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ObjectiveTo investigate the effect of kinesin family member 15 （KIF15） on the proliferation of hepatocellular carcinoma （HCC） cells and its mechanism of action. MethodsTCGA and GEPIA datasets were analyzed to determine the expression of KIF15 in HCC and its effect on tumor stage and survival. Quantitative real-time PCR and Western blot were used to measure the expression level of KIF15 in human-derived HCC cell lines （HepG2， Hep3B， MHCC-97H， and LM3） and human normal liver cell line L02 cultured in vitro， and Hep3B and HepG2 were selected for subsequent studies. CCK-8 assay， plate colony formation assay， and EdU staining were performed for Hep3B cells transfected with shRNA-NC or shRNA-KIF15 and HepG2 cells transfected with LV-vector or LV-KIF15 to evaluate the viability and proliferative capacity of these cells. GSEA was used to analyze the potential signaling pathways associated with KIF15 in HCC， and Western blot was used for detection. The independent-samples t test was used for comparison of continuous data between two groups； a one-way analysis of variance was used for comparison between multiple groups， and the least significant difference t-test was used for further comparison between two groups. ResultsThe analysis of TCGA and GEPIA datasets showed that in HCC patients， the expression of KIF15 in HCC tissue was significantly higher than that in normal tissue， and the HCC patients with high KIF15 expression tended to have a poorer prognosis. Compared with sh-NC-Hep3B， sh3-Hep3B showed significant reductions in the mRNA and protein levels of KIF15 （P<0.05）， cell viability， clone formation number， and EdU positive rate （all P<0.05）. Compared with vector-HepG2， LV-KIF15-HepG2 showed significant increases in the mRNA and protein levels of KIF15 （P<0.05）， cell viability， clone formation number， and EdU positive rate （all P<0.05）. Subcutaneous tumor assay showed that compared with sh-NC-Hep3B， sh3-Hep3B showed reductions in tumor volume and tumor weight， as well as a significant reduction in the immunohistochemical score of Ki67 and a significant increase in the immunohistochemical score of TUNEL （P<0.05）. GSEA analysis showed that the PI3K/AKT/mTOR pathway was positively correlated with KIF15 in HCC （NES=1.59， P<0.001）. Western blot showed that LY294002 could inhibit the PI3K/AKT/mTOR pathway upregulated in LV-KIF15-HepG2， and compared with LV-KIF15-HepG2， LY294002+LV-KIF15-HepG2 showed significant reductions in cell viability， clone formation number， and EdU positive rate （all P<0.05）. ConclusionKIF15 enhances the viability and proliferative capacity of HCC cells by upregulating the PI3K/AKT/mTOR signaling pathway.
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Objective To explore the key molecular targets and possible mechanisms of Aidi injection in the treatment of ovarian cancer using network pharmacology and cell experiments.Methods TCMSP database was used to screen the active ingredients and tar-gets of Aidi injection,and the abnormal expressed genes of ovarian cancer were screened,and the possible targets of Aidi injection in o-varian cancer were obtained after intersection analysis.Then,protein-protein interaction analysis,drug-compact-target network con-struction and enrichment analysis of possible targets were performed.The target was further screened,and the key genes related to the prognosis of ovarian cancer were experimentally verified.After treated with 50mg/ml Aidi injection,the cell proliferation ability was ob-served by CCK-8 assay,and the expression of core target genes was detected by real-time quantitative polymerase chain reaction.Results A total of 13 possible targets of Aidi injection in ovarian cancer were screened.These targets were mainly enriched in signaling pathways closely related to the occurrence and development of tumors,such as apoptosis,platinum resistance and interleukin-17.Among the 13 genes,claudin 4(CLDN4),secretory leukocyte peptidase inhibitor(SLPI)and baculoviral IAP repeat containing 5(BIRC5)were associated with the prognosis of ovarian cancer.Cell experiments showed that Aidi injection significantly inhibited the proliferation of ovari-an cancer cell,promoted the expression of BIRC5,a protective target of ovarian cancer,while significantly decreased the levels of ovarian cancer risk factors CLDN4 and SLPI.Conclusion Aidi injection may achieve multi-component,multi-target and multi-pathway anti-ovarian cancer and combination chemotherapy by affecting the expression of CLDN4,SLPI and BIRC5.
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AIM:One of the important characteristics of the occurrence and development of triple-negative breast cancer(TNBC)is dysregulated cell metabolism.The aim of this study is to investigate the mechanism of pyruvate dehydrogenase E1 subunit alpha 1(PDHA1),a key enzyme component in aerobic glycolysis,affecting the proliferation,metastasis and invasion of TNBC.METHODS:(1)The expression levels of PDHA1 in breast cancer tissues and adja-cent tissues were analyzed by UALCAN database,KM-plotter database,Gene MANIA database and TCGA database.The expression of PDHA1 was compared according to tumor pathological stage,subtype classification and breast cancer bio-markers.The function of PDHA1 in TNBC was explored by gene enrichment analysis.(2)Immunohistochemistry assays were used to detect the expression of PDHA1 in human TNBC tissue and adjacent tissue samples.(3)Stable PDHA1 knockout and PDHA1 rescue TNBC MDA-MB-231 cells were constructed.The proliferation of MDA-MB-231 cells was de-tected by colony formation assay and cell counting assay.The regulatory effect of PDHA1 on the invasion and migration of MDA-MB-231 cells was detected by in vitro scratch assay and Transwell migration assay.RESULTS:Database analysis showed that the group with high PDHA1 expression in breast cancer had shorter survival and worse prognosis.In clinical specimens,the expression of PDHA1 in cancer tissues was higher than that in adjacent normal tissues.Knockout of PDHA1 inhibited the proliferation,metastasis,invasion and epithelial-mesenchymal transition of MDA-MB-231 cells.CONCLUSION:PDHA1 is overexpressed in TNBC,and it promotes cell proliferation and facilitates TNBC metastasis through the epithelial-mesenchymal transition pathway.
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Objective To investigate the effects of circDNMT1 on the proliferation,apoptosis and epithelial-mesenchymal transition(EMT)of triple-negative breast cancer(TNBC)cells by regulating miR-377-3p/PUM1 axis.Methods The TNBC tissues and adjacent normal tissues of 24 patients with TNBC treated in Danzhou People's Hospital from 2018 to 2021 were collected.qRT-PCR and Western blot were used to detect the expression of circDNMT1,miR-377-3p,and PUM1 protein in tissue and mouse normal breast epithelial cell line HC11 and TNBC cell lines 4T1,Eph41424,and JC.4T1 cells were divided into the 4T1 group(untransfected),the si-NC group(transfected with si-NC),the si-DNMT1 group(transfected with si-DNMT1),the si-DNMT1+anti-NC group(simultaneously transfected with si-DNMT1 and anti-NC),and the si-DNMT1+anti-miR-377-3p group(simultaneously transfected with si-DNMT1 and anti-miR-377-3p).qRT-PCR was used to detect the expression of circDNMT1 and miR-377-3p of 4T1 cells in each group;CCK-8 was used to detect the proliferation of 4T1 cells in each group;flow cytometry was used to detect the apoptosis of 4T1 cells in each group;Western blot was used to detect the expression of PUM1,EMT-related proteins and apoptosis-related proteins of 4T1 cells in each group;TargetScan website was used to predict the binding sites of miR-377-3p with circDNMT1 and PUM1;dual luciferase report was used to verify the targeting relationships of miR-377-3p with circDNMT1 and PUM1.After inoculation with 4T1 cells,BALB/c mice were randomly divided into the blank control group(injected with equal amount of normal saline),the negative control group(injected with si-NC via tail vein),the DNMT1 silencing group(injected with si-DNMT1 via tail vein),the combined control group(injected with si-DNMT1 and anti-NC via tail vein),and the combined silencing group(injected with si-DNMT1 and anti-miR-377-3p via tail vein),the tumor massess of mice were recorded and the morphological changes of tumors were observed by hematoxylin-eosin staining.Results circDNMT1 and PUM1 were up-regulated in TNBC tissues and cells,and miR-377-3p was down-regulated.The expression difference was most obvious in 4T1 cells,so 4T1 cells were selected for subsequent experiments.Compared with the 4T1 group and the si-NC group,the expression of miR-377-3p,the apoptosis rate of 4T1 cells,the expression levels of Bax,cleaved caspase-3 and E-cadherin protein of 4T1 cells in the si-DNMT1 group were significantly increased(P<0.05),the circ-DNMT1 level,the expression level of PUM1 protein,OD values at 24 hours and 48 hours,the expression level of Bcl-2,N-cadherin,Vimentin protein were significantly decreased(P<0.05).Compared with the si-DNMT1 group and the si-DNMT1+anti-NC group,the expression of miR-377-3p,the apoptosis rate of 4T1 cells,the expression levels of Bax,cleaved caspase-3 and E-cadherin proteins of 4T1 cells in the si-DNMT1+anti-miR-377-3p group were significantly decreased(P<0.05),the expression level of PUM1 protein,OD values at 24 hours and 48 hours,the expression levels of Bcl-2,N-cadherin,Vimentin proteins were significantly increased(P<0.05).Compared with the miR-NC+WT-circDNMT1 group,the cell luciferase activity in the miR-377-3p mimic+WT-circDNMT1 group was significantly decreased(P<0.05);compared with the miR-NC+WT-PUM1 group,the cell luciferase activity in the miR-377-3p mimic+WT-PUM1 group was significantly decreased(P<0.05).The tumor cells in the blank control group and the negative control group were densely arranged with clear boundary;the tumor cells in the DNMT1 silencing group and the combined control group were loosely arranged,the nuclei were pyknotic,and the cell fragments were increased;the tumor cells in the combined silencing group were densely arranged and the boundaries tended to be clear.Compared with the blank control group and the negative control group,the tumor mass of mice in the DNMT1 silencing group was significantly decreased(P<0.05).Compared with the DNMT1 silencing group and the combined control group,the tumor mass of mice in the combined silencing group was significantly increased(P<0.05).Conclusion Silencing circDNMT1 may inhibit the expression of PUM1 by up-regulating miR-377-3p,thereby inhibiting the proliferation and EMT of TNBC cells,and promoting cell apoptosis.
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Objective To explore the effects of long non-coding RNA(lncRNA)MIR4435-2HG(MIR4435-2HG)on the proliferation,migration,invasion and apoptosis of cholangiocarcinoma cells and its regulatory effect on microRNA-376a-3p(miR-376a-3p).Methods qRT-PCR method was used to detect the expression of MIR4435-2HG and miR-376a-3p in human intrahepatic bile duct epithelial cells HIBEpic and human cholangiocarcinoma cells RBE.si-NC,si-MIR4435-2HG,miR-NC,miR-376a-3p mimics,si-MIR4435-2HG and anti-miR-NC,and si-MIR4435-2HG and anti-miR-376a-3p were transfected into RBE cells,respectively,as the si-NC group,the si-MIR4435-2HG group,the miR-NC group,the miR-376a-3p group,the si-MIR4435-2HG+anti-miR-NC group,the si-MIR4435-2HG+ anti-miR-376a-3p group.MTT method,Transwell chamber method and flow cytometry were used to detect cell proliferation,migration,invasion and apoptosis;dual luciferase reporter gene assay was used to verify the targeting relationship between MIR4435-2HG and miR-376a-3p.Western blot was used to detect the expression of related proteins.Results The expression of MIR4435-2HG was increased in RBE cells,while the expression of miR-376a-3p was decreased(P<0.05).Compared with the si-NC group,the MIR4435-2HG expression,cell viability,and protein levels of CyclinD1,MMP-2,MMP-9 in the si-MIR4435-2HG group were reduced(P<0.05),the numbers of migrating and invading cells were reduced(P<0.05),while the MIR4435-2HG expression and apoptosis rate were increased(P<0.05).Compared with the miR-NC group,the cell viability and protein levels of CyclinD1,MMP-2,MMP-9 in the miR-376a-3p group were decreased(P<0.05),the numbers of migrating and invading cells were decreased(P<0.05),while the MIR4435-2HG expression and apoptosis rate were increased(P<0.05).MIR4435-2HG was of targeted regulation on miR-376a-3p.Compared with the si-MIR4435-2HG+ anti-miR-NC group,the cell viability and protein levels of CyclinD1,MMP-2,MMP-9 in the si-MIR4435-2HG+anti-miR-376a-3p group were increased(P<0.05),the numbers of migrating and invading cells were increased(P<0.05),while the MIR4435-2HG expression and apoptosis rate were decreased(P<0.05).Conclusion Knockdown of MIR4435-2HG can inhibit the proliferation,migration,invasion and induce apoptosis of RBE cells by targeting miR-376a-3p.
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Cardiovascular disease is a health hazard to humans and systolic heart failure due to myocardial infarction is a major cause of death.It was previously thought that myocardial cells of the adult mammalian heart possess a limited ability to proliferate and self-renew.However,it has been widely reported that mammals have the ability to regenerate the myocardium,which is restricted to early postnatal life,and that it is strong enough to repair damaged heart tissue.The discovery of myocardial regeneration in neonatal hearts has provided an ideal animal model to investigate the mechanisms that affect myocardial regeneration,and many mechanisms that reverse myocardial cell cycle arrest and promote myocardial regeneration have been revealed.In this article,we review the factors affecting gene expression for myocardial regeneration(e.g.,ncRNAs and transcription factors),myocardial regeneration-related signaling pathways,and the regulation of myocardial regeneration by non-myocardial cells(e.g.,extracellular matrix,immune response,and epicardium)to provide directions for achieving myocardial regeneration after myocardial injury in adult mammals.
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Objective To investigate the effect of Fuzhengquxie prescription on the proliferation,apoptosis,invasion,and migration of ovarian cancer cells and its associated mechanism.Methods After Fuzhengquxie prescription was applied to human ovarian cancer SKOV3 cells,the effects on cell proliferation,apoptosis,invasion,and migration were detected by 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyl tetrazolium bromide,cell cloning,cell scratch,and Transwell assay experiments.Quantitative reverse transcription-polymerase chain reaction(qRT-PCR)and Western blotting were used to determine the expression levels of the negative epigenetic regulatory protein,EZH2;its related protein,E-cadherin;and the apoptosis-related proteins,Bax and Bcl-2.Results Fuzhengquxie prescription inhibited the growth rate of SKOV3 cells in a concentration-and time-dependent manner,and significantly inhibited the proliferation,invasion,and migration of SKOV3 cells.Western blotting and qRT-PCR results showed that Fuzhengquxie prescription combined with GSK126 inhibited the transcription of EZH2and Bcl-2,promoted the transcription of Baxand E-cadherin,down-regulated the expression of EZH2 and Bcl-2 proteins,and promoted the expression of Bax and E-cadherin proteins.Conclusion Fuzhengquxie prescription inhibited the proliferation,invasion,and migration of SKOV3 cells and induced their apoptosis.It may be involved in regulating the E-cadherin-mediated proliferation,invasion,and migration of ovarian cancer cells by inhibiting the epigenetic regulatory protein EZH2,and regulating the apop-tosis of ovarian cancer cells mediated by Bcl-2 and Bax.
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Objective @#To investigate the effect of autophagy activation on cell proliferation in human umbilical vein endothelial cells (HUVECs) .@*Methods @#HUVECs were treated with rapamycin (Rapa) . Western blot assay was performed to examine the expression of protein of microtubule associated protein 1 light chain 3 (LC3) , Beclin 1 and unc-51-like kinase 1 (ULK1) . Autophagosomes were detected by transmission electron microscopy ( TEM) , and autophagy fluorescence was detected by monodansylcadaverine staining(MDC) assay . The effect of autophagy activation on cell proliferation was assessed by CCK-8 assay and EdU assay . Vascular formation experiments were used to detect vasculogenic ability .@*Results @#After Rapa treatment , LC3 , Beclin1 and ULK1 expressions were en- hanced , while the green autophagy fluorescence expression in the experimental group was stronger than that in the control group , and autophagosomes were visible by TEM ; CCK-8 and EdU results showed that compared with the control group , the cell proliferation ability was weakened and tubes formation ability was reduced after the activation of autophagy in experimental cells . @*Conclusion @#Rapa upregulates autophagy activity in HUVECs to inhibit cell proliferation under certain time .
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ObjectiveTo investigate the effects of microRNA (miRNA, miR)-887-3p on the proliferation and apoptosis of rat intervertebral disc annulus fibrosus cells and its underlying molecular mechanism. MethodsAnnulus fibrosus tissues were obtained from 8-week-old SPF-grade SD male rats, centrifuged to prepare and identify annulus fibrosus cells. Rats in the experiment were randomly divided into four groups: a Normal group consisting of primary annulus fibrosus cells without any treatment; a Control group treated with 10 ng/mL interleukin-1β (IL-1β) for 24 hours to establish a degenerative cell model; an interference group (miR-887-3p inhibitor) transfected with miR-887-3p inhibitor using Lipo3000 based on the Control group; and an overexpression group (miR-887-3p mimics) transfected with miR-887-3p mimics using Lipo3000 based on the Control group. CCK-8 assay was used to assess cell viability; flow cytometry was used to measure cell apoptosis rates; real-time fluorescence quantitative PCR (qPCR) was used to detect the expression levels of miR-887-3p and murine double minute 4 (MDM4) mRNA; Western blotting was used to measure the protein expression levels of MDM4, Bcl-2, and Caspase-3. ResultsImmunofluorescence staining of isolated and cultured cells revealed a Collagen I positive rate of over 90% in rat intervertebral disc annulus fibrosus cells, indicating a cell purity level greater than 90%. Real-time fluorescence qPCR results showed that after establishing an annulus fibrosus degenerative cell model using IL-1β, the expression level of miR-887-3p significantly increased compared to the Normal group (P<0.001). Compared to the Control group, transfection with miR-887-3p inhibitor resulted in a significant decrease in its expression level (P<0.001). The CCK-8 assay showed that compared to the Normal group, cell viability significantly decreased in the Control group (P<0.001). Compared to the Control group, cell proliferation ability significantly increased after miR-887-3p inhibition, and significantly decreased after overexpression of miR-887-3p. Flow cytometry results revealed that compared to the Normal group, the apoptosis rate in the Control group significantly increased (P<0.001). Compared to the Control group, the cell apoptosis rate significantly decreased in the miR-887-3p interference group (P<0.001) and increased in the overexpression group (P<0.001). Western blotting analysis showed that compared to the Normal group, Bcl-2 expression level significantly decreased (P<0.001) and Caspase-3 expression level significantly increased (P<0.001) in the Control group. Compared to the Control group, Bcl-2 and MDM4 expression levels significantly increased (P<0.01), and Caspase-3 expression level significantly decreased (P<0.01) in the miR-887-3p interference group; whereas in the overexpression group, Bcl‑2 and MDM4 expression levels significantly decreased (P<0.05), and Caspase-3 levels significantly increased (P<0.05). Real-time fluorescence qPCR and protein immunoblotting results showed that after interfering with miR-887-3p, the expression of MDM4 protein and mRNA increased (P<0.001); after overexpressing miR-887-3p, their expression decreased (protein, P<0.01; mRNA, P<0.001). ConclusionMiR-887-3p may modulate the cell proliferation and apoptosis of rat intervertebral disc annulus fibrosus cells by regulating MDM4 expression, thereby influencing the development and progression of disc degeneration.
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Objective To explore the regulatory relationship between CCAAT enhancer binding protein beta(CEBPB)and four-jointed box kinase 1(FJX1)in colon cancer and their effect on colon cancer(CC)malignant progression and angiogenesis.Methods Bioinformatics was used to analyze the expression of FJX1 and CEBPB in CC and the regulatory relationship between them.Real-time quantitative reverse transcription polymerase chain reaction(qRT-PCR)was used to verify the expression of FJX1 and CEBPB in CC cells,and chromatin immunoprecipitation(CHIP)and dual luciferase assay were used to verify the binding relationship between FJX1 and CEBPB.The effects of FJX1 and CEBPB on the viability,migration,invasion and angiogenesis of CC cells were detected by cell counting kit-8(CCK-8),scratch test,Transwell and angiogenesis test.Results This study revealed that FJX1 was highly expressed in CC.Inhibiting the expression of FJX1 could significantly inhibit the cell viability,migration,invasion and angiogenesis of CC cells.Subsequently,we found that CEBPB was an upstream regulatory gene of FJX1,and CEBPB was highly expressed in CC.CHIP and dual luciferase experiments showed that CEBPB could bind to FJX1.The results of cell experiments showed that the transcription factor CEBPB could promote the proliferation,migration,invasion and angiogenesis of CC cells by activating FJX1.Conclusion CEBPB/FJX1 axis played a cancer-promoting role in the progression of CC,suggesting that CEBPB and FJX1 may be potential therapeutic targets for CC.
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Objective To investigate the correlation between Yes-associated protein(YAP)nuclear expression and tumor size with prognosis of patients with epithelial ovarian cancer(EOC)and to study the role of YAP in EOC.Methods 120 patients with EOC were selected as the experimental group,including 38 patients with early stage(Ⅰ+Ⅱ)EOC and 8 2 patients with advanced stage(Ⅲ+Ⅳ)EOC.3 0 normal ovarian tissues obtained from patients with uterine leiomyoma were enrolled as the control group.Immunohistochemical(IHC)assay was em-ployed to determine YAP expression and sub-location.The relationship between YAP expression and the pathologi-cal parameters of the 120 patients with EOC was analyzed,so as to the prognosis of these patients.EOC cells(C13K and OV2008)were cultured with varying initial cell volumes.Ki67 expression and cell proliferation were tested by immunofluorescence and cloning assay respectively.YAP expression at mRNA and protein levels were de-tected by q-PCR and Western blot respectively when the cell conference of EOC cells reached to low(60％)and high(90％)cell density.Results The YAP nuclear expression was significantly higher in the EOC group com-pared to the control group(P＜0.05).The average diameter of stage Ⅰ+Ⅱ EOC was larger than that of stage Ⅲ+Ⅳ EOC(P＜0.01).The high nuclear expression of YAP was positively associated with pathological grade,clinical stage and the level of Ca125＞1 000 IU/ml,while negatively correlated with tumor size(all P＜0.05).Survival analyses showed that smaller tumor size(＜10 cm)and higher YAP nuclear expression were negatively as-sociated with the 3-year overall survival rate of EOC patients(P＜0.01).C13K and OV2008 cells cultured in the low density group exhibited a high number of clone formation,high Ki67 and YAP expression(P＜0.01).The down-regulation of YAP expression could decrease the cell viability of EOC cells in the low-and high-density groups(P＜0.05).Conclusion Higher level of YAP nuclear expression and smaller tumour size are inversely associated with the clinical prognosis of patients with EOC.Inhibiting YAP nuclear expression leads to a decrease in the prolif-eration capacity of EOC cells.
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Objective:To discuss the expression of programmed cell death-ligand 1(PD-L1)in the oral squamous cell carcinoma(OSCC)cells and its effect on biological behavior of the OSCC CAL27 cells,and to clarify the possible mechanism.Methods:Western blotting method was used to detect the expression levels of PD-L1 protein in the oral epithelial HOK cells and OSCC CAL27,TCA8113,and SCC15 cells;immunofluorescence staining method was used to detect the expression and localization of PD-L1 protein in the CAL27 cells.The CAL27 cells were divided into control group(transfected with si-NC)and si-PD-L1 group(transfected with si-PD-L1).Western blotting method was used to detect the interference efficiency of the cells in two groups;CCK-8 assay was used to detect the proliferative activities of the cells in two groups at different time points;plate clone formation assay was used to detect the numbers of clone formation of the cells in two groups;cell scratch healing assay was used to detect the scratch healing rates of the cells in two groups;Transwell chamber assay was used to detect the numbers of migration and invasion cells in two groups.Results:The expression level of PD-L1 protein in the OSCC cells was higher than that in the HOK cells(P<0.05 or P<0.01);PD-L1 expressed in the cytoplasm and nucleus of the CAL27 cells.The CCK-8 assay and plate clone formation assay results showed that compared with control group,the proliferative activities of the CAL27 cells in si-PD-L1 group at different time points were significantly decreased(P<0.05 or P<0.01),and the numbers of clone formation were significantly decreased(P<0.01).The cell scratch healing assay results showed that compared with control group,the scratch healing rates of the cells in si-PD-L1 group were significantly decreased(P<0.05 or P<0.01).The Transwell chamber assay results showed that compared with control group,the numbers of migration and invasion cells in si-PD-L1 group were significantly decreased(P<0.01).Conclusion:The expression of PD-L1 in the OSCC cells is higher than that in normal oral epithelial cells,and knocking down PD-L1 expression can inhibit the proliferation,clone formation,migration and invasion capabilities of the OSCC cells.
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Objective:To discuss the effect of apolipoprotein C1(APOC1)expression on the proliferation and apoptosis of the hepatocellular carcinoma cells,and to preliminarily clarify the related molecular mechanism.Methods:The expression level of APOC1 mRNA in hepatocellular carcinoma tissue and its relationship with the prognosis of the patient were analyzed by The Cancer Genome Atlas(TCGA)Database;real-time fluorescence quantitative PCR(RT-qPCR)method was used to detect the expression levels of APOC1 mRNA in different hepatocellular carcinoma cells;the human liver cancer HepG2 cells with low APOC1 expression were selected as the subjects.The HepG2 cells were transfected with pcDNA3.1-APOC1 plasmid to over-express APOC1(APOC1 over-expression group),and the HepG2 cells transfected with empty vector pcDNA3.1 were regarded as control group.MTS assay and 5-ethynyl-2'-deoxyuridine(EdU)staining were used to detect the proliferative activities and proliferation rates of the cells in two groups;Transwell chamber assay was used to detect the numbers of migration cells in two groups;flow cytometry and TUNEL assay were used to detect the percentages of the cells at different cell cycles and apoptotic rates in two groups;Western blotting method was used to detect the expression levels of extracellular regulated protein kinase(ERK),phosphorylated ERK(p-ERK),protein kinase B(AKT),phosphorylated AKT(p-AKT),B-cell lymphoma-2(Bcl-2),and cleaved cysteinyl aspartate specific proteinase-3(cleaved caspase-3)proteins in the cells in two groups.Results:The TCGA Database results showed that the expression level of APOC1 mRNA in hepatocellular carcinoma tissue was lower than that in normal liver tissue(P<0.05),and the patients with low expression of APOC1 mRNA had poor prognosis.The RT-qPCR results showed that the expression level of APOC1 mRNA in the HepG2 cells was the lowest,and the HepG2 cells were chosen for the subsequent research.Compared with control group,the proliferative activity and proliferation rate of the cells in APOC1 over-expression group were decreased(P<0.05 or P<0.01),the number of migration cells was decreased(P<0.01),and the percentage of the cells at S phase and the apoptotic rate were significantly increased(P<0.01).Compared with control group,the expression levels of p-ERK,p-AKT,and Bcl-2 proteins in the cells in APOC1 over-expression group were significantly decreased(P<0.05),and the expression level of cleaved caspase-3 protein was increased(P<0.01).Conclusion:High expression of APOC1 can inhibit the proliferation of the human liver cancer HepG2 cells and induce the apoptosis,and its mechanism may be related to inhibition of the expressions of p-ERK,p-AKT,Bcl-2 proteins and promotion of the expression of cleaved caspase-3 protein.
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OBJECTIVE The expression of cancerous inhibitor of protein phosphatase 2A(CIP2A)in hypopharyngeal carcinoma FaDu cells(FaDu cells)was reduced by shRNA to understand its role in the occurrence and development of hypopharyngeal carcinoma.METHODS Specific shRNA sequence was designed,lentivirus was packaged and transfected into hypopharyngeal carcinoma FaDu cells,and CIP2A expression was specifically knocked down.The expression of CIP2A was detected by RT-PCR and Western blot.RESULTS 1.After shRNA knocked down CIP2A in FaDu cells,the CIP2A mRNA expression in the experimental group(CIP2A knocked down group)was significantly lower than that in the blank group,and the CIP2A protein expression in the experimental group was also significantly lower than that in the blank group.2.Cell cloning and CCK8 experiments showed that the cell proliferation ability of the experimental group was significantly decreased compared with that of the blank group(t=50.86,P<0.01;t=12.406,P<0.001);The results of cell scratch test showed that the transverse migration ability of the experimental group was significantly decreased compared with the blank group,and the longitudinal migration ability of the experimental group was significantly decreased compared with the blank group by Transwell test(t=40.038,P<0.01;t=12.247,P<0.001).CONCLUSION After knockdown CIP2A expression in hypopharyngeal carcinoma FaDu cells,the proliferation and migration ability of hypopharyngeal cancer cells decreased,suggesting that CIP2A is involved in regulating the biological behavior of hypopharyngeal cancer cells and can be used as a potential anticancer target.
摘要
Objective:To investigate the expression of long non-coding RNA(lncRNA) ZFP36-AS1 in bladder cancer and the effect of ZFP36-AS1/miR-221 axis on the proliferation and immune escape of bladder cancer cells.Methods:The expression difference of ZFP36-AS1 in bladder cancer tissues was analyzed by cBioPortal database. Real-time fluorescence quantitative polymerase chain reaction (RT-qPCR) was used to analyze the expression difference of ZFP36-AS1 in bladder cancer cell lines (J82, RT-4, MGH-U3, 5637). MGH-U3 cells were randomly divided into negative control (NC) group and ZFP36-AS1 group, which were transfected with pcDNA3.1-NC plasmid and pcDNA3.1-ZFP36-AS1 plasmid, respectively. Colony formation assay and flow cytometry were used to analyze the proliferation activity and cell cycle of MGH-U3 cells, respectively. T lymphocytes were co-cultured with MGH-U3 cells in each group, and the levels of interleukin-10 (IL-10), γ-interferon (IFN-γ), and interleukin-4 (IL-4) in the supernatants of each group were detected by enzyme-linked immunosorbent assay (ELISA). The dual-luciferase reporter gene assay verified the targeting relationship between ZFP36-AS1 and miR-221. The effect of ZFP36-AS1 on the expression of miR-221 in MGH-U3 cells was detected by RT-qPCR. Western blotting was used to detect the effect of ZFP36-AS1/miR-221 axis on the protein expression of CDK3, Cyclin C, CDK5, Cyclin D1 and Cyclin D3 in MGH-U3 cells.Results:Compared with normal bladder tissue, ZFP36-AS1 was abnormally low-expressed in bladder cancer tissue ( P<0.01). Compared with SV-HUC-1 cells, ZFP36-AS1 was abnormally low-expressed in bladder cancer cell lines (J82, RT-4, MGH-U3, 5637) ( P<0.01), and the expression was lowest in MGH-U3 cells ( P<0.01). The number of MGH-U3 cell colonies formed in the NC group and the ZFP36-AS1 group were (220.80±34.65) and (77.84±19.11), respectively, and the number of MGH-U3 cell colonies formed in the ZFP36-AS1 group was significantly down-regulated, the difference was statistically significant ( P<0.01). The proportions of G 0/G 1 phase cells in NC group and ZFP36-AS1 group were (48.04±2.89)% and (72.89±3.46)%, respectively, and the proportion of S phase cells were (35.38±2.98)% and (20.62±2.56)%, respectively. The proportion of G 2/M stage cells was (16.59±1.46)% and (6.48±1.50)%, respectively. The proportion of cells in G 0/G 1 phase were up-regulated in ZFP36-AS1 group ( P<0.01), and the proportion of cells in S phase and G 2/M phase were both down-regulated ( P<0.01). Compared with the NC group, the levels of IL-4 and IFN-γ in the ZFP36-AS1 group were significantly up-regulated ( P<0.01), and the level of IL-10 was significantly down-regulated ( P<0.01). ZFP36-AS1 can target miR-221 ( P<0.01). The relative expression of miR-221 in the NC group and the ZFP36-AS1 group was 6.84±1.35 and 1.00±0.21, respectively. Compared with the NC group, overexpression of ZFP36-AS1 could significantly inhibit the expression of miR-221 ( P<0.01). Compared with the NC group, the expressions of CDK3, Cyclin C, CDK5, Cyclin D1, and Cyclin D3 in the ZFP36-AS1 group were significantly decreased. Conclusion:ZFP36-AS1 is abnormally low-expressed in bladder cancer, and it reduces the proliferation activity of bladder cancer cells and inhibits their immune escape by inhibiting the expression of miR-221.
摘要
Monopolar spindle 1, also known as threonine and tyrosine kinase (TTK), is a key component of spindle assembly checkpoint (SAC). It is considered to be a monitoring mechanism to ensure mitotic fidelity and genomic stability. TTK is overexpressed in a variety of malignant tumors, and patients with low expression of TTK tend to have a longer survival time, suggesting that it may be used as a biomarker for diagnosis and prognosis. Abnormal expression of TTK often impairs the function of SAC, resulting in irregular mitosis, increased aneuploidy and mitotic disaster, thus promoting the occurrence of tumors. Current studies have shown that TTK inhibitors can inhibit the proliferation of tumor cells and increase the sensitivity of tumor cells to therapy in combination with chemotherapy or radiotherapy to achieve sensitization and attenuated effects. This article will review the research and application of TTK and its inhibitors in malignant tumors.
摘要
Objective:To study the effects and potential mechanisms of the combination of dihydroartemisinin and carfilzomib on the activity, proliferation, and apoptosis of multiple myeloma ARD cell lines.Methods:In vitro cultivation of multiple myeloma ARD cells involved treating the cells with dihydroartemisinin at concentrations of 0, 5, 10, 20, 40, and 80 μg/ml, and with carfilzomib at concentrations of 0, 5, 10, 20, 40, and 80 nmol/L. The ARD cells were divided into a control group (no treatment) , a dihydroartemisinin group (2 μg/ml) , a carfizomib group (8 nmol/L) , and a combination group (dihydroartemisinin 2 μg/ml + carfizomib 8 nmol/L) . Cell activity and proliferation were assessed by MTT assay and EdU-488 assay; cell apoptosis was evaluated using live cell/dead cell dual staining and flow cytometry. The expression levels of apoptosis-related proteins were examined using Western blotting analysis. Results:The cell survival rates of ARD cells treated with 0, 5, 10, 20, 40, and 80 μg/ml dihydroartemisinin were (100.00±2.18) %, (50.22±3.09) %, (37.39±2.34) %, (30.42±1.79) %, (23.80±1.12) %, and (18.04±0.79) %, respectively, and there was a statistically significant difference ( F=653.30, P<0.001) . With the increase of drug concentration, ARD cell activity decreased gradually (all P<0.05) . The cell survival rates of ARD cells treated with 0, 5, 10, 20, 40, and 80 nmol/L carfilzomib were (100.00±1.12) %, (83.98±2.95) %, (67.27±2.10) %, (58.24±2.02) %, (46.34±1.14) %, and (37.47±1.36) %, respectively, and there was a statistically significant difference ( F=227.40, P<0.001) . With the increase of drug concentration, ARD cell activity decreased gradually (all P<0.05) . The cell survival rates for the control group, dihydroartemisinin group, carfilzomib group, and combination group were (100.00±2.67) %, (67.23±0.57) %, (76.23±2.83) %, and (27.06±1.09) %, respectively, and there was a statistically significant difference ( F=655.60, P<0.001) . There were statistically significant differences in the dihydroartemisinin group, carfilzomib group, and combination group compared with control group (all P<0.001) . There were statistically significant differences in the dihydroartemisinin group and carfilzomib group compared with combined group (both P<0.001) . The EdU-488 experiment showed that the EdU-positive rates of ARD cells in the control group, dihydroartemisinin group, carfilzomib group, and combination group were (100.00±8.17) %, (68.07±6.14) %, (85.04±2.78) %, and (19.62±3.83) %, respectively, and there was a statistically significant difference ( F=115.20, P<0.001) . There were statistically significant differences in the dihydroartemisinin group, carfilzomib group, and combination group compared with control group ( P<0.001; P=0.047; P<0.001) . There were statistically significant differences in the dihydroartemisinin group and carfilzomib group compared with combined group (both P<0.001) . The live cell/dead cell dual staining experiment showed, under bright-field observation, the cell morphology was intact in the control group. In all the drug groups, the cell morphology became irregular, reduced in size with condensed cytoplasmic, and apoptotic vesicles with irregular morphology were seen around the cells, among which the most obvious changes were seen in the combination group. Under fluorescence observation, the cells in the control group only displayed green fluorescence. In all drug-treated groups, cells with red fluorescence were observed, with the combination group having the highest percentage of cells with red fluorescence among the total cell population. The apoptosis rates for the control group, dihydroartemisinin group, carfilzomib group, and combination group were (9.06±2.95) %, (29.50±1.34) %, (20.77±3.00) %, and (58.23±5.13) %, respectively, and there was a statistically significant difference ( F=115.80, P<0.001) . There were statistically significant differences in the dihydroartemisinin group, carfilzomib group, and combination group compared with control group ( P<0.001; P=0.012; P<0.001) . There were statistically significant differences in the dihydroartemisinin group and carfilzomib group compared with combined group (both P<0.001) . There were statistically significant differences in the relative expression levels of P53, Cleaved-Caspase-3, Bcl-2, and Bax proteins among the control group, dihydroartemisinin group, carfilzomib group, and combination group ( F=21.76, P<0.001; F=42.87, P<0.001; F=44.27, P<0.001; F=163.50, P<0.001) . There were statistically significant differences in the dihydroartemisinin group, carfilzomib group, and combination group compared with control group (all P<0.05) . There were statistically significant differences in the dihydroartemisinin group and carfilzomib group compared with combined group (both P<0.05) . Conclusion:The combination of dihydroartemisinin and carfilzomib can synergistically inhibit the activity and proliferation of multiple myeloma ARD cells, and promote apoptosis, and the underlying mechanism may be associated with the mitochondrial apoptosis pathway.
摘要
Objective:To explore the effects of Jianpi Bushen Jiedu Prescription on the proliferation and migration of hepatocellular carcinoma cells; To discuss its possible mechanism.Methods:Using human highly metastatic liver cancer cell line (HCCLM3) as the research object, they were randomly divided into control group and TCM group (100, 200, 400, 800, 1 600, 3 200 μg/ml Jianpi Bushen Jiedu Prescription) and Western medicine group (2.5, 5, 10, 20, 40 μmol/L sorafenib) using a random number table method. Cell viability was detected using cell counting reagent (CCK-8) method; HCCLM3 cells were divided into control group and TCM (Jianpi Bushen Jiedu Prescription 800 μg/ml) group and combined group (Jianpi Bushen Jiedu Prescription 800 μg/ml +sorafenib 20 μmol/L). Western blot method was used to detect the protein expressions of kinase/signaling transducer and transcriptional activator (JAK2/STAT3) pathway related proteins (p-JAK2, JAK2, p-STAT3, STAT3) in each group.Results:Compared with the control group, viability and mobility of HCCLM cell in TCM group and Western medicine group decreased ( P<0.01 or P<0.05); compared with the control group, the protein expressions of P-JAK2, JAK2, P-STAT3 and STAT3 in the TCM group and the combined group decreased ( P<0.05), and the JAK2 protein expression in the combined group was lower than that in the TCM group ( P<0.05). Conclusion:Jianpi Bushen Jiedu Prescription can inhibit the proliferation and migration of HCC cells by regulating JAK2/STAT3 pathway.
摘要
In this article,the mechanism of Shanxian Granule in inhibiting liver cancer,lung cancer,sarcoma,melanoma and other tumors was reviewed,with a view to providing a theoretical basis for the clinical research of Shanxian Granules in the treatment of malignant tumors.Shanxian Granule are the pure Chinese medicine preparation for counteracting malignant tumor developed by the Oncology Research Team of Shaanxi University of Chinese Medicine on the basis of the theory of traditional Chinese medicine syndrome differentiation and treatment combined with decades of clinical experience as well as the achievements of modern pharmacological research.Shanxian Granule are mainly composed of Crataegi Fructus,Agrimoniae Herba,Panacis Quinquefolii Radix,Curcumae Rhizoma,Testudinis Carapax et Plastrum,Trionycis Carapax,Corydalis Rhizoma,and Polyporus,and have the actions of benefiting qi and nourishing yin,supporting healthy-qi and cultivating the essence,activating blood and removing stasis,and eliminating swelling and counteracting cancer.The compatibility of Shanxian Granule embodies the principle of supporting healthy-qi but avoiding maintaining pathogens,and eliminating pathogens but avoiding injuring healthy-qi.The granules can effectively inhibit the growth and metastasis of liver cancer,lung cancer,sarcoma,melanoma and other tumors both in vivo and in vitro,alleviate the clinical symptoms of tumor patients,and improve their prognosis.The anti-tumor mechanism of Shanxian Granules is related to the enhancement of body immune function,inhibition of tumor cell proliferation,enhancement of tumor cell apoptosis,inhibition of tumor cell invasion and metastasis as well as the tumor angiogenesis.
摘要
Objective To investigate the expression of N6 methyladenine(m6A)demethylase human fat mass and obesity-associated(FTO)protein in nasopharyngeal carcinoma(NPC),and the effect of over-expression of FTO on the proliferation of nasopharyngeal carcinoma in vitro and in vivo.Methods Immunohistochemistry method was used to detect the expression of FTO protein in nasopharyngeal carcinoma tissues and para-cancerous tissues;The dominant expression cell line of FTO was screened,the over-expression FTO cell line was constructed.The cell pro-liferation was examined by soft-agar method.A mouse tumor model was developed for measurement of tumor growth.ResultsThe expression of FTO in nasopharyngeal carcinoma tissues was lower than that in adjacent tissues.Low ex-pression of FTO promoted proliferation of NPC cells,while over-expression of FTO reversed this effect.Conclusions FTO inhibits proliferation of nasopharyngeal carcinoma and this result may provide an experimental technology in searching therapeutic targets of chemotherapy for nasopharyngeal carcinoma.