High expression of ErbB2 contributes to cholangiocarcinoma cell invasion and proliferation through AKT/p70S6K

AIM:To compare the impact of ErbB2 on cell invasion and proliferation in cholangiocarcinoma(CCA) cell lines.METHODS:Level of endogenous ErbB2 expression in three CCA cell lines,namely HuCCA-1,KKU-100 and KKU-M213,was determined by real-time reversetranscriptase polymerase chain reaction.Two ErbB2 inhibitory methods,a small molecule ErbB2 kinase inhibitor(AG825) and siRNA,were used to disrupt ErbB2 function in the cell lines.CCA cell invasion,motility and proliferation under ErbB2-disrupted conditions were detected using Transwell and 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide assays.In addition,ErbB2 downstream effectors were investigated by Western blotting analysis.RESULTS:Suppression of ErbB2 activity,using a specific kinase inhibitor(AG825),reduced invasion,motility and proliferation of all three CCA cell lines.The ability of this drug to inhibit neoplastic properties(invasion,motility and proliferation) increased concomitantly with the level of ErbB2 expression.Similarly,knockdown of ErbB2 level by siRNA inhibited cell invasion and proliferation of KKU-M213,a high-ErbB2-expressing cell,better than those of the lower-ErbB2-expressing cells,HuCCA-1 and KKU-100.Thus,both inhibitory methods indicated that there is more ErbB2-dependency for malignancy of the high-ErbB2-expressing cell,KKU-M213,than for that of low-ErbB2-expressing ones.In addition,interrupting ErbB2 activity decreased phosphorylation of AKT and p70S6K,but not extracellular signal-regulated kinase 1/2,in the high-ErbB2-expressing CCA cell line.CONCLUSION:Our data indicated that high ErbB2 expression enhances CCA invasion,motility and proliferation via the AKT/p70S6K pathway,which suggests the possibility of targeting these molecules for CCA therapy.

Glycosylation-independent binding to extracellular domains 11-13 of mannose-6-phosphate/insulin-like growth factor-2 receptor mediates the effects of soluble CREG on the phenotypic proliferation of vascular smooth muscle cells

Background The present study aimed to investigate the detailed mode and specific sites for their binding as well as the functional relevance of this binding in the phenotypic proliferation of vascular smooth muscle cells(SMCs). Methods CREG knocked-down SMCs were employed to evaluate the biological activity of wtCREG and mCREG.Expressions of SMC differentiation markers SM myosin heavy chain(SM-MHC),SM-actin,heavy caldesmon and myocardin were determined by Western blotting using specific antibodies. Cellular growth of SMCs was assessed by bromide dewuridine (BrdU) incorporation and cell cycle analysis on fluorescence-activated cell sorting(FACS).A solid-phase binding assay was used to study the binding of CREG to extracellular domains of M6P/IGF2R.The cellular co-localization of the two recombinant CREGs with M6P/IGF2R was detected on SMC surface by immunoprecipitation and immunofluorescence analysis.Results The molecular weight of wtCREG was around 30 kD while that of the mCREG was～25 kD.Treatment of wtCREG with PNGase F reduced its molecular weight from～30 kD to～25 kD,whereas PNGase F treatment had no effect on the molecular weight of mCREG.Both wtCREG and mCREG proteins enhanced SMC differentiation,inhibited BrdU incorporation,and arrested cell cycle progression when added to the culture medium.In CREG knocked-down SMCs,the amount of CREG detected by immunoblotting in M6P/IGF2R immunoprecipitates was significantly reduced when compared to normal cells.Both recombinant CREGs co-immunoprecipitated with M6P/IGF2R, although slightly reduced amount of the mutant CREG was detected in M6P/IGF2R immunoprecipitates.Immunostaining revealed that His-tagged CREGs co-localized with IGF2R on the cell surface in a glycosylation-independent manner.In vitro binding assay showed that CREGs bound to M6P/ IGF2R extracellular domains 7-10 and 11-13 in a glycosylation -dependent and -independent manner,respectively.Further blocking experiments using soluble M6P/IGF2R fragments and M6P/IGF2R neutralizing antibody indicated that the biological activities of recombinant CREGs in SMC growth and the up-regulation of SMC differentiation markers were all abolished by treatment with the M6P/IGF2R neutralizing antibody. However,although the growth inhibitory effect of wtCREG was nearly abolished by D7-10 or D11-13,the effect of mCREG was only reversed by Dll-13,indicating that the binding to domains 11-13 is required for CREG to modulate the proliferation of SMCs.Conclusions These data suggest that solubleCREG proteins can exert their biological function via binding to the extracellular domains 7-10 and 11-13 of cell surface M6P/IGF2R in both a glycosylation-dependent and -independent manner.

Effectof the Local Strain CN5 of Human Herpesvirus 6 on CD Molecule Expression and PHA-Induced Proliferation of Cord Blood or Peripheral Blood Mononuclear Cells

Cord blood mononuclear cells (CBMC's) and adult peripheral blood mononuclear cells (PBMCs), cultured with RPMI 1640 medium containing 20% fetal calf serum plus 5 mg/L PHA and 10 mg/L IL-Z, were inoculated with CN5 strain of human herpesvirus 6 (HHV 6 ) isolated from a Chinese patient with exanthem subitum and the CN5 infected cell lysates were added to cultures of CBMCs and PBMCs, so as to observe the effects of the local strain CN5 on expression of CD molecule or on proliferation of mononuclear cells by the methods of APAAP staining and MTT assay.The results were as follows: ①Expressions of some CD antigens of CBMCs and PBMCs could change after CN5 strain infection. In both cases, CD3 expresion was down-regulated while CD4 expression was up-regulated. There were no significant differences of CD2, CD8 and CD45RA expressions between the two groups with and without CN5 infection. But the ratio of CD4 to CD8 sigmificantly rose because of the increasing of CD4 positiveity. ②The lysates of CB5-infected CBMCs inhibited the liferation of PBMCs, not of CBMCs, in a protein concentration-dependent pattern. This inhibition was partially neutralized by specific antiserum to CN5, not by antisera to INF-α and TNF-α.

Effects of selective cyclooxygenase-2 inhibitor on C6 glioma cell proliferation and apoptosis

BACKGROUND： Studies have shown that cyclooxygenase-2 is associated with proliferation and apoptosis of glioma cells. OBJECTIVE： To investigate the effects of selective cyclooxygenase-2 inhibitor celecoxib on proliferation and apoptosis of C6 glioma cells in vitro. DESIGN, TIME AND SETTING： A cellular, molecular, controlled study was performed at the Central Laboratory and Room of Electron Microscope, Medical School, Xi＇an Jiaotong University, China from March 2007 to March 2008. MATERIALS： C6 glioma cells during in vitro log phase were assigned to control and experimental groups. Celecoxib （Pfizer, USA）, dimethyl sulfoxide （Sigma, USA）, and MTT （Sigma, USA） were used for this study. METHODS： The control group was subdivided into blank control and dimethyl sulfoxide control groups. C6 glioma cells in the blank control and dimethyl sulfoxide control groups were incubated in Dulbecco＇s modified Eagle＇s medium supplemented with 10% calf serum and 0.3% dimethyl sulfoxide respectively. C6 glioma cells in the experimental group were separately treated with 60, 80 and 100 μmol/L celecoxib. MAIN OUTCOME MEASURES： Activity of C6 glioma cells was examined by MTT assay. C6 glioma cell cycle and apoptosis were determined by annexin V-fluorescein isothiocyanate/propidium iodide double-staining, followed by flow cytometry. Morphology and ultrastructure of C6 glioma cells were observed with an inverted microscope and a transmission electron microscope, respectively. RESULTS： Compared with the blank control group, cell density was reduced, adherence ability weakened, and irregular nuclei were visible, with the presence of chromatin condensation, margination, and some apoptotic bodies in the experimental group. Activity of C6 glioma cells was significantly decreased （P 〈 0.05）, cell number was reduced during S phase, cell number was significantly increased during G2/M phase （P 〈 0.01 ）, and the apoptotic rate was significantly increased （P 〈 0.05） in the experimental group. These results were displayed in a dose- and time-dependent fashion. The outcomes were obvious in the 100 IJmol/L celecoxib group following 72 hours of treatment. CONCLUSION： Celecoxib blocked proliferation and induced apoptosis of C6 glioma cells in a dose- and time-dependent fashion.

Induction of apoptosis and inhibition of proliferation of C6 glioma cells in vitro by tamoxifen

Objective To investigate the anti-tumor effect and mechanism of tamoxifen on rat C6 glioma cells. Methods C6 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) with 3% fetal calf serum (FCS), and treated with tamoxifen of different concentrations, i.e. group A (1.25μmol/L), group B (2.50 μmol/L), group C (5.00 μmol/L), group D (10.00 μmol/L), group E (20.00 μmol/L) and control group (0.00 μmol/L). Morphological changes, MTT assay and 5-bromo-2’-deoxyuriding labeling ratio were assessed. Apoptosis was observed by flow cytometry. Results C6 cells treated with different doses of tamoxifen for 24, 48, and 72 hours became irregular in shape, while cells treated with vehicle grew normally. MTT assay showed that tamoxifen did not suppress C6 cell growth until 72 hours after treatment. Seventy-two hours after treatment, there were significant differences in cell viable rate between group A versus groups C, D and E; so did group B versus group D as well as group E (P<0.05). BrdU incorporation assay indicated significant difference of BrdU labbled index (BrdU LI) among groups A, C, E and control group 48 hours after treatment (P<0.05). And the BrdU LI decreased with the increased concentration of tamoxifen. Flow cytometry (FCM) showed significant difference between treated group and control group at 24, 48, and 72 hours after treatment (P<0.05). Conclusion Tamoxifen significantly suppresses the growth of C6 glioma cells in a time-and dose-dependent manner. The mechanism of tamoxifen suppressing C6 glioma cells may be inhibiting proliferation and inducing apoptosis. Therefore, tamoxifen can be a candidate as a chemotherapy agent for glioma.

HPV16 E6 gene mutation promotes the proliferation of cervical cancer cells by regulating the expression of BDNF/TrKB

Objective:Study on the mechanism of HPV16 E6 gene mutation promoting the proliferation of cervical cancer cells by influencing the expression of BDNF/TrkB.Methods:The expression levels of HPV16 E6 T350G,BDNF,TrkB and p53 mRNA in cervical cancer tissue samples and CINII cervical tissues were detected by Real-time PCR.HPV16 E6 T350G lentivirus(pLV5-HPV16 E6 T350G)and empty vector(pLV5-vector)were designed and constructed,and transfected with HCerEpiC cells,the expression levels of HPV16 E6 T350G,BDNF,TrKB and p53 mRNA were detected by Real-time PCR,and the expression levels of BDNF,TrKB,PI3K,pPI3K,AKT and pAKT protein were detected by western blot;cell proliferation was detected by MTT experiments.Results:Compared with cinii cervical tissue,HPV16 E6 T350G,BDNF and TrkB mRNA expression levels were all positive,while p53 mRNA expression was negative.After overexpression of HPV16 E6 T350G in HCerEpiC cells,it can up-regulate the expression levels of BDNF and TrKB protein and mRNA,and activate the PI3K/AKT signaling pathway which is the downstream of BDNF/TrKB,and reduce p53 protein expression levels;HPV16 E6 T350G overexpression can enhance the proliferation capacity of HCerEpiC cells.Conclusion:Overexpression of HPV16 E6 T350G can promote the proliferation of cervical cancer cells,which may be related to the upregulation of BDNF/TrKB expression,the activation of PI3K/AKT signaling pathway,and the decrease of p53 expression.

Colon cancer-associated B2 Escherichia coli colonize gut mucosa and promote cell proliferation

AIM: To provide further insight into the characterization of mucosa-associated Escherichia coli (E. coli) isolated from the colonic mucosa of cancer patients.

Meclofenamic acid represses spermatogonial proliferation through modulating m^6A RNA modification

Background: N6-Methyladenosine(m^6A), the most prevalent modification in mammalian m RNA, plays important roles in numerous biological processes. Several m^6A associated proteins such as methyltransferase like 3(METTL3),methyltransferase like 14(METTL14), α-ketoglutarate-dependent dioxygenase Alk B homolog 5(ALKBH5) and YTH domain containing 2(YTHDC2) are involved in the regulation of spermatogenesis and oogenesis. However, the role of the first detected m^6A demethylase, fat mass and obesity associate protein(FTO), in germ cells remains elusive.Elucidation of FTO roles in the regulation of germ cell fate will provide novel insights into the mammalian reproduction.Methods: Mouse GC-1 spg cells were treated with the ester form of meclofenamic acid(MA2) to inhibit the demethylase activity of FTO. The cellular m^6A and m^6Amlevel were analyzed through high performance liquid chromatography combined with tandem mass spectrometry(HPLC/MS-MS). The cell apoptosis was detected via TUNEL and flow cytometry. The cell proliferation was detected through Ed U and western blot. The m RNA level of core cyclin dependent kinases(CDKs) was quantified via q-PCR. RNA decay assay were performed to detect RNA stability. Dual fluorescence assay was conducted to study whether MA2 affects the expression of CDK2 dependent on the m^6A modification at 3’UTR.Results: MA2 significantly increased the cellular m^6A level and down-regulated the expression of CDK1, CDK2, CDK6 and Cd C25 a, resulting in arrest of G1/S transition and decrease of cell proliferation. MA2 downregulated CDK2 m RNA stability. Additionally, mutation of the predicted m^6A sites in the Cdk2–3’UTR could mitigated the degradation of CDK2 m RNA after MA2 treatment.Conclusion: MA2 affected CDKs expression through the m^6A-dependent m RNA degradation pathway, and thus repressed spermatogonial proliferation.

Torin 1,TOR Inhibitor Enhances Cellular Proliferation in NT-1 Tobacco Suspension Cell Cultures

Torin 1 is an ATP-competitive TOR inhibitor which inhibits the signaling of TOR and S6K kinase in mammals and plants.The objective of this research is to determine the effect of Torin 1 in a relatively simple and homogeneous plant system such as the NT-1 tobacco suspension cell cultures.Cultures of NT-1 cells were tested with 5,50,150 and 250 nM of Torin 1.During kinetics growth of NT-1 tobacco suspension cell cultures,150 and 250 nM Torin 1 inhibits the early growth and later enhanced the cellular proliferation during exponential growth by means of an increased expression of E2F1 and cyclin B.Furthermore,Torin 1 stimulates the growth of NT-1 cells during log phase with small shaped cell,characteristic of tobacco suspension cell cultures with high mitotic activity.

miR-216a和CAPN6基因过表达人宫颈癌细胞系HeLa增殖迁移侵袭变化及靶向关系

目的观察微小RNA-216a(miR-216a)和钙蛋白酶6(CAPN6)基因过表达的人宫颈癌细胞系HeLa增殖、迁移、侵袭变化及靶向关系,探讨miR-216a对HeLa细胞增殖迁移侵袭影响的作用机制。方法取对数生长期HeLa细胞,分为一、二、三、四、五组,一组转染miR-216a mimic,二组转染pcDNA3.1-CAPN6质粒,三组顺序转染miR-216a mimic、pcDNA3.1-CAPN6,四组转染pcDNA3.1,五组转染miR-216a mimic NC,培养48 h时分别采用CCK-8法、划痕愈合实验、Transwell试验观察各组细胞的增殖迁移侵袭情况,培养24 h时采用qRT-PCR法检测各组细胞miR-216a、采用WesternBlotting法检测各组细胞CAPN6及信号转导子与激活子3(STAT3)蛋白。取对数生长期HeLa细胞分为四组:A组细胞顺序转染miR-216a mimic、pGL3-CAPN6-WT质粒,B组细胞顺序转染miR-216a mimic、pGL3-CAPN6-MUT质粒,C组细胞顺序转染miR-216amimicNC、pGL3-CAPN6-WT,D组细胞顺序转染miR-216amimicNC、pGL3-CAPN6-MUT,培养36 h时收集各组细胞,采用双荧光素酶报告基因检测试剂盒测算各组细胞相对荧光素酶活性。结果与五组相比,培养48 h时一组细胞增殖活性及划痕前缘迁移距离百分比低、侵袭细胞数少(P均<0.01);与四组相比,二组细胞增殖活性及划痕前缘迁移距离百分比高、侵袭细胞数多(P均<0.01);与一组相比,三组细胞增殖活性及划痕前缘迁移距离百分比高、侵袭细胞数多(P均<0.01);与二组相比,三组细胞增殖活性及划痕前缘迁移距离百分比低、侵袭细胞数少(P均<0.01)。与五组相比,一组细胞miR-216a相对表达量高(P<0.01)。与四组相比,培养24 h时二组细胞CAPN6蛋白、p-STAT3/STAT3相对表达量高,三组细胞表达CAPN6蛋白、p-STAT3/STAT3相对表达量低;与五组相比,一组细胞CAPN6蛋白、p-STAT3/STAT3相对表达量低;与一组相比,三组细胞CAPN6蛋白、p-STAT3/STAT3相对表达量高。与B组相比,A组细胞荧光素酶活性低(P<0.05)。结论miR-216a过表达可抑制HeLa细胞的增殖侵袭和迁移。HeLa细胞中miR-216a与CAPN6基因存在靶向关系。miR-216a可能通过调控CAPN6基因表达,抑制HeLa的增殖、迁移及侵袭。

B细胞淋巴瘤6对HTR-8/SVneo滋养细胞增殖、凋亡、侵袭和迁移的影响

目的:探讨B细胞淋巴瘤6(B cell lymphoma 6,BCL6)基因过表达对人绒毛膜滋养细胞HTR-8/SVneo增殖、凋亡、侵袭、迁移的影响。方法:利用慢病毒介导的过表达LV5-BCL6(OE-BCL6)技术构建BCL6过表达的HTR-8/SVneo细胞系,同时设阴性对照组(OE-NC),并采用实时荧光定量聚合酶链反应(real-time fluorescence quantitative polymerase chain reaction,qRT-PCR)和蛋白质印迹(Western blotting)检测BCL6的mRNA和蛋白表达水平。随后进行CCK8法、流式细胞术、Transwell实验以及Transwell小室侵袭实验,对比2组HTR-8/SVneo细胞增殖、凋亡、侵袭和迁移的能力。结果:荧光显微镜观察结果显示,OE-BCL6组和OE-NC组HTR-8/SVneo细胞的慢病毒转染后细胞生长状态良好,感染效率都达到了70%以上,慢病毒转染成功。mRNA水平检测显示,OE-BCL6组中BCL6表达高于OE-NC组(P<0.001)。蛋白水平检测显示,OE-BCL6组中BCL6表达高于OE-NC组(P<0.001)。转染48 h和72 h后,OEBCL6组细胞的增殖活性较OE-NC组降低,OE-BCL6组细胞的凋亡率较OE-NC组升高(均P<0.05)。OE-BCL6组细胞侵袭和迁移的细胞数均较OE-NC组降低(均P<0.05)。结论:BCL6基因可抑制HTR-8/SVneo细胞增殖、侵袭及迁移,促进细胞凋亡,提示BCL6可能是临床病理妊娠诊疗的一个潜在靶点,但还需进一步研究阐明其分子机制。

Leptin activates the JAK/STAT pathway to promote angiogenesis in RF/6A cells in vitro

AIM: To investigate the effect of leptin on the angiogenesis of RF/6 A cells(monkey retinal choroidal endothelial cells) in vitro and test the cellular signaling in the mechanism.METHODS: RF/6 A cells were cultured in vitro and randomly divided into four groups: normal control, with leptin at 50, 100, 200 ng/m L for cell counting kit-8(CCK8). RF/6 A cell proliferation and migration were examined by Transwell assays, while RF/6 A cell tube formation by Matrigel assay. JAK2, p-JAK2, STAT3, and p-STAT3 protein expression was measured by Western blotting. Cells were then divided into the following treatment groups: control, 100 ng/m L leptin and AG-490(100 ng/m L leptin+10 μmol/L AG-490) for examinations of RF/6 A cellular behaviour again. Analysis of differences was carried out using one-way ANOVA and least significant difference(LSD).RESULTS: RF/6 A cell proliferation, migration and cell tube formation were promoted significantly by leptin in a dose-dependent manner(P<0.05). Western blotting showed that leptin up-regulated p-JAK2 and p-STAT3 expression levels. Treatment with the JAK/STAT pathway inhibitor, AG-490, decreased leptin-induced p-JAK2 and p-STAT3 expression, and inhibited cell proliferation, migration and cell tube formation induced by leptin(P<0.05). CONCLUSION: Leptin can promote RF/6 A cell angiogenesis in vitro via activation of the JAK2/STAT3 signaling pathway.

Frequent Down-regulation and Deletion of KLF6 in Primary Hepatocellular Carcinoma

Kruppel-like factor 6 (KLF6) was reported as tumor suppressor in multiple cancers. However, loss of chromosomal locus spanning KLF6 is relatively infrequent in previous published studies. To explore the role of KLF6 in hepatocellular carcinoma (HCC), we examined the gene for expression change, loss of heterozygosity (LOH) and mutation in 26 HCC samples. The expression levels of KLF6 were significantly down-regulated in HCCs, as detected by qRT-PCR. LOH occurred in 11 (52%) of 21 tumors, and all the samples with LOH showed KLF6 down-regulation. The mutational frequency was 24%, and sequence changes located in activation domain of KLF6. Furthermore, MTT assay showed a significant antiproliferative effect of the wt KLF6 transfected in HepG2 hepatoblastoma cells. Fluorescence-activated cell sorting analysis revealed that KLF6 could induce apoptosis. These findings indicate that deregulation of KLF6, together with genetic abnormalities of allelic imbalance and mutations, may play a role in HCC pathogenesis.

Deregulated expression and subcellular localization of CPSF6,a circRNA-binding protein,promote malignant development of esophageal squamous cell carcinoma

Objective:Cleavage and polyadenylation specific factor 6(CPSF6)has been documented as an oncoprotein in different types of cancer.However,functions of CPSF6 have not been investigated yet in esophageal squamous cell carcinoma(ESCC).Here,we aimed to investigate the potential clinical values and biological functions of CPSF6 in ESCC.Methods:For determining the expression level of CPSF6 in ESCC patients,we analyzed published data,performed quantitative real-time polymerase chain reaction(RT-qPCR)and immunohistochemistry assays.Kaplan-Meier curves and log-rank tests were used for survival analyses.GO and KEGG analyses were done for CPSF6-related genes.Cell proliferation,colony formation and xenograft assays were conducted to verify the effects of CPSF6 on ESCC.In addition,cell cycle and apoptosis assays were also performed to manifest the functions of CPSF6 and circCPSF6.RNA pulldown and radioimmunoprecipitation(RIP)assays were used for confirming the interaction between circCPSF6(hsa_circ_0000417)and CPSF6 protein.The regulatory relationship between CPSF6 protein and circCPSF6 was determined by RT-qPCR.Results:We found that CPSF6 was upregulated in ESCC tissues and overexpression of cytoplasmic CPSF6 was associated with poor prognosis.GO and KEGG analyses suggested that CPSF6 could mainly affect cell division in ESCC.Further experiments manifested that CPSF6 promoted cell proliferation and colony formation in vitro.Xenograft assay showed that knockdown of CPSF6 significantly decreased tumor growth rate in vivo.Subsequently,we verified that depletion of CPSF6 led to cell cycle arrest and apoptosis.Finally,we validated that CPSF6,as a circRNA-binding protein,interacted with and regulated its circular isoform circCPSF6(hsa_circ_0000417),of which depletion also resulted in cell cycle arrest and cell apoptosis in ESCC.Conclusions:These findings gave us insight that overexpression of cytoplasmic CPSF6 protein is associated with poor prognosis in ESCC and CPSF6 may function as an oncoprotein,at least in part,through regulating circCPSF6 expression.

6-姜辣素对人多发性骨髓瘤细胞的抑制作用及分子机制

目的研究6-姜辣素对人多发性骨髓瘤细胞的抑制作用及分子机制。方法体外培养人多发性骨髓瘤RPMI 8226、ARH77细胞,加入不同浓度(50、100、200、300、400μmol/L)的6-姜辣素处理RPMI 8226、ARH77细胞,CCK-8法测定细胞增殖抑制率;流式细胞术检测细胞凋亡和周期;qRT-PCR和Western blotting法检测基因和蛋白表达水平。结果6-姜辣素以剂量和时间依赖的方式抑制RPMI8226、ARH77细胞的增殖,并诱导其凋亡,差异有统计学意义(P<0.05);进一步机制研究发现6-姜辣素处理RPMI 8226细胞后,将细胞阻滞在G_(0)/G_(1)期,显著增加Bax mRNA水平,降低Bcl-2 mRNA和c-Myc mRNA的水平(P<0.05);同时显著增加Bax、Cleaved-PARP、Cleaved-caspase3、P53、p-AKT蛋白的表达,而降低Bcl-2蛋白的表达(P<0.05)。结论6-姜辣素可抑制MM细胞增殖及诱导其凋亡,并阻滞细胞周期在G_(0)/G_(1)期,其机制可能与抑制AKT信号通路并通过影响Bcl-2家族蛋白表达以及c-Myc表达受抑等密切有关。

PROMOTION OF IN VITRO GROWTH OF HUMAN MEDULLOBLASTOMA CELLS BY EXOGENEOUS IL－6

In vivo and in vitro expression of IL-6 and its signal transducer genes, IM-6R and gp130, in human medulloblastoma cells were investigated by the approaches of molecular biologr and cellular immunology.The results revealed that 12 out of 13 samples examined were found to express IL-6R and gp 130, but none of them showcd IL-6 expression. Thcn, the Potential effccts of cxogenous IL-6 on the proliferation of medulloblastoma cell line, Med-3 were evaluated, which showed that IL-6could enhance cell outgrowth dramatically. Our data thus for the first t'me demonstrate the important role of IL-6as paracrine growth factor in the proliferat'on of medulloblastoma cells.

基于VEGFA/TNF/IL-6信号通路探讨灵泽片对良性前列腺增生大鼠的治疗作用

目的:基于VEGFA/TNF/IL-6通路探讨灵泽片对良性前列腺增生大鼠的干预机制。方法:将30只SPF级SD大鼠随机等分为空白对照组、模型对照组、灵泽片低剂量组、灵泽片中剂量组、灵泽片高剂量组。除空白对照组外,余各组大鼠采用非去势丙酸睾酮诱导法建立前列腺增生大鼠模型。造模结束后,空白对照组及模型对照组选用生理盐水,灵泽片低、中、高剂量组分别选用相应药物,连续灌胃给药28 d。灌胃结束后处死大鼠,取前列腺组织,观察各组大鼠前列腺指数、组织结构及VEGFA/TNF/IL-6信号通路相关蛋白的变化。结果:与空白对照组比较,模型对照组大鼠前列腺组织增生明显;前列腺指数明显上升(0.84±0.01,P<0.05),组织学观察形态可见前列腺上皮组织厚度扩大和管腔区浸润现象,VEGFA(0.60±0.02,P<0.05)、TNF(0.76±0.02,P<0.05)、IL-6(0.64±0.02,P<0.05)蛋白表达水平显著上升,差异具有统计学意义。与模型对照组比较,灵泽片低剂量组(0.76±0.02,P<0.05)、中剂量组(0.58±0.02,P<0.05)、高剂量组(0.52±0.01,P<0.05)大鼠前列腺指数均有所下降,差异有统计学意义;组织形态可见显著改善,WB结果显示灵泽片干预各组的VEGFA蛋白(低剂量组:0.45±0.01,中剂量组:0.35±0.01,高剂量组:0.31±0.02,各组P均<0.05)、TNF蛋白(低剂量组:0.45±0.01,中剂量组:0.33±0.01,高剂量组:0.27±0.01,各组P均<0.01)、IL-6蛋白(低剂量组:0.44±0.01,中剂量组:0.36±0.01,高剂量组:0.30±0.01,各组P均<0.01)表达水平均有所下降,差异有统计学意义,且改善情况与灵泽片剂量呈正比。结论:灵泽片可能通过负向调控VEGFA/TNF/IL-6信号通路,下调VEGFA/TNF/IL-6蛋白表达水平,调控细胞增殖与凋亡,调节炎症反应,从而发挥对BPH的治疗作用。

PRMT6促进乳腺癌细胞的增殖和迁移

目的·研究蛋白质精氨酸甲基转移酶6 (protein arginine methyltransferase 6,PRMT6)在乳腺癌中的表达及其对乳腺癌细胞增殖和迁移能力的影响。方法·通过R语言分析癌症基因组图谱(The Cancer Genome Atlas,TCGA)数据库中PRMT6mRNA在多种癌症中的表达情况。采用基因表达谱交互分析(GeneExpressionProfilingInteractive Analysis,GEPIA2)在线数据库分析PRMT6在正常乳腺组织和乳腺癌组织中的表达差异。利用人类蛋白质图谱(The Human Protein Atlas,HPA)数据库获得人正常乳腺组织和乳腺癌组织的免疫组织化学数据,分析PRMT6的蛋白表达情况。使用免疫组织化学法(immunohistochemistry,IHC)检测27例乳腺癌组织及配对癌旁组织中PRMT6的表达。通过小干扰RNA (small interfering RNA,siRNA)转染技术在MDA-MB-231和MCF-7细胞系中敲低PRMT6,实时荧光定量PCR (quantitative realtime PCR,qRT-PCR)及Western blotting在转录和蛋白水平验证PRMT6的敲低效率。通过细胞计数试剂盒8 (cell counting kit-8,CCK-8)、克隆形成实验探究PRMT6对乳腺癌细胞增殖能力的影响。通过细胞划痕实验和Transwell实验探究PRMT6对乳腺癌细胞迁移能力的影响。利用基因表达综合(Gene Expression Omnibus,GEO)数据库中GSE210948数据集的转录组测序数据分析对照组和PRMT6低表达组的差异基因,并进行京都基因与基因组数据库(Kyoto Encyclopedia of Genes and Genomes,KEGG)通路富集分析。使用流式细胞术进行细胞周期分析。采用Western blotting技术检测增殖和迁移相关靶蛋白细胞周期蛋白D1 (cyclin D1)、E-钙黏蛋白(E-cadherin)、N-钙黏蛋白(N-cadherin)和波形蛋白(Vimentin)的表达。结果·生物信息学相关分析显示,PRMT6在乳腺癌组织中的表达高于正常乳腺组织(P=0.000)。IHC结果显示,PRMT6在乳腺癌组织中的表达显著高于配对癌旁组织(P=0.001)。qRT-PCR及Western blotting验证MDA-MB-231和MCF-7细胞系中PRMT6的mRNA及蛋白质表达水平,发现与对照组相比,siRNA (siPRMT6#1、siPRMT6#2)显著降低两种细胞中PRMT6mRNA (P=0.006, P=0.004;P=0.001, P=0.043)和蛋白(P=0.035, P=0.001;P=0.003, P=0.002)的表达水平。敲低PRMT6显著降低MDA-MB-231和MCF-7细胞的增殖(P=0.014,P=0.000;P=0.003,P=0.003)和迁移能力(P=0.000,P=0.000;P=0.000,P=0.002)。KEGG通路富集分析显示,PRMT6的表达影响细胞周期通路。Western blotting结果显示,敲低PRMT6后,与细胞周期通路相关的cyclin D1蛋白水平下降(P=0.021,P=0.000;P=0.034,P=0.014);qRT-PCR结果显示,敲低PRMT6后,cyclin D1转录水平明显下降(P=0.036,P=0.001;P=0.044,P=0.000)。流式细胞术结果显示,敲低PRMT6后,G0/G1期细胞增加(P=0.000;P=0.003), G2/M期细胞减少。下调PRMT6表达后,与细胞迁移相关的Ecadherin表达增加(P=0.002, P=0.012;P=0.043, P=0.003), N-cadherin (P=0.004, P=0.041;P=0.032, P=0.034)和Vimentin (P=0.028,P=0.005;P=0.024,P=0.001)的蛋白表达减少。结论·PRMT6在乳腺癌组织中表达升高,可促进乳腺癌的增殖和迁移。

基于IL-6/JAK2/STAT3通路探讨化瘀消痞汤对胃癌前病变大鼠胃黏膜损伤的作用及机制

目的基于白细胞介素6(IL-6)/Janus激酶2(JAK2)/信号转导和转录激活因子3(STAT3)通路探讨化瘀消痞汤对胃癌前病变(PLGC)大鼠胃黏膜损伤的作用及机制。方法随机选取10只大鼠作为空白组,其余50只大鼠作为造模组,采用多因素综合造模。将模型复制成功的大鼠随机分为模型组、叶酸组(0.002 g·kg^(-1))及化瘀消痞汤低、中、高剂量组(6.2、12.4、24.8 g·kg^(-1))。灌胃给药,每日1次,连续干预3个月。观察大鼠一般状况并测定体质量、3 h进食量;采用HE染色法观察胃组织病理变化;ELISA法检测血清中IL-6、肿瘤坏死因子α(TNF-α)、IL-1β水平;免疫荧光法检测胃组织中B细胞淋巴瘤/白血病-xL(Bcl-xL)、血管内皮生长因子(VEGF)蛋白表达水平;WesternBlot法检测胃组织中IL-6、p-JAK2、p-STAT3、细胞周期素D1(CyclinD1)、原癌基因(c-Myc)蛋白表达水平。结果与空白组比较,模型组大鼠体质量、3 h进食量明显降低(P<0.05);胃黏膜明显变薄且不完整,腺体排列紊乱且数量减少,部分区域可见胃上皮细胞肠化的杯状细胞及炎性细胞浸润;血清IL-6、TNF-α、IL-1β水平明显升高(P<0.05);胃组织中Bcl-xL、VEGF、IL-6、p-JAK2、p-STAT3、CyclinD1、c-Myc蛋白表达明显上调(P<0.05)。与模型组比较,化瘀消痞汤中、高剂量组大鼠的体质量、3 h进食量明显升高(P<0.05);胃黏膜损伤有不同程度改善,腺体排列趋于整齐,胃上皮细胞肠化情况有所改善,炎性细胞较少;血清IL-6、TNF-α、IL-1β水平明显降低(P<0.05);胃组织中Bcl-xL、VEGF、IL-6、p-JAK2、p-STAT3、CyclinD1、c-Myc蛋白表达明显下调(P<0.05)。结论化瘀消痞汤能明显改善PLGC大鼠的胃黏膜病理损伤,可能与其抑制炎症反应及调控IL-6/JAK2/STAT3通路抑制细胞增殖有关。

LSM6 promotes cell proliferation and migration regulated by HMGB1 in laryngeal squamous cell carcinoma

Elevated levels of high mobility group protein B1(HMGB1)play a significant role in the pathogenesis of many diseases,but is particularly important for the formation of malignant tumors.Nonetheless,the function of HMGB1 and the underlying mechanism of laryngeal squamous cell carcinoma(LSCC)remain incompletely understood,causing uncertainty.Here we found immunohistochemistry from 97 LSCC tissues showed HMGB1 was upreg-ulated,which was associated with poor differentiation.HMGB1 knockdown could significantly inhibit wound closure and colony formation.The full-genome gene expression microarray was performed to investigate the mechanism.After knockdown of HMGB1 by siRNA,among the expressed differential genes,10 genes were ran-domly selected for validation.Then,shRNA lentivirus targeting these genes were constructed to explore their role in LSCC by cell proliferation assay.LSM6 downregulation was dramatically promoted by HMGB1 knockdown,resulting in higher expression in LSCC tissues.Furthermore,downregulation of LSM6 could significantly suppress cell proliferation,migration and colony formation.This study indicated that HMGB1 promoted LSCC cell malig-nant phenotypes through regulation of LSM6.We anticipate that HMGB1-LSM6 could be a putative therapeutic target for LSCC.