Cell Transwell Invasion Assays Research Articles

Knockdown of CPEB4 expression suppresses cell migration and invasion via Akt pathway in non-small cell lung cancer.

Cytoplasmic polyadenylation element binding protein 4 (CPEB4) could be an important regulator in variety of cancers. However, the biological function and the underlying molecular mechanism of CPEB4 in non-small cell lung cancer (NSCLC) remains unknown. In this study, we investigated the metastasis role of CPEB4 in NSCLC cells, we knocked down CPEB4 using siRNA. Transwell migration assay and cell invasion assay on Matrigel were presented, and cell migration was also determined by scratch-healing assay. ROS generation were determined by fluorescence probe DCFH2-DA. The protein expression was assessed by ELISA and Western blot analysis. LY294002 were used to inhibit PI3 K/Akt signaling. The data showed that knockdown of CPEB4 inhibited the migration and invasion of NSCLC. Moreover, silencing of CPEB4 reduced Snail and MMP-3 expression in vitro. We also indicated that CPEB4 knockdown increased the ROS expression. Furthermore, we found that silencing of CPEB4 decreased pAkt expression. Taken all together, our data demonstrated that silencing of CPEB4 induces ROS generation, thus suppressing the Akt expression, which finally prevents NSCLC cells invasion and migration. Therefore, CPEB4 may regard as a target to inhibit NSCLC invasion and metastasis through Akt pathway.

Ginsenoside Rg3 inhibits keloid fibroblast proliferation, angiogenesis and collagen synthesis in vitro via the TGF‑β/Smad and ERK signaling pathways.

A wide range of therapeutic options exists for the treatment of keloids, all of which have their own strengths; however, a high risk of side-effects and frequent recurrence remains. Therefore, the present study aimed to identify improved therapeutic approaches or drugs for the treatment of keloids. Ginsenoside Rg3 (Rg3) has been reported to exert numerous antitumor effects, thus indicating that Rg3 may be a potential therapeutic agent that targets keloids. The present study determined the effects of Rg3 on human keloid fibroblasts (KFs) in vitro, and further explored the associated molecular and cellular mechanisms. Keloid scar specimens were obtained from patients, aged between 22 and 35 years, without systemic diseases and primary cells were isolated from keloid tissues. In each assay, KFs were divided into three groups and were cultured in medium with or without various concentrations of Rg3 (50 or 100 μg/ml). Cell viability assay, flow cytometry, quantitative polymerase chain reaction, cell migration assay, immunofluorescence staining, western blot analysis, Transwell cell invasion assay and immunohistochemical analysis were used to analyze the KFs and keloid explant cultures. The results of the present study demonstrated that Rg3 was able to exert an inhibitory effect on the transforming growth factor-β/Smad and extracellular signal-regulated kinase signaling pathways in KFs. The proliferation, migration, invasion, angiogenesis and collagen synthesis of KFs were markedly suppressed following treatment with Rg3. Furthermore, the results of an ex vivo assay indicated that Rg3 inhibited angiogenesis and reduced collagen accumulation in keloids. Significant statistical differences existed between the control and Rg3-treated groups (P<0.05). All of these experimental results suggested that Rg3 may serve as a reliable drug for the treatment of patients with keloids.

Effect of TAGLN2 in the regulation of meningioma tumorigenesis and development.

To explore the role of transgelin-2 TAGLN2 in the development and progression of meningioma and the potential regulatory. TAGLN2 knockdown expression and overexpression in vitro models were constructed using lentivirus in meningioma cell line CH157; their corresponding transfection efficiencies were verified by qRT-PCR and Western Blot. Actions of TAGLN2 on the proliferation of meningioma cells were explored by CCK8 and colony formation assays. The effect of TAGLN2 on invasion of meningioma cells was analyzed by transwell cell invasion assay. Biological function of TAGLN2 on apoptosis of meningioma cells was determined by flow cytometry. Finally, Western Blot was used to investigate the detailed mechanism of TAGLN2 on regulating the biological functions of meningioma cells. After down-regulating the expression of TAGLN2, there were significantly decreased capacities of cells proliferation and colony formation of meningioma cells, meanwhile, cell invasion was significantly decreased but the apoptosis rate was increased. On the contrary, up-regulation of TAGLN2 expression, the proliferation, colony formation ability were significantly increased as well as the invasion capacity, whilst apoptosis rate was decreased. Western Blot showed that expressions of p-PI3K and p-AKT were inhibited after knockdown of TAGLN2, which were significantly increased after TAGLN2 was overexpressed. TAGLN2 can affect the proliferation, invasion and apoptosis of meningioma cells and may participate in the development of meningioma through regulating the PI3K/AKT signaling pathway.

Downregulation of SNHG1 suppresses cell proliferation and invasion by regulating Notch signaling pathway in esophageal squamous cell cancer.

Long non-coding RNAs (lncRNAs) exert important functions involved in tumorigenesis and cancer progression including esophageal squamous cell cancer (ESCC), however, the clinical role and underlying biological function of Small Nucleolar RNA Host Gene 1 (SNHG1) in ESCC is not well known. Quantitative Real-time polymerase chain reaction (QRT-PCR) was used to detect the SNHG1 expression levels in ESCC tissues and adjacent non-cancerous tissues. Chi-square test was used to evaluate the association between clinicopathological features and SNHG1 expression in ESCC patients, Kaplan-Meier curve and log rank test was performed to analyze the association between overall survival and SNHG1 expression. Cell proliferation and invasion was assessed by MTT assay, colony formation, and transwell cell invasion assays. Western blot were also performed to examine protein expression levels of E-cadherin, Vimentin and N-cadherin, Notch 1 and Hes-1. We demonstrated that lncRNA SNHG1 was significantly up-regulated in ESCC tissues compared with adjacent non-cancerous tissues in ESCC patients. Meanwhile, increased lncRNA SNHG1 expression levels markedly correlated with lymph node metastasis, depth of invasion, TNM stage and reduced over survival time in ESCC patients. Furthermore, MTT assay, colony formation, transwell cell invasion, qRT-PCR and Western-blot assays demonstrated that knockdown of lncRNA SNHG1 could inhibit cell proliferation and cell invasion capacity and cell Epithelial-Mesenchymal Transition (EMT) phenomenon by up-regulation E-cadherin and down-regulating Vimentin and N-cadherin in ESCC cells. Besides, we demonstrated that knockdown of SNHG1 suppressed the Notch signaling pathway by reducing the Notch1 and Hes-1 expression levels in ESCC cells. These results indicated that lncRNA SNHG1 may be a potential predictor of prognosis in ESCC patients and a novel target for ESCC treatment.

NEAT1 negatively regulates miR-218 expression and promotes breast cancer progression.

Studies have shown thatlncRNA nuclear paraspeckle assembly transcript 1 (NEAT1) is related to breast cancer progression, however, the role of NEAT1 remains largely unknown. The aim of the current study was to further investigate the function of NEAT1 involved in breast cancer. QRT-PCR was used to analyze lncRNA NEAT1 expression in breast cancer tissues and determine the association between NEAT1 and clinicopathologic features in patients. Kaplan-Meier analysis and the log-rank test were used to establish the relationship between NEAT1 and overall survival. Cell proliferation and invasion were evaluated based on CCK8 cell proliferation, cell colony formation and Transwell cell invasion assays results. Bioinformatics analysis and the luciferase reporter assay were performed to demonstrate the association between NEAT1 and miR-218. NEAT1 expression was significantly up-regulated in breast cancer tissues compared to adjacent normal tissues, and higher NEAT1 was positively associated with lymph node metastasis and TNM stage. Patients with higher NEAT1 had a poor prognosis. Furthermore, miR-218 was shown to be a direct target of NEAT1 in breast cancer cells. In addition, NEAT1 promoted cell invasion and proliferation by negatively regulating miR-218 in breast cancer. NEAT1 is a potential biomarker for prognosis and the target of treatment in breast cancer patients.

Abstract 1385: New insights into the roles of histone lysine-specific demethylase 2 (LSD2) in breast cancer

Abstract Background: Epigenetic modifiers have been emerging as new players in breast cancer development. FAD-dependent histone demethylases have been proved to play important roles in regulating breast tumor initiation and development. While the role of histone lysine-specific demethylase 1 (LSD1) in breast cancer progression has been well characterized, the activity of its homolog, lysine-specific demethylase 2 (LSD2), in breast cancer is still a riddle that needs to be uncovered. Methods: To explore the precise role of LSD2 in breast cancer, we overexpressed LSD2 protein in MDA-MB-231 cells (LSD2-OE). The phenotypic effect of LSD2-OE on MM231 cells was characterized by proliferation assay, soft agar assay (3D growth), trans-well cell migration and invasion assay and mammosphere formation assay. To extend stem cell-like features study of LSD2 overexpressed MM231 cells, we examined CD44+/CD24- subpopulation by flow cytometry and the expression of several stem cell markers by qPCR and western blot. In addition, we also investigated the potential altered phenotypes of LSD2 knockdown in MM231 cells via RNAi. Results: Results from Oncomine database show that LSD2 expression level is greatly increased in invasive ductal carcinoma (IDC) vs. non-invasive breast cancers. The cellular proliferation assay revealed that LSD2-OE promoted MM231 cell growth. Similarly, LSD2 overexpressed MM231 cells were capable of forming large colonies in soft agar. Trans-well cell migration and invasion assay results indicated LSD2-OE significantly decreased both migration and invasion in MM231 cells. Mammosphere formation assay results showed LSD2 overexpression increased the number of primary and tertiary mammospheres in MM231 cells. Consistent with increase of mammosphere forming efficiency, NANOG and SOX2 were increased by LSD2 overexpression at the protein level. However, LSD2-OE significantly decreased CD44+/CD24- subpopulation. Moreover, siRNA mediated LSD2 depletion significantly decreased cell growth in multiple breast cancer cell lines without altering migration and invasion in MM231 cells. Finally, no obvious phenotypic changes were observed in MM231 cells expressing stable LSD2 shRNA. Conclusion: The multifaceted effects of LSD2 on breast cancer phenotypes may reflect the profound influence of LSD2 activity on chromatin structure and gene expression in the breast cancer genome. Our studies suggest that overexpression of LSD2 facilitates growth and confers stem cell-like traits to breast cancer cells, and transient LSD2 inhibition by siRNA hinders breast cancer cell growth. These results suggest that LSD2 might be a valuable therapeutic target in breast cancer. Citation Format: Lin Chen, Shauna N. Vasilatos, Ye Qin, Steffi Oesterreich, Nancy E. Davidson, Yi Huang. New insights into the roles of histone lysine-specific demethylase 2 (LSD2) in breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1385. doi:10.1158/1538-7445.AM2017-1385

Abstract 4848: The functional synergism and pro-metastatic role of FOXM1 and CENPF in hepatocellular carcinoma

Abstract Hepatocellular carcinoma (HCC) is the second leading cause of cancer death worldwide and metastasis is regarded as the major cause of HCC-associated lethality. In this study, we have analysed the TCGA whole-transcriptome sequencing data of paired human HCC samples (n=50), and identified Forkhead Box M1 (FOXM1) and Centromere Protein F (CENPF) to be the top-listing upregulated genes. Interestingly, both of them are the essential components in cell-cycle progression. FOXM1 encodes for the cell-cycle-dependent transcription factor that regulates genes for DNA replication and mitosis, while CENPF encodes for the centromere protein that is required for kinetochore function and chromosome segregation in mitosis. We hypothesized that the upregulation of FOXM1-CENPF signaling axis may drive hepatocarcinogenesis. In our human HCC cohort (n=34), FOXM1 and CENPF were shown to be upregulated compared with the non-tumorous liver tissues, and their mRNA expressions were positively correlated (p&amp;lt;0.0001). Co-upregulation of FOXM1 and CENPF in patient samples was demonstrated to be positively correlated with the absence of tumor encapsulation (p=0.035) in our clinico-pathological correlation analysis, whereas the samples without their co-upregulation was less likely to be correlated with venous invasion (p =0.045). This indicated that FOXM1 and CENPF are likely to be associated with HCC metastasis synergistically. Stable single and co-knockdown clones of FOXM1 and CENPF in four HCC cell lines (BEL7402, SMMC-7721, MHCC-97L, Huh7) were established using short-hairpin (sh) RNA approach for subsequent functional characterization. Both single and co-knockdown of FOXM1 and CENPF cells have shown reduced proliferation rate (p&amp;lt;0.05) in cell proliferation assay. Moreover, all single and co-knockdown of FOXM1 and CENPF cell lines were found to have a at least 2-fold reduction in migration and invasion rate (p&amp;lt;0.0001) in transwell cell migration and invasion assay. These suggested that FOXM1 and CENPF are critical for HCC cell proliferation, migration and invasion. To assess the translational significance of targeting FOXM1 and CENPF, seven HCC cell lines (BEL7402, SMMC-7721, MHCC-97L, Huh7, Hep3B, HepG2, PLC/PRF/5) were challenged with thiostrepton and zoledronic acid, which are inhibitors against FOXM1 and CENPF, respectively. Intriguingly, both individual and combined treatments of these inhibitors effectively inhibited HCC cell growth. It was also demonstrated that such inhibition acts via suppressing the respective endogenous transcript and protein expression of FOXM1 and CENPF. Taken together, our study has demonstrated that FOXM1 and CENPF play a critical oncogenic role in HCC and they may function as an attractive molecular therapeutic target. This study is supported in part by Health and Medical Research Fund (03142836). Citation Format: Wai Ling Macrina Lam, Lo Kong Chan, Daniel Wai-Hung Ho, Charles Shing Kam, Irene Oi-Lin Ng. The functional synergism and pro-metastatic role of FOXM1 and CENPF in hepatocellular carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4848. doi:10.1158/1538-7445.AM2017-4848

Effect of microRNA-106b on the invasion and proliferation of trophoblasts through targeting MMP-2

Objective: To investigate the expression of microRNA-106b (miR-106b) in the placentas of patients with pre-eclampsia and its relationship with matrix metallopeptidase (MMP) -2, and its effect on the invasion and proliferation of trophoblasts. Methods: (1) Placental tissues were collected from patients with mild pre-eclampsia (mPE, n=30), severe pre-eclampsia (sPE, n=30) and normal pregnant women (n=40). Human choriocarcinoma cell lines JAR and JEG3 were assigned to the miR-106b mimics group, the mimics negative control group, the miR-106b inhibitor group and the inhibitor negative control group, respectively. (2) The target gene of miR-106b(such as MMP-2) was predicted by bioinformatics. Dual-luciferase reporting system was used to verify the regulation of miR-106b on the expression of MMP-2. (3) The expressions of miR-106b and MMP-2 were measured by quantitative real-time PCR (qRT-PCR) and western blot. (4) Cell proliferation was determined by MTT assay. (5) Invasive activities in each group were assessed by cell transwell invasion assays. Results: (1) Predicting result of bioinformatics indicated that MMP-2 was one of the target genes of miR-106b. Dual-luciferase activity assay demonstrated that MMP-2 was the direct target of miR-106b (P<0.01) .(2) The results of qRT-PCR.①The expression of miR-106b in the placentas of mPE, sPE, normal pregnant women were 2.89±0.04, 1.96±0.03, 1.01±0.03, respectively (P<0.05). And the expression of MMP-2 mRNA in the placentas of mPE, sPE, normal pregnant women were 1.87±0.05, 0.69±0.03, 2.78±0.03, respectively (P<0.05). ②The expression of miR-106b in the JAR cell line in the miR-106b mimics group, the mimics negative control group, the miR-106b inhibitor group and the inhibitor negative control group were 2.39±0.03, 1.03±0.04, 0.73±0.03, 1.11±0.04, respectively (P<0.05). And its expression in the JEG3 cell line were 2.17±0.04, 1.18±0.04, 0.61±0.03 and 1.22±0.03, respectively (P<0.05). ③The expression of MMP-2 mRNA in the JAR cell line in the miR-106b mimics group, the mimics negative control group, the miR-106b inhibitor group and the inhibitor negative control group were 0.45±0.15, 1.02±0.03, 2.28±0.03, 1.11±0.03, respectively (P<0.05). And its expression in the JEG3 cell line were 0.58±0.03, 1.25±0.15, 2.25±0.03, 1.21±0.03, respectively (P<0.05). (3) The results of western blot. ①The expression of MMP-2 protein in the placentas of mPE, sPE, normal pregnant women were 1.63±0.04, 0.55±0.03, 2.82±0.03, respectively (P<0.05). ②The expression of MMP-2 protein in the JAR cell line in the miR-106b mimics group, the mimics negative control group, the miR-106b inhibitor group and the inhibitor negative control group were 0.41±0.03, 0.97±0.03, 2.25±0.03, 1.01±0.03, respectively (P<0.05). And its expression in the JEG3 cell line were 0.53±0.03, 1.20±0.03, 2.31±0.04, 1.19±0.03, respectively (P<0.05). (4) miR-106b could inhibit the proliferation of JAR and JEG3 cells, cell proliferation rates in the miR-106b mimics group were lower than that in the mimics negative control group (P<0.05). And cell proliferation rate in the miR-106b inhibitor group was higher than the inhibitor negative control group (P<0.05). (5) The numbers of JAR cell that passed the membrane in the miR-106b mimics group, the mimics negative control group. The miR-106b inhibitor group and the inhibitor negative control group were 61±15, 79±13, 134±13, 80±12, respectively(P<0.05). And the numbers of JEG3 cell that passed were 57±12, 71±15, 128±15, 70±14, respectively (P<0.05). Conclusion: The miR-106b could inhibit the invasion and proliferation of JAR and JEG3 cells through targeting MMP-2, and have a relationship with the pathogenesis of pre-eclampsia.

Moesin Expression Is Associated with Glioblastoma Cell Proliferation and Invasion.

To investigate the effect of moesin expression on cell proliferaton and invasion of human glioblastoma cell lines in vitro. Glioblastoma LN229 and U87 cells were transfected with the H4645-plenti-EGFP-moesin expression vector for moesin up-regulation. Moesin and β-catenin expression levels in the transfected cells were analyzed by real-time polymerase chain reaction (PCR) and Western blotting. Cell proliferation was measured using the CCK8 assay. Cell invasion and migration ability were assessed using a transwell cell invasion and wound-healing assay. Moesin mRNA and protein expression were significantly increased in the two transfected LN229-H4645 and U87-H4645 cell lines. β-catenin expression was increased by moesin up-regulation in the transfected LN229-H4645 and U87-H4645 cell lines. The CCK-8 assay revealed that up-regulating moesin results in a significant increase in glioblastoma cell proliferation. Glioblastoma cell invasion and migration were increased by moesin up-regulation. Up-regulation of moesin expression in glioblastoma cells correlated with increases in cell proliferation, invasion and migration, suggesting moesin's role in glioblastoma progression.

Effect of PKC inhibition on cell migration and invasion of oral squamous cell carcinoma

By using HN4 cells, the correlation among protein kinase C(PKC), P120-catenin(P120ctn) and E-cadherin (E-cad) was investigated, and the role of them in migration and invasion of oral squamous-cell cancer(OSCC) was evaluated. Plasmid pGFP-V-RS-P120ctn shRNA was used to transfect HN4 cells to significantly reduce the expression of P120ctn and PKC inhibitor staurosporine (STS) was added. mRNA and protein expression of PKC, P120ctn and E-cad were tested and Transwell cell invasion and cell migration assay was used to test the invasion and migration capacity before and after PKC inhibition. Statistical analysis was performed using SPSS 18.0 software package. When PKC was inhibited by STS, the expression of P120ctn and E-cad in OSCC cells were increased and the migration and invasion capacity of tumor cells decreased significantly (P<0.05). CONCLUSIONS：PKC may be involved in the process of HN4 migration and invasion by regulating cell adhesion through the effect of P120ctn and E-cad.

Electrophilic Nitro‐oleic Acid Inhibits Triple Negative Breast Cancer Cell Migration via Suppression of NF‐kB activity

Triple negative breast cancer (TNBC) is an aggressive and therapy‐resistant metastatic cancer. Patients with metastatic TNBC are difficult to treat due to the absence of effective target therapies, thus leading to poor clinical outcome. Constitutive activation of the nuclear factor kB (NF‐kB) pathway is a main driver to promote TNBC tumor aggressive phenotypes including cell survival, migration, and invasion. This study investigates the anticancer effects of electrophilic anti‐inflammatory mediators nitroalkene fatty acids in TNBC in vitro and in vivo. Decreased inflammatory metastatic potential of TNBC cells by an exemplary electrophilic nitrolipid, nitro‐oleic acid (NO2‐OA), was shown through inhibition of cell motility using transwell cell migration and invasion assays. NO2‐OA treatment inhibited NF‐kB transcriptional activity and expression of two TNFa‐induced NF‐kB‐regulated genes involved in tumor metastasis, intercellular adhesion molecule‐1 (ICAM‐1) and urokinase‐type plasminogen activator (uPA) in TNBC cells. The mechanism of NF‐kB signaling inhibition by NO2‐OA was due to suppression of IKKb phosphorylation and IkBa degradation. Notably, NO2‐OA also caused polyubiquitination and degradation of NF‐kB RelA protein via protein nitroalkylation in TNBC cells. In an MDA‐MB‐231 xenograft model, NO2‐OA inhibited tumor burden resulted in reduced tumor growth and incidence of metastasis in mice. Taken together, electrophilic NO2‐OA shows potential as a therapeutic agent that may display high selectivity for inhibition of TNBC progression and metastasis.

Cadherin switching induced by P120-catenin can promote the migration and invasion of oral squamous cell cancer cells

The main goal is to investigate the role of P120-catenin (P120ctn) in cadherin switching, as well as migration and invasion, of oral squamous cell cancer (OSCC) cells. The plasmid pGFP-V-RS-P120ctn shRNA was used to transfect TSCCA cells and significantly reduce the expression of P120ctn in these cells. Real-time fluorescent quantitative polymerase chain reaction and Western blot were conducted to determine the mRNA and protein expression levels of P120ctn, E-cadherin (E-cad), and N-cadherin (N-cad). By contrast, the Transwell cell invasion and cell migration assay was used to determine the invasion and migration capacities before and after the transfection. After the plasmid pGFP-V-RS-P120ctn shRNA was transfected into the TSCCA cells, we found that as the P120ctn expression significantly decreased, E-cad mRNA and protein expression decreased significantly. Moreover, N-cad mRNA and protein expression increased significantly (P<0.05). Lastly, the cell migration and invasion capacities were augmented significantly (P<0.05). In OSCC cells, P120ctn may be involved in cadherin switching and promote metastasis and invasion.

MicroRNA-1258: An invasion and metastasis regulator that targets heparanase in gastric cancer.

Numerous microRNAs (miRNAs/miRs) are involved in suppressing or promoting the formation of cancer. However, to the best of our knowledge, the role of miRNA-1258 in gastric cancer (GC) has not previously been investigated. Our previous study demonstrated an increased expression of heparanase (HPSE) in GC tissues and HPSE-facilitated invasion and metastasis of GC cells. Consequently, in the present study, the function of miR-1258 in the invasion and metastasis of GC cells was investigated to determine whether miR-1258 is associated with GC through HPSE. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) was used to determine miR-1258 expression in GC cell lines and tissues. A Transwell cell invasion assay and an MTT proliferation assay were used to investigate the effects of miR-1258 on the invasion and proliferation of one of the cell lines, SGC-7901 cells, in vitro. The effect of miR-1258 on SGC-7901 cell metastasis was investigated using an in vivo tumor metastasis formation assay. Western blot analysis and RT-qPCR were used to investigate whether HPSE was a target of miR-1258 in GC. The expression of miR-1258 was significantly downregulated in 3 GC cell lines and 116 GC tissues compared with controls (all P<0.001). An association was identified between decreased miR-1258 expression level and increased age (P=0.042), advanced pathological tumor stage (P=0.027) and positive lymphatic vessel invasion (P=0.044) in patients with GC. Furthermore, the upregulation of miR-1258 expression suppressed SGC-7901 cell invasion in vitro (P<0.001) and inhibited SGC-7901 cell metastasis in vivo (P=0.016). The western blot analysis and RT-qPCR results indicated that miR-1258 downregulated the expression of HPSE at the translational level. The results of the present study indicate that miR-1258 acts as a tumor suppressor to inhibit invasion and metastasis by targeting HPSE. Therefore, miR-1258 may serve as a novel biomarker and therapeutic target in the treatment of GC.

RETRACTED ARTICLE: miR-484 suppresses proliferation and epithelial–mesenchymal transition by targeting ZEB1 and SMAD2 in cervical cancer cells

BackgroundMicroRNAs (miRNAs) play important roles in cancer initiation and development. Epithelial–mesenchymal transition (EMT) is a form of cellular plasticity that is critical for embryonic development and metastasis. The purpose of the study was to determine the function and mechanism of miR-484 in initiation and development of cervical cancer (CC).MethodsWe determined the expression levels of miR-484 in cervical cancer tissues and cell lines with RT-qPCR. Prediction algorithms and EGFP reporter assay were performed to evaluate the targets for miR-484. MTT assay, colony formation assay, flow cytometric analysis, transwell cell migration and invasion assays, and detection of EMT markers were employed to investigate the roles of miR-484 and the targets in regulation of cell proliferation and EMT process. We also used rescue experiments to confirm the effect of miR-484 on CC cells through directly regulating the expression of its targets.ResultsFirstly we found miR-484 was down-regulated in cervical cancer tissues and cell lines compared with their matched non-cancerous tissues or normal cervical keratinocytes cells. Further studies revealed that overexpression of miR-484 suppressed the cell proliferation, while exacerbates apoptosis. Besides, miR-484 suppressed cellular migration, invasion and EMT process of CC cells. EGFP reporter assay showed that miR-484 binds to ZEB1 and SMAD2 3′UTR region and reduced their expression. The expression of miR-484 had reverse correlation with SMAD2/ZEB1, and SMAD2/ZEB1 had positive correlation with each other in cervical cancer tissues and cell lines. Furthermore, the ectopic expression of ZEB1 or SMAD2 could rescue the malignancies suppressed by miR-484, suggesting that miR-484 down-regulates ZEB1 and SMAD2 to repress tumorigenic activities.ConclusionWe found miR-484 inhibits cell proliferation and the EMT process by targeting both ZEB1 and SMAD2 genes and functions as a tumor suppressor, which may served as potential biomarkers for cervical cancer.

α2,3-sialyltransferase type I regulates migration and peritoneal dissemination of ovarian cancer cells.

Epithelial ovarian cancer (EOC) has the highest mortality rate among gynecologic cancers due to advanced stage presentation, peritoneal dissemination, and refractory ascites at diagnosis. We investigated the role of α2,3-sialyltransferase type I (ST3GalI) by analyzing human ovarian cancer datasets and human EOC tissue arrays. We found that high expression of ST3GalI was associated with advanced stage EOC. Transwell migration and cell invasion assays showed that high ST3GalI expression enhanced migration of EOC cells. We also observed that there was a linear relation between ST3GalI expression and epidermal growth factor receptor (EGFR) signaling in EOC patients, and that high ST3GalI expression blocked the effect of EGFR inhibitors. Co-Immunoprecipitation experiments demonstrated that ST3GalI and EGFR were present in the same protein complex. Inhibition of ST3GalI using a competitive inhibitor, Soyasaponin I (SsaI), inhibited tumor cell migration and dissemination in the in vivo mouse model with transplanted MOSEC cells. Further, SsaI synergistically enhanced the anti-tumor effects of EGFR inhibitor on EOC cells. Our study demonstrates that ST3GalI regulates ovarian cancer cell migration and peritoneal dissemination via EGFR signaling. This suggests α2,3-linked sialylation inhibitors in combination with EGFR inhibitors could be effective agents for the treatment of EOC.

Morin Inhibits Proliferation, Migration, and Invasion of Bladder Cancer EJ Cells via Modulation of Signaling Pathways, Cell Cycle Regulators, and Transcription Factor-Mediated MMP-9 Expression.

Preclinical Research Previous studies have shown that morin exerts diverse pharmacological activities. In this study, we investigated the inhibitory activity of morin on bladder cancer EJ cells. Morin significantly inhibited EJ cell proliferation, which was related to the G1-phase cell cycle arrest together with the reduced expression of cyclin D1, cyclin E, CDK2, and CDK4 via increased expression of p21WAF1. Morin also increased ERK1/2 phosphorylation and decreased JNK and AKT phosphorylation without altering the p38MAPK phosphorylation levels. Morin treatment suppressed the migration and invasion of EJ cells in wound-healing and transwell cell invasion assays. Zymographic and electrophoretic mobility shift assays showed that morin suppressed the expression of matrix metalloproteinase-9 (MMP-9) via repression of the binding activity of AP-1, Sp-1, and NF-κB. Collectively, these results demonstrate that morin reduced cyclin D1, cyclin E, CDK2 and CDK4 expression via the induction of p21WAF1 expression, increased ERK1/2 phosphorylation and decreased JNK, and AKT phosphorylation, and prevented MMP-9 expression via the inhibition of transcription factors AP-1, Sp-1, and NF-κB, thereby resulting in the inhibition of growth, migration, and invasion of bladder cancer EJ cells. These results provide a novel insight into the use of morin in the prevention of bladder cancer. Drug Dev Res 78 : 81-90, 2017. © 2017 Wiley Periodicals, Inc.

Silencing of Apurinic/Apyrimidinic Endonuclease 1 Inhibits the Growth and Migration in Ovarian Cancer Cell via Activator-Protein-1 Signaling

We hypothesized that apurinic/apyrimidinic endonuclease 1 (APE1) may regulate cell activity via activator protein 1 (AP-1) signaling pathway through its redox function in the development of ovarian cancer. Ovarian cancer and paired paracarcinoma tissues were collected for determining APE1 expression with immunohistochemistry. Cell transfection was performed to silence APE1. For mRNA and protein expressions of genes after silencing, real-time reverse transcription polymerase chain reaction and Western blot assay were conducted. Transwell cell proliferation experiment and clone formation assay were conducted for cell proliferation; Transwell cell migration and cell invasion assays were conducted for cell migration and invasion. Most cancer tissues had APE1 cytoplasmic expression and a few had nuclear/cytoplasmic expression while few had only nuclear expression. A significantly higher APE1 expression was identified in cancer tissues than in paracarcinoma tissues and it was correlated with the clinicopathologic features. SKOV3 was used for cell experiments. After silencing of APE1, both mRNA and protein expressions of APE1 and AP-1 signaling-related genes were downregulated, and the cell activity in proliferation, migration and invasion was remarkably suppressed. Overexpressed APE1 promotes ovarian cancer growth and metastasis. Downregulated APE1 could suppress cell activity via AP-1 pathway, suggesting that APE1 gene may be a potential therapeutic target for ovarian cancer.

BCAT1 promotes tumor cell migration and invasion in hepatocellular carcinoma.

Branched-chain amino acid transaminase 1 (BCAT1) has been associated with numerous types of tumors; however, few previous studies have evaluated the expression and role of BCAT1 in hepatocellular carcinoma (HCC). In the present study, the expression of BCAT1 was detected by reverse transcription-quantitative polymerase chain reaction and immunoblotting in six HCC cell lines and 74 pairs of HCC and adjacent non-cancerous liver tissues. In addition, the correlation between the expression levels of c-Myc and BCAT1 was analyzed using immunohistochemistry. Furthermore, RNA silencing was performed using c-Myc-specific or BCAT1-specific small interfering RNA, after which wound healing and Transwell cell invasion assays were performed. Finally, the clinicopathological characteristics of BCAT1 in patients with HCC were analyzed. It was shown that the expression of BCAT1 was significantly higher in HCC tissues compared with adjacent non-tumor tissues (P<0.001), and in HCC cell lines compared within the L-02 hepatic cell line (P<0.001). In addition, immunohistochemical analyses indicated that the expression of BCAT1 was positively correlated with c-Myc (r=0.706, P<0.001). BCAT1 expression was shown to be downregulated in c-Myc-knockdown cells, and silencing of BCAT1 expression reduced the invasion and migration of HCC cells. Furthermore, a clinical analysis indicated that BCAT1 expression in HCC tissues was significantly associated with the tumor-node-metastasis stage, tumor number and tumor differentiation (all P<0.05), and that BCAT1 was able to predict the 5-year survival and disease-free survival rates of patients with HCC (both P<0.001). The results of the present study suggested that BCAT1 expression is upregulated in patients with HCC, and that BCAT1 may serve as a potential molecular target for the diagnosis and treatment of HCC.

Effect of shRNA-Mediated Gene Silencing of Bmi-1 Expression on Chemosensitivity of CD44+ Nasopharyngeal Carcinoma Cancer Stem-Like Cells.

In this study, we investigate the effect of short hairpin RNA-mediated gene silencing of Bmi-1 expression on chemosensitivity of CD44(+) nasopharyngeal carcinoma cancer stem-like cells. The sequence-specific short hairpin RNA lentivirus targeting at human Bmi-1 was synthesized and used to infect CD44(+) nasopharyngeal cells that were sorted by flow cytometry. We also employed flow cytometry to detect transfection efficiency. Real-time polymerase chain reaction was used to detect Bmi-1 and its downstream repressor genes p16(INK4a) and p14(ARF) messenger RNA, while each protein expression level of Bmi-1, p16(INK4a), p14(ARF), and p53 was confirmed by Western blotting protocol. Tumor spheroid assay was used to evaluate the self-renewal capacity. 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide assay and colony formation assay were applied to detect proliferation capacity and colony-forming capacity under different concentrations of chemotherapeutic drugs 5-fluorouracil or cisplatin. Transwell cell migration and invasion assay were employed to observe migration and invasion capacity after cells were exposed to cisplatin for 24 hours. The constructed short hairpin RNA lentivirus targeting Bmi-1 gene successfully infected into the CD44(+) nasopharyngeal carcinoma cells and effectively inhibited the Bmi-1 messenger RNA and protein expression level, while the expression level of Bim-1 target genes, p16(INK4a), p14(ARF), and p53 was significantly increased (P < .05). Notably, the proliferation, colony formation, migration, and invasion capabilities of the sequence-specific short hairpin RNA lentivirus-infected CD44(+) nasopharyngeal carcinoma cells reduced significantly under chemotherapeutic treatments (P < .05). Our results indicated that Bmi-1 may play an important role in the chemosensitivity of CD44(+) nasopharyngeal carcinoma cancer stem-like cells. Bmi-1 may be a potential new target for the treatment of nasopharyngeal carcinoma displaying chemotherapy resistance.

Oncogenic role of mortalin contributes to ovarian tumorigenesis by activating the MAPK-ERK pathway.

Mortalin is frequently overexpressed in human malignancies. Previous studies have suggested that mortalin contributes to ovarian cancer development and progression, but further investigation is warranted. The aim of this study is to elucidate the mechanism of mortalin in ovarian cancer development and progression. In this study, lentivirus‐delivered mortalin short hairpin RNA (shRNA) was used to knockdown mortalin expression in A2780 and A2780/cis ovarian cancer cell lines, and lentiviral mortalin‐pLVX‐AcGFP was used to generate mortalin‐overexpressing cell lines. The results demonstrated that decreased mortalin expression reduced ovarian cancer cell proliferation, colony formation, migration and invasion by Cell Counting Kit‐8 assay, colony formation assay, wounding healing assay and Transwell cell invasion assay, respectively. Flow cytometry results suggested that mortalin promotes the G1 transition, leading to faster restoration of a normal cell‐cycle distribution. Cell‐cycle proteins, including C‐myc and Cyclin‐D1, significantly increased, and Cyclin‐B1 remarkably decreased upon mortalin down‐regulation. Western blot analysis showed that mortalin knockdown significantly decreased p‐c‐Raf and phospho‐extracellular–regulated protein kinases (p‐ERK1/2) pathways but not the Jun N‐terminal kinase pathway, whereas mortalin overexpression had the opposite effect. Taken together, these results indicate that mortalin is an oncogenic factor, and mitogen‐activated protein kinase‐ERK signalling pathway activation by mortalin may contribute to ovarian cancer development and progression.