LIV-1 suppression inhibits HeLa cell invasion by targeting ERK1/2-Snail/Slug pathway

It was reported that expression of the estrogen-regulated zinc transporter LIV-1 was particularly high in human cervical cancer cell line HeLa. This result prompted us to study the role that LIV-1 played in human cervical cancer. The results of real-time PCR showed that LIV-1 mRNA was significantly higher in cervical cancer in situ than in normal tissues. RNAi mediated suppression of LIV-1 in HeLa cells significantly inhibited cell proliferation, colony formation, migration, and invasive ability, but had no effect on cell apoptosis. Furthermore, LIV-1 suppression is accompanied by down-regulation of p44/42 MAPK, phospho-p44/42 MAPK, Snail and Slug expression levels. Hence, our data provide the first evidence that LIV-1 mRNA is overexpressed in cervical cancer in situ and is involved in invasion of cervical cancer cells through targeting MAPK-mediated Snail and Slug expression.     

Long noncoding RNA DLEU1 promotes proliferation and glycolysis of gastric cancer cells via APOC1 upregulation by recruiting SMYD2 to induce trimethylation of H3K4 modification

ObjectivesAPOC1 has been reported to promote tumor progression. Nevertheless, its impact on cell proliferation and glycolysis in gastric cancer (GC) remains to be probed. Hence, this study explored the related impacts and mechanisms.MethodsDLEU1, SMYD2, and APOC1 expression was detected in GC cells. Afterward, ectopic expression and knockdown experiments were conducted in GC cells, followed by measurement of cell proliferation, glucose uptake capability, lactic acid production, ATP content, extracellular acidification rate (ECAR), oxygen consumption rate (OCR), and GLUT1, HK2, and LDHA expression. In addition, interactions between DLEU1 and SMYD2 were analyzed with RIP and RNA pull down assays, and the binding of SMYD2 to APOC1 promoter and the methylation modification of SMYD2 in H3K4me3 were assessed with a ChIP assay. The ectopic tumor formation experiment in nude mice was conducted for in vivo validation.ResultsDLEU1, SMYD2, and APOC1 were highly expressed in GC cells. The downregulation of DLEU1 or APOC1 inhibited glucose uptake capability, lactic acid production, ECAR, the expression of GLUT1, HK2, and LDHA, ATP contents, and proliferation but augmented OCR in GC cells, which was also verified in animal experiments. Mechanistically, DLEU1 interacted with SMYD2 and recruited SMYD2 to APOC1 promoter to promote H3K4me3 modification, thus facilitating APOC1 expression. Furthermore, the effects of DLEU1 silencing on GC cell proliferation and glycolysis were negated by overexpressing SMYD2 or APOC1.ConclusionLncRNA DLEU1 recruited SMYD2 to upregulate APOC1 expression, thus boosting GC cell proliferation and glycolysis.     

CXCL3 overexpression promotes the tumorigenic potential of uterine cervical cancer cells via the MAPK/ERK pathway

CXCL3 belongs to the CXC-type chemokine family and is known to play a multifaceted role in various human malignancies. While its clinical significance and mechanisms of action in uterine cervical cancer (UCC) remain unclear. This investigation demonstrated that the UCC cell line HeLa expressed CXCL3, and strong expression of CXCL3 was detected in UCC tissues relative to nontumor tissues. In addition, CXCL3 expression was strongly correlated with CXCL5 expression in UCC tissues. In vitro, HeLa cells overexpressing CXCL3, HeLa cells treated with exogenous CXCL3 or treated with conditioned medium from WPMY cells overexpressing CXCL3, exhibited enhanced proliferation and migration activities. In agreement with these findings, CXCL3 overexpression was also associated with the generation of HeLa cell tumor xenografts in athymic nude mice. Subsequent mechanistic studies demonstrated that CXCL3 overexpressing influenced the expression of extracellular signal-regulated kinase (ERK) signaling pathway associated genes, including ERK1/2, Bcl-2, and Bax, whereas the CXCL3-induced proliferation and migration effects were attenuated by exogenous administration of the ERK1/2 blocker PD98059. The data of the current investigation support that CXCL3 appears to hold promise as a potential tumor marker and interference target for UCC.     

氢分子对宫颈癌细胞HeLa的影响

氢分子对多种疾病具有良好的治疗或改善效果,并且使用简便无副作用,多项实验结果也证明氢分子对肿瘤的防治具有良好的效果。从细胞增殖、细胞成瘤、细胞活性、细胞周期和凋亡、细胞转移侵袭等方面研究了氢分子对宫颈癌细胞HeLa的作用效果,结果显示：克隆球实验中,加入氢分子后,HeLa细胞集落数显著降低,集落的直径也明显减小。平板克隆形成中,加入氢分子后,细胞克隆的数量和直径均显著降低。细胞活性实验显示,氢分子对HeLa细胞活性具有明显抑制效果,对细胞内中间丝波形蛋白的表达具有一定的抑制作用。此外,氢分子对HeLa细胞周期的影响显著,且具有促凋亡的作用和抑制细胞迁移与浸润的效果。研究结果表明,氢分子抑制了细胞波形蛋白的表达,降低了HeLa细胞的增殖速率,同时抑制了细胞侵袭及迁移的能力,为氢分子对宫颈癌的防治提供了一定的实验基础。     

Ubiquitin B in Cervical Cancer: Critical for the Maintenance of Cancer Stem-Like Cell Characters

Cervical cancer cells exhibit an increased requirement for ubiquitin-dependent protein degradation associated with an elevated metabolic turnover rate. Ubiquitin, which is a small, highly conserved protein expressed in all eukaryotic cells, can be covalently linked to certain target proteins to mark them for degradation by the ubiquitin-proteasome system. Previous studies highlight the essential role of Ubiquitin B (UbB) and UbB-dependent proteasomal protein degradation in histone deacetylase inhibitor (HDACi) -induced tumor selectivity. We hypothesized that UbB plays a critical role in the function of cervical cancer stem cells. We measured endogenous UbB levels in mammospheres in vitro by real-time PCR and Western blotting. The function of UbB in cancer stem-like cells was assessed after knockdown of UbB expression in prolonged Trichostatin A-selected HeLa cells (HeLa/TSA) by measuring in vitro cell proliferation, cell apoptosis, invasion, and chemotherapy resistance as well as by measuring in vivo growth in an orthotopic model of cervical cancer. We also assessed the cancer stem cell frequency, tumorsphere formation, and in vivo growth of human cervical cancer xenografts after UbB silencing. We found that HeLa/TSA were resistant to chemotherapy, highly expressed the UbB gene and the stem cell markers Sox2, Oct4 and Nanog. These cells also displayed induced differentiation abilities, including enhanced migration/invasion/malignancy capabilities in vitro and in vivo. Furthermore, an elevated expression of UbB was shown in the tumor samples of chemotherapy patients. Silencing of UbB inhibited tumorsphere formation, lowered the expression of stem cell markers and decreased cervical xenograft growth. Our results demonstrate that UbB was significantly increased in prolonged Trichostatin A-selected HeLa cells and it played a key role in the maintenance of cervical cancer stem-like cells.     

Exogenous interleukin-1 beta promotes the proliferation and migration of HeLa cells via the MEK/ERK signaling pathway

Objective

Interleukin-1 beta (IL-1β) is a crucial cytokine that has been implicated in cancer and metastasis development. However, its possible mechanistic role in cervical cancer remains unclear. This study aimed to investigate the functions of exogenous IL-1β in cervical cancer cell proliferation and migration.

Methods

HeLa cell proliferation and migration were measured using MTT and Transwell assays. A lentivirus-mediated packaging system was used to construct an IL-1β overexpressing cell line. MEK/ERK signal transduction was inhibited by pretreatment with the MEK inhibitor PD98059. qRT–PCR and Western blotting were used to test the expression of relevant genes.

Results

Exogenous IL-1β promoted the proliferation and migration of HeLa cells. In addition, overexpression of IL-1β in HeLa cells promoted cell proliferation. Mechanistically, exogenous IL-1β increased the phosphorylated MEK and ERK levels in HeLa cells and the expression of JUN, RELB, and NF-κB2. Alternatively, blockade of MEK inhibited the promoting proliferation effects of IL-1β and the expression of JUN, RELB, and NF-κB2.

Conclusions

Our data suggest that exogenous IL-1β regulates HeLa cell functions by regulating the MEK/ERK signaling pathway and by targeting JUN, RELB, and NF-κB2. Our study uncovered a potential association across IL-1β, cervical tumor development, and cancer progression.

     

RNA interference targeting tNOX attenuates cell migration via a mechanism that involves membrane association of Rac

tNOX, a tumor-associated NADH oxidase, is a growth-related protein present in transformed cells. In this study, we employed RNA interference (RNAi)-mediated down-regulation of tNOX protein expression to explore the role of tNOX in regulating cell growth in human cervical adenocarcinoma (HeLa) cells. In this first reported use of RNAi to decrease tNOX expression, we found that HeLa cell growth was significantly inhibited by shRNA-knockdown of tNOX. Furthermore, cell migration and membrane association of Rac were decreased concomitantly with the reduction in tNOX protein expression. These results indicate that shRNA targeting of tNOX inhibits the growth of cervical cancer cells, and reduces cell migration via a decrease in the membrane association of Rac. We propose that tNOX is a potential upstream mediator of Rho activation that plays a role in regulating cell proliferation, migration, and invasion.     

Plexin-B1 is a target of miR-214 in cervical cancer and promotes the growth and invasion of HeLa cells

Plexin-B1, the receptor for Sema4D, has been reported to trigger multiple and sometimes opposing cellular responses in various types of tumor cells. It has been implicated in the regulation of tumor-cell survival, proliferation, angiogenesis, invasion and metastasis. However, the plexin-B1 gene expression and its regulatory mechanism in cervical cancer remain unclear. The present study shows that plexin-B1 is over-expressed in cervical tumor tissues compared to normal cervical tissues by immunohistochemistry, Western blotting and quantitative RT-PCR. The expression of plexin-B1 is significantly associated with cervical tumor metastasis and invasion according to the analysis of the clinicopathologic data. Plexin-B1 also promotes proliferation, migration and invasion in human cervical cancer HeLa cells. We also found that the plexin-B1 levels are inversely correlated with miR-214 amounts in both cervical cancer tissues and HeLa cells. And miR-214 expression level is also associated with metastasis and invasion of cervical tumor. Furthermore, we demonstrate that plexin-B1 is inhibited by miR-214 through a miR-214 binding site within the 3'UTR of plexin-B1 in HeLa cells. Ectopic expression of miR-214 could inhibit the proliferation capacity, migration and invasion ability of HeLa cells. Our findings suggest that plexin-B1, a target of miR-214, may function as an oncogene in human cervical cancer HeLa cells.     

SMYD3 promotes hepatocellular carcinoma progression by methylating S1PR1 promoters

Hepatocellular carcinoma (HCC) is one of the most common malignancies worldwide. SET and MYND domain-containing protein 3 (SMYD3) has been shown to promote the progression of various types of human cancers, including liver cancer; however, the detailed molecular mechanism is still largely unknown. Here, we report that SMYD3 expression in HCC is an independent prognostic factor for survival and promotes the proliferation and migration of HCC cells. We observed that SMYD3 upregulated sphingosine-1-phosphate receptor 1 (S1PR1) promoter activity by methylating histone 3 (H3K4me3). S1PR1 was expressed at high levels in HCC samples, and high S1PR1 expression was associated with shorter survival. S1PR1 expression was also positively correlated with SMYD3 expression in HCC samples. We confirmed that SMYD3 promotes HCC cell growth and migration in vitro and in vivo by upregulating S1PR1 expression. Further investigations revealed that SMYD3 affects critical signaling pathways associated with the progression of HCC through S1PR1. These findings strongly suggest that SMYD3 has a crucial function in HCC progression that is partially mediated by histone methylation at the downstream gene S1PR1, which affects key signaling pathways associated with carcinogenesis and the progression of HCC.Subject terms: Liver cancer, Oncogenes     

乳酸菌发酵液联合瑞琳他抗对HeLa细胞活性及HPV18病毒载量影响的实验

研究乳酸菌发酵液联合瑞琳他抗对宫颈癌细胞系HeLa细胞的增殖、凋亡、迁移及HPV18 DNA病毒载量的影响,为2种药物联合治疗HPV感染提供新的选择。

采用CCK8法检测不同浓度的单药、组合药对HeLa细胞增殖变化的影响,得出乳酸菌发酵液联合瑞琳他抗对HeLa细胞的半数抑制浓度（IC₅₀）;采用流式细胞术、Transwell实验和杂交捕获—化学发光法（DH3）分别检测乳酸菌发酵液联合瑞琳他抗对HeLa细胞凋亡、细胞迁移、HPV18 DNA病毒载量的影响。

通过CCK-8法检测乳酸菌发酵液IC₅₀为5.5×10⁸ CFU/mL,瑞琳他抗IC₅₀为160 mg/mL。增殖实验结果显示作用48 h后,单药组均对HeLa细胞抑制生长作用明显,且乳酸菌发酵液+瑞琳他抗组对细胞的抑制作用强于瑞琳他抗组（F=164.810,P＜0.05）;流式细胞术检测发现单药组均可诱导HeLa细胞凋亡,乳酸菌发酵液+瑞琳他抗组对细胞的凋亡作用增加且大于瑞林他抗组（F=83.823,P＜0.05）;Transwell实验发现单药组均可抑制细胞迁移,乳酸菌发酵液+瑞琳他抗组对细胞迁移的抑制作用大于瑞林他抗组（F=492.242,P＜0.05）;DH3法显示单药组均可明显下调HeLa细胞HPV18 DNA病毒载量,乳酸菌发酵液+瑞琳他抗组对细胞HPV18 DNA病毒载量的作用大于瑞林他抗组（F=284.769,P＜0.05）。

乳酸菌发酵液联合瑞琳他抗对抑制HeLa细胞增殖、诱导细胞凋亡、抑制细胞迁移和下调细胞HPV18 DNA病毒载量的作用优于瑞琳他抗,为临床治疗HPV感染提供了实验依据和借鉴。

     

circ_0000218通过靶向吸附miR-1182对宫颈癌HeLa细胞增殖、迁移和侵袭的影响

目的探讨环状RNA 0000218(circ_0000218)是否通过靶向吸附miR-1182从而影响宫颈癌HeLa细胞增殖、迁移和侵袭。方法采用实时荧光定量PCR(RT-qPCR)技术分析43例宫颈癌患者癌组织、癌旁组织中circ_0000218和miR-1182的表达水平。根据转染序列不同分为si-NC组、si-circ_0000218组、miR-NC组、miR-1182组、pcDNA组、pcDNAcirc_0000218组、si-circ_0000218+anti-miR-NC组、si-circ_0000218+anti-miR-1182组。运用细胞计数试剂盒(CCK-8)法、Transwell实验分析circ_0000218和miR-1182表达对HeLa细胞增殖、迁移和侵袭的影响。蛋白质印迹法检测Ki-67、基质金属蛋白酶2(MMP-2)和MMP9蛋白表达。双荧光素酶报告实验和RT-qPCR分析circ_0000218和miR-1182的靶向关系。癌旁组织与宫颈癌组织比较采用配对t检验,两组间比较采用独立样本t检验进行统计学分析。结果宫颈癌组织中circ_0000218表达量高于癌旁组织(4.17±0.32比1.00±0.05),而miR-1182表达量低于癌旁组织(0.33±0.03比1.00±0.05),差异具有统计学意义(P均<0.001)。与si-NC组比较,si-circ_0000218组HeLa细胞增殖活力(0.86±0.04比0.37±0.03)、迁移数量[(86.73±7.13)个比(38.52±3.19)个]和侵袭数量[(66.80±4.95)个比(26.58±2.55)个]以及Ki-67(0.57±0.05比0.18±0.02)、MMP-2(0.74±0.07比0.28±0.03)和MMP-9蛋白表达量(0.64±0.04比0.22±0.02)降低,差异有统计学意义(P均<0.001).与miR-NC组比较,miR-1182组HeLa细胞增殖活力(0.88±0.04比0.46±0.04)、迁移数量[(89.74±5.53)个比(46.63±3.79)个]和侵袭数量[(68.03±4.34)个比(34.63±3.37)个]以及Ki-67(0.59±0.04比0.24±0.02)、MMP-2(0.76±0.05比0.33±0.03)和MMP-9蛋白表达量(0.66±0.04比0.29±0.03)降低,差异有统计学意义(P均<0.001)。circ_0000218靶向负调控miR-1182表达。与si-circ_0000218+anti-miR-NC组比较,si-circ_0000218+anti-miR-1182组HeLa细胞增殖活力(0.35±0.03比0.76±0.04)、迁移数量[(35.58±3.11)个比(77.04±4.08)个]和侵袭数量[(25.44±2.29)个比(57.61±3.47)个]以及Ki-67(0.16±0.02比0.46±0.04)、MMP-2(0.26±0.02比0.65±0.04)和MMP-9蛋白表达量(0.20±0.02比0.57±0.04)升高,差异有统计学意义(P均<0.001)。结论circ_0000218通过靶向吸附miR-1182可促进宫颈癌HeLa细胞增殖、迁移和侵袭。     

HMQ‐T‐F2 exert antitumour effects by upregulation of Axin in human cervical HeLa cells

Looking for novel, effective and less toxic therapies for cervical cancer is of significant importance. In this study, we reported that HMQ‐T‐F2(F2) significantly inhibited cell proliferation and transplantable tumour growth. Mechanistically, HMQ‐T‐F2 inhibited HeLa cell growth through repressing the expression and nuclear translocation of β‐catenin, enhancing Axin expression, as well as downregulating the Wnt downstream targeted proteins. Knock‐down of a checkpoint β‐catenin by siRNA significantly attenuated HeLa cell proliferation. Furthermore, XAV939, an inhibitor of β‐catenin, was used to treat HeLa cells and the results demonstrated that HMQ‐T‐F2 inhibited proliferation and migration via the inhibition of the Wnt/β‐catenin pathway.     

线粒体分裂蛋白1在人宫颈癌细胞中过表达能促进线粒体裂变并降低细胞的增殖和迁移能力

线粒体是持续进行分裂和融合的动态细胞器。近年来,除了线粒体代谢作用相关的研究之外,线粒体动力学也开始逐渐引起研究的关注。越来越多的研究表明,线粒体动力学与肿瘤细胞生物学行为具有相关性。线粒体分裂蛋白1(mitochondrial fission protein 1, FIS1)介导线粒体分裂复合物的组装,参与线粒体分裂,是线粒体融合分裂过程中重要的蛋白质。然而,鲜有研究揭示FIS1在人宫颈癌中的表达及其作用。本研究对比了宫颈癌组织以及癌旁组织的转录物组数据,结果显示,与癌旁组织相比,人宫颈癌组织中的FIS1 mRNA水平明显降低(P<0.01)。进一步进行宫颈癌组织FIS1高表达组与低表达组的差异基因分析,发现差异基因主要与线粒体功能相关。随后,进行FIS1过表达后HeLa细胞增殖、迁移、线粒体裂变以及ROS水平的相关分析。结果显示,过表达FIS1基因,HeLa细胞增殖及迁移能力显著降低,细胞内线粒体裂变程度加剧并且细胞内ROS水平升高。综合以上结果,FIS1在人宫颈癌细胞中表达水平较低,而过表达FIS1可促使宫颈癌细胞因线粒体动力学失衡而发生一系列生物学功能异常。因此,本研究为进一步研究FIS1在宫颈癌治疗中的作用奠定了重要基础。     

Synergistic antitumor activity of reversine combined with aspirin in cervical carcinoma in vitro and in vivo

A recent report showed that reversine treatment could induce murine myoblasts dedifferentiation into multipotent progenitor cells and inhibit proliferation of some tumors, and other reports showed that apoptosis of lung adenocarcinoma cells could be induced by aspirin. The aim of the present study was to evaluate the synergistic antitumor effects of reversine and aspirin on cervical cancer. The inhibition rate of reversine and aspirin on cervical cancer cell lines’ (HeLa and U14) was determined by MTT method, cell cycle of HeLa and U14 cells was analyzed by FACS, mitochondrial membrane potential of HeLa and U14 was detected using a JC-1 kit. HeLa and U14 colony formation was analyzed by soft agar colony formation assay. The expression of caspase-3, Bcl-2/Bax, cyclin D1 and p21 was detected by qRT-PCR and Western Blotting. Moreover, tumor weight and tumor volume was assessed using a murine model of cervical cancer with U14 cells subcutaneously (s.c.) administered into the neck, separately or combined with drug administration via the intraperitoneal (i.p.) route. The inhibition rate of cells in the combination group (10 μmol/L reversine, 10 mmol/L aspirin) increased significantly in comparison to that when the drugs were used alone (P < 0.05); moreover, this combination could synergistically inhibit the proliferation of five cervical cancer cell lines (HeLa, U14, Siha, Caski and C33A). In the therapeutic mouse model, tumor weight and tumor volume of cervical cancer bearing mice was more reduced when compared with the control agents (P < 0.05) in tumor-bearing mice. The combination of reversine and aspirin exerts synergistic growth inhibition and apoptosis induction on cervical cancers cells.