[Plectin Promotes the Migration of Hepatocellular Carcinoma Cells Through Inducing F-actin Polymerization]. / Plectin通过诱导F-actin聚合增强肝癌细胞的迁移能力.

Objective: To explore the relationship between the expression of plectin and the migration of hepatocellular carcinoma (HCC) cells and to elucidate the molecular mechanisms by which plectin expression affects the migration of HCC cells. Methods: First of all, Western blot was performed to determine the expression of plectin in normal hepatocytes and HCC cells. Secondly, a plectin-downregulated HCC cell strain was established and the control group (shNC group) and shPLEC group were set up. Each group was divided into a vehicle control group (shNC+DMSO group or shPLEC+DMSO group) and a F-actin cytoskeleton polymerization inducer Jasplakinolide group (shNC+Jasp group or shPLEC+Jasp group). Western blot was performed to determine the expression of plectin and epithelial-mesenchymal transition (EMT)-related proteins, including N-cadherin, vimentin, and E-cadherin. HCC cell migration was evaluated by Transwell assay. KEGG (Kyoto Encyclopedia of Genes and Genomes) was used to analyze the signaling pathways related to plectin gene. The polymerization of F-actin was analyzed by immunofluorescence assay. Results: Compared with the normal hepatocytes, HCC cells showed high expression of plectin. Compared with those in the shNC group, the expression of plectin in the shPLEC group was decreased (P<0.05), the migration ability of HCC cells was weakened (P<0.05), and the EMT process was inhibited (with the expression of N-cadherin and vimentin being decreased and the expression of E-cadherin being increased) (P<0.05). KEGG analysis showed that the regulation of cytoskeletal F-actin was most closely associated with plectin and cytoskeletal F-actin depolymerized in the shPLEC group. After treatment with Jasplakinolide, an inducer of F-actin cytoskeleton polymerization, the migration ability of HCC cells in the shPLEC+Jasp group was enhanced compared with that of shPLEC+DMSO group (P<0.05) and the EMT process was restored (with the expression of N-cadherin and vimentin being increased and the expression of E-cadherin being decreased) (P<0.05). In addition, the polymerization of cytoskeletal F-actin in HCC cells was also restored. Conclusion: Plectin is highly expressed in HCC cells. Plectin promotes the migration and the EMT of HCC cells through inducing F-actin polymerization.

PRP significantly promotes the adhesion and migration of vascular smooth muscle cells on stent material.

BACKGROUND: The adhesion and survival state of cells on scaffold material is a major problem in tissue-engineered blood vessel (TEBV) culture. Platelet-rich plasma (PRP) contains a large amount of biologically active factors and fibrin, which is expected to play an important role in TEBV culture. PURPOSE: To combine PRP with cells and scaffold material to promote cell adhesion and biological activity on the scaffold material. METHODS: The adhesion status and migration of SMCs under the optimal concentration suitable for SMC growth and the optimal concentration of PRP were examined by scanning electron microscopy, HE staining, CCK-8 assays, qPCR, WB, and other experimental methods and compared with those under the conventional culture (20% FBS); finally, the effect of PRP on the deposition of ECM in vascular tissue engineering culture was verified by three-dimensional culture. RESULTS: PRP at 20% is a suitable concentration for SMCs. Compared with the control group, the 20% PRP group had better migration, and the number of SMC adhesions was significantly higher than that of the control group. In addition, collagen deposition in the experimental group was significantly higher than that in the control group. CONCLUSION: PRP (20%) can promote SMC adhesion, migration, and collagen deposition on the scaffold material.

Assessment of the effects of hydrogen water on human gingival fibroblast cell culture in patients with chronic periodontitis.

Background: Activated inflammatory cells produce reactive oxygen species (ROS) to eliminate pathogens. Under normal conditions, the pathogens are taken care of, and tissues are repaired. However, in periodontal disease, persistent inflammation causes increased ROS release and impaired healing. Therefore, removal of overproduced ROS using antioxidants is necessary. Hydrogen water has an antioxidative effect on cells and impedes oxidative stress-related disorders. Aim: To study the effect of hydrogen water on cell viability, migration, and its antioxidative potential in fibroblasts obtained from chronic periodontitis patients. Materials and Methods: The gingival tissue samples were obtained from 26 subjects (13 periodontally healthy individuals and 13 chronic periodontitis patients) and processed. The human gingival fibroblasts were cultured and the assays were commenced once adequate growth was detected. The effect of hydrogen water on cell viability was checked by neutral red assay, while the migration potential was assessed by transwell migration assay. The antioxidative potential of hydrogen water was evaluated by CUPRAC assay. Statistical Analysis: Intergroup comparison was done using Mann-Whitney U-test. Intragroup comparison was done using Wilcoxon signed-rank test. Results: Hydrogen water was nontoxic to the fibroblasts at 24 h and 48 h. The intergroup comparison of the cell viability between hydrogen water-treated periodontally healthy gingival fibroblasts (HF) and fibroblasts from patients with chronic periodontitis (CF) showed a statistically significant (P = 0.00) difference at 24 h and 48 h. Hydrogen water also positively influenced the migratory capacity. Hydrogen water-treated fibroblasts obtained from chronic periodontitis patients showed more migration in comparison to the healthy group (P = 0.00). Hydrogen water showed an antioxidative potential. The maximum potential was seen in relation to the fibroblasts obtained from chronic periodontitis patients at 48 h. Conclusion: Hydrogen water was nontoxic, increased the migratory capacity, and showed an antioxidative potential on human fibroblasts obtained from periodontally healthy individuals and patients with chronic periodontitis.

[Antiproliferative, apoptotic, and antimigratory activities of kafirins on cervical cancer-derived cell lines]. / Actividades antiproliferativas, apoptóticas y antimigratorias de las kafirinas en líneas celulares derivadas de cáncer cervical.

Background: Cervical cancer is one of the leading causes of death in women worldwide, both in developed and developing countries. Therefore, effective treatment of cervical cancer with potential anti-tumor drugs is important. However, new treatments inspired by nutritional medicine are needed. Objective: To use the human cervical cancer cell lines HeLa and SiHa to evaluate the antiproliferative, apoptotic, and migratory activity of sorghum (kafirins). Materials and methods: The anticancer effects of the kafirins were examined by counting cells, MTT assays, apoptosis, and migration assays. Results: This investigation showed that sorghum induced growth inhibition of HeLa and SiHa cells at a significant level. The growth inhibition is dose-dependent and irreversible. When HeLa and SiHa cells were treated with sorghum due to the activity of kafirins, morphological changes were observed, which were identified through the formation of apoptopic bodies. And the kafirins at concentrations of 37.5, 75, 150, and 300 µg/mL decreased the migration of HeLa cells and SiHa cells. Conclusion: This paper demonstrates the induction of antiproliferative, apoptotic, and anti-migratory activity in HeLa and SiHa cells by kafirins. Sorghum may be used as a nutraceutical with potential cancer-prevention benefits.

Introducción: el cáncer de cuello uterino es una de las principales causas de muerte en las mujeres de todo el mundo, tanto en los países desarrollados como en los países en desarrollo. Por lo tanto, es importante un tratamiento eficaz de este cáncer con posibles fármacos antitumorales. Sin embargo, se necesitan nuevos tratamientos inspirados en la medicina nutricional. Objetivo: usar las líneas celulares de cáncer cervicouterino humano HeLa y SiHa para evaluar la actividad antiproliferativa, apoptótica y migratoria del sorgo (kafirinas). Material y métodos: los efectos anticancerígenos de las kafirinas se examinaron mediante recuento de células, ensayos de MTT, apoptosis y ensayos de migración. Resultados: la presente investigación demostró que el sorgo induce la inhibición del crecimiento de las células HeLa y SiHa a un nivel significativo. La inhibición del crecimiento es dependiente de la dosis e irreversible. Cuando las células HeLa y SiHa fueron tratadas con sorgo debido a la actividad de las kafirinas, se observaron cambios morfológicos, los cuales se identificaron mediante la formación de cuerpos apoptóticos. Y las kafirinas en concentraciones de 37.5, 75, 150 y 300 µg/mL disminuyeron la migración de las células HeLa y SiHa. Conclusiones: este trabajo demuestra la inducción de actividad antiproliferativa, apoptótica y antimigratoria en las células HeLa y SiHa por parte de las kafirinas. El sorgo puede utilizarse como nutracéutico con potenciales beneficios en la prevención del cáncer.

Expression of glucose transporter 3 in cutaneous squamous cell carcinoma and its effect on the proliferation, invasion and migration of A431 cells / 中华皮肤科杂志

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.

Expression of miR-4783-3p in hepatocellular carcinoma tissue and its effect on the proliferation and migration of hepatocellular carcinoma cells / 国际外科学杂志

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.

The role of B3GNT3 as an oncogene in the growth, invasion and migration of esophageal cancer cells

Purpose: To investigate the role and mechanism of ß1,3-N-acetylglucosaminyltransferase-3 gene (B3GNT3) in esophageal cancer (ESCA). Methods: The starBase database was used to evaluate the expression of B3GNT3. B3GNT3 function was measured using KYSE-30 and KYSE-410 cells of esophageal squamous cell carcinoma (ESCC) cell lines. The mRNA levels were detected by quantitative real-time polymerase chain reaction (qRT-PCR). Cell counting kit-8, clone formation assay and transwell assay were used to detect the changes of proliferation, invasion and migration. Results: B3GNT3 expression was higher in ESCA tissues than in normal tissues. The overall survival rate of ESCA patients with high B3GNT3 expression was lower than that of ESCA patients with low B3GNT3 expression. In vitro functional experiments showed that the proliferation ability, migration and invasion ability of KYSE-30 and KYSE-410 cells with B3GNT3 interference were lower than those of the control, and the overexpression of B3GNT3 had the opposite effect. After silencing B3GNT3 expression in ESCC cell lines, the growth of both cell lines was inhibited and the invasiveness was decreased. Knockdown of B3GNT3 reduced the growth rate and Ki-67 expression level. Conclusion: B3GNT3, as an oncogene, may promote the growth, invasion and migration of ESCC cell.

miRNA-522-3p expression in gastric cancer and the effect of its targeted inhibition of RSU1 expression on the biological function of gastric cancer cells / 肿瘤研究与临床

Objective:To investigate the expression of miRNA-522-3p (miR-522-3p) in gastric cancer tissues/cells and its regulation of RSU1 expression and to analyze the effect of miR-522-3p on biological function of gastric cancer cells in vitro.Methods:miR-522-3p relative expression levels in tumor tissues and adjacent tissues of 50 patients clinically diagnosed as gastric cancer in Shanxi Bethune Hospital from May 2019 to June 2019 were detected by using real-time quantitative polymerase chain reaction (qRT-PCR). MGC-803 cells and BGC-823 cells in gastric cancer were divided into miR-522-3p inhibitor group (transfected with miR-522-3p inhibitor) and empty vector group (transfected with empty vector). The cell proliferation was detected by using CCK-8 assay, cell scratch assay was used to detect the scratch healing ability of cells, and flow cytometry was used to detect the apoptosis. The target correlation between miR-522-3p and RSU1 was detected by using double luciferase reporter gene assay, the change of RSU1 protein was detected by using Western blot.Results:qRT-PCR showed that compared with adjacent cancer tissues, the relative expression level of miR-522-3p was up-regulated, and the difference was statistically significant (0.84±0.31 vs. 0.48±0.22, t = 2.93, P < 0.05). There were no statistically significant differences in the relative expression level of miR-522-3p in gastric cancer tissues with different tumor diameter and pathological grade (all P < 0.05). In vitro experimental assay showed that the cell proliferation rates of MGC-803 and BGC-823 cells in miR-522-3p inhibitor group at 48 h and 72 h were decreased (all P < 0.05). Furthermore, MGC-803 and BGC-823 tranfected with miR-522-3p inhibitor could effectively inhibit the scratch healing ability of MGC-803 and BGC-823 cells. Dual-luciferase reporter gene assay verified that miR-522-3p targeted to RSU1 3'UTR and affected its fluorescence activity. Western blot results showed that miR-522-3p could promote RSU1 protein expression. Conclusions:miR-522-3p is involved in the progression of gastric cancer probably via the regulation of RSU1 expression, and it may be a potential therapeutic target.

lncRNA CALCOCO1 inhibits the proliferation and migration of bladder cancer cells by regulating miR-200a-3p / 国际外科学杂志

Objective:To investigate the expression of long non-coding RNA (lncRNA) CALCOCO1 in bladder cancer tissue and its effect on the proliferation and migration of bladder cancer cells by regulating miR-200a-3p.Methods:The relative expression levels of CALCOCO1 in bladder cancer tissues and adjacent tissues were analyzed by TCGA database. Human bladder cancer cells UM-UC-3 were selected, and the cells were divided into negative control group and CALCOCO1 group, and NC plasmid and CALCOCO1 plasmid were transfected into UM-UC-3 cells respectively. The expression level of CALCOCO1 in each group was detected by quantitative real-time polymerase chain reaction (qRT-PCR). The proliferation and migration ability of UM-UC-3 cells were detected by MTT assay and Transwell migration assay. Bioinformatics technology was used to predict and dual-luciferase reporter gene experiments to verify the targeting relationship between CALCOCO1 and miR-200a-3p. The expression levels of miR-200a-3p in UM-UC-3 cells in each group were detected by qRT-PCR. Western blotting was used to detect the expression of UM-UC-3 cells proliferation and migration phenotype in each group. Measurement data were expressed as mean ± standard deviation ( ± s), t-test was used for comparison between two groups, and repeated measurement analysis of variance was used for comparison at different time. Results:Compared with adjacent tissues, the relative expression level of CALCOCO1 in bladder cancer tissues was significantly lower, the difference was statistically significant( P<0.01). The relative expression of CALCOCO1 in UM-UC-3 cells in CALCOCO1 group and negative control group was 9.66±2.51 and 1.07±0.59, respectively. The relative expression level of CALCOCO1 in CALCOCO1 group was significantly higher than that in negative control group, the difference was statistically significant ( P<0.01). Compared with the negative control group, the proliferation activity of UM-UC-3 cells in the CALCOCO1 group was decreased ( P<0.05), and the migration number of UM-UC-3 cells was significantly decreased ( P<0.01). CALCOCO1 had a binding site with miR-200a-3p ( P<0.01). The relative expression of miR-200a-3p in UM-UC-3 cells in CALCOCO1 group and negative control group was 1.02 ± 0.31 and 5.79 ± 1.68, respectively, the difference was statistically significant ( P<0.01). Compared with the negative control group, the expression levels of proliferation phenotype proteins CCNB1, CCNE1 and CCND2 in UM-UC-3 cells in CALCOCO1 group decreased, and the expression levels of migration phenotype proteins FOXC2 and Fibronectin decreased. Conclusion:The expression of CALCOCO1 is down-regulated in bladder cancer tissue, promoting the expression of CALCOCO1 can inhibit the proliferation and migration of bladder cancer UM-UC-3 cells through targeted down-regulation of miR-200a-3p expression.

Expression of miR-128-3p in gastric cancer and its effect on migration and invasion of gastric cancer cells / 中华普通外科杂志

Objective:To study the effects of miR-128-3p on the migration and invasion of the gastric cancer cells.Methods:qRT-PCR was used to detect the expression of miR-128-3p in 126 gastric cancer tissues and adjacent tissues from Jan 2014 to Jan 2016 at He'nan Cancer Hospital. The effect of miR-128-3p on the invasion and migration of gastric cancer cell line was detected.The expression of miR-128-3p related proteins was detected by Western blotting, miRNA on-line target prediction tool for the prediction of miR-128-3p directly regulated downstream target genes.Results:the expression of miR-128-3p in gastric cancer was significantly higher than that in adjacent non-tumor tissues ( P<0.05). The expression of miR-128-3p was correlated with the vascular tumor thrombus, pN staging and pTNM staging, the prognosis of patients with high expression of miR-128-3p was poor (all P<0.05). MiR-128-3p expression was significantly higher in gastric cancer cell lines ( P<0.05). Online target prediction tool and double luciferase reporter gene showed that CLDN18 was a downstream target gene directly regulated by mir-128-3p. Conclusion:The high expression of miR-128-3p is related to the poor prognosis of gastric cancer patients.

Role of long non-coding RNA 068 in the migration of melanoma cells / 中华皮肤科杂志

Objective:To investigate the effect of long non-coding RNA 068 (lncRNA 068) on the migration of a melanoma cell line A375, and to explore its mechanism of action.Methods:From December 2015 to November 2020, 21 patients with pathologically confirmed cutaneous melanoma were collected from Department of Dermatology, Affiliated Hospital of Nantong University, and quantitative PCR (qPCR) was performed to determine the expression of lncRNA 068 in melanoma and paracancerous tissues. LncRNA 068 was overexpressed or knocked down via lentiviral transfection in A375 human melanoma cells in the following experiments. Specifically, A375 cells were divided into lentiviral vector (LV) -green fluorescent protein (GFP) group and LV-lncRNA 068 group to be transfected with a GFP-expressing LV and a LV containing lncRNA 068 respectively in the overexpression experiment, and were divided into LV-LacZ short hairpin RNA (shRNA) group and LV-lncRNA 068 shRNA group to be transfected with a LV containing the reporter gene LacZ-specific shRNA and a LV containing the lncRNA 068-targeting shRNA respectively in the low-expression experiment, with the LV-GFP group and LV-LacZ shRNA group serving as the control groups. Transwell and scratch assays were performed to evaluate cell migration, EdU cell proliferation assay and cell counting kit-8 (CCK8) assay to determine the proportion of proliferative cells and cell viability respectively, and immunofluorescence staining was conducted to evaluate epithelial-mesenchymal transformation in the above groups. Lentivirus-transfected A375 cells from the above groups were inoculated into the axillae of BALB/c nude mice, and tumor volume was measured and calculated every 3 days. After 30 days, all mice were sacrificed, and tumor tissues were resected to measure the tumor volume and weight. Cultured B16F10 cells were subcutaneously inoculated into the back and foot of BALB/c nude mice to construct mouse models of subcutaneously transplanted B16F10 melanoma. After 2 weeks, the mice were sacrificed, and qPCR and Western blot analysis were performed to determine the mRNA expression of inflammatory factors in transplanted B16F10 melanoma and paracancerous tissues, and expression of IκB kinase (IKK) /P65 signaling pathway-related proteins, respectively. Comparisons between 2 groups were done by t test, and comparisons of tumor volume and weight at different time points among groups were done by repeated measures analysis of variance. Results:qPCR showed that the relative expression of lncRNA 068 was significantly lower in human melanoma tissues and transplanted B16F10 murine melanoma tissues (0.414 ± 0.109, 0.717 ± 0.041, respectively) than in the corresponding paracancerous tissues (1.050 ± 0.103, 1.011 ± 0.023, t = 19.48, 10.83, respectively, both P < 0.001). Transwell and scratch assays both showed that the cellular migratory ability was significantly lower in the LV-lncRNA 068 group than in the LV-GFP group (both P < 0.01), and significantly higher in the LV-lncRNA 068 shRNA group than in the LV-LacZ shRNA group (both P < 0.05). Immunofluorescence assay showed significantly increased fluorescence intensity of E-cadherin and decreased fluorescence intensity of N-cadherin in the LV-lncRNA 068 group compared with the LV-GFP group (both P < 0.001), but significantly decreased fluorescence intensity of E-cadherin and increased fluorescence intensity of N-cadherin in the LV-lncRNA 068 shRNA group compared with the LV-LacZ shRNA group (both P < 0.05). In vivo tumor formation experiment in nude mice showed that there were no significant differences in the volume or weight of melanoma between the LV-lncRNA 068 group and LV-GFP group (both P > 0.05), as well as between the LV-lncRNA 068 shRNA group and LV-LacZ shRNA group (both P > 0.05). As qPCR and Western blot analysis showed, the mRNA and protein expression of interleukin-10 (IL-10) and claudin-1 in A375 cells were significantly higher in the LV-lncRNA 068 group than in the LV-GFP group (both P < 0.05), but significantly lower in the LV-lncRNA 068 shRNA group than in the LV-LacZ shRNA group (both P < 0.05). Compared with the paracancerous tissues, B16F10 melanoma tissues showed significantly decreased mRNA expression of IL-10 ( P < 0.01), but significantly increased mRNA expression of IL-6 and tumor necrosis factor-α, as well as protein expression of phosphorylated P65 and phosphorylated IKK ( P < 0.01) . Conclusion:Overexpression of lncRNA 068 can inhibit the migration of A375 melanoma cells, and may affect the development of inflammation and inhibit the epithelial-mesenchymal transformation by inhibiting the IKK/P65 signaling pathway.

Mechanism of silencing miR-4320 expression in inhibiting proliferation and migration of gastric cancer cells / 国际外科学杂志

Objective:To explore the effect and mechanism of microRNA (miRNA)-4320 on the proliferation and migration of gastric cancer MGC803 cells.Methods:Real-time quantitative polymerase chain reaction (qRT-PCR) was used to detect the expression of miR-4320 in four gastric cancer cell lines(MGC803, HS-746T, SGC7901, BGC823) MGC803 cells were infected with recombinant lentivirus carrying miR-4320 interference fragments or blank lentivirus, and set as si-miR-4320 group and NC group. Thiazole blue colorimetry and Transwell small box experiment were used to detect the proliferation and migration of MGC803 cells after miR-4320 was down-regulated. The bioinformatics software RNAhybrid was used to predict the target gene of miR-4320. The targeting relationship between miR-4320 and target gene was verified by dual-luciferase reporter gene experiment. qRT-PCR and Western blot were used to detect the expression of miR-4320 target gene. Measurement data were expressed as mean ± standard deviation ( ± s), and t-test or one-way ANOVA was used for comparison between groups. Results:The expression of miR-4320 in the four gastric cancer cell lines was significantly higher than that of normal gastric mucosal epithelial cells ( P<0.01). The expression of miR-4320 in MGC803 cells in the NC group and the si-miR-4320 group were 8.19±1.00 and 1.09±0.31, respectively. The miR-4320 interference fragment significantly reduced the expression of miR-4320 ( P<0.01). The absorbance of MGC803 cells in the si-miR-4320 group was significantly lower than that of the NC group ( P<0.05), and the migration ability was significantly lower than that of the NC group ( P<0.01). Suppressor of cytokine signaling1 ( SOCSI) is the target gene of miR-4320. Compared with the NC group, the SOCS1 gene expression in the si-miR-4320 group was significantly up-regulated ( P<0.01). Conclusions:The expression of miR-4320 is increased in gastric cancer cell lines. Down-regulating the expression of miR-4320 can inhibit the proliferation and migration of gastric cancer MGC803 cells by inducing the expression of SOCS1 gene.

[Chronic toxicity of Multi-walled carbon nanotubes in human pleural mesothelial cells].

Objective: To explore the chronic toxicity and its potential mechanism of multi-walled carbon nanotube (MWCNT) in human pleural mesothelial cells. Methods: A sustainable exposure of MeT-5A cells to MWCNT at 10 µg/cm(2) for one year was conducted in 2016. During the exposure, the cell images and cell proliferation was recorded every 4 weeks. The cell apoptosis, cell cycle, cell migration and cell invasion were compared between the control cells and the cells after MWCNT exposure. Finally, the gene expression was screened with Affymetrix clariom D assay, and some of the significantly differential expressed genes was verified by RT-PCR. Results: Compared with the control group, the proliferation ability of the cells in the 1-year exposed group was significantly increased, and the rate of proliferation was about 2-3 times as that in the Control Group (F=481.32, P<0.05) . MeT-5A cells all showed cell cycle arrest effect, which showed the increase of G1 phase and the decrease of s phase and G2 phase (F=14.94, P<0.05) . The apoptosis rate of cells in the treated group was significantly higher than that in the control group after 6 months (F=15.12, P<0.05) , but the early apoptosis rate and the total apoptosis rate of cells in the treated group were not significantly different from those in the control group after 1 year (F=3.97, P<0.05) . The cell migration and invasion were both promoted by MWCNT. Furthermore, the differentially expressed genes was screened, to find 2, 878 genes with more than 2 folds changes. To further verified, RT-PCR was conducted with PIK3R3、WNT2B、VANGL2、ANXA1, and their expression changes were consistent with above. Conclusion: MWCNT might have a carcinogenic potential to MeT-5A cells after the long term exposure.

[Effects of B-cell lymphoma-2/adenovirus E1B 19 000 interacting protein 3 on the migration and motility of human dermal microvascular endothelial cells under hypoxia and the mechanism].

Objective: To explore the effects of B-cell lymphoma-2/adenovirus E1B 19 000 interacting protein 3 (BNIP3) on the migration and motility of human dermal microvascular endothelial cells (HDMECs) under hypoxia and the mechanism. Methods: The experimental research method was applied. (1) HDMECs were divided into normoxia group received routine culture and hypoxia 6, 12, 24 h groups treated under hypoxia with oxygen volume fraction of 2% for corresponding time according to the random number table (the same grouping method below). Western blotting was used to detect the protein expressions of BNIP3 and microtubule-associated protein 1 light chain 3Ⅱ (LC3Ⅱ) in HDMECs. (2) HDMECs were divided into normoxia+ unloaded group, normoxia+ BNIP3 knockdown group, hypoxia+ unloaded group, and hypoxia+ BNIP3 knockdown group which were transfected with unloaded virus or BNIP3 knockdown virus and were subjected to normoxic or hypoxic treatment. The BNIP3 protein expression was detected by Western blotting and immunofluorescence staining. The scratch area at 24 h post scratching was detected by scratch test, and the healing rate of scratch was calculated. The curve distance of cell movement was measured with the living cell workstation, and the speed of movement was calculated within 3 hours. (3) HDMECs were grouped and treated as experiment (2). Western blotting and immunofluorescence staining were performed to detect the protein expression of LC3Ⅱ. The number of sample was 3 in the above-mentioned experiments. Data were statistically analyzed with one-way analysis of variance and least significant difference test. Results: (1) Compared with those of normoxia group, the protein expressions of BNIP3 and LC3Ⅱ of cells in hypoxia 6, 12, 24 h groups were significantly increased (P<0.01). (2) After 6 hours of culture, compared with that of hypoxia+ unloaded group, the BNIP3 protein expressions of cells in normoxia+ unloaded group and hypoxia+ BNIP3 knockdown group were significantly decreased (P<0.05 or P<0.01). The red fluorescence denoting BNIP3 protein expression of cells in normoxia+ unloaded group and normoxia+ BNIP3 knockdown group was weak, the red fluorescence of cells in hypoxia+ unloaded group was strong, and the red fluorescence of cells in hypoxia+ BNIP3 knockdown group was significantly decreased compared with that in hypoxia+ unloaded group. After scratching for 24 hours, the scratch of cells in hypoxia+ unloaded group basically healed, while the remaining scratch area in the other three groups were large. The healing rates of scratch of cells in normoxia+ unloaded group, normoxia+ BNIP3 knockdown group, hypoxia+ unloaded group, and hypoxia+ BNIP3 knockdown group were (61±4)%, (58±4)%, (88±4)%, and (57±4)%, respectively. The healing rate of scratch of cells in hypoxia+ unloaded group was significantly higher than that in normoxia+ unloaded group (P<0.01) and hypoxia+ BNIP3 knockdown group (P<0.05). Within 3 hours of observation, the range of cell movement in hypoxia+ unloaded group was significantly larger than that in normoxia+ unloaded group, the range of cell movement in hypoxia+ BNIP3 knockdown group was significantly smaller than that in hypoxia+ unloaded group, and the curve movement velocity of cells in hypoxia+ unloaded group was significantly higher than that in normoxia+ unloaded group and hypoxia+ BNIP3 knockdown group (P<0.01). (3) After 6 hours of culture, compared with hypoxia+ unloaded group, the LC3Ⅱ protein expressions of cells in hypoxia+ unloaded group and hypoxia+ BNIP3 knockdown group were decreased significantly (P<0.05 or P<0.01). After 6 hours of culture, the red fluorescence denoting LC3 protein expressions of cells was weak in normoxia+ unloaded group and normoxia+ BNIP3 knockdown group, the red fluorescence of cells was significantly enhanced in hypoxia+ unloaded group, and the red fluorescence of cells was significantly inhibited in hypoxia+ BNIP3 knockdown group. Conclusions: BNIP3 can promote the migration and motility of HDMECs under hypoxia, and autophagy may be involved in the regulation migration of HDMECs by BNIP3.

Effects of microRNA-26a targeted high-mobility group A2 protein antibody on the proliferation and migration of hepatoma cells / 中国基层医药

Objective:To investigate the effects of miRNA-26a (miR-26a) on the target gene HMGA2 on the proliferation and migration of hepatoma cells and the underlying mechanism. Methods:Liver cancer tissue samples ( n = 30) and adjacent normal tissue samples ( n = 30) pathologically confirmed by Wenzhou Hospital of Traditional Chinese Medicine between September 2018 and September 2019 were collected. MiR-26a mimics, control mimics (miR-Control), high-mobility group A2 protein (HMGA2) siRNA or negative control siRNA (Control) were transfected into human hepatoma cell lines HepG2 or Huh-7 cells. The expression of miR-26a in hepatocellular carcinoma tissue was detected by reverse transcription quantitative polymerase chain reaction (RT-qPCR). MTT assay and scratch test were performed to determine the ability of cell proliferation and migration. RT-qPCR and western blotting were performed to detect miR-26a and HMGA2 mRNA expression. The relationship between miR-26a and HMGA2 mRNA was analyzed using Bioinformatics and luciferase reporter gene assay. Results:RT-qPCR results showed that the expression level of miR-26a in hepatocellular carcinoma tissue was 0.11 ± 0.02, which was significantly lower than that in normal tissues (0.25 ± 0.03, t = 21.268, P < 0.05). The expression level of miR-26a in stage III + IV was 0.05 ± 0.01, which was significantly lower than that in stage I + II (0.09 ± 0.01, t = 15.491, P < 0.05). Cell experiment showed that in the miR-26a group, the proliferation ability of Huh-7 cells was (3.10 ± 0.30) and (4.10 ± 0.40), and the proliferation ability of HepG2 cells was (3.08 ± 0.31) and (4.11 ± 0.40), which was significantly lower than that in the control group [(3.90 ± 0.40), (5.50 ± 0.60), (3.92 ± 0.41), (5.49 ± 0.58), t = 8.764, 10.634, 11.148, 10.728, all P < 0.05]. In the miR-26a group, the migration ability was (0.50 ± 0.06), (0.65 ± 0.07), which was significantly lower than that in the control group [(1.00 ± 0.10), (0.96 ± 0.10), t = 23.483, 13.910, both P < 0.05]. Bioinformatics and in vitro experiments showed that HMGA2 was a direct target of miR-26a. Restoring the expression of HMGA2 in miR-26a mimics-transfected cells, compared with that in the miR-26a group [(0.24 ± 0.02), (0.31 ± 0.03);(0.45 ± 0.05)], could significantly reverse the inhibitory effect of miR-26a on tumor cell proliferation and migration [(0.31 ± 0.03), (0.40 ± 0.04);(0.93 ± 0.08), t = 10.634, 9.859, 27.868, all P < 0.05). Conclusion:miR-26a inhibits the proliferation and migration of hepatoma cells by directly targeting HMGA2. The abnormal decrease of miR-26a and the increase of HMGA2 may be the important factors that participate in the occurrence and development of liver cancer.

[Effects and mechanism of norepinephrine on the migration of bone marrow mesenchymal stem cells in mice].

Objective: To investigate the effects and mechanism of sympathetic neurotransmitter norepinephrine (NE) on the migration of bone marrow mesenchymal stem cells (BMSCs) in mice. Methods: (1) Twenty 3-week-old male C57BL/6 mice were sacrificed for isolating, culturing, and identifying BMSCs from the femur and tibia. Cells of the second or third passages were divided into phosphate buffer solution (PBS) group, 1 µmol/L NE group, 10 µmol/L NE group, and 100 µmol/L NE group, with 8 wells in each group. Cells in 1 µmol/L NE group, 10 µmol/L NE group, and 100 µmol/L NE group were cultured in low-sugar Dulbecco's modified eagle medium containing 1% volume fraction of fetal bovine serum (hereinafter referred to as low-serum medium) added with NE in final molarity of 1 µmol/L, 10 µmol/L, and 100 µmol/L, respectively. Cells in PBS group were cultured in low-serum medium added with the same volume of PBS. Before stimulation (0 d) and on stimulation day 1, 3, 5, cell counting kit 8 method was used to detect cell proliferation activity (expressed as the absorbance value). (2) In cell scratch test 1, cells were divided into PBS group and simple NE group. After the scratch test, cells in simple NE group were cultured with low-serum medium+ NE in final molarity of 10 µmol/L, and cells in PBS group were cultured with low-serum medium+ the same volume of PBS. In cell scratch test 2, cells were divided into PBS group, propranolol+ NE group, and phentolamine+ NE group. After the scratch test, cells in propranolol+ NE group were pretreated with low-serum medium+ propranolol in final molarity of 1 µmol/L for 30 minutes each day, cells in phentolamine+ NE group were pretreated with low-serum medium+ phentolamine in final molarity of 10 µmol/L for 30 minutes each day, and then they were cultured with low-serum medium+ NE in final molarity of 10 µmol/L. Cells in PBS group were cultured with low-serum medium+ the same volume of PBS. In cell scratch test 3, cells were divided into simple NE group, simple (2E, 6E)-2, 6-bis (4-pyridylmethylene) cyclohexanone (SC-66) group, and SC-66+ NE group. After the scratch test, cells in simple NE group was cultured with low-serum medium+ NE in final molarity of 10 µmol/L, cells in simple SC-66 group were cultured with low-serum medium after being pretreated with SC-66 in final molarity of 30 mmol/L for 30 minutes every day, cells in SC-66+ NE group were cultured with low-serum medium+ NE in final molarity of 10 µmol/L after being pretreated with SC-66 in final molarity of 30 mmol/L for 30 minutes every day. In the above 3 cell scratch tests, the sample numbers in each group were all 6, and the scratch healing rates at post scratch hour (PSH) 24, 48, and 72 were all calculated. (3) Cells were divided into PBS group, simple NE group, propranolol+ NE group, and phentolamine+ NE group, with 3 wells in each group. The lower chamber treatment methods of PBS group and simple NE group were the same as those of the same groups in cell scratch test 1. The lower chamber treatment of propranolol+ NE group and phentolamine+ NE group were the same as those of the same groups in cell scratch test 2. After the Transwell experiment was performed and the cells were routinely cultured for 24 hours, the migrated cells were counted. (4) Cells were divided into PBS group, simple NE group, propranolol+ NE group, and phentolamine+ NE group, with 2 dishes in each group. The cell treatment of PBS group and simple NE group were the same as those of the same groups in cell scratch test 1. The cell treatment of propranolol+ NE group and phentolamine+ NE group were the same as those of the same groups in cell scratch test 2. After 24 hours of routine culture, the phosphorylation level of protein kinase B (Akt) of cells was detected by Western blotting. Data were statistically analyzed with analysis of variance for repeated measurement, one-way analysis of variance, independent sample t test, least significant difference t test, and Bonferroni correction. Results: (1) After 1 day of stimulation, the absorbance value of cells in 100 µmol/L NE group was significantly lower than that in PBS group (t=2.986, P<0.05). After 5 days of stimulation, the absorbance value of cells in 10 µmol/L NE group was significantly higher than that in PBS group (t=3.547, P<0.01). (2) In cell scratch test 1, at PSH 24, 48, and 72, the scratch healing rates of cells in simple NE group were (34.4±3.4)%, (52.5±4.7)%, and (70.0±3.8)%, which were significantly lower than (44.1±4.2)%, (80.0±3.6)%, and (95.9±2.2)% in PBS group (t=19.320, 128.319, 221.575, P<0.01). In cell scratch test 2, at PSH 24, 48, and 72, the scratch healing rates of cells in propranolol+ NE group were significantly lower than those in PBS group (t=4.073, 9.618, 15.272, P<0.01). In cell scratch test 3, at PSH 72, the scratch healing rates of cells in NE group was significantly lower than that in simple SC-66 group (t=8.862, P<0.01). At PSH 24, 48, and 72, the scratch healing rates of cells in SC-66+ NE group were significantly lower than those in simple SC-66 group (t=3.862, 4.290, 10.357, P<0.01). (3) The Transwell experiment showed that after 24 hours of culture, the numbers of migrated cells in simple NE group, propranolol+ NE group, and phentolamine+ NE group were significantly less than the number in PBS group (t=11.895, 10.196, 3.222, P<0.01). (4) After 24 hours of culture, the phosphorylation levels of Akt of cells in simple NE group and propranolol+ NE group were significantly higher than the level in PBS group (t=8.186, 5.996, P<0.01). Conclusions: NE can inhibit the migration of BMSCs in mice, a process in which the signal pathway of Akt is involved in its regulation.

Razor scrape assay, an alternative variation to wound and healing assays.

Cell migration is the process by which cells move through tissues, and it is crucial to carry out a wide variety of physiological and pathological processes. The study methods to evaluate cell migration are very useful tools for biomedical research. Among these methods, the wound and healing assay is one of the simplest, most economical and is widely used in research. However, one of its disadvantages is that the width and shape of the wound can vary among experimental samples since the scraping is carried out manually, representing a difficult variable to control. In the present article a variant of the razor scrape assay is addressed, which eliminates this variation in the width of the wound, thus facilitating the measurement and comparison using the total area of cell migration.•A method that can be carried out under standard culture conditions.•Avoids the disadvantage of variation in width and shape of the wound.•It constitutes a simple, cheap option and multiple advantages over the traditional method.

[Mechanism of transcriptional regulation of Meox1 by transforming growth factor ß (1) and its effect on cell migration of adult human dermal fibroblasts].

Objective: To explore the transcriptional regulation mechanism of transforming growth factor ß(1) (TGF-ß(1)) on Meox1 and its effect on cell migration of adult human dermal fibroblasts (HDF-a). Methods: (1) HDF-a cells were cultured in RPMI 1640 complete medium (hereinafter referred to as routinely cultured). The cells were divided into TGF-ß(1) stimulation group and blank control group. The cells in TGF-ß(1) stimulation group were stimulated with 10 µL TGF-ß(1) in the mass concentration of 1 mg/µL, while the cells in blank control group were stimulated with the equal volume of phosphate buffer solution. After 72 hours in culture, partial cells in both groups were collected for transcriptome sequencing. The genes with differential expression ratio greater than or equal to 2 and P<0.01 between the two groups were selected to perform enrichment analysis and analysis of metabolic pathways of the Kyoto Gene and Genome Encyclopedia with, and the expression value of Meox1 per million transcripts (TPM) was recorded (n=3). Partial cells from the two groups were used to detect the Meox1 mRNA expression by real-time fluorescent quantitative reverse transcription polymerase chain reaction (RT-PCR) (n=3). (2) Cultured HDF-a cells in the logarithmic growth phase (the same growth phase of cells below) were divided into empty plasmid group, Smad2 overexpression (OE) group, Smad3 OE group, and Smad4 OE group, which were transfected respectively with 2 µg empty pcDNA3.1 plasmid and pcDNA3.1 plasmids separately carrying Smad2, Smad3, and Smad4 for 6 hours, and then were routinely cultured for 48 hours. The Meox1 mRNA expression in the transfected cells of each group was detected by real-time fluorescent quantitative RT-PCR (n=3). (3) HDF-a cells were routinely cultured and grouped the same as in experiment (1). After 72 hours in culture, the enrichment of Smad2, Smad3, and Smad4 protein on the Meox1 promoter in the cells of each group was detected by chromatin immunoprecipitation-quantitative PCR (ChIP-qPCR) (n=3). (4) HDF-a cells were routinely cultured and divided into negative interference group, small interference RNA (siRNA)-Smad2 group, siRNA-Smad3 group, siRNA-Smad4 group, empty plasmid group, Smad2 OE group, Smad3 OE group, and Smad4 OE group, which were transfected respectively with 50 µmol/L random siRNA, siRNA-Smad2, siRNA-Smad3, siRNA-Smad4, 2 µg empty pcDNA3.1 plasmid and pcDNA3.1 plasmids separately carrying Smad2, Smad3, and Smad4 for 6 hours and then routinely cultured for 48 hours. The enrichment of Smad2, Smad3, and Smad4 protein on the Meox1 promoter in the cells of corresponding group was detected by ChIP-qPCR (n=3). (5) Two batches of HDF-a cells were cultured and divided into negative interference group, siRNA-Meox1 group, empty plasmid group, and Meox1 OE group, which were transfected respectively with 50 µmol/L random siRNA, siRNA-Meox1, 2 µg empty pcDNA3.1 plasmid and pcDNA3.1 plasmid carrying Meox1 for 6 hours and then routinely cultured for 24 hours. One batch of cells were subjected to scratch test with the scratch width being observed 24 hours after scratching and compared with the initial width for scratch wound healing; the other batch of cells were subjected to Transwell assay, in which the migrated cells were counted after being routinely cultured for 24 hours (n=3). (6) From January 2018 to June 2019, 3 hypertrophic scar patients (2 males and 1 female, aged 35-56 years) were admitted to the First Affiliated Hospital of Army Medical University (the Third Military Medical University) 8-12 months after burns. The scar tissue and normal skin tissue along the scar margin resected during surgery were taken, and immunohistochemical staining was performed to observe the distribution of Meox1 protein expression. Data were statistically analyzed with one-way analysis of variance and independent sample t test. Results: (1) After 72 hours in culture, a total of 843 genes were obviously differentially expressed between the two groups, being related to tissue repair, cell migration, inflammatory cell chemotaxis induction process and potential signaling pathways such as tumor necrosis factor, interleukin 17, extracellular matrix receptor. The TPM value of Meox1 in the cells of blank control group was 45.9±1.9, which was significantly lower than 163.1±29.5 of TGF-ß(1) stimulation group (t=6.88, P<0.01) with RNA-sequencing. After 72 hours in culture, the Meox1 mRNA expression levels in the cells of blank control group was 1.00±0.21, which was significantly lower than 11.00±3.61 of TGF-ß(1) stimulation group (t=4.79, P<0.01). (2) After 48 hours in culture, the Meox1 mRNA expression levels in the cells of Smad2 OE group, Smad3 OE group, and Smad4 OE group were 198.70±11.02, 35.47±4.30, 20.27±2.50, respectively, which were significantly higher than 1.03±0.19 of empty plasmid group (t=31.07, 13.80, 13.12, P<0.01). (3) After 72 hours in culture, the enrichment of Smad2, Smad3, and Smad4 protein on the promoter of Meox1 in the cells of TGF-ß(1) stimulation group was significantly higher than that of blank control group respectively (t=12.99, 41.47, 29.10, P<0.01). (4) After 48 hours in culture, the enrichment of Smad2 protein on the promoter of Meox1 in the cells of negative interference group was (0.200 000±0.030 000)%, significantly higher than (0.000 770±0.000 013)% of siRNA-Smad2 group (t=11.67, P<0.01); the enrichment of Smad2 protein on the promoter of Meox1 in the cells of empty plasmid group was (0.200 000±0.040 000)%, significantly lower than (0.700 000±0.090 000)% of Smad2 OE group (t=8.85, P<0.01). The enrichment of Smad3 protein on the promoter of Meox1 in the cells of negative interference group was (0.500 0±0.041 3)%, significantly higher than (0.006 0±0.001 3)% of siRNA-Smad3 group (t=17.79, P<0.01); the enrichment of Smad3 protein on the promoter of Meox1 in the cells of empty plasmid group was (0.470 0±0.080 0)%, which was significantly lower than (1.100 0±0.070 0)% of Smad3 OE group (t=9.93, P<0.01). The enrichment of Smad4 protein on the promoter of Meox1 in the cells of negative interference group was similar to that of siRNA-Smad4 group (t=2.11, P>0.05); the enrichment of Smad4 protein on the promoter of Meox1 in the cells of empty plasmid group was similar to that of Smad4 OE group (t=0.60, P>0.05). (5) Twenty-four hours after scratching, the scratch healing width of cells in siRNA-Meox1 group was narrower than that of negative interference group, while that of Meox1 OE group was wider than that of empty plasmid group. After 24 hours in culture, the number of migration cells in negative interference group was significantly higher than that in siRNA-Meox1 group (t=9.12, P<0.01), and that in empty plasmid group was significantly lower than that in Meox1 OE group (t=8.99, P<0.01). (6) The expression of Meox1 protein in the scar tissue was significantly higher than that in normal skin of patients with hypertrophic scars. Conclusions: TGF-ß(1) transcriptionally regulates Meox1 expression via Smad2/3 in HDF-a cells, thus promoting cell migration.

Assessment of the Photobiomodulation Effect of a Blue Diode Laser on the Proliferation and Migration of Cultured Human Gingival Fibroblast Cells: A Preliminary In Vitro Study.

Introduction: Photobiomodulation therapy (PBM) is emerging as an effective strategy for the management of wound healing. The application of red and near infra-red light sources in laser therapy has been the subject of most researches in recent literature. Considering the lack of sufficient evidence in assessing the blue light in PBM, we aimed to investigate the photobiomodulation effect of a blue diode laser on the proliferation and migration of cultured human gingival fibroblast cells as a preliminary in vitro study. Methods: Human gingival fibroblast cells were irradiated with a blue diode laser at a 445 nm wavelength. Irradiation was done using three different powers of 200 mW (irradiation times of 5, 10,15, and 20 seconds); 300 mW (irradiation times of 5, 10, and 15 seconds); and 400 mW (irradiation times of 5 and 10 seconds). The fibroblast cells without laser exposure were considered as control. After 24 hours of incubation, the MTT assay and the wound scratch test were performed on the cells to investigate the biomodulation effect of the blue laser on the proliferation and migration of the cells respectively. The results were analyzed by one-way ANOVA and a post-hoc Tukey test with a P value <0.05 as a statistical significance level. Results: PBM with blue diode laser at power densities of 400 mW/cm2 with irradiation times of 10 and 15 seconds corresponding to energy densities of 4 and 6 J/cm2 exerted the statistically significant positive effect on both proliferation and migration of gingival fibroblast cells. Conclusion: Considering the encouraging findings of this study, PBM with blue diode laser can promote proliferation and migration of human gingival fibroblasts, the key cells involved in the process of oral wound healing.

Effect of Beclin1 overexpression on biological behaviors of SK-MEL-2 human malignant melanoma cells / 中华皮肤科杂志

Objective@#To evaluate the effect of overexpression of the autophagy marker gene Beclin1 on biological behaviors of SK-MEL-2 human malignant melanoma cells.@*Methods@#Western blot analysis was performed to determine the protein expression of Beclin1 in melanoma cell lines A375 and SK-MEL-2. SK-MEL-2 cells with low Beclin1 protein expression were selected as research objects, and divided into 3 groups: blank group receiving no treatment, negative control group transfected with pcDNA.3.1/myc-His (-) A, and experimental group transfected with pcDNA3.1-Beclin1 plasmid. After 2-week culture, cell counting kit-8 (CCK-8) assay was conducted to evaluate the effect of Beclin1 on cell proliferation at 24, 48 and 72 hours, and Transwell assay and wound-healing assay were performed to assess the effect of Beclin1 overexpression on the invasion and migration abilities of SK-MEL-2 cells. Repeated measures analysis of variance and completely randomized analysis of variance were used to analyze differences in indices among groups, and least significant difference (LSD) -t test was used for multiple comparisons.@*Results@#The protein expression of Beclin1 was significantly lower in the SK-MEL-2 cells (0.037 ± 0.010) than in the A375 cells (0.670 ± 0.150, F = 46.62, P<0.05) . The experimental group showed significantly increased protein expression of Beclin1 (0.32 ± 0.04) compared with the negative control group (0.06 ± 0.02, P < 0.05) and blank group (0.07 ± 0.02, P < 0.05) . CCK-8 assay revealed a significant difference in the cell proliferation rate among different groups and different time points (F = 1 077.36, 4 903.04 respectively, both P<0.05) , and there was a significant interaction between the transfection treatment and time (F= 205.20, P<0.05) . Transwell assay showed that the number of SK-MEL-2 cells crossing the chamber per high-power field (× 200) after 24-hour treatment was significantly lower in the experimental group (18.67 ± 1.19) than in the negative control group (87.89 ± 6.05, P<0.05) and blank group (86.78 ± 5.93, P<0.05) . In the wound-healing assay, the cell migration distance was significantly shorter in the experimental group than in the blank group and negative control group at 24 and 48 hours (all P < 0.05) .@*Conclusion@#Beclin1 overexpression can markedly inhibit the proliferation, invasion and migration of SK-MEL-2 cells.