ObjectiveTo investigate the role of Peptidase domain containing associated with muscle regeneration 1 (PAMR1) in the proliferation, migration, and prognosis of hepatocellular carcinoma (HCC) through cellular experiments and clinical sample validation. Methods① Bioinformatics analysis was performed on datasets from the GEO public database to identify and screen for key genes, ultimately selecting PAMR1 for further study. Findings were validated using data from the TCGA database and six primary HCC surgical specimens prospectively obtained from the Army Characteristic Medical Center of Army Medical University from February 2024 to June 2024. ② PAMR1-overexpressing cell lines were established using HCC cell lines Huh7 and Lm3. Cells were transfected with either the recombinant plasmid pcDNA3.1(+)-PAMR1 as overexpression group or the empty vector pcDNA3.1(+) as negative control group. The effects of PAMR1 on HCC cell proliferation and migration were assessed using the Cell Counting Kit-8 (CCK-8) assay and wound healing assay, respectively. ③ Pathological specimens and clinical data were retrospectively collected from 61 patients with primary HCC who underwent surgical resection at the Army Characteristic Medical Center of Army Medical University between May 2019 and April 2020. The impact of PAMR1 expression on disease-free survival was evaluated. Results① PAMR1 was identified as a candidate gene through GEO database screening and was found to be downregulated in HCC tissues based on both TCGA data and the six HCC surgical specimens. ② The CCK-8 assay revealed that cell proliferation was significantly inhibited in the PAMR1 overexpression group compared to the negative control group (P<0.05). Similarly, the wound healing assay demonstrated reduced migratory capability in PAMR1 overexpression group (P<0.05). ③ Multivariate cox proportional hazards regression analysis of patient data indicated that high PAMR1 expression serves as an independent protective factor for disease-free survival in HCC (HR= 0.335, P=0.026). ConclusionPAMR1 serves as a crucial gene in HCC, high PAMR1 expression can significantly suppressing tumor cell proliferation and migration and indicates a favorable prognosis.
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.
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.
To investigate the effects of Snail1 gene silence on the expression of tight junction proteins and the migration ability of Hep-2 cells, Hep-2 cells were transfected with plasmids which is containing the shRNA of Snail1 gene, and cultured till the cells could be passaged stably (named Sh-snail1 cells). The expression of tight junction proteins (ZO-1, Occludin, Claudin-5) were detected by Western blot. The migration ability of Sh-snail1 cells was investigated by wound healing assay, and the protein expression of members of RhoGTPase family (RhoA, Cdc42) was detected by Western blot, which is closely related to the migration ability. Our results showed that the expression of tight junction proteins (ZO-1, Occludin, Claudin-5) was significantly increased; the migration ability of Sh-snail1 cell was inhibited; the expression of RhoA and Cdc42 was downregulated. All of these indicated that silencing the gene of Snail1 in Hep-2 cells can up-regulate the expression of tight junction proteins and down regulate the expression of Cdc42 and RhoA, and further inhibit the migration of Hep-2 cells. Furthermore, opening of the tight junctions between cells and the stronger migration ability of cancer cells are important processes in cancer metastasis. It is confirmed that the Snail1 gene is closely related to the two processes, providing an experimental basis for targeted therapy of laryngeal squamous cell carcinoma.
Endogenous adult neural stem cells are closely related to the normal physiological functions of the brain and many neurodegenerative diseases. Neurons are affected by factors such as extracellular microenvironment and intracellular signaling. In recent years, some specific signaling pathways have been found that affect the occurrence of neural stem cells in adult neural networks, including proliferation, differentiation, maturation, migration, and integration with host functions. In this paper, we summarize the signals and their molecular mechanisms, including the related signaling pathways, neurotrophic factors, neurotransmitters, intracellular transcription factors and epigenetic regulation of neuronal differentiation from both the extracellular and intracellular aspects, providing basic theoretical support for the treatment of central nervous system diseases through neural stem cells approach.
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.
ObjectiveTo explore the potential role of WNT6 in the proliferation, differentiation, and migration of bone marrow mesenchymal stem cells (BMSCs). MethodsMouse BMSCs were cultured to the cell fusion of 30%-50%, and divided into different groups. WNT6 knockdown included 3 experiment groups:cells transfected with WNT6 specific short hairpin RNA (shRNA) (group A1), cells transfected with control shRNA group (group B1), and nontransfected cells (group C1). WNT6 over-expression included 3 groups:cells transfected with WNT6 recombinant plasmid (group A2), cells transfected with blank vector (group B2), and non-transfected cells (group C2). After transfection, the stably transfected cells were cultured for 48 hours. Cell morphology was observed under inverted microscope; real-time fluorescent quantitative PCR was used to analyze WNT6 mRNA levels; Western blot was used to detect WNT6 and Ki67 protein expressions; cell proliferation was assayed by MTT method, and cell migration was detected by Transwell assay. After cells were cultured in osteogenic differentiation medium for 12 days, the alkaline phosphatase (ALP) activity and calcium deposits were detected by biochemical determination. ResultsThe inverted microscope observation showed that the cell morphology were similar among groups A1, B1, C1, and A2, B2, C2. The WNT6 mRNA and protein levels, Ki67 protein level, cell proliferation, cell migration, ALP activity, and calcium deposition in group A1 were all significantly lower than those in groups B1 and C1 (P<0.05), but there was no significant difference between groups B1 and C1 (P>0.05). On the contrary, the above indexes in group A2 were all significantly higher than those in groups B2 and C2 (P<0.05), but no significant difference was shown between groups B2 and C2 (P>0.05). ConclusionWNT6 can promote the proliferation and migration, as well as can enhance osteogenic differentiation ability in mouse BMSCs.
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.
Objective To investigate the effects of 4-methylcatechol (4MC) on the migration and inflammatory response in rheumatoid arthritis (RA) fibroblast-like synoviocytes (FLS), as well as its underlying mechanisms of action. Methods RA-FLS was isolated from synovial tissue donated by RA patients, and the optimal concentration of 4MC was determined by cell counting kit 8 method for subsequent experiments, and the effect of 4MC on the migratory ability of RA-FLS was evaluated via a cell scratch assay. An inflammation model of RA-FLS was induced by tumor necrosis factor α (TNF-α). Real-time fluorescence quantitative PCR and ELISA were employed to detect the gene and protein expression levels of interleukin-1β (IL-1β) and IL-6 in RA-FLS and their culture supernatants, respectively, thereby investigating the anti-inflammatory effects of 4MC. Western blot was used to examine the expressions of nuclear factor κB (NF-κB) signaling pathway-related proteins, including inhibitor of NF-κB-α (IKBα), phosphorylated (P)-IκBα, NF-κB-inducing kinase α (IKKα), P-IKKαβ, P-p65, and p65. Cellular immunofluorescence was utilized to detect the expression and localization of p65 in RA-FLS, exploring whether 4MC exerts its anti-inflammatory effects by regulating the NF-κB signaling pathway. Finally, a collagen-induced arthritis (CIA) mouse model was established. The anti-RA effect of 4MC in vivo was evaluated by gross observation and histological examination. Results 4MC inhibited RA-FLS migration in a concentration-dependent manner. In the TNF-α-induced RA-FLS inflammation model, 4MC significantly decreased the gene and protein expression levels of IL-1β and IL-6. Furthermore, 4MC markedly reduced the ratios of P-IΚBα/IΚBα, P-IKKαβ/IKKα, and P-p65/p65, thereby blocking the transcriptional activity of p65 by inhibiting its nuclear translocation. This mechanism effectively suppressed the activation of the TNF-α-mediated NF-κB signaling pathway. Animal studies demonstrated that 4MC [10 mg/(kg·day)] significantly lowered serum levels of IL-1β, IL-6, and TNF-α, and alleviated arthritis severity and bone destruction in CIA mice. Conclusion 4MC not only inhibits the migration of RA-FLS but also mitigates their inflammatory response by suppressing the NF-κB signaling pathway, thereby effectively exerting its anti-RA effects.
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.