ObjectiveTo investigate the regulatory mechanism of thioredoxin binding protein (TXNIP)/nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3) pathway in the occurrence and development of breast cancer.MethodsThe resected 15 cases of breast cancer tissues and their adjacent tissues in our hospital from September 2019 to June 2020 were selected, and the immunohistochemistry was used to detect the expression levels of TXNIP and NLRP3 in breast cancer and its adjacent tissues. Three kinds of breast cancer cell lines (MDA-MB231, MCF-7 and SKBR3) and normal breast epithelial cell line (HMEC) were collected. Western blot was used to detect the relative expression levels of TXNIP and NLRP3 in three kinds of breast cancer cell lines and HMEC cell line. MDA-MB231 cancer cells were divided into blank control group (normal culture without any treatment), TXNIP overexpression group (Ad-TXNIP group, transfected with adenovirus vector carrying TXNIP overexpression sequence), Ad-TXNIP negative control group (Ad-eGFP1 group, transfected of empty adenovirus vector without TXNIP overexpression sequence), NLRP3 overexpression group (Ad-NLRP3 group, transfected with adenovirus vector containing NLRP3 overexpression sequence), TXNIP and NLRP3 overexpression co-transfection group (Ad-TXNIP+Ad-NLRP3 group, co-transfection of adenovirus vector carrying TXNIP and NLRP3 overexpression sequence), TXNIP overexpression and Ad-NLRP3 negative control (Ad-eGFP2) co-transfection group (Ad-TXNIP+Ad-eGFP2 group,co-transfection of adenovirus vector carrying TXNIP overexpression sequence and empty adenovirus without NLRP3 overexpression sequence). After 24 hours of transfection and culture, CCK-8 method was used to detect the MDA-MB231 cells proliferation. Transwell chamber method was used to detect MDA-MB231 cells migration and invasion. Nude mice tumorigenicity test was used to detect the tumorigenicity of the MDA-MB231 cells in vivo. Western blot was used to detect the expressions of TXNIP, NLRP3, proliferation marker protein (Ki-67), caspase-1, vascular endothelial growth factor (VEGF), interleukin (IL)-1β, IL-18 and caspase-1 precursor protein (pro-caspase-1) in the MDA-MB231 cells.ResultsCompared with the adjacent tissues, the relative expression level of TXNIP decreased (P<0.05) and the relative expression level of NLRP3 increased (P<0.05) in breast cancer tissues. Compared with normal breast epithelial cell line (HMEC cell line), the relative expression levels of TXNIP in MDA-MB231, MCF-7 and SKBR3 breast cancer cell lines were decreased (P<0.05), and the relative expression levels of NLRP3 were increased (P<0.05). Compared with the blank control group, the relative expression levels of TXNIP, NLRP3, IL-1β, IL-18, pro-caspase-1 and caspase-1 were increased (P<0.05), the relative expression levels of Ki-67 and VEGF, the proliferation activity, invasion and migration ability of MDA-MB231 cells and tumor weight were decreased (P<0.05) in the Ad-TXNIP group and the Ad-NLRP3 group. Compared with the Ad-TXNIP group and the Ad-NLRP3 group, the relative expression levels of TXNIP, NLRP3, IL-1β, IL-18, pro-caspase-1 and caspase-1 were further increased (P<0.05), the relative expression levels of Ki-67 and VEGF, the proliferation activity, invasion and migration ability of MDA-MB231 cells and tumor weight were further decreased (P<0.05) in the Ad-TXNIP+Ad-NLRP3 group.ConclusionsIn breast cancer tissues and breast cancer cell lines, TXNIP is low expression and NLRP3 is high expression. They can interact with each other to promote pyroptosis and inhibit the proliferation, invasion and migration of breast cancer cells.
Objective To explore the effects of DNA cross-linking repair 1B (DCLRE1B) gene on the migration and invasion ability of hepatocellular carcinoma cell. Methods Bioinformatics analysis was used to analyze the expression of DCLRE1B mRNA in hepatocellular carcinoma, and its relationship with the prognosis and related influencing factors of patients. Immunohistochemical staining was used to detect the expression of DCLRE1B protein in resected hepatocellular carcinoma tissues and their corresponding normal liver tissues. The DCLRE1B gene silenced Huh7 and HepG2 hepatocellular carcinoma cell lines were constructed by lentivirus, and the transfected effect was detected by Western blot. The migration and invasion of DCLRE1B silenced hepatocellular carcinoma cells were detected by scratch test and Transwell method. The changes of genes related to epithelial mesenchymal transformation (EMT) after DCLRE1B silencing were detected by quantitative real-time polymerase chain reaction (qRT-PCR). Results ① The biological information analysis results showed that: The mRNA expression of DCLRE1B was highly expressed in a variety of tumors including hepatocellular carcinoma (P<0.05). The mRNA expression of DCLRE1B was associated with the TNM staging of tumor (P<0.05). The relative expression level of DCLRE1B mRNA in hepatocellular carcinoma patients was related to their prognosis. The overall survival situation (P=0.038) and progression free survival situation (P=0.005) of hepatocellular carcinoma patients in the high expression group were worse than those in the low expression group. Univariate and multivariate Cox analysis showed that the expression of DCLRE1B gene was an independent factor affecting the prognosis of hepatocellular carcinoma (P<0.05). ② The positive rate of DCLRE1B protein expression in resected hepatocellular carcinoma tissues was higher than that in normal liver tissues (P<0.05). ③ Cell experiment results showed that: After stable silencing DCLRE1B gene of hepatocellular carcinoma cell (Huh7 and HepG2) constructed by lentivirus, the expression of DCLRE1B protein was significantly down regulated (P<0.05). After silencing DCLRE1B gene, the migration and invasion ability of hepatocellular carcinoma cells were significantly decreased (P<0.05). After silencing DCLRE1B, the mRNA expressions of E-cadherin, matrix metalloproteinase 9, and β-catenin were up regulated (P<0.05), and the mRNA expressions of N-cadherin and Vimentin were down regulated (P<0.05), but the mRNA expression of zinc finger transcription factor had no significant change, and the difference was not statistically significant (P＞0.05). Conclusion Silencing DCLRE1B gene can inhibit the migration and invasion ability of hepatocellular carcinoma cells, and its mechanism may be related to the process of EMT.
ObjectiveTo investigate the influence of heat shock protein A2 (HSPA2) on the biological behavior of pancreatic adenocarcinoma cells and its mechanism. MethodsThe expressions of HSPA2 were determined in the human pancreatic adenocarcinoma cell lines (PANC-1, BxPC-3, and AsPC-1) using the Western blot. Subsequently, the cells with the lowest and highest HSPA2 expressions among these three lines were selected for conducting overexpression and knockdown experiments targeting HSPA2, respectively. The cellular proliferation, cell clonogenesis, migration, and invasion capabilities were assessed using MTT, clonogenic assay, and Transwell assay, respectively. Additionally, the impact of HSPA2 on the expression of key markers of epithelial-mesenchymal transition (EMT) was examined using the Western blot. The potential target molecules of HSPA2 were identified through immunoprecipitation assay and mass spectrometry. The rescue experiments further explored the regulatory relation between the HSPA2 and its target molecules. The influence of HSPA2 on pancreatic adenocarcinoma growth was investigated through establishment of xenograft tumor model in nude mice. ResultsThe HSPA2 exhibited the lowest expression in the PANC-1 cells and the highest expression in the AsPC-1 cells among the three cell lines. Subsequent functional studies demonstrated that the overexpression of HSPA2 in the PANC-1 cells markedly promoted proliferation, cell clonogenesis, migration, and invasion, while the knockdown of HSPA2 expression in the AsPC-1 cells markedly inhibited these processes. The Western blot analysis further showed that the HSPA2 overexpression downregulated E-cadherin expression and upregulated N-cadherin and Vimentin expressiones, whereas the HSPA2 knockdown produced opposite effects. The rescue experiments indicated that the HSPA2 promoted the EMT in pancreatic adenocarcinoma cells by upregulating Yes associated protein (YAP). The subcutaneous xenograft tumor experiments in the nude mice showed that the HSPA2 knockdown inhibited tumor growth. ConclusionThe results of this study suggest that HSPA2 promotes EMT via upregulating YAP, which facilitates proliferation, migration, and invasion of pancreatic adenocarcinoma cells.
ObjectiveTo investigate the effect of cells in the epimysium conduit (EMC) on the regeneration of sciatic nerve of mice.MethodsThe epimysium of the 8-week-old male C57BL/6J enhanced green fluorescent protein (EGFP) mouse was trimmed to a size of 5 mm×3 mm, and prepared in a tubular shape (ie, EMC). Some epimysia were treated with different irradiation doses (0, 15, 20, 25, 30, 35 Gy) to inhibit cells migration. Then the number of migrating cells were counted, and the epimysia with the least migrating cells were selected to prepare EMC. Some epimysia were subjected to decellularization treatment and prepared EMC. HE and Masson staining were used to identify the decellularization effect. Twenty-four C57BL/6J wild-type mice were used to prepare a 3-mm-long sciatic nerve defect of right hind limb model and randomly divided into 3 groups (n=8). EMC (group A), EMC after cell migration inhibition treatment (group B), and decellularized EMC (group C) were used to repair defects. At 16 weeks after operation, the midline of the regenerating nerve was taken for gross, toluidine blue staining, immunofluorescence staining, and transmission electron microscopy.ResultsAt 15 days, the number of migrating cells gradually decreased with the increase of irradiation dose. There was no significant difference between 30 Gy group and 35 Gy group (P>0.05); there were significant differences between the other groups (P<0.05). The epimysium after treatment with 35 Gy irradiation dose was selected for thein vivo experiment. After the decellularization of the epimysium, no nucleus was found in the epimysium and the epimysium could be sutured to prepare EMC. At 16 weeks after operation, the nerves in all groups were recanalized. The sciatic nerve was the thickest in group A, followed by group B, and the finest in group C. Immunofluorescence staining showed that the EGFP cells in group A were surrounded by regenerated axons. Toluidine blue staining and transmission electron microscopy observation showed that the number of regenerated axons and the thickness of regenerated myelin sheath in group A were significantly better than those in groups B and C (P<0.05). There was no significant difference between groups B and C (P>0.05).ConclusionThe cellular components of the epimysium participate in and promote the regeneration of the sciatic nerve in mice.
Objective To study the function and mechanism of G protein coupled receptor kinase interacting protein 1(GIT1) RNA hairpin (GIT1-RNAh) in osteoblast migration. Methods The sixth passage osteoblasts were divided into 2 groups and were infected by GIT1-RNAh (experimental group) and green fluoresence protein RNA hairpin (GFP-RNAh) (control group) adenovirus for 12 hours respectively. Each group was further classfied into two groups according to with or without platelet-drived growth factor (PDGF) stimulation. The GIT1 expression and Paxillindistribution was analyzed by immunofluorescence staining. Paxillin phosphorylation was detected by Western Blot. The localization of Paxillin was determined by co-immunofluorescence staining after transfection with cyanine fluorescence protein tagged GIT1RNAh (CFP-GIT1-RNAh)(experimental group) and GFP-RNAh (CFP-GFP-RNAh)(control group). The role of GIT1-RNAh (experimental group) and GFP-RNAh (control group) adenovirus in osteoblasts migration was determined by wound healing assay. Results Immunofluorescence staining results showed that the GIT1-RNAh significantly inhibited endogenous GIT1 expression, interfered Paxillin distribution.Western Blot results showed that Paxillin phosporylation was obviously inhibited in osteoblasts infected with GIT1-RNAh adenovirus (P＜0.05). The wound healing assay results howed that GIT1-RNAh adenovirus significantly inhibited osteoblast migration induced by PDGF. Conclusion GIT1-RNAh inhibits osteoblasts migration by interfering paxillin distribution and decrease Paxillin phosphorylation.
ObjectiveTo explored the effect of stromal cell-derived factor 1α (SDF-1α) on promoting the migration ability of rat adipose derived stem cells (rADSCs) by constructed the rADSCs overexpression SDF-1α via adenovirus transfection.MethodsrADSCs were isolated from adipose tissue of 6-week-old SPF Sprague Dawley rats. Morphological observation, multi-directional differentiations (osteogenic, adipogenic, and chondrogenic inductions), and flow cytometry identification were performed. Transwell cell migration experiment was used to observe and screen the optimal concentration of exogenous SDF-1α to optimize the migration ability of rADSCs; the optimal multiplicity of infection (MOI) of rADSCs was screened by observing the cell status and fluorescence expression after transfection. Then the third generation of rADSCs were divided into 4 groups: group A was pure rADSCs; group B was rADSCs co-cultured with SDF-1α at the best concentration; group C was rADSCs infected with recombinant adenovirus-mediated green fluorescent protein (Adv-GFP) with the best MOI; group D was rADSCs infected with Adv-GFP-SDF-1α overexpression adenovirus with the best MOI. Cell counting kit 8 (CCK-8) and Transwell cell migration experiment were preformed to detect and compare the effect of exogenous SDF-1α and SDF-1α overexpression on the proliferation and migration ability of rADSCs.ResultsThe cell morphology, multi-directional differentiations, and flow cytometry identification showed that the cultured cells were rADSCs. After screening, the optimal stimulating concentration of exogenous SDF-1α was 12.5 nmol/L; the optimal MOI of Adv-GFP adenovirus was 200; the optimal MOI of Adv-GFP-SDF-1α overexpression adenovirus was 400. CCK-8 method and Transwell cell migration experiment showed that compared with groups A and C, groups B and D could significantly improve the proliferation and migration of rADSCs (P<0.05); the effect of group D on enhancing the migration of rADSCs was weaker than that of group B, but the effect of promoting the proliferation of rADSCs was stronger than that of group D (P<0.05).ConclusionSDF-1α overexpression modification on rADSCs can significantly promote the proliferation and migration ability, which may be a potential method to optimize the application of ADSCs in tissue regeneration and wound repair.
Objective To explore the effects of Zhaoke defibrase and anti alpha;vbeta;3mAb (23C6) on the adhesion and immigration of bovine retinal vascular endothelial cells. Methods The culture dishes coated with vitronectin (Vn) and collagen，assays of adhesion and immigration were performed 60 minutes after different concentration of Zhaoke defibrase and anti-alpha;vbeta;3 mAb was added to the bovine retinal vascular endothelial cells. The apoptosis of bovine retinal vascular endothelial cells induced by Zhaoke defibrase and anti-alpha;vbeta;3 mAb was detected by electron microscopy. Results Both Zhaoke defibrase and anti-alpha;vbeta;3 mAb inhibited the adhesion and immigration of bovine retinal vascular endothelial cells in a dose-dependent manner. The inhibited concentration (IC50) of Zhaoke defibrase was less than 0.05 mu;mol/L, while (IC50) of anti-alpha;vbeta;3 mAb was more than 2.5 mu;mol/L. 81.8% endothelial cells adhering to Vn were inhibited by 0.1 mu;mol/L Zhaoke defibrase, while 76.3% by endothelial cells adhering to Vn were inhibited by 10 mu;mol/L anti-alpha;vbeta;3 mAb. Typical apoptosis cells were found in bovine retinal vascular endothelial cells after affected by Zhaoke defibrase and anti-alpha;vbeta;3 mAb. Conclusion Both Zhaoke defibrase and anti- alpha;vbeta;3mAb can significantly inhibit the adhesion and immigration of bovine retinal vascular endothelial cells to extracellular matrix， and the mechanism may lie in inducing the apoptosis of endothelial cells. (Chin J Ocul Fundus Dis, 2005,21:118-121)
Objective To investigate the effect of monocyte chemoattractant protein 1 (MCP-1) on the migration of the induced and differentiated mouse bone marrow mesenchymal stem cells (BMSCs) for raising the efficacy of intravenous transplantation of BMSCs. Methods The BMSCs were cultured with the method of differential adhesion and density gradient centrifugation of C57/BL10 mice, and were identified by alkal ine phosphatase Gomori modified staining after osteogenic inducing. At the 3rd passage, the BMSCs were induced to the myoblasts with 5-azacytidine (5-Aza). The chemotaxis of MCP-1 in the induced and differentiated BMSCs in vitro at concentrations of 25, 50, 100, 200, and 400 ng/mL was observed through the migration test, by counting the number of the migrated cells. The expression of the chemokine receptor 2 (CKR-2) in the induced and differentiated BMSCs was detected with the flow cytometry. Results The cells could be cultured with the methods of differential adhesion and density gradient centrifugation and still had higher prol iferative and differentiative potency; the induced cells at the 3rd passage could differenciate to the osteoblasts, confirming that the cells were BMSCs; the myogenic induced BMSCs possesed the sarcotubule structure. The number of the migrating BMSCs at MCP-1 concentrations of 25-400 ng/ mL were respectively 35.066 7 ± 6.584 2, 43.200 0 ± 6.460 8, 44.466 7 ± 4.823 5, 45.600 0 ± 8.650 3, and 50.733 3 ± 7.582 5; showing significant difference when compared with control group (28.333 3 ± 8.917 6, P lt; 0.05), and presenting significant difference among 25, 50, 400 ng/mL groups compared with each other (P lt; 0.05). The expression of CKR-2 in the mouse BMSCs (48.0%) was significantly higher (P lt; 0.001) than those of blank control (0.6%) and negative control (17.0%). Conclusion The results indicate that the MCP-1 can induce the migration of mouse BMSCs by MCP-1/CKR-2 pathway.
ObjectiveTo investigate the effects of overexpression of alpha/beta hydrolase domain-containing protein 5 (ABHD5) on the invasion and migration of human colon cancer cell line HCT116 and the pathway of adenosine monophosphate-activated protein kinase (AMPK)/mechanistic target of rapamycin (mTOR).MethodsThe expression of ABHD5 in colon cancer tissues and its relationship with clinicopathological features was analyzed by UALCAN database. HCT116 cells were cultured in vitro and transfected with ABHD5 recombinant plasmid, then they were divided into control group, negative transfection group and ABHD5 transfection group. Real time quantitative PCR (qRT-PCR) was used to detect the expression of ABHD5 mRNA in HCT116 cells. The proliferation of HCT116 cells was detected by CCK-8 method. Transwell assay was used to detect the invasion and migration of HCT116 cells. The expression of matrix metalloprotein 9 (MMP-9), E-cadherin, Snail, and AMPK/mTOR pathway proteins p-AMPK, AMPK, p-mTOR and mTOR were detected by Western blot.ResultsThe results of the UALCAN showed that compared with normal colon tissues, the expression of ABHD5 mRNA in colon cancer tissues was decreased (P<0.05), and which in the adenocarcinoma and the N1 stage was lower than that of the mucinous adenocarcinoma (P<0.05) and N0 stage (P<0.05), respectively. Compared with the control group and the negative transfection group, the expression of ABHD5 mRNA in the ABHD5 transfection group was increased (P<0.05), the proliferation inhibition rate of HCT116 cells in the ABHD5 transfection group was increased (P<0.05), the numbers of migration and invasion cells in the ABHD5 transfection group were decreased (P<0.05), the expressions of MMP-9, Snail, p-mTOR and mTOR were reduced, and the expressions of E-cadherin, p-AMPK and AMPK were increased (P<0.05).ConclusionsThe overexpression of ABHD5 can inhibit the invasion and migration of colon cancer HCT116 cells, activate AMPK, and inhibit the expression of mTOR. It suggests that ABHD5 may play a role in inhibiting colon cancer by affecting AMPK/mTOR pathway.
Lysophosphatidic acid (LPA) is a pluripotent lipid mediator and acts via different G-protein-couple LPA receptors. LPA has significant effects on several cellular biological behaviours, such as cell migration, invasion, proliferation and differentiation, etc. Cell migration is essential for tumor progression, and vital for stem cell to repair injured tissues. Increasing evidences have demonstrated that LPA dramatically affects migration capacity of various cells, particularly cancer cells and stem cells. In this paper, we review the effect of LPA on migration of cancer cells and stem cells, and discuss the underlying mechanisms. A better understanding of this process will shed new light on tissue regeneration and the prevention of tumor progression.