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ObjectiveTo explore the mechanism of Qigesan （QGS） in intervening in the migration and invasion of esophageal carcinoma TE-1 cells. MethodMicroarray technology was used to screen differentially expressed genes （DEGs） in the normal group and the QGS group， and the ontological functions and signaling pathways of DEGs were analyzed. The thiazolyl tetrazolium （MTT） assay was used to detect the effect of QGS on the viability of TE-1 cells. In the subsequent experiments for verification， a blank group， a transforming growth factor-β1 （TGF-β1） group， a TGF-β1 + QGS group， and a TGF-β1 + SB431542 group were set up. The cell morphology in each experimental group was observed by microscopy. The migration and invasion abilities of cells were detected by wound healing assay， and the mRNA expression levels of E-Cadherin， vimentin， Smad2， and Smad7 were detected by Real-time quantitative polymerase chain reaction （Real-time PCR）. The protein expression of E-Cadherin， vimentin， p-Smad2/3， Smad2/3， and Smad7 was detected by Western blot. ResultThere were 1 487 DEGs between the QGS group and the blank group， including 1 080 down-regulated ones （accounting for 72.63%） and 407 up-regulated ones. The down-regulated genes were mainly involved in biological processes such as cytoskeletal protein binding， ATP binding， adenylate nucleotide binding， and adenylate ribonucleotide binding， and the involved Kyoto Encyclopedia of Genes and Genomes （KEGG） pathways included TGF-β signaling pathway， cell cycle， extracellular matrix-receptor interaction protein， tumor pathways， and oocyte meiosis. The up-regulated genes were mainly involved in RNA binding， DNA binding， transcriptional regulator activity， transcriptional activator activity， and nucleotide binding， and the KEGG pathways involved mainly included mitogen-activated protein kinase （MAPK） signaling pathway， bladder cancer， renal cell carcinoma， cancer pathways， and p53 signaling pathway. Compared with the blank group， the inhibition rate of cell viability of TE-1 cells increased after QGS （20， 30， 40， 60， 80 mg·L-1） intervention for 12， 24， 36， 48， 60 h （P<0.05）， and the inhibition rate was time- and dose-dependent. Compared with the blank group， the TGF-β1 group showed lengthened cells with fibroblast phenotype. Compared with the TGF-β1 group， the TGF-β1 + QGS group showed shortened cells with normal morphology and epithelial phenotype. The cell morphology in the TGF-β1 + SB431542 group was similar to that of the TGF-β1 + QGS group. Compared with the blank group， the TGF-β1 group showed potentiated ability of cell migration and invasion （P<0.05）. Compared with the TGF-β1 group， the TGF-β1 + QGS group and the TGF-β1 + SB431542 group showed inhibited and weakened migration and invasion abilities of cells （P<0.05）. However， there was no significant difference in migration and invasion abilities between the TGF-β1 + QGS group and the TGF-β1 + SB431542 group. The mRNA expression levels of vimentin and Smad2 in the TGF-β1 group were higher （P<0.05）， and the mRNA expression levels of E-Cadherin and Smad7 were lower （P<0.05） than those in the blank group. Compared with the TGF-β1 group， the TGF-β1 + QGS group and the TGF-β1+ SB431542 group exhibited decreased expression levels of vimentin and Smad2 mRNA （P<0.05）， and elevated expression levels of E-Cadherin and Smad7 mRNA （P<0.05）. Compared with the blank group， the TGF-β1 group showed up-regulated protein expression levels of vimentin， p-Smad2/3， and Smad2/3 （P<0.05）， and reduced protein expression levels of E-Cadherin and Smad7 （P<0.05）. Compared with the TGF-β1 group， the TGF-β1 + QGS group and the TGF-β1 + SB431542 group displayed decreased protein expression levels of vimentin， p-Smad2/3， and Smad2/3 （P<0.05）， and increased protein expression levels of E-Cadherin and Smad7 （P<0.05）. ConclusionThe ethyl acetate extract of QGS inhibits the epithelial-mesenchymal transition （EMT） of TE-1 cells through the TGF-β1 pathway to reduce the migration and invasion of TE-1 cells.
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@#【Objective】To investigate the effect of miR- 127-3p on proliferation，apoptosis，migration and invasion of uveal melanoma cells.【Methods】The expression of miR- 127- 3p and MAPK4 mRNA in human uveal melanoma tissues and cells，normal tissues and cells were detected by RT-qPCR. The mimic-NC，miR-127-3p mimic，pc-MAPK4 plasmids were transfected into SP6.5 or OM431 cells，respectively，by Lipofectamine 2000. The relationship between miR-127-3p and MAPK4 counterstaining was detected by dual luciferase assay. Cell proliferation was detected by CCK- 8 method，apoptosis was detected by flow cytometry，cell migration ability was detected by scratch test，cell invasion ability was detected by Transwell method，and relative expression level of AKT/mTOR pathway protein was detected by Western blot.【Results】In uveal melanoma tissues and cell lines，the expression of miR- 127-3p was down-regulated（P < 0.01）while that of MAPK4 expression was significantly up-regulated（P < 0.01）. The binding site of miR-127-3p and MAPK4 3′UTR region，the high expression of miR-127-3p significantly inhibited the luciferase activity of wild-type MAPK4 plasmid（P < 0.01），but the mutant MAPK4 plasmid Luciferase activity has no effect. Compared with the Control group ，the proliferation of SP6.5 cells and OM431 cells in miR- 127-3p mimic group were significantly decreased（P < 0.01），and the apoptotic rate was significantly increased（P < 0.01）. The scratch closure rate was obvious. The decrease（P < 0.01）， the number of invading cells per field was significantly decreased（P < 0.01），and the expression of p-AKT（T308）/AKT and p-mTORr（S473）/mTOR protein were significantly down-regulated（P < 0.01）. Transfection of pc-MAPK4 reversed the above changes.【Conclusion】MiR-127-3p inhibits proliferation，migration and invasion of uveal melanoma cells and induces apoptosis by down-regulating MAPK4，which may be involved in the inhibition of AKT/mTOR pathway activation.
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@# Objective: To investigate the effect and mechanism of AMP-activated protein kinase α (AMPKα) over-expression on proliferation, migration, invasion and epithelial mesenchymal transition (EMT) of bladder cancer T24 cells. Methods: A bladder cancer T24 cells over-expressing AMPKα was established and divided into T24 group, pc-DNA group and pc-AMPKα group according to different plasmid transfection. Western blotting was used to verify the over-expression ofAMPKα and detect the expressions of EMT-related proteins and EMT pathway-related molecules. Hoechst staining was used to detect apoptosis of transfected T24 cells. CCK8 assay was used to detect cell proliferation. Cell scratch test was used to detect cell migration. Transwell assay was used to detect cell invasion. Results: The bladder cancer cell line T24 over-expressingAMPKα was successfully constructed. Compared with the T24 group and the pc-DNA group, the level of E-cadherin in the pc-AMPKα group was significantly increased (P<0.01) while the levels of Vimentin and N-cadherin were significantly decreased (all P<0.01), and the activities of P38 and STAT3 which related to EMT pathway were significantly inhibited (all P<0.01); cell proliferation, migration and invasion were significantly decreased while cell apoptosis was obviously enhanced (all P<0.01). Conclusion: Over-expression of AMPKα can inhibit the activity of EMT pathway-related molecules, which leads to obvious apoptosis, limited proliferation, reduced invasion and migration of bladder cancer T24 cells, and accompanied by the reversal of EMT.
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@#【Objective】To clarify the role of neuro-cadherin（N-cadherin）in epithelial-mesenchymal transition of diabetic retinopathy，and to investigate the effect of N-cadherin on proliferation ，migration and invasion of retinal pigment epithelial cells.【Methods】Cells were turned into over-expressed or silenced N-cadherin by using Ad-N-cadherin （Ad-N-cad）and Ad-si N-cadherin（si N-cad）. Glucose（25 mmol/L）was used to simulate high glucose（HG）condi⁃ tions. Cell Counting Kit-8（CCK-8）kit was used to detect cell proliferation. Transwell chamber was used to detect the vertical migration and invasion of cells.【Results】Transwell assay showed N-cadherin over-expression increased the num⁃ ber of cells migrated to the transwell subventricular chamber. The difference was statistically significant（P < 0.05）. The number of ARPE19 cells that migrated to or invaded the transwell subventricular chamber increased after high glucose treatment. N-cadherin knockdown suppressed high glucose-induced migration and invasion（P < 0.05）. CCK8 results showed N-cadherin knockdown could inhibit cell proliferation induced by high glucose（P < 0.05）.【Conclusion】N-cad⁃ herin may promote cell migration，and down-regulation of N-cadherin can inhibit cell proliferation，migration and inva⁃ sion induced by high glucose.
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@#Objective: To investigate the expression of ciRS-7 in esophageal squamous cell carcinoma (ESCC) and its effect on the cellular proliferation, migration and invasion. Methods: The cancer tissues and paired adjacent normal tissues from 60 ESCC patients treated in the Fourth Hospital of Hebei Medical University between May, 2016 andApril, 2017 were selected for this study. The expressions of ciRS-7 were detected by qRT-PCR. After over-expressing or silencing of ciRS-7, the proliferation of ESCC cell line TE1 was measured by CCK-8 assay; and the migration and invasion were tested by wound healing assay and Transwell invasion assay，respectively. Finally, the effect was validated via animal experiment. Results: CiRS-7 was highly expressed in ESCC tissues (P<0.05), and its expression level was closely related to pathological grade and lymph node metastasis (P<0.05). Over-expression of ciRS-7 significantly increased the proliferation, migration and invasion (all P<0.05) of TE1 cells; while silencing of ciRS-7 remarkably suppressed the proliferation, migration and invasion (all P<0.05). Conclusion: CiRS-7 was up-regulated in ESCC and could enhance ESCC cell proliferation, migration and invasion, suggesting that ciRS-7 could be used as a potential target for the diagnosis and treatment of ESCC.
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@# Objective: To observe the effects of miR-204 on the proliferation and invasion of retinoblastoma (RB) cells and to explore the potential regulatory mechanism. Methods: The expression level of miR-204 in RB cell lines (Y79, SO-RB50, and HXO-Rb44) as well as in normal human retinal pigment epithelial cell line hTERT RPE-1 was detected using qRT-PCR. The Y79 cells were divided into two groups (negative control group and miR-204 group) by respectively transfecting Y79 cells with NC-mimics and miR-204 mimics using liposome transfection method. The effects of miR-204 on Y79 cell proliferation was detected with CCK-8 assay; while the effect of miR-204 on migration and invasion of Y79cellsweredeterminedbycellscratchassayandTranswellassay,respectively.Besides, thepotentialtargetgeneofmiR-204waspredictedbybioinformatics;and the influence of miR-204 on the expression of high mobility group AT-hook 2 gene (HMGA2) at both mRNA and protein levels was detected using qRT-PCR and Western blotting, respectively. Results: miR-204 expression in RB cell lines Y79, SO-RB50 and HXO-Rb44 was remarkably lower than that in normal human retinal pigment epithelial cell line hTERT RPE-1 (P<0.01). miR-204 expression in Y79 cells was markedly up-regulated after transfection with miR-204 mimics (P<0.01) along with significantly reduced cell proliferation, migration and invasion capacities (all P<0.01), and mRNA and protein expressions of HMGA2 were also outstandingly reduced (P<0.01). Conclusion: miR-204 is lowly expressed in RB cell lines; in addition, miR-204 over-expression can suppress RB cell proliferation, migration and invasion, the mechanism of which might be related to down-regulation of the expression of HMGA2.
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@# Objective: To study the expression of H O X A 1 0 gene in endometrial carcinoma and its effect on the apoptosis, migration and invasion of Ishikawa cells. Methods: Twenty-one cases of endometrial carcinoma tissue samples and 25 cases of normal endometrial tissue samples from patients treated at the Department of Obstetrics and Gynecology, Nanjing Drum Tower Hospital from 2012 to 2013 were collected for this study. The mRNA and protein expressions of H O X A 1 0 in endometrial carcinoma and normal endometrial tissues were separately tested by Realtime-qPCR (qRT-PCR) and Western blotting. Ishikawa cells were infected with adenovirus-flagHOXA10 at different multiplicity (5, 10, 20 MOI), and infected by adenovirus-flag-lacz (20 MOI) as control; And the cell apoptosis was tested by Flow Cytometry. Ishikawa cells were transfected with 50 nmol/L si-HOXA10 plasmids and 50 nmol/L si-NC plasmids, as down-regulation group and down-regulation control group, respectively. Ishikawa cells were infected with 20 MOI adenovirus-flagHOXA10 and 20 MOI adenovirus-flag-lacz, as up-regulation group and up-regulation control group, respectively. The ability of migration and invasion was detected by transwell assay. Results: The results of qRT-PCR and Western blotting showed that the expressions of H O X A 1 0 mRNA and protein in endometrial carcinoma samples were both significantly lower than normal samples [mRNA: (0.56± 0.14)vs (1.36±0.33), P<0.01; protein: (1.01±0.25) vs (2.10±0.71), P<0.001]. After the up-regulation of H O X A 1 0 gene in Ishikawa cell line, the cell apoptosis rate in ad-flag-HOXA10 groups (5, 10, 20 MOI) was significantly raised, and most of which was in the early apoptosis [(50.92±8.79)%, (55.17±4.07)%, (76.10±3.65)% vs (7.74 ± 0.15)%, all P <0.01]. The number of migrated cells was markedly up-regulated in si-HOXA10 group [(248±25) vs (135±15), P <0.01] but markedly down-regulated in ad-flag-HOXA10 group [(50±6) vs (100±13), P <0.01]. The number of invasive cells was markedly up-regulated in si-HOXA10 group [(131±18) vs (66±9), P <0.01] but markedly down-regulated in ad-flag-HOXA10 group [(34±8) vs (60±4), P <0.01]. Conclusions: Both mRNAand protein expressions of H O X A 1 0 were down-regulated in endometrial carcinoma samples than in normal endometrium. Up-regulation of H O X A 1 0 gene in Ishi kawa cell line can promote cell apoptosis and inhibit cell migration and invasion.
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OBJECTIVES: MicroRNAs are important modulators of the cellular epithelial‑mesenchymal transition (EMT) process and are associated with metastasis in human nonsmall cell lung cancer (NSCLC). In this study, we tried to investigate the role of miR‑338‑3p in NSCLC cells. MATERIALS AND METHODS: Real‑time polymerase chain reaction was applied to quantify the expression levels of miR‑338‑3p, as well as EMT‑associated molecules in NSCLC cells. Wound healing and transwell assays were performed to evaluate the migration and invasion capacities, respectively. Dual‑luciferase reporter assay was finally performed to determine the targeting of zinc finger E‑box‑binding protein 2 (ZEB2) by miR‑338‑3p. RESULTS: We found that miR‑338‑3p was significantly reduced in NSCLC cell lines. Forced expression of miR‑338‑3p in A549 cells led to the suppression of migration/invasion capacity and inhibition of epithelial markers. In addition, we proved that miR‑338‑3p could directly target ZEB2. CONCLUSIONS: In general, we summarized that miR‑338‑3p could inhibit EMT and metastasis of human NSCLC cells, which probably via directly targeting ZEB2 expression.