Molecular cloning,expression, and characterization of bovine tissue factor pathway inhibitor-2

Human tissue factor pathway inhibitor-2 (TFPI-2) is a matrix-associated Kunitz-type serine proteinase inhibitor that is secreted by all cells of the vasculature, and presumably plays a role in the regulation of plasmin-mediated matrix remodeling. In this report, we describe the cloning and expression of a full-length cDNA for bovine TFPI-2 that exhibits 72% sequence identity with that of human TFPI-2. Following a 22 residue signal peptide, the mature protein contains 212 amino acids with 18 cysteines, three putative N-glycosylation sites, and one putative O-glycosylation site. The deduced sequence of mature bovine TFPI-2 revealed a short acidic amino-terminal region, three tandem Kunitz-type domains, and a carboxy-terminal tail highly enriched in basic amino acids. Recombinant bovine TFPI-2 was expressed in HEK 293 cells and resolved into two isoforms, designated as alpha-TFPI-2 (M(r) 33 kDa) and beta-TFPI-2 (M(r) 31 kDa), which presumably represent differentially glycosylated forms of the inhibitor. Similar to human TFPI-2, both bovine TFPI-2 isoforms exhibited strong inhibitory activity towards trypsin and plasmin, and weak inhibitory activity towards the factor VIIa-tissue factor complex.     

PTBP1 promotes the growth of breast cancer cells through the PTEN/Akt pathway and autophagy

Invasion and migration is the hallmark of malignant tumors as well as the major cause for breast cancer death. The polypyrimidine tract binding, PTB, protein serves as an important model for understanding how RNA binding proteins affect proliferation and invasion and how changes in the expression of these proteins can control complex programs of tumorigenesis. We have investigated some roles of polypyrimidine tract binding protein 1 (PTBP1) in human breast cancer. We found that PTBP1 was upregulated in breast cancer tissues compared with normal tissues and the same result was confirmed in breast cancer cell lines. Knockdown of PTBP1 substantially inhibited tumor cell growth, migration, and invasion. These results suggest that PTBP1 is associated with breast tumorigenesis and appears to be required for tumor cell growth and maintenance of metastasis. We further analyzed the relationship between PTBP1 and clinicopathological parameters and found that PTBP1 was correlated with her‐2 expression, lymph node metastasis, and pathological stage. This will be a novel target for her‐2(⁺) breast cancer. PTBP1 exerts these effects, in part, by regulating the phosphatase and tensin homolog‐phosphatidylinositol‐4,5‐bisphosphate 3‐kinase/protein kinase B (PTEN‐PI3K/Akt) pathway and autophagy, and consequently alters cell growth and contributes to the invasion and metastasis.     

CXC趋化因子配体8促进结直肠癌微环境中M2型巨噬细胞趋化及浸润

CXC趋化因子配体8(CXC chemokine ligand 8,CXCL8)在结直肠癌等多种肿瘤中高表达,并促进肿瘤恶性进展。研究发现,结直肠癌微环境中有大量M2型巨噬细胞浸润,但CXCL8是否影响M2型巨噬细胞的浸润及其潜在机制尚未可知。本文旨在探讨CXCL8对结直肠癌中M2型巨噬细胞浸润及趋化作用的影响。本研究首先分析了TCGA数据库结直肠癌样本中CXCL8表达水平及免疫细胞浸润情况,并在临床组织中进行验证。随后Western 印迹及qRT-PCR检测5种结直肠癌细胞株CXCL8的表达情况。佛波酯(PMA)及IL-4诱导THP-1至M2型巨噬细胞后,与HCT116、SW480细胞及过表达CXCL8的HCT116(CXCL8/HCT116)、SW480(CXCL8/SW480)共培养,检测M2型巨噬细胞趋化情况。白细胞介素1β(IL-1β)处理HCT116、SW480细胞,检测CXCL8表达情况,与M2型巨噬细胞共培养,分析趋化结果。结果显示,患者癌组织CXCL8表达高于癌旁组织,CXCL8高表达癌组织中存在更多M2型巨噬细胞浸润;IL-1β作用于HCT116或SW480后,CXCL8的mRNA及蛋白质表达水平升高(P<0.05)。Transwell实验证实,CXCL8趋化M2型巨噬细胞(P<0.05)。综上所述,结直肠癌细胞中CXCL8可由IL-1β诱导产生,CXCL8表达增加能够促进M2型巨噬细胞的趋化,结直肠癌微环境中M2型巨噬细胞大量浸润可能与CXCL8表达升高有关。     

Tissue factor pathway inhibitor-2 induced hepatocellular carcinoma cell differentiation

To investigate the effect of over-expression of tissue factor pathway inhibitor-2 (TFPI-2) on the differentiation of hepatocellular carcinoma (HCC) cells (Hep3B and HepG2). The TFPI-2 recombinant adenovirus (pAd-TFPI-2) was constructed using the pAdeasy-1 vector system. Transfected by pAd-TFPI-2, the cell proliferation of HCC cells was evaluated by CCK-8 assay, flow cytometry was used to detect cell apoptosis and CD133 expression. Real-time PCR and Western blot were used to detect the expression levels of markers of hepatocellular cancer stem cells (CSC) and hepatocytes. The over-expression of TFPI-2 significantly suppressed cell proliferation, induced apoptosis, and dramatically decreased the percentage of CD133 cells, which was considered as CSC in HCC. Real-time PCR and Western blot showed that the expression of markers of CSC in Hep3B cells and HepG2 cells infected with pAd-TFPI-2 was markedly lower than those of the control group (P < 0.05), while the expression of markers of hepatocytes was significantly increased (P < 0.05). Hence, TFPI-2 could induce the differentiation of hepatocellular carcinoma cells into hepatocytes, and is expected to serve as a novel way for the treatment of HCC.     

VMP1 related autophagy and apoptosis in colorectal cancer cells: VMP1 regulates cell death

Vacuole membrane protein 1 (VMP1) is an autophagy-related protein and identified as a key regulator of autophagy in recent years. In pancreatic cell lines, VMP1-dependent autophagy has been linked to positive regulation of apoptosis. However, there are no published reports on the role of VMP1 in autophagy and apoptosis in colorectal cancers. Therefore, to address this gap of knowledge, we decided to interrogate regulation of autophagy and apoptosis by VMP1. We have studied the induction of autophagy by starvation and rapamycin treatment in colorectal cell lines using electron microscopy, immunofluorescence, and immunoblotting. We found that starvation-induced autophagy correlated with an increase in VMP1 expression, that VMP1 interacted with BECLIN1, and that siRNA mediated down-regulation of VMP1-reduced autophagy. Next, we examined the relationship between VMP1-dependent autophagy and apoptosis and found that VMP1 down-regulation sensitizes cells to apoptosis and that agents that induce apoptosis down-regulate VMP1. In conclusion, similar to its reported role in other cell types, VMP1 is an important regulator of autophagy in colorectal cell lines. However, in contrast to its role in pancreatic cell lines, in colorectal cancer cells, VMP1-dependent autophagy appears to be pro-survival rather than pro-cell death.     

Therapeutic implications of GIPC1 silencing in cancer

GIPC1 is a cytoplasmic scaffold protein that interacts with numerous receptor signaling complexes, and emerging evidence suggests that it plays a role in tumorigenesis. GIPC1 is highly expressed in a number of human malignancies, including breast, ovarian, gastric, and pancreatic cancers. Suppression of GIPC1 in human pancreatic cancer cells inhibits in vivo tumor growth in immunodeficient mice. To better understand GIPC1 function, we suppressed its expression in human breast and colorectal cancer cell lines and human mammary epithelial cells (HMECs) and assayed both gene expression and cellular phenotype. Suppression of GIPC1 promotes apoptosis in MCF-7, MDA-MD231, SKBR-3, SW480, and SW620 cells and impairs anchorage-independent colony formation of HMECs. These observations indicate GIPC1 plays an essential role in oncogenic transformation, and its expression is necessary for the survival of human breast and colorectal cancer cells. Additionally, a GIPC1 knock-down gene signature was used to interrogate publically available breast and ovarian cancer microarray datasets. This GIPC1 signature statistically correlates with a number of breast and ovarian cancer phenotypes and clinical outcomes, including patient survival. Taken together, these data indicate that GIPC1 inhibition may represent a new target for therapeutic development for the treatment of human cancers.     

乳腺癌细胞QSOX1的CRISPR/Cas9基因编辑及其对增殖侵袭的影响研究*

目的:乳腺癌细胞的恶性增殖和易于侵袭转移特性与其对患者的危害直接相关,因此,探究其产生的分子机制,对其有效防治具有重要意义。静息巯基氧化酶-1(QSOX1)是巯基氧化酶家族成员之一,有研究证明其对细胞内蛋白质折叠过程中二硫键形成及细胞外基质的形成发挥重要作用。由于QSOX1在乳腺癌和胰腺癌等多种癌细胞中过表达,将探索QSOX1对乳腺癌细胞过度增殖和侵袭转移方面的可能作用。方法:通过利用CRISPR/Cas9技术构建QSOX1基因敲除和敲入的乳腺癌细胞模型,检测分析QSOX1对乳腺癌细胞MCF-7的分裂增殖、侵袭迁移能力等方面的影响。结果:利用CRISPR/Cas9基因编辑技术成功构建了QSOX1基因敲除和敲入的乳腺癌MCF-7细胞株,其与对照野生型组细胞相比,QSOX1基因敲除株的增殖能力显著下降,癌细胞在体外的迁移和侵袭能力受到明显抑制;而QSOX1基因敲入株的增殖能力和体外迁移侵袭能力却明显有提高。结论:初步揭示了QSOX1在癌症发生与发展中的作用,为进一步阐明其作用的分子机制和设计靶向药物奠定了重要基础。     

GJA1在结直肠癌组织中表达及促进侵袭转移的研究

摘要 目的：间隙连接Alpha-1蛋白（Gap Junction Alpha-1,GJA1）是间隙连接中分布最广泛的蛋白,并在多种肿瘤中起促癌作用,但其在结直肠癌发生、发展的作用研究甚少。本实验旨在探究GJA1在结直肠癌组织中的表达情况及其对结直肠癌细胞系侵袭、转移能力的影响,以期为结直肠癌的诊断和预后寻找新的生物标志物。方法：收集92对结直肠癌及其癌旁组织样本,提取组织RNA,利用qRT-PCR检测GJA1相对表达量,并分析GJA1表达与临床病理特征及预后的相关性。在HCT116和HCT8两种结直肠癌细胞系中分别构建GJA1过表达载体和敲减载体,利用qRT-PCR和、Western Blot检测上皮间充质转化（epithelial-mesenchymal transition, EMT）相关蛋白E-Cadherin、N-Cadherin、Vimentin和Snail的表达变化,利用Wound healing和Transwell实验观察其迁移、侵袭能力的变化。结果：相对于癌旁组织,GJA1在结直肠癌组织中显著低表达。并且结直肠癌中低表达的GJA1与肿瘤分化程度、浸润深度、淋巴血管转移相关,低表达GJA1结直肠癌患者显示更差的总体生存率和更低的无病生存率。此外,过表达GJA1后,结直肠癌细胞E-cadherin的表达升高,N-cadherin、Vimentin和Snail的表达降低,划痕愈合减慢,Transwell转移细胞减少;而敲减GJA1后,结直肠癌细胞E-Cadherin的表达降低,N-Cadherin、Vimentin和Snail的表达升高,划痕愈合加快,Transwell转移细胞增多。结论：GJA1在结直肠癌中低表达,其表达降低可通过EMT促进结直肠癌的侵袭、转移并影响病人预后。     

The scaffold protein MEK Partner 1 is required for the survival of estrogen receptor positive breast cancer cells

ABSTRACT: MEK Partner 1 (MP1 or MAPKSP1) is a scaffold protein that has been reported to function in multiple signaling pathways, including the ERK, PAK and mTORC pathways. Several of these pathways influence the biology of breast cancer, but MP1's functional significance in breast cancer cells has not been investigated. In this report, we demonstrate a requirement for MP1 expression in estrogen receptor (ER) positive breast cancer cells. MP1 is widely expressed in both ER-positive and negative breast cancer cell lines, and in non-tumorigenic mammary epithelial cell lines. However, inhibition of its expression using siRNA duplexes resulted in detachment and apoptosis of several ER-positive breast cancer cell lines, but not ER-negative breast cancer cells or non-tumorigenic mammary epithelial cells. Inhibition of MP1 expression in ER-positive MCF-7 cells did not affect ERK activity, but resulted in reduced Akt1 activity and reduced ER expression and activity. Inhibition of ER expression did not result in cell death, suggesting that decreased ER expression is not the cause of cell death. In contrast, pharmacological inhibition of PI3K signaling did induce cell death in MCF-7 cells, and expression of a constitutively active form of Akt1 partially rescued the cell death observed when the MP1 gene was silenced in these cells. Together, these results suggest that MP1 is required for pro-survival signaling from the PI3K/Akt pathway in ER-positive breast cancer cells.     

Quiescin sulfhydryl oxidase 1 promotes invasion of pancreatic tumor cells mediated by matrix metalloproteinases

Quiescin sulfhydryl oxidase 1 (QSOX1) oxidizes sulfhydryl groups to form disulfide bonds in proteins. We previously mapped a peptide in plasma from pancreatic ductal adenocarcinoma (PDA) patients back to an overexpressed QSOX1 parent protein. In addition to overexpression in pancreatic cancer cell lines, 29 of 37 patients diagnosed with PDA expressed QSOX1 protein in tumor cells, but QSOX1 was not detected in normal adjacent tissues or in a transformed, but nontumorigenic cell line. To begin to evaluate the advantage QSOX1 might provide to tumors, we suppressed QSOX1 protein expression using short hairpin (sh) RNA in two pancreatic cancer cell lines. Growth, cell cycle, apoptosis, invasion, and matrix metalloproteinase (MMP) activity were evaluated. QSOX1 shRNA suppressed both short and long isoforms of the protein, showing a significant effect on cell growth, cell cycle, and apoptosis. However, QSOX1 shRNA dramatically inhibited the abilities of BxPC-3 and Panc-1 pancreatic tumor cells to invade through Matrigel in a modified Boyden chamber assay. Mechanistically, gelatin zymography indicated that QSOX1 plays an important role in activation of MMP-2 and MMP-9. Taken together, our results suggest that the mechanism of QSOX1-mediated tumor cell invasion is by activation of MMP-2 and MMP-9.     

基于生物信息学分析KCNQ1OT1在胃癌中的作用

长非编码RNA KCNQ1OT1在多种肿瘤中高表达,但是在胃癌中的研究较少并且研究结果不一致,其在胃癌中具体的作用机制也缺乏相关研究。通过癌症基因组图谱(The Cancer Genome Atlas, TCGA)公共数据库分析发现:KCNQ1OT1在胃癌中普遍高表达,且高表达KCNQ1OT1的胃癌病人预后不良,它与胃癌多种临床因素密切相关,尤其是与TP53的突变有明显的相关性,而且其表达与免疫细胞浸润明显相关;KCNQ1OT1在胃癌肿瘤细胞系中普遍高表达,敲低后可抑制胃癌肿瘤细胞的增殖能力,共表达网络分析发现,其表达与肿瘤代谢有密切的相关性;谷氨酰胺酶1(glutaminase 1, GLS1)在胃癌中普遍高表达,与预后不良密切相关,KCNQ1OT1与GLS1的表达具有明显的相关性,敲低KCNQ1OT1的表达可抑制GLS1 mRNA的表达,而过表达GLS1可以部分逆转敲低KCNQ1OT1造成的胃癌细胞增殖能力的下降,因此推测KCNQ1OT1可能通过GLS1调控胃癌肿瘤细胞的生长。本研究通过大数据及实验验证了KCNQ1OT1在胃癌中的表达及功能,提示KCNQ1OT1有可能通过调控谷氨酰胺代谢来促进了胃癌的发生发展,这为分子靶向治疗胃癌的临床研究提供了新的靶点和思路。