OBJECTIVE: To explore the influence of long non-coding (lnc) RNA Gm15645 on the podocyte injury in mice with diabetic nephropathy. METHODS: Male db/db mice (with Type 2 diabetes) with a genetic background of C57BLKs/J and db/m mice (healthy) born in littermates were randomly divided into three groups. db/db group was injected with lncRNAGm15645 shRNA lentivirus with a podocyte-specific marker NPHS2; db/db blank group was injected with saline, and db/db control group was injected withnon-sense lentivirus. The results of PAS staining, pathological changes of renal tissue, relative expression of GSK-3beta, and podocin expression were compared. RESULTS: lncRNAGm15 645 was overexpressed and podocin was down-regulated in the lentivirus overexpressed group. Mesangial cell proliferation, mesangial matrix hyperplasia, thickened basement membrane, widely fused foot process, and podocyte injury were observed by PAS staining. The expression of Gm15645 in the db/db group was significantly lower than that of the db/db blank group and db/db control group (P< 0.05), while the expression of podocin was higher (P< 0.05). Gm15645 was co-stained with podocin in renal tissue, and the target gene was GSK-3beta. CONCLUSION lncRNAGm15645 may provide an early biomarker for the occurrence of podocyte injury in diabetic nephropathy. The mechanism may be related to the feedback regulation of GSK-3beta gene.
Animals ; Diabetes Mellitus, Type 2 ; Diabetic Nephropathies/genetics* ; Glycogen Synthase Kinase 3 beta ; Male ; Mice ; Podocytes ; RNA, Long Noncoding/genetics*

OBJECTIVE: To explore the role of Long noncoding RNA UFC1 (lincRNA-UFC1) in modulating the metastasis and invasion of hepatocellular carcinoma (HCC) cells and the underlying mechanism. METHODS: Human HCC cell line Huh7 was infected with the lentiviral vector carrying lincRNA-UFC1 to obtain a cell line with lincRNA-UFC1 overexpression. A short hairpin RNA (shRNA) targeting lincRNA-UFC1 was delivered in human HCC BEL-7402 cells via a lentiviral vector to obtain a cell line with lincRNA-UFC1 knockdown. Expression levels of lincRNA-UFC1 in the two HCC cell lines were detected using real-time PCR, and the changes in the cell invasion and migration in response to lincRNA-UFC1 overexpression or knockdown were analyzed using Transwell and wound-healing assays. The expressions of GSK-3β/β-catenin-related proteins in the cells were detected with Western blotting. XAV-939, a GSK-3β/β-catenin inhibitor, was used for assessing the impact of lincRNAUFC1 overexpression on the invasion and migration of the HCC cells through Transwell and wound-healing assays. RESULTS: Overexpression of lincRNA-UFC1 significantly promoted the invasion and migration of Huh7 cells as compared with the control cells ( < 0.001), while lincRNA-UFC1 knockdown obviously suppressed the invasion and migration of BEL-7402 cells ( < 0.001). The results of Western blotting showed that the expressions of proteins associated with the cell invasion and migration, namely β-catenin and P-GSK-3β, were significantly upregulated in response to lincRNA-UFC1 overexpression, and were obviously lowered after lincRNA-UFC1 knockdown. Treatment of the cells with XAV-939 significantly reversed the effect of lincRNA-UFC1 overexpression on the cell invasion and migration ( < 0.001). CONCLUSIONS lincRNA-UFC1 overexpresison promotes cell invasion and migration through the GSK-3β/β-catenin axis in HCC cells .
Carcinoma, Hepatocellular ; genetics ; Cell Line, Tumor ; Glycogen Synthase Kinase 3 beta ; Humans ; Liver Neoplasms ; genetics ; Neoplasm Invasiveness ; Neoplasm Metastasis ; RNA, Long Noncoding ; beta Catenin

The recombinant adenoviruses expressing miR-22 (Ad-miR-22) was constructed and the effect of Ad-miR-22 on insulin signal pathway and glucose uptake in HepG2 cells was analyzed. MiR-22 gene was amplified by PCR from human hepatocytes and cloned into the pAdTrack-CMV vector to generate the shuttle plasmid pAdT-22. The positive colonies were confirmed by PCR and sequencing. The resultant shuttle plasmid was linearized with Pme I, followed by co-transformation into competent BJ5183 cells containing an adenoviral backbone plasmid (pAdEasy-1) to create the recombinant plasmid pAd-miR-22. After digested with Pac I, the linearized pAd-miR-22 was transfected into 293A packaging cell line to generate recombinant adenoviruses Ad-miR-22. HepG2 cells were infected with Ad-miR-22 or control Ad-GFP (adenoviruses expressing green fluorescent protein), and then the miR-22 expression levels were analyzed by qPCR. The result shows that adenovirus-mediated overexpression of miR-22 significantly decreased insulin-induced glucose uptake in HepG2 cells. Moreover, overexpression of miR-22 markedly decreased insulin-induced phosphorylation of GSK-3β. miR-22 also increased the mRNA levels of gluconeogenic genes in HepG2 cells. Furthermore, Western blotting results indicate that the protein expression of SIRT1 decreased in Ad-miR-22 infected HepG2 cells as compared with Ad-GFP infected HepG2 cells. In summary, overexpressing of miR-22 significantly increased gluconeogenesis while decreased glucose uptake in HepG2 cells. The effect of miR-22 on glucose metabolism may be mediated by SIRT1.
Adenoviridae ; genetics ; Glucose ; metabolism ; Glycogen Synthase Kinase 3 beta ; metabolism ; Hep G2 Cells ; Humans ; MicroRNAs ; genetics ; metabolism ; Signal Transduction ; genetics ; Transfection

The present study explored the main active ingredients and the underlying mechanism of Spatholobi Caulisin the treatment of ovarian cancer(OC) by network pharmacology, molecular docking, and in vitro cell experiments. The active ingredients and their predicted targets(AITs) were first acquired online with the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform(TCMSP). Theoretical disease targets(DTs) were obtained through professional databases including GeneCards, OMIM, PharmGkb, TTD, and DrugBank. The common targets in the intersection of AITs and DTs were used for the construction of a "drug-ingredient-disease-target" network by Cytoscape 3.7.1. STRING database was used to construct a protein-protein interaction(PPI) network. R 4.0.5 was used for GO and KEGG functional enrichment analyses. Schr9 dinger Maestro was used to perform and optimize the molecular docking and virtual screening.Twenty-three active ingredients of Spatholobi Caulis were screened out, involving 75 OC targets and 178 signaling pathways.Network analysis revealed that Spatholobi Caulis presumedly exerted an anti-OC effect by acting on key protein targets such as GSK-3β, Bcl-2, and Bax. Molecular docking showed that GSK-3β possessed goodbinding activity to prunetin. In vitro cell experiments preliminarily verified the core targets and pathways of prunetin, the active ingredient of Spatholobi Caulis against human OC SKOV3 cells.CCK-8 assay was used to detect the cell proliferation, and flow cytometry was used to detect the effect of prunetin on apoptosis of human OC SKOV3 cells.The expression of prunetin targets and related regulatory proteins was detected by Western blot.In vitro cell experiments demonstrated that prunetindisplayed significant inhibitory effects on the proliferation of OC cells and could induce apoptosis of SKOV3 cells. Western blot showed that prunetin could induce SKOV3 cell apoptosis by inhibiting GSK-3β phosphorylation and regulating the expression of downstream Bcl-2 and Bax proteins. This study reveals the scientific nature of network pharmacology in the prediction and guidance of experimental design, confirming that prunetin can treat OC by blocking the GSK-3β/Bcl-2/Bax cell signal transduction pathway. The findings are expected to provide a basis for the investigation of the mechanism of Spatholobi Caulis in the treatment of OC.
Drugs, Chinese Herbal/pharmacology* ; Glycogen Synthase Kinase 3 beta/genetics* ; Humans ; Medicine, Chinese Traditional ; Molecular Docking Simulation ; Network Pharmacology ; Ovarian Neoplasms/genetics*

OBJECTIVE: To observe the effects of Yizhi Tiaoshen (benefiting mental health and regulating the spirit) acupuncture on learning and memory function, and the expression of phosphorylated tubulin-associated unit (tau) protein in the hippocampus of Alzheimer's disease (AD) model rats, and explore the effect mechanism of this therapy on AD. METHODS: A blank group and a sham-operation group were randomly selected from 60 male SD rats, 10 rats in each one. AD models were established in the rest 40 rats by the intraperitoneal injection of D-galactose and okadaic acid in the CA1 region of the bilateral hippocampus. Thirty successfully-replicated model rats were randomly divided into a model group, a western medication group and an acupuncture group, 10 rats in each one. In the acupuncture group, acupuncture was applied to "Baihui" (GV 20), "Sishencong" (EX-HN 1), "Neiguan" (PC 6), "Shenmen" (HT 7), "Xuanzhong" (GB 39) and "Sanyinjiao" (SP 6); and the needles were retained for 10 min. Acupuncture was given once daily. One course of treatment was composed of 6 days, with the interval of 1 day; the completion of treatment included 4 courses. In the western medication group, donepezil hydrochloride solution (0.45 mg/kg) was administrated intragastrically, once daily; it took 7 days to accomplish one course of treatment and a completion of intervention was composed of 4 courses. Morris water maze (MWM) and novel object recognition test (NORT) were used to assess the learning and memory function of the rats. Using HE staining and Nissl staining, the morphological structure of the hippocampus was observed. With Western blot adopted, the protein expression of the tau, phosphorylated tau protein at Ser198 (p-tau Ser198), protein phosphatase 2A (PP2A) and glycogen synthase kinase-3β (GSK-3β) in the hippocampus was detected. RESULTS: There were no statistical differences in all of the indexes between the sham-operation group and the blank group. Compared with the sham-operation group, in the model group, the MWM escape latency was prolonged (P<0.05), the crossing frequency and the quadrant stay time in original platform were shortened (P<0.05), and the NORT discrimination index (DI) was reduced (P<0.05); the hippocampal cell numbers were declined and the cells arranged irregularly, the hippocampal neuronal structure was abnormal and the numbers of Nissl bodies decreased; the protein expression of p-tau Ser198 and GSK-3βwas increased (P<0.05) and that of PP2A decreased (P<0.05). When compared with the model group, in the western medication group and the acupuncture group, the MWM escape latency was shortened (P<0.05), the crossing frequency and the quadrant stay time in original platform were increased (P<0.05), and DI got higher (P<0.05); the hippocampal cell numbers were elevated and the cells arranged regularly, the damage of hippocampal neuronal structure was attenuated and the numbers of Nissl bodies were increased; the protein expression of p-tau Ser198 and GSK-3β was reduced (P<0.05) and that of PP2A was increased (P<0.05). There were no statistically significant differences in the above indexes between the acupuncture group and the western medication group (P>0.05). CONCLUSION Acupuncture therapy of "benefiting mental health and regulating the spirit" could improve the learning and memory function and alleviate neuronal injure of AD model rats. The effect mechanism of this therapy may be related to the down-regulation of GSK-3β and the up-regulation of PP2A in the hippocampus, and then to inducing the inhibition of tau protein phosphorylation.
Male ; Animals ; Rats ; Rats, Sprague-Dawley ; Glycogen Synthase Kinase 3 beta ; Tubulin ; Alzheimer Disease/therapy* ; tau Proteins/genetics* ; Acupuncture Therapy ; Hippocampus

BACKGROUND: Histone deacetylase 4 (HDAC4) regulates chondrocyte hypertrophy and bone formation. The aim of the present study was to explore the effects of HDAC4 on Interleukin 1 beta (IL-1β)-induced chondrocyte extracellular matrix degradation and whether it is regulated through the WNT family member 3A (WNT3A)/β-catenin signaling pathway. METHODS: Primary chondrocytes (CC) and human chondrosarcoma cells (SW1353 cells) were treated with IL-1β and the level of HDAC4 was assayed using Western blotting. Then, HDAC4 expression in the SW1353 cells was silenced using small interfering RNA to detect the effect of HDAC4 knockdown on the levels of matrix metalloproteinase 3 (MMP3) and MMP13 induced by IL-1β. After transfection with HDAC4 plasmids, the overexpression efficiency was examined using Real-time quantitative polymerase chain reaction (qRT-PCR) and the levels of MMP3 and MMP13 were assayed using Western blotting. After incubation with IL-1β, the translocation of β-catenin into the nucleus was observed using immunofluorescence staining in SW1353 cells to investigate the activation of the WNT3A/β-catenin signaling pathway. Finally, treatment with WNT3A and transfection with glycogen synthase kinase 3 beta (GSK3β) plasmids were assessed for their effects on HDAC4 levels using Western blotting. RESULTS: IL-1β downregulated HDAC4 levels in chondrocytes and SW1353 cells. Furthermore, HDAC4 knockdown increased the levels of MMP3 and MMP13, which contributed to the degradation of the extracellular matrix. Overexpression of HDAC4 inhibited IL-1β-induced increases in MMP3 and MMP13. IL-1β upregulated the levels of WNT3A, and WNT3A reduced HDAC4 levels in SW1353 cells. GSK-3β rescued IL-1β-induced downregulation of HDAC4 in SW1353 cells. CONCLUSION HDAC4 exerted an inhibitory effect on IL-1β-induced extracellular matrix degradation and was regulated partially by the WNT3A/β-catenin signaling pathway.
Cell Line, Tumor ; Cells, Cultured ; Chondrocytes/metabolism* ; Glycogen Synthase Kinase 3 beta/genetics* ; Histone Deacetylases/genetics* ; Humans ; Interleukin-1beta/pharmacology* ; Matrix Metalloproteinase 13/metabolism* ; Matrix Metalloproteinase 3 ; Repressor Proteins ; Wnt Signaling Pathway ; Wnt3A Protein/genetics* ; beta Catenin/metabolism*

OBJECTIVETo investigate the effect of glycogen synthase kinase-3beta (GSK-3beta) on the proliferation of human ovarian cancer cells.

METHODSTwo human ovarian cancer cell lines SKOV3 and ES-2 were analysed for the expression of GSK-3beta and phosphorylated GSK-3beta (pGSK-3beta) by Western blot analysis. Cell growth curve analysis done by cell count was used to investigate the effect of GSK-3beta inhibitors on the growth of SKOV3 and ES-2 cells. Four plasmids, namely, GSK-3betaS9A, GID5-6, GID5-6LP and the control vector, were cotransfected respectively with the green fluorescent protein (GFP) into SKOV3 cells by electroporation, and then BrdU incorporation assay was adopted to analyse the role of GSK-3beta activity in the proliferation of ovarian cancer cells. After transfection, G418 was added to the medium to select those stably transfected cells, which were used to investigate the long term effect of GSK-3beta activity change on the proliferation of ovarian cancer cells by colony formation assay.

RESULTSBoth SKOV3 and ES-2 cells expressed GSK-3beta, though the expression level of pGSK-3beta was lower in SKOV3 than in ES-2 cells. GSK-3beta inhibitors attenuated the growth of SKOV3 and ES-2 cells. Transfection with GSK-3betaS9A to upregulate the GSK-3beta activity resulted in the increase of BrdU incorporation in SKOV3 cells compared with that in the control vector. On the contrary, transfection with GID5-6 to downregulate GSK-3beta activity decreased the BrdU incorporation in SKOV3 cells, compared with that in GID5-6LP, which is a control vector of GID5-6. Stable transfection with GSK-3betaS9A increased the colony number while stable transfection with GID5-6 decreased the colony number, compared with each control vector.

CONCLUSIONGSK-3beta can promote the proliferation of ovarian cancer cells. Inhibition of GSK-3 p may become a potential theraputic

Blotting, Western ; Cell Line, Tumor ; Cell Proliferation ; Female ; Glycogen Synthase Kinase 3 ; antagonists & inhibitors ; genetics ; metabolism ; Glycogen Synthase Kinase 3 beta ; Green Fluorescent Proteins ; genetics ; metabolism ; Humans ; Indoles ; pharmacology ; Lithium Chloride ; pharmacology ; Maleimides ; pharmacology ; Microscopy, Fluorescence ; Ovarian Neoplasms ; enzymology ; genetics ; pathology ; Phosphorylation ; drug effects ; Plasmids ; genetics ; Serine ; genetics ; metabolism ; Time Factors ; Transfection ; beta Catenin ; metabolism

BACKGROUND: Sjögren's syndrome (SS) is an autoimmune disorder characterized by sicca syndrome and/or systemic manifestations. The treatment is still challenging. This study aimed to explore the therapeutic role and mechanism of exosomes obtained from the supernatant of stem cells derived from human exfoliated deciduous teeth (SHED-exos) in sialadenitis caused by SS. METHODS: SHED-exos were administered to the submandibular glands (SMGs) of 14-week-old non-obese diabetic (NOD) mice, an animal model of the clinical phase of SS, by local injection or intraductal infusion. The saliva flow rate was measured after pilocarpine intraperitoneal injection in 21-week-old NOD mice. Protein expression was examined by western blot analysis. Exosomal microRNA (miRNAs) were identified by microarray analysis. Paracellular permeability was evaluated by transepithelial electrical resistance measurement. RESULTS: SHED-exos were injected into the SMG of NOD mice and increased saliva secretion. The injected SHED-exos were taken up by glandular epithelial cells, and further increased paracellular permeability mediated by zonula occluden-1 (ZO-1). A total of 180 exosomal miRNAs were identified from SHED-exos, and Kyoto Encyclopedia of Genes and Genomes analysis suggested that the phosphatidylinositol 3 kinase (PI3K)/protein kinase B (Akt) pathway might play an important role. SHED-exos treatment down-regulated phospho-Akt (p-Akt)/Akt, phospho-glycogen synthase kinase 3β (p-GSK-3β)/GSK-3β, and Slug expressions and up-regulated ZO-1 expression in SMGs and SMG-C6 cells. Both the increased ZO-1 expression and paracellular permeability induced by SHED-exos were abolished by insulin-like growth factor 1, a PI3K agonist. Slug bound to the ZO-1 promoter and suppressed its expression. For safer and more effective clinical application, SHED-exos were intraductally infused into the SMGs of NOD mice, and saliva secretion was increased and accompanied by decreased levels of p-Akt/Akt, p-GSK-3β/GSK-3β, and Slug and increased ZO-1 expression. CONCLUSION Local application of SHED-exos in SMGs can ameliorate Sjögren syndrome-induced hyposalivation by increasing the paracellular permeability of glandular epithelial cells through Akt/GSK-3β/Slug pathway-mediated ZO-1 expression.
Mice ; Animals ; Humans ; Sjogren's Syndrome/therapy* ; Proto-Oncogene Proteins c-akt/metabolism* ; Tight Junctions/metabolism* ; Glycogen Synthase Kinase 3 beta ; Mice, Inbred NOD ; Phosphatidylinositol 3-Kinases/metabolism* ; Exosomes/metabolism* ; Xerostomia ; Phosphatidylinositol 3-Kinase ; MicroRNAs/genetics*

OBJECTIVEThe goal of this study is to investigate the inappropriate activation of Wnt pathway in the hepatocarcinogenesis.

METHODSWe analyzed the alterations of three key components of Wnt pathway, beta-catenin, glycogen synthase kinase 3beta (GSK-3beta) and T cell factor 4 (Tcf-4), in 34 samples of hepatocellular carcinoma (HCC) and paracancerous normal liver by immunohistochemistry, polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP), direct sequencing, semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) and in situ hybridization.

RESULTSWe found 61.8% (21/34) of all the HCCs examined showed an abnormal beta-catenin protein accumulation in the cytoplasm or nuclei. RT-PCR-SSCP and direct sequencing showed that beta-catenin exon 3 mutations existed in 44.1% (15/34) of the HCCs. No mutations of GSK-3beta or Tcf-4 were detected in HCCs. Moreover, mRNA of beta-catenin and Tcf-4 but not GSK-3beta was found to be over expressed in HCCs. On analyzing the relationship between alterations of beta-catenin or Tcf-4 and C-myc or Cyclin D1 expression, we found that the mutations of beta-catenin as well as over expression of beta-catenin or Tcf-4 gene were independently correlated with C-myc gene over expression in HCCs.

CONCLUSIONSOur present findings strongly suggest mutations of beta-catenin as well as over expression of beta-catenin and Tcf-4 gene activate the Wnt pathway in HCC independently with the target gene most likely to be C-myc.

Carcinoma, Hepatocellular ; metabolism ; Cytoskeletal Proteins ; genetics ; physiology ; Glycogen Synthase Kinase 3 ; genetics ; physiology ; Glycogen Synthase Kinase 3 beta ; Humans ; Immunohistochemistry ; Liver Neoplasms ; metabolism ; Polymerase Chain Reaction ; Polymorphism, Single-Stranded Conformational ; Proto-Oncogene Proteins ; metabolism ; Reverse Transcriptase Polymerase Chain Reaction ; TCF Transcription Factors ; Trans-Activators ; genetics ; physiology ; Transcription Factor 7-Like 2 Protein ; Transcription Factors ; genetics ; physiology ; Wnt Proteins ; Zebrafish Proteins ; beta Catenin

OBJECTIVETo investigate correlation between polymorphisms of rs3755557 and rs1880481 located in glycogen synthase kinase 3beta gene and beta-catenin gene respectively, the products of which are components of Wnt signalling pathway, and risk of gastric carcinoma.

METHODSPCR and denaturing high-performance liquid chromatography combining with DNA sequencing were used to analyse genotype polymorphism of rs3755557 and rs1880481 of the subjects including 26 patients of gastric carcinoma and 33 patients of chronic superficial gastritis.

RESULTSChi-square analysis revealed that there was no significant difference in the frequencies of alleles and genotypes of rs3755557 polymorphic site between gastric carcinoma group and control group. As to the rs1880481 polymorphic site, there was no significant difference in the frequencies of alleles between gastric carcinoma group and the corresponding control group. The frequency of heterozygous genotype in male control group was 68.18% and it was significantly higher than 26.67% in male gastric carcinoma group, OR=5.893, 95%CI: 1.377-25.226 (P=0.013); but the frequencies of AA genotype of the site in male control group and male gastric carcinoma group were 9.09% and 40.00% respectively. There was statistical significance, OR=6.667, 95% CI: 1.121-39.660 (P=0.025).

CONCLUSIONThe above results suggest that the genotypes and alleles of rs3755557 site do not contribute to the risk of gastric carcinoma. Low level of the heterozygous genotype or high level of AA genotype of rs1880481 polymorphic site in male patients might cause a higher risk of developing gastric carcinoma.

Adult ; Aged ; Base Sequence ; Case-Control Studies ; Female ; Gene Frequency ; Genotype ; Glycogen Synthase Kinase 3 ; genetics ; metabolism ; Glycogen Synthase Kinase 3 beta ; Humans ; Male ; Middle Aged ; Polymorphism, Genetic ; Risk Factors ; Sequence Analysis, DNA ; Sex Factors ; Signal Transduction ; Stomach Neoplasms ; enzymology ; genetics ; Wnt Proteins ; genetics ; metabolism ; beta Catenin ; genetics ; metabolism