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Objective To explore the mechanism of Golgi phosphoprotein 3(GOLPH3) regulating lysosomal biogenesisviamTORC1 signaling.Methods GOLPH3 knock-out(GOLPH3 KO) stable cell line was constructed by CRISPR Cas9. mTORC1 activity and the levels of TFEB and p-TFEB in the control cells and GOLPH3 KO cells were compared through Western blot. Further, lysosome-associated membrane protein LAMP1 was labelled by the means of immunofluorescence and the number of lysosomes in the control cells and GOLPH3 KO cells was compared.Results GOLPH3 KO suppressed mTORC1 activity significantly, decreased the cytoplasm level of pTFEB, increased the nuclear localization of active TFEB, and promoted the lysosome biogenesis.Conclusion Golgi protein GOLPH3 regulates lysosome biogenesis through mTORC1 signaling.

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Objective To investigate the effects of 3 types of trihalomethanes(THMs), the by-products in treatment of drinking water disinfection, on the development and reproductive capacity of germ cells ofCaenorhabditis elegans.Methods Tribromomethane( TBM), trichloromethane( TCM) and triiodomethane( TIM), 3 types of THMs were selected and wild typeCaenorhabditis eleganswas used as a model to detect germ cell apoptosis, oocyte number and the final brood size after exposure to different concentrations of THMs.Results The results of germ cell apoptosis showed that the exposure to 50 μmol/L TCM and TIM caused elevated levels of germ cell apoptosis in a dose-dependent manner. Oocyte counting results showed that exposure to 50 μmol/L all three THMs significantly reduced the number of oocytes. The brood size results showed that only 50 μmol/L TIM exposure significantly decreased the brood size of nematodes.Conclusion THMs showed negative impacts on the reproductive system of Caenorhabditis elegans, and the toxicity is related to the halogenated elements of THMs.

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Objective To investigate the effects of aflatoxin B1(AFB1) on the biophysical properties and cytoskeleton structure of human hepatocellular carcinoma cells(HCCs). Methods HepG2 cells were respectively treated with 0, 0.01, 0.1, 1, 5, 10 μmol/L AFB1 for 24 h and 48 h, and the cell viability was measured by CCK-8 kit. Based on this result, the influences of 10 μmol/L AFB1 on the osmotic fragility, membrane fluidity, electrophoretic mobility(EPM) and F-actin structure of cells were analyzed. Subsequently, total RNAs were extracted and the transcription-level changes of twelve main cytoskeleton binding proteins were detected by real-time quantitative PCR. Results The increased viability of HepG2 cells was induced by AFB1 in a dose-dependent manner after 48 h treatment. After treated with 10 μmol/L AFB1, the anti-hypotonic ability and EPM of HepG2 cells were enhanced. The content of F-actin in HepG2 cells increased obviously, while the mRNA expression levels of the main cytoskeleton binding proteins were altered. Conclusion AFB1 can affect the biophysical properties, cytoskeleton structure and its binding proteins of HepG2 cells, which may be directly related to its toxic action.

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Objective To construct a high-throughput in vitro method for the detection of botulinum toxin A enzyme activity by using fluorescence resonance energy transfer(FRET). Methods Recombinant expression plasmid based on double fluorescent labeled substrate was constructed to recognize botulinum toxin type A only, and the constructed plasmid was transferred into the E.coli expression system for expression. The expressed recombinant protein of fluorescent labeled substrate was purified, dialyzed and stored for standby; The activity of the recombinant protein was detected by digestion of botulinum toxin type A light chain(ALc); The conditions of this detection method were optimized; The enzyme kinetic parameters Kmand Kcatwere determined by cutting the fluorescent labeled substrate with ALc. Results The recombinant expression plasmid was successfully constructed. After being expressed in the E. coli expression system, the target band appeared obviously. The purity of the purified recombinant protein was about 90%. The recombinant protein was named CYA. CYA was identified by enzyme digestion of ALc and Botulinum toxin type B light chain(BLc). The results showed that CYA could only be digested by ALc to produce two protein fragments that were consistent with expectations, but could not be digested by BLc. By optimizing the conditions based on FRET substrate, it was obtained that the filter sensitivity was set between 65-110; The real-time dynamic detection interval was 2 min/time, the dynamic detection time was 30-120 min, and the appropriate concentration range of substrate CYA was 0.5-32 μmol/L. The ratio of the time change of CYA at any time under the action of ALc enzyme to the fluorescence value 528 and 485 was plotted to be about 0.5 at the minimum and 0.9 at the maximum. The enzyme kinetic parameters determined that the value of ALC cleaving CYA Kcatwas(5±0.4) s-1and Kmwas(2.33±0.21) μmol/L. Conclusion A high-throughput in vitro method for the detection of botulinum toxin type A activity based on FRET technology is successfully constructed.

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Objective To explore the effect of(-)-meptazinol-serotonin hybrid, [(-)-Mep-S] on scopolamine-induced learning and memory impairment in mice. Methods The binding of(-)-Mep-S and hAChE was analyzed by molecular docking. The effect of(-)-Mep-S on AChE activity was observed in vivo and in vitro. The effect of(-)-Mep-S on learning and memory impairment induced by scopolamine was examined in Morris water maze test in mice. The effect of(-)-Mep-S on the motor behaviors of mice was detected by open field test. Results Molecular docking analysis revealed that(-)-Mep-S binded to human AChE. In parallel,(-)-Mep-S overtly inhibited the activity of AChE derived from mouse forebrain and SH-SY5Y neuronal cells, and IC50 values were lower than those of the positive control drug rivastigmine. In the Morris water maze test, mice treated with(-)-Mep-S(2.5 mg/kg) showed significantly reduced latency on day 4(P<0.05). Moreover, they exhibited significantly greater percentage of distance travelled and percentage of time spent in the target quadrant in the probe trial on day 5(P<0.05).(-)-Mep-S at 0.5 mg/kg did not show any significant effects. In addition,(-)-Mep-S inhibited the enhancement of forebrain AChE activity induced by scopolamine(P<0.05). The open field test showed that the effect of(-)-Mep-S on scopolamine-induced learning and memory impairment was not due to the difference on the motor behaviors of mice. Conclusion(-)-Mep-S can effectively inhibit AChE activity and ameliorate scopolamine-induced learning and memory impairment in mice.

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Objective To investigate the immunomodulatory effects of umbilical cord mesenchymal stem cells(UC-MSCs) modified by miR-125b-5p through JAK2/STAT3 pathway on systemic lupus erythematosus(SLE). Methods UC-MSCs were isolated and cultured under aseptic conditions; Peripheral blood mononuclear cells(PBMCs) were separated from SLE patients by density gradient centrifugation. The relative expressions of IL-17A, Foxp3, IFN-γ, IL-4 genes in PBMCs cultured with UC-MSCs for 48 h were detected by RT-qPCR; The relative expressions of JAK2, p-JAK2, STAT3, p-STAT3 and IL-18 proteins in kidney tissues of MRL/lpr mice were detected by western blot. Results Compared with the PBMCs culture group, the expression of IFN-γ and IL-17A and the proportion of Th17/Treg cells were significantly down-regulated in UC-MSCs+miR-125b-5p group, UC-MSCs+miR-NC group and UC-MSCs group(P<0.01), the expression of the proportion of Th1/Th2 cells(P<0.05) was down-regulated in UC-MSCs+miR-125b-5p group and UC-MSCs+miR-NC group; Compared with the untreated group of MRL/lpr mice, the relative expressions of JAK2, p-JAK2, STAT3, p-STAT3 and IL-18 proteins in kidney tissues of MRL/lpr mice were significantly down-regulated in UC-MSCs+miR-125b-5p group(P<0.01), and the relative expressions of IL-18 proteins was significantly down-regulated in UC-MSCs+miR-NC group and UC-MSCs group(P<0.01). Conclusion miR-125b-5p plays a synergistic role in UC-MSCs, which regulates the differentiation of Th2 cells in PBMCs of SLE patients and the relative expressions of p-JAK2/JAK2, p-STAT3/STAT3, and IL-18 proteins in kidney tissues of MRL/lpr mice. UC-MSCs modified by miR-125b-5p may play immunomodulatory effect on SLE by JAK2/STAT3 signaling pathway.

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Objective To investigate the expression of CD155/TIGIT in patients with osteosarcoma and its significance. Methods The expression differences of CD155 and TIGIT in tumor tissues of distant metastatic osteosarcoma patients and non-metastatic osteosarcoma patients were analyzed by cancer Genome Atlas(TCGA) database. The surgically removed tissues of osteosarcoma patients were collected to prepare pathological sections and tissue chips, and the tumor tissue and cell morphology were observed by HE staining. Immunohistochemical staining was used to detect the expression of CD155 and TIGIT, and the scores were divided into high expression group and low expression group according to the scores. Chi-square test was used to analyze the relationship between CD155/TIGIT expression and clinical features and prognosis. Results TCGA database data showed higher expression of CD155 and TIGIT in patients with osteosarcoma accompanied by metastasis. HE staining of pathological sections revealed that tumor tissues with high expression of CD155/TIGIT contained more binuclear or multinucleated, hyperchromatic and obviously heteromorphic tumor cells. Immunohistochemical staining and score analysis of tissue chip showed that the expression of CD155 and TIGIT was correlated with clinical stage and distant metastasis(P<0.05), but not with age, sex, tumor size, pathological type and tumor necrosis rate. Conclusion CD155 and TIGIT may be one of the prognostic indicators of osteosarcoma.

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Objective To explore whether the NLRP3 inflammasome is activated after Newcastle disease virus (NDV) exposure to esophageal cancer ECA109 cells, whether its activation is related to K+efflux, and the effect of ATP/P2X7 axis on the activation of NLRP3 inflammasome.Methods The expression of NLRP3 and IL-1β was detected by Western blot; the content of IL-1β in the supernatant was detected by ELISA; the formation of ASC spots was detected by fluorescence immunoassay; the change of intracellular K+concentration was detected by fluorescent probe technology; Interventions with ATPase, ATP and P2X7 receptor inhibitors were used to investigate their role in NLRP3 inflammasome activation.Results Compared with the control group, the expression of NLRP3, IL-1β and ASC protein in cells was up-regulated after NDV F3 infection; the intracellular potassium concentration decreased with the prolongation of infection time(P<0. 05). After the intervention of P2X7 receptor inhibitor, the efflux of intracellular K+was blocked. With the increase of inhibitor concentration, the efflux of K+was maximally inhibited at 10 μmol/L(P<0. 05). The results of ATPase and ATP intervention showed that ATPase inhibited K+efflux, while ATP promoted K+efflux. Western blot results showed that compared with the control group, P2X7 receptor was inhibited, and the expressions of NLRP3 and IL-1β were down-regulated; after ATPase intervened cells, the expressions of NLRP3 and IL-1β decreased; After ATP intervention in cells, the protein expressions of NLRP3 and IL-1β were up-regulated(P<0. 05).Conclusion NDV F3 infection of ECA109 cells can activate the NLRP3 inflammasome,the mechanism may be related to the ATP/P2X7 axis.

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Objective To investigate the effect of silencing human leukocyte-associated antigen-G(HLA-G) expression in the chorionic trophoblast cell line JEG-3 cells under hypoxic conditions on the biological function of JEG-3 cells and through the hypoxia response pathway of endothelial PAS1 region protein 1(EPAS1)is involved in the molecular mechanism of preeclampsia under high altitude hypoxia.Methods The expression of HLA-G in JEG-3 cells was inhibited by transfection of small interfering RNA(siRNA). The JEG-3 cells after transfection were divided into four groups: normoxic control group, hypoxic control group, normoxic inhibition group, and hypoxic inhibition group.CCK-8 test and Transwell test were used to detect the proliferation and invasion ability of the cells in four groups; The effects of four groups of apoptosis and cell cycle were detected by flow cytometry; HLA-G and EPAS1 mRNA and protein expression levels were detected by real-time fluorescence quantitative PCR(qPCR) and Western blot. Results(1) Compared with the normoxic control group, hypoxic control group, normoxic inhibition group, and hypoxic inhibition group could reduce the proliferation activity and invasion ability of JEG-3 cells, and significantly increase the apoptosis rate. The hypoxic control group and hypoxic inhibition group also produced an obvious necrotic cell population; Under the condition of hypoxia, after reducing the expression of HLA-G, the cell necrosis rate was further aggravated; Whether under normoxia or hypoxia, inhibition of HLA-G expression caused the cells to be blocked in the G1 phase.(2) Compared with the normoxic control group, hypoxia control group, normoxic inhibition group, and hypoxia inhibition group decreased the expression of HLA-G protein, and hypoxia and inhibition of HLA-G had a synergistic effect; Hypoxia-inducible factors-2α(HIF-2α),vascular endothelial growth factor(VEGF) and endothelin-1(ET-1) protein expression could be added, inhibition of HLA-G decreased the expression of inducible nitric oxide synthase(NOS2). Conclusion In the hypoxic environment, silencing HLA-G may affect the biological behavior of trophoblast through the EPAS1 hypoxic response pathway and participate in the occurrence and development of preeclampsia.

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Objective To investigate whether GIV, a coiled helix structural domain protein containing 88A, has an effect on the neuroinflammatory response in a model of cerebral ischemia-reperfusion injury. Methods A middle cerebral artery embolization-reperfusion model(MACO/R) and an oxygen glucose deprivation/reoxygenation model(OGD 6 h+R 24 h) of BV2 microglia were constructed in C57BL/6 mice, and the area of cerebral infarction was detected by TTC staining; the Longa neurobiological score was used to evaluate the degree of neurological deficit in mice; ELISA was used to detect the release of IL-6 and TNF-α in the supernatant of peripheral blood and cell cultures, and Western blot was used to detect the protein expression of GIV, TREM2 and TLR4 in the cortical area around the infarct foci in mice; different concentrations of lipopolysaccharide(LPS, 1, 5, 10 μg/ml) were used to stimulate BV2 cells for 24 h to establish a neuroinflammation model, qRT-PCR was performed to detect the mRNA levels of IL-6, TNF-α and IL-1β, and Western blot was used to detect the expression of GIV; OGD/R culture treatment was performed after knocking down the expression of GIV gene using siRNA interference technique; ELISA was performed to detect the release concentration of IL-6 and TNF-α in cell culture medium supernatant; protein immunoblotting was performed to detect the knockdown efficiency of GIV. Results Both the successfully constructed MCAO/R and OGD/R models activated the neuroinflammatory response and induced a decrease in protein expression of GIV; MCAO/R induced increased concentrations of IL-6 and TNF-α release in peripheral blood of mice and promoted the protein expression of TREM2 and TLR4; LPS activated IL-6, IL-1β and TNF-α expression in BV2 cells, but did not affect GIV expression; siRNA interference with GIV gene expression further increased the expression of inflammatory factors IL-6 and TNF-α.Conclusion The GIV gene may be characteristically involved in regulating the neuroinflammatory response induced by cerebral ischemia-reperfusion injury, and it may be a potential therapeutic target for cerebral ischemia-reperfusion injury.

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Objective To investigate the function of FAK/Twist1 signaling pathway during craniosynostosis closure. Methods Ten days old rats were divided into a control group(n=50) and a rotation group(n=50). Both of them were made a approximately 0.5 cm circle bone window at the midpoint of the lambdoid suture of the rat. The bone flaps were left free without damaging the dura mater. The bone flaps in the control group were repositioned in situ, and the bone flaps in the rotation group were rotated 180° and repositioned 3 weeks later.Then the experiments were performance as followed: open field test, measurement of body weight, head circumference, bone flap area, and thickness of bone flap in the two groups, observation of cranial suture closure by microscopy and HE staining, FAK/Twist1 expression determined by Western blot, real-time PCR, and immunohistochemical staining in the bone flap and dure, respectively. Results The cranial sutures was completely closed in the rotation groupand that was open in the control group through detecting by microscopic examination and HE staining. The thickness of the bone flap in the derotation group was greater than that in the control group, with statistical significance(P<0.01). There were no significant differences between two groups in head circumference, weight, bone flap area, and operative area. The results of behavioral test showed that after the closure of cranial suture, the action ability of rats decreased and showed a tendency of depression.Compared with that in control group, the expression of FAK was significantly increased in the calvaria and dura as well as Twsit1 was significantly decreased in the dura in rotating group measuring by Western blot, real-time PCR, and immunohistochemical staining(P<0.05). Conclusion FAK/Twist1 may play an important role in craniosynostosis after rotation.

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Objective To investigate the correlation between CD47 and cervical lesions. Methods The expression and overall survival of CD47 in cervical cancer was analyzed using TCGA and TCGA Target GTEx databases.Based on GEO databases, the expression of CD47 was analyzed between cervical sample infected by HPV 16/18 and non-HPV.The differences of CD47 expression in the degrees of cervical lesions infected by HPV 16/18 were compared.The patholgical sections of colposcopy biopsy came from 320 patients who were admitted cervical cancer screening, including HPV typing and E6/E7 mRNA detection. Immunohistochemistry were used to determine CD47 expression, and histochemistry score(H-score) was used to quantify the levels of CD47.The correlation among HPV 16/18, pathological grades and CD47 was analyzed. Results Bioinformatics tool analysis and immunohistochemistry showed that CD47 was up-regulated in HSIL and CC, associated with the prognosis of CC. The expression levels of CD47 in cervical tissues increased significantly after HPV 16/18 infection(t=2.494, P<0.05), and in HPV 16/18 infected tissues, with the increase of pathological grades.The expression levels of CD47 significantly increased(F=4.351, P<0.05; rs=0.278, P<0.001).The expression of CD47 in the E6/E7 mRNA(+) was higher than that in the E6/E7 mRNA(-)(t=5.710, P<0.000 1), and the copies of E6/E7 were related to the degree of cervical lesions, and were positively correlated with the expression level of CD47(F=15.557, P<0.000 1; rs=0.649, P<0.000 1)(rs=0.73, P<0.000 1). Conclusion CD47 is overexpressed in HSIL and CC after HPV 16/18 infection. The expression levels of CD47 increases with the degree of cervical lesions, and is correlated with E6/E7 mRNA. CD47 may be a potential target for cervical disease progression and therapy.

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Objective To investigate the neuroprotective effect of mesenchymal stem cells(MSC) combined with low-intensity transcranial ultrasound(LITUS) treatment on traumatic brain injury(TBI). Methods Seventy-two SD rats were randomly divided into four groups, namely, control group, TBI group, MSC injection group, and combined treatment group, with 18 rats in each group. TBI model was established by applying a pneumatic controlled cortical impingement instrument. Within 24 h after surgery, MSC was injected into the injury site by microinjector and microinjector pump using in situ injection. After injection, the injury site was treated with LITUS for 28 consecutive days using an ultrasound stimulator. The modified neurological functioning score(mNSS) was performed on rats in each group at 1, 3, 7, 14, 21 and 28 days postoperatively, and then the brains were extracted to detect pathological changes at the injury site and the mRNA and protein expression of brain-derived neurotrophic factor(BDNF), growth associated protein-43(GAP-43), postsynaptic density protein-95(PSD-95) and glial fibrillary acidic protein(GFAP) by HE staining, immunohistochemistry, Western blot and RT-PCR.Results Compared with the control group, the mNSS score increased in the TBI group(P<0. 05), the expression of GAP-43 and PSD-95 decreased, and the expression of GFAP increased(P<0. 05); Compared with the TBI group, the mNSS score of MSC group was lower(P<0. 05), the expression of BDNF, GAP-43, PSD-95 increased, and the expression of GFAP decreased(P<0. 05); mNSS scores were lower in the combined treatment group than those in the MSC group(P<0. 05), the expression of BDNF, GAP-43, PSD-95 increased, and the expression of GFAP decreased(P<0. 05).Conclusion The mechanism by which MSC combined with LITUS exerts neuroprotective effects in TBI may be related to the promotion of BDNF, GAP-43, and PSD-95 expression and reduction of GFAP expression.

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Objective To investigate the effects of sulforaphane(SFN) on proliferation, migration and invasion of human renal carcinoma cells and its mechanism. Methods The cultured human renal carcinoma cells 786-O were divided into control group(0 μmol/L) and SFN group(5, 10, 20 μmol/L). The activated proliferation of cells was detected by CCK-8; the effect of SFN on migration of 786-O cells was detected by scratch healing assay and Transwell cell migration assay; the effect of SFN on the invasion ability of 786-O cells was detected by Transwell cell invasion ability assay; Western blot and qRT-PCR were used to detect the effects of SFN on the expression of epithelial-mesenchymal transition(EMT)-related proteins and mRNA. The effect of SFN on the expression of NF-κB signaling pathway was detected by Western blot. Results After SFN treatment for 24, 48 and 72 h, the proliferation activity of 786-O cells decreased with the increase of SFN concentration; compared with the control group, the cell migration and invasion ability of SFN-treated group were significantly reduced; with the increase of SFN concentration, the mRNA and protein expression levels of E-cadherin in 786-O cells increased, while the mRNA and protein expression levels of N-cadherin and Vimentin decreased; the levels of NF-κB signaling pathway related protein phosphorylated p65 and phosphorylated IκBα decreased with the increase of SFN concentration. Conclusion SFN may inhibit the proliferation, migration and invasion of human renal carcinoma cells by regulating the EMT process of renal carcinoma through inhibition of NF-κB signaling pathway.

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Objective To explore the mechanism of advanced glycation end products(AGEs) on diabetic endothelial cell damage based on monocyte-macrophage exosomes(Exos)/microRNA-92a(miR-92a). Methods Twenty apolipoprotein E-deficient(ApoE-/-) mice were randomly divided into two groups: injury group(n=10) and injury +STZ group(n=10). The injury+STZ group established a diabetes model induced by streptozotocin(STZ). All animals underwent partial left carotid artery(PLCA) ligation. The carotid arteries were collected, the number of M1 macrophages was detected by immunohistochemistry, and the level of AGEs was analyzed by ELISA. Microvascular endothelial cell line bEnd.3 cells were treated with conditioned medium(CM) of AGEs treated RAW264. 7 cells or Exos derived from RAW264. 7, followed by evaluations of the cell barrier function and mitochondrial function.Results There was an increased number of M1 macrophages in carotid atherosclerotic tissues of diabetic mice and in AGEs treated RAW264. 7 cells. CM or Exos significantly induced barrier dysfunction, reactive oxygen species(ROS) accumulation and mitochondrial dysfunction in vascular endothelial cellsin vitro. In addition, bioinformatics analysis showed that miR-92a was up-regulated in Exos derived from macrophages stimulated by AGEs. Experimentally, Exos participated in CM-induced barrier dysfunction, ROS accumulation and mitochondrial dysfunction in bEnd. 3 cells by transferring miR-92a. Finally, a series of rescue experiments further confirmed that Exos regulated the barrier dysfunction and mitochondrial function in vascular endothelial cells through miR-92a.Conclusion The expression of AGEs and the number of M1 macrophages in diabetic ApoE-/-mice increase,and AGEs stimulates Exos from macrophages could impair the barrier function and mitochondrial function in vascular endothelial cells by delivering miR-92a in vitro.

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Objective To investigate the effect and mechanism of phosphoprotein phosphatase 5 catalytic(PPP5C) on the migration, invasion and tumor stemness of human lung adenocarcinoma H1299 cells. Methods The PPP5C-pcDNA3.1 overexpression vector was constructed. PPP5C-pcDNA3.1 and pcDNA3.1 were transfected into H1299 cells, and H1299 stable cell lines were screened with G418. The mRNA and protein expression levels of PPP5C were identified by qRT-PCR and Western blot. The proliferation activity of H1299 cells was detected by drawing cell growth curve and cell clonal formation assay. The wound-healing assay and transwell assay were used to test the migration and invasion abilities of H1299 cells, respectively. The stemness of H1299 cells was evaluated by sphere formation assay. Results The PPP5C-pcDNA3.1 eukaryotic expression vector was successfully constructed and the expression levels of PPP5C significantly increased after transfection into H1299 cells. After overexpression of PPP5C in H1299 cells, the cell growth curve and clonal formation assay displayed that the proliferation ability was not affected, the migration and invasion of cells were significantly enhanced through wound-healing assay and transwell assay, accompanied by an increase in the expression of MMP9, stem cell spheroid assay showed a significant increase in stemness of cells, accompanied by increased expression of SOX2. Conclusion The proliferation ability of cells is not affected, the migration and invasion and the stemness of cells are enhanced by regulating MMP9 and SOX2 respectively, after overexpression of PPP5C in human lung adenocarcinoma H1299 cells.

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Objective To screen the differential expression profile of circular RNA(circRNA) in pancreatic cancer and analyze its potential function. Methods Four pairs of pancreatic cancer tissues and corresponding adjacent pancreatic tissues were selected for high-throughput sequencing, and the differentially expressed circRNA was screened according to fold change>2 and P<0.05. qRT-PCR was used to detect the expression of 5 randomly selected differentially expressed circRNA in 20 pairs of pancreatic cancer tissue samples. GO and KEGG enrichment analysis of differentially expressed circRNA was performed, and the top 50 circRNA-microRNA interaction network was constructed. The relationship between hsa＿circ＿0046523 and the clinicopathological features of pancreatic cancer was further analyzed, and the effects of hsa＿circ＿0046523 on the proliferation, migration and invasion of pancreatic cancer cells were observed by functional experiments. Results A total of 17 182 circRNA were predicted by high-throughput sequencing, of which 302 circRNA were differentially expressed including 137 circRNA were upregulated and 165 circRNA were downregulated in pancreatic cancer tissues. The qRT-PCR results showed that the expression levels of hsa＿circ＿0046523, hsa＿circ＿0004220 and hsa＿circ＿0000690 in pancreatic cancer tissues were significantly upregulated(P<0.05), while the expression levels of hsa＿circ＿0008676 and hsa＿circ＿0004416 were significantly downregulated(P<0.05), which was consistent with the sequencing results. GO analysis indicated that differentially expressed circRNA were mainly involved in substrate-dependent cell migration, protein kinase complex and cytoskeletal protein binding. KEGG pathway enrichment analysis showed that differentially expressed circRNA were mainly involved in protein digestion and absorption, ABC transporters, central carbon metabolism in cancer, and glycine, serine and threonine metabolism pathways. The expression level of hsa＿circ＿0046523 was closely correlated with tumor differentiation(P=0.002), T stage(P=0.006), lymph node metastasis(P=0.011) and TNM stage(P=0.001). Compared with HPDE6-C7 cells, the expression of hsa＿circ＿0046523 significantly increased in pancreatic cancer SW1990, Mia PaCa-2, BxPC-3 and Capan-2 cells(P<0.05). hsa＿circ＿0046523 knockdown significantly inhibited the proliferation, migration and invasion of pancreatic cancer Mia PaCa-2 BxPC-3 cells(P<0.05). Conclusion There are characteristic differentially expressed circRNAs in pancreatic cancer tissues, and these differentially expressed circRNAs may be involved in the occurrence and development of pancreatic cancer.

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Objective To explore the growth characteristics of rat calvaria by detecting the calvaria of SD rats in different periods. Methods The calvaria of SD rats at 1, 4, 7, 10, and 12 weeks from the same littermate were selected(3 rats per week). Real-time PCR and Western blot techniques were used to detect the expression of focal adhesion kinase(FAK)-phosphatidylinositol 3 kinase/protein kinase B(PI3K/AKT)signal pathway in the calvaria, and the role of FAK-PI3K/AKT in the growth and development of the calvaria was analyzed by correlation.Results The increase of brain volume and the thickness of calvaria increased synchronously, the expression of FAK was positively correlated with the changes of meridians, and the expression of FAK was positively correlated with the expression of PI3K/AKT.Conclusion The expression of FAK is related to the growth and development of rat skull. FAK plays a role in calvaria by activating PI3K/AKT signal pathway. FAK may be used as a marker of rapid skull growth and development, which provides a basic theoretical basis for the timing of clinical skull defect repair and treatment.

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Objective To investigate the associations between the single nucleotide polymorphism(SNP) in vestigial like family member 4(VGLL4) gene and Helicobacter pylori(H. pylori) infection. Methods The blood samples of 450 normal physical examiners were collected, and the samples were divided into H. pylori negative group(n=220) and H. pylori positive group(n=230) using enzyme-linked immunosorbent assay(ELISA). SNP rs1803489, rs7617620, and rs13078528 in VGLL4 gene were genotyped using polymerase chain reaction(PCR)-restriction fragment length polymorphism(RFLP) technology. Results SNP rs1803489, rs7617620, and rs13078528 in VGLL4 gene were not associated with H. pylori infection in the Han population in Baotou, Inner Mongolia. Conclusion SNP rs1803489, rs7617620, and rs13078528 in VGLL4 gene may not play a major role in H. pylori infection in Baotou Han population.

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Objective To explore the effects of Kruppel-like factor 7(KLF7) on the proliferation, migration and cycle of esophageal squamous cell carcinoma cell line Eca109. Methods Bioinformatics analysis was used to study the expression pattern of KLF7 in esophageal squamous cell carcinoma and its relationship with prognosis and pathological grade. The expression pattern of KLF7 in esophageal squamous cell carcinoma cells was studied by real-time PCR, Western blot and cellular immunofluorescence in vitro. The KLF7 overexpression in Eca109 cells was carried out by transfection of the plasmid of pcDNA-3.10-KLF7, and the effects of KLF7 on the proliferation and migration of ESCC cells were detected by CCK-8 assay, plate clone formation assay and Transwell assay, and the cell cycle was studied by Flow cytometric analysis. Results KLF7 was expressed in the tissues and cells of esophageal cancer, and its expression was significantly associated with the pathological grade of esophageal cancer patients(P<0.05), however, there was no significant associaition of KLF7 expression with the survival rate of patients. The expression level of KLF7 in esophageal cancer cells(Eca109 and EC9706) was significantly higher than that in normal esophageal epithelial cells(Het-1A,P<0. 05). In addition, the expression of KLF7 was higher in the cytoplasm and nucleus of Het-1A cells, while only in the nucleus of esophageal cancer cells(Eca109 and EC9706).Additionally, the overexpression of KLF7 in Eca109 cells changed the cell cycle, promoted cell proliferation and migration, and up-regulated the expression levels of CCND1 and P53(P<0. 05).Conclusion Overexpression of KLF7 might promote the proliferation and migration of esophageal cancer cells.

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Objective To explore the type and control measure of black dot-like contaminants in cell cultures.Methods The growth state of bacteria was investigated under an inverted microscope; Their morphological characteristics were analyzed by Gram and auramine O staining as well as electron microscopy; 16S rDNA gene sequencing was used to analyze bacterial species; Drug sensitivity test was used to screen antibiotics against the bacteria;Cryopreserved SH-SY5Y cells were resuscitated by cell culture supernatant of RAW264 cells.Results Inverted microscopic real-time observations showed that black dot-like substances had two growth states: static and moving.They were negative for Gram staining while positive for auramine O staining. Electron microscopy revealed that they were short rod-shaped bacteria with a polar flagellum during moving phase. 16S rDNA gene sequencing showed that these bacteria werephenylobacterium zucineum HLK1.Ceftriaxone, carboxycillin and imipenem were screened by drug sensitivity test to have inhibitory effects on the bacteria, but cell culture experiments showed that they could not remove the bacteria from SH-SY5Y cells. Contaminated cells could not be cryopreserved for a long time, but resuscitation with RAW264. 7 cell culture supernatant significantly improved the survival rate of cells.Conclusion The black dot-like contaminants in cell cultures are a special type of oligotrophic bacterium with strong viability that can invade the cells and cannot be cleared with antibiotic treatment. RAW264. 7 cell culture supernatant seems contain some substances against bacteria, and resuscitating frozen cells with RAW264. 7 cell culture supernatant may significantly improve the survival rate of cells.

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Objective To investigate the activation level of NLRP3 inflammasome in immune thrombocytopenia(ITP) and the effect of inhibiting NLRP3 mediated inflammasome activation on polarization and immune function of M1 macrophages. Methods The expression of NLRP3 mRNA in peripheral blood mononuclear cells(PBMC) and CD14+monocytes of ITP patients(ITP group) and Control group(Control group) was detected by RT-qPCR. The levels of IL-1β and IL-18 in serum of the two groups were determined by ELISA. M0 macrophages(MDMs) from ITP group were divided into 4 groups: IgG control group(IgG group), MCC950 treatment group(MCC950 group), LPS, IFN-γ and IgG treatment group(LPS+IFN-γ+IgG group) and LPS, IFN-γ and MCC950 treatment group(LPS+IFN-γ+MCC950 group); mRNA and protein levels of M1 macrophage markers CD86, iNOS and MCP-1 were detected by RT-qPCR and Western blot. Western blot was used to detect the expression of NLRP3 inflammasome associated protein, ASC, Cleaved caspase-1 and IL-β. Flow cytometry was used to detect the phagocytosis of MDMs on platelets in each group, and CFSE was used to detect the proliferation of CD4+T and CD8+T. Results Compared with the control group, the expression of NLRP3 mRNA in PBMC and CD14+monocytes, and the concentration of IL-1β and IL-18 in serum of ITP group increased significantly(P<0.05). Platelet counts were negative correlated with NLRP3 mRNA expression in CD14+monocyte and the concentration of IL-1β, IL-18 in serum in patients with ITP(P<0.05).Compared with IgG group, the mRNA and protein expressions of M1 macrophage markers CD86, iNOS, MCP-1, and the protein expression level of NLRP3, ASC, cleaved caspase-1 and IL-β, the platelet phagocytosis and the proliferation promoting ability of CD4+T and CD8+T cells significantly increased in LPS+IFN-γ+IgG group and LPS+IFN-γ+MCC950 group( all P<0.05). Compared with LPS+IFN-γ+IgG group, the above indexes significantly decreased in LPS+IFN-γ+MCC950 group(P<0.05). Conclusion The activation level of NLRP3 inflammasome in ITP is abnormally elevated, which was related to the excessive M1 polarization of MDMs. Inhibiting NLRP3 mediated inflammasome activation could attenuate the M1 polarization and immune function of macrophages.

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Objective To explore the factors associated with the transplant outcomes after the treatment of preimplantation genetic testing(PGT) for infertile patients. Methods A retrospective analysis of data of frozen-thawed embryo transfers in PGT for structural rearrangements/PGT for monogenic(PGT-SR/PGT-M) cycles was performed. According to the transplant outcomes, it was divided into the pregnancy group(74 cases) and the non-pregnancy group(74 cases). The factors including male age, female age, duration of infertility, number of abortion, female BMI, follicle-stimulating hormone(FSH), luteinizing hormone(LH), estradiol(E2), progesterone(P), blood glucose, insulin, albumin, total cholesterol, triglycerides, high-density lipoprotein(HDL), parents′ causes of infertility, type of chromosomal abnormality, number of retrieved oocyte, number of meiosis II(MII), number of fertilization, number of two pronuclei(2PN), endometrial thickness on embryo transfer day, endometrial preparation, day of embryo development, presence of a top quality embryo(TQE) or not were analyzed by the Mann-Whitney U test and Pearson′s chi-square test. The probability of transplant outcomes in correlation with the relevant parameters analyzed was measured with multivariate logistic regression analysis. Results Univariate analysis indicated that E2, insulin, total cholesterol, triglycerides and endometrial preparation were significantly associated with transplant outcomes in PGT-SR, PGT-M cycles(P<0.05). Multiple logistic regression analysis revealed that lower levels of insulin(OR=0.14, 95% CI=0.03-0.27) and triglycerides(OR=1.38, 95% CI=0.14-2.78) in women were beneficial to transplant outcomes in PGT cycles. For endometrial preparation, compared with natural cycles, hormone replacement therapy(HRT) cycles(OR=3.52, 95% CI=2.54-4.65) were another factor to improve transplant outcomes. Conclusion There is a correlation between the levels of insulin and triglycerides, endometrial preparation and transplant outcomes in PGT cycles. In detail, lower levels of insulin and triglycerides, and the HRT cycle can lead to a better transplant outcome.

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Objective To establish a three-dimensional cephalometric standardized value database for Anhui province, to study the craniofacial characteristics of normal adult population in Anhui, China and to provide effective information for making orthodontic and orthognathic surgery treatment plans of the craniofacial deformity. Methods 160 normal occlusal volunteers were selected according to the inclusive and exclusive criteria and were photographed by Cone Beam CT(CBCT). The coordinate system was established for the 3D model reconstructed with Dolphin based on CBCT data, and 38 hard tissue landmarks were located. 40 lines and 20 angles were defined and measured, and measurements were analyzed with SPSS 17.0, to evaluate craniofacial features of the overall, midface, maxilla, mandible, alveolar bone, and dentition of the standard population. Results The 3D cephalometric measurements of craniofacial hard tissues of normal adults in Anhui were obtained. The symmetry of craniofacial hard tissues was good both in males and females with the maximum asymmetry ratio of 2.2% and the minimum of 0.01%. The gender comparisons showed that most of lines measured of males were larger than those of females(P<0.01), while there was no significant difference in the measurements of the midline deviation of upper and lower mandible, the relative deviation of the maxillary and mandible and the protrusion of upper and lower incisor. Additionally, there was no statistical significance in angles measured between genders, except for the occlusal plane angle. The results also showed that Chinese had unique facial features based on the measured data, compared with other ethnic populations. Conclusion The normal occlusal and maxillofacial hard tissue of Anhui Han has good symmetry.The gender obviously influences line measurements, with generally the measurements of males larger than females, but it has no effect on most angle measurements.

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Objective To identify the associated factors of peripheral blood lymphocyte counts(PBLC) in gastric cancer patients. Methods In this cross-sectional study, data were collected from patients underwent gastric cancer surgery. Univariate analysis, multiple linear regression and variable importance metrics were used to assess factors associating PBLC in pre-operative patients. The findings were further investigated by fitting locally weighted regression(loess) and robust linear model(rlm). Results Patients with pre-operative PBLC<1.1×109/L(group A) were 138(20.72%), whereas patients with PBLC ≥ 1.1×109/L(group B) were 528(79.28%). Compared to group B, patients in group A were older[(64.61±10.42 years) vs(62.18±10.41 years), P<0.05] and showed lower neutrophils [(3.21±1.41) ×109/L vs(3.59±1.31) ×109/L, P<0.01]. Lymphopenia was associated with later stage of gastric cancer, P<0.01. A multiple linear regression model with randomly distributed residuals found that age(β=-0.01, t=-3.70, P<0.01) and tumor stage [β(Ⅱ vs Ⅰ)=-0.16, t=-2.79, P<0.01; β(Ⅲ vs Ⅰ)=-0.18, t=-3.86, P<0.01; β(Ⅳ vs Ⅰ)=-0.21, t=-2.16, P<0.05] were to be statistically significant factors for lymphopenia. Neutrophil increase(β=0.05, t=3.61, P<0.01) was associated with a higher PBLC. The relative importance analysis for continuous independent variables showed that relative importance metric LMGs in variable age, neutrophil and CEA were 55.55%, 44.14% and 0.31% respectively. Loess and rlm models showed that age was the negatively influencing factor of PBLC. Conclusion PBLC in gastric cancer patients were positively associated with neutrophils, and negatively associated with age and tumor stages.

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Objective To study the relationship between the single nucleotide polymorphism of HLA-A rs3132682 and the risk of liver cancer. Methods The author selected 291 cases of liver cancer patients in Yanbian area as the experimental group and 272 healthy people as the control group. The genotypes and allele frequencies of the two loci were detected by MassARRAY SNPS mass spectrometry. Odds ratio(OR) and 95% confidence interval(CI) were calculated by unconditional logistic regression to evaluate the risk of liver cancer in patients with different genotypes. Results There were G and C alleles in HLA-A rs3132682 locus, GG, GC and CC genotype. After adjusting for confounding factors in HLA-A rs3132682 locus, there was correlation between CC genotype and the risk of liver cancer compared with GG genotype(P<0. 05). Stratification analysis showed that compared with the population with CG + GG genotype, the Korean population with CC genotype increased the risk of liver cancer by 3. 331 times.Conclusion The single nucleotide polymorphism ofHLA-Ars3132682 is correlated with the risk of liver cancer in Yanbian area.

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Objective To investigate the relationship between monokine induced by interferon-gamma(MIG) level and coronary slow flow phenomenon(CSFP). Methods 80 patients diagnosed with CSFP and 54 patients with normal CAG were selected as the CSFP group and no-CSFP group respectively in this study. Coronary slow flow was determined quantitatively by thrombolysis in myocardial infarction(TIMI) frame count(TFC) method. The clinical characteristics and biochemical indexes, including serum CD40L and Interferon-γ(IFN-γ), monokine induced by interferon-gamma(MIG) levels were measured, and the relationship between interferon-γ induced monocyte level and CSFP were analyzed. Results The serum levels of CD40L, IFN-γand MIG in the CSFP group were higher than those in the no-CSFP group(P=0.001). The MIG levels were positive correlated with mean TFC(r=0.879,P=0.009). Multivariate logistic regression analysis showed that MIG was an important risk factor for CSFP(β=0.874,P=0.011). The ROC curve analyses indicated that the MIG levels had diagnostic value in patients with CSFP, the area under the curve(AUC) was 0.793, the sensitivity was 0.79% and the specificity was 76.0%, and 95% CI 0.714-0.872. Conclusion Chemokine CD40L, IFN-γ and MIG may be involved in the process of vascular inflammation and arteriosclerosis. MIG is an important influencing factor of CSFP and participated in the occurrence and development of CSFP.