Inhibitor of growth 4 inhibits cell proliferation,migration, and induces apoptosis of renal cell carcinoma cells

The inhibitor of growth 4 (ING4) is known as a tumor suppressor. The expressions of ING4 were markedly reduced in human renal clear cell carcinoma (ccRCC) tissues. However, the role of ING4 in renal cell carcinoma (RCC) remains unknown. The aim of the current study was to detect the ING4 expression level and its potential role in human RCC cell lines. Our results showed that ING4 was lowly expressed in human RCC cell lines compared with that in proximal tubular cell line. Ectopic overexpression of ING4 inhibited the proliferation, migration, and invasion properties, and as well as prevented epithelial-mesenchymal transition (EMT) phenotype of RCC cells. In addition, ING4 overexpression induced cell apoptosis and autophagy in RCC cells. Furthermore, ING4 overexpression suppressed the activation of PI3K/Akt pathway in RCC cells. The activator of PI3K/Akt, insulin-like growth factor 1, abolished the effects of ING4 on RCC cells. These findings indicated that ING4 presented anticancer activity in RCC cells. The effects of ING4 on RCC cells were mediated by regulating the PI3K/Akt pathway. These findings suggested that ING4 could be used for gene therapy of RCC.     

Retracted: Targeting of SPP1 by microRNA-340 inhibits gastric cancer cell epithelial–mesenchymal transition through inhibition of the PI3K/AKT signaling pathway

Gastric cancer (GC) is a common heterogeneous disease. The critical roles of microRNA-340 (miR-340) in the development and progression of GC were emphasized in accumulating studies. This study aims to examine the regulatory mechanism of miR-340 in GC cellular processes. Initially, microarray technology was used to identify differentially expressed genes and regulatory miRs in GC. After that, the potential role of miR-340 in GC was determined via ectopic expression, depletion, and reporter assay experiments. Expression of secreted phosphoprotein 1 (SPP1), miR-340, phosphatidylinositol 3-kinase/protein kinase B (PI3K/AKT) pathway, and epithelial–mesenchymal transition (EMT)-related genes was measured. Moreover, to further explore the function of miR-340 in vivo and in vitro, proliferation, apoptosis, migration, invasion, and tumorigenic capacity were evaluated. SPP1 was a target gene of miR-340 which could then mediate the PI3K/AKT signaling pathway by targeting SPP1 in GC. Furthermore, miR-340 levels were reduced and SPP1 was enriched in GC tissues and cells, with the PI3K/AKT signaling pathway being activated. Inhibitory effects of upregulated miR-340 on SPP1 and the PI3K/AKT signaling pathway were confirmed in vivo and in vitro. Overexpression of miR-340 or the silencing of SPP1 inhibited GC cell proliferation, invasion, migration, and EMT process, but promoted apoptosis of GC cells. Typically, targeting of SPP1 by miR-340 may contribute to the inhibition of proliferation, migration, invasion, and EMT of GC cells via suppression of PI3K/AKT signaling pathway.     

芳姜黄酮对人皮肤鳞状细胞癌A431细胞迁移、侵袭及凋亡的影响和机制研究

为研究芳姜黄酮(Ar-Turmerone)对人鳞状细胞癌A431细胞增殖、迁移、侵袭和凋亡的影响及机制。实验采用CCK-8法检测抑制率,吉姆萨染色观察细胞形态,划痕实验和Transwell小室实验研究细胞迁移和侵袭能力的变化,流式细胞仪检测细胞凋亡率。此外,通过实时荧光定量聚合酶链反应(Real-time PCR)与蛋白质印迹法(western blot)法检测mRNA和蛋白表达。siRNA阻断Notch1,Hes1和PTEN,检测相应的下游mRNA和蛋白的表达变化,流式细胞仪检测细胞凋亡率。结果发现,芳姜黄酮可以抑制A431细胞增殖,使细胞形态发生改变,抑制细胞体外迁移和侵袭能力,促进细胞凋亡。经过芳姜黄酮处理后,Notch1,Hes1,PTEN的mRNA和蛋白表达升高。沉默Notch1,Hes1 mRNA和蛋白表达低于单纯给药组,而沉默Hes1,PTEN mRNA和蛋白表达也低于单纯给药组;沉默PTEN后,与单纯给药组相比,细胞死亡率降低。总之,芳姜黄酮可以抑制人鳞状细胞癌A431细胞的增殖并促进其凋亡,且具有抑制体外迁移和侵袭的作用,其促进细胞凋亡的机制是通过Notch1/Hes1/PTEN途径实现的。     

沉默脂肪细胞增强子结合蛋白2通过PI3K/Akt途径抑制肝癌细胞增殖和迁移

脂肪细胞增强子结合蛋白2(AEBP2)作为多梳抑制复合物2(PRC2)的组成蛋白质,参与多种肿瘤细胞的增殖和迁移,然而其在肝癌中的作用尚不清楚。本研究基于UALCAN和Kaplan-Meier Plotter数据库分析发现,AEBP2在肝癌组织中高表达,并且与患者的不良预后呈正相关。实时荧光定量PCR和蛋白质印迹结果证实,AEBP2在肝癌细胞中的表达高于正常肝细胞。在HepG2和Huh-7细胞中转染AEBP2 siRNA,平板克隆、CCK-8、流式细胞术、划痕愈合和Transwell结果显示,沉默AEBP2可以抑制肝癌细胞增殖、迁移和侵袭,并促进细胞凋亡(P＜0.05)。免疫荧光检测和蛋白质印迹结果显示,沉默AEBP2能够抑制肝癌细胞上皮-间质转化(EMT)(P＜0.05)。生物信息学分析结果表明,AEBP2参与调控PI3K/Akt信号通路。蛋白质印迹结果证实,沉默AEBP2能下调PI3K、p-AKT (S473)、mTOR、MMP-2和MMP-9的蛋白质表达水平(P＜0.05)。此外,沉默AEBP2对HepG2细胞迁移和侵袭的影响可被PI3K/Akt通路激动剂胰岛素样生长因子1(IGF-1)部分逆转(P＜0.01)。综上所述,AEBP2可能通过调节PI3K/Akt途径促进肝癌细胞增殖和迁移。本研究为AEBP2在肝癌中的作用提供理论依据。     

Retracted: Smad4 gene silencing enhances the chemosensitivity of human lymphoma cells to adriamycin via inhibition of the activation of transforming growth factor β signaling pathway

There are diverse investigations focused on the therapies of lymphoma. Our research was taken to identify the effects of lentiviral-mediated Smad4 gene silencing on chemosensitivity of human lymphoma cells to adriamycin (ADM) via transforming growth factor β (TGFβ) signaling pathway. Raji/ADM cells were cultured and infected with lentiviral particles Smad4-short hairpin (shRNA) and control-shRNA. Then, the messenger RNA (mRNA) and protein levels of TGFβ signaling pathway–related factors (Smad4, Smad3, cyclinE, cyclinD1, and p21) in Raji/ADM cells were determined. The effect of Smad4-shRNA on cell viability, invasion and migration, and apoptosis were also detected. Compared with the Raji group, increased mRNA and protein levels of Smad4, Smad3, cyclinE, cyclinD1, enhanced cell proliferation, migration and invasion as well as decreased mRNA, and protein levels of p21 and cell apoptosis rate were found in the Raji/ADM and control-shRNA groups. However, Smad4 gene silencing resulted in decreased mRNA and protein levels of Smad4, Smad3, cyclinE, and cyclinD1 along with inhibited cell proliferation, migration and invasion but increased expression of p21 together with cell apoptosis. Collectively, Smad4 gene silencing can inhibit the activation of TGFβ signaling pathway, thereby enhancing the chemosensitivity of human lymphoma cells to ADM.     

The role of focal adhesion kinase-phosphatidylinositol 3-kinase-akt signaling in hepatic stellate cell proliferation and type I collagen expression

Following a fibrogenic stimulus, the hepatic stellate cell (HSC) undergoes a complex activation process associated with increased cell proliferation and excess deposition of type I collagen. The focal adhesion kinase (FAK)-phosphatidylinositol 3-kinase (PI3K)-Akt signaling pathway is activated by platelet-derived growth factor (PDGF) in several cell types. We investigated the role of the FAK-PI3K-Akt pathway in HSC activation. Inhibition of FAK activity blocked HSC migration, cell attachment, and PDGF-induced PI3K and Akt activation. Both serum- and PDGF-induced Akt phosphorylation was inhibited by LY294002, an inhibitor of PI3K. A constitutively active form of Akt stimulated HSC proliferation in serum-starved HSCs, whereas LY294002 and dominant-negative forms of Akt and FAK inhibited PDGF-induced proliferation. Transforming growth factor-beta, an inhibitor of HSC proliferation, did not block PDGF-induced Akt phosphorylation, suggesting that transforming growth factor-beta mediates its antiproliferative effect downstream of Akt. Expression of type I collagen protein and alpha1(I) collagen mRNA was increased by Akt activation and inhibited when PI3K activity was blocked. Therefore, FAK is important for HSC migration, cell attachment, and PDGF-induced cell proliferation. PI3K is positioned downstream of FAK. Signals for HSC proliferation are transduced through FAK, PI3K, and Akt. Finally, expression of type I collagen is regulated by the PI3K-Akt signaling pathway.     

LINC00961 restrains cancer progression via modulating epithelial–mesenchymal transition in renal cell carcinoma

Recently, long noncoding RNA have been identified as new gene regulators and prognostic biomarkers in various cancers, including renal cell carcinoma (RCC). The expression and biological roles of LINC00961 have been reported in many human cancers. However, up to date, no study of LINC00961 has been shown in RCC. Currently, we aimed to investigate the function of LINC00961 in RCC progression. Interestingly, we observed that LINC00961 could act as a novel biomarker in predicting the diagnosis of RCC. Then, we found that LINC00961 was greatly downregulated in RCC cell lines (Caki-1, Caki-2, 786-O, A498, and ACHN cells) compared with normal renal cell lines (HK-2 cells). Then, 786-O cells and ACHN cells were infected with LV-LINC00961. As displayed in our current study, LINC00961 overexpression could obviously suppress the proliferation and survival of RCC cells in vitro. In addition, RCC cell apoptosis was greatly induced and cell cycle progression was blocked in G1 phase by upregulation of LINC00961 in 786-O cells and ACHN cells. Subsequently, we found that LV-LINC00961 was able to restrain RCC cell migration and cell invasion capacity. Meanwhile, the messenger RNA and protein expression levels of epithelial–mesenchymal transition (EMT)-associated markers Slug and N-cadherin in RCC cell lines were dramatically inhibited by overexpressing LINC00961. Finally, the in vivo experiment was carried out and we observed that LINC00961 could inhibit RCC development through modulating EMT process. Taken these together, it was indicated in our study that LINC00961 was involved in RCC progression through targeting EMT pathway.     

Role of PI3K and AKT specific isoforms in ovarian cancer cell migration, invasion and proliferation through the p70S6K1 pathway

Ovarian cancer is the leading cause of death from gynecological malignancy for women. The amplification of the PI3K catalytic subunit (p110) and the lost function of PTEN are frequently detected in ovarian cancer cells. PI3K plays an important role in tumorigenesis. To specifically inhibit PI3K activity in ovarian cancer cells, we constructed small interfering RNA (siRNA) against p110. The expression of p110 siRNA significantly decreased cell migration, invasion, and proliferation compared to the siSCR control cells. The expression of p110 siRNA induced CDK inhibitor p27^KIP1 levels, and decreased levels of cyclin D1, CDK4, and phosphorylated retinoblastoma protein. PI3K transmits the mytogenic signal through AKT. AKT has three isoforms in the cells: AKT1, AKT2 and AKT3. We found that inhibition of AKT1 is sufficient to affect cell migration, invasion, and proliferation. Expression of AKT1 siRNA had a similar effect as p110 siRNA in the cells. We showed the roles of specific PI3K and AKT isoforms in the cells, which are important to understanding the mechanism of PI3K/AKT signaling in ovarian cancer cells. Both p110 and AKT1 siRNA-expressing cells decreased the activation of p70S6K1. Inhibition of p70S6K1 activity by its siRNA also decreased cell migration, invasion, and proliferation associated with the induction of p27^KIP1 levels, and with the inhibition of cell cycle-associated proteins including cyclin D1, CDK2, and phosphorylated retinoblastoma protein. This study demonstrates the important role of the PI3K/AKT/mTOR/p70S6K1 pathway in cell proliferation, migration, and invasion in ovarian cancer cells by using siRNA-mediated gene silencing as a reverse genetic method.     

Downregulation of LAT1 expression suppresses cholangiocarcinoma cell invasion and migration

Currently, there is no effective treatment for cholangiocarcinoma (CCA), which is the most prevalent in the northeastern part of Thailand. A new molecular target for the treatment of CCA is, therefore, urgently needed. Although L-type amino acid transporter 1 (LAT1) is highly expressed in CCA cells, its role in malignant phenotypes of CCA cells remains unclear. This study aimed to investigate the impact of LAT1 on proliferation, migration, and invasion of KKU-M213 cells, the CCA cells derived from Thai patients with intrahepatic cholangiocarcinoma. Results showed that KKU-M213 cells expressed all LAT isoforms (LAT1, LAT2, LAT3 and LAT4). The expressions of LAT1 and its associated protein 4F2hc were highest whereas those of LAT2 and LAT4 were extremely low. Treatment with 2-aminobicyclo-(2,2,1)-heptane-2-carboxylic acid (BCH) reduced l-leucine uptake concomitant with an inhibition of cell motility and, to a lesser extent, on cell proliferation. It also induced a time dependent up-regulation of LAT1 and 4F2hc expressions. Similarly, cell migration and invasion, but not proliferation, were reduced in LAT1 knockdown KKU-M213 cells. In addition, silencing of LAT1 inhibited the expressions of 4F2hc mRNA and protein whereas the expression of microRNA-7, the 4F2hc down-regulator, was increased. Furthermore, the phosphorylation levels of ERK1/2 and p70S6K were reduced after LAT1 knockdown. Collectively, these results suggest that suppression of cell invasion and migration in LAT1 knockdown KKU-M213 cells may be partly mediated through the inhibition of the 4F2hc-signaling pathway by the up-regulation of microRNA-7. Based on this finding, LAT1 may be a potential therapeutic target for treating CCA.     

PAK1 promotes proliferation,migration and invasion of hepatocellular carcinoma by facilitating EMT via directly up-regulating Snail

BackgroundHepatocellular carcinoma (HCC) is a primary cause of cancer mortality. PAK1 plays key roles in many types of cancers. However, the role of PAK1 in HCC is not clear.MethodsqRT-PCR and Western blotting were used to determine expressions of PAK1, Snail and epithelial mesenchymal transition (EMT)-related proteins. Luciferase reporter assay was used to measure the interaction between PAK1 and Snail. Wound healing, transwell, colony formation assays and flow cytometry were used to assess cell migration, invasion, proliferation and apoptosis. Mouse tumor xenograft model was used to determine the effect of PAK1 on tumor growth in vivo.ResultsPAK1 and Snail were up-regulated in HCC cells. PAK1 knockdown suppressed cell proliferation, migration and invasion, and increased apoptosis of HCC cells. PAK1 knockdown also inhibited tumor growth in vivo. Mechanistically, PAK1 promoted EMT by targeting Snail. Knockdown of PAK1 could up-regulate pro-apoptotic proteins but down-regulate proliferation-related proteins via suppressing β-catenin signaling pathway.ConclusionPAK1 promotes EMT process by increasing Snail, and facilitates progression of HCC by activating β-catenin pathway.