Objective To investigate the effect of resveratrol on the proliferation,apoptosis,and migration of breast cancer cells and its mechanism and correlation with immune cell infiltration.Methods The network pharmacology and bioinformatics(GEPIA and KM-PLOT databases)were used to predict the target genes of breast cancer regulated by resveratrol,and their expression levels in breast cancer and correlations with immune cell infiltration were verified.The effects of resveratrol on the proliferation,cell cycle and apopto-sis,and migration of breast cancer cells were determined by the CCK-8 assay,flow cytometry,and cell scratch test,respectively.The mRNA expression levels of related target genes were detected by real-time fluorescence quantitative polymerase chain reaction(qRT-PCR).Pearson correlation analysis was used to analyze the correlations of the expression levels of related genes with immune cell infiltration.Results The expression levels of TP53 and VEGFA genes in breast cancer tissues were significantly higher than those in normal tissues(Z=-4.181 and-8.763,P＜0.05).In the GEPIA database,the expression level of CDKN1A gene in breast cancer tis-sues was significantly higher than that in normal tissues(Z=-2.135,P＜0.05).In the KMPLOT database,the expression level of HIF1α gene in breast cancer tissues was significantly higher than that in normal tissues(Z=-2.269,P＜0.05).Pearson correlation a-nalysis showed that the expression level of TP53 gene was positively correlated with B cell infiltration(partial.cor=0.102,P＜0.01).The expression level of HIF-1α was positively correlated with the infiltration of CD8+T cells,CD4+T cells,monocytes,neutrophils,and dendritic cells(partial.cor=0.39,0.159,0.284,0.325,and 0.294,P＜0.01).The expression level of VEGFA gene was positive-ly correlated with neutrophil infiltration(partial.cor=0.141,P＜0.01).The expression level of CDKN1A gene was positively correlated with the infiltration of CD8+T cells,monocytes,neutrophils,and dendritic cells(partial.cor=0.081,0.209,0.11,and 0.09,P＜0.01).Resveratrol could inhibit the proliferation of breast cancer cells,and the IC50 and IC25 values of breast cancer cells were 150 μmol/L and 50 μmol/L,respectively,after 48 hours of treatment with resveratrol.In addition,resveratrol could also arrest breast cancer cells in G1 phase,induce their apoptosis,and inhibit their migration.After treating breast cancer cells with resveratrol for 24 hours,with the increase of resveratrol concentration from 50 μmol/L to 150 μmol/L,the mRNA expression levels of TP53 and HIF1α genes increased gradually,with a significant difference between the two groups(P＜0.05).After treating breast cancer cells with res-veratrol for 48 hours,the expression level of VEGFA mRNA was significantly decreased,and there was a significant difference between the two groups(P＜0.05).Conclusion TP53,HIF-1α,VEGFA,and CDKN1A genes are the action targets of resveratrol in breast cancer,which are mainly involved in the regulation of immune cell infiltration in breast cancer.Resveratrol inhibits the proliferation of breast cancer cells,induces cell cycle arrest and apoptosis,and inhibits cell migration by regulating the P53-HIF1α/CDKN1A-VEGFA signaling pathway.

Objective To investigate the effect of resveratrol on the proliferation,apoptosis,and migration of breast cancer cells and its mechanism and correlation with immune cell infiltration.Methods The network pharmacology and bioinformatics(GEPIA and KM-PLOT databases)were used to predict the target genes of breast cancer regulated by resveratrol,and their expression levels in breast cancer and correlations with immune cell infiltration were verified.The effects of resveratrol on the proliferation,cell cycle and apopto-sis,and migration of breast cancer cells were determined by the CCK-8 assay,flow cytometry,and cell scratch test,respectively.The mRNA expression levels of related target genes were detected by real-time fluorescence quantitative polymerase chain reaction(qRT-PCR).Pearson correlation analysis was used to analyze the correlations of the expression levels of related genes with immune cell infiltration.Results The expression levels of TP53 and VEGFA genes in breast cancer tissues were significantly higher than those in normal tissues(Z=-4.181 and-8.763,P＜0.05).In the GEPIA database,the expression level of CDKN1A gene in breast cancer tis-sues was significantly higher than that in normal tissues(Z=-2.135,P＜0.05).In the KMPLOT database,the expression level of HIF1α gene in breast cancer tissues was significantly higher than that in normal tissues(Z=-2.269,P＜0.05).Pearson correlation a-nalysis showed that the expression level of TP53 gene was positively correlated with B cell infiltration(partial.cor=0.102,P＜0.01).The expression level of HIF-1α was positively correlated with the infiltration of CD8+T cells,CD4+T cells,monocytes,neutrophils,and dendritic cells(partial.cor=0.39,0.159,0.284,0.325,and 0.294,P＜0.01).The expression level of VEGFA gene was positive-ly correlated with neutrophil infiltration(partial.cor=0.141,P＜0.01).The expression level of CDKN1A gene was positively correlated with the infiltration of CD8+T cells,monocytes,neutrophils,and dendritic cells(partial.cor=0.081,0.209,0.11,and 0.09,P＜0.01).Resveratrol could inhibit the proliferation of breast cancer cells,and the IC50 and IC25 values of breast cancer cells were 150 μmol/L and 50 μmol/L,respectively,after 48 hours of treatment with resveratrol.In addition,resveratrol could also arrest breast cancer cells in G1 phase,induce their apoptosis,and inhibit their migration.After treating breast cancer cells with resveratrol for 24 hours,with the increase of resveratrol concentration from 50 μmol/L to 150 μmol/L,the mRNA expression levels of TP53 and HIF1α genes increased gradually,with a significant difference between the two groups(P＜0.05).After treating breast cancer cells with res-veratrol for 48 hours,the expression level of VEGFA mRNA was significantly decreased,and there was a significant difference between the two groups(P＜0.05).Conclusion TP53,HIF-1α,VEGFA,and CDKN1A genes are the action targets of resveratrol in breast cancer,which are mainly involved in the regulation of immune cell infiltration in breast cancer.Resveratrol inhibits the proliferation of breast cancer cells,induces cell cycle arrest and apoptosis,and inhibits cell migration by regulating the P53-HIF1α/CDKN1A-VEGFA signaling pathway.

Objective To investigate the feasibility of green fluorescent protein (GFP) as a marker to trace the transplanted um-bilical cord mesenchymal stem cells (ucMSCs) in rats with cerebral ischemia-reperfusion .Methods ucMSCs were transfected by GFP-adenovirus .The rats were subjected to left middle cerebral artery occlusion using suture method .1 × 106 GFP-ucMSCs were transplanted with cerebral stereotaxic technique .Frozen sections of brain tissue were made at 7 d after cerebral ischemia .The ex-pression of GFP was observed by fluorescence microscope .Results In vitro ,the morphology of GFP in ucMSCs was fibrous ,and when fused ,the clusters were arranged in a radial or whirlpool shape ,The morphological feature of transfected ucMSCs was similar to that un-transfected ucMSCs .Under the fluorescence microscope ,the positive rate of GFP was more than 80% .In addition ,GFP could spread to the whole cells and show the completed form .No rejection was observed in the rats after transplantation ,and the GFP was found near the site of transplantation after 7 d ,and the fluorescence was not attenuated .Conclusion GFP is an effective tracer maker for ucMSCs transplantation in the treatment of ischemia-reperfusion ,which could provide experimental method for fur-ther study .

Objective To compare the transfection efficiency of cationic polyethylenimine(PEI)with Lipofectamine 2000TM by using the plasmid DNA encoding vascular endothelial cell growth factor (VEGF165 )gene in human embryonic kidney cell line 293T.Methods PEI of different N/P ratio and Lipofectamine 2000TM were used to deliver the vector containing VEGF165 to 293T cells,respectively.Green fluorescent protein(GFP)gene was inserted into the vector as a report gene.Evaluation of cytoactive was performed by CCK-8 assay 24 h after transfection.The cells were observed by fluorescent microscope and the presence of VEGF165 in cell supernatant was detected by ELISA 48 h after transfection.The transfection efficiency was calculated and com-pared.Results Similar cytoactive and best transfection efficiency could be obtained when N/P ratio was 9,the transfection efficien-cy was around 70%.Furthermore,the presence of VEGF165 increased significantly after transfection(P <0.05),but there was no significant difference between the two groups in which different transfection methods were adopted.Conclusion PEI as a novel oli-gofectamine reagent could mediate more efficient transfection compared with lipofectamine.It also has low cell-toxicity and low price and could be an ideal vector in gene delivery technology.

Objective To construct a recombinant interfering RNA(miRNA)plasmid vector targeting annexinA3 and observe its impact on the growth and apoptosis of human gallbladder cancer cell line SGC-996.Methods Four small fragments of miRNA and one negative control were sequenced and cloned into vector pcDNATM 6.2-GW/EmGFPmiR.The recombinant plasmids were then transfected into gallbladder cancer cell line SGC-996 by positive liposomal transfection.The transcription and translation level of annexinA3 expression were detected by RT-PCR and Western Blot.Meanwhile, the proliferation and apoptosis of transfected tumor cells were determined by MTT and flow cytometry.Results The recombinant plasmids containing annexinA3 miRNA were successfully constructed.AnnexinA3 expression was significantly knocked down after miRNA plasmid transfection.Transfection of annexinA3 gene miRNA could effectively inhibit the proliferation of gallbladder cancer cell and promote apoptosis.Conclusion AnnexinA3 gene silenced by miRNA can induce the apoptosis of human gallbladder cancer cell line SGC-996.The process might serve as a potential approach for cancer gene therapy.

OBJECTIVETo observe the effect of grape seed extract (GSE) on breast cancer cell MCF-7 about the of proliferation and the gene expression of survivin, and to investigate the molecular biological mechanism of inhibition by GSE.

METHODMTT was used to measure the role of proliferated inhibition of GSE at the dosage of 40, 80, 120, 160, 200 mg x L(-1) with different time. To calculate the IC50 of GSE and to select the suitable concentration and treatment time. The change of cell cycle was detected by flow cytometry. mRNA expression of Survivin was observed by RT-PCR. Luciferase kit was used to observe the change of core promoter of survivin which was inserted in the flourescence report vector. And further to analyse the bind side of transcription factor on the sequence of core promoter of survivin was further analysed by using the microsoft of bioinformatics.

RESULTGSE could inhibit the proliferation of breast cancer cell MCF-7 with time and dosage-dependent (P < 0.05). GSE could arrest the cell cycle in S periods (P < 0.05) and also inhibit the mRNA expression of survivin effectively (P < 0.01); GSE could down-regulate the activity of core promoter of survivin significantly (P < 0.01).

CONCLUSIONGSE can inhibit the proliferation of breast cancer cell MCF-7 through arresting the cell cycle in S periods. The mechanism may be that GSE regulate the activity of transcription factor which are related to the activity of core promoter of survivin and decrease the gene expression of survivin.

Antineoplastic Agents, Phytogenic ; pharmacology ; Breast Neoplasms ; genetics ; metabolism ; pathology ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Cloning, Molecular ; Down-Regulation ; drug effects ; Female ; Gene Expression ; drug effects ; Gene Expression Regulation, Neoplastic ; Genetic Vectors ; Humans ; Inhibitor of Apoptosis Proteins ; Microtubule-Associated Proteins ; genetics ; metabolism ; Transfection ; Xenograft Model Antitumor Assays

Objective Investigate the effect of TrKA variation on the expression of neucleoprotein NF-?B P65 and apoptosis.Methods To construct the expression vector of TrKA small interfering RNA,the recombinant was transfected into MCF-7 cells.the stable cell line expressing TrKA small interfering RNA were selected by G418.The mRNA and protein of TrKA were tested by real-time PCR,Western-blot and Immunohistochemistry.The change of neucleoprotein NF-?B P65 was detected by WB,Flow cytometry was used to observe the cell apoptosis.ResultsThe expression vector of TrKA-siRNA was successfully constructed.The mRNA and protein of TrKA were decreased by 74.7% and 80.5% respectively(P