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Objective:To determine the expression of glucose transporter 3 (GLUT3) in cutaneous squamous cell carcinoma (cSCC), and to evaluate its effect on the cSCC cell line A431.Methods:From June 2016 to December 2020, 22 paraffin-embedded tissue specimens were collected from patients with pathologically confirmed cSCC in the Department of Dermatology, Peking University Third Hospital, and 20 discarded normal skin tissues after dermatological surgeries served as controls. Immunohistochemical assay was performed to determine the GLUT3 expression in cSCC tissues and normal skin tissues. Cultured A431 cells were divided into two groups: GLUT3 overexpression group transfected with a lentiviral vector carrying the SLC2A3 gene, and negative control group transfected with an empty lentiviral vector. Real-time fluorescence-based quantitative PCR and Western blot analysis were conducted to determine the mRNA and protein expression of GLUT3 in A431 cells in different groups, the cell proliferation assay (MTS assay) was performed to estimate the cell proliferative activity, and the live-cell analysis system Incucyte S3 was used for real-time evaluation of the migratory and invasive abilities of A431 cells in different groups. The relative glucose consumption and lactic acid production in A431 cells at 48 hours were measured by using glucose and lactic acid assay kits, retrospectively. Two independent samples t-test was used for comparisons between two groups, and one-way analysis of variance was used for comparisons among multiple groups. Results:The GLUT3 expression was significantly higher in the cSCC tissues than in the normal skin tissues (immunohistochemical assay score: 9.39 ± 2.56 points vs. 2.30 ± 2.60 points; t = 8.91, P < 0.05). Compared with the negative control group, the mRNA and protein expression of GLUT3 markedly increased in the GLUT3 overexpression group. MTS assay showed significantly increased proliferative activity of A431 cells in the GLUT3 overexpression group compared with the negative control group after 24- and 96-hour treatment ( t = 2.49, 3.54, P = 0.048, 0.012, respectively); cell fusion rates in the scratched area were significantly higher in the GLUT3 overexpression group than in the negative control group in the cell migration assay at 6, 12 18, and 24 hours and cell invasion assay at 12, 18, and 24 hours (all P < 0.05). At 48 hours, the relative glucose consumption and lactic acid production in A431 cells were significantly higher in the GLUT3 overexpression group than in the negative control group ( t = 2.98, 2.20, P = 0.011, 0.038, respectively) . Conclusion:GLUT3 was highly expressed in the cSCC tissues, and may participate in the occurrence and development of cSCC by providing energy to cSCC cells via promoting glucose uptake in cells to enhance their proliferative, migratory and invasive abilities.
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Objective:To study the expression of microRNA (miRNA)-4783-3p in liver cancer tissue and its effect on the proliferation and migration of liver cancer Huh-7 cells.Methods:The cBioPortal database was used to analyze the expression of miR-4783-3p in liver cancer tissues and adjacent tissues. In strict accordance with the instructions of Lipofectamine? 2000 transfection kit, miR-4783-3p overexpression mimics or overexpression control mimics were transfected into Huh-7 cells respectively, namely overexpression group and control group. The proliferation of Huh-7 cells was analyzed by CCK-8 assay, and the migration of Huh-7 cells was analyzed by cell scratch method. The targeting relationship between miR-4783-3p and insulin-like growth factor binding protein 2 ( IGFBP2) mRNA was detected by dual-luciferase reporter gene assay. RT-qPCR was used to detect the expression of IGFBP2 mRNA. Western-blotting was used to detect the expression of IGFBP2 protein and EGFR-STAT3 molecular pathway proteins. Results:The expression of miR-4783-3p in liver cancer tissues was significantly lower than that in adjacent tissues ( P<0.01). Compared with the control group, the proliferation ability of Huh-7 cells in the overexpression group was significantly decreased ( P<0.05). The scratch healing rates of Huh-7 cells in the control group and the overexpression group were (67.71±9.04)% and (29.58±10.51)%, respectively, and the scratch healing rate in the overexpression group was significantly lower ( P<0.01). miR-4783-3p targeted and bound to IGFBP2 mRNA ( P<0.01). The expression of IGFBP2 mRNA in the control and overexpression groups was 5.76±1.44 and 0.99±0.47, respectively, and miR-4783-3p negatively regulated the expression of IGFBP2 mRNA ( P<0.01). Compared with the control group, the expressions of IGFBP2 protein and EGFR-STAT3 molecular pathway protein were decreased in the overexpression group. Conclusions:miR-4783-3p is lowly expressed in liver cancer tissues. miR-4783-3p can attenuate the proliferation and invasion ability of liver cancer Huh-7 cells by inducing the low expression of IGFBP2 gene.
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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.
Subject(s)
Oncogenes , N-Acetylglucosaminyltransferases/analysis , Cell Migration Assays , Transcriptome , Esophageal Squamous Cell Carcinoma , Esophageal Neoplasms/physiopathologyABSTRACT
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.
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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.
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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.
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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.
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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.
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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.
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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.
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Objective To evaluate the effect of overexpression of the autophagy marker gene Beclin I on biological behaviors of SK-MEL-2 human malignant melanoma cells.Methods Western blot analysis was performed to determine the protein expression of Beclin 1 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 Beclin 1 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 Beclin 1 overexpression can markedly inhibit the proliferation,invasion and migration of SK-MEL-2 cells.
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Objective@#To evaluate the effect of water-soluble components of atmospheric fine particulate matter PM2.5 on proliferation, migration, tyrosinase activity and melanin content of a human melanocyte line PIG1.@*Methods@#PM2.5 was collected during haze weather in heating seasons, and processed into suspensions. PIG1 melanocytes were cultured and divided into 5 experimental groups and 1 control group. PIG1 melanocytes in the 5 experimental groups were treated with 10, 20, 50, 100 and 200 mg/L PM2.5 suspensions respectively for 48 hours, while cells in the control group were not treated with PM2.5 suspensions. In cell migration assay, there was only 1 experimental group treated with 10 mg/L PM2.5 suspensions. After treatment, methyl thiazol tetrazolium (MTT) assay, micropore filtration assay, DOPA oxidase assay and NaOH lysis method were performed to determine the cell proliferation rate, migration rate, tyrosinase activity and melanin content respectively. Statistical analysis was carried out by using t test for comparison of means of two samples, one-way analysis of variance for means of multiple samples, Student-Newman-Keuls (SNK) -q test for multiple comparisons, and linear correlation analysis for analysis of correlations.@*Results@#Compared with the control group ([100 ± 1.41]%) , the proliferation rate of PIG1 cells significantly decreased in the 20-, 50-, 100- and 200-mg/L PM2.5 groups ([93.41 ± 2.13]%, [88.31 ± 1.3557]%, [79.75 ± 1.89]%, [69.83 ± 2.50]% respectively, all P < 0.05) . Linear correlation analysis showed that the proliferation rate and tyrosinase activity of PIG1 cells decreased with the increase in PM2.5 concentrations (r = -0.98, -0.93, respectively, both P < 0.01) . After the treatment with 10 mg/L PM2.5, the migration rate of PIG1 cells significantly decreased (66.23% ± 1.11%) compared with the control group ([76.86 ± 1.81]%, t = 7.55, P < 0.01) . With the increase in PM2.5 concentrations (50-200 mg/L) , the melanin content of PIG1 cells gradually decreased (r = -0.97, P < 0.01) .@*Conclusion@#Atmospheric fine particulate matter PM2.5 can affect the normal functions of melanocytes by inhibiting their proliferation and migration, and reducing their tyrosinase activity and melanin content.
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Objective@#To observe content of cytokine in human stromal vascular fraction gel (SVF-GEL) and effect of SVF-GEL on biological behaviors of epidermal and dermal cells in vitro and clinical efficacy of SVF-GEL.@*Methods@#(1) SVF-GEL was prepared using liposuction aspirates harvested from females who received abdomen liposuction in author′s unit. SVF-GEL (1 mL) and high-glucose Dulbecco′s modified eagle medium (DMEM, 1 mL) were respectively cultured for 24 h with high-glucose DMEM containing 10% fetal calf serum, 10 g/L penicillin, and 10 g/L streptomycin, denoted as SVF-GEL group and negative control group, with 6 samples in each group. Content of epidermal growth factor (EGF) and vascular endothelial growth factor (VEGF) in the supernatant was determined by enzyme-linked immunosorbent assay. (2) A number of 5×105 human skin fibroblasts (HSF) and HaCaT cells in logarithmic phase were inoculated and cultured in Transwell chambers for 12 h. All Transwell chambers containing cells were divided into SVF-GEL group (0.5 mL SVF-GEL was added for co-culture) and control group (0.5 mL high-glucose DMEM was added for co-culture), with 9 samples in each group for HSF and HaCaT cells. Scratch assay was performed after culture for 24 h, and residual scratch width was observed at post scratch hour (PSH) 0 (immediately), 24, and 48. Cell migration distance was measured at PSH 24 and 48. After culture for 24, 48, and 72 h, the number of living cell was counted using cell counter. (3) From June 2018 to June 2019, SVF-GEL was applied clinically to treat 15 patients with depressed scars on face, including 2 males and 13 females, aged 19 to 42 years. Survival condition of SVF-GEL and whether complications or not were observed 6 months after surgery. Before surgery and 6 months after surgery, depressed degree, color, and pliability of scar were compared. Vancouver Scar Scale (VSS) was employed to access color, vascularity, and pliability before surgery and 6 months after surgery, and total score was calculated. The number of patients with complete satisfaction or satisfaction was counted six months after surgery. Data were processed with analysis of variance of factorial design, paired samples t test, and Wilcoxon rank sum test.@*Results@#(1) The content of EGF in SVF-GEL group and negative control group was (316.6±12.8) and (3.4±0.6) pg/mL, and the content of VEGF in SVF-GEL group and negative control group was (568.67±12.19) and (4.93±0.16) pg/mL, with statistically significant differences between the two groups (t=48.777, 92.485, P<0.01). (2) Residual scratch widths of HSF and HaCaT in SVF-GEL group and control group were decreased gradually along with time elapse, in which those in SVF-GEL group at PSH 24 and 48 were less than those in control group. At PSH 24 and 48, cell migration distances of HSF and HaCaT in SVF-GEL group were more than those in control group (tHSF=-20.304, -43.516, tHaCaT=-15.060, -8.684, P<0.01). After culture for 24, 48, and 72 h, the number of living cell of HSF and HaCaT in SVF-GEL group was significantly more than that in control group (tHSF=-3.374, -6.809, -18.036, tHaCaT=-4.793, -6.028, -8.141, P<0.05 or P<0.01). (3) Six months after surgery, SVF-GEL grafted into patients survived well without complications, and depressed degree of scar ameliorated obviously with lightened pigmentation and softer texture as compared with before surgery. Compared with those before surgery, VSS scores of color, vascularity, and pliability, and total score of 15 patients with depressed scars on face were obviously decreased 6 months after surgery (Z=-2.06, -2.07, -2.07, t=-15.811, P<0.05 or P<0.01). One patient was satisfied with the clinical outcome, and the rest 14 patients were completely satisfied with the clinical outcomes.@*Conclusions@#SVF-GEL contains cytokines EGF and VEGF, which can enhance cell migration ability and proliferation ability of HSF and HaCaT cells and have obvious effects on depressed scars for clinical application.
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Objective To investigate the regulation of miR-939-5p on USP22 gene expression and its effect on liver cancer migration and proliferation.Methods The expression of miR-939-5p in hepatoma cell lines (HepG2,MHCC-97H,SMMC-7721,BEL-7404 and Huh7) and normal liver cell line LO2 was detected by realtime PCR (qPCR).The liver cancer cells with the lowest expression were used as experimental subjects,and transfected with miR-939-5p (Experimental group) or miR-NC (Control group).qPCR was used to detect the transfection efficiency of miR-939-5p.Transwell migration assay and MTT proliferation assay were used to detect the migration and proliferation of hepatoma cells after transfected miR-939-5p.Bioinformatics software predicted target genes for miR-939-5p.The dual luciferase reporter gene verified the interaction of miR-939-5p with the target gene.qPCR and Western blotting were used to detect the expression of target genes at mRNA and protein levels after over-expression of miR-939-5p.Measurement data were expressed as (Mean ± SD),and t test was used for comparison between groups.Results The expression of miR-939-5p was significantly lower in hepatoma cell lines than in normal hepatocytes (P ＜0.01),and the expression of miR-939-5p was the lowest in SMMC-7721 cells (P＜0.01).miR-939-5p was efficiently transfected into SMMC-7721 cells [(1.01 ±0.07) vs (20.12 ± 1.27),P ＜0.01].High expression of miR-939-5p inhibited the migration ability (P ＜ 0.01) and proliferative capacity of liver cancer SMMC-7721 cells (P ＜0.05).The USP22 gene may be a target gene of miR-939-5p.The luciferase reporter gene confirmed that miR-939-5p specifically binds to the 3'-UTR of USP22 mRNA (P ＜ 0.01).USP22 expression was decreased at mRNA and protein levels after high expression of miR-939-5p (P ＜ 0.01).Conclusions The expression of miR-939-5p was down-regulated in hepatocellular carcinoma cell lines,and miR-939-5p inhibited the migration and proliferation of liver cancer SMMC-7721 cells.The molecular mechanism was to interfere with the expression of USP22 gene.
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Objective To explore the application of neutrophil migration distance for wound age estimation of skeletal muscles in rats, and to provide methodological basis for follow-up study in future. Methods The skeletal muscle contusion model was established in rats, and the control group and the 2, 4, 6 h post-traumatic groups were set. The law of response of neutrophils that participated in the inflammation after injury was detected by immunohistochemical staining, and the relationship between neutrophil migration distance and injury time was detected by TissueFAXS PLUS software. Results The skeletal muscle was obviously infiltrated with neutrophils 2-6 h after injury. The positive rate of neutrophil was （28.75±0.94）% at 2 h post-traumatic, and reached the peak （45.50±3.63）% at 4 h post-traumatic, then decreased to （31.92±1.56）% at 6 h post-traumatic. The neutrophil migration distances increased with the progress of inflammation, and reached （124.80±12.32） μm, （229.03±21.45） μm and （335.04±16.75） μm at 2 h, 4 h and 6 h, respectively. Conclusion There is a relationship of neutrophil infiltrated number and migration distance and wound age within the 2-6 h after skeletal muscle injury, which could be used for the inference of skeletal muscle wound age.
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Animals , Rats , Contusions/metabolism , Follow-Up Studies , Muscle, Skeletal/pathology , Neutrophil Infiltration , Neutrophils , Rats, Sprague-Dawley , Time FactorsABSTRACT
Objective@#To preliminarily evaluate the effect of exosomes from human adipose-derived stem cells (hADSCs) on the migration of epidermal cells.@*Methods@#hADSCs were isolated from adipose tissues obtained from a healthy woman after liposuction, and subjected to culture. Exosomes were collected from the culture supernatant of hADSCs by using a modified method combining ultracentrifugation with ultrafiltration, and identified and isolated by transmission electron microscopy (TEM) , dynamic light scattering (DLS) and Western blot analysis. Some cultured HaCaT cells were divided into several groups to be treated with hydrogen peroxide (H2O2) at different concentrations of 0, 50, 100, 200, 250, 300, 400, 500, 600, 800 μmol/L for 1 hour, and cell counting kit-8 (CCK8) assay was performed to evaluate the effect of H2O2 on the survival rate of HaCaT cells. Some HaCaT cells were classified into 2 groups to be pretreated with phosphate-buffered saline (PBS) (normal group) or 100 μmol/L H2O2 (injury group) for 0.5 hour; then, HaCaT cells in the 2 groups were separately divided into treatment group and control group to be co-cultured with exosomes or not. Confocal fluorescence microscopy was conducted to confirm the uptake of PKH26-labelled exosomes by HaCaT cells, scratch assay to estimate the wound healing potential, and Transwell assay to evaluate the migratory activity of HaCaT cells. Statistical analysis was carried out by using one-way analysis of variance for comparison among groups, least significant difference (LSD) -t test for multiple comparisons, and Spearman correlation analysis for analyzing correlations.@*Results@#TEM showed that the exosomes isolated from hADSCs were saucer-like nanovesicles with diameters of 60-80 nm. DLS revealed that the purity of the isolated exosomes was 65.88%, and they were stained positively for CD63, Alix and TSG101, which coincided with the basic characteristics of exosomes. CCK8 assay showed that survival rates of HaCaT cells gradually decreased along with the increase of H2O2 concentrations after 1-hour treatment, and were negatively correlated with the concentration of H2O2 (r= -0.91, P < 0.01) . Confocal fluorescence microscopy showed that the isolated exosomes could be endocytosed into impaired HaCaT cells. Scratch assay showed that the gap-filling rates at 12 hours were 40.26% ± 0.64%, 69.57% ± 0.69%, 32.28% ± 0.31% and 69.62% ± 1.68% in the normal control group, normal treatment group, injury control group and injury treatment group respectively, and the injury treatment group showed a significantly increased gap-filling rate compared with the injury control group (t = 37.33, P < 0.01) . Transwell assay showed that the number of migratory cells per × 10 field was 20.85 ± 4.84, 44.8 ± 5.24, 14.95 ± 2.58 and 40.05 ± 7.66 in the normal control group, normal treatment group, injury control group and injury treatment group respectively, and was significantly larger in the injury treatment group than in the injury control group (t = 25.10, P < 0.01) .@*Conclusion@#Exosomes isolated from hADSCs can improve the migration of HaCaT cells after oxidative injury.
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Objective@#To explore the mechanism of shRNA Twist gene on proliferation and invasion of lung adenocarcinoma NCI-H1299 cells.@*Methods@#Twist siRNA interference expression vector was constructed and NCI-H1299 cells were divided into three groups: blank control group, negative control group and experimental group. The blank control group was the untreated cell group, while the negative control group was the lentivirus transfected cell group by the blank vector. The experimental group was the lentivirus transfected cell group constructed by the lentivirus interference vector of shRNA Twist. The siRNA interference expression vector of Twist was constructed by quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot to detect the expression of Twist. Transwell kit was used to detect cell invasion. Cell counting kit-8 (CCK-8) kit was used to detect cell proliferation.@*Results@#⑴ The titer of lentivirus was detected. The transfection titer of lentivirus vector: shRNA-Twist vector was 3×108 TU/ml. ⑵ The results of qRT-PCR test showed that compared with the negative control group, the mRNA expression of Twist in the experimental group was decreased (q=3.177, P=0.0234). ⑶ The results of Western blot showed that compared with the negative control group, the protein expression of Twist in the experimental group was decreased (q=4.071, P=0.0304). ⑷ The results of Transwell test showed that there was significant statistical difference among the three groups (F=19.472, P=0.000). Compared with the negative control group, the number of cell imigration in the experimental group was decreased (q=3.567, P=0.0318). ⑸ The results of CCK-8 showed that there was significant statistical difference among the three groups (F=20.983, P=0.000). Compared with the negative control group, the proliferation rate in the experimental group was decreased (q=5.272, P=0.0157).@*Conclusions@#ShRNA Twist gene can significantly inhibit the proliferation and invasion of lung adenocarcinoma NCI-H1299 cells.
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Objective To explore the mechanism of shRNA Twist gene on proliferation and invasion of lung adenocarcinoma NCI-H1299 cells.Methods Twist siRNA interference expression vector was constructed and NCI-H1299 cells were divided into three groups:blank control group,negative control group and experimental group.The blank control group was the untreated cell group,while the negative control group was the lentivirus transfected cell group by the blank vector.The experimental group was the lentivirus transfected cell group constructed by the lentivirus interference vector of shRNA Twist.The siRNA interference expression vector of Twist was constructed by quantitative real-time polymerase chain reaction(qRT-PCR) and Western blot to detect the expression of Twist.Transwell kit was used to detect cell invasion.Cell counting kit-8 (CCK-8) kit was used to detect cell proliferation.Results (1) The titer of lentivirus was detected.The transfection titer of lentivirus vector:shRNA-Twist vector was 3 × 108 TU/ml.(2)The results of qRT-PCR test showed that compared with the negative control group,the mRNA expression of Twist in the experimental group was decreased (q =3.177,P =0.0234).(3) The results of Western blot showed that compared with the negative control group,the protein expression of Twist in the experimental group was decreased (q =4.071,P =0.0304).(4) The results of Transwell test showed that there was significant statistical difference among the three groups (F =19.472,P =0.000).Compared with the negative control group,the number of cell imigration in the experimental group was decreased (q =3.567,P =0.0318).(5) The results of CCK-8 showed that there was significant statistical difference among the three groups (F =20.983,P =0.000).Compared with the negative control group,the proliferation rate in the experimental group was decreased (q =5.272,P =0.0157).Conclusions ShRNA Twist gene can significantly inhibit the proliferation and invasion of lung adenocarcinoma NCI-H1299 cells.
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Objective@#To explore the effects of hypoxic exosomes secreted from hepatocellular carcinoma Huh7 cells on the proliferation, migration and invasion under co-cultured normoxic condition.@*Methods@#Hypoxic exosomes secreted from Huh7 cells under hypoxic conditions were extracted by differential ultracentrifugation. Transmission electron microscopy, nanoparticles tracking analysis and western blot were used for the identification of hypoxic exosomes. Hypoxic exosomes were co-cultured with Huh7 cells under normoxic conditions. CCK8, cell scratch and transwell assay were used to detect the changes of cell proliferation, migration and invasion. Statistical analysis was performed by one-way ANOVA and t-test.@*Results@#Hypoxic exosomes secreted from Huh7 cells ranged in size from 30 to 150 nm in diameter, and expressed exosome surface markers CD9, CD63 and TSG101. Hypoxic exosomes significantly enhanced the proliferation of normoxic Huh7 cells (A value of hypoxic exosomes and control group at 48 and 72 h were 2.131 ± 0.092 and 1.760 ± 0.104,t= 3.740,P<0.01, 3.121 ± 0.157 and 2.298 ± 0.085,t= 8.289,P< 0.01). The migration distance between hypoxic exosome and control group at 48 and 72 h were (0.37 ± 0.06 cm)and(0.19 ± 0.05 cm),t= 4.813,P< 0.05, (1.15 ± 0.07 cm) and(0.62 ± 0.08 cm),t= 8.874,P< 0.05, and invasion ability [hypoxic exosomes and control group were (123 ± 18), (44 ± 12),t= 6.203,P< 0.01].@*Conclusion@#Hypoxic exosomes secreted from hepatocellular carcinoma Huh7 cells can promote cell proliferation, migration and invasion in hypoxic environment, suggesting that intercellular information transmission mediated by hypoxic exosomes may be one of the key mechanisms for the amplification of malignancy of hepatocellular carcinoma cells in hypoxic microenvironment.
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Objective To study the effect and mechanism of Momordica anti-HIV protein of 30 ku (MAP30) on the migration of bladder cancer.Methods The IC50 of human bladder cancer 5637 and T24 cells was calculated by methyl thiazolyl tetrazolium (MTT) method.The migration ability of these two cells was evaluated by scratch migration test and Transwell cell migration test.The expression of migrating proteins such as matrix metalloproteinases (MMPs) and adhesion molecule N-cadherin were compared by Western blot.Results Scratch migration test:there were significant differences in migration rates of 5637 cells at 8 h and 22 h (P ＜ 0.05).There were significant differences in migration rates of T24 cells at 22 h (P ＜ 0.05),but no significant differences in migration rates at 8 h (P ＞ 0.05).The expression of Vimentin,Fibronectin,MMP-2,MMP-9 and N-Cadherin in 5637 cells and T24 cells of human bladder cancer decreased significantly after adding MAP30.The E-Cadherin expression in human bladder cancer 5637 cells were decreased,but no target band was detected in human bladder cancer T24 cells.Conclusions The ribosome-inactivating protein MAP30 can effectively inhibit the migration of human bladder cancer 5637 and T24 cells by inhibiting the EMT pathway and inhibiting the expression of MMPs.