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AIM: To investigate the effects of agkis-trodon halys venom anti-tumor component (AHVAC-) on the biological behavior of gastric cancer MKN-28 cells. METHODS: Gastric cancer MKN-28 cells were treated with the experimental concentrations (5, 10, 15 μg/mL) of AHAVC- for 24 h. Cell proliferation and toxicity assay (cell counting kit-8, CCK-8) was used to detect the inhibition rates of the cells in different concentrations of AHVAC-. The migration ability of the cells was evaluated by wound-healing and Transwell assay. The apoptosis were observed by laser confocal microscopy with annexin V-mCherry/DAPI double staining, and the apoptosis rates were analyzed by flow cytometry with annexin V-FITC/PI double fluorescence staining. The protein level of Caspease-3 was determined by Western blot. RESULTS: Compared with normal control group, the results of AHVAC- concentration groups showed that with the increase of AHVAC- concentration, the proliferative activity of MN-28 cells decreased gradually (P<0.01), the cell migration ability decreased gradually (P<0.01), and the cell apoptosis rate increased (P<0.05). The expression of apoptosis-related protein Caspease-3 was up-regulated (P<0.01). CONCLUSION: AHVAC- inhibits proliferation and migration of gastric cancer MSN-28 cells and induces apoptosis.
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Objective@#To investigate the effects of PssL-NAC reactive oxygen species (ROS)-responsive nanoparticles on intracellular ROS production, inflammatory factor levels, collagen production, cell function and Toll-like receptor 4 (TLR4), NF-κB nuclear factor-κB (p65) pathway protein expression in human gingival fibroblasts (HGFs) induced by Porphyromonas gingivalis-lipopolysaccharide (P.g-LPS).@*Methods@#This study was reviewed and approved by the ethics committee. PssL-NAC microspheres containing oil soluble antioxidant N-acetylcysteine (NAC) were obtained by connecting the hydrophobic end of polycaprolactone (PCL) and the hydrophilic end of polyethylene glycol (PEG) via thioketal (TK) bonds in response to ROS, and self loading in the aqueous and oil phases. After preparation of the PssL-NAC microspheres and aqueous NAC solution, successful synthesis of the nanoparticles was verified by transmission electron microscopy. Then, HGFs were exposed to P.g-LPS (0, 5, or 10 μg/mL), P.g-LPS (0, 5, or 10 μg/mL)+NAC, and P.g-LPS (0, 5, or 10 μg/mL)+PssL-NAC, and the ROS levels in the different groups were observed under confocal microscopy to determine the concentration of P.g-LPS for use in subsequent experiments. The groups were as follows: control group (no treatment), P.g-LPS group (HGFs treated with P.g-LPS), NAC group (HGFs treated with P.g-LPS and NAC), and PssL-NAC group (HGFs treated with P.g-LPS and PssL-NAC). Cell counting kit-8 (CCK-8) assays verified the biosafety of PssL-NAC. The ROS levels in the different groups were detected by DCFH-DA probes and observed via confocal microscopy. Real-time qPCR (RT-qPCR) was used to monitor the gene expression levels of the intracellular inflammatory cytokines interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), collagen 1 (COL1) and collagen 3 (COL3). The effect of PssL-NAC on the migration of HGFs was observed via the scratch test. The protein expression of TLR4-NF-κB, and phosphorylated p65 (p-p65) in the TLR4-NF-κB pathway was evaluated by Western blot.@*Results@#PssL-NAC had no significant effect on HGF proliferation (P>0.05). At elevated P.g-LPS concentrations, PssL-NAC maintained intracellular ROS levels approximately twice those in the control group (P<0.001). PssL-NAC significantly decreased P.g-LPS-induced IL-6 (P<0.001) and TNF-α (P<0.001) gene expression and increased COL1 gene expression (P<0.001). After P.g-LPS stimulation, PssL-NAC restored cell migration to the control level (P>0.05) and decreased the protein expression of TLR4 (P<0.001), p65 (P = 0.006), and p-p65 (P = 0.017) in the TLR4-NF-κB pathway.@*Conclusion@#PssL-NAC maintains the appropriate intracellular ROS concentration, alleviates P.g-LPS-induced inflammation in HGFs through the TLR4-NF-κB pathway, and restores the cell functions of collagen production and migration in an inflammatory environment.
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Objective:To explore the effects of knocking down glycine cleavage system H protein (GCSH) on proliferation, apoptosis, oxidative stress and migration of gastric cancer SNU-1 cells in vitro. Methods:SNU-1 cells were cultured in vitro and divided into control group (no transfection) , negative control group (transfection of negative control siRNA) and GCSH knockdown group (transfection of GCSH siRNA) . Quantitative PCR was used to detect the knockdown effect. Immunofluorescence was used to observe the morphology of cells in each group. CCK-8 was used to test the proliferation of SNU-1 cells. Flow cytometry was used to detect the apoptosis and oxidative stress level, and scratch test was used to detect the cell migration. Results:Quantitative PCR experiment showed that the relative expression levels of GCSH in the control group, negative control group and GCSH knockdown group were 1.29±0.16, 1.36±0.17 and 0.32±0.04, respectively ( F=90.32, P<0.001) . There was no significant difference between the control group and negative control group ( P=0.497) . Compared to the negative control group, the GCSH knockdown group was significantly decreased ( P<0.001) . Immunofluorescence experiment showed no significant difference in the morphology of cells among the groups. The CCK-8 experiment results showed that the cell proliferation activities of the control group, negative control group and GCSH knockdown group were 2.63±0.12, 2.61±0.14, 2.45±0.14, respectively ( F=6.35, P=0.005) . There was no significant difference between the control group and negative control group ( P=0.751) , and the GCSH knockdown group significantly decreased compared to the negative control group ( P=0.011) . The results of flow cytometry showed that the early stage apoptosis rates of SNU-1 cells in the control group, negative control group and GCSH knockdown group were (13.38±0.45) %, (12.86±0.65) %, (20.04±3.61) %, respectively ( F=15.37, P<0.001) . There was no significant difference between the control group and negative control group ( P=0.559) . Compared to the negative control group, the GCSH knockdown group significantly increased ( P=0.002) . The late stage apoptosis rates of the three groups were (2.21±0.25) %, (2.68±0.45) %, (5.67±1.67) %, respectively ( F=18.24, P<0.001) . There was no significant difference between the control group and negative control group ( P=0.356) . Compared to the negative control group, the GCSH knockdown group showed a significant increase ( P=0.024) . The reactive oxygen species positive rates in the control group, negative control group and GCSH knockdown group were (26.98±8.79) %, (28.27±5.63) %, (48.41±0.94) %, respectively ( F=22.56, P<0.001) . There was no significant difference between the control group and negative control group ( P=0.950) . Compared to the negative control group, the GCSH knockdown group significantly increased ( P<0.001) . The cell migration rates of the control group, negative control group and GCSH knockdown group were (48.29±5.79) %, (51.66±2.29) %, (14.01±1.56) %, respectively ( F=148.80, P<0.001) . There was no significant difference between the control group and negative control group ( P=0.328) . Compared with the negative control group, the GCSH knockdown group significantly decreased ( P<0.001) . Conclusion:Knock down of GCSH gene can inhibit the proliferation and migration, increase cell apoptosis rate and oxidative stress of SNU-1 cells in vitro. GCSH gene may be a potential target for the treatment of gastric cancer.
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Objective:To study the expression of microRNA (miRNA)-4783-3p in liver cancer tissue and its effect on the proliferation and migration of liver cancer Huh-7 cells.Methods:The cBioPortal database was used to analyze the expression of miR-4783-3p in liver cancer tissues and adjacent tissues. In strict accordance with the instructions of Lipofectamine? 2000 transfection kit, miR-4783-3p overexpression mimics or overexpression control mimics were transfected into Huh-7 cells respectively, namely overexpression group and control group. The proliferation of Huh-7 cells was analyzed by CCK-8 assay, and the migration of Huh-7 cells was analyzed by cell scratch method. The targeting relationship between miR-4783-3p and insulin-like growth factor binding protein 2 ( IGFBP2) mRNA was detected by dual-luciferase reporter gene assay. RT-qPCR was used to detect the expression of IGFBP2 mRNA. Western-blotting was used to detect the expression of IGFBP2 protein and EGFR-STAT3 molecular pathway proteins. Results:The expression of miR-4783-3p in liver cancer tissues was significantly lower than that in adjacent tissues ( P<0.01). Compared with the control group, the proliferation ability of Huh-7 cells in the overexpression group was significantly decreased ( P<0.05). The scratch healing rates of Huh-7 cells in the control group and the overexpression group were (67.71±9.04)% and (29.58±10.51)%, respectively, and the scratch healing rate in the overexpression group was significantly lower ( P<0.01). miR-4783-3p targeted and bound to IGFBP2 mRNA ( P<0.01). The expression of IGFBP2 mRNA in the control and overexpression groups was 5.76±1.44 and 0.99±0.47, respectively, and miR-4783-3p negatively regulated the expression of IGFBP2 mRNA ( P<0.01). Compared with the control group, the expressions of IGFBP2 protein and EGFR-STAT3 molecular pathway protein were decreased in the overexpression group. Conclusions:miR-4783-3p is lowly expressed in liver cancer tissues. miR-4783-3p can attenuate the proliferation and invasion ability of liver cancer Huh-7 cells by inducing the low expression of IGFBP2 gene.
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The matrix metalloproteinases family (MMPs) are proteins related to tumor formation and metastasis that have attracted the attention of scholars in recent years. Tumor cells can secrete MMPs during malignant transformation, and the expression of MMPs in different malignant tumors is diverse, and different members of MMPs do not have exactly the same biological properties. Matrix metalloproteinase-19 (MMP-19) is a new member of MMPs whose secretion increases rapidly during the malignant transformation of cells and is released into the extracellular space to participate in biological processes such as proliferation, adhesion, invasion, migration, and angiogenesis of tumor cells. In this paper, the progress of research on the biological properties of MMP-19 in tumors was reviewed to provide a theoretical basis for exploring the development of tumors, especially for studying the invasion and metastasis of tumor cells.
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Objective:To investigate the effects of cytoplasmic fragile X mental retardation protein 1 binding protein 2 (CYFIP2) overexpression on the biological functions and Wnt/β-catenin signaling pathways of bladder cancer T24 cells.Methods:The control group was T24 cells transfected with the empty pcDNA3 vector, and the overexpression group was T24 cells transfected with the CYFIP2 overexpression vector. The expression of CYFIP2 mRNA and protein was detected by reverse transcriptase, quantitative polymerase chain reaction, and Western Blot. The effect of CYFIP2 overexpression on T24 cell proliferation was detected by CCK-8. The effect of CYFIP2 overexpression on T24 cell migration and invasion was detected by Transwell. The effects of CYFIP2 overexpression on Wnt/β-catenin signaling pathway in T24 cells were detected by Western Blot.Results:Compared with the control group, the expression levels of CYFIP2 mRNA and protein were increased in the overexpression group (all P < 0.001), and the cell proliferation, migration, and invasion abilities were reduced (all P < 0.01). β-catenin, c-Myc, and Cyclin D1 protein expression were down-regulated in CYFIP2 overexpressed T24 cells (all P < 0.05), while the protein levels of p-β-catenin were increased ( P < 0.05). Conclusions:CYFIP2 overexpression can inhibit T24 cell proliferation, migration, and invasion, and its possible molecular mechanism is related to the inhibition of Wnt/β-catenin signaling pathway.
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Objective:To investigate the effect of kaempferol on proliferation, migration, and invasion of cervical cancer SiHa cells by regulating circFBXW7.Methods:SiHa cells were treated with kaempferol at low, medium, and high doses (15, 30, and 60 μmol/L) for 24 h. Untreated SiHa cells were used as the control group. CCK-8 was used to detect the effect of kaempferol on the proliferation of SiHa cells. Transwell was used to detect the effect of kaempferol on the migration and invasion of SiHa cells. Real-time fluorescence quantitative polymerase chain reaction was used to detect the expression of circFBXW7 in SiHa cells. pcDNA and pcDNA-circFBXW7 were transfected into SiHa cells, respectively, and si-NC and si-circFBXW7 were transfected into SiHa cells after adding 60 μmol/L kaempferol treatment for 24 h. The effects of circFBXW7 and its knockdown, circFBXW7, on the proliferation, migration, and invasion of SiHa cells were investigated, and the effects of E-cadherin and N-cadherin protein expression levels were detected by Western Blot. Results:Compared with the control group, the cell value-added, migration, and invasion abilities of the low, medium, and high dose groups were decreased (all P < 0.05) and were dose-related, and the expression of circFBXW7 was increased ( P < 0.05). After transfection with pcDNA-circFBXW7, the expression of circFBXW7 increased ( P < 0.05), while promoting the proliferation, cell migration, and invasion of kaempferol on SiHa cells (all P < 0.05). After transfection with si-circFBXW7, the expression of circFBXW7 decreased ( P < 0.05), while inhibiting the proliferation, cell migration, and invasion of kaempferol on SiHa cells (all P < 0.05). That indicated that the transfection of si-circFBXW7 could attenuate the inhibitory effects of kaempferol on the above oncogenic phenotypes of SiHa cells. Compared with the control group, E-cadherin expression was upregulated, and N-cadherin expression was downregulated in the low, medium, and high dose groups (all P < 0.05) in a dose-related manner. After transfection of pcDNA-circFBXW7 with SiHa cells, the expression of E-cadherin was increased, and the expression of N-cadherin was decreased (all P < 0.05). After transfection of si-circFBXW7 with SiHa cells, the expression of E-cadherin decreased, and the expression of N-cadherin increased (all P < 0.05). Conclusions:Kaempferol can reduce the proliferation, migration, and invasion abilities of SiHa cells by promoting circFBXW7 expression.
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@#Objective To investigate the effects of miR-130b-5p targeting E26 transformation specific-1(ETS1)on proliferation,migration and invasion of prostatic cancer(PCa)cells and its mechanism. Methods The mRNA transcription level of miR-130b-5p gene in PCa tissues,adjacent tissues,(LNCap,PC-3,DU-145)and normal prostate cells(RPWE-1)PCa cells was measured by qRT-PCR,and the expression of ETS1 protein in PCa cells was detected by Western blot. Bioinformatics,fluorescein experiment,qRT-PCR and Western blot were used to predict and verify the targeting relationship between miR-130b-5p and ETS1. PC-3 cells were divided into control group(without any treatment),mimic group(transfected with miR-130b-5p mimic)and mimic + ETS1 group(transfected with miR-130b-5p mimic + pcDNA-ETS1). The cells were detected for the proliferation and viability by clone formation assay and CCK-8 respectively,measured for the migration and invasion by scratch test and Transwell chamber assay,and detected for the expression of invasion-related proteins and PI3K/AKT/mTOR pathway-related proteins by Western blot. Results The transcription level of miR-130b-5p mRNA in PCa tissues was significantly lower than that in adjacent tissues(t = 12. 450,P < 0. 001);Compared with RPWE-1 cells,the transcription level of miR-130b-5p mRNA in LNCap,PC-3 and DU-145 cells decreased significantly(t = 4. 463,7. 103 and 5. 741,P = 0. 001 2,< 0. 001 and < 0. 001,respectively),while the expression level of ETS1protein increased significantly(t = 4. 850,9. 325 and 7. 723,P = 0. 008,< 0. 001 and = 0. 002,respectively). miR-130-5p targeted and negatively regulated the expression of ETS1. Compared with the control group,the cloning rate,viability and scratch healing rate of cells in mimic group decreased significantly(t = 11. 370,10. 640 and 15. 660,respectively,each P < 0. 001),the number of invasive cells decreased significantly(t = 10. 160,P < 0. 001),the expression levels of matrix metalloproteinase-2(MMP-2),MMP-9 and vimentin decreased significantly(t = 15. 120,9. 992 and 12. 600,P < 0. 001,< 0. 001 and = 0. 002,respectively),while the expression level of E-cadherin increased significantly(t = 6. 928,P < 0. 001),and the phosphorylation levels of phosphatidylinositol-3 kinase(PI3K),protein kinase B(AKT)and mammalian target of rapamycin(mTOR)decreased significantly(t = 7. 746,8. 041 and 11. 510,P = 0. 002,0. 002,and < 0. 001,respectively);Compared with mimic group,the cell cloning rate,viability,scratch healing rate significantly increased in mimic + ETS1 group(t = 6. 988,6. 642 and 6. 660,respectively,each P < 0. 001),the number of invasive cells significantly increased(t = 4. 082,P = 0. 002),the expression levels of MMP-2,MMP-9 and vimentin proteins were significantly up-regulated(t = 10. 410,6. 754 and 8. 521,P = 0. 002,0. 003 and 0. 002,respectively),however,the expression level of E-cadherin was significantly down-regulated(t = 4. 648,P < 0. 01),and the phosphorylation levels of PI3K,AKT and mTOR were significantly up-regulated(t = 4. 850,4. 323 and 10. 840,P = 0. 008,0. 008 and < 0. 001,respectively)Conclusion miR-130b-5p targets ETS1 to inhibit the proliferation,migration and invasion of PC-3 cells,which may be through the regulation of PI3K/AKT/mTOR signaling pathway.
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@#Objective To evaluate the effect of tyrosine kinase inhibitor BGJ398 on the proliferation,apoptosis and migration of human hepatocellular cancer Huh-7 cells and explore the mechanism.Methods The effects of 10 tyrosine kinase inhibitors on the survival of Huh-7 cells were detected by MTT assay,and the sensitivity of Huh-7 cells to BGJ398 was analyzed by single-target kinetic equation and biphasic kinetic equation respectively.Huh-7 cells were added with 10,30 and 90 nmol/L BGJ398 respectively,and the control group(without drugs)was set.The effects of BGJ398 on the apoptosis and cell cycle of Huh-7 cells were detected by flow cytometry after culturing at 37℃for 24 h,the effect on the migration ability was detected by wound healing assay and the effect on the expression of multiple pathway-related proteins was detected by Western blot.Results All of 10 tyrosine kinase inhibitors inhibited the proliferation of Huh-7 cells,among which Huh-7 cells were most sensitive to BGJ398 and the IC_(50)was(0.020±0.013)μmol/L;The response of Huh-7 cells to BGJ398 was composed of two phases with F_1 accounted for 92.8%(K_(d1)was 36 nmol/L)and F_2 accounted for 7.2%(K_(d2)>1 000μmol/L).Compared with the control group,the apoptosis rate and the percentage of Huh-7 cells in G1 phase increased significantly in 30 and 90 nmol/L BGJ398 groups(t=-6.407～-4.459,each P<0.05),while the percentage of Huh-7 cells in S phase decreased significantly in 10,30 and 90 nmol/L BGJ398 groups(t=2.982,7.859 and 12.425,respectively,each P<0.05);After 24 and 48 h of scratching,the scratch area of 30 and 90 nmol/L BGJ398groups decreased significantly(t=5.376～18.197,each P<0.05);The expression levels of phosphorylated fibroblast growth factor receptor(FGFR)and phosphorylated extracellular signal-regulated kinase 1/2(Erk1/2)protein decreased significantly in 30 and 90 nmol/L BGJ398 groups(t=4.015～6.729,each P<0.01).Conclusion BGJ398 can inhibit the proliferation and migration of human hepatocellular cancer Huh-7 cells,induce apoptosis and cell cycle arrest,which might be achieved by inhibiting FGFR phosphorylation and MAPK signaling pathway.BGJ398 is expected to be a potential agent for the treatment of hepatocellular cancer.
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@#Objective To the effects of VALD-3,a derivative of o-vanilla Schiff base ligand,on proliferation,migration and apoptosis of colorectal cancer cells and evaluate its mechanism.Methods HT-29 and HCT116 cells were cultured in vitro,and the inhibitory effects of VALD-3(5,10,20 and 40 mg/L) on proliferation of the two kinds of cells were detected by MTT assay;The effects of VALD-3(10,20 and 40 mg/L) on the morphological changes of the cells were observed by inverted microscope,while the effects on the migration ability of HT-29 cells were detected by cell scratch test,and the effects on the apoptosis of HT-29 cells were detected by flow cytometry and Hoechst 33258 fluorescence staining;The effects of VALD-3(5,10,20 and 40 mg/L) on the expression of apoptosis-related proteins in HT-29 cells were detected by Western blot.Negative control groups were set up(with no VALD-3).Results Compared with the negative control group,the survival rates of HT-29 and HCT116 cells in 10,20 and 40 mg/L VALD-3 treated groups significantly decreased(t=7.717～2 006.148,each P <0.05);the number of HT-29 and HCT116 cells in 10,20 and 40 mg/L VALD-3groups decreased significantly with the increase of VALD-3 concentration,the cells appeared irregular morphology and gradually became round and smaller,and cell fragments increased;In 10,20 and 40 mg/L VALD-3 treated groups,the migration rate of HT-29 cell scratches decreased significantly(t=7.596～73.780,each P <0.01),the apoptosis rate increased significantly(t=7.092～8.057,each P <0.01),and the number of apoptotic cells increased significantly with strong bright blue fluorescence,chromatin concentration and nuclear fragmentation;The levels of cleaved caspase-3 and Bax protein in HT-29 cells treated with 5,10,20 and 40 mg/L VALD-3 significantly increased(t=2.998～24.901,each P <0.05),the level of Bcl-2 protein in 40 mg/L VALD-3 group decreased significantly(t=10.035,P <0.05),and the levels of cleaved caspase-8 in 20 and 40 mg/L VALD-3 group significantly increased(t=12.630 and 8.064 respectively,each P <0.01).Conclusion VALD-3 inhibited the proliferation and migration of colorectal cancer cells and induced apoptosis by regulating the expression of cleaved caspase-3,cleaved caspase-8,Bax and Bcl-2 proteins.
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@#Objective To investigate the expression of C-C chemokine ligand 5(CCL5) in head and neck squamous cell carcinoma(HNSCC),and explore the effect of CCL5 on the biological characteristics of laryngeal carcinoma cells.Methods Gene Expression Profiling Interactive Analysis(GEPIA) database was used to investigate the expression of CCL5 in HNSCC.The laryngeal carcinoma cells TU177 were transfected with siRNA(siRNA group),and the control(NC) group was set up.The cell proliferation,migration,cycle and apoptosis of each group were detected by CCK8 assay,cell scratch test and flow cytometry respectively.RT-PCR and Western blot were used to detect the knock-down efficiency of CCL5 and the mRNA transcription and protein expression of multidrug resistance protein 2(MRP2) and bcl-2-associated x protein(Bax).Results The expression of CCL5 in HNSCC was higher than that in normal tissues(P <0.05).Compared with NC group,siRNA showed higher knock-down efficiency(t=12.898 and 22.656 respectively,each P <0.01);siRNA interference with CCL5 inhibited the proliferation and migration of laryngeal carcinoma cells,and promoted the late apoptosis of laryngeal carcinoma cells and the expression of apoptosis protein Bax(t=2.600～11.667,each P <0.05).Conclusion CCL5 was highly expressed in HNSCC,while siRNA interference with CCL5 inhibited the proliferation,migration and promoted apoptosis of laryngeal carcinoma cells TU177 by up-regulating the expression of Bax,which laid a foundation of the possibility of CCL5 as a new target for the treatment of laryngeal carcinoma.
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Objective:To observe the effects of p21 activated kinase 4 (PAK4) on the mitochondrial function and biological behavior in retinal vascular endothelial cells.Methods:The experimental study was divided into two parts: in vivo animal experiment and in vitro cell experiment. In vivo animal experiments: 12 healthy C57BL/6J male mice were randomly divided into normal control group and diabetes group, with 6 mice in each group. Diabetes mice were induced by streptozotocin to establish diabetes model. Eight weeks after modeling, quantitative real-time polymerase chain reaction and Western blots were performed to detect the expression of PAK4 in diabetic retinas. In vitro cell experiments: the human retinal microvascular endothelial cells (hRMEC) were divided into three groups: conventional cultured cells group (N group), empty vector transfected (Vector group); pcDNA-PAK4 eukaryotic expression plasmid transfected group (PAK4 group). WB and qPCR were used to detect transfection efficiency, while scratching assay, cell scratch test was used to detect cell migration in hRMEC of each group. In vitro white blood cell adhesion experiment combined with 4 ', 6-diamino-2-phenylindole staining was used to detect the number of white blood cells adhering to hRMEC in each group. The Seahorse XFe96 cell energy metabolism analyzer measures intracellular mitochondrial basal respiration, adenosine triphosphate (ATP) production, maximum respiration, and reserve respiration capacity. The t-test was used for comparison between the two groups. Single factor analysis of variance was used for comparison among the three groups. Results:In vivo animal experiments: compared with normal control group, the relative expression levels of PAK4 mRNA and protein in retina of diabetic mice were significantly increased, with statistical significance ( t=25.372, 22.419, 25.372; P<0.05). In vitro cell experiment: compared with the N group and Vector group, the PAK4 protein, mRNA relative expression and cell mobility in the hRMEC of PAK4 group were significantly increased, with statistical significance ( F=36.821, 38.692, 29.421; P<0.05). Flow cytometry showed that the adhesion number of leukocytes on hRMEC in PAK4 group was significantly increased, and the difference was statistically significant ( F=39.649, P<0.01). Mitochondrial pressure measurement results showed that the capacity of mitochondrial basic respiration, ATP production, maximum respiration and reserve respiration in hRMEC in PAK4 group was significantly decreased, with statistical significance ( F=27.472, 22.315, 31.147, 27.472; P<0.05). Conclusion:Over-expression of PAK4 impairs mitochondrial function and significantly promotes leukocyte adhesion and migration in retinal vascular endothelial cells.
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Objective:To determine the expression of glucose transporter 3 (GLUT3) in cutaneous squamous cell carcinoma (cSCC), and to evaluate its effect on the cSCC cell line A431.Methods:From June 2016 to December 2020, 22 paraffin-embedded tissue specimens were collected from patients with pathologically confirmed cSCC in the Department of Dermatology, Peking University Third Hospital, and 20 discarded normal skin tissues after dermatological surgeries served as controls. Immunohistochemical assay was performed to determine the GLUT3 expression in cSCC tissues and normal skin tissues. Cultured A431 cells were divided into two groups: GLUT3 overexpression group transfected with a lentiviral vector carrying the SLC2A3 gene, and negative control group transfected with an empty lentiviral vector. Real-time fluorescence-based quantitative PCR and Western blot analysis were conducted to determine the mRNA and protein expression of GLUT3 in A431 cells in different groups, the cell proliferation assay (MTS assay) was performed to estimate the cell proliferative activity, and the live-cell analysis system Incucyte S3 was used for real-time evaluation of the migratory and invasive abilities of A431 cells in different groups. The relative glucose consumption and lactic acid production in A431 cells at 48 hours were measured by using glucose and lactic acid assay kits, retrospectively. Two independent samples t-test was used for comparisons between two groups, and one-way analysis of variance was used for comparisons among multiple groups. Results:The GLUT3 expression was significantly higher in the cSCC tissues than in the normal skin tissues (immunohistochemical assay score: 9.39 ± 2.56 points vs. 2.30 ± 2.60 points; t = 8.91, P < 0.05). Compared with the negative control group, the mRNA and protein expression of GLUT3 markedly increased in the GLUT3 overexpression group. MTS assay showed significantly increased proliferative activity of A431 cells in the GLUT3 overexpression group compared with the negative control group after 24- and 96-hour treatment ( t = 2.49, 3.54, P = 0.048, 0.012, respectively); cell fusion rates in the scratched area were significantly higher in the GLUT3 overexpression group than in the negative control group in the cell migration assay at 6, 12 18, and 24 hours and cell invasion assay at 12, 18, and 24 hours (all P < 0.05). At 48 hours, the relative glucose consumption and lactic acid production in A431 cells were significantly higher in the GLUT3 overexpression group than in the negative control group ( t = 2.98, 2.20, P = 0.011, 0.038, respectively) . Conclusion:GLUT3 was highly expressed in the cSCC tissues, and may participate in the occurrence and development of cSCC by providing energy to cSCC cells via promoting glucose uptake in cells to enhance their proliferative, migratory and invasive abilities.
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Objective:To investigate the effect of the key glycolysis enzyme 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (PFKFB3) on the biological activity of hemangioma-derived endothelial cells (HemECs) .Methods:Totally, 4 proliferating infantile hemangioma (IH) tissues and 4 involuting IH tissues were collected. Primary HemECs were isolated from the proliferating IH tissues, and human umbilical vein endothelial cells (HUVECs) served as controls. Immunohistochemical study and Western blot analysis were performed to determine the expression of PFKFB3 in the IH tissues and HemECs, respectively. Cell counting kit-8 (CCK8) assay was conducted to evaluate the effect of PFK15 (a specific inhibitor of PFKFB3) at concentrations of 0 - 10 μmol/L on the proliferation of HemECs, and HemECs treated without PFKFB3 served as the control group. Some in vitro cultured HemECs were treated with 5 μmol/L PFK15, and served as a PFK15 intervention group, while HemECs treated without PFK15 served as a control group; then, the migratory ability of HemECs was assessed by Transwell assay, and the apoptosis level of HemECs was detected by flow cytometry. Comparisons between groups were performed by using t test or analysis of variance. Results:Immunohistochemical study showed that the positive rate of PFKFB3 was significantly higher in the proliferating IH tissues (74.34% ± 5.26%) than in the involuting IH tissues (41.46% ± 2.99%, t = 9.40, P < 0.001). Western blot analysis showed that the relative expression level of PFKFB3 was also significantly higher in HemECs (0.73 ± 0.05) than in HUVECs (0.45 ± 0.04, t = 8.50, P < 0.001). CCK8 assay revealed significantly decreased proliferative activity of HemECs in the 0.625-, 1.25-, 2.5-, 5-, and 10-μmol/L PFK15 groups compared with the control group (all P < 0.01). Compared with the control group, the PFK15 intervention group showed significantly decreased number of migratory HemECs (297 ± 15 vs. 422 ± 8, t = 12.59, P < 0.001), but significantly increased apoptosis rates of HemECs (6.69% ± 0.64% vs. 0.34% ± 0.07%, t = 17.07, P < 0.001) . Conclusion:The key glycolytic enzyme PFKFB3 was highly expressed in the proliferating IH tissues and HemECs, and the PFKFB3 inhibitor PFK15 could suppress the proliferation, migration, and increase the apoptosis of HemECs.
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Aim To investigate the effects of HXL130 on the proliferation, invasion and migration of prostate cancer PC3 cells and its molecular mechanism. Methods MTT assay was used to detect the effect of HXL130 on the proliferation of prostate cancer PC3 cells. Hoechst 33258 staining and flow cytometry were used to detect the effects on apoptosis and cell cycle of cancer cells. Transwell was used to detect the effects of compounds on the invasion and migration of cancer cells. Proteomic sequencing was employed to detect differentially expressed proteins (DEPs) induced by compound treatment of cancer cells. Bioinformatics was used to analyze the functions of DEPs and the related signaling pathways regulated by DEPs, and Western blot was used to verify the result. Results The survival rate of PC3 cells decreased with the increase of HXL130 concentration and treatment time. HXL130 could significantly induce cell apoptosis and block G
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Aim To investigate the effect of casticin (CAS) on the migration and invasion of MHCC97H cells and preliminarily explore the molecular mechanism. Methods CCK-8 kit was used to detect the effect of different concentrations of CAS on the viability of MHCC97H cells; cell migration and invasion assays were carried out in groups to assess the migration and invasion ability of MHCC97H cells; reverse transcription fluorescence quantitative PCR (RT-qPCR) was performed to detect the miR-148a-3p and Wnt1 mRNA expression in MHCC97H cells after CAS treatment; migration-invasion related proteins (MMP2, MMP9) and Wnt1 protein expression were detected by Western blot; Dual-Luciferase reporter gene was used to detect the binding of miR-148a-3p to Wnt1 3′-UTR. Results CAS significantly inhibited the viability of MHCC97H cells. The IC
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Aim To investigate the effects of realgar on the proliferation, invasion and ferroptosis of esophageal cancer cells. Methods Different concentrations of realgar(0, 10,20, 40, 60, 80, 100 μmol·L-1)or realgar 1/2IC
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Aim To clarify the regulatory effect of Artesunate(ART) on tumor cell function and cell cycle in the pathological process of esophageal squamous cell carcinoma(ESCC). Methods KYSE450 and TE14 cells were treated with different concentrations of ART. The cells treated with 0 mg •L
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Aim To explore the effect of artesunate (ART) on the function of breast cancer cells during the progression of breast cancer and the possible mechanism of action. Methods MCF-7 (30 μmol • L-
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OBJECTIVE@#To study the role of apolipoprotein E (APOE) in regulating endometrial cancer metastasis and explore the signaling pathway in the regulatory mechanism.@*METHODS@#Human endometrial cancer cell line HEC-1B was transfected with a control siRNA (siCtrl) or a specific siRNA targeting APOE (siAPOE) or with either pEGFP-N1 plasmid or an APOEoverexpressing plasmid. The changes in migration, proliferation, apoptosis and cell cycle of the transfected cells were examined using wound healing assay, Transwell migration assay, MTT assay, flow cytometry, and Hoechst staining. The activity of the ERK/MMP9 signaling pathway in the transfected cells was assessed using RT-qPCR and Western blotting. The expression level of APOE in clinical specimens of endometrial cancer tissues were detected using immunohistochemistry and its correlation with differentiation of endometrial cancer tissues was analyzed.@*RESULTS@#Wound healing assay and Transwell migration assay showed that compared with those in siCtrl group, HEC-1B cells transfected with siAPOE showed significantly reduced migration ability (P < 0.05), whereas APOE overexpression significantly promoted the migration of the cells (P < 0.05). Neither APOE knockdown nor overexpression produced significant effects on HEC-1B cell proliferation as shown by MTT assay and flow cytometry. Hoechst staining revealed that transfection with siAPOE did not significantly affect apoptosis of HEC-1B cells. APOE knockdown obviously reduced and APOE overexpression enhanced ERK phosphorylation and MMP9 expression in HEC-1B cells (P < 0.05). Treatment with U0126 partially reversed the effects of APOE overexpression on ERK phosphorylation, migration and MMP9 expression in HEC-1B cells (P < 0.05). APOE is highly expressed in clinical samples of endometrial cancer tissues as compared with the adjacent tissues.@*CONCLUSION@#APOE is highly expressed in endometrial cancer tissues to promote cancer cell migration by enhancing ERK phosphorylation and MMP9 expression.