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Objective:To investigate the role of embryonic stem cell pluripotent factor NANOG in mediating the activity and invasion of breast cancer cells via AMPK/mTOR pathway.Methods:A total of 58 breast cancer patients were collected from Jul. 2019 to Aug. 2020, and the clinical data of each patient at admission were collected for comparative analysis. qRT-PCR was used to detect the expression level of NANOG in adjacent tissues and cancer tissues, and Western blot was used to verify the regulation of AMPK/mTOR pathway by NANOG. Cells were treated with NANOG specific plasmid or AMPK inhibitor Compound C. Cell viability was detected by MTT and invasion ability was detected by Transwell.Results:The expression of NANOG was increased in breast cancer tissues (adjacent to cancer tissue: 1.00±0.31, cancer tissue: 1.45±0.27, t=8.34, P<0.004) and cell lines (MCF-10A: 1.00±0.12, BT474: 2.64±0.25, t=10.24, P=0.001; MCF-7: 1.56±0.13, t=5.48, P=0.005; ZR-75-30:1.84±0.16, t=7.28, P=0.002), which could be used as a specific biomolecule for predicting breast cancer (all P<0.05). The expression level of NANOG may be related to lymph node metastasis, histological grade and pathological type. Compared with patients with non-lymph node metastasis (1.36±0.23) or non-invasive patients (1.35±0.25), patients with lymph node metastasis (1.54±0.27, t=2.61, P=0.012) or invasive patients (1.53±0.26, t=2.60, P=0.012) had higher expression of NANOG. After NANOG knockdown, AMPK protein and phosphorylation levels were increased, while mTOR and p70S6K protein and phosphorylation levels were decreased (all P<0.05). Knockdown of NANOG in cells inhibited the activity and invasion of breast cancer cells (activity: si-RNA: 100±8.65, si-NANOG: 58.36±4.58, t=7.37, P=0.002; invasion: si-RNA: 121.41±10.34, si-NANOG: 58.34±8.41, t=8.20, P=0.001), and the effect of knockdown of NANOG was relieved after AMPK inhibitor was used in cells (all P<0.05) . Conclusions:Embryonic stem cell pluripotent factor NANOG promotes the activity and invasion of breast cancer cells by inhibiting the activation of AMPK/mTOR pathway. NANOG can be used as an effective biomolecule for predicting breast cancer.
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Objective:To explore the effect of miR-1249-5p on the proliferation, metastasis and cell cycle of PC-3 cell in prostate cancer.Methods:The relationship between the expression level of miR-1249-5p and the overall survival of prostate cancer patients was analyzed using OncoMir Cancer Database (OMCD). The human prostate cancer cell line PC-3 was divided into two groups: miR-1249-5p group and negative control group. Mediated by Lipofectamine 2000, miR-1249-5p mimics liposome complex or negative miRNA liposome complex were transfected into PC-3 cell at logarithmic growth stage. Real-time quantitative polymerase chain reaction (RT-qPCR) was used to detect the expression of miR-1249-5p in PC-3 cell of two groups. Colony formation assay was used to detect the changes of the proliferation ability of PC-3 cell in the two groups. Transwell experiment was used to detect the changes of PC-3 cell invasion in the two groups, and the cell cycle changes of the two groups of PC-3 were detected by flow cytometry. The miRNA prediction software miRGator was used to predict the target gene of miR-1249-5p. RT-qPCR and Western blotting were used to detect the target gene expression of miR-1249-5p. Measurement data were expressed as mean±standard deviation ( ± s), and t-test was used for comparison between two groups. Results:Compared with prostate cancer patients with low miR-1249-5p expression, prostate cancer patients with higher miR-1249-5p expression had longer overall survival, and the difference was statistically significant ( P<0.01). The expression level of miR-1249-5p in the miR-1249-5p group (10.74±1.19) was significantly higher than that of the negative control group (1.56±0.27), the difference was statistically significant ( P<0.01). The number of colonies formed in the miR-1249-5p group (35.86±6.94) was significantly less than that in the negative control group (88.94±11.66), and the difference was statistically significant ( P<0.01). The number of transmembrane cells [(25.01±6.83)/high power field of view] in the miR-1249-5p group was significantly less than that of the negative control group [(82.76±8.35)/high power field of view], and the difference was statistically significant ( P<0.01). The proportion of cells in the G 0-G 1 phase in the miR-1249-5p group [(50.79±6.61)%] was significantly higher than that in the negative control group [(27.09±2.30)%], the difference was statistically significant ( P<0.01), and PC-3 cell were inhibited in the G 0-G 1 phase. Neural precursor cell expressed developmentally down-regulated 9 ( NEDD9) may be the target gene of miR-1249-5p. Compared with the negative control group, the NEDD9 gene expression in the miR-1249-5p group was significantly lower than that of the negative control group, the difference was statistically significant ( P<0.01). Conclusion:miR-1249-5p can inhibit the proliferation, metastasis and cell cycle of PC-3 cell in prostate cancer, which may be achieved by negatively regulating the expression of proto-oncogene NEDD9.
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Objective:To investigate the effect of lncRNA AC132217.4 on the proliferation and invasion of liver cancer MHCC97-H cells and its molecular mechanism.Methods:The TCGA database was used to analyze the differential expression of AC132217.4 in liver cancer tissue and adjacent tissue, and to analyze the relationship between the expression level of AC132217.4 and the overall survival of liver cancer patients. Transfection of pcDNA-AC132217.4 plasmid into MHCC97-H cells was defined as AC132217.4 group, transfection of pcDNA plasmid into MHCC97-H cells was defined as negative control (NC) group, respectively. The proliferation and invasion ability of MHCC97-H cells were detected by MTT method and Matrigel invasion assay. The binding site between AC132217.4 and miR-18a-5p was analyzed by starBase v2.0 software and dual luciferase reporter gene assay. Real-time quantitative PCR (RT-qPCR) detected the differential expression of miR-18a-5p in the two groups of MHCC97-H cells. The expression of epithelial-mesenchymal transition protein was detected by Western-blotting. Measurement data with normal distribution were expressed as mean±standard deviation ( ± s), and t-test was used for comparison between the two groups. Results:Compared with adjacent tissues, the expression of AC132217.4 was down-regulated in liver cancer tissues ( P<0.01). Compared with liver cancer patients with low expression of AC132217.4, the overall survival of liver cancer patients with high AC132217.4 expression was longer ( P<0.05). The pcDNA-AC132217.4 plasmid significantly inhibited the proliferation of MHCC97-H cells ( P<0.05). The number of invasive cells in the NC group and AC132217.4 group were (131.30±12.55) and (37.45±7.77), respectively. The pcDNA-AC132217.4 plasmid significantly inhibited the invasive ability of MHCC97-H cells ( t=6.36, P<0.01). AC132217.4 directly complemented miR-18a-5p ( P<0.01). The expression of miR-18a-5p in MHCC97-H cells in AC132217.4 group (1.04±0.30) was significantly lower than that in NC group (6.13±0.75) ( t=6.27, P<0.01). Compared with the NC group, the expressions of epithelial phenotype proteins Cytokeratin and Claudin-1 in MHCC97-H cells in AC132217.4 group were up-regulated, while the expressions of mesenchymal phenotype proteins Vimentin, Slug and Snail were down-regulated. Conclusions:The expression of AC132217.4 is low in liver cancer tissue, and it is related to the overall survival of liver cancer patients. AC132217.4 might inhibit the proliferation and invasion of liver cancer MHCC97-H cells by sponge miR-18a-5p.
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The matrix metalloproteinases family (MMPs) are proteins related to tumor formation and metastasis that have attracted the attention of scholars in recent years. Tumor cells can secrete MMPs during malignant transformation, and the expression of MMPs in different malignant tumors is diverse, and different members of MMPs do not have exactly the same biological properties. Matrix metalloproteinase-19 (MMP-19) is a new member of MMPs whose secretion increases rapidly during the malignant transformation of cells and is released into the extracellular space to participate in biological processes such as proliferation, adhesion, invasion, migration, and angiogenesis of tumor cells. In this paper, the progress of research on the biological properties of MMP-19 in tumors was reviewed to provide a theoretical basis for exploring the development of tumors, especially for studying the invasion and metastasis of tumor cells.
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Objective:To investigate the effects of cytoplasmic fragile X mental retardation protein 1 binding protein 2 (CYFIP2) overexpression on the biological functions and Wnt/β-catenin signaling pathways of bladder cancer T24 cells.Methods:The control group was T24 cells transfected with the empty pcDNA3 vector, and the overexpression group was T24 cells transfected with the CYFIP2 overexpression vector. The expression of CYFIP2 mRNA and protein was detected by reverse transcriptase, quantitative polymerase chain reaction, and Western Blot. The effect of CYFIP2 overexpression on T24 cell proliferation was detected by CCK-8. The effect of CYFIP2 overexpression on T24 cell migration and invasion was detected by Transwell. The effects of CYFIP2 overexpression on Wnt/β-catenin signaling pathway in T24 cells were detected by Western Blot.Results:Compared with the control group, the expression levels of CYFIP2 mRNA and protein were increased in the overexpression group (all P < 0.001), and the cell proliferation, migration, and invasion abilities were reduced (all P < 0.01). β-catenin, c-Myc, and Cyclin D1 protein expression were down-regulated in CYFIP2 overexpressed T24 cells (all P < 0.05), while the protein levels of p-β-catenin were increased ( P < 0.05). Conclusions:CYFIP2 overexpression can inhibit T24 cell proliferation, migration, and invasion, and its possible molecular mechanism is related to the inhibition of Wnt/β-catenin signaling pathway.
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Objective:To investigate the effect of kaempferol on proliferation, migration, and invasion of cervical cancer SiHa cells by regulating circFBXW7.Methods:SiHa cells were treated with kaempferol at low, medium, and high doses (15, 30, and 60 μmol/L) for 24 h. Untreated SiHa cells were used as the control group. CCK-8 was used to detect the effect of kaempferol on the proliferation of SiHa cells. Transwell was used to detect the effect of kaempferol on the migration and invasion of SiHa cells. Real-time fluorescence quantitative polymerase chain reaction was used to detect the expression of circFBXW7 in SiHa cells. pcDNA and pcDNA-circFBXW7 were transfected into SiHa cells, respectively, and si-NC and si-circFBXW7 were transfected into SiHa cells after adding 60 μmol/L kaempferol treatment for 24 h. The effects of circFBXW7 and its knockdown, circFBXW7, on the proliferation, migration, and invasion of SiHa cells were investigated, and the effects of E-cadherin and N-cadherin protein expression levels were detected by Western Blot. Results:Compared with the control group, the cell value-added, migration, and invasion abilities of the low, medium, and high dose groups were decreased (all P < 0.05) and were dose-related, and the expression of circFBXW7 was increased ( P < 0.05). After transfection with pcDNA-circFBXW7, the expression of circFBXW7 increased ( P < 0.05), while promoting the proliferation, cell migration, and invasion of kaempferol on SiHa cells (all P < 0.05). After transfection with si-circFBXW7, the expression of circFBXW7 decreased ( P < 0.05), while inhibiting the proliferation, cell migration, and invasion of kaempferol on SiHa cells (all P < 0.05). That indicated that the transfection of si-circFBXW7 could attenuate the inhibitory effects of kaempferol on the above oncogenic phenotypes of SiHa cells. Compared with the control group, E-cadherin expression was upregulated, and N-cadherin expression was downregulated in the low, medium, and high dose groups (all P < 0.05) in a dose-related manner. After transfection of pcDNA-circFBXW7 with SiHa cells, the expression of E-cadherin was increased, and the expression of N-cadherin was decreased (all P < 0.05). After transfection of si-circFBXW7 with SiHa cells, the expression of E-cadherin decreased, and the expression of N-cadherin increased (all P < 0.05). Conclusions:Kaempferol can reduce the proliferation, migration, and invasion abilities of SiHa cells by promoting circFBXW7 expression.
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OBJECTIVE@#To investigate the effects of expression levels of S100 calcium-binding protein A10 (S100A10) in lung adenocarcinoma (LUAD) on patient prognosis and the regulatory role of S100A10 in lung cancer cell proliferation and metastasis.@*METHODS@#Immunohistochemistry was used to detect the expression levels of S100A10 in LUAD and adjacent tissues, and the relationship between S100A10 expression and clinicopathological parameters and prognosis of the patients was statistically analyzed. The lung adenocarcinoma expression dataset in TCGA database was analyzed using gene enrichment analysis (GSEA) to predict the possible regulatory pathways of S100A10 in the development of lung adenocarcinoma. Lactate production and glucose consumption of lung cancer cells with S100A10 knockdown or overexpression were analyzed to assess the level of glycolysis. Western blotting, CCK-8 assay, EdU-594 assay, and Transwell assays were performed to determine the expression level of S100A10 protein, proliferation and invasion ability of lung cancer cells. A549 cells with S100A10 knockdown and H1299 cells with S100A10 overexpression were injected subcutaneously in nude mice, and tumor growth was observed.@*RESULTS@#The expression level of S100A10 was significantly upregulated in LUAD tissues as compared with the adjacent tissues, and an elevated S100A10 expression level was associated with lymph node metastasis, advanced tumor stage and distant organ metastasis (P < 0.05), but not with tumor differentiation or the patients' age or gender (P > 0.05). Survival analysis showed that elevated S100A10 expressions in the tumor tissue was associated with a poor outcome of the patients (P < 0.001). In the lung cancer cells, S100A10 overexpression significantly promoted cell proliferation and invasion in vitro (P < 0.001). GSEA showed that the gene sets of glucose metabolism, glycolysis and mTOR signaling pathway were significantly enriched in high expressions of S100A10. In the tumor-bearing nude mice, S100A10 overexpression significantly promoted tumor growth, while S100A10 knockdown obviously suppressed tumor cell proliferation (P < 0.001).@*CONCLUSION@#S100A10 overexpression promotes glycolysis by activating the Akt-mTOR signaling pathway to promote proliferation and invasion of lung adenocarcinoma cells.
Subject(s)
Animals , Mice , Humans , Adenocarcinoma of Lung/pathology , Cell Proliferation , Lung Neoplasms/pathology , Mice, Nude , Proto-Oncogene Proteins c-akt , Signal Transduction , TOR Serine-Threonine Kinases , S100 Proteins/geneticsABSTRACT
@#Objective To investigate the effects of miR-130b-5p targeting E26 transformation specific-1(ETS1)on proliferation,migration and invasion of prostatic cancer(PCa)cells and its mechanism. Methods The mRNA transcription level of miR-130b-5p gene in PCa tissues,adjacent tissues,(LNCap,PC-3,DU-145)and normal prostate cells(RPWE-1)PCa cells was measured by qRT-PCR,and the expression of ETS1 protein in PCa cells was detected by Western blot. Bioinformatics,fluorescein experiment,qRT-PCR and Western blot were used to predict and verify the targeting relationship between miR-130b-5p and ETS1. PC-3 cells were divided into control group(without any treatment),mimic group(transfected with miR-130b-5p mimic)and mimic + ETS1 group(transfected with miR-130b-5p mimic + pcDNA-ETS1). The cells were detected for the proliferation and viability by clone formation assay and CCK-8 respectively,measured for the migration and invasion by scratch test and Transwell chamber assay,and detected for the expression of invasion-related proteins and PI3K/AKT/mTOR pathway-related proteins by Western blot. Results The transcription level of miR-130b-5p mRNA in PCa tissues was significantly lower than that in adjacent tissues(t = 12. 450,P < 0. 001);Compared with RPWE-1 cells,the transcription level of miR-130b-5p mRNA in LNCap,PC-3 and DU-145 cells decreased significantly(t = 4. 463,7. 103 and 5. 741,P = 0. 001 2,< 0. 001 and < 0. 001,respectively),while the expression level of ETS1protein increased significantly(t = 4. 850,9. 325 and 7. 723,P = 0. 008,< 0. 001 and = 0. 002,respectively). miR-130-5p targeted and negatively regulated the expression of ETS1. Compared with the control group,the cloning rate,viability and scratch healing rate of cells in mimic group decreased significantly(t = 11. 370,10. 640 and 15. 660,respectively,each P < 0. 001),the number of invasive cells decreased significantly(t = 10. 160,P < 0. 001),the expression levels of matrix metalloproteinase-2(MMP-2),MMP-9 and vimentin decreased significantly(t = 15. 120,9. 992 and 12. 600,P < 0. 001,< 0. 001 and = 0. 002,respectively),while the expression level of E-cadherin increased significantly(t = 6. 928,P < 0. 001),and the phosphorylation levels of phosphatidylinositol-3 kinase(PI3K),protein kinase B(AKT)and mammalian target of rapamycin(mTOR)decreased significantly(t = 7. 746,8. 041 and 11. 510,P = 0. 002,0. 002,and < 0. 001,respectively);Compared with mimic group,the cell cloning rate,viability,scratch healing rate significantly increased in mimic + ETS1 group(t = 6. 988,6. 642 and 6. 660,respectively,each P < 0. 001),the number of invasive cells significantly increased(t = 4. 082,P = 0. 002),the expression levels of MMP-2,MMP-9 and vimentin proteins were significantly up-regulated(t = 10. 410,6. 754 and 8. 521,P = 0. 002,0. 003 and 0. 002,respectively),however,the expression level of E-cadherin was significantly down-regulated(t = 4. 648,P < 0. 01),and the phosphorylation levels of PI3K,AKT and mTOR were significantly up-regulated(t = 4. 850,4. 323 and 10. 840,P = 0. 008,0. 008 and < 0. 001,respectively)Conclusion miR-130b-5p targets ETS1 to inhibit the proliferation,migration and invasion of PC-3 cells,which may be through the regulation of PI3K/AKT/mTOR signaling pathway.
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Objective:To investigate the effect of transcription factor nuclear factor IB (NFIB) on cell proliferation and invasion in breast cancer.Methods:The lentivirus pLKO.1-shNFIB plasmid was constructed, packaged and infected with human estrogen receptor positive (ER + ) breast cancer cell line MCF-7 and triple-negative breast cancer (TNBC) cell line MDA-MB-231, respectively, NFIB was stably knocked down and verified by Western blot method; Cell count test (CCK-8) and clone formation test were used to investigate the effect of knockdown NFIB on the growth and proliferation of breast cancer cells; The transwell experiment and Western blot method were performed to detect the expression of epithelial mesenchymal transition protein markers. The effect of knockdown NFIB on the invasive ability of triple-negative breast cancer cells was explored; Kaplan-Meier survival was used to analyze web data (http: //kmplot.com/analysis/) to explore the effect of NFIB on the prognosis of ER + breast cancer and triple-negative breast cancer patients. Results:In MCF-7 and MDA-MB-231 breast cancer cells, knocking down NFIB inhibited cell growth and proliferation; In triple-negative breast cancer MDA-MB-231 cells, knocking down NFIB promoted the expression of interstitial marker fibronectin and promoted cell invasion; The lower the expression of NFIB, the worse the prognosis of triple negative breast cancer patients, while the expression of NFIB had no effect on the prognosis of ER + breast cancer patients. Conclusions:Knocking down NFIB inhibits the proliferation of MCF-7 cells, and the expression level of NFIB is not related to the prognosis of ER + breast cancer patients; Knocking down NFIB inhibits the proliferation of MDA-MB-231 cells but promotes their invasion; The low expression of NFIB is associated with the poor prognosis of triple-negative breast cancer patients.
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OBJECTIVE@#To evaluate the effects of CLEC5A expression level on cell proliferation, migration and invasion and epithelial-mesenchymal transition (EMT) in hepatocellular carcinoma (HCC) and explore the role of CLEC5A in the tumorigenesis and progression of HCC.@*METHODS@#The expression level of CLEC5A was detected in 50 pairs of HCC and adjacent tissues using immunohistochemical staining, and its association with clinicopathological parameters of HCC patients was analyzed. Cultured HCC cell line SK-HEP-1 was transfected with a lentiviral vector overexpressing CLEC5A, and the transfection efficiency was verified using real-time fluorescence quantitative PCR and Western blotting. The changes in proliferation, migration and invasion abilities of the transfected cells were analyzed using CCK-8, 5-ethynyl-29-deoxyuridine (EdU) and Transwell assays, and EMT of the cells was determined using Western blotting.@*RESULTS@#The protein expression level of CLEC5A was significantly lower in HCC tissues than in the adjacent tissues (P < 0.001). The expression level of CLEC5A was significantly correlated with tumor size (P=0.008), tumor number (P=0.010), histological differentiation (P=0.016), microvascular invasion (P=0.024) and BCLC stage (P=0.040). In SK-HEP-1 cells, overexpression of CLEC5A obviously inhibited the cell proliferation, migration and invasion and reversed EMT phenotype of the cells.@*CONCLUSION@#CLEC5A is a potential HCC suppressor gene and may serve as a promising therapeutic target for HCC.
Subject(s)
Humans , Carcinoma, Hepatocellular/genetics , Epithelial-Mesenchymal Transition , Liver Neoplasms/genetics , Cell Proliferation , Cell Differentiation , Receptors, Cell Surface/genetics , Lectins, C-Type/geneticsABSTRACT
Objective:To explore the expression of long non-coding RNA (lncRNA) CDK5RAP3 in gastric cancer tissue and its regulatory effect on gastric cancer cell proliferation and invasion.Methods:The expression differences of CDK5RAP3 in gastric cancer tissues and adjacent tissues were analyzed by TCGA database. By transfecting the pcDNA3.1-CDK5RAP3 plasmid into Hs-746T cells, a gastric cancer cell line overexpressing CDK5RAP3 (CDK5RAP3 group) was constructed, and the pcDNA3.1 plasmid was transfected into Hs-746T cells as a control group. The changes of CDK5RAP3 expression in the two groups of cells were detected by real-time quantitative PCR (qRT-PCR). The effects of overexpression of CDK5RAP3 on the proliferation and invasion of Hs-746T cells were detected by CCK-8 assay and Transwell assay, respectively. The binding sites of CDK5RAP3 and miR-223-3p were predicted by the starBase v2.0 database. The direct binding of CDK5RAP3 and miR-223-3p was verified by dual-luciferase reporter gene experiment. The expression levels of miR-223-3p in Hs-746T cells in each group were detected by qRT-PCR. Western blot was used to detect the expression levels of proliferation proteins and invasion proteins in Hs-746T cells in each group. The experimental data were analyzed by SPSS 17.0 software, and the measurement data conforming to the normal distribution were expressed as Mean±SD. The t-test was used to compare between two groups, and the one-way analysis of variance was used to compare the means of multiple groups. Results:Compared with adjacent tissues, the expression level of CDK5RAP3 in gastric cancer tissues was significantly lower ( P<0.01). The expressions of CDK5RAP3 in Hs-746T cells in the control group and CDK5RAP3 group were (1.08±0.77) and (10.63±2.14), respectively, and the difference was statistically significant ( P<0.01). Up-regulation of CDK5RAP3 significantly decreased the proliferation activity of Hs-746T cells ( P<0.05). The number of invasive cells in the control group and CDK5RAP3 group were (137.80±28.72) and (57.76±24.95), respectively, and the difference was statistically significant ( P<0.01). CDK5RAP3 could directly bind miR-223-3p ( P<0.01). The expression of miR-223-3p in Hs-746T cells in control group and CDK5RAP3 group were (6.22±1.20) and (1.01±0.98), respectively, and the difference was statistically significant ( P<0.01). Compared with the control group, up-regulation of CDK5RAP3 significantly reduced the expression levels of proliferation and invasive proteins. Conclusion:The expression of CDK5RAP3 is low in gastric cancer tissue, and CDK5RAP3 inhibits the proliferation and invasion of gastric cancer Hs-746T cells by targeting miR-223-3p.
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Objective To investigate the effects of Tspan8 gene knockout combined with anlotinib on the proliferation, migration, invasion, and apoptosis of colon cancer SW480 cells. Methods The plasmid was constructed by CRISPR/Cas9 technique, and Tspan8 gene was knocked out in SW480 cells. The knockout effect was detected by Western blot. The IC50 of anlotinib was calculated by MTT assay. The experiment was divided into control group, anlotinib group, Tspan8 knockout group, and combined group. Cell proliferation, migration, invasion, and apoptosis were detected by cell proliferation assay, clonal formation assay, scratch assay, Transwell chamber assay, and flow cytometry. Western blot was used to detect the effect of anlotinib on Tspan8 expression in SW480 cells. Results SW480-KO-Ⅲ cells had the highest knockout efficiency in the Tspan8 knockout group. They were used in subsequent experiments. Different concentrations of anlotinib could inhibit the proliferation of SW480 cells at different times (P < 0.01) in a concentration-dependent and time-dependent manner (P < 0.01). According to IC50, 14 μmol/L was selected as the subsequent experimental concentration. Compared with those in the control group, the cell proliferation, migration, and invasion abilities in the anlotinib group, Tspan8 knockout group, and combined group were significantly decreased, and the cell apoptosis level was significantly increased (P < 0.05). The above changes in the combined group were more significant than those in the anlotinib group or Tspan8 knockout group (P < 0.05). Compared with that in the control group, the expression level of Tspan8 in SW480 cells in the anlotinib group was significantly decreased (P < 0.01). Conclusion Tspan8 gene knockout combined with anlotinib can synergistically inhibit the proliferation, migration and invasion of SW480 cells. This combination can also promote the apoptosis of these cells.
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AIM: To investigate the effect and mechanism of matrix metalloproteinase(MMPs)inhibitor AG3340 on the migration and invasion ability of retinal pigment epithelial cells-19(ARPE-19)cultured in high glucose(CHG). METHODS: ARPE-19 cells cultured in vitro were divided into four groups: Control group, the glucose at the concentration of 5.6mmol/L in DMEM/F12 medium; HG group, the glucose at the concentration of 30mmol/L was cultured with DMEM/F12 medium; HG+AG3340 group, the cells were pretreated with AG3340 for 12h, and then cultured in DMEM/F12 medium containing 30mmol/L glucose; The mannitol(MA)group, cultured with DMEM/F12 medium of 5.6mmol/L glucose and 24.4mmol/L mannitol, which used as hypertonic control group. The migration ability of cells was detected by wound healing assay, the invasion ability of cells was detected by Transwell assay, and the relative expression levels of MMP-9, MMP-2, fibronectin and collagen Ⅳ were detected by Western blot.RESULTS: The results of wound healing assay showed that compared with the Control group, the cell migration rate of scratching after 24h and 48h in the HG group was significantly increased(all P<0.001).After pretreated by AG3340, the cell migration rate was significantly lower than that in the HG group(all P<0.01).Transwell assay showed that compared with the Control group, the number of cell invasion in the HG group was significantly higher than that in the Control group(all P<0.001). After pretreated by AG3340, the number of cell invasion was decreased than the HG group(all P<0.01). Western blot results showed that compared with the Control group, the relative expression levels of MMP-9 and MMP-2 of the cells in the HG group were increased, and the relative expression levels of Fibronectin and Collagen Ⅳ were decreased(all P<0.001). Compared with the HG group, the relative expression levels of MMP-9 and MMP-2 protein in AG3340 pretreatment group were decreased, and the relative expression levels of Fibronectin and Collagen Ⅳ were increased(all P<0.05). CONCLUSION: High glucose induced ARPE-19 cells with enhanced migration and invasion ability, and AG3340 partially reversed this effect, which was related to the inhibition of MMP-9 and MMP-2 expression and the stability of extra-cellular matrix components.
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Objective:To investigate the effects of solute carrier family 39 (SLC39) A14 on proliferation, migration and invasion of diffuse large B-cell lymphoma (DLBCL) OCI-LY3 cells.Methods:The human DLBCL cell line OCI-LY3 was divided into Vector group (transfected with empty control plasmid) and SLC39A14 group (transfected with SLC39A14 plasmid). The proliferation of OCI-LY3 cells in the two groups was detected by CCK-8 method, the migration and invasion of cells were detected by Transwell method, and the expression level of SLC39A14 protein and the expressions of PI3K-AKT-mTOR signaling pathway-related proteins in OCI-LY3 cells were detected by Western blotting.Results:Compared with the Vector group, the cell proliferation ability in the SLC39A14 group was increased from day 3 to day 5 (all P < 0.05).The results of Transwell cell migration assay showed that the number of migrating cells after 36 h in the Vector group was (64±4) cells, and that in the SLC39A14 group was (236±25) cells. The cell migration ability in the SLC39A14 group was increased, and the difference was statistically significant ( t = 15.02, P < 0.05). The results of Transwell cell invasion assay showed that the number of invasive cells in the Vector group was (32±2) cells, and that in the SLC39A14 group was (127±17) cells. The cell invasion ability in the SLC39A14 group was increased, and the difference was statistically significant ( t = 8.33, P < 0.05).The results of Western blotting showed that the expression levels of pmTOR, pAKT and pPI3K proteins in the SLC39A14 group were all increased. Conclusions:SLC39A14 may be involved in the occurrence and development of DLBCL through PI3K-AKT-mTOR signaling pathway.
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Objective:To investigate the effects of miRNA-628-3p (miR-628-3p) on the proliferation, apoptosis and invasion of non-small cell lung cancer H1299 cells and its targeting relationship with insulin-like growth factor 1 receptor (IGF-1R).Methods:The blank control group (untreated H1299 cells), miR-NC group (H1299 cells transfected with empty plasmid), miR-628-3p-M group (H1299 cells transfected with miR-628-3p mimic sequence plasmid) and miR-628-3p-I group (H1299 cells transfected with miR-628-3p inhibitory sequence plasmid) were established. The cells in each group were cultured for 72 h, and the cell proliferation ability was detected by methyl thiazol tetrazolium (MTT) method, the number of cell monoclonal formation was determined by crystal violet staining, the level of cell apoptosis was determined by flow cytometry, and the cell invasion ability was determined by Transwell method. The mRNA levels of miR-628-3p and IGF-1R in cells were determined by real-time fluorescence quantitative reverse transcription polymerase chain reaction (qRT-PCR), and the protein level of IGF-1R in cells was determined by Western blotting.Results:Compared with the blank control group and miR-NC group, the cell survival rate [(42±7)% vs. (78±6)%, (76±7)%], the number of monoclonal formation [235±35 vs. 614±89, 618±75], the number of invasive cells [(265±85) cells vs. (693±185) cells, (703±119) cells], relative expression of IGF-1R mRNA (2.17±0.14 vs. 3.38±0.15, 3.37±0.13) and relative expression of IGF-1R protein (0.34±0.13 vs. 0.89±0.19, 0.88±0.18) in the miR-628-3p-M group were lower (all P < 0.05), but the apoptosis rate [(9.30±3.51)% vs. (3.30±1.54)%, (3.10±1.94)%] and relative expression of miR-628-3p (6.93±0.17 vs. 3.29±0.15, 3.30±0.16) were higher (all P < 0.05); the cell survival rate [(90±6)%], the number of monoclonal formation (1 063±102), the number of invasive cells [(1 985±426) cells], relative expression of IGF-1R mRNA (4.30±0.18) and relative expression of IGF-1R protein (1.47±0.17) in the miR-628-3p-I group were higher (all P < 0.05), but the apoptosis rate [(0.90±0.20)%] and the relative expression of miR-628-3p (1.93±0.18) were lower (both P < 0.05). Compared with the miR-628-3p-M group, the miR-628-3p-I group had higher cell survival rate, the number of monoclonal formation, the number of invasive cells, and the relative expressions of IGF-1R mRNA and protein (all P < 0.05), but the apoptosis rate and relative expression of miR-628-3p were lower (both P < 0.05). Conclusions:After regulation of miR-628-3p level, the proliferation, migration and invasion of H1299 cells are affected. miR-628-3p may have a targeting relationship with IGF-1R.
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Objective:To investigate the expression difference of miR-3189-3p in renal cancer tissue and adjacent tissue and its effect on the biological function of renal cancer cells.Methods:quantitative real-time polymerase chain reaction (qRT-PCR) was used to detect the expression level of miR-3189-3p in renal cancer tissues and adjacent tissues, renal cancer cell lines (Caki-1, ACHN, A498, OS-RC-2) and normal renal tubular epithelial cells HK-2. miR-NC or miR-3189-3p mimics were transfected into renal cancer cells with the lowest expression of miR-3189-3p, respectively, named miR-NC group and miR-3189-3p group. The effects of miR-3189-3p on the proliferation and invasion of renal cancer cells were detected by CCK-8 method and Transwell migration experiment. miRanda and miRTarBase software was used to predict the downstream gene of miR-3189-3p. The dual luciferase reporter gene experiment was used to verify the downstream gene of miR-3189-3p. qRT-PCR and Western blotting were used to detect the expression of miR-3189-3p downstream gene. Measurement data were expressed as mean ± standard deviation ( ± s), and t-test was used for comparison between groups. Results:The relative expression of miR-3189-3p in renal cancer tissue and paracancerous tissue was 1.97±0.61 and 6.19±0.73, respectively, and the relative expression of miR-3189-3p in renal cancer tissue was lower than that in paracancerous tissue ( P<0.01). The relative expression of miR-3189-3p in renal cancer cell lines was lower than that in HK-2 cells ( P<0.05). The relative expression of miR-3189-3p in OS-RC-2 cells was the lowest ( P<0.01). The relative expression levels of miR-3189-3p in OS-RC-2 cells in the miR-NC group and miR-3189-3p group were 1.01±0.11 and 9.27±1.43, respectively, and the relative expression levels of miR-3189-3p in the miR-NC group significantly lower than the miR-3189-3p group ( P<0.01). Compared with the miR-NC group, the proliferation ability of OS-RC-2 cells with high expression of miR-3189-3p was significantly reduced ( P<0.05). The numbers of penetrating cells in the miR-NC group and miR-3189-3p group were 165.40±17.02 and 41.07±6.36, respectively, and the invasive ability of OS-RC-2 cells in the miR-3189-3p group was significantly reduced ( P<0.01). The target gene of miR-3189-3p is Aquaporin 3 ( AQP3) and miR-3189-3p can target AQP3 mRNA ( P<0.01). Compared with the miR-NC group, the expression of AQP3 gene in the high-expressing miR-3189-3p cells was significantly reduced at both the mRNA level and the protein level ( P<0.01). Conclusions:The expression of miR-3189-3p is down-regulated in renal cell carcinoma. High expression of miR-3189-3p can significantly inhibit the proliferation and invasion of renal cell carcinoma OS-RC-2 cells. The molecular mechanism is that miR-3189-5p targets and inhibits the expression of AQP3 gene.
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The aim of this study was to explore the effect of hsa_circ_0002162 on regulating cell proliferation, apoptosis, and invasion, and investigate its potential target microRNA (miRNA) in tongue squamous cell carcinoma (TSCC). Hsa_circ_0002162 expression was detected in human TSCC cell lines and human oral keratinocytes (HOK) cell line. Cell proliferation, apoptosis, invasion, and candidate target miRNA expressions were detected in hsa_circ_0002162 knockdown-treated CAL-27 cells and hsa_circ_0002162 overexpression-treated SCC-9 cells. In the rescue experiment, miR-33a-5p knockdown plasmid was transfected into hsa_circ_0002162 knockdown-treated CAL-27 cells, while miR-33a-5p overexpression plasmid was transfected into hsa_circ_0002162 overexpression-treated SCC-9 cells. Subsequently, cell proliferation, apoptosis, and invasion were detected, and then luciferase reporter assay was performed. Hsa_circ_0002162 expression was increased in human TSCC cell lines SCC-9, CAL-27, HSC-4, and SCC-25 compared with HOK. In CAL-27 cells, hsa_circ_0002162 knockdown inhibited cell proliferation and invasion and promoted apoptosis. In SCC-9 cells, hsa_circ_0002162 overexpression enhanced cell proliferation and invasion and suppressed apoptosis. Furthermore, a negative regulation of hsa_circ_0002162 on miR-33a-5p (but not miR-302b-5p and miR-545-5p) was observed. In the rescue experiment, miR-33a-5p knockdown increased cell proliferation and invasion, and decreased apoptosis in hsa_circ_0002162 knockdown-treated CAL-27 cells, whereas miR-33a-5p overexpression decreased cell proliferation and invasion, but increased apoptosis in hsa_circ_0002162 overexpression-treated SCC-9 cells. The luciferase reporter assay showed the direct binding of hsa_circ_0002162 to miR-33a-5p. In conclusion, hsa_circ_0002162 had an important role in malignant progression of TSCC through targeting miR-33a-5p.
Subject(s)
Humans , Tongue Neoplasms/genetics , Carcinoma, Squamous Cell/genetics , MicroRNAs/genetics , Tongue , Cell Line, Tumor , RNA, CircularABSTRACT
Objective:To explore the molecular mechanism of microRNA (miRNA, miR)-1914-3p regulating the expression of ARL4C and affecting the invasion and proliferation of renal cancer cells.Methods:Real-time fluorescent quantitative polymerase chain reaction (qRT-PCR) was used to detect the expression level of miR-1914-3p in tumor tissues and adjacent tissues of 53 renal cancer patients, 4 types of renal cancer cell lines (ACHN, OS-RC-2, 786-O, A498) and normal proximal renal tubular epithelial cell line (HK-2). The nonsense sequence (NC) and miR-1914-3p mimic were transiently transfected into renal cancer cells with the lowest miR-1914-3p expression by liposome method, namely the NC group and miR-1914-3p group. qRT-PCR was used to detect the expression level of miR-1914-3p in transfected cells. Transwell invasion test and cell counting kit-8 (CCK-8) were used to detect the invasion and proliferation ability of each group of cells. Bioinformatics software and dual luciferase gene report experiment were used to predict and test the targeted regulation mechanism of miR-1914-3p on target genes. qRT-PCR and Western blot was conducted to analyze the target gene expression level in cells of each group.Results:The expression level of miR-1914-3p in renal cancer tissue was significantly lower than that in adjacent tissues ( P<0.01). The expression level of miR-1914-3p in renal cancer cell lines was significantly lower than that in HK-2 cell lines ( P<0.01), and the expression of miR-1914-3p in OS-RC-2 cells was the lowest ( P<0.01). The expression of miR-1914-3p in the NC group and the miR-1914-3p group were (1.04±0.17) and (11.40±0.91), respectively. The expression level of miR-1914-3p in the miR-1914-3p group was significantly increased ( P<0.01), indicating that the transfection was successful. Overexpression of miR-1914-3p can significantly inhibit the invasion ( P<0.01) and proliferation ( P<0.05) of renal cancer OS-RC-2 cells. Dual luciferase gene report experiment indicated that the target gene of miR-1914-3p may be ADP-ribosylation factor-like 4C (ARL4C); miR-1914-3p can significantly inhibit the luciferase activity of wild-type ARL4C-3′UTR ( P<0.01). Overexpression of miR-1914-3p decreased the expression of ARL4C mRNA and protein in OS-RC-2 cells ( P<0.01), and decreased the expression of cell invasion phenotype proteins (Snail, Slug) and cell proliferation phenotype proteins (Mcm2, Mcm7) ( P<0.01). Conclusions:miR-1914-3p is low-expressed in renal cell carcinoma. It inhibits the invasion and proliferation of renal cell carcinoma OS-RC-2 cells through targeted interference with the expression of the oncogene ARL4C, and participates in the occurrence and development of renal cell carcinoma.
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Objective:To investigate the effect of propofol on proliferation, invasion and migration of human melanoma cell line A375 via miR-137/fibroblast growth factor 9 (FGF9) pathway.Methods:A375 cells were cultured in vitro. The half inhibitory concentration and half inhibitory time of propofol on A375 cells were determined by methyl thiazolyl tetrazolium (MTT) method. miR-137 mimics and miR-137 inhibitors were transfected into A375 cells by lipofectamine method. A375 cells were divided into control group, propofol group, miR-137 mimics group, propofol+ miR-137 mimics group, miR-137 inhibitors group and propofol+ miR-137 inhibitors group. After treated with the optimal time and concentration, the mRNA expression of miR-137 and FGF9 was detected by real time fluorescence quantitative reverse transcription polymerase chain reaction (qRT-PCR), and the invasion and migration ability of cells in vitro was detected by transwell method. Results:With the increase of propofol concentration, the proliferation rate of A375 cells was gradually decreased, and 80 μmol/L was selected as the half inhibition concentration. With the increase of propofol action time, the proliferation rate of A375 cells was gradually decreased, and 48 hours was selected as the half inhibition time. Compared with the control group, propofol could promote the expression of miR-137 mRNA, inhibit the expression of FGF9 mRNA, and inhibit the invasion and migration of A375 cells ( P<0.05). miR-137 mimics could promote the expression of miR-137 mRNA, inhibit the expression of FGF9 mRNA, and inhibit the invasion and migration of A375 cells. At the same time, after propofol intervention, the effect of promoting the expression of miR-137 mRNA, inhibiting the expression of FGF9 mRNA and inhibiting the invasion and migration of A375 cells was more significant ( P<0.05); miR-137 inhibitors could inhibit the expression of miR-137 mRNA, promote the expression of FGF9 mRNA, and promote the invasion and migration of A375 cells ( P<0.05). At the same time, after propofol intervention, the effects of inhibiting the expression of miR-137 mRNA, promoting the expression of FGF9 mRNA and promoting the invasion and migration of A375 cells were inhibited ( P<0.05). Conclusions:Propofol can inhibit the proliferation, invasion and migration of human melanoma cell line A375. The mechanism may be related to the inhibition of miR-137/FGF9 pathway activation by propofol.
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Objective:To observe the expression of microRNA (miRNA, miR)-5787 in breast cancer tissues and various cell lines, analyze the effect of overexpression of miR-5787 on breast cancer cell invasion and proliferation, and explore its possible molecular mechanism.Methods:Real-time fluorescence quantitative polymerase chain reaction (qRT-PCR) method was used to detect the expression of miR-5787 in 47 breast cancer tissues and adjacent tissues, 4 breast cancer cell lines and normal breast epithelial cells. Breast cancer cell lines with the lowest miR-5787 expression were selected and transfected with miR-5787 mimics (experimental group) and control NC mimics (control group), respectively. qRT-PCR was used to detect the expression of miR-5787 in cells of the two groups. Transwell invasion experiment and cell counting kit-8 (CCK-8) were used to detect the effect of miR-5787 overexpression on breast cancer cell invasion and proliferation. Bioinformatics software and dual luciferase reporter gene experiments were used to predict and verify the target genes that miR-5787 could complementally bind. qRT-PCR and Western blot were used to detect the expression of target gene mRNA and protein.Results:Compared with adjacent tissues (5.05±0.82), the expression of miR-5787 in breast cancer tissues (1.32±0.33) was significantly reduced ( P<0.01). Compared with normal breast epithelial cells, the expression of miR-5787 in the four breast cancer cell lines was reduced ( P<0.05), and the expression in HCC1937 cells was the lowest ( P<0.01). After transfection of miR-5787 mimics, the expression of miR-5787 in HCC1937 cells in the experimental group was significantly higher than that in the control group ( P<0.01). Overexpression of miR-5787 could inhibit the invasion ( P<0.05) and proliferation ( P<0.05) of breast cancer HCC1937 cells. Bioinformatics software showed that the target gene of miR-5787 might be heparan sulfate proteoglycan 2 (HSPG2), and miR-5787 could complement HSPG2 mRNA ( P<0.01). qRT-PCR and Western blot indicated that overexpression of miR-5787 could significantly inhibit the expression of HSPG2 gene ( P<0.01), with the decreased expression of Vimentin, N-cadherin, Ki67 and PCNA. Conclusions:miR-5787 expression was low in breast cancer tissues and cell lines. Overexpression of miR-5787 could inhibit the invasion and proliferation of breast cancer HCC1937 cells by interfering with the expression of HSPG2 gene.