TCTP在肝癌细胞增殖过程中的作用及机制研究

目的:研究翻译控制肿瘤蛋白(TCTP)在肝癌细胞增殖过程中的作用及相关机制。方法:通过western blot技术检测14对肝癌与癌旁组织中TCTP的蛋白表达水平。通过siRNA(small interference RNA)技术在肝癌细胞系SMMC-7721和BEL-7404中下调TCTP的表达,然后通过CCK-8实验、克隆形成实验和EdU实验观察下调TCTP对肝癌细胞增殖的影响。通过western blot技术分析TCTP促进肝癌发生这一过程中可能涉及的分子通路。结果:相比于对应的癌旁组织,TCTP在肝癌组织中显著高表达。用siRNA技术下调TCTP水平后能够明显抑制肝癌细胞的增殖能力。下调TCTP的表达之后,AKT和ERK蛋白的磷酸化水平也随之降低。结论:TCTP在肝癌组织中显著高表达,并且在肝癌细胞的增殖过程中发挥着极其重要作用,其作用机制可能与AKT和ERK通路的磷酸化激活有关。     

泛素相关蛋白样因子2在肝癌中的表达及意义

目的:探讨肝癌中泛素相关蛋白样因子2(UBAP2L)的表达水平与预后的关系及其对肝癌细胞侵袭、转移能力的影响。方法:挖掘oncomine数据库,提取UBAP2L在肝癌与正常组织转录水平的变化及相关临床资料,采用Kaplan-Meier法分析UBAP2L表达水平与肝癌患者预后的关系。采用实时定量PCR、Western blot检测HCCLM3、MHCC97H、Hep3B、Huh7肝癌细胞株及正常肝细胞LO2中UBAP2L mRNA及蛋白水平,免疫组织化学染色法检测本院80例肝癌组织与癌旁组织中UBAP2L的表达水平加以验证。通过慢病毒载体使UBAP2L高表达的肝癌细胞株HCCLM3表达下调,采用克隆形成和划痕实验检测UBAP2L对肝癌细胞增殖能力和侵袭、转移的影响。结果:UBAP2L在oncomine数据库大多数队列呈高表达。UBAP2L在肝癌细胞株中的mRNA和蛋白的表达水平显著高于正常肝细胞(P0.05)。肝癌组织中UBAP2L阳性、强阳性表达率高于癌旁组织(P0.05),下调UBAP2L表达可明显抑制HCCLM3肝癌细胞的克隆形成能力和运动能力(P0.05)。UBAP2L高表达组的中位生存时间与UBAP2L低表达组比较差异无统计学意义(P0.05)。结论:UBAP2L在肝癌组织中呈高表达,下调UBAP2L表达可抑制肿瘤增殖和侵袭、转移表型,其可能成为肝癌早期诊断的生物标志物和治疗的潜在靶点。     

Lnc01089在肝癌细胞中的表达分析

为探讨Lnc01089与肝癌细胞侵袭的关系,本研究分析了Lnc01089在肝癌细胞株和肝癌组织中的表达情况。从肝癌细胞株HepG2、Huh7、SMMC-7721和15例成对的肝癌组织及其癌旁组织中提取总的RNA,经逆转录得到cDNA,通过qRT-PCR实验,分析Lnc01089在肝癌组织和这三种肝癌细胞株中的表达水平。结果发现Huh7、SMMC-7721中Lnc01089的表达量明显低于HepG2;肝癌组织中Lnc01089的平均表达量也明显低于癌旁组织,差异均有统计学意义(p0.05)。结果提示Lnc01089表达水平的降低与肝细胞的恶性程度有关。因此Lnc01089可能与肝癌的发生、发展有密切联系,有望为肝癌治疗提供新的靶点。     

人肝细胞癌中PCNA和RAB10水平上调miR-224表达下调

目的为了进一步揭示肝癌发生的分子机理,探讨肝癌中细胞增殖与miR-224、RAB10表达之间的关系。方法选取确诊的肝细胞癌患者8例,手术后分别获得其肝细胞癌和癌旁组织,免疫组织化学检测代表细胞增殖的PCNA和癌基因产物RAB10的表达,实时定量PCR检测miR-224的表达。结果在肝细胞癌中,细胞增殖水平非常显著地高于癌旁组织;miR-224表达显著地低于癌旁组织,但RAB10水平显著高于癌旁组织。结论肝细胞癌发生可能在于肝细胞中下调的miR-224对其靶基因rab10沉默作用降低,使其高水平表达RAB10;高水平RAB10作为细胞癌基因产物促进细胞的增殖、发生肝癌。     

Smad7对肝癌细胞增殖和迁移的影响及其临床意义

目的:探究Smad7对肝癌细胞增殖和迁移的影响及其临床意义。方法:通过转染pcDNA3.1(+)-Smad7质粒或Smad7的小干扰RNA使得Smad7在肝癌细胞系HepG2和Huh7中过表达或敲减,应用MTT法检测Smad7对肝癌细胞增殖的影响,采用细胞划痕实验以及Transwell实验探讨Smad7对肝癌细胞迁移的影响。采用qRT-PCR检测9例肝癌癌患者手术切除的组织样本中Smad7的表达。结果:过表达Smad7的肝癌细胞增殖能力与对照组相比有明显的下降,而敲减smad7能够促进肝癌细胞的增殖。过表达smad7的肝癌细胞穿过Transwell小室底膜的能力显著下降,而敲减Smad7能够促进这种能力。Smad7在肝癌癌旁组织中的表达显著高于癌组织。结论:Smad7能够在肝细胞肝癌的进展中发挥负向调控作用。     

miR-375 在HBV相关性肝细胞癌肿瘤组织与癌旁组织中的表达

目的:比较HBV相关性肝细胞癌患者的肝癌组织与癌旁组织中miR-375的表达。方法:以西京医院进行肝癌切除术治疗的HBs Ag阳性的25例患者为研究对象,收集肝癌组织、癌旁组织及术前外周血标本,并采集相关临床信息。采用反转录和实时荧光定量PCR的方法检测组织中miR-375的表达水平。结果:本研究共收集的25例肝癌患者HBs Ag均为阳性,在肝癌组织中miR-375的表达水平为2.51×10-3±3.61×10-3,显著低于在癌旁组织中的表达水平(15.23×10-3±20.85×10-3;t=3.12,P0.05)。结论:miR-375在HBV相关性肝细胞癌肝癌组织中表达显著下调。     

MKi67-siRNA可抑制QGY-7703细胞的生长

为了探讨MKI67在肝癌细胞发生发展中的作用,采用实时定量 PCR 方法检测人肝细胞癌 QGY 7703 细胞中MKI67 基因表达水平, 以及 MKI67在肝细胞癌组织和癌旁正常组织中的表达情况,设计并合成针对MKI67 的siRNA,利用脂质体转染法将其转入QGY-7703 细胞内,通过MTT和细胞集落形成实验观察MKI67-siRNA 对QGY-7703细胞生长活性和增殖能力的影响.实时定量PCR结果表明,MKI67在肝细胞癌组织中的表达水平明显高于癌旁正常组织（P< 0.01）. MTT和细胞集落形成实验结果显示,转染MKI67-siRNA 的QGY-7703细胞生长活性和集落形成率明显低于对照组（P< 0.01）.由此得出结论：MKI67 在肝癌细胞系QGY-7703细胞中的表达水平较高,且它在肝癌组织中的表达水平明显上调. 同时,MKI67-siRNA 可以有效抑制QGY-7703细胞的生长活性和增殖能力,提示MKI67可能与肝细胞癌的发生、发展相关.     

MFN1在肝癌转移中的调控作用研究

目的:探讨线粒体融合蛋白MFN1(mito-fusion 1)在肝癌转移中的作用及其机制。方法:1).采用免疫组化实验检测15对肝癌转移灶组织与原发灶组织中MFN1的表达,以明确肝癌转移时是否伴有MFN1表达的改变。2).采用si RNA (small interference RNA)下调肝癌细胞中MFN1的表达后,提高Transwell迁移实验和Transwell侵袭实验分别检测其迁移和侵袭能力,通过实时荧光定量PCR (Quantitative Real-time PCR,qRT-PCR)和Western blot实验分别检测基质金属蛋白酶1 (matrix metalloproteinase 1,MMP1)、MMP2、MMP7及MMP9的m RNA和蛋白表达。结果:1)肝癌转移灶组织中MFN1表达显著低于原发灶组织(P0.05)。2).下调MFN1表达后,肝癌细胞的迁移和侵袭能力显著升高,MMP7的表达显著增加,而MMP1、MMP2与MMP9的表达无明显变化。结论:线粒体融合蛋白MFN1在肝癌转移组织中表达显著降低,可能通过激活MMP7表达,促进肝癌细胞侵袭和转移。     

MTFP1 overexpression promotes the growth of oral squamous cell carcinoma by inducing ROS production

Mitochondrial fission process 1 (MTFP1) is a novel nuclear‐encoded protein that promotes mitochondrial fission. Increasing lines of evidence indicate that increased mitochondrial fission is involved in carcinogenesis and tumor progression. However, the expression and biological effects of MTFP1 in cancer development is still unclear, especially in oral squamous cell carcinoma (OSCC). In this study, we first evaluated the expression of MTFP1 in 12‐paired OSCC tumor and peritumor tissues. We then explored the effects of MTFP1 knockdown or overexpression on cell growth by cell proliferation, colony formation, cell cycle, and cell apoptosis assays. Furthermore, the mechanisms by which MTFP1 promoted OSCC cell growth were explored. Our results showed that MTFP1 is frequently overexpressed in OSCC tissues. Functional experiments revealed that MTFP1 promoted the growth of OSCC cells by inducing the progression of cell cycle and suppressing cell apoptosis. Mechanistically, MTFP1 overexpression‐mediated mitochondrial fragmentation and subsequent ROS production was found to be involved in the promotion of OSCC cell growth. Collectively, our study demonstrates that MTFP1 plays a critical oncogenic role in OSCC carcinogenesis, which may serve as a potential therapeutic target in the treatment of this malignance.     

肉毒碱棕榈酰基转移酶CPT2促进肝癌细胞迁移侵袭的作用研究

目的:探讨肉毒碱棕榈酰基转移酶2(CarnitinePalmitoyltransferase2, CPT2)在肝癌细胞迁移侵袭中的调控作用。方法:1).用免疫组化实验,检测62对肝癌癌组织与癌周组织中CPT2表达,以明确肝癌组织细胞中CPT2表达是否发生异常改变。2).细胞划痕实验,在人肝癌细胞HLE中分析干涉CPT2表达对肝癌细胞迁移能力的影响。3).Transwell侵袭实验,在人肝癌细胞HLE中分析干涉CPT2表达对肝癌细胞侵袭能力的影响。结果:1). CPT2主要为胞浆染色且染色呈线粒体蛋白染色典型的颗粒状分布;与癌周组织相比,肝癌组织中CPT2表达显著升高。2).细胞划痕实验证实,干涉人肝癌细胞HLE中CPT2表达后,细胞的相对迁移距离显著变短(si Ctrl VS si CPT2#1 VS si CPT2#2=1.00±0.8 VS 0.67±0.42 VS 0.64±0.31)。3).Transwell侵袭实验证实,干涉人肝癌细胞HLE中CPT2表达后,侵袭至小室底部的细胞数目显著变少(si Ctrl VS si CPT2#1 VS si CPT2#2=23.34±3.51 VS 8.00±2.00 VS8.67±1.53)。结论:CPT2在肝癌细胞中表达显著上调,CPT2表达上调促进了肝癌细胞的迁移与侵袭。     

MARCH5 restores endothelial cell function against ischaemic/hypoxia injury via Akt/eNOS pathway

MARCH5 is a critical regulator of mitochondrial dynamics, apoptosis and mitophagy. However, its role in cardiovascular system remains poorly understood. This study aimed to investigate the role of MARCH5 in endothelial cell (ECs) injury and the involvement of the Akt/eNOS signalling pathway in this process. Rat models of myocardial infarction (MI) and human cardiac microvascular endothelial cells (HCMECs) exposed to hypoxia (1% O₂) were used in this study. MARCH5 expression was significantly reduced in ECs of MI hearts and ECs exposed to hypoxia. Hypoxia inhibited the proliferation, migration and tube formation of ECs, and these effects were aggravated by knockdown of MARCH5 but antagonized by overexpressed MARCH5. Overexpression of MARCH5 increased nitric oxide (NO) content, p-eNOS and p-Akt, while MARCH5 knockdown exerted the opposite effects. The protective effects mediated by MARCH5 overexpression on ECs could be inhibited by eNOS inhibitor L-NAME and Akt inhibitor LY294002. In conclusion, these results indicated that MARCH5 acts as a protective factor in ischaemia/hypoxia-induced ECs injury partially through Akt/eNOS pathway.     

Increased expression of MARCH8, an E3 ubiquitin ligase,is associated with growth of esophageal tumor

Background

Herein, for the first time, we report aberrant expression of membrane-associated RING-CH8 (MARCH8) in human esophageal squamous cell carcinoma. MARCH8 is a member of the recently discovered MARCH family of really interesting new genes (RING) E3 ligases. Though initial studies primarily focused on its immunomodulatory role, the newly discovered targets of this E3 ligase point towards its possible role in other biological processes such as embryogenesis and inhibition of apoptosis. However, its relevance in cancers is yet to be elucidated.

Methods

We carried out quantitative real time PCR and immunohistochemistry to examine the levels of MARCH8 mRNA and protein in esophageal squamous cell carcinoma tissues. The role of MARCH8 in esophageal cancer cells was evaluated by cell proliferation, clonogenic and migration/invasion assays and flow cytometry with MARCH8 gene knockdown.

Results

Significantly increased expression of MARCH8 mRNA was found in esophageal squamous cell carcinoma as compared to distant matched non-malignant tissues (p = 0.024, AUC = 0.654). Immunohistochemical analysis revealed overexpression of MARCH8 protein in 86% of esophageal squamous cell carcinoma tissues (p < 0.001, AUC = 0.908). Interestingly, intense nuclear staining of MARCH8 protein was detected in cancer cells in addition to its cytoplasmic expression. Knockdown of MARCH8 resulted in decreased proliferation, migration, invasion and clonogenic potential of esophageal cancer cells. In addition to this, silencing of MARCH8 induced apoptosis in esophageal cancer cells which was measured by cell cycle distribution assay which showed increase in sub G0 and G2/M populations (cell death) and decrease in S-phase population. To further check the type of apoptosis induced by MARCH8 silencing, annexin assay was performed which showed significant increase in the number of cells in early apoptotic phase.

Conclusions

Overall, increased expression of MARCH8 gene in preneoplastic and neoplastic esophageal tissues and its knockdown effect on cancer cell properties demonstrated herein points towards the potential role of this protein in esophageal tumorigenesis.

     

Circ-CSPP1 knockdown suppresses hepatocellular carcinoma progression through miR-493-5p releasing-mediated HMGB1 downregulation

BackgroundHepatocellular carcinoma (HCC) accounts for over 80% of primary liver cancers and leads to a high death rate. Research on circular RNAs (circRNAs) suggests that circRNAs are promising biomarkers for cancer treatment. This study aimed to explore the function of a novel circRNA (circ-CSPP1) in HCC.MethodsCirc-CSPP1 was obtained from the microarray data downloaded from the Gene Expression Omnibus (GEO) database. The expression of circ-CSPP1, miR-493-5p and high mobility group box 1 (HMGB1) was measured by quantitative real-time polymerase chain reaction (qRT-PCR). Cell proliferation, colony formation ability, migration and invasion were monitored using cell counting kit-8 (CCK-8) assay, colony formation assay, wound healing assay and transwell assay, respectively. The protein levels of CyclinD1, Vimentin, matrix metallopeptidase 9 (MMP-9) and HMGB1 were detected by western blot. Xenograft models were established to investigate the function of circ-CSPP1 in vivo. The association between miR-493-5p and circ-CSPP1 or HMGB1 was predicted by the online tool starBase and ensured by dual-luciferase reporter assay.ResultsThe expression of circ-CSPP1 and HMGB1 was elevated, while the expression of miR-493-5p was declined in HCC tissues and cells. Circ-CSPP1 knockdown not only depleted HCC cell proliferation, formation, migration and invasion in vitro but also inhibited tumor growth in vivo. MiR-493-5p was a target of circ-CSPP1, and HMGB1 was a target of miR-493-5p. Rescue experiments presented that miR-493-5p deficiency reversed the effects of circ-CSPP1 knockdown, and HMGB1 overexpression reversed the effects of miR-493-5p restoration. Circ-CSPP1 sponged miR-493-5p to regulate HMGB1 expression.ConclusionKnockdown of circ-CSPP1 suppressed HCC development both in vitro and in vivo by upregulation of miR-493-5p and downregulation of HMGB1, hinting that circ-CSPP1 participated in HCC pathogenesis.     

lncRNA CEBPA-AS1靶向miR-455-3p调控胃癌细胞生物学行为的分子机制研究

摘要 目的：探讨lncRNA CEBPA-AS1对胃癌细胞生物学行为的影响及其可能作用机制。方法：qRT-PCR法检测胃癌组织、癌旁组织与正常人胃上皮GES1细胞和人胃癌SNU-1、AGS、HS-746T细胞系中lncRNA CEBPA-AS1、miR-455-3p的表达量。si-NC、si-lncRNA CEBPA-AS1、miR-NC、miR-455-3p mimics、si-lncRNA CEBPA-AS1与anti-miR-NC、si-lncRNA CEBPA-AS1与anti-miR-455-3p分别转染至SNU-1细胞（分别命名为si-NC组、si-lncRNA CEBPA-AS1组、miR-NC组、miR-455-3p组、si-lncRNA CEBPA-AS1+anti-miR-NC组和si-lncRNA CEBPA-AS1+anti-miR-455-3p组）后,MTT实验与平板克隆形成实验分别检测细胞增殖及克隆形成能力,Transwell小室实验检测细胞迁移及侵袭能力,双荧光素酶报告基因实验与qRT-PCR实验验证lncRNA CEBPA-AS1与miR-455-3p的靶向调控关系,Western blot法检测MMP2、MMP9蛋白表达情况。结果：与癌旁组织比较,胃癌组织中lncRNA CEBPA-AS1的表达量显著升高,miR-455-3p的表达量显著降低,差异均有统计学意义（均P＜0.05）。与GES1细胞比较,SNU-1、AGS、HS-746T细胞中lncRNA CEBPA-AS1的表达量显著升高,miR-455-3p的表达量显著降低,其中SNU-1细胞的lncRNA CEBPA-AS1表达量最高,差异均有统计学意义（均P＜0.05）。与si-NC组比较,si-lncRNA CEBPA-AS1组细胞活力降低,细胞克隆形成数、迁移及侵袭细胞数减少,MMP2、MMP9蛋白表达水平降低,差异均有统计学意义（P＜0.05）。与miR-NC组比较,miR-455-3p组细胞活力降低,细胞克隆形成数、迁移及侵袭细胞数减少,MMP2、MMP9蛋白表达水平降低,差异均有统计学意义（P＜0.05）。lncRNA CEBPA-AS1可靶向结合miR-455-3p,并可负调控miR-455-3p的表达。与si-lncRNA CEBPA-AS1+anti-miR-NC组比较,si-lncRNA CEBPA-AS1+anti-miR-455-3p组细胞活力升高,细胞克隆形成数、迁移及侵袭细胞数增多,MMP2、MMP9蛋白表达水平升高,差异均有统计学意义（P＜0.05）。结论：干扰lncRNA CEBPA-AS1表达可通过靶向调控miR-455-3p而抑制胃癌细胞增殖、克隆形成、迁移及侵袭。     

长非编码RNA SNHG18对人胃癌细胞BGC823增殖和凋亡的影响

目的:探究长非编码RNA SNHG18对胃癌细胞增殖和凋亡的影响。方法:采用实时定量PCR(qRT-PCR)技术检测人胃癌组织及癌旁组织和胃癌细胞系中lncRNA SNHG18的表达;采用MTT和克隆形成试验观察转染SNHG18过表达质粒后胃癌细胞BGC823增殖活力的变化;通过流式细胞术检测lncRNA SNHG18对胃癌细胞BGC823凋亡的影响。结果:相较于癌旁组织和胃正常粘膜上皮细胞系GSE-1,胃癌组织及胃癌细胞系中SNHG18的表达水平显著降低(P0.05);胃癌细胞过表达SNHG18增殖活力以及克隆形成的能力均显著降低(P0.05),而细胞凋亡率明显升高(P0.05)。结论:胃癌组织中长非编码RNA SNHG18呈低表达,可促进胃癌细胞增殖并抑制其凋亡,可能在胃癌发生发展过程中发挥重要作用。     

CHPF promotes gastric cancer tumorigenesis through the activation of E2F1

Chondroitin polymerizing factor (CHPF) is an important glycosyltransferase involved in the biosynthesis of chondroitin sulfate. However, the relationship between CHPF and gastric cancer has not been fully investigated. CHPF expression in gastric cancer tissues was detected by immunohistochemistry and correlated with gastric cancer patient prognosis. Cultured gastric cancer cells and human gastric epithelial cell line GES1 were used to investigate the effects of shCHPF and shE2F1 on the development and progression of gastric cancer by MTT, western blotting, flow cytometry analysis of cell apoptosis, colony formation, transwell and gastric cancer xenograft mouse models, in vitro and in vivo. In gastric cancer tissues, CHPF was found to be significantly upregulated, and its expression correlated with tumor infiltration and advanced tumor stage and shorter patient survival in gastric cancer. CHPF may promote gastric cancer development by regulating cell proliferation, colony formation, cell apoptosis and cell migration, while knockdown induced the opposite effects. Moreover, the results from in vivo experiments demonstrated that tumor growth was suppressed by CHPF knockdown. Additionally, E2F1 was identified as a potential downstream target of CHPF in the regulation of gastric cancer, and its knockdown decreased the CHPF-induced promotion of gastric cancer. Mechanistic study revealed that CHPF may regulate E2F1 through affecting UBE2T-mediated E2F1 ubiquitination. This study showed, for the first time, that CHPF is a potential prognostic indicator and tumor promoter in gastric cancer whose function is likely carried out through the regulation of E2F1.Subject terms: Gastric cancer, Cell biology