Human umbilical cord mesenchymal stem cells ameliorate liver fibrosis in vitro and in vivo: From biological characteristics to therapeutic mechanisms

Liver fibrosis is a wound-healing response to chronic injuries, characterized by the excessive accumulation of extracellular matrix or scar tissue within the liver;in addition, its formation is associated with multiple cytokines as well as several cell types and a variety of signaling pathways. When liver fibrosis is not well controlled, it can progress to liver cirrhosis, but it is reversible in principle. Thus far, no efficient therapy is available for treatment of liver fibrosis. Although liver transplantation is the preferred strategy, there are many challenges remaining in this approach, such as shortage of donor organs, immunological rejection, and surgical complications. Hence, there is a great need for an alternative therapeutic strategy. Currently, mesenchymal stem cell (MSC) therapy is considered a promising therapeutic strategy for the treatment of liver fibrosis;advantageously, the characteristics of MSCs are continuous self-renewal, proliferation, multipotent differentiation, and immunomodulatory activities. The human umbilical cord-derived (hUC)-MSCs possess not only the common attributes of MSCs but also more stable biological characteristics, relatively easy accessibility, abundant source, and no ethical issues (e.g., bone marrow being the adult source), making hUC-MSCs a good choice for treatment of liver fibrosis. In this review, we summarize the biological characteristics of hUC-MSCs and their paracrine effects, exerted by secretion of various cytokines, which ultimately promote liver repair through several signaling pathways. Additionally, we discuss the capacity of hUC-MSCs to differentiate into hepatocyte-like cells for compensating the function of existing hepatocytes, which may aid in amelioration of liver fibrosis. Finally, we discuss the current status of the research field and its future prospects.

Hepatogenic differentiation of human mesenchymal stem cells from adipose tissue in comparison with bone marrow mesenchymal stem cells

AIM: To investigate and compare the hepatogenic transdifferentiation of adipose tissue-derived stem cells (ADSC) and bone marrow-derived mesenchymal stem cells (BMSC) in vitro. Transdifferentiation of BMSC into hepatic cells in vivo has been described. Adipose tissue represents an accessible source of ADSC, with similar characteristics to BMSC.METHODS: BMSCs were obtained from patients under-going total hip arthroplasty and ADSC from human adi-pose tissue obtained from lipectomy. Cells were grown in medium containing 15% human serum. Cultures were serum deprived for 2 d before cultivating under similar pro-hepatogenic conditions to those of liver develop-ment using a 2-step protocol with sequential addition of growth factors, cytokines and hormones. Hepatic differ-entiation was RT-PCR-assessed and liver-marker genes were immunohistochemically analysed. RESULTS: BMSC and ADSC exhibited a fibroblastic mor-phology that changed to a polygonal shape when cells differentiated. Expression of stem cell marker Thy1 de-creased in differentiated ADSC and BMSC. However, the expression of the hepatic markers, albumin and CYPs increased to a similar extent in differentiated BMSC and ADSC. Hepatic gene activation could be attributed to in-creased liver-enriched transcription factors (C/EBPβ and HNF4α), as demonstrated by adenoviral expression vec-tors.CONCLUSION: Mesenchymal stem cells can be induced to hepatogenic transdifferentiation in vitro. ADSCs have a similar hepatogenic differentiation potential to BMSC, but a longer culture period and higher proliferation ca-pacity. Therefore, adipose tissue may be an ideal source of large amounts of autologous stem cells, and may become an alternative for hepatocyte regeneration, liver cell transplantation or preclinical drug testing.

Key nodes of a micro RNA network associated with the integrated mesenchymal subtype of high-grade serous ovarian cancer

Metastasis is the main cause of cancer mortality. One of the initiating events of cancer metastasis of epithelial tumors is epithelial-to-mesenchymal transition(EMT), during which cells dedifferentiate from a relatively rigid cell structure/morphology to a flexible and changeable structure/morphology often associated with mesenchymal cells. The presence of EMT in human epithelial tumors is reflected by the increased expression of genes and levels of proteins that are preferentially present in mesenchymal cells. The combined presence of these genes forms the basis of mesenchymal gene signatures, which are the foundation for classifying a mesenchymal subtype of tumors. Indeed, tumor classification schemes that use clustering analysis of large genomic characterizations, like The Cancer Genome Atlas(TCGA), have defined mesenchymal subtype in a number of cancer types, such as high-grade serous ovarian cancer and glioblastoma. However, recent analyses have shown that gene expression-based classifications of mesenchymal subtypes often do not associate with poor survival. This "paradox" can be ameliorated using integrated analysis that combines multiple data types. We recently found that integrating m RNA and micro RNA(mi RNA) data revealed an integrated mesenchymal subtype that is consistently associated with poor survival in multiple cohorts of patients with serous ovarian cancer. This network consists of 8 major mi RNAs and 214 m RNAs. Among the 8 mi RNAs, 4 are known to be regulators of EMT. This review provides a summary of these 8 mi RNAs, which were associated with the integrated mesenchymal subtype of serous ovarian cancer.

Re-evaluating the role of epithelial-mesenchymal-transition in cancer progression

Epithelial-mesenchymal transition（EMT） and mesenchymal-epithelial transition（MET） are essential for embryonic development and also important in cancer progression. In a conventional model, epithelial-like cancer cells transit to mesenchymal-like tumor cells with great motility via EMT transcription factors; these mesenchymallike cells migrate through the circulation system, relocate to a suitable site and then convert back to an epithelial-like phenotype to regenerate the tumor. However, recent findings challenge this conventional model and support the existence of a stable hybrid epithelial/mesenchymal（E/M） tumor population. Hybrid E/M tumor cells exhibit both epithelial and mesenchymal properties, possess great metastatic and tumorigenic capacity and are associated with poorer patient prognosis. The hybrid E/M model and associated regulatory networks represent a conceptual change regarding tumor metastasis and organ colonization. It may lead to the development of novel treatment strategies to ultimately stop cancer progression and improve disease-free survival.

Targeting of RhoE inhibits epithelial-mesenchymal transition during colorectal cancer cell migration

Objective Despite microRNA(miR-200b) being proved to promote the proliferation of colorectal cancer(CRC) cells, the relationship between mi R-200 b and epithelial-mesenchymal transition(EMT) of CRC cells remains poorly understood. The aim of the study was to investigate the relationship between miR-200 b and EMT during CRC cell migration.Methods The effect of miR-200 b on EMT-associated markers E-cadherin and vimentin was evaluated by western blot in CRC cells(SW620 and HT-29) by treatment with mi R-200 b mimics and inhibitors. A luciferase reporter assay was employed to detect downstream targets of mi R-200 b. Transwell migration assays were used to detect CRC cell migration. Results Western blots revealed that treatment with miR-200 b mimics led to up-regulation of E-cadherin and down-regulation of vimentin, metalloproteinase(MMP)-9, and MMP-2, whereas treatment with mi R-200 b inhibitor exhibited opposite effects on expression of E-cadherin and vimentin. Luciferase reporter assays demonstrated that Rho E(RND3) was targeted by miR-200 b. Two predicted target sites of miR-200 b were present in the 3'-UTR of Rho E. Predicted target site 1 was from nucleotides 1584 to 1591, and site 2 was from nucleotides 1729 to 1735. Rho E knockdown cell lines were also established to investigate the impact of Rho E and mi R-200 b on EMT and cell migration. Rho E knockdown enhanced the effect of miR-200 b mimics, up-regulating E-cadherin and down-regulating vimentin. Rho E knockdown also inhibited cell migration. Furthermore, miR-200 b mimic treatment further promoted the inhibitory effect of Rho E knockdown on cell migration. Conclusion miR-200 b inhibited EMT and CRC cell migration partly via inhibiting Rho E expression in CRC. Rho E and miR-200 b might therefore be promising target genes in the management of CRC.

Review: Neuronal Differentiation Protocols of Mesenchymal Stem Cells

Mesenchymal stem cells (MSCs) are self-renewing cells found in almost all postnatal organs and tissues in the perivascular region. These cells present multiple characteristics that make them candidates to be applied in cell therapy for neurodegenerative diseases, such as their secretory action, migration to the lesion area, and immunomodulatory potential. These cells have a high capacity for mesodermal differentiation;however, numerous studies have shown that MSCs can also differentiate into neurons. However, despite positive results in multiple trials in which undifferentiated MSCs transplanted into animal models of neurodegenerative diseases, some studies suggest that the therapeutic effects obtained are enhanced by the use of MSCs differentiated towards the neuronal lineage before transplant. In this sense, there are several methods to induce in vitro reprogramming of MSCs towards the neuronal lineage, including chemical substances, growth factors, cocultures with neural lineage cells, transfection of genes, miRNAs, etc., and small molecules stand out. Therefore, this article compares multiple experimental tests in which these inducers promote neuronal differentiation of MSCs and identify those methods that originate an optimal neuronal differentiation. The analysis includes the percentage of differentiation, maturation, expression of neuronal markers, functionality, and cell survival considering the intrinsic characteristics of the MSCs used as the tissue of origin and the species from which they were isolated.

白藜芦醇通过Hippo-YAP信号通路在TGF-β1诱导胃癌细胞EMT过程中的作用及其机制

目的探讨白藜芦醇(RSVL)通过Hippo-YAP信号通路在TGF-β1诱导胃癌细胞上皮间充质转化(EMT)过程中的作用及其机制。方法选取胃癌细胞SGC-7901为研究对象,首先将其随机分为空白组、RSVL低剂量组(5μM)、RSVL中剂量组(10μM)、RSVL高剂量组(20μM),通过细胞增殖(MTT)实验确定RSVL浓度,然后对细胞进行转染,分为si-NC组、TGF-β1+10μM RSVL组、TGF-β1+si-YAP组、TGF-β1+pcDNA3.1-YAP组、TGF-β1+10μM RSVL+pcDNA3.1-YAP组。采用MTT、细胞侵袭(Transwell)、划痕实验,分别检测细胞的增殖、侵袭和迁移;采用蛋白质印迹法(WB)和荧光定量PCR检测E-钙粘连蛋白(E-cadherin)、神经型钙黏附蛋白(N-cadherin)、波形蛋白(Vimentin)、Snali 1、HIP-PO/Yes相关蛋白(YAP)和mRNA。其次,选取24只裸鼠,将其分为模型组、RSVL组(10μM)、RSVL+pcDNA3.1组、RSVL+pcDNA3.1-YAP组,每组各六只。计算小鼠肿瘤的重量和体积;采用免疫组化检测Ki67蛋白。结果RSVL对细胞增殖有抑制作用(P<0.05);与si-NC组相比,其余各组侵袭细胞数、迁移率较低(P<0.05);与si-NC组相比,其余各组E-cadherin蛋白及mRNA表达较高,N-cadherin、Vimentin、Snali 1蛋白及mRNA以及YAP蛋白表达较低(P<0.05);与Model组相比,其余各组小鼠肿瘤的重量和体积均较低(P<0.05);与Model组比较,其余各组Ki67染色强度均显著减弱。结论RSVL可以通过抑制YAP相关通路的激活,来发挥抑制肿瘤细胞SGC-7901上皮间充质转化、增殖、迁移、侵袭的作用。

Regulation of autophagy on epithelial mesenchymal transformation in oral squamous cell carcinoma

Objective: To investigate the role and mechanism of autophagy in epithelial-mesenchymal transition (EMT) of oral squamous cell carcinoma cells induced by CQ (chloroquine) and rapamycin (RAPA). Methods: TGF-β (transforming growth factor β) was used to induce EMT in Cal-27 cell line. At the same time, RAPA was used to enhance and CQ was used to inhibit autophagy. The ability of cell migration was detected by scratch distribution test and the ability of cell migration was detected by Transwell chamber test. Western blot was used to detect the changes of ZO-1, vimentin, FN1 and other EMT-related proteins after 3 d induction, and SPSS 22.0 statistical software was used to analyze the data. Results: After 3 d of induction with 5 ng/mL TGF-β, E-cadherin decreased significantly and Vimentin increased significantly. Compared with the control group, the wound healing rate increased significantly (P<0.05) and the number of penetrating cells increased significantly (P<0.05) after 3 d induction with 5 ng/mL TGF-β, and then the cells were co-induced with 100 ng/mL RAPA and 100 ng/mL CQ and 5 ng/mL TGF-β for 3 d. Compared with TGF-β group. The healing rate of the RAPA co-induced with 5 ng/mL TGF-β group decreased significantly (P<0.05) and the number of penetrating cells decreased significantly (P<0.05). Compared with TGF-β group. The healing rate of the CQ co-induced with 5 ng/mL TGF-β group increased significantly (P<0.05) and the number of penetrating cells increased significantly (P<0.05). Compared with the control group, FN1 and Vimentin expression increased and ZO-1 expression decreased 3 d after induction with 5 ng/mL TGF-β. And then induced Cal-27 cells with 100 ng/mL RAPA and 100 ng/mL CQ and 5 ng/mL TGF-β respectively for 3 d. Compared with TGF-β group, FN1 and Vimentin expression decreased in RAPA co-induction group. Compared with TGF-βgroup, the expression of FN1 and Vimentin increased and the expression of ZO-1 decreased in CQ co-induction group. Conclusion: TGF-β can induce Cal-27 cells to establish EMT model. In EMT model, promoting autophagy can inhibit EMT, inhibiting autophagy can promote EMT.

Effect of Rapamycin on TGF-β_1-induced epithelial-mesenchymal transition in LoVo colonic adenocarcinoma cells

Objective： To investigate the effect of Rapamycin on epithelial-mesenchymal transition（EMT） of LoVo colonic adenocarcinoma cells in vitro. Methods：Cultured LoVo colonic adenocarcinoma cells were divided into three groups： negative control group, EMT-inducing group（TGF-β1） and EMT-interfering group（TGF-β1 plus Rapamycin）. E-cadherin expression in LoVo cells was detected by Western Blot, while the expression of vimentin was evaluated through immunocytochemistry. The Snail mRNA in LoVo cells was examined by RT- PCR. Results：TGF-β1 induced LoVo cell switching from polygonal to spindle-shaped. TGF-β1 enhanced the expression of vimentin, but lowered the level of E-cadhefin. In contrast, Rapamycin impaired the transition induced by TGF-β1. Rapamycin dramatically abrogated TGF-β1-induced vimentin expression and restored E-cadherin expression in LoVo cells. Rapamycin significantly repressed the upregulation of Snail mRNA expression induced by TGF-β1. Conclusion：Rapamycin dramatically abrogated TGF-β1 induced Snail mRNA expression in LoVo cells, hence inhibiting EMT of these cells in vitro.

Strategies for Synchronous and Multiple Metastatic Liver Tumors Designed from Epithelial-Mesenchymal Transition Concept

At some point in the natural course of colorectal cancer up to 50% of patients will develop metastasis to the liver and it is one of the most critical effects for patient prognosis. The incidence of synchronous liver metastasis has been detected at around 20% - 25%, but the optimal timing of surgical resection remains controversial. Neoadjuvant chemotherapy has also been found to be beneficial not only for initially unresectable but also resectable synchronous metastases. Then, traditional surgical strategies of hepatic resection in accordance with past chemotherapeutic regimens have been used decreasingly over the past several years. This review will primarily discuss treatments in association with the recent developed chemotherapeutic regimens and surgical procedure from the clinical data and the concept for epithetlial-mesenchymal transition, which has recently been studied to elucidate mechanisms of the liver metastatic process.

INHBA-AS1通过c-Myc/SCD通路调控宫颈癌HeLa细胞的鸟氨酸代谢和EMT进程

目的:探讨抑制素β亚基A反义RNA1(INHBA-AS1)对宫颈癌HeLa细胞EMT和鸟氨酸代谢途径的影响及其机制。方法:体外常规培养HeLa细胞,实验分为10组:对照组、阴性对照(NC)组、sh-INHBA-AS1组、PluriSIn 1[硬脂酰辅酶A去饱和酶(stearyl CoA desaturase,SCD)抑制剂]组、NC+PluriSIn 1组、sh-INHBA-AS1+PluriSIn 1组、10058-F4(c-Myc抑制剂)组、NC+10058-F4组、sh-INHBA-AS1+10058-F4组、sh-INHBA-AS1+OE-c-Myc组。平板克隆实验检测各组细胞的增殖能力,FCM检测各组细胞的凋亡情况,Transwell小室实验检测各组细胞的侵袭、迁移能力,qPCR法检测各组细胞中INHBA-AS1、c-Myc、SCD和EMT相关基因(N-cadherin、TGF-β、ZEB1)mRNA的表达,WB法检测各组细胞中c-Myc、SCD、EMT相关(N-cadherin、TGF-β、ZEB1)、S-腺苷-甲硫氨酸脱羧酶(SAMDC)和亚精胺/精胺N1-乙酰转移酶(SSAT)蛋白的表达,ELISA检测各组细胞上清液中鸟氨酸脱羧酶(ODC)的含量。结果:敲减INHBA-AS1表达使HeLa细胞的增殖、侵袭和迁移能力显著降低(均P<0.05)而细胞凋亡率显著升高(P<0.05),q PCR、WB法检测结果显示,敲减INHBA-AS1均可显著抑制HeLa细胞中c-Myc、SCD、N-cadherin、TGF-β、ZEB1和SAMDC的表达(均P<0.05),而促进SSAT的表达(P<0.05),并降低HeLa细胞上清液中ODC的含量(P<0.05)。与c-Myc抑制剂和SCD抑制剂单独处理相比,其联合敲减INHBA-AS1后上述作用更加显著(均P<0.05);与sh-INHBA-AS1组相比,进一步过表达c-Myc后HeLa细胞的增殖能力显著升高(P<0.05)、SCD和N-cadherin蛋白表达水平显著升高(P<0.05)、细胞上清液中ODC含量显著升高(P<0.05)。结论:INHBA-AS1可通过c-Myc调控SCD的表达,从而影响HeLa细胞鸟氨酸代谢和EMT进程,进而促进HeLa细胞的增殖、侵袭和迁移能力。

虎杖苷通过Hippo/YAP通路影响甲状腺癌8505C细胞的恶性生物学行为和顺铂敏感性

目的:探究虎杖苷通过Hippo/Yes相关蛋白(YAP)通路对人甲状腺癌8505C细胞的恶性生物学行为和顺铂(DDP)敏感性的影响。方法:体外培养8505C细胞,构建其DDP耐药细胞8505C/DDP,用CCK-8法检测0、25、50、75、100 nmol/L虎杖苷处理8505C和8505C/DDP细胞的增殖能力,以筛选虎杖苷的最佳作用浓度。将8505C细胞分为对照组、虎杖苷组、空载组、虎杖苷+YAP1过表达组;将8505C/DDP细胞分为对照组、DDP组、DDP+虎杖苷组、DDP+空载组、DDP+虎杖苷+YAP1过表达组。WB法检测各组8505C细胞中Hippo/YAP通路[YAP1、转录辅激活因子(TAZ)]和EMT(E-cadherin、N-cadherin)相关蛋白,8505C/DDP细胞中YAP1、TAZ、耐药相关蛋白[P-糖蛋白(P-gp)、多药耐药相关蛋白1(MRP1)]、凋亡相关蛋白(C-caspase-3、BAX、Bcl-2)的表达。Transwell小室和细胞划痕实验分别检测各组8505C、8505C/DDP细胞的侵袭、迁移能力。结果:虎杖苷可显著抑制8505C细胞的增殖活性(P<0.05)明显抑制8505C细胞中YAP1、TAZ蛋白、N-cadherin的表达(均P<0.05),提升E-caderin蛋白的表达(P<0.05),显著抑制8505C细胞的迁移和侵袭能力(均P<0.05),而8505C/DDP细胞对低浓度的虎杖苷具有耐药性(P<0.05);过表达YAP1则可逆转虎杖苷对8505C细胞的影响。50 nmol/L虎杖苷明显抑制DDP处理的8505C/DDP细胞中YAP1、TAZ、P-gp、MRP1、Bcl-2的蛋白的表达(均P<0.05),提升cleaved caspase-3、BAX蛋白的表达(均P<0.05)并诱导其细胞凋亡(P<0.05),过表达YAP1则可逆转虎杖苷对8505C/DDP细胞的影响。结论:虎杖苷抑制Hippo/YAP信号通路,从而抑制8505C细胞的恶性生物学行为和增强其对的DDP敏感性。

低氧微环境通过TGFBI调控Wnt/β-catenin通路介导胰腺癌化疗耐药及机制研究

目的:研究低氧微环境通过TGFBI调控胰腺癌耐药的作用及分子机制。方法:以CCK-8方法检测细胞增殖;以Western blotting技术检测蛋白表达水平;以ImageJ软件分析蛋白灰度值;RNAi技术用于敲减TGFBI基因。结果:TGFBI在Panc-1细胞中表达比正常细胞增强;低氧促进胰腺癌细胞Panc-1增殖并减弱顺铂对Panc-1的抑制作用,而高氧抑制Panc-1细胞增殖并加强顺铂的杀伤作用;低氧促进TGFBI表达及EMT行为;低氧通过TGFBI调控Panc-1细胞增殖及顺铂的杀伤作用;低氧通过TGFBI调控Wnt/β-catenin信号,进而促进EMT行为。结论:低氧微环境通过增强TGFBI表达促进胰腺癌细胞增殖及耐药;低氧微环境通过TGFBI激活Wnt/β-catenin信号通路,促进EMT标志分子表达。

Yes相关蛋白(YAP)通过激活PI3K/AKT通路促进皮肤鳞状细胞癌细胞侵袭和迁移

目的探讨Yes相关蛋白(YAP)在皮肤鳞状细胞癌(cSCC)中表达及与cSCC侵袭和迁移中的作用。方法通过免疫组织化学染色法检测cSCC、鲍温病(BD)、癌旁正常皮肤组织中YAP的表达水平,并分析与临床病理参数之间的关系;利用慢病毒转染构建YAP基因敲低的A431稳定细胞株,利用四甲基罗丹明标记的鬼笔环肽检测A431细胞微丝分布和数量,Transwell TM实验检测细胞侵袭能力,划痕实验检测A431细胞的迁移能力;免疫荧光细胞化学染色法观察敲低YAP后上皮间质转化(EMT)相关标志物上皮钙黏素(E-cadherin)、锌指转录因子Snail的表达;Western blot法检测E-cadherin、Snail、β-catenin、磷脂酰肌醇3激酶(PI3K)、蛋白激酶B(AKT)、磷酸化的蛋白激酶B(p-AKT)、核糖体蛋白S6(S6)、磷酸化S6(p-S6)、4E结合蛋白1(4EBP1)、磷酸化的4EBP1(p-4EBP1)的表达。结果YAP在cSCC和BD中表达显著高于癌旁正常皮肤组织;cSCC中YAP高表达与肿瘤大小、分化程度、侵袭程度密切相关,与患者的性别、年龄、发病部位、形态类型、是否神经脉管侵犯不相关;敲低A431细胞中YAP后,肿瘤细胞的侵袭、迁移能力降低,细胞微丝变细、伪足变少;E-cadherin表达增加,Snail和β-catenin蛋白表达降低,p-AKT、p-S6及p-4EBP1蛋白表达降低。结论YAP在cSCC中高表达,YAP激活PI3K/AKT信号通路促进cSCC的侵袭、迁移及EMT过程。

BMP4通过诱导EMT促进肝癌迁移侵袭

目的:检测BMP4在肝癌中的表达并探讨BMP4在诱导肝癌EMT中的作用,进而研究其对肝癌细胞迁移侵袭能力的影响。方法:采用免疫组织化学方法检测肝癌组织中BMP4的表达,分析其与肝癌临床病理资料之间的关系。将BMP4表达质粒转染至肝癌细胞系Hep G2中,诱导BMP4外源性过表达。观察BMP4转染前、后Hep G2的细胞形态学改变;Western blot检测转染前、后Hep G2中BMP4、EMT相关蛋白(E-cadherin、Vimentin)表达变化情况;划痕和侵袭实验检测BMP4对细胞迁移侵袭能力的影响。结果:BMP4与患者的年龄、病理分级、临床分期、不良预后密切相关。BMP4过表达后Hep G2呈现典型的EMT形态学改变,E-cadherin表达下调、Vimentin表达上调、细胞的迁移侵袭能力显著增强。结论:BMP4与肝癌临床病理资料密切相关,并可能通过诱导EMT促进肝癌细胞的迁移侵袭能力。

CCL20通过AKT/MMP3轴而非EMT途径诱导结肠癌SW480细胞的侵袭和转移

目的:探讨趋化因子CCL20/CCR6促进结肠癌SW480细胞侵袭和迁移的分子机制。方法:筛选高表达CCR6的结肠癌SW480细胞,加入外源性重组人CCL20后,采用Transwell、划痕愈合实验检测其侵袭和迁移能力,以免疫荧光、WB实验检测SW480细胞EMT标志蛋白、AKT信号蛋白以及靶标蛋白MMP3的表达;通过MK2206阻断实验验证AKT信号是其作用机制,通过TCGA数据库资源(https://portal.gdc.cancer.gov/)分析CCL20和MMP3在结直肠癌组织中的表达水平及其相关性。结果:趋化因子CCL20能够明显促进结肠癌SW480细胞侵袭和迁移(均P<0.01),其间并不伴随细胞的EMT变化,而是通过AKT信号的激活及下游靶标蛋白MMP3表达上调是其诱因之一;阻断AKT信号能够明显抑制SW480细胞侵袭和迁移能力,且下调MMP3的表达水平(P<0.05或P<0.01)。TCGA平台数据提示,结肠癌组织中CCL20和MMP3的表达明显高于正常肠黏膜组织,且两者呈明显正相关(r=0.051,P<0.01)。结论:趋化因子CCL20通过AKT/MMP3信号轴而非EMT机制促进结肠癌SW480细胞的侵袭和迁移。

EMT分子标志物在肝癌细胞系中的表达及其意义

目的探讨人肝癌细胞系EMT相关分子标志物的表达情况,为确定相关的肝癌细胞研究模型奠定基础。方法利用实时定量PCR方法检测5株人肝癌细胞系(SNU368、SNU739、HLF、Huh-1和MHCC97L)中E-cadherin、ZO-1、N-cadherin、Vimentin、Snail和Slug的mRNA表达,Westernblot和免疫荧光检测E-cadherin、ZO-1、N-cadherin、Vimentin、Snail和Slug的蛋白表达。结果E-cadherin、ZO-1、N-cadherin、Vimentin、Snail和Slug的mRNA在5种肝癌细胞中均有不同程度表达,E-cadherin、ZO-1、N-cadherin和Slug蛋白在5个细胞株中亦有表达,Vimentin蛋白主要在SNU368、SNU739、HLF和MHCC97L细胞中表达明显,Snail蛋白在SNU368,SNU739,Huh-1和MHCC97L细胞中表达较高。结论HLF细胞系表现出更明显的间质细胞表型特点,Huh-1和SNU368则表现为较典型的上皮细胞表型特点。

ADAM8通过激活STAT3信号通路促进结肠癌的上皮间质转化

目的:探讨去整合素样金属蛋白酶8(A disintegrin and metalloproteinase 8,ADAM8)对结肠癌细胞增殖和迁移的影响及其机制。方法:通过TCGA数据库分析ADAM8在结肠癌癌组织和正常组织中的表达水平。采用荧光定量PCR检测ADAM8在结肠癌细胞(CaCO-2、HCT8、HT29、HCT116、SW620、LOVO)中的相对表达。通过慢病毒稳转构建过表达ADAM8的结肠癌细胞系。通过实时定量聚合酶链反应(real-time quantitative polymerase chain reaction,RT-qPCR)和蛋白质印迹法(Western blot)检测ADAM8的表达水平。通过CCK-8实验和集落克隆实验检测过表达ADAM8对结肠癌细胞体外增殖能力的影响。通过Transwell实验和划痕实验检测过表达ADAM8对结肠癌细胞体外迁移能力的影响。Western blot法检测EMT相关蛋白Snail、Vimentin、N-cadherin和E-cadherin以及信号转导和转录激活因子3(STAT3)信号通路相关蛋白STAT3和p-STAT3的相对表达水平。结果:与正常组织相比,ADAM8在结肠癌癌组织中表达升高(P<0.001);ADAM8在结肠癌细胞HCT116和LOVO中相对表达量较低(P<0.05);CCK-8实验和集落克隆实验结果显示过表达ADAM8促进了结肠癌细胞增殖能力(P<0.05);Transwell和划痕实验结果显示过表达ADAM8促进了结肠癌细胞迁移能力(P<0.001);Western blot结果显示过表达ADAM8能够下调EMT相关蛋白E-cadherin表达,上调Snail、N-cadherin和Vimentin表达并激活STAT3信号通路(P<0.05)。结论:ADAM8可通过激活STAT3信号通路促进结肠癌细胞的增殖和迁移,提示ADAM8可能是结肠癌治疗的潜在靶点。

宫颈癌细胞系中EMT相关基因的表达及其意义

目的上皮-间质转化(EMT)是上皮来源的恶性肿瘤侵袭转移的重要机制之一,其特征为上皮细胞标志物表达下调(E-cadherin等)而间质细胞标志物(vimentin等)表达上调。本研究旨在探讨人宫颈癌细胞系EMT相关标志物的表达情况,以确定宫颈癌细胞是否发生EMT,并为确定相关的细胞研究模型奠定基础。方法采用逆转录PCR方法检测4株人宫颈癌细胞系(SiHa、HeLa、RJC-1、CS1213)中E-cadherin、vimentin、Snail和Slug mRNA的表达;Western blotting检测E-cadherin和vimentin的蛋白表达;免疫细胞化学方法检测Snail和Slug的蛋白表达。结果vimentin和Snail mRNA在4种细胞中均有表达,E-cadherin和Slug mRNA在SiHa、HeLa和RJC-1细胞表达。E-cadherin蛋白在SiHa和RJC-1细胞表达,vimentin蛋白在HeLa和CS1213细胞表达。Snail蛋白在4个细胞株都有表达,Slug蛋白在SiHa、HeLa和RJC-1细胞表达。结论 HeLa和CS1213表现出间质细胞表型,SiHa和RJC-1则表现为上皮细胞表型。