Claudin-15 overexpression inhibits proliferation and promotes apoptosis of Schwann cells in vitro

Our previous experiments have discovered that Claudin-15 was up-regulated in Schwann cells of the distal nerve stumps of rat models of sciatic nerve injury.However,how Claudin-15 affects Schwann cell function is still unknown.This study aimed to identify the effects of Claudin-15 on proliferation and apoptosis of Schwann cells cultured in vitro and explore the underlying mechanisms.Primary Schwann cells were obtained from rats.Claudin-15 in Schwann cells was knocked down using siRNA(siRNA-1 group)compared with the negative control siRNA transfection group(negative control group).Claudin-15 in Schwann cells was overexpressed using pGV230-Claudin-15 plasmid(pGV230-Claudin-15 group).The pGV230 transfection group(pGV230 group)acted as the control of the pGV230-Claudin-15 group.Cell proliferation was analyzed with EdU assay.Cell apoptosis was analyzed with flow cytometric analysis.Cell migration was analyzed with Transwell inserts.The mRNA and protein expressions were analyzed with quantitative polymerase chain reaction assay and western blot assay.The results showed that compared with the negative control group,cell proliferation rate was up-regulated;p-AKT/AKT ratio,apoptotic rate,p-c-Jun/c-Jun ratio,mRNA expression of protein kinase C alpha,Bcl-2 and Bax were down-regulated;and mRNA expression of neurotrophins basic fibroblast growth factor and neurotrophin-3 were increased in the siRNA-1 group.No significant difference was found in cell migration between the negative control and siRNA-1 groups.Compared with the pGV230 group,the cell proliferation rate was down-regulated;apoptotic rate,p-c-Jun/c-Jun ratio and c-Fos protein expression increased;mRNA expression of protein kinase C alpha and Bax decreased;and mRNA expressions of neurotrophins basic fibroblast growth factor and neurotrophin-3 were up-regulated in the pGV230-Claudin-15 group.The above results demonstrated that overexpression of Claudin-15 inhibited Schwann cell proliferation and promoted Schwann cell apoptosis in vitro.Silencing of Claudin-15 had the reverse effect and provided neuroprotective effect.This study was approved by the Experimental Animal Ethics Committee of Jilin University of China(approval No.2016-nsfc001)on March 5,2016.

Adenoviral 15-lipoxygenase-1 gene transfer inhibits hypoxia-induced proliferation of retinal microvascular endothelial cells in vitro

AIM: To investigate whether 15-Lipoxygenase-1 (15-LOX-1) plays an important role in the regulation of angiogenesis, inhibiting hypoxia-induced proliferation of retinal microvascular endothelial cells (RMVECs) and the underlying mechanism. METHODS: Primary RMVECs were isolated from the retinas of C57/BL63 mice and identified by an evaluation for FITC-marked CD31. The hypoxia models were established with the Bio-bag and evaluated with a blood-gas analyzer. Experiments were performed using RMVECs treated with and without transfer. Ad-15-LOX-1 or Ad-vector both under hypoxia and normoxia condition at 12, 24, 48, 72 hours. The efficacy of the gene transfer was assessed by immunofluorescence staining. Cells proliferation was evaluated by the CCK-8 method. RNA and protein expressions of 15-LOX-1, VEGF-A, VEGFR-2, eNOs and PPAR-r were analyzed by real-time reverse transcription polymerase chain reaction (RT-PCR) and Western blot. RESULTS: Routine evaluation for FITC-marked CD31 showed that cells were pure. The results of blood-gas analysis showed that when the cultures were exposed to hypoxia for more than 2 hours, the Po2 was 4.5 to 5.4 Kpa. We verified RMVECs could be infected with Ad-LS-LOX-for Ad-vectorvia Fluorescence microscopy. CCK-8 analysis revealed that the proliferative capacities of RMVECs in hypoxic group were significantly higher at each time point than they were in normoxic group (P <0.05). In a hypoxic condition, the proliferative capacities of RMVECs in 15-LOX-1 group were significantly inhibited (P<0.05). Real-time RT-PCR analysis revealed that the expressions of VEGF-A, VEGF-R2 and eNOs mRNA increased in hypoxia group compared with normoxia group (P<0.01). However, the expressions of 15-LOX-1, PPAR-r mRNA decreased in hypoxia group compared with normoxia group (P<0.01). It also showed that in a hypoxic condition, the expressions of VEGF-A, VEGF-R2 and eNOs mRNA decreased significantly in 15-LOX-1 group compared with hypoxia group (P<0.01). However, 15-LOX-1 and PPAR-r mRNA increased significantly in 15-LOX-1 group compared with hypoxia group (P<0.01). There was no significant difference of the mRNA expressions between vector group and hypoxia group (P>0.05). Western blot analysis revealed that the expressions of relative proteins were also ranked in that order. CONCLUSION: Our results suggested that 15-LOX-1 and PPAR-r might act as a negative regulator of retinal angiogenesis. And the effect of 15-LOX-1 overexpression is an anti-angiogenic factor in hypoxia-induced retinal neovascularization (RNV). Overexpression 15-LOX-1 on RMVECs of hypoxia-induced RNV blocked signaling cascades by inhibiting hypoxia-induced increases in VEGF family. PPAR-r effect on VEGFR(2) could be an additional mechanism whereby 15-LOX-1 inhibited the hypoxia-induced RNV.

Expression of Interleukin-15 and Its Receptor on the Surface of Stimulated Human Umbilical Vein Endothelial Cells

Summary： Human interleukin-15 （hIL-15） is an important cytokine to activate endothelial cells and can be regulated by many other cytokines. The aim of this study is to examine the ability of interferon-γ （IFN-γ）, and tumor necrosis factor-or （TNF-α to induce the production of human interleukin-15 （hIL-15） and IL-15 receptor （IL-15Rα by human umbilical vein endothelial cells （HUVECs）. The data are summarized as follows： 1. Northern blot revealed that IL-15 mRNA was up-regulated by IFN-γ and TNF-α 2. Intracellular IL-15 protein was visualized by fluorescence microscopy, whereas the expression of IL-15 on the surface of HUVECs was detected by fluorescence activated cell sorting （FACS）, and no detectable IL-15 in the medium was verified by ELISA. 3. IL-15Rα was detected on the surface of HUVECs by FACS after IFN-α and TNF-α stimulation, whereas Western blotting revealed that the elevated expression on surface IL-15Rα was not due to the increased protein expression. The conclusion demonstrated from our results is that IFN-α and TNF-α play an important role in regulating the expression of IL-15 and IL-15Rα on the surface of HUVECs.

Inhibition of the Growth of Raji Cells by Precursor MicroRNA-15a

OBJECTIVE To investigate the effects of microRNA-15a （miR- 15a） on the growth of the lymphoma Raji cell line. METHODS A eukaryotic expression vector of precursor mLR-15a （pre-miR-15a） was constructed. Paired oligonucleotides for pre- miR-15a expression were designed and synthesized chemically. Annealed oligonucleotides were inserted into a pGCSIL- GFP vector under a U6 promoter to construct a pre-miR-15a expression plasmid. Oligonucleotides with a scrambled sequence which were used as a negative control were also constructed into the pGCSIL-GFP vector. A recombinant expression vector was identified through PCR and sequencing. A pre-miR-15a expression plasmid and a negative control plasmid were all transferred into Raji cells with lipofectamine 2000. Quantitative reverse-transcription polymerase chain reaction （qRT-PCR） was employed to explore expression of miR-15a. The expression levels of the protein were assayed via immunofluorescence. The growth effect of Raji cells was measured by CCK8 assay. Apoptosis was determined using Hoechst dyeing and flow cytometry. The growth-inhibitory effect on Raji cells was measured using a CCK8 assay. RESULTS The results showed that the insertion sequence was correct. The expression level of miR-15a was obviously higher in Raji cells transferred with pre-miR-15a plasmid than in the blank group and in the negative control group. After Raji cells were transferred with the pre-miR-15a expression plasmid for 48 h, Bcl-2 protein expression levels were obviously decreased, which indicated that there was a significant difference as compared with the negative control group and blank group （P 〈 0.05）. The CCK8 assay showed that transfection of pre-miR-15a expression plasmid decreased the cell growth at 24, 48 and 72 h post-transfection. After Raji cells were transferred with pre-miR-15a expression plasmid, apoptotic cells can be seen with Hoechst dyeing, and the cell apoptotic rate was obviously higher than that in blank group and negative control group. CONCLUSION Pre-miR-15a can induce apoptosis and inhibit the growth of Raji cells.

Combined transfection of Bcl-2 siRNA and miR-15a oligonucleotides enhanced methotrexate-induced apoptosis in Raji cells

Objective: B-cell lymphoma 2 (Bcl-2) is an important member of the Bcl-2 family of proteins that regulate the induction of apoptosis. This study aims to investigate whether Bcl-2 small interfering RNA (siRNA) combined with miR-15a oligonucleotides (ODN) could enhance methotrexate (MTX)-induced apoptosis in Raji cells. Methods: Chemically synthesized miR-15a ODN and Bcl-2 siRNA were transfected in Raji cells by using a HiPerFect Transfection Reagent and then combined with MTX. Expression levels of Bcl-2 protein were detected by Western blot. Cell proliferation was determined by CCK8 assay. The rate of cell apoptosis was determined by Annexin V/PI double staining. The morphology of apoptotic cells was observed by Hoechst-33 258 staining. Results: After the cells were transfected with miR-15a ODN combined with Bcl-2 siRNA, Bcl-2 protein levels were evidently decreased. CCK8 assay showed that cell proliferation was significantly decreased and was significantly lower in miR-15a ODN combined with Bcl-2 siRNA plus MTX group than in miR-15a ODN with methotrexate group, Bcl- 2 siRNA with MTX group, and single MTX group (P<0.05). Hoechst 33258 staining revealed numerous apoptotic cells. AnnexinV/PI double staining showed that the apoptotic rates were (13.13±1.60)%, (34.47±2.96)%, (32.87±3.48)%, and (45.47±2.16)% in MTX, Bcl-2 siRNA plus MTX, miR-15a ODN plus MTX, and miR-15a ODN combined with Bcl- 2 siRNA plus MTX groups, respectively. Among these groups, the apoptotic rate of miR-15a ODN combined with Bcl-2 siRNA plus MTX group was the highest; this apoptotic rate was also significantly different from that of miR-15a ODN or Bcl-2 siRNA plus MTX (P<0.05). Conclusions: Bcl-2 siRNA combined with miR-15a ODN could enhance MTX-induced apoptosis in Raji cells. Bcl-2 siRNA and miR-15a combined with MTX may be a useful approach to improve the treatment effects on lymphoma.

TNFSF15 facilitates the differentiation of CD11b^(+) myeloid cells into vascular pericytes in tumors

Objective:Immature vasculature lacking pericyte coverage substantially contributes to tumor growth,drug resistance,and cancer cell dissemination.We previously demonstrated that tumor necrosis factor superfamily 15(TNFSF15)is a cytokine with important roles in modulating hematopoiesis and vascular homeostasis.The main purpose of this study was to explore whether TNFSF15 might promote freshly isolated myeloid cells to differentiate into CD11b^(+) cells and further into pericytes.Methods:A model of Lewis lung cancer was established in mice with red fluorescent bone marrow.After TNFSF15 treatment,CD11b^(+) myeloid cells and vascular pericytes in the tumors,and the co-localization of pericytes and vascular endothelial cells,were assessed.Additionally,CD11b^(+) cells were isolated from wild-type mice and treated with TNFSF15 to determine the effects on the differentiation of these cells.Results:We observed elevated percentages of bone marrow-derived CD11b^(+)myeloid cells and vascular pericytes in TNFSF15-treated tumors,and the latter cells co-localized with vascular endothelial cells.TNFSF15 protected against CD11b^(+)cell apoptosis and facilitated the differentiation of these cells into pericytes by down-regulating Wnt3a-VEGFR1 and up-regulating CD49e-FN signaling pathways.Conclusions:TNFSF15 facilitates the production of CD11b^(+) cells in the bone marrow and promotes the differentiation of these cells into pericytes,which may stabilize the tumor neovasculature.

Siglec-15与CD4^(+)、CD8^(+)在肝细胞癌中的表达关系及其临床意义

目的研究在肝细胞癌(hepatocellutar carcinoma,HCC)中唾液酸免疫球蛋白型凝集素-15(Sialic acid immunoglobulin type lectin 15,Siglec-15)与CD4^(+)、CD8^(+)的表达及相关性,并分析其与临床病理特征及预后的影响。方法通过TCGA数据库分析HCC与正常组织中Siglec-15、CD4^(+)T、CD8^(+)T mRNA的表达水平。收集我院2019年1月至2022年1月行肝癌根治切除术的27例新鲜HCC组织及正常组织标本和56例HCC组织及正常组织石蜡切片标本,qRT-PCR检测Siglec-15和CD4^(+)T、CD8^(+)T mRNA表达水平,IHC检测Siglec-15和CD4^(+)、CD8^(+)蛋白表达水平,并对Siglec-15与CD4^(+)、CD8^(+)蛋白之间的关系及临床病理因素和预后进行分析。结果HCC患者中Siglec-15在HCC组织表达高于正常组织(P<0.05),CD4^(+)、CD8^(+)在HCC组织表达低于正常组织(P<0.05);在HCC组织中,Siglec-15表达与CD4^(+)、CD8^(+)蛋白表达呈负相关(P<0.05)。在HCC组织中,Siglec-15蛋白的表达与患者的TNM分期、肿瘤大小相关(P<0.05),CD4^(+)蛋白的表达与患者的肿瘤大小相关(P<0.05),CD8^(+)蛋白的表达与患者的TNM分期、远处转移相关(P<0.05)。Siglec-15及CD4^(+)的表达与患者的无进展生存期具有相关性(P<0.05)。结论HCC组织中Siglec-15高表达、CD4^(+)、CD8^(+)低表达,且Siglec-15与CD4^(+)、CD8^(+)表达呈负相关。Siglec-15及CD4^(+)的表达高低与HCC患者进展及不良预后相关,可作为预测HCC患者的独立预后因素。

The study of miR-15a oligonucleotide inhibiting cell growth and enhancing Ara-C-induced apoptosis in Raji cells

Objective:The aim of the research was to study whether microRNA-15a(miR-15a) oligonucleotide could inhibit cell growth and enhance cytarabine(Ara-C)-induced apoptosis in Raji cells.Methods:Transfecting miR-15a oligonucleotide into Raji cells with LipofectamineTM 2000,and then combined with Ara-C.IC50 value and cell proliferation were detected by CCK8 assay;the expression levels of Bcl-2 mRNA and protein were evaluated by RT-PCR and indirect immuno-fluorescence.The apoptotic cells were observed by Hoechst Dyeing;AnnexinV/PI double dyeing method was used to detect the cell apoptotic rate by Flow Cytometry(FCM).Results:After Raji cells were transfected with miR-15a oligonucleotide for 48 h,Bcl2 protein expression levels obviously decreased,however,there was no difference in Bcl-2 mRNA levels,as compared with the control group and blank group(P < 0.05).CCK8 assay showed that miR-15a oligonucleotide decreased the cell growth at 24,48 and 72 h,moreover,miR-15a oligonucleotides combined with Ara-C obviously decreased the cell growth than miR-15a group,Ara-C group and scrambled oligonucleotides(SODN) + Ara-C group.Meanwhile,miR-15a oligonucleotides combined with Ara-C significantly decreased IC50 of Ara-C(10.41 μg/mL),which were obviously lower than those of Ara-C group(15.43 μg/mL) and SODN plus Ara-C group(14.92 μg/mL).Plenty of apoptotic cells could be seen with Hoechst dyeing.AnnexinV/PI double dying assays by FCM indicated that the cell apoptotic rates in earlier period and late period of miR-15a + Ara-C group were 20.93% and 25.27%,respectively,which were obviously higher than those of miR-15a group,Ara-C group and SODN plus Ara-C group.Conclusion:miR-15a oligonucleotides can inhibit cell growth and enhance Ara-C-induced apoptosis in Raji cells.

Correlation of serum GDF-15 and MIF contents with the malignant behaviors of cancer cells in patients with NSCLC

Objective:To study the correlation of serum GDF-15 and MIF contents with the malignant behaviors of cancer cells in patients with NSCLC.Methods: Patients with non-small cell lung cancer who underwent surgical resection in Kashgar Prefecture First People's Hospital in the Xinjiang Uygur Autonomous Region between January 2015 and November 2017 were selected as lung cancer group for the research, and healthy volunteers who received physical examination in Kashgar Prefecture First People's Hospital in the Xinjiang Uygur Autonomous Region during the same period were selected as the control group. Serum was collected from the lung cancer group before surgery and from the control group during physical examination respectively to determine the contents of GDF-15 and MIF;lung cancer tissue and adjacent tissue were collected from lung cancer group after surgery to determine the expression of tumor suppressor genes, proliferation genes and invasion genes.Results:Serum GDF-15 and MIF contents of lung cancer group were significantly higher than those of control group;TCF21, Bax, GRPC5A and PTEN mRNA expression in lung cancer tissue were significantly lower than those in the adjacent tissue whereas Bcl-2, AQP4, c-myc, CyclinD1, SIRT1, CatL, MMP9, N-cadherin and Vimentin mRNA expression were significantly higher than those in adjacent tissue;serum GDF-15 and MIF contents of patients with lung cancer were negatively correlated with TCF21, Bax, GRPC5A and PTEN mRNA expression, and positively correlated with Bcl-2, AQP4, c-myc, CyclinD1, SIRT1, CatL, MMP9, N-cadherin and Vimentin mRNA expression in lung cancer tissue.Conclusion:The abnormal increase of GDF-15 and MIF in patients with NSCLC is closely related to the abnormal proliferation and invasion of cancer cells.

Interleukin-15 Promotes the Commitment of Cord Blood CD34^+ Stem Cells into NK Cells

To explore the effect of rhIL-15 on CB-CD34 + stem cells committing to NK cells, CD34 + stem cells were obtained from cord blood (CB) by magnetic-assisted cell sorting (MACS) method. CD3, CD16 and CD56 molecules expressed on cell surface were detected by flow cytometer. MTT method was used to test the cytotoxicity of NK cells. The results were that stem cell factor (SCF) alone has no effect on CD34 + stem cells. IL-15 stimulated CD34 + stem cells commit to NK cells, and SCF showed strong synergistic effect with IL-15. It was concluded that IL-15 and SCF played different roles during NK cell development, IL-15 promoted CD34 + stem cells differentiate to NK cell precursor and SCF improved the effects of IL-15 on NK cell differentiation.

喉鳞状细胞癌组织中SIGLEC-15表达及其与患者临床病理特征及预后的关系

目的分析喉鳞状细胞癌组织中SIGLEC-15表达及其与患者临床病理特征及预后的关系。方法选取106例喉鳞状细胞癌组织标本及30例癌旁正常组织标本,免疫组化染色检测SIGLEC-15蛋白表达,分析SIGLEC-15表达及其与患者临床病理特征的关系,Kaplan-Meier生存曲线分析SIGLEC-15蛋白阳性表达与患者预后的关系。结果癌组织中SIGLEC-15阳性表达率(61.32%)高于癌旁组织(P<0.05)。SIGLEC-15蛋白阳性表达与喉鳞状细胞癌的分化程度、TNM分期、淋巴结转移相关(P<0.05)。随访截止至2024年3月,106例喉鳞状细胞癌患者死亡36例、生存70例。SIGLEC-15蛋白阳性表达患者累积生存率为49.23%(32/65),低于阴性表达患者的累积生存率92.68%(38/41)(Log Rankχ^(2)=15.955,P<0.001)。结论喉鳞状细胞癌组织中SIGLEC-15的阳性表达率较高,与患者的肿瘤分化程度、TNM分期、淋巴结转移有关。SIGLEC-15的阳性表达可能预示着患者预后较差,可能成为喉鳞状细胞癌患者预后评估的一个重要标志物。

Anti-tumor effects of p53N15-based fusion peptide in the transfected H1299 lung cancer cells

Objective Loss-of-function mutation of p53,a tumor suppressor gene,is an important mechanism for the development of human cancers. In this study we tried to transfect p53N15-based fusion peptide into H1299,a lung cancer cell line,and evaluate the anti-tumor effects of the fusion peptide. Methods Adeno-associated virus (AAV) vectors were used for transfecting p53N15 fusion peptide into p53-null lung adenocarcinoma H1299 cells.

二甲双胍对慢性暴露于高糖及高游离脂肪酸的HIT-T15细胞胰岛素受体酪氨酸蛋白激酶活性的影响

目的观察二甲双胍(MF)对慢性高糖和高脂处理后的HIT-T15细胞(β细胞系)胰岛素受体(IRc)酪氨酸蛋白激酶(TPK)活性的影响,探讨MF对β细胞糖脂毒性,即β细胞胰岛素抵抗的改善作用机制。方法实验分为对照组、对照+MF组、高糖组、高糖+MF组、高脂组、高脂+MF组。将HIT-T15细胞分别接种于含有5.5mmol/L、16.7 mmol/L葡萄糖(G)及0.5 mmol/L软脂酸的培养液中,培养48 h后,加入2.5μg/mL MF干预24h。用放射性酶分析法测定β细胞IRc TPK活性。结果高糖〔(52.5±18.6)pmol/(min.μg)〕和高脂组〔(54.6±14.0)pmol/(min.μg)〕HIT-T15细胞IRc TPK活性分别较对照组〔(119.4±29.1)pmol/(min.μg)〕降低(P<0.01);予2.5μg/mL MF干预24 h后,高糖和高脂组IRc TPK活性较干预前增高〔(113.0±29.8)vs(52.5±18.6)pmol/(min.μg),P<0.01;(98.6±26.1)vs(54.6±14.0)pmol/(min.μg),P<0.01〕,且与对照组相比差异无统计学意义。MF对对照组HIT-T15细胞IRc TPK活性无明显影响。结论高糖和高脂可抑制β细胞IRcTPK活性。MF能明显改善受高糖及高脂所抑制的HIT-T15细胞IRc TPK活性,且恢复到接近正常水平。提示MF对糖脂毒性所致β细胞胰岛素抵抗的改善作用可能与增加IRc TPK活性有关。

单纯疱疹病毒2型ICP27_(377-513)核酸疫苗联合IL-15核酸疫苗免疫效果的观察

目的构建表达单纯疱疹病毒2型感染细胞蛋白27(infected cells protein 27,ICP27)的重组质粒pcDNA3.1-ICP27377-513并观察白细胞介素(IL)-15核酸疫苗联合HSV-2-ICP27377-513核酸疫苗的免疫效果。方法采用分子克隆技术构建pcDNA3.1-ICP27377-513重组质粒。将BALB/c雌性小鼠随机分为pcDNA3.1-ICP27377-513联合pcDNA3.1-IL-15(pIL-15)组、pcDNA3.1-ICP27377-513组、pcDNA3.1组和pIL-15组,共免疫3次,每次间隔2周。末次免疫后28 d取血,用微量中和实验法检测血清中特异性中和抗体,ELISA法检测血清中IL-4、IL-2和干扰素-γ(IFN-γ)水平。阴道给予致死量HSV-2攻击小鼠,分别于接种后3 d、7 d和14 d收集阴道冲洗液,用荧光定量PCR法检测生殖道病毒载量。结果pcDNA3.1-ICP27377-513联合pIL-15组和pcDNA3.1-ICP27377-513组中和抗体效价分别为40.00±8.16、28.67±4.47,但两组间差异无统计学意义(P>0.05)。pcDNA3.1-ICP27377-513联合pIL-15组IFN-γ、IL-4水平明显高于pcDNA3.1-ICP27377-513组(P<0.05),但IL-2水平两组间差异无统计学意义(P>0.05)。阴道给予致死量HSV-2攻击后,pcDNA3.1-ICP27377-513联合pIL-15组和pcDNA3.1-ICP27377-513组小鼠阴道冲洗液中病毒载量随着时间延长逐渐减低,pcDNA3.1-ICP27377-513联合pIL-15组和pcDNA3.1-ICP27377-513组相比差异有统计学意义(P<0.05)。结论IL-15核酸疫苗联合HSV-2-ICP27377-513核酸疫苗对小鼠有较好的免疫保护作用。

唾液酸结合免疫球蛋白样凝集素-15在食管鳞癌组织中的表达及意义

目的探讨免疫检查点唾液酸结合免疫球蛋白样凝集素-15(Siglec-15)在食管鳞癌中的表达及意义。方法免疫组化检测食管鳞癌组织、癌旁正常食管组织中Siglec-15表达情况,并比较分析其与食管鳞癌患者临床病理特征的关系。结果Siglec-15在食管鳞癌组织中表达阳性率为60.0%(48/80),高于癌旁正常食管组织中的23.75%(19/80)(χ^(2)=21.595,P<0.001)。食管鳞癌组织中Siglec-15蛋白表达与患者肿瘤直径、T分期、TNM分期、N分期及分化程度有关(χ^(2)=7.500,P=0.006;χ^(2)=10.342,P=0.001;χ^(2)=22.547,P=0.016;χ^(2)=20.508,P<0.001;χ^(2)=12.586,P=0.002)。结论Siglec-15在食管鳞癌组织中高表达,与患者的疾病进展显著相关。

Sequencing of p53 mutation in established human hepatocellular carcinoma cell line of HHC4 and HHC15 in nude mice

AIM To set up cell lines of human hepatocellular carcinoma in nude mice for the research of cell biology and gene therapy. METHODS Xenotransplantation of human hepatoma into nude mice was carried out and the growth rate, histopathology and immunology of the nude mice were studied. The DNA from xenografts were analyzed by HBV gene and PCR amplification of a fragment of p 53 gene exon 7, which were identified by dot blot hybridization, restriction fragments length polymorphism and DNA sequencing. RESULTS hHCC4 and hHCC415 cell lines could be successively transplanted in nude mice and the population doubling time was 7 and 5 days respectively. These strains retained the original characteristics of histopathology, secreting AFP and heteroploid karyotypes in human hepatocellular carcinoma. The fragment of HBV gene was detected in the genomic DNA of both hHCC4 and hHCC15, however only hHCC4 secreted HBsAg. The mutation at 250 code (C→A) and 249 code (G→T) were detected respectively in the genomic DNA of hHCC4 and hHCC15. CONCLUSION The two cell lines are useful material for studying cell biology and gene therapy in human hepatocellular carcinoma and provide molecular biological trace of the relationship between high mortality of hepatoma and AFB1 severe pollution of the daily common foods in this district.

15-PGDH is reduced and induces apoptosis and cell cycle arrest in gastric carcinoma

AIM: To investigate the expression of 15-hydroxyprostaglandin dehydrogenase (15-PGDH) in human gastric cancer and it's mechanism in apoptosis and cell cycle arrest. METHODS: Expression of 15-PGDH mRNA and protein was examined by immunohistochemistry, immunocytochemistry, reverse transcriptase polymerase chain reaction (RT-PCR) and Western blotting in tissue from human gastric cancer, gastric precancerous state (gastric polyps and atrophic gastritis), normal stomach, and gastric cancer cell lines. The relationship between gastric cancer, gastric precancerous state and 15-PGDH expression was determined. The association between expression of 15-PGDH and various clinicopathological parameters in gastric cancer was evaluated. Human gastric cancer cell line SGC-7901 was transfected with 15-PGDH expression plasmids. The effect of 15-PGDH on the cell cycle was examined by flow cytometry. The effect of 15-PGDH on apoptosis was examined by transmission electron microscopy, flow cytometry and transferasemediated nick end labeling (TUNEL) assay. Expression of cell cycle (p21, p27, p16 and p53) and apoptosis (Survivin, BCL-2, BCL-XL, BAK and BAX) genes was analyzed by RT-PCR. RESULTS: Expression of 15-PGDH mRNA and protein in human gastric cancer tissues was significantly lower than in normal gastric tissues (P < 0.01). Expression in human gastric cancer cell lines MKN-28 and MKN-45 was reduced, and absent in SGC-7901 cells (P < 0.05). Reduction of 15-PGDH expression was also found in precancerous tissues, such as gastric polyps and atrophic gastritis (P < 0.01). There was a significant difference in expression of 15-PGDH among various gastric cancer pathological types (P < 0.05), with or without distant metastasis (P < 0.05) and different TNM stage (P < 0.01). Flow cytometry demonstrated a significant increase in apoptotic cells in SGC-7901 cells transfected with pcDNA3/15-PGDH plasmid for 24 and 48 h (P < 0.01), and an increased fraction of sub-G1 phase after transfection (P < 0.05). TUNEL assay showed an increased Apoptotic Index in cells overexpressing 15-PGDH (P < 0.01). After transfection, expression of proapoptotic genes, such as BAK (P < 0.05), BAX and p53 (P < 0.01), was increased. Expression of antiapoptotic genes was decreased, such as Survivin, BCL-2 and BCL-XL (P < 0.01). Expression of cyclin-dependent kinase inhibitors p21 and p16 (P < 0.01) was significantly upregulated in cells overexpressing 15-PGDH. CONCLUSION: Reduction of 15-PGDH is associated with carcinogenesis and development of gastric carcinoma. 15-PGDH induces apoptosis and cell cycle arrest in SGC-7901 cells.

糖类抗原15-3、糖类抗原242在弥漫大B细胞淋巴瘤患者中的表达及临床意义

目的探讨糖类抗原15-3(CA15-3)、糖类抗原242(CA242)在弥漫大B细胞淋巴瘤(DLBCL)患者中的表达及临床意义。方法选取173例疑似DLBCL患者,根据病理结果的不同将104例DLBCL患者纳入观察组,69例非DLBCL患者纳入对照组。比较两组患者及不同临床特征DLBCL患者的血清CA15-3、CA242水平。绘制受试者工作特征(ROC)曲线,计算曲线下面积(AUC),评估血清CA15-3、CA242单独及联合检测对DLBCL的诊断价值。结果观察组患者血清CA15-3、CA242水平均明显高于对照组,差异均有统计学意义(P﹤0.01)。Ⅲ~Ⅳ期DLBCL患者血清CA15-3、CA242水平均明显高于Ⅰ~Ⅱ期患者,差异均有统计学意义(P﹤0.01)。ROC曲线显示,血清CA15-3、CA242联合检测诊断DLBCL的AUC为0.925,高于血清CA15-3、CA242单独检测的0.860、0.807。结论DLBCL患者血清CA15-3、CA242水平均升高,CA15-3联合CA242检测对DLBCL具有较好的诊断价值。临床分期越高,DLBCL患者血清CA15-3、CA242水平就越高。

TATA-box-binding protein-associated factor 15 is a novel biomarker that promotes cell proliferation and migration in gastrointestinal stromal tumor

BACKGROUND Gastrointestinal stromal tumor(GIST)is a common neoplasm with high rates of recurrence and metastasis,and its therapeutic efficacy is still not ideal.There is an unmet need to find new molecular therapeutic targets for GIST.TATA-boxbinding protein-associated factor 15(TAF15)contributes to the progress of various tumors,while the role and molecular mechanism of TAF15 in GIST progression are still unknown.AIM To explore new molecular therapeutic targets for GIST and understand the biological role and underlying mechanisms of TAF15 in GIST progression.METHODS Proteomic analysis was performed to explore the differentially expressed proteins in GIST.Western blotting and immunohistochemical analysis were used to verify the expression level of TAF15 in GIST tissues and cell lines.Cell counting kit-8,colony formation,wound-healing and transwell assay were executed to detect the ability of TAF15 on cell proliferation,migration and invasion.A xenograft mouse model was applied to explore the role of TAF15 in the progression of GIST.Western blotting was used to detect the phosphorylation level and total level of RAF1,MEK and ERK1/2.RESULTS A total of 1669 proteins were identified as differentially expressed proteins with 762 upregulated and 907 downregulated in GIST.TAF15 was selected for the further study because of its important role in cell proliferation and migration.TAF15 was significantly over expressed in GIST tissues and cell lines.Overexpression of TAF15 was associated with larger tumor size and higher risk stage of GIST.TAF15 knockdown significantly inhibited the cell proliferation and migration of GIST in vitro and suppressed tumor growth in vivo.Moreover,the inhibition of TAF15 expression significantly decreased the phosphorylation level of RAF1,MEK and ERK1/2 in GIST cells and xenograft tissues,while the total RAF1,MEK and ERK1/2 had no significant change.CONCLUSION TAF15 is over expressed in GIST tissues and cell lines.Overexpression of TAF15 was associated with a poor prognosis of GIST patients.TAF15 promotes cell proliferation and migration in GIST via the activation of the RAF1/MEK/ERK signaling pathway.Thus,TAF15 is expected to be a novel latent molecular biomarker or therapeutic target of GIST.

NT4(Si)-p53(N15)-antennapedia induces cell death in a human hepatocellular carcinoma cell line

AIM： To construct the recombinant lentivirus expression plasmid, pLenti6/V5-NT4 p53（N 15）-antennapedia （Ant）, and study its effect on HepG2 cells. METHODS： Plasmid pLenti6/V5-NT4 p53（N15）-Ant was constructed incorporating the following functional regions, including signal peptide sequence and proregion of neurotrophin 4, N-terminal residues 12-26 of p53 and 17 amino acid drosophila carrier protein, Ant. Hepatocellular carcinoma （HepG2） cells were used for transfection. 3-[4,5-climethyl-thiazol-2yl]-2,5 diphenyl tetrazolium bromide （MI-I） assay, lactate dehydrogenase （LDH） release assay, transmission electron microscopy （TEM） and flow cytometric analysis （FCM） were employed to investigate the effects of LV-NT4（Si）- p53（N15）-Ant in vitro on HepG2 cells. In vivo experiment was also performed to investigate the inhibitory effect of LV-NT4（Si）-p53（N15）-Ant on tumor growth in nude mice.RESULTS： LV-NT4（Si）-p53（N15）-Ant significantly suppressed the growth of HepG2 cells. MTT assay showed that the growth of HepG2 cells was mucj more significantly inhibited by LV-NT4（Si）-p53（N15）-Ant than by LV-EGFP. The inhibition rate for HepG2 cell growth in the two groups was 46.9% and 94.5%, respectively, 48 h after infection with LV-NT4（Si）-p53（N15）-Ant, and was 33.9% and 95.8%, respectively, 72 h after infection with LV-NT4（Si）-p53（N15）-Ant （P 〈 0.01）. Light microscopy and TEM showed morphological changes in HepG2 cells infected with LV-NT4（Si）-p53（N15）-Ant, but no significant changes in HepG2 cells infected with LV-EGFR Changes were observed in ultra-structure of HepG2 cells infected with LV-NT4（Si）-p53（N15）-Ant, with degraded membranes, resulting in necrosis. LDH release from HepG2 cells was analyzed at 24, 48, 72 and 96 h after infection with LV-NT4（Si）-p53（N15）-Ant and LV-EGFP, which showed that LDH release was significantly higher in LV-NT4（Si）-p53（N15）-Ant treatment group （682 IU/L） than in control group （45 IU/L, P 〈 0.01）. The longer the time was after infection, the bigger the difference was in LDH release. FCM analysis showed that LV-NT4（Si）- p53（N15）-Ant could induce two different kinds of cell death： necrosis and apoptosis, with apoptosis being the minor type and necrosis being the main type, suggesting that LV-NT4（Si）-p53（N15）-Ant exerts its anticancer effect on HepG2 cells by inducing necrosis. The in vivo study showed that LV-NT4（Si）-p53（N15）-Ant significantly inhibited tumor growth with an inhibition rate of 66.14% in terms of tumor size and weight. CONCLUSION： LV-NT4（Si）-p53（N15）-Ant is a novel recombinant lentivirus expression plasmid and can be used in gene therapy for cancer.