Long noncoding RNA TCL6 binds to miR-106a-5p to regulate hepatocellular carcinoma cells through PI3K/AKT signaling pathway.

Long noncoding RNAs (lncRNAs) have been reported to dysregulate and involve in the pathology of hepatocellular carcinoma (HCC). Nonetheless, the functional role of lncRNA T cell leukemia/lymphoma 6 (TCL6) and its underlying mechanism in HCC remain unclear. Herein, we analyzed the expression of TCL6 and elucidated its mechanistic involvement in HCC. Bioinformatics analyses indicated TCL6 was evidently downregulated in HCC tissues compared with normal controls. TCL6 was downregulated while microRNA-106a-5p (miR-106a-5p) was upregulated in HCC cell lines. Moreover, knockdown or overexpression of TCL6 significantly raised or diminished the expression level of miR-106a-5p in HCC cells, similar to the effect of miR-106a-5p on TCL6 expression. Functionally, TCL6 inhibited the proliferative, migratory, and invasive potentials of HCC cells as analyzed by cell counting kit-8, scratch wound healing, and transwell assays, respectively. Conversely, miR-106a-5p exerted an opposite effect on the proliferative, migratory, and invasive potentials of HCC. RNA immune precipitation and luciferase reporter assays revealed TCL6 directly bound to miR-106a-5p and luciferase reporter assay verified phosphatase and tensin homolog (PTEN) was a target gene of miR-106a-5p. Mechanistically, TCL6 knockdown evidently reduced PTEN expression at both messenger RNA and protein levels, and miR-106a-5p inhibitor partially rescued this reduction effect in HCC cells. Additionally, western blot assays demonstrated miR-106a-5p downregulation or TCL6 overexpression promoted the protein level of PTEN, and suppressed the phosphorylation level of AKT, the protein level of phosphatidylinositol 3-kinase (PI3K). Collectively, these results revealed TCL6 as a tumor-suppressive lncRNA regulates PI3K/AKT signaling pathway via directly binding to miR-106a-5p in HCC. This mechanism provides a theoretical basis for HCC pathogenesis and a potential therapeutic strategy for HCC treatment.

Long non-coding RNA NKILA inhibited angiogenesis of breast cancer through NF-κB/IL-6 signaling pathway.

OBJECTIVE: The relationship between NF-κB Interacting lncRNA (NKILA) and angiogenesis in breast cancer has never been studied. Our study aimed to investigate effect of NKILA on proliferation, migration, apoptosis, as well as angiogenesis in breast cancer. METHODS: NKILA was over-expressed in MDA-MB-231 cells by transfection of pcDNA3.1-NKILA vector. Cell viability, apoptosis and migration were measured by MTT, flow cytometry and wound healing assays, respectively. Angiogenesis of human umbilical vein endothelial cells (HUVEC) was measured using tube formation assay. The expression levels of NKILA, IL-6, VEGFA, VEGFR, apoptosis and epithelial-mesenchymal transition (EMT) and NF-κB/IL-6 signaling-related markers were determined using qRT-PCR or Western blotting. RESULTS: Cell viability and migration of MDA-MB-231 cells were significantly inhibited, while cell apoptosis was obviously promoted by overexpression of NKILA. Overexpression of NKILA could also inhibit the phosphorylation of IκBα and the nuclear transposition of p65, as well as induce cell apoptosis-related proteins and inhibit epithelial-mesenchymal transition-related proteins. Cell viability and migration of HUVEC were also significantly inhibited when treated with supernatant of cells overexpressed NKILA or treated with BAY11-7028. Exogenous IL-6 significantly increased the cell viability and migration of HUVEC, and overexpression of NKILA could reverse these effects induced by IL-6. Overexpression of NKILA significantly inhibited the protein levels of IL-6 and VEGFA in supernatant, as well as VEGFR in HUVEC, thus inhibited the angiogenesis of HUVEC. NKILA also reversed the above effects on protein levels of IL-6 and VEGFA in supernatant and angiogenesis induced by exogenous IL-6. CONCLUSION: Overexpression of NKILA could inhibit cell proliferation, migration and promote apoptosis of breast cancer cells. It could also inhibit cell proliferation, migration and angiogenesis of HUVEC through inhibiting IL-6 secretion via NF-κB signaling pathway.

Inhibiting Hypoxia and Chemotherapy-Induced Cancer Cell Metastasis under a Valid Therapeutic Effect by an Assistance of Biomimetic Oxygen Delivery.

Tumor metastasis is the most dangerous stage in tumorigenesis and its evolution, which causes about 80% clinical death. However, common therapies including chemotherapy may increase the risk of tumor metastasis while killing cancer cells. Tumor metastasis is closely related to many factors in the tumor microenvironment, especially hypoxia. As one of the characteristics of a malignant tumor microenvironment, hypoxia plays an important role in the growth, metabolism, and metastasis of tumors. Upregulation of the hypoxia-inducible factor (HIF) would stimulate the metastasis and migration of cancer cells. In this study, we developed an artificial oxygen carrier system, a hemoglobin-loaded liposome (Hb@lipo), which was capable of effectively delivering oxygen to tumor. The way of providing oxygen not only alleviated tumor hypoxia but also downregulated the expression of HIF, which is conducive to reducing tumor malignancy. Alleviating the tumor hypoxic microenvironment alone is not enough to inhibit tumor metastasis; thus, we prepared the liposome containing a chemotherapeutic agent cabazitaxel (CBZ@lipo). Our data indicated that the combination therapy of Hb@lipo and CBZ@lipo can efficiently kill cancer cells and inhibit tumor growth. At the same time, it can effectively entrap cancer cells in tumor sites by relieving the hypoxic microenvironment of tumors and reduce the metastasis of cancer cells during and after the chemotherapy. Our research may provide a clinical cancer chemotherapy reference that reduces the risk of cancer cell metastasis while inhibiting tumor growth.

Tumescence Anesthesia Solution-Assisted Laser Ablation Treatment of Lower Limb Varicose Veins: The Effect of Temperature of the Tumescence Anesthesia Solution on Intraoperative and Postoperative Pain, Clinical Observations, and Comprehensive Nursing Care.

PURPOSE: To investigate the effect of cold and room temperature tumescence anesthesia solution (TAS) on the treatment of lower limb varicose veins via endovenous laser ablation. DESIGN: On the basis of the TAS temperature, patients were divided into two groups: group A (n = 26) received room temperature (24°C) TAS, and group B (n = 25) received cold (4°C) TAS. METHODS: A numerical rating scale was used to evaluate pain. Perioperative and intraoperative nursing care and clinical observations were performed following a generalized standard. FINDINGS: Percentages of patients who felt pain in groups A and B were 69.2% and 36.0%. Average numerical rating scale scores of patients in the two groups (A and B) on the day of surgery and on postoperative days 1, 2, and 3 were 4.3 versus 2.1, 3.5 versus 1.0, 3.0 versus 0.8, and 1.6 versus 0.3, respectively. CONCLUSIONS: Cold TAS reduces intraoperative and postoperative pain more effectively than room temperature.

Tim3/galectin-9 alleviates the inflammation of TAO patients via suppressing Akt/NF-kB signaling pathway.

Thyroid-associated ophthalmopathy (TAO) is an autoimmune disease. Studies showed that T helper 1 (Th1), Th2, and Th17 cells play important roles in the pathology of TAO. Tim-3 and its only known ligand Galectin-9 (Gal-9) is related to the suppression of Th1 and Th17 cytokine secretion. This study aims to investigate the role of Tim3/Gal-9 in the inflammatory response of TAO. In this study, the levels of Tim3, Gal-9, and cytokines of Th1 (TNF-α and IFN-γ), Th2 (IL-4), and Th17 (IL-17) cells were analyzed in the blood samples of TAO patients and healthy controls as well as in orbital fibroblasts. Tim3 overexpression and Gal-9 neutralizing antibody were used in TAO and LPS-stimulated control orbital fibroblasts to further investigate the role and mechanism of Tim3/Gal-9 on the inflammation of TAO. We found Tim3 and Gal-9 expression was significantly downregulated in TAO patients and further lower in active TAO than inactive TAO or controls. Th1, Th2, and Th17 cytokines were all increased in TAO patients. Th1 and Th17 cytokines were higher in active TAO patients than in inactive TAO patients, while Th2 cytokines were enhanced in inactive TAO. Tim3 overexpression decreased the levels of Th1 and Th17 cytokines, but not Th2 cytokine in TAO or LPS-stimulated control orbital fibroblasts. These effects were abrogated by Gal-9 neutralizing antibody. Moreover, Tim3 reduced the levels of p-Akt and p-p65 in TAO or LPS-induced control orbital fibroblasts that were reversed by Gal-9 blocking. In conclusion, Tim3/Gal-9 alleviates the inflammation of TAO patients via suppressing Akt/NF-κB signaling pathway.

Alteration of Th17 and Treg cells in patients with unexplained recurrent spontaneous abortion before and after lymphocyte immunization therapy.

BACKGROUND: Several types of T cells have been associated with the pathogenesis of unexplained recurrent spontaneous abortion (URSA), including Th1/Th2/Th17/Tregs cell. It has been appreciated that immunotherapy with paternal or third party lymphocytes is an effective method of treatment for URSA patients. The balance of Th1/Th2 cells could be maintained and an increase of Treg cells would be beneficial after immunotherapy; however, the mechanism by which the Th17/Treg balance affects URSA has not yet been fully elucidated. METHODS: Here, we used flow cytometry, liquid chip technology and quantitative real-time PCR (qPCR) methods to characterize Th17/Treg cell populations after immunotherapy. We found that after immunotherapy in URSA patients, the percentage of Th17 cells decreased and the percentage of Treg cells in peripheral blood mononuclear cells (PBMC) increased, as detected by flow cytometry. RESULTS: Immunotherapy may induce a decrease in the Th17/Treg ratio and the Treg bias, which may be beneficial for the maintenance of pregnancy. The expression level of ROR gamma t, a transcription factor found in Th17 cells, decreased and the expression of the Treg-specific transcription factor Foxp3 increased in peripheral blood as detected by qPCR. Immunotherapy may induce a decrease in the ratio of ROR gamma t to Foxp3 and a Treg cell bias, which would be beneficial for pregnancy maintenance. The secretion of the Treg-associated cytokine TGF-beta, as well as Th2 cytokines, was increased in serum, while the secretion of Th17-associated cytokine IL-17A and Th1 cytokine production was decreased. The Th1/Th2 cytokine ratio significantly decreased. Similarly, the Th17/Treg ratio significantly decreased in the total patient after immunotherapy. CONCLUSIONS: These results indicate that in patients with URSA, immunotherapy with mononuclear cells derived from the baby's father could affect both Th1/Th2 and Th17/Treg balance, and we found that the Th2 and Treg bias would be beneficial for pregnancy, which may lead to a balancing of the Th17/Treg ratio in URSA patients after immunotherapy.

Spatiotemporal multi-omics: exploring molecular landscapes in aging and regenerative medicine.

Aging and regeneration represent complex biological phenomena that have long captivated the scientific community. To fully comprehend these processes, it is essential to investigate molecular dynamics through a lens that encompasses both spatial and temporal dimensions. Conventional omics methodologies, such as genomics and transcriptomics, have been instrumental in identifying critical molecular facets of aging and regeneration. However, these methods are somewhat limited, constrained by their spatial resolution and their lack of capacity to dynamically represent tissue alterations. The advent of emerging spatiotemporal multi-omics approaches, encompassing transcriptomics, proteomics, metabolomics, and epigenomics, furnishes comprehensive insights into these intricate molecular dynamics. These sophisticated techniques facilitate accurate delineation of molecular patterns across an array of cells, tissues, and organs, thereby offering an in-depth understanding of the fundamental mechanisms at play. This review meticulously examines the significance of spatiotemporal multi-omics in the realms of aging and regeneration research. It underscores how these methodologies augment our comprehension of molecular dynamics, cellular interactions, and signaling pathways. Initially, the review delineates the foundational principles underpinning these methods, followed by an evaluation of their recent applications within the field. The review ultimately concludes by addressing the prevailing challenges and projecting future advancements in the field. Indubitably, spatiotemporal multi-omics are instrumental in deciphering the complexities inherent in aging and regeneration, thus charting a course toward potential therapeutic innovations.

Application of early enteral nutrition nursing based on enhanced recovery after surgery theory in patients with digestive surgery.

BACKGROUND: Postoperative nursing can improve the restlessness and gastrointestinal function of patients with tracheal intubation under general anesthesia in digestive surgery. Wide application of various nursing methods and routine nursing in perioperative nursing of patients with general anesthesia in digestive surgery. AIM: To investigate the impact of early postoperative enteral nutrition nursing based on the enhanced recovery after surgery (ERAS) theory on postoperative agitation and gastrointestinal recovery in patients undergoing general anesthesia that experienced tracheal intubation. METHODS: The data of 126 patients with digestive surgery from May 2019 to February 2022 were retrospectively analyzed. According to different nursing methods, they were divided into control group and observation group, with 63 cases in observation group and 63 cases in control group. The patients in the control group had standard perioperative nursing care, whereas those in the observation group got enteral nourishment as soon as possible after surgery in accordance with ERAS theory. Both the rate and quality of gastrointestinal function recovery were compared between the two groups after treatment ended. Postoperative anesthesia-related adverse events were tallied, patients' nutritional statuses were monitored, and the Riker sedation and agitation score (SAS) was used to measure the incidence of agitation. RESULTS: When compared to the control group, the awake duration, spontaneous breathing recovery time, extubation time and postoperative eye-opening time were all considerably shorter (P < 0.05). There was no significant difference in the recovery time of orientation force between the two groups (P > 0.05); however, the observation group had a lower SAS score than the control group (P < 0.05). The recovery time for normal intestinal sounds, the time it took to have the first postoperative exhaust, the time it took to have the first postoperative defecation, and the time it took to have the first postoperative half-fluid feeding were all faster in the observation group than in the control group (P < 0.05); Fasting blood glucose was lower in the observation group compared to the control group (P < 0.05), while the albumin and hemoglobin levels were higher on the first and third postoperative days; however, there was no statistically significant difference in the incidence of anesthesia-related adverse reactions between the two groups (P > 0.05). CONCLUSION: The extremely early postoperative enteral nutrition nursing based on ERAS theory can reduce the degree of agitation, improve the quality of recovery, promote the recovery of gastrointestinal function, and improve the nutritional status of patients in the recovery period after tracheal intubation under general anesthesia.

Umbilical cord blood-derived exosomes from healthy term pregnancies protect against hyperoxia-induced lung injury in mice.

Bronchopulmonary dysplasia (BPD) is a chronic, devastating disease primarily occurring in premature infants. To date, intervention strategies to prevent or treat BPD are limited. We aimed to determine the effects of umbilical cord blood-derived exosomes (UCB-EXOs) from healthy term pregnancies on hyperoxia-induced lung injury and to identify potential targets for BPD intervention. A mouse model of hyperoxia-induced lung injury was created by exposing neonatal mice to hyperoxia after birth until the 14th day post birth. Age-matched neonatal mice were exposed to normoxia as the control. Hyperoxia-induced lung injury mice were intraperitoneally injected with UCB-EXO or vehicle daily for 3 days, starting on day 4 post birth. Human umbilical vein endothelial cells (HUVECs) were insulted with hyperoxia to establish an in vitro model of BPD to investigate angiogenesis dysfunction. Our results showed that UCB-EXO alleviated lung injuries in hyperoxia-insulted mice by reducing histopathological grade and collagen contents in the lung tissues. UCB-EXO also promoted vascular growth and increased miR-185-5p levels in the lungs of hyperoxia-insulted mice. Additionally, we found that UCB-EXO elevated miR-185-5p levels in HUVECs. MiR-185-5p overexpression inhibited cell apoptosis, whereas promoted cell migration in HUVECs exposed to hyperoxia. The luciferase reporter assay results revealed that miR-185-5p directly targeted cyclin-dependent kinase 6 (CDK6), which was downregulated in the lungs of hyperoxia-insulted mice. Together, these data suggest that UCB-EXO from healthy term pregnancies protect against hyperoxia-induced lung injuries via promoting neonatal pulmonary angiogenesis partially by elevating miR-185-5p.

Umbilical cord blood exosomes from very preterm infants with bronchopulmonary dysplasia aggravate lung injury in mice.

Bronchopulmonary dysplasia (BPD) is characterized by abnormal development of the blood vessels and alveoli in lungs, which largely occurs in premature infants. Exosomes (EXO) from very preterm infants (VPI) with BPD (BPD-EXO) impair angiogenic activities of human umbilical vein endothelial cells (HUVECs) via EXO-miRNAs cargo. This study aimed to determine whether and how BPD-EXO affect the development of BPD in a mouse model. We showed that treating BPD mice with BPD-EXO chronically and irreversibly aggravated lung injury. BPD-EXO up-regulated 139 and down-regulated 735 genes in the mouse lung tissue. These differentially expressed genes were enriched to the MAPK pathway (e.g., Fgf9 and Cacna2d3), which is critical to angiogenesis and vascular remodeling. BPD-EXO suppressed expression of Fgf9 and Cacna2d3 in HUVECs and inhibited migration, tube formation, and increased cell apoptosis in HUVECs. These data demonstrate that BPD-EXO aggravate lung injury in BPD mice and impair lung angiogenesis, plausibly leading to adverse outcomes of VPI with BPD. These data also suggest that BPD-EXO could serve as promising targets for predicting and treating BPD.

The marriage of immunomodulatory, angiogenic, and osteogenic capabilities in a piezoelectric hydrogel tissue engineering scaffold for military medicine.

BACKGROUND: Most bone-related injuries to grassroots troops are caused by training or accidental injuries. To establish preventive measures to reduce all kinds of trauma and improve the combat effectiveness of grassroots troops, it is imperative to develop new strategies and scaffolds to promote bone regeneration. METHODS: In this study, a porous piezoelectric hydrogel bone scaffold was fabricated by incorporating polydopamine (PDA)-modified ceramic hydroxyapatite (PDA-hydroxyapatite, PHA) and PDA-modified barium titanate (PDA-BaTiO3, PBT) nanoparticles into a chitosan/gelatin (Cs/Gel) matrix. The physical and chemical properties of the Cs/Gel/PHA scaffold with 0-10 wt% PBT were analyzed. Cell and animal experiments were performed to characterize the immunomodulatory, angiogenic, and osteogenic capabilities of the piezoelectric hydrogel scaffold in vitro and in vivo. RESULTS: The incorporation of BaTiO3 into the scaffold improved its mechanical properties and increased self-generated electricity. Due to their endogenous piezoelectric stimulation and bioactive constituents, the as-prepared Cs/Gel/PHA/PBT hydrogels exhibited cytocompatibility as well as immunomodulatory, angiogenic, and osteogenic capabilities; they not only effectively induced macrophage polarization to M2 phenotype but also promoted the migration, tube formation, and angiogenic differentiation of human umbilical vein endothelial cells (HUVECs) and facilitated the migration, osteo-differentiation, and extracellular matrix (ECM) mineralization of MC3T3-E1 cells. The in vivo evaluations showed that these piezoelectric hydrogels with versatile capabilities significantly facilitated new bone formation in a rat large-sized cranial injury model. The underlying molecular mechanism can be partly attributed to the immunomodulation of the Cs/Gel/PHA/PBT hydrogels as shown via transcriptome sequencing analysis, and the PI3K/Akt signaling axis plays an important role in regulating macrophage M2 polarization. CONCLUSION: The piezoelectric Cs/Gel/PHA/PBT hydrogels developed here with favorable immunomodulation, angiogenesis, and osteogenesis functions may be used as a substitute in periosteum injuries, thereby offering the novel strategy of applying piezoelectric stimulation in bone tissue engineering for the enhancement of combat effectiveness in grassroots troops.

Fertilization rates are improved after IVF if the corona radiata is left intact in vitrified-warmed human oocytes.

BACKGROUND: Before human MII oocytes are vitrified they are usually denuded from their cumulus cells. In this study we wanted to investigate the effects of an intact corona radiata on the vitrification and fertilization of human oocytes. METHODS: The study comprised two different parts. In Part 1, 36 MII stage oocytes, from 6 patients, were randomly assigned into a control group, a group of vitrified-warmed oocytes without a corona radiata and a group of vitrified-warmed oocytes with an intact corona radiata. In each group of 12, 6 oocytes were used for evaluation of the zona pellucida solubility (hardening) and another 6 oocytes were used for the analysis of their ultrastructure. In addition, six polyspermically fertilized oocytes were used as positive controls for zona pellucida hardening. In Part 2, 16 patients in total produced 107 fresh and 98 vitrified-warmed oocytes, with or without an intact corona radiata. All oocytes were fertilized via conventional IVF and embryos were transferred according to our standard ET routines. The oocyte survival and fertilization rates, embryo quality and pregnancy and implantation rates were evaluated. RESULTS: There were no differences in oocyte survival, zona pellucida solubility (hardening) or the number of cortical granules between the vitrified-warmed and fresh oocytes. There were also no differences in the zona pellucida solubility and the number of cortical granules between vitrified-warmed oocytes with or without an intact corona radiata. However, the oocytes with an intact corona radiata had a higher fertilization rate after conventional IVF insemination. No differences were seen in the survival and cleavage rates, the percentage of high-quality embryos or the clinical outcome. CONCLUSIONS: Zona hardening and ultrastructural damage do not seem to occur in vitrified human oocytes. An intact corona radiata in vitrified-warmed oocytes retains their fertilization capacity in conventional IVF, but does not improve the embryo quality. Poor fertilizing capacities of vitrified-warmed oocytes without an intact corona radiata seem to have been due to the complete removal of the cumulus cells.

Health Protection of CT Radiographers During the Outbreak of COVID-19: Application of Automatic Positioning Technology for Relocatable CT in the Fang Cang Hospital.

Background: To investigate the value of automatic positioning technology in improving the protection of radiographers in the relocatable CT room of a Fang Cang hospital during the outbreak of coronavirus disease 2019 (COVID-19). Methods: The National Emergency Medical Team of our hospital assumed command of Wuchang Fang Cang Hospital and treated confirmed COVID-19 patients with mild symptoms. Relocatable CT was used to examine patients in this hospital. Automatic positioning technology was applied to avoid close contact between medical staff and patients and to protect medical staff more effectively. Results: Seven hundred lung CT scans acquired from 269 patients were completed from February 17 to 26, 2020 with automatic positioning technology for relocatable CT in a Fang Cang hospital. All scans were conducted successfully using automatic positioning technology. All patients entered the scanning room from a separate door. All the position lines were accurate, and all images met the requirement for diagnosis of COVID-19, with satisfied quality. None of our medical staff had any close contact with patients. Conclusion: Automatic positioning technology applied to relocatable CT can minimize the close contact between technologists and patients and effectively improve the protection of medical staff without sacrificing image quality.

MiR-543/Numb promotes proliferation, metastasis, and stem-like cell traits of prostate cancer cells.

MiR-543 and Numb are associated with various malignancies, including prostate cancer (PCa). However, whether miR-543 regulates Numb in PCa development remains unclear. In this study, we identified Numb as a direct target of miR-543. The role of miR-543 was examined both in vitro and in vivo. The in vivo effects of miR-543 were investigated using tumor transplantation experiments and a lung metastasis model. The in vitro effects of miR-543 on proliferation, migration, invasion, and cancer stem-like cell (CSC)-associated properties were also examined. The binding sites of Numb were predicted using bioinformatics tools and confirmed by luciferase and rescue assays. QRT-PCR and western blot analyses were used to detect target expression levels. Expression levels of both miR-543 and Numb were manipulated in CD44+ and CD44-PCa cells followed by a series of functional assays. The results demonstrated that miR-543 promoted PCa growth and metastasis both in vivo and in vitro. Luciferase reporter assays, qRT-PCR, and western blot analyses revealed Numb as a direct target of miR-543. The function of miR-543 was abolished by Numb, as shown in rescue experiments. Moreover, miR-543 was verified to promote CSC properties, whereas Numb elicited the opposite effects. MiR-543 also influenced the expression of several stem-like factors, including Dll4, NF-κB, c-myc, and Oct-4, and the Numb/p53 signaling pathway. Taken together, these results demonstrate that miR-543 plays an oncogenic role by negatively controlling Numb, revealing the existence of an miR-543/Numb/p53 regulatory pathway in PCa tumorigenesis and development.

SKIL facilitates tumorigenesis and immune escape of NSCLC via upregulating TAZ/autophagy axis.

Immune escape is an important mechanism in tumorigenesis. The aim of this study was to investigate roles of SKIL in tumorigenesis and immune escape of non-small-cell lung cancer (NSCLC). SKIL expression levels in NSCLC cell line, clinical sample, and adjacent normal tissue were measured by quantitative PCR, western blot, or immunohistochemistry. Lentivirus was used to overexpress/silence SKIL or TAZ expression. Malignant phenotypes of NSCLC cells were evaluated by colony formation, transwell, and MTT assays, and in xenograft mice model. Syngeneic mice model and flow cytometry were used to evaluate T cell infiltration. Quantitative PCR and western blot were applied to evaluate relevant mRNA and protein levels, respectively. Co-immunoprecipitation was applied to unveil the interaction between SKIL and TAZ. SKIL expression was higher in NSCLC tissue compared to adjacent normal tissue. Silencing of SKIL inhibited malignant phenotypes of NSCLC cells and promoted T cell infiltration. SKIL-knockdown inhibited autophagy and activated the STING pathway in NSCLC cells through down-regulation of TAZ. Silencing of TAZ cancelled the effects of SKIL overexpression on malignant phenotypes and autophagy of NSCLC cells. Inhibition of autophagy reversed the effects of SKIL/TAZ overexpression on the STING pathway. In conclusion, SKIL promoted tumorigenesis and immune escape of NSCLC cells through upregulation of TAZ/autophagy axis and inhibition on downstream STING pathway.

LncRNA NEAT1 Interacted With DNMT1 to Regulate Malignant Phenotype of Cancer Cell and Cytotoxic T Cell Infiltration via Epigenetic Inhibition of p53, cGAS, and STING in Lung Cancer.

PURPOSE: Lung cancer is the main cause of cancer-related mortality worldwide. We report here the biological role of nuclear paraspeckle assembly transcript 1 (NEAT1) in the pathogenesis of lung cancer and the underlying mechanisms. METHODS: Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and Western blotting analysis were used to evaluate expression of mRNA and protein. RNA immunoprecipitation (RIP) assay, chromatin immunoprecipitation followed by qPCR analysis, and reporter assay were used to detect DNA/RNA and protein binding. Tumor-infiltrating lymphocytes were assessed with hematoxylin-eosin staining. Cytotoxic T cell infiltration was evaluated with flow cytometric analysis and immunohistochemistry (IHC) staining. The changes of cell viability and cell invasive and migratory ability were analyzed by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide), colony formation, and Transwell assays, respectively. Syngeneic tumor model was set up to evaluate antitumor effect. RESULTS: The results showed that NEAT1 was overexpressed in lung cancer tissues and cancer cell lines. This aberrant expression was closely related with tumor stage and lymph node metastasis. Tumor sample with high CD8+ showed lower NEAT1 expression. In vitro studies displayed that inhibition of NEAT1 with shRNA resulted in suppression of survival and migration/invasion of lung cancer cells. On the other side, NEAT1 was found to promote tumor growth via inhibiting cytotoxic T cell immunity in syngeneic models. Finally, NEAT1 was found to interact with DNMT1, which in turn inhibited P53 and cyclic GMP-AMP synthase stimulator of interferon genes (cGAS/STING) expression. CONCLUSION: Our findings demonstrated that NEAT1 interacted with DNMT1 to regulate cytotoxic T cell infiltration in lung cancer via inhibition of cGAS/STING pathway. The results provided the novel mechanistic insight into the pathogenesis of lung cancer.

[Application studies on the gene diagnosis and carrier detection of hemophilia A by using polymerase chain reaction-conformation sensitive gel electrophoresis].

OBJECTIVE: To establish a simple, rapid and easy method for screening the gene mutation in hemophilia A, which was further applied to a direct diagnosis and carrier detection at gene level. METHODS: Twenty-four clinically diagnosed hemophilia pedigrees, including all the hemophilia patients and female members, were tested for the introns 22 and 1 in factor VIII gene by using inversion polymerase chain reaction (PCR) and regular PCR techniques. All the 26 exons of factor VIII gene were consecutively screened in the 17 patients manifesting non-inverted sequences in intron 22 by using PCR, subsequently all the 37 amplicons resulted from 26 exons were analyzed by conformation sensitive gel electrophoresis (CSGE), finally the mutated exons were subjected to sequencing verification. According to the mutation results, mothers and twin sisters of the hemophilia probands were tested by CSGE or subjected to nucleotide sequencing directly, to ascertain if those individuals had the same mutation or were the carriers of disease-causing gene. RESULTS: Intron 22 inversion was detected in 7 hemophilia probands out of 24 hemophilia pedigrees, intron 1 inversion was not detected in these pedigrees. Single-base mutations distributed in different exons of factor VIII gene were detected in 13 pedigrees with family history and 3 sporadic pedigrees, diagnosed as non-inverted 22 intron patients. By comprehensive usage of PCR-CSGE and nucleotide sequencing, the positive rate and the diagnosable rate of gene diagnosis or carrier detection in the 24 hemophilia pedigrees was 94.12% and 100% respectively. CONCLUSION: PCR-CSGE is a highly sensitive and special assay for detecting single base mutation. By integrated utilization of introns 22 and 1 of factor VIII gene detection and PCR-CSGE genotyping, combining with nucleotide sequencing, a direct diagnosis of all hemophilia pedigrees be could nearly make at gene level, including the sporadic families. This method might be used to screen new mutation theoretically and ascertain the mutation type. It is a simple, rapid and low-cost method, possessing unique advantages in direct diagnosis of hemophilia A and carrier screening. It should have important application value in hemophilia diagnosis.

Sp1-induced upregulation of the long noncoding RNA TINCR inhibits cell migration and invasion by regulating miR-107/miR-1286 in lung adenocarcinoma.

Long non-coding RNA tissue differentiation-inducing non-protein coding (TINCR) is associated with the carcinogenesis of several cancers. However, little is known about the function and mechanism of TINCR in lung adenocarcinoma (LUAD). Here, we aimed to analyze expression of TINCR and elucidate its mechanistic involvement in the progression of LUAD. The expression of TINCR was investigated according to Gene Expression Profiling Interactive Analysis at first and then detected in 29 LUAD tissues and paired adjacent normal tissues using qRT-PCR. Results indicated that TINCR was evidently downregulated in LUAD. The association between TINCR and clinicopathological parameters was analyzed by Pearson's chi-square test, suggesting TINCR was closely correlated with TNM stage and lymph mode metastasis. Subsequently, the function role of TINCR was examined by gain- and loss-of-function studies in LUAD (A549 and NCI-H292) cells. As analyzed by the scratch wound-healing and transwell assays, results revealed that TINCR suppressed the migration and invasion of A549 and NCI-H292 cells. However, TINCR exerted no effects on the cell proliferation as determined by CCK8 assay. Furthermore, we reported that loss of Sp1 could inhibit TINCR expression. Expressions of miR-107/miR-1286 were detected by qRT-PCR assay in A549 and NCI-H292 cells after TINCR knockdown or overexpression. In addition, the direct binding ability of the predicted miR-107 or miR-1286 binding site on TINCR was validated by luciferase activity assay. Results indicated TINCR could constrain the expression of miR-107/miR-1286, and was a target of them in LUAD cells. Bioinformatics analyses showed that BTRC and RAB14 was the potential target gene of miR-107 and miR-1286, respectively. These data revealed a possible regulatory mechanism in which upregulation of TINCR induced by Sp1 could constrain the migration and invasion through regulating miR-107 or miR-1286 in LUAD cells. Conjointly, our findings provide a valuable insight into the regulatory mechanism of TINCR in LUAD, supportive to its potential of therapeutic target for LUAD patients.

[The measurement of proportion and function of regulatory T cells in unexplained recurrent spontaneous abortion].

OBJECTIVE: To investigate the proportion and function of CD(4)(+)CD(25)(+) regulatory T cells (CD(4)(+)CD(25)(+) Tr) in unexplained recurrent spontaneous abortion (URSA). METHODS: (1) Proportion measurement: the proportion of CD(4)(+)CD(25)(+) Tr cells in peripheral blood was measured by double-label flow cytometric analysis. The samples were taken from 15 URSA women, 15 normal non-pregnancy women and 13 normal pregnancy women. (2) Function measurement: CD(4)(+)CD(25)(+) Tr cells and CD(4)(+)CD(25)(-) T cells were extracted from peripheral blood lymphocytes by the microbeads separation. The purity of CD(4)(+)CD(25)(+) Tr cells and CD(4)(+)CD(25)(-) T cells was measured by flow cytometry. The growth inhibitory effect of CD(4)(+)CD(25)(+) Tr cells on CD(4)(+)CD(25)(-) T cells was assessed in vitro. RESULTS: The proportion of CD(4)(+)CD(25)(+) Tr cells was decreased significantly in URSA women (6.9 +/- 1.8)% than that in normal non-pregnancy women [(10.8 +/- 1.1)%] (P<0.05) and normal pregnancy women [(11.2 +/- 1.4)%] (P<0.01). Moreover, the suppressive rate of CD(4)(+)CD(25)(+) Tr cells was decreased significantly in URSA women (75 +/- 6)% than that in normal non-pregnancy women [(89 +/- 4)%] (P<0.05) and normal pregnancy women [(90 +/- 4)%] (P<0.01). However, with respect to the proportion and function of CD(4)(+)CD(25)(+) Tr cells, there was no significant difference between normal non-pregnancy women and normal pregnancy women (P > 0.05). CONCLUSION: The results suggest that decrease in proportion and function of CD(4)(+)CD(25)(+) Tr cells may be associated with URSA.

Pharmacokinetics and tissue distribution of docetaxel liposome mediated by a novel galactosylated cholesterol derivatives synthesized by lipase-catalyzed esterification in non-aqueous phase.

The purpose of this study is to synthesize a novel galactosylated cholesterol derivative, cholesterol-diethenyl decanedioate-lactitol (CHS-DD-LA) through lipase-catalyzed esterification in non-aqueous and to evaluate the preparation, pharmacokinetics and biodistribution of docetaxel (DOC) liposomes modified with CHS-DD-LA (G-DOC-L), which may actively gather at the liver compared with the conventional DOC liposomes (DOC-L) and commercial dosage form of DOC injection (DOC-i). A rapid and simple liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay was developed for the determination of the DOC concentration in plasma and tissues with Taxol as the internal standard (IS). To measure the liver-targeting effect of the G-DOC-L, relative uptake rate (Re), peak concentration ratio (Ce), targeting efficiency (Te) and relative targeting efficiency (RTe) were reduced as the evaluation parameters. The results showed that the entrapment efficiency, particle size and Zeta potential of G-DOC-L was 76.8 ± 3.5%, 95.6 nm and 27.19 mV, respectively. After i.v. administration at the dose of 2.5 mg/kg in rats, a decrease in the AUC, MRT and an increase in CL (p < 0.05) were observed in the G-DOC-L group compared with DOC-L. All these results suggested that galactose-anchored liposomes could rapidly be removed from the circulation in vivo. The tissue distribution of G-DOC-L was widely different from that of DOC-L. The Re of G-DOC-L, DOC-L on liver was 4.011, 0.102; Ce was 3.391, 0.111; Te was 55.01, 3.08, respectively, demonstrating that G-DOC-L had an excellent effect on liver-targeting, which may help to improve the therapeutic effect of hepatic diseases.