OBJECTIVE: To investigate the effect and possible mechanism of hydroxysafflor yellow A (HSYA) on human immortalized keratinocyte cell proliferation and migration. METHODS: HaCaT cells were treated with HSYA. Cell proliferation was detected by the cell counting kit-8 assay, and cell migration was measured using wound healing assay and Transwell migration assay. The mRNA and protein expression levels of heparin-binding epidermal growth factor (EGF)-like growth factor (HBEGF), EGF receptor (EGFR), phosphatidylinositol 3-kinase (PI3K), protein kinase B (AKT), mammalian target of rapamycin (mTOR), and hypoxia-inducible factor-1α (HIF-1α) were detected by quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot, respectively. Circ_0084443-overexpressing HaCaT cells and empty plasmid HaCaT cells were constructed using the lentiviral stable transfection and treated with HSYA. The expression of circ_0084443 was detected by qRT-PCR. RESULTS: HSYA (800 µmol/L) significantly promoted HaCaT cell proliferation and migration (P<0.05 or P<0.01). It also increased the mRNA and protein expression levels of HBEGF, EGFR, PI3K, AKT, mTOR and HIF-1α, and increased the phosphorylation levels of PI3K and AKT (P<0.05 or P<0.01). Furthermore, HSYA promoted HaCaT cell proliferation and migration via the HBEGF/EGFR and PI3K/AKT/mTOR signaling pathways (P<0.01). Circ_0084443 attenuated the mRNA expression levels of HBEGF, EGFR, PI3K, AKT, mTOR and HIF-1α (P<0.05). HSYA inhibited the circ_0084443 expression, further antagonized the inhibition of circ_0084443 on HBEGF, EGFR, PI3K, AKT, mTOR and HIF-1α, and promoted the proliferation of circ_0084443-overexpressing HaCaT cells (P<0.05 or P<0.01). However, HSYA could not influence the inhibitory effect of circ_0084443 on HaCaT cell migration (P>0.05). CONCLUSION HSYA played an accelerative role in HaCaT cell proliferation and migration, which may be attributable to activating HBEGF/EGFR and PI3K/AKT signaling pathways, and had a particular inhibitory effect on the keratinocyte negative regulator circ_0084443.
Humans ; Proto-Oncogene Proteins c-akt/metabolism* ; Phosphatidylinositol 3-Kinase ; Phosphatidylinositol 3-Kinases/metabolism* ; ErbB Receptors/genetics* ; TOR Serine-Threonine Kinases/metabolism* ; Cell Proliferation ; RNA, Messenger/genetics* ; Cell Movement ; Cell Line, Tumor ; Chalcone/analogs & derivatives* ; Quinones

OBJECTIVE: To explore the mechanism of electroacupuncture (EA) in promoting recovery of the facial function with the involvement of autophagy, glial cell line-derived neurotrophic factor (GDNF), and phosphatidylinositol-3-kinase (PI3K)/mammalian target of rapamycin (mTOR) signaling pathway. METHODS: Seventy-two male Sprague-Dawley rats were randomly allocated into the control, sham-operated, facial nerve injury (FNI), EA, EA+3-methyladenine (3-MA), and EA+GDNF antagonist groups using a random number table, with 12 rats in each group. An FNI rat model was established with facial nerve crushing method. EA intervention was conducted at Dicang (ST 4), Jiache (ST 6), Yifeng (SJ 17), and Hegu (LI 4) acupoints for 2 weeks. The Simone's 10-Point Scale was utilized to monitor the recovery of facial function. The histopathological evaluation of facial nerves was performed using hematoxylin-eosin (HE) staining. The levels of Beclin-1, light chain 3 (LC3), and P62 were detected by immunohistochemistry (IHC), immunofluorescence, and reverse transcription-polymerase chain reaction, respectively. Additionally, IHC was also used to detect the levels of GDNF, Rai, PI3K, and mTOR. RESULTS: The facial functional scores were significantly increased in the EA group than the FNI group (P<0.05 or P<0.01). HE staining showed nerve axons and myelin sheaths, which were destroyed immediately after the injury, were recovered with EA treatment. The expressions of Beclin-1 and LC3 were significantly elevated and the expression of P62 was markedly reduced in FNI rats (P<0.01); however, EA treatment reversed these abnormal changes (P<0.01). Meanwhile, EA stimulation significantly increased the levels of GDNF, Rai, PI3K, and mTOR (P<0.01). After exogenous administration with autophagy inhibitor 3-MA or GDNF antagonist, the repair effect of EA on facial function was attenuated (P<0.05 or P<0.01). CONCLUSIONS EA could promote the recovery of facial function and repair the facial nerve damages in a rat model of FNI. EA may exert this neuroreparative effect through mediating the release of GDNF, activating the PI3K/mTOR signaling pathway, and further regulating the autophagy of facial nerves.
Rats ; Male ; Animals ; Rats, Sprague-Dawley ; Electroacupuncture ; Phosphatidylinositol 3-Kinase/metabolism* ; Facial Nerve Injuries/therapy* ; Phosphatidylinositol 3-Kinases/metabolism* ; Beclin-1 ; Glial Cell Line-Derived Neurotrophic Factor ; Signal Transduction ; TOR Serine-Threonine Kinases/metabolism* ; Autophagy ; Mammals/metabolism*

OBJECTIVE: Programmed cell death 6 (PDCD6), a Ca ²⁺-binding protein, has been reported to be aberrantly expressed in all kinds of tumors. The aim of this study was to explore the role and mechanism of PDCD6 in hepatocellular carcinomas (HCCs). METHODS: The expression levels of PDCD6 in liver cancer patients and HCC cell lines were analyzed using bioinformatics and Western blotting. Cell viability and metastasis were determined by methylthiazol tetrazolium (MTT) and transwell assays, respectively. And Western blotting was used to test related biomarkers and molecular pathway factors in HCC cell lines. LY294002, a PI3K inhibitor inhibiting AKT, was used to suppress the AKT/GSK3β/β-catenin pathway to help evaluate the role of this pathway in the HCC carcinogenesis associated with PDCD6. RESULTS: The analysis of The Cancer Genome Atlas Database suggested that high PDCD6 expression levels were relevant to liver cancer progression. This was consistent with our finding of higher levels of PDCD6 expression in HCC cell lines than in normal hepatocyte cell lines. The results of MTT, transwell migration, and Western blotting assays revealed that overexpression of PDCD6 positively regulated HCC cell proliferation, migration, and invasion. Conversely, the upregulation of PDCD6 expression in the presence of an AKT inhibitor inhibited HCC cell proliferation, migration, and invasion. In addition, PDCD6 promoted HCC cell migration and invasion by epithelial-mesenchymal transition. The mechanistic investigation proved that PDCD6 acted as a tumor promoter in HCC through the AKT/GSK3β/β-catenin pathway, increasing the expression of transcription factors and cellular proliferation and metastasis. CONCLUSION PDCD6 has a tumor stimulative role in HCC mediated by AKT/GSK3β/β-catenin signaling and might be a potential target for HCC progression.
Humans ; Carcinoma, Hepatocellular/pathology* ; Liver Neoplasms/pathology* ; Proto-Oncogene Proteins c-akt/metabolism* ; beta Catenin/metabolism* ; Phosphatidylinositol 3-Kinases/metabolism* ; Glycogen Synthase Kinase 3 beta/metabolism* ; Cell Line ; Cell Proliferation ; Cell Line, Tumor ; Gene Expression Regulation, Neoplastic ; Calcium-Binding Proteins/metabolism* ; Apoptosis Regulatory Proteins/genetics*

OBJECTIVE: To investigate the anti-angiogenic activity of Kunxian Capsule (KX) extract and explore the underlying molecular mechanism using zebrafish. METHODS: The KX extract was prepared with 5.0 g in 100 mL of 40% methanol followed by ultrasonication and freeze drying. Freeze dried KX extract of 10.00 mg was used as test stock solution. Triptolide and icariin, the key bioactive compounds of KX were analyzed using ultra-high performance liquid chromatography. The transgenic zebrafish Tg(flk1:GFP) embryos were dechorionated at 20-h post fertilization (hpf) and treated with PTK 787, and 3.5, 7, 14 and 21 µg/mL of KX extract, respectively. After 24-h post exposure (hpe), mortality and malformation (%), intersegmental vessels (ISV) formation, and mRNA expression level of angiogenic pathway genes including phosphoinositide 3-kinase (PI3K), protein kinase B (AKT), extracellular signal-regulated kinases (ERKs), mitogen-activated protein kinase (MAPK), vascular endothelial growth factor (VEGF) and fibroblast growth factor (FGF-2) were determined. Further, the embryos at 72 hpf were treated with KX extract to observe the development of sub-intestinal vein (SIV) after 24 hpe. RESULTS: The chromatographic analysis of test stock solution of KX extract showed that triptolide and icariin was found as 0.089 mg/g and 48.74 mg/g, respectively, which met the requirements of the national drug standards. In zebrafish larvae experiment, KX extract significantly inhibited the ISV (P<0.01) and SIV formation (P<0.05). Besides, the mRNA expression analysis showed that KX extract could significantly suppress the expressions of PI3K and AKT, thereby inhibiting the mRNA levels of ERKs and MAPK. Moreover, the downstream signaling cascade affected the expression of VEGF and its receptors (VEGFR and VEGFR-2). FGF-2, a strong angiogenic factor, was also down-regulated by KX treatment in zebrafish larvae. CONCLUSION KX extract exhibited anti-angiogenic effects in zebrafish embryos by regulating PI3K/AKT-MAPK-VEGF pathway and showed promising potential for RA treatment.
Animals ; Fibroblast Growth Factor 2 ; Human Umbilical Vein Endothelial Cells ; Mitogen-Activated Protein Kinases/metabolism* ; Phosphatidylinositol 3-Kinase ; Phosphatidylinositol 3-Kinases/metabolism* ; Proto-Oncogene Proteins c-akt/metabolism* ; Vascular Endothelial Growth Factor A/metabolism* ; Zebrafish

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*

Objective To investigate the effect of viral myocarditis serum exosomal miR-320 on apoptosis of cardiomyocytes and its mechanism. Methods The model of viral myocarditis mice was established by intraperitoneal injection of Coxsackie virus B3. Serum exosomes were extracted by serum exosome extraction kit and co-cultured with cardiomyocytes. The uptake of exosomes by cardiomyocytes was detected by laser confocal microscopy. Cardiomyocytes were transfected with miR-320 inhibitor or mimic, and the expression level of miR-320 was detected by real-time quantitative PCR. Flow cytometry was used to detect cardiomyocyte apoptosis rate, and the expression levels of B cell lymphoma 2 (Bcl2) and Bcl2-related X protein (BAX) were tested by Western blot analysis. The prediction of miR-320 target genes and GO and KEGG enrichment analysis were tested by online database. The relationship between miR-320 and its target gene phosphoinositide-3-kinase regulatory subunit 1(Pik3r1) was examined by luciferase reporter gene. The effect of miR-320 on AKT/mTOR pathway protein was detected by Western blot analysis. Results Viral myocarditis serum exosomes promoted cardiomyocyte apoptosis, and increased the level of BAX while the level of Bcl2 was decreased. miR-320 was significantly up-regulated in myocardial tissue of viral myocarditis mice, and both pri-miR-320 and mature of miR-320 were up-regulated greatly in cardiomyocytes. The level of miR-320 in cardiomyocytes treated with viral myocarditis serum exosomes was significantly up-regulated, while transfection of miR-320 inhibitor counteracted miR-320 overexpression and reduced apoptosis rate caused by exosomes. Pik3r1 is the target gene of miR-320, and its overexpression reversed cardiomyocyte apoptosis induced by miR-320 up-regulation. The overexpression of miR-320 inhibited AKT/mTOR pathway activation. Conclusion Viral myocarditis serum exosome-derived miR-320 promotes apoptosis of mouse cardiomyocytes by inhibiting AKT/mTOR pathway by targeting Pik3r1.
Mice ; Animals ; Myocytes, Cardiac ; Phosphatidylinositol 3-Kinase/metabolism* ; Proto-Oncogene Proteins c-akt/metabolism* ; Myocarditis/pathology* ; Exosomes/metabolism* ; bcl-2-Associated X Protein/metabolism* ; MicroRNAs/metabolism* ; TOR Serine-Threonine Kinases/metabolism* ; Apoptosis/genetics*

Activated fibroblasts and M2-polarized macrophages may contribute to the progression of pulmonary fibrosis by forming a positive feedback loop. This study was aimed to investigate whether fibroblasts and macrophages form this loop by secreting SDF-1 and TGF-β and the impacts of neotuberostemonine (NTS) and tuberostemonine (TS). Mice were intratracheally injected with 3 U·kg^-1 bleomycin and orally administered with 30 mg·kg^-1 NTS or TS. Primary pulmonary fibroblasts (PFBs) and MH-S cells (alveolar macrophages) were used in vitro. The animal experiments showed that NTS and TS improved fibrosis related indicators, inhibited fibroblast activation and macrophage M2 polarization, and reduced the levels of TGF-β and SDF-1 in alveolar lavage fluid. Cell experiments showed that TGF-β1 may activated fibroblasts into myofibroblasts secreting SDF-1 by activating the PI3K/AKT/HIF-1α and PI3K/PAK/RAF/ERK/HIF-1α pathways. It was also found for the first time that SDF-1 was able to directly polarize macrophages into M2 phenotype secreting TGF-β through the same pathways as mentioned above. Moreover, the results of the cell coculture confirmed that fibroblasts and macrophages actually developed a feedback loop to promote fibrosis, and the secretion of TGF-β and SDF-1 was crucial for maintaining this loop. NTS and TS may disturb this loop through inhibiting both the PI3K/AKT/HIF-1α and PI3K/PAK/RAF/ERK/HIF-1α pathways to improve pulmonary fibrosis. NTS and TS are stereoisomeric alkaloids with pyrrole[1,2-a]azapine skeleton, and their effect on improving pulmonary fibrosis may be largely attributed to their parent nucleus. Moreover, this study found that inhibition of both the AKT and ERK pathways is essential for maximizing the improvement of pulmonary fibrosis.
Animals ; Mice ; Pulmonary Fibrosis/metabolism* ; Transforming Growth Factor beta/pharmacology* ; Proto-Oncogene Proteins c-akt/metabolism* ; Phosphatidylinositol 3-Kinases/metabolism* ; MAP Kinase Signaling System ; Alkaloids/pharmacology* ; Fibroblasts ; Macrophages/metabolism*

OBJECTIVE: To explore the protective effect of Huoxin Pill (HXP) on acute myocardial ischemia-reperfusion (MIRI) injury in rats. METHODS: Seventy-five adult SD rats were divided into the sham-operated group, model group, positive drug group (diltiazem hydrochloride, DH), high dose group (24 mg/kg, HXP-H) and low dose group (12 mg/kg, HXP-L) of Huoxin Pill (n=15 for every group) according to the complete randomization method. After 1 week of intragastric administration, the left anterior descending coronary artery of the rat's heart was ligated for 45 min and reperfused for 3 h. Serum was separated and the levels of creatine kinase (CK), creatine kinase isoenzyme (CK-MB) and lactate dehydrogenase (LDH), superoxide dismutase (SOD), and malondialdehyde (MDA), hypersensitive C-reactive protein (hs-CRP) and interleukin-1β (IL-1β) were measured. Myocardial ischemia rate, myocardial infarction rate and myocardial no-reflow rate were determined by staining with Evans blue and 2,3,5-triphenyltetrazolium chloride (TTC). Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCMSP) and Bioinformatics Analysis Tool for Molecular mechANism of Traditional Chinese Medicine (BATMAN) databases were used to screen for possible active compounds of HXP and their potential therapeutic targets; the results of anti-inflammatory genes associated with MIRI were obtained from GeneCards, Drugbank, Online Mendelian Inheritance in Man (OMIM), and Therapeutic Target Datebase (TTD) databases was performed; Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment were used to analyze the intersected targets; molecular docking was performed using AutoDock Tools. Western blot was used to detect the protein expression of Toll-like receptor 4 (TLR4)/nuclear factor kappa-B (NFκB)/NOD-like receptor protein 3 (NLRP3). RESULTS: Compared with the model group, all doses of HXP significantly reduced the levels of LDH, CK and CK-MB (P<0.05, P<0.01); HXP significantly increased serum activity of SOD (P<0.05, P<0.01); all doses of HXP significantly reduced the levels of hs-CRP and IL-1β (P<0.05, P<0.01) and the myocardial infarction rate and myocardial no-reflow rate (P<0.01). GO enrichment analysis mainly involved positive regulation of gene expression, extracellular space and identical protein binding, KEGG pathway enrichment mainly involved PI3K-Akt signaling pathway and lipid and atherosclerosis. Molecular docking results showed that kaempferol and luteolin had a better affinity with TLR4, NFκB and NLRP3 molecules. The protein expressions of TLR4, NFκB and NLRP3 were reduced in the HXP group (P<0.01). CONCLUSIONS HXP has a significant protective effect on myocardial ischemia-reperfusion injury in rats, and its effect may be related to the inhibition of redox response and reduction of the inflammatory response by inhibiting the TLR4NFκB/NLRP3 signaling pathway.
Humans ; Rats ; Animals ; NF-kappa B/metabolism* ; Myocardial Reperfusion Injury/drug therapy* ; NLR Family, Pyrin Domain-Containing 3 Protein ; Rats, Sprague-Dawley ; C-Reactive Protein ; Toll-Like Receptor 4 ; Phosphatidylinositol 3-Kinases/metabolism* ; Molecular Docking Simulation ; Signal Transduction ; Myocardial Infarction/drug therapy* ; Creatine Kinase ; L-Lactate Dehydrogenase/metabolism* ; Superoxide Dismutase/metabolism*

Objective: To investigate the effects of regenerating islet-derived protein 3A (REG3A) on the proliferation and invasion of glioma cells and its molecular mechanism. Methods: Five low-grade, five high-grade glioma tissues and ten adjacent tissues from glioma patients who underwent surgery at Linyi People's Hospital from October 17, 2017 to October 18, 2018 were collected. Human glioma cell lines (SF295, U251, TG905, A172, CRT) and a primary glioma cell line PT-1 were cultured in vitro. The protein and mRNA expressions of REG3A in these tissues and glioma cell lines were detected by Western blot and reverse transcription-quantitative real-time polymerase chain reaction (RT-qPCR). SF295 cells were infected with lentivirus and labeled as REG3A plasmid transfection group, and the TG905 cells were transfected with si-REG3A by liposome transfection reagent and labeled as si-REG3A transfection group. At the same time, the empty transfection control and blank control groups were set up. Glioma cells were treated with REG3A recombinant protein alone or in combination with Akt1/2 inhibitors. Cell counting kit-8 (CCK-8) and cell scratch assay were used to detect cell proliferation and invasion, respectively. Western blot was used to detect the protein expression of N-cadherin, vimentin and phosphorylation of Akt (p-Akt) in REG3A overexpressed and knockdown glioma cells. Results: RT-qPCR results showed that the mRNA expression levels of REG3A in glioma cells in each group were U251 (2.129±0.13), TG905 (2.22±0.59), CRT (5.02±0.31), A172 (6.62±1.34) and PT-1 (9.18±0.61), respectively, higher than its expression in SF295 cells (1.00±0.18, P<0.001). The mRNA expression level of REG3A in high-grade glioma tissue samples (3.18±2.92) was higher than that in the control group (1.00±1.14, P=0.031) and low-grade glioma group (0.90±0.67, P=0.014). The results of western blot and immunohistochemical staining were consistent with that of RT-qPCR. The migration rate of cells in si-REG3A transfection group [(60.57±5.30)%] was lower than that of the empty transfection group [(84.18±13.63)% (P=0.038)] and blank control group [(79.65±12.09)% (P=0.076)]. The results of the scratch experiment showed that the migration rate of cells in REG3A plasmid transfected cells in the SF295 group was (96.05±6.41)%, which was significantly higher than that of empty transfected cells [(74.47±8.23)%, P=0.021)]. REG3A recombinant protein could up-regulate the expression of N-cadherin, vimentin and p-Akt in SF295 cells. Compared with the control group [(100.00±2.53)%], the proliferation rate in the REG3A recombinant protein group [(117.70±10.24)%] was significantly up-regulated, and the proliferation rate in the REG3A recombinant protein+ Akt inhibitor group [(98.31±3.64)%] was significantly lower than that of the REG3A recombinant protein group (P=0.017). The migration rate of the REG3A recombinant protein+ Akt inhibitor group was (63.35±4.06)%, which was significantly lower than (89.26±11.07)% of the REG3A recombinant protein group (P=0.019). Conclusion: REG3A can promote the proliferation and invasion of human glioma cells by activating the PI3K/Akt signaling pathway.
Humans ; Cell Line, Tumor ; Cell Movement/genetics* ; Cell Proliferation/genetics* ; Glioma/genetics* ; Phosphatidylinositol 3-Kinases/metabolism* ; Protein Kinase Inhibitors ; Proto-Oncogene Proteins c-akt/metabolism* ; RNA, Messenger/metabolism* ; Signal Transduction ; Vimentin/metabolism*

Objective: To investigate the effects of regenerating islet-derived protein 3A (REG3A) on the proliferation and invasion of glioma cells and its molecular mechanism. Methods: Five low-grade, five high-grade glioma tissues and ten adjacent tissues from glioma patients who underwent surgery at Linyi People's Hospital from October 17, 2017 to October 18, 2018 were collected. Human glioma cell lines (SF295, U251, TG905, A172, CRT) and a primary glioma cell line PT-1 were cultured in vitro. The protein and mRNA expressions of REG3A in these tissues and glioma cell lines were detected by Western blot and reverse transcription-quantitative real-time polymerase chain reaction (RT-qPCR). SF295 cells were infected with lentivirus and labeled as REG3A plasmid transfection group, and the TG905 cells were transfected with si-REG3A by liposome transfection reagent and labeled as si-REG3A transfection group. At the same time, the empty transfection control and blank control groups were set up. Glioma cells were treated with REG3A recombinant protein alone or in combination with Akt1/2 inhibitors. Cell counting kit-8 (CCK-8) and cell scratch assay were used to detect cell proliferation and invasion, respectively. Western blot was used to detect the protein expression of N-cadherin, vimentin and phosphorylation of Akt (p-Akt) in REG3A overexpressed and knockdown glioma cells. Results: RT-qPCR results showed that the mRNA expression levels of REG3A in glioma cells in each group were U251 (2.129±0.13), TG905 (2.22±0.59), CRT (5.02±0.31), A172 (6.62±1.34) and PT-1 (9.18±0.61), respectively, higher than its expression in SF295 cells (1.00±0.18, P<0.001). The mRNA expression level of REG3A in high-grade glioma tissue samples (3.18±2.92) was higher than that in the control group (1.00±1.14, P=0.031) and low-grade glioma group (0.90±0.67, P=0.014). The results of western blot and immunohistochemical staining were consistent with that of RT-qPCR. The migration rate of cells in si-REG3A transfection group [(60.57±5.30)%] was lower than that of the empty transfection group [(84.18±13.63)% (P=0.038)] and blank control group [(79.65±12.09)% (P=0.076)]. The results of the scratch experiment showed that the migration rate of cells in REG3A plasmid transfected cells in the SF295 group was (96.05±6.41)%, which was significantly higher than that of empty transfected cells [(74.47±8.23)%, P=0.021)]. REG3A recombinant protein could up-regulate the expression of N-cadherin, vimentin and p-Akt in SF295 cells. Compared with the control group [(100.00±2.53)%], the proliferation rate in the REG3A recombinant protein group [(117.70±10.24)%] was significantly up-regulated, and the proliferation rate in the REG3A recombinant protein+ Akt inhibitor group [(98.31±3.64)%] was significantly lower than that of the REG3A recombinant protein group (P=0.017). The migration rate of the REG3A recombinant protein+ Akt inhibitor group was (63.35±4.06)%, which was significantly lower than (89.26±11.07)% of the REG3A recombinant protein group (P=0.019). Conclusion: REG3A can promote the proliferation and invasion of human glioma cells by activating the PI3K/Akt signaling pathway.
Humans ; Cell Line, Tumor ; Cell Movement/genetics* ; Cell Proliferation/genetics* ; Glioma/genetics* ; Phosphatidylinositol 3-Kinases/metabolism* ; Protein Kinase Inhibitors ; Proto-Oncogene Proteins c-akt/metabolism* ; RNA, Messenger/metabolism* ; Signal Transduction ; Vimentin/metabolism*