x 10(6) nucleated cells (NC) from C57BL/6 (H-2(b)) fetal and neonatal peripheral blood (FNPB) were separately transfused into lethally cyclophosphamide (380 mg/kg, ip) treated BALB/C (H-2(d)) recipients, 15 mg.kg(-1).d(-1) and 5 mg.kg(-1).d(-1) RA (15 mg and 5 mg RA) were administrated respectively 2 days before and after UCBT. Hematopoiesis and immune recovery, graft versus host disease (GVHD), engraftment and survival rates were then observed. RESULTS: Hematopoiesis and immune recovery occurred faster in RA treated than in untreated mice (P < 0.05). Acute GVHD was absent. The levels of engraftment were higher in both 15 mg and 5 mg RA treated mice than those in untreated controls (P < 0.05). In 1 x 10(6) NC transfused mice, 15 mg and 5 mg RA could significantly increased the 30 and 60 days survival rates from 41.67% (without RA) to 72.23% and 70.83%, respectively (P < 0.05). In 0.5 x 10(6) cells transfused mice, 15 mg and 5 mg RA increased the survival rate from 14.29% (without RA) to 42.86% and 43.48%, respectively (P < 0.05), which were comparable to that of being transfused 1 x 10(6) cells without RA treatment (P > 0.05). CONCLUSION: RA enhances the engraftment of umbilical cord blood stem/progenitor cells in murine model for UCBT. This might provide an experimental evidence of RA in clinical UCBT.

[Establishment of a murine model for allogeneic umbilical cord blood transplantation].

This study was undertaken to establish a murine model for unrelated allogeneic umbilical cord blood transplantation (UCBT). The characteristics and percentage of hematopoietic stem/progenitor cells between near-term fetal and neonatal murine peripheral blood (FNPB) and bone marrow (BM) were evaluated by flow cytometry and semisolid methylcellulose culture. BABL/c (H-2(d)) recipient mice conditioned with high dose CTX were transplanted with FNPB form C57BL/6 (H-2(b)) mice and the survival rate, hematopoietic and immunological reconstruction, graft versus host disease (GVHD) and engraftment level were observed. The results showed that the numbers of day 14 CFU-GM and CFU-GEMM in FNPB (176.40 +/- 78.39)% and (141.40 +/- 56.57)%, respectively were much higher than those in BM (75.20 +/- 26.41)% and (68.80 +/- 23.95)%, respectively. Moreover the percentage of Sca-1(+) CD34(+) cell subsets in FNPB (3.63 +/- 1.13)% was also higher than that in BM (1.41 +/- 0.8 7)%. FNPB transplantation improved survival rate and reconstituted hematopoietic and immune function in recipients. There was no evidence of GVHD. Chimeric analysis showed that the proportion of donor cells in BM of recipients was 27.94% at 21 days after transplantation. It was concluded that FNPB contains more hematopoietic stem and progenitor cells with high expansion ability and weak allogeneic immunity, which was similar to human UCB. The murine model for allogeneic UCBT (C57BL/6-->BALB/c) was established successfully.