Lentivirus-mediated siRNA targeting SAE1 induces cell cycle arrest and apoptosis in colon cancer cell RKO

Deregulated expression of proteins involved in the SUMOylation pathway has been detected in several tumors. SUMO1-activating enzyme subunit 1 (SAE1) plays an important role in this process. We found that SAE1 was highly expressed in the colon cancer cell line RKO and used lentivirus-mediated siRNA to suppress SAE1 expression in RKO cells. RNA-interference efficiently and specifically downregulated the target gene expression in RKO on both mRNA and protein levels. Silencing of SAE1 inhibited proliferation and reduced colony formation of RKO cells. Furthermore, as it has been shown by flow cytometry analysis, specific knockdown of SAE1 slowed down the cell population at G0/G1 phase and induced apoptosis of RKO cells. On the base of results obtained, one can suppose the biological significance of SAE1 in colon tumorigenesis and a use of this protein as a novel molecular target for colon cancer therapy.     

RNA interference-mediated silencing of iASPP induces cell proliferation inhibition and G0/G1 cell cycle arrest in U251 human glioblastoma cells

iASPP is an evolutionally conserved inhibitory member of the ASPP (apoptosis-stimulating protein of p53) protein family. Overexpression of iASPP was observed in several types of human tumors, however, its role in tumorigenesis has not been fully clarified. To investigate the role of iASPP in human glioblastoma multiforme (GMB) progression, the authors employed lentivirus-mediated shRNA to silence endogenous iASPP expression and elucidated iASPP function by analysis of viability, colony formation, DNA synthesis, and cell cycle in p53-mutant glioblastoma cell line U251. iASPP was significantly and sustainably knocked down by iASPP-specific shRNA in U251 cells. Stable down-regulation of iASPP expression-induced cell proliferation inhibition and G0/G1 cell cycle arrest by down-regulation of cyclin D1 and up-regulation of p21(Waf1/Cip1). Thus, the findings not only provide a molecular basis for the role of iASPP in cell cycle progression of glioblastoma cells but also suggest a novel therapeutic target for the treatment of GBM.     

FOXG1在结直肠癌侵袭及转移中的作用及机制

构建叉头框G1(Forkhead box G1,FOXG1)的慢病毒干扰(shRNA)质粒及表达质粒,通过敲低和过表达FOXG1探讨其对结直肠癌细胞上皮-间质转化EMT的作用及其机制。应用Western blotting检测FOXG1在RKO、SW480、SW620、LOVO、DLD-1五种结直肠癌细胞中蛋白的表达水平,设计并合成FOXG1的shRNA片段(shFOXG1),运用DNA重组技术获得重组质粒,经双酶切技术及测序方法鉴定后进行慢病毒的包装、纯化及稳定转染,经筛选后获得稳定的结直肠癌细胞株,通过Western blotting和qRT-PCR技术检测FOXG1敲低和过表达效率及EMT关键因子E-cadherin、Vimentin、Fibronectin、Snail、Twist mRNA和蛋白的变化,光学显微镜观察敲低后细胞形态学变化,通过划痕实验检测迁移能力变化,Transwell检测侵袭迁移能力的变化。5种结直肠癌细胞中,FOXG1在RKO细胞中蛋白表达量最高,而在DLD-1细胞中表达量最低,与对照组相比较,在RKO细胞中敲低FOXG1,细胞形态由长梭型变成了类圆形或者多边形,细胞极性和紧密连接增加,细胞迁移距离明显降低,侵袭转移穿过小室的细胞数也明显减少,EMT关键因子E-cadherin表达增高,Vimentin、Fibronectin、Snail、Twist表达降低,过表达FOXG1组则相反。FOXG1在结直肠癌中高表达,这种基因的高表达能够促进结直肠癌细胞的侵袭和转移,对结直肠癌细胞的EMT起着重要的调控作用。     

CD24 regulates cell proliferation and transforming growth factor β-induced epithelial to mesenchymal transition through modulation of integrin β1 stability

To determine the role of CD24 in breast cancer cells, we knocked down CD24 in MCF-7 human breast cancer cells by retroviral delivery of shRNA. MCF-7 cells with knocked down CD24 (MCF-7 hCD24 shRNA) exhibited decreased cell proliferation and cell adhesion as compared to control MCF-7 mCD24 shRNA cells. Decreased proliferation of MCF-7 hCD24 shRNA cells resulted from the inhibition of cell cycle progression from G1 to S phase. The specific inhibition of MEK/ERK signaling by CD24 ablation might be responsible for the inhibition of cell proliferation. Phosphorylation of Src/FAK and TGF-β1-mediated epithelial to mesenchymal transition was also down-regulated in MCF-7 hCD24 shRNA cells. Reduced Src/FAK activity was caused by a decrease in integrin β1 bound with CD24 and subsequent destabilization of integrin β1. Our results suggest that down-regulation of Raf/MEK/ERK signaling via Src/FAK may be dependent on integrin β1 function and that this mechanism is largely responsible for the CD24 ablation-induced decreases in cell proliferation and epithelial to mesenchymal transition.     

Utidelone inhibits growth of colorectal cancer cells through ROS/JNK signaling pathway

Utidelone (UTD1), a novel microtubule stabilizing agent, is an epothilone B analogue which was produced by genetic engineering. UTD1 has exhibited broad antitumor activity in multiple solid tumors. However, its activity and mechanism in colorectal cancer (CRC) remain to be studied. In this study, UTD1 dramatically inhibited CRC cell proliferation (with 0.38 µg/ml, 0.77 µg/ml IC50 in RKO and HCT116, respectively) in vitro. Immunofluorescence staining showed that UTD1 induced the formation of microtubule bundling and asters in RKO cells. Flow cytometry analysis demonstrated that UTD1 induced cell cycle to arrest in G2/M phase, subsequent apoptosis. Significantly, UTD1 exhibited stronger effect on inducing apoptosis than paclitaxel and 5-FU, especially in HCT15 cells which is ABCB1 high-expression. UTD1 exposure cleaved caspase-3 and poly ADP-ribose polymerase (PARP), decreased mitochondrial membrane potential, released cytochrome c, increased the production of active oxygen and activated c-Jun N-terminal kinase (JNK), suggesting ROS/JNK pathway was involved in this process. Moreover, UTD1 inhibited tumor growth and was more effective and safer compared with paclitaxel and 5-FU in RKO xenograft in nude mice. Taken together, our findings first indicate that UDT1 inhibits tumor growth in CRC xenograft model and may be a promising agent for CRC treatment.Subject terms: Drug development, Preclinical research     

Gastrin mediates resistance to hypoxia-induced cell death in xenografts of the human colorectal cancer cell line LoVo

Aim: Gastrins act as growth factors for the normal and neoplastic colorectal mucosa. The aim of this study was to determine the role of gastrins in the response of human colorectal cancer (CRC) cells to hypoxia in vitro and in vivo. Methods: Expression of the gastrin gene in the human CRC cell line LoVo was examined under normoxia and hypoxia by quantitative PCR and by radioimmunoassay. Gastrin expression was knocked down with shRNA, and the effect on cell proliferation was measured by cell counting, on cell apoptosis by annexin V staining, and on cell migration by Boyden chamber assay. The effect of gastrin knockdown on tumourigenesis in mouse xenografts was analysed by measurement of tumour volumes and weights, and by immunohistochemistry. Results: Gastrin gene expression in LoVo cells was stimulated by hypoxia via binding of hypoxia-inducible factor-1α to the gastrin promoter. The viability of gastrin knockdown cells exposed to hypoxia (1% O₂) in vitro was diminished because of loss of resistance against hypoxia-induced apoptosis, and the effect was partly reversed by treatment with non-amidated, but not amidated, gastrin. Conditioned medium from control LoVo cells under hypoxia simulated proliferation but not migration, and the effect was blocked by an inhibitor of non-amidated gastrins, but not by an inhibitor of amidated gastrins. In xenografts in mice exposed to hypoxia (10% O₂) for 21 days, tumour necrosis was significantly increased by knocking down gastrin expression. Conclusion: These results provide evidence that non-amidated gastrins are involved in the adaptation of CRCs to hypoxic microenvironments through increasing resistance to apoptosis.     

LncRNA ABHD11‐AS1 promotes the development of endometrial carcinoma by targeting cyclin D1

To investigate the expression, role and mechanism of action of long non‐coding RNA (lncRNA) ABHD11‐AS1 in endometrial carcinoma. The expression of lncRNA ABHD11‐AS1 was quantified by qRT‐PCR in human endometrial carcinoma (n = 89) and normal endometrial tissues (n = 27). LncRNA ABHD11‐AS1 was stably overexpressed or knocked‐down in endometrial carcinoma cell lines to examine the cellular phenotype and expression of related molecules. Compared to normal endometrial tissue, lncRNA ABHD11‐AS1 was significantly overexpressed in endometrial carcinoma. Overexpression of lncRNA ABHD11‐AS1 promoted the proliferation, G1‐S progression, invasion and migration of endometrial cancer cells; inhibited apoptosis; up‐regulated cyclin D1, CDK1, CDK2, CDK4, Bcl‐xl and VEGFA; and down‐regulated p16, while ABHD11‐AS1 down‐regulation has the opposite effect. RNA pull down demonstrated that lncRNA ABHD11‐AS1 binds directly to cyclin D1. Knockdown of cyclin D1 can reverse the effect of ABHD11‐AS1. Overexpression of lncRNA ABHD11‐AS1 increased the tumorigenicity and up‐regulated cyclin D1 in an in vivo model of endometrial cancer in nude mice. LncRNA ABHD11‐AS1 functions as an oncogene to promote cell proliferation and invasion in endometrial carcinoma by positively targeting cyclin D1.     

SSRP1 promotes colorectal cancer progression and is negatively regulated by miR‐28‐5p

In this study, microarray data analysis, real‐time quantitative PCR and immunohistochemistry were used to detect the expression levels of SSRP1 in colorectal cancer (CRC) tissue and in corresponding normal tissue. The association between structure‐specific recognition protein 1 (SSRP1) expression and patient prognosis was examined by Kaplan‐Meier analysis. SSRP1 was knocked down and overexpressed in CRC cell lines, and its effects on proliferation, cell cycling, migration, invasion, cellular energy metabolism, apoptosis, chemotherapeutic drug sensitivity and cell phenotype‐related molecules were assessed. The growth of xenograft tumours in nude mice was also assessed. MiRNAs that potentially targeted SSRP1 were determined by bioinformatic analysis, Western blotting and luciferase reporter assays. We showed that SSRP1 mRNA levels were significantly increased in CRC tissue. We also confirmed that this upregulation was related to the terminal tumour stage in CRC patients, and high expression levels of SSRP1 predicted shorter disease‐free survival and faster relapse. We also found that SSRP1 modulated proliferation, metastasis, cellular energy metabolism and the epithelial‐mesenchymal transition in CRC. Furthermore, SSRP1 induced apoptosis and SSRP1 knockdown augmented the sensitivity of CRC cells to 5‐fluorouracil and cisplatin. Moreover, we explored the molecular mechanisms accounting for the dysregulation of SSRP1 in CRC and identified microRNA‐28‐5p (miR‐28‐5p) as a direct upstream regulator of SSRP1. We concluded that SSRP1 promotes CRC progression and is negatively regulated by miR‐28‐5p.     

Low NLRP3 expression predicts a better prognosis of colorectal cancer

Background: NOD-like receptor pyrin domain-3 (NLRP3) inflammasome activation is a double-edged sword in tumorigenesis. Whether NLRP3 is involved in the progression and prognosis of colorectal cancer (CRC) remains elucidated and is the focus of the present study.Methods: Immunohistochemistry (IHC) was applied on tissue microarray (TMA) to determine the expression of NLRP3 in CRC patients. All 100 patients were divided into the low NLRP3 group and the high NLRP3 group according to their NLRP3 IHC scoring. Additionally, CRC xenografts were established by injecting HCT116 or RKO cells subcutaneously into nude mice. Cell proliferation and apoptosis were determined in HCT116 cells after treatment with NLRP3 inhibitor MCC950.Results: NLRP3 expression was up-regulated in colon adenocarcinoma tissues compared with that in paracancerous tissues in CRC patients, HCT116 xenograft, and RKO xenograft. High NLRP3 level correlated with the advanced TNM classification of malignant tumors, the occurrence of distant metastasis, vascular invasion, and positive lymph nodes. Furthermore, Kaplan–Meier survival analysis revealed that a high NLRP3 level was associated with a low 5-year survival rate and even a low 10-year survival rate. Moreover, the multivariable Cox proportional hazards regression model implied that NLRP3 expression level was an independent risk factor for CRC prognosis. Inhibition of NLRP3 by MCC950 suppressed cell proliferation, induced cell apoptosis, and decreased mRNA levels of interleukin 1β (IL1β) and interleukin 18 (IL18) in HCT116 cells.Conclusions: High level of NLRP3 predicts poor survival in CRC patients. NLRP3 is a putative prognostic biomarker and a potential therapeutic target in CRC treatments.     

The role of nucleolar spindle-associated protein 1 in human ovarian cancer

Ovarian cancer (OC) is one of the deadliest malignancies of the female reproductive system. The present study focused on the role of Nucleolar spindle-associated protein 1 (NuSAP1) in OC. Relative expression of NuSAP1 was detected in OC tissues as well as cells. After knocking down NuSAP1 with lentivirus-mediated shRNA and verifying the knockdown efficiency via quantitative real-time polymerase chain reaction and Western blot assays, the cell proliferation, apoptosis, and cell cycle were determined using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, colony formation, and flow cytometry, respectively. Transwell assay was conducted to detect the migration and invasion of OC cells. It was showed that NuSAP1 was abundantly expressed in OC tissues and cell lines. After knocking down NuSAP1 in OC cells, in addition to significantly inhibiting proliferation and colony forming ability, it also promotes apoptosis and affects cell cycle distribution. Moreover, cells in the shNuSAP1 group showed significantly suppressed migration and invasion ability compared with that in the shCtrl group. In conclusion, NuSAP1 may act as an oncogenic factor in OC and therefore might serve as an indicator for prognosis and therapeutic target for OC treatment.     

Valosin-containing protein (VCP) promotes the growth,invasion, and metastasis of colorectal cancer through activation of STAT3 signaling

Valosin-containing protein (VCP) was previously shown to exhibit high expression in colorectal cancer (CRC) tissues as compared with that in normal tissues; however, the role of VCP in human CRC cells has remained to be elucidated. Two colorectal cancer cell lines HCT116 and RKO were used in the experiment. We introduced lentiviral constructs expressing VCP to infect RKO cells and lenti-shRNA targeting VCP into HCT116 cells, respectively. Cell proliferation, invasion, apoptosis, and cell cycle arrest were subsequently examined by MTT assay, transwell chamber assay, flow cytometry, and western blot analysis, respectively. Furthermore, a subcutaneous tumor mouse model and lung metastasis model was used to investigate the effects of VCP on the growth and metastasis of CRC cells in vivo. VCP knockdown was shown to inhibit cell proliferation, chemoresistance and invasion, and induce apoptosis in the HCT116 CRC cells, whereas VCP over-expression suppressed apoptosis and chemoresponse, promoted proliferation and invasion of the RKO CRC cells. In addition, in the subcutaneous tumor and lung metastasis mouse model, VCP knockdown in HCT116 cells suppressed carcinogenesis and metastasis in vivo. The findings of the present study indicated that VCP is very important for the proliferation and metastasis of CRC; therefore, targeting VCP and its downstream targets may represent novel therapies for the treatment of CRC.

     

WTAP facilitates progression of endometrial cancer via CAV-1/NF-κB axis

The N⁶-methyladenosine (m⁶A) modification is one of the most prevalent methylations in eukaryotic messenger RNA (mRNA), and it is essential for the development of many important biological processes such as multiple types of tumors. One of the most important enzymes catalyzing generation of m⁶A on mRNA is Wilms' tumor 1-associating protein (WTAP); however, the potential role of WTAP in endometrial cancer (EC) still remains unknown. Here, we investigated WTAP expression level in cancer tissue and paracancerous tissue from an EC patient. Subsequently, WTAP was knocked down by small interfering RNA in EC cell line of Ishikawa and HEC-1A, respectively. Cell proliferation, migration, and invasion were studied. The expression of caveolin-1 (CAV-1) was detected by quantitative polymerase chain reaction (qPCR). The enrichments of m⁶A and METTL3 on CAV-1 were detected using RNA immunoprecipitation-qPCR. The activity of nuclear factor-κB (NF-κB) was studied using Western blot. We observed that WTAP was dramatically upregulated in the cancer tissue, and there was an enhancement in cell proliferation, migration, and invasion and a decrease in EC apoptosis in vivo and in vitro, which indicated higher tumor malignancy and worse survival outcome. After WTAP was knocked down in EC cells, CAV-1 was significantly upregulated and the enrichments of m⁶A and METTL3 at 3′-untranslated region (UTR) region of CAV-1 were decreased. Moreover, the activity of NF-κB signaling pathway was inhibited by its regulator CAV-1. Taken together, we concluded that WTAP could methylate 3′-UTR of CAV-1 and downregulate CAV-1 expression to activate NF-κB signaling pathway in EC, which promoted EC progression.     

Possible role of Pax-6 in promoting breast cancer cell proliferation and tumorigenesis

Pax 6, a member of the paired box (Pax) family, has been implicated in oncogenesis. However, its therapeutic potential has been never examined in breast cancer. To explore the role of Pax6 in breast cancer development, a lentivirus based short hairpin RNA (shRNA) delivery system was used to knockdown Pax6 expression in estrogen receptor (ER)-positive (MCF-7) and ER-negative (MDA-MB-231) breast cancer cells. Effect of Pax6 silencing on breast cancer cell proliferation and tumorigenesis was analyzed. Pax6-RNAi-lentivirus infection remarkably downregulated the expression levels of Pax6 mRNA and protein in MCF-7 and MDA-MB-231 cells. Accordingly, the cell viability, DNA synthesis, and colony formation were strongly suppressed, and the tumorigenesis in xenograft nude mice was significantly inhibited. Moreover, tumor cells were arrested at G0/G1 phase after Pax6 was knocked down. Pax6 facilitates important regulatory roles in breast cancer cell proliferation and tumor progression, and could serve as a diagnostic marker for clinical investigation.     

FOXQ1促进肝癌细胞系SMMC-7721细胞的增殖

Forkhead Box Q1 (FOXQ1)是FOX家族的重要成员之一,在许多肿瘤中异常高表达,而FOXQ1在肝癌中的研究甚少。本研究通过重组慢病毒载体介导的FOXQ1 shRNA感染肝癌SMMC-7721细胞,敲减FOXQ1的表达,研究FOXQ1对SMMC-7721细胞增殖的影响。CCK8法、倍增时间及集落形成实验显示,敲减FOXQ1导致细胞生长减慢,倍增时间延长,细胞集落形成能力减弱。流式细胞技术检测证明,与对照比较,敲减FOXQ1的表达可显著增加G1期细胞、减少S期细胞,提示G1期阻滞。qRT-PCR和Western印迹法显示,cyclinD1和c-Myc表达下调,其可能与G1阻滞有关。上述结果提示,沉默FOXQ1的表达能够抑制SMMC-7721细胞增殖,其机制可能与cyclinD1和c-Myc的下调有关。     

Schlafen 1 Inhibits the Proliferation and Tube Formation of Endothelial Progenitor Cells

Endothelial progenitor cells (EPCs) are the major source of cells that restore the endothelium during reendothelialization. This study was designed to investigate whether Schlafen 1 (Slfn1) has an effect on the proliferation and tube formation of EPCs in vivo. Slfn1 was expressed in rat EPCs. The overexpression of Slfn1 suppressed the proliferation and tube formation of EPCs; conversely, the knockdown of Slfn1 by shRNA promoted the proliferation and tube formation of EPCs. Furthermore, when Slfn1 was overexpressed, the EPCs were arrested in the G1 phase of the cell cycle. In contrast, when Slfn1 was knocked down, the EPCs progressed into the S phase of the cell cycle. Additionally, the overexpression of Slfn1 decreased the expression of Cyclin D1, whereas the knockdown of Slfn1 increased the expression of Cyclin D1; these findings suggest that Cyclin D1 is downstream of Slfn1 in Slfn1-mediated EPC proliferation. Taken together, these results indicate a key role for Slfn1 in the regulation of EPC biological behavior, which may provide a new target for the use of EPCs during reendothelialization.     

Downregulation of PKD1 by shRNA results in defective osteogenic differentiation via cAMP/PKA pathway in human MG-63 cells

Mutations and/or deletions of Pkd1 in mouse models resulted in attenuation of osteoblast function and defective bone formation; however, the function of PKD1 in human osteoblast and bone remains uncertain. In the current study, we used lentivirus-mediated shRNA technology to stably knock down PKD1 in the human osteoblastic MG-63 cell line and to investigate the role of PKD1 on human osteoblast function and molecular mechanisms. We found that a 53% reduction of PKD1 by PKD1 shRNA in stable, transfected MG-63 cells resulted in increased cell proliferation and impaired osteoblastic differentiation as reflected by increased BrdU incorporation, decreased alkaline phosphatase activity, and calcium deposition and by decreased expression of RUNX2 and OSTERIX compared to control shRNA MG-63 cells. In addition, knockdown of PKD1 mRNA caused enhanced adipogenesis in stable PKD1 shRNA MG-63 cells as evidenced by elevated lipid accumulation and increased expression of adipocyte-related markers such as PPARγ and aP2. The stable PKD1 shRNA MG-63 cells exhibited lower basal intracellular calcium, which led to attenuated cytosolic calcium signaling in response to fluid flow shear stress, as well as increased intracellular cAMP messages in response to forskolin (10 μM) stimulation. Moreover, increased cell proliferation, inhibited osteoblastic differentiation, and osteogenic and adipogenic gene markers were significantly reversed in stable PKD1 shRNA MG-63 cells when treated with H89 (1 μM), an inhibitor of PKA. These findings suggest that downregulation of PKD1 in human MG-63 cells resulted in defective osteoblast function via intracellular calcium-cAMP/PKA signaling pathway.