Tumor-Derived Exosomal Circular RNA Pinin Induces FGF13 Expression to Promote Colorectal Cancer Progression through miR-1225-5p.

Background/Aims: : Colorectal cancer (CRC) is a common malignant tumor, and circular RNAs (circRNAs) are abnormally expressed in CRC. However, the function and underlying mechanism of circRNA pinin (circ-PNN; hsa_circ_0101802) in CRC remain unclear. Methods: : Exosomes were isolated from the plasma of CRC patients and identified by transmission electron microscopy and Western blotting. The RNA expression levels of circ-PNN, miR-1225-5p, and fibroblast growth factor 13 (FGF13) were measured by quantitative real-time polymerase chain reaction. Cell proliferation was detected by Cell Counting K-8, colony formation, and 5-ethynyl-2'-deoxyuridine assays. Cell apoptosis was assessed by flow cytometry. The expression of apoptosis and metastasis-related proteins was evaluated by Western blotting. The associations among circ-PNN, miR-1225-5p, and FGF13 were confirmed by dual-luciferase report assay and RNA immunoprecipitation assay. A xenograft model was used to verify the function of circ-PNN in tumor formation in vivo. Results: : circ-PNN expression was upregulated in plasmic exosomes derived from CRC patients. The expression of circ-PNN and FGF13 was upregulated, while miR-1225-5p expression was downregulated in CRC cells incubated with plasmic exosomes derived from CRC patients. Tumor-derived exosomes promoted the proliferation, migration, and invasion but inhibited apoptosis of CRC cells. Moreover, the addition of tumor-derived exosomes partly reversed the inhibitory effect of circ-PNN knockdown on CRC tumor progression in vitro and in vivo. Thus, circ-PNN acts as a sponge for miR-1225-5p to regulate FGF13 expression. Conclusions: : Tumor-derived exosomal circ-PNN promoted CRC progression through the regulation of the miR-1225-5p/FGF13 pathway, providing a potential therapeutic target for CRC.

Sodium sulfite triggered hepatic apoptosis, necroptosis, and pyroptosis by inducing mitochondrial damage in mice and AML-12 cells.

Sodium sulfite (SS) is a biological derivative of the air pollutant sulfur dioxide, and is often used as a food and pharmaceutical additive. Improper or excessive SS exposure in liver cell death. The phenomenon of simultaneous regulation of apoptosis, necroptosis, and pyroptosis is defined as PANoptosis. However, the specific types of programmed cell death (PCD) caused by SS and their interconnections remain unclear. In the present study, C57BL/6 mice were orally administered SS for 30 d, consecutively, to establish an in vivo mouse exposure model. AML-12 cells were treated with SS for 24 h to establish an in vitro exposure model. The results showed that SS-induced mitochondrial reactive oxygen species (mtROS) accumulation activated the BAX/Bcl-2/caspase 3 pathway to trigger apoptosis and RIPK1/RIPK3/p-MLKL to trigger necroptosis. Interestingly, ROS-activated p-MLKL perforated not the cell membrane as well as the lysosomal membrane. We determined that p-MLKL mediates lysosomal membrane permeabilization (LMP), resulting in cathepsin B (CTSB) release. Furthermore, knockdown of MLKL, a CTSB inhibitor (CA074-ME) and an NLRP3 inhibitor (MCC950) alleviated SS-induced pyroptosis. In summary, our study showed that SS induced apoptosis and necroptosis though mtROS accumulation, whereas the activation of p-MLKL mediated NLRP3-dependent pyroptosis by causing CTSB leakage through LMP. This study comprehensively explored the mechanism unerlying SS-induced PCD and provided an experimental basis for p-MLKL as a potential regulatory protein in PANoptosis.

Speculation on optimal numbers of examined lymph node for early-stage epithelial ovarian cancer from the perspective of stage migration.

Introduction: In early-stage epithelial ovarian cancer (EOC), how to perform lymphadenectomy to avoid stage migration and achieve reliable targeted excision has not been explored in depth. This study comprehensively considered the stage migration and survival to determine appropriate numbers of examined lymph node (ELN) for early-stage EOC and high-grade serous ovarian cancer (HGSOC). Methods: From the Surveillance, Epidemiology, and End Results database, we obtained 10372 EOC cases with stage T1M0 and ELN ≥ 2, including 2849 HGSOC cases. Generalized linear models with multivariable adjustment were used to analyze associations between ELN numbers and lymph node stage migration, survival and positive lymph node (PLN). LOESS regression characterized dynamic trends of above associations followed by Chow test to determine structural breakpoints of ELN numbers. Survival curves were plotted using Kaplan-Meier method. Results: More ELNs were associated with more node-positive diseases, more PLNs and better prognosis. ELN structural breakpoints were different in subgroups of early-stage EOC, which for node stage migration or PLN were more than those for improving outcomes. The meaning of ELN structural breakpoint varied with its location and the morphology of LOESS curve. To avoid stage migration, the optimal ELN for early-stage EOC was 29 and the minimal ELN for HGSOC was 24. For better survival, appropriate ELN number were 13 and 8 respectively. More ELNs explained better prognosis only at a certain range. Discussion: Neither too many nor too few numbers of ELN were ideal for early-stage EOC and HGSOC. Excision with appropriate numbers of lymph node draining the affected ovary may be more reasonable than traditional sentinel lymph node resection and systematic lymphadenectomy.

Establishment of Prognosis Model in Acute Myeloid Leukemia Based on Hypoxia Microenvironment, and Exploration of Hypoxia-Related Mechanisms.

Acute myeloid leukemia (AML) is a highly heterogeneous hematologic neoplasm with poor survival outcomes. However, the routine clinical features are not sufficient to accurately predict the prognosis of AML. The expression of hypoxia-related genes was associated with survival outcomes of a variety of hematologic and lymphoid neoplasms. We established an 18-gene signature-based hypoxia-related prognosis model (HPM) and a complex model that consisted of the HPM and clinical risk factors using machine learning methods. Both two models were able to effectively predict the survival of AML patients, which might contribute to improving risk classification. Differentially expressed genes analysis, Gene Ontology (GO) categories, and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis were performed to reveal the underlying functions and pathways implicated in AML development. To explore hypoxia-related changes in the bone marrow immune microenvironment, we used CIBERSORT to calculate and compare the proportion of 22 immune cells between the two groups with high and low hypoxia-risk scores. Enrichment analysis and immune cell composition analysis indicated that the biological processes and molecular functions of drug metabolism, angiogenesis, and immune cell infiltration of bone marrow play a role in the occurrence and development of AML, which might help us to evaluate several hypoxia-related metabolic and immune targets for AML therapy.

Development and identification of a prognostic nomogram model for patients with mixed cell adenocarcinoma of the ovary.

BACKGROUND: Mixed cell ovarian adenocarcinoma (MCOA) is a malignant gynecologic tumor consisting of serous, mucous, and papillary tumor cells. However, the clinical features and prognosis of MCOA patients are unclear. METHODS: In this study, univariate and multivariate Cox proportional risk models were performed to identify independent prognostic factors. The Kaplan-Meier method was used to assess the relationship between clinical characteristics and patient survival. Finally, a nomogram was constructed and validated to predict patient survival time, and the C-index was used to evaluate the efficacy of the nomogram. RESULTS: A total of 2,818 patients diagnosed with MCOA were identified, and the 5-year survival rate was 62%. Univariate and multivariate Cox models suggested that age (HR=1.28, 95% CI[1.15,1.44]), grade (HR=1.26, 95% CI[1.12,1.41]), SEER stage (HR=1.63, 95% CI[1.25,2.13]) and AJCC (American Joint Committee on Cancer) stage (HR=1.59, 95% CI[1.36,1.86]) were independent prognostic factors for MCOA patients. After propensity score matching for age, grade, SEER stage, and AJCC stage, the 5-year survival rate was 69.7% for ovarian serous cystadenocarcinoma and 62.9% for ovarian papillary serous cystadenocarcinoma. These results mean that serous adenocarcinoma had the best prognosis of the three pathologic types of ovarian carcinoma (p<0.0001), with no significant difference between papillary serous cystadenocarcinoma and MCOA (p=0.712). Finally, a nomogram consisting of age, grade, SEER stage, and AJCC stage was established and validated to predict the survival time, with C-indices of 0.743 and 0.731, respectively. CONCLUSIONS: In summary, MCOA is uncommon, and age, grade, SEER stage, and AJCC stage are independent prognostic factors. Compared with other common malignant ovarian tumors, MCOA has a poor prognosis.

Human Amniotic Mesenchymal Stem Cells Inhibit aGVHD by Regulating Balance of Treg and T Effector Cells.

BACKGROUND: Acute graft versus host disease (aGVHD) remains a leading cause of transplant-related mortality following allogeneic haematopoietic cell transplantation (allo-HCT). Human amniotic mesenchymal stem cells (hAMSCs) are a novel mesenchymal stem cells (MSCs), which have stronger proliferation and immunomodulatory ability compared with bone marrow mesenchymal stem cells (BM-MSCs). Besides, as the amniotic membrane is often treated as medical waste after delivery, hAMSCs can be obtained conveniently and noninvasively. The aim of this study was to explore the therapeutic efficacy and underlying mechanisms of hAMSCs transplantation for the humanized aGVHD mouse model. METHODS: We established a humanized aGVHD mouse model by transplanting human peripheral blood mononuclear cells (PBMCs) into NOD-PrkdcscidIL2rγnull (NPG) mice, human amniotic membrane collected from discarded placenta of healthy pregnant women after delivery and hAMSCs were extracted from amniotic membrane and expanded in vitro. Mice were divided into untreated group (Control), aGVHD group (aGVHD), and hAMSCs treatment group (aGVHD+hAMSCs), the hAMSCs labeled with GFP were administered to aGVHD mice to explore the homing ability of hAMSCs. T effector and regulatory T cells (Tregs) levels and cytokines of each group in target organs were detected by flow cytometry and cytometric bead array (CBA), respectively. RESULTS: We successfully established a humanized aGVHD mouse model using NPG mice. The hAMSCs have the ability to inhibit aGVHD in this mouse model through reduced villous blunting and lymphocyte infiltration of the gut while reducing inflammatory edema, tissue destruction and lymphocyte infiltration into the parenchyma of the liver and lung. hAMSCs suppressed CD3+CD4+ T and CD3+CD8+ T cell expression and increased the proportion of Tregs, and besides, hAMSCs can reduce the levels of IL-17A, INF-γ, and TNF in aGVHD target organs. CONCLUSION: The NPG murine environment was capable of activating human T cells to produce aGVHD pathology to mimic aGVHD as in humans. The hAMSCs controlled aGVHD by decreasing inflammatory cytokine secretion within target organs by modulating the balance of Tregs and T effector cells in humanized mice.

Immunomodulating functions of human leukocyte antigen-G and its role in graft-versus-host disease after allogeneic hematopoietic stem cell transplantation.

Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is a potentially curative therapeutic strategy to treat several hematological malignancies and non-hematological malignancies. However, graft-versus-host disease (GVHD) is a frequent and serious transplant-related complication which dramatically restrains the curative effect of allo-HSCT and a significant cause of morbidity and mortality in allogeneic HCT recipients. Effective prevention of GVHD mainly depends on the induction of peripheral immune tolerance. Human leukocyte antigen-G (HLA-G) is a non-classical MHC class I molecule with a strong immunosuppressive function, which plays a prominent role in immune tolerance. HLA-G triggers different reactions depending on the activation state of the immune cells and system. It also exerts a long-term immune tolerance mechanism by inducing regulatory cells. In this present review, we demonstrate the immunomodulatory properties of human leukocyte antigen-G and highlight the role of HLA-G as an immune regulator of GVHD. Furthermore, HLA-G could also serve as a good predictor of GVHD and represent a new therapeutic target for GVHD.

Palmitoyl acyltransferase, Zdhhc13, facilitates bone mass acquisition by regulating postnatal epiphyseal development and endochondral ossification: a mouse model.

ZDHHC13 is a member of DHHC-containing palmitoyl acyltransferases (PATs) family of enzymes. It functions by post-translationally adding 16-carbon palmitate to proteins through a thioester linkage. We have previously shown that mice carrying a recessive Zdhhc13 nonsense mutation causing a Zdhcc13 deficiency develop alopecia, amyloidosis and osteoporosis. Our goal was to investigate the pathogenic mechanism of osteoporosis in the context of this mutation in mice. Body size, skeletal structure and trabecular bone were similar in Zdhhc13 WT and mutant mice at birth. Growth retardation and delayed secondary ossification center formation were first observed at day 10 and at 4 weeks of age, disorganization in growth plate structure and osteoporosis became evident in mutant mice. Serial microCT from 4-20 week-olds revealed that Zdhhc13 mutant mice had reduced bone mineral density. Through co-immunoprecipitation and acyl-biotin exchange, MT1-MMP was identified as a direct substrate of ZDHHC13. In cells, reduction of MT1-MMP palmitoylation affected its subcellular distribution and was associated with decreased VEGF and osteocalcin expression in chondrocytes and osteoblasts. In Zdhhc13 mutant mice epiphysis where MT1-MMP was under palmitoylated, VEGF in hypertrophic chondrocytes and osteocalcin at the cartilage-bone interface were reduced based on immunohistochemical analyses. Our results suggest that Zdhhc13 is a novel regulator of postnatal skeletal development and bone mass acquisition. To our knowledge, these are the first data to suggest that ZDHHC13-mediated MT1-MMP palmitoylation is a key modulator of bone homeostasis. These data may provide novel insights into the role of palmitoylation in the pathogenesis of human osteoporosis.

[Brain mitochondrial DNA damage of newborn piglets following hypoxia-ischemia].

OBJECTIVE: This study investigated the 8003 base pair (bp) fragmentation damage of brain mitochondrial DNA in newborn piglets at different times after hypoxic-ischemic brain damage (HIBD) so as to explore the biomolecular foundation of neonatal neuronal metabolic disorders. METHODS: Fifty 3-day-old piglets were randomly assigned into Control and HIBD groups. The HIBD group was subdivided into groups sacrificed at 0, 24, 48 and 72 hrs post-HIBD (n=10). HIBD was induced by left carotid ligation and exposure to 8% oxygen for 2 hours. The Control group was exposed to air and was sham-operated. The left hippocampal cortexes of all subjects were obtained to amplify the fragments of 200 bp and 8003 bp by the LX-PCR method. The PCR products were electrophoresed on agaros gels to obtain the integral optical density (IOD). RESULTS: The IOD of 8003 bp fragment was markedly reduced in the HIBD 0 hr group (22.616 +/- 2.276) when compared with that of the Control group (56.995 +/- 0.317) (P < 0.05). The IOD value remained lower at 24 hrs (27.719 +/- 0.309) and 48 hrs post-HIBD (49.491 +/- 3.233) (P < 0.05). Until 72 hrs post-HIBD, the IOD (55.972 +/- 2.236) restored to the control value. CONCLUSIONS: The brain mitochondrial DNA was fragmented in newborn piglets following brain hypoxia-ischemia. It did not recover to normal until 72 hrs post-HIBD. The fragmentation damage of mitochondrial DNA may be related to the depression of mitochondrial respiratory enzymes activity and neuron apoptosis.

[Study of beta 1-integrin in neonatal rats with ischemic renal injury].

OBJECTIVE: To study the change of beta 1-integrin in neonatal rats with ischemic renal injury. METHODS: Twenty-four neonatal rats weighing from 30 to 40 g were chosen. Anesthesia was carried out by peritoneal injection of nembutal-sodium solution (4 mg/100 g). Renal ischemic injury model was induced by clamping the left renal pedicle for different periods of time. The kidneys of normal neonatal rats were functioned as the control group. Immunofluorescence technique was used to detect the location of beta 1-integrin in kidney, especially in cortex. RESULTS: In control group, beta 1-integrin subunit was found to be located mainly in the basal membrane of tubular epithelial cell. After 10 minutes of ischemia, immunostaining highlighted the broken basement membranes of the experiment groups. The longer the ischemia continued, the more the broken points we observed. There were significant differences between the experiment groups and the control group. Yet, no significant difference in the IOD value of beta 1-integrin was seen between the 10-min-ischemia and 30-min-ischemia groups. CONCLUSION: Under physiological condition, beta 1-integrin is distributed over the basal membrane of the tubular epithelial cell of neonatal rat. Ischemic injury can cause the changes of beta 1-integrin which may be the reason of exfoliation and depolarization of tubular epithelia cell.

[The changes of beta 1-integrin and its mRNA distribution in renal tubular epithelial cells during kidney ischemia/reperfusion injury in neonatal rats].

OBJECTIVE: By studying the distribution and expression of beta 1-integrin and its mRNA in renal tubular epithelial cell at different ischemia/reperfusion time phases we probe the role of beta 1-integrin in I/R injury of neonatal animals. METHODS: The neonatal SD rat ischemia/reperfusion model was set up. Immunofluorescent staining and in situ hybridization (ISH) were used to show the distribution and expression of beta 1-integrin with its mRNA in renal tubular epithelial cells and the quantities of beta 1-integrin and its mRNA were counted by ImagePlus-Pro system. RESULTS: 1. beta 1-integrin was located at the basal plasma membrane in normal renal tubular epithelial cells. After ischemia for 0.5 h, the redistribution was not significant. After reperfusing for 0.5 h, beta 1-integrin began to move onto the lateral and apex surfaces of cells. This kind of change was most apparent after reperfusion for 2 h and was accompanied with reduced expression and destruction of renal tubular. No positive dying was seen in the lumen. The regeneration started 24 hours post-ischemia. beta 1-integrin was redistributed onto the basal plasma membrane. After reperfusion for 120 hours, the regeneration ended, but the expression did not gain the normal level. 2. The mRNA of beta 1-integrin was mainly expressed in the cell plasma. Its expression was increased while reperfusion for 0.5 h to 2 h and was still above normal level after 24 h to 120 h of reperfusion. CONCLUSION: The distribution of beta 1-integrin was "depolarized" and its expression was reduced. But the level of mRNA was raised, which indicated that during ischemia/reperfusion injury, there were mechanisms that influenced the distribution and synthesis of beta 1-integrin at the same time.

[Injury of myocardial mitochondria in neonatal swines with hypoxic-ischemic brain damage].

OBJECTIVE: To clarify the effect of hypoxia and ischemia on the concentration of Ca2+ in myocardial mitochondria, and on the activity of respiratory chain complex IV--cytochrome oxidase (CCO) in neonatal swines, and to find out the change of myocardial mitochondria during hypoxic-ischemic brain damage (HIBD). METHODS: The study was performed on neonatal swines. For establishment of HIBD model, the left carotid was ligated and the swine was placed under hypoxia for two hours. Then the concentration of Ca2+ and the activity of respiratory chain complex IV in myocardial cell mitochondria were measured at 0 hour and 24, 48, 72 hours after reoxygenation. The measured values from these 0-, 24-, 48-, 72-hour-HIBD-groups (n = 10 per group) and from the normal control group (n = 10) were statistically analyzed by means of ANOVA, Newman-Keuls test and linear correlation. RESULTS: After the swines were placed under hypoxia-ischemia for two hours, 1. the concentration of Ca2+ in myocardial mitochondria increased, till 24 hours after re-oxygenation, and then it descended at 48 hours and 72 hours after reoxygenation; 2. the activity of myocardial mitochondrial respiratory chain complex IV descended, though activity recovery was seen at 48 and 72 hours, it did not come back to normal level at 72 hours after reoxygenation; 3. the concentration of Ca2+ in myocardial mitochondria was negatively correlated with the activity of respiratory chain complex IV. CONCLUSION: The results showed that in the neonatal swines with HIBD, the mitochondrial respiratory chain in myocardial cell was damaged by hypoxia-ischemia, yet it could be rehabilitated after reoxygenation. Calcium overload in myocardial mitochondria after hypoxia-ischemia might be one of the causes of the descending activity of respiratory chain complex IV.

[The changes of filamin distribution in renal tubular epithelial cells during kidney ischemia/reperfusion injury in neonatal rats].

OBJECTIVE: By investigating the changes of filamin distribution in renal tubular epithelial cells at different ischemia/reperfusion time stages we studied the role of filamin in kidney ischemia/reperfusion injury. METHODS: The neonatal SD rat ischemia/reperfusion model was set up. Immunofluorescent staining was used to show the distribution of filamin in renal tubular epithelial cells and the quantities of filamin were counted by ImagePlus-Pro system. RESULTS: Filamin was located around the basal plasma membrane in normal renal tubular epithelial cells. After ischemia for 0.5 h, the redistribution was not significant. After reperfusion for 0.5 h, filamin came into the cell plasma, appeared at the apex of cells and in the lumen. This kind of change was most apparent after reperfusion for 2 h and accompanied with destruction of renal tubules. The regeneration started 24 h post-ischemia. Filamin was redistributed to the basal plasma membrane. After reperfusion for 120 h, the regeneration ended and the structure of renal tubules became normal. CONCLUSION: The distribution of filamin was changed during ischemia/reperfusion injury, this change was prior to the change of actin cytoskeleton and integrin.