Jinfeng Bai

Knockdown of c-Myc expression by RNAi inhibits MCF-7 breast tumor cells growth in vitro and in vivo

IntroductionBreast cancer is the leading cause of cancer death in women worldwide. Elevated expression of c-Myc is a frequent genetic abnormality seen in this malignancy. For a better understanding of its role in maintaining the malignant phenotype, we used RNA interference (RNAi) directed against c-Myc in our study. RNAi provides a new, reliable method to investigate gene function and has the potential for gene therapy. The aim of the study was to examine the anti-tumor effects elicited by a decrease in the protein level of c-Myc by RNAi and its possible mechanism of effects in MCF-7 cells.MethodA plasmid-based polymerase III promoter system was used to deliver and express short interfering RNA (siRNA) targeting c-myc to reduce its expression in MCF-7 cells. Western blot analysis was used to measure the protein level of c-Myc. We assessed the effects of c-Myc silencing on tumor growth by a growth curve, by soft agar assay and by nude mice experiments in vivo. Standard fluorescence-activated cell sorter analysis and TdT-mediated dUTP nick end labelling assay were used to determine apoptosis of the cells.ResultsOur data showed that plasmids expressing siRNA against c-myc markedly and durably reduced its expression in MCF-7 cells by up to 80%, decreased the growth rate of MCF-7 cells, inhibited colony formation in soft agar and significantly reduced tumor growth in nude mice. We also found that depletion of c-Myc in this manner promoted apoptosis of MCF-7 cells upon serum withdrawal.Conclusionc-Myc has a pivotal function in the development of breast cancer. Our data show that decreasing the c-Myc protein level in MCF-7 cells by RNAi could significantly inhibit tumor growth both in vitro and in vivo, and imply the therapeutic potential of RNAi on the treatment of breast cancer by targeting overexpression oncogenes such as c-myc, and c-myc might be a potential therapeutic target for human breast cancer.

Overexpression of human pituitary tumor transforming gene (hPTTG), is regulated by β‐catenin /TCF pathway in human esophageal squamous cell carcinoma

Overexpression of human pituitary tumor transforming gene (PTTG) is wildly detected in many tumors, including esophageal cancer. Besides overexpression of PTTG in esophageal squamous cell carcinoma (ESCC) tissues and cells, we detected accumulation of cytoplasmic β‐catenin in ESCC. In our study, a putative TCF4‐binding element (TBE) was identified in PTTG promoter region. The activity of PTTG promoter containing the TBE was activated by S37Aβ‐catenin and inhibited by dominant‐negative TCF. Furthermore, the activation by S37Aβ‐catenin was mostly abrogated among PTTG promoter region without the TBE or with a mutant one. By using biotin‐streptavidin pull‐down assay, we also found that the TBE among PTTG promoter bound to TCF‐4 protein. Moreover, levels of PTTG mRNA and protein were increased by S37Aβ‐catenin. Finally, it is noticeable that we detected a correlation between β‐catenin localization and PTTG expression in 69 primary ESCC (p<0.01). In brief, our study shows that overexpression of PTTG in ESCC is likely due to the activation of β‐catenin/WNT signaling. As a target gene of β‐catenin/TCF pathway, PTTG may play an important role in tumorigenesis of human ESCC.

Overexpression of EB1 in human esophageal squamous cell carcinoma (ESCC) may promote cellular growth by activating β -catenin/TCF pathway

Esophageal squamous cell carcinoma (ESCC) has a multifactorial etiology involving environmental and/or genetic factors. End-binding protein 1 (EB1), which was cloned as an interacting partner of the adenomatous polyposis coli (APC) tumor suppressor protein, was previously found overexpressed in ESCC. However, the precise role of EB1 in the development of this malignancy has not yet been elucidated. In this study, we analysed freshly resected ESCC specimens and demonstrated that EB1 was overexpressed in approximately 63% of tumor samples compared to matched normal tissue. We report that overexpression of EB1 in the ESCC line EC9706 significantly promotes cell growth, whereas suppression of EB1 protein level by RNA interference significantly inhibited growth of esophageal tumor cells. In addition, EB1 overexpression induced nuclear accumulation of β-catenin and promoted the transcriptional activity of β-catenin/T-cell factor (TCF). These effects were partially or completely abolished by coexpression of APC or ΔN TCF4, respectively. Also, we found that EB1 affected the interaction between β-catenin and APC. Furthermore, EB1 overexpression was correlated with cytoplasmic/nuclear accumulation of β-catenin in primary human ESCC. Taken together, these results support the novel hypothesis that EB1 overexpression may play a role in the development of ESCC by affecting APC function and activating the β-catenin/TCF pathway.

miR‐106a is frequently upregulated in gastric cancer and inhibits the extrinsic apoptotic pathway by targeting FAS

Emerging evidence has shown the association of aberrantly expressed miR‐106a with cancer development, however, little is known about its potential role in gastric carcinogenesis. In our present study, obviously overexpressed miR‐106a was found in gastric cancer tissues compared with their nontumor counterparts. Suppression of miR‐106a significantly inhibited gastric cancer cell proliferation and triggered apoptosis. Bioinformatic analysis combining with validation experiments identified FAS as a direct target of miR‐106a. Rescue experiments and examination of caspase‐8, PARP and caspase‐3 further approved that miR‐106a could inhibit gastric cancer cell apoptosis through interfering with FAS‐mediated apoptotic pathway. Moreover, a significant inverse correlation was found between miR‐106a and FAS expression not only in gastric cancer cell lines but also in gastric cancer specimens. Taken together, these findings suggest that ectopicly overexpressed miR‐106a may play an oncogenic role in gastric carcinogenesis and impair extrinsic apoptotic pathway through targeting FAS.

Identification of genes regulated by Wnt/β-catenin pathway and involved in apoptosis via microarray analysis

BackgroundWnt/β-catenin pathway has critical roles in development and oncogenesis. Although significant progress has been made in understanding the downstream signaling cascade of this pathway, little is known regarding Wnt/β-catenin pathway modification of the cellular apoptosis.MethodsTo identify potential genes regulated by Wnt/β-catenin pathway and involved in apoptosis, we used a stably integrated, inducible RNA interference (RNAi) vector to specific inhibit the expression and the transcriptional activity of β-catenin in HeLa cells. Meanwhile, we designed an oligonucleotide microarray covering 1384 apoptosis-related genes. Using oligonucleotide microarrays, a series of differential expression of genes was identified and further confirmed by RT-PCR.ResultsStably integrated inducible RNAi vector could effectively suppress β-catenin expression and the transcriptional activity of β-catenin/TCF. Meanwhile, depletion of β-catenin in this manner made the cells more sensitive to apoptosis. 130 genes involved in some important cell-apoptotic pathways, such as PTEN-PI3K-AKT pathway, NF-κB pathway and p53 pathway, showed significant alteration in their expression level after the knockdown of β-catenin.ConclusionCoupling RNAi knockdown with microarray and RT-PCR analyses proves to be a versatile strategy for identifying genes regulated by Wnt/β-catenin pathway and for a better understanding the role of this pathway in apoptosis. Some of the identified β-catenin/TCF directed or indirected target genes may represent excellent targets to limit tumor growth.

Downregulation of Survivin by RNAi Inhibits the Growth of Esophageal Carcinoma Cells

Esophageal squamous cell carcinoma ranks among one of the most frequent cause of cancer death in the world. Understanding of the molecular mechanisms involved in the pathogenesis of esophageal cancer becomes critical to develop more effective treatments. Elevated expression of survivin in esophageal carcinoma has been reported before and suppression of survivin expression leads to many tumor cells growth inhibition. We hypothesized that down-regulation of survivin would inhibit the growth of human esophageal cancer cells. RNA interference directed against survivin was introduced into a human esophageal squamous cell carcinoma cell line KYSE510. Stable clones were selected and western blot analysis was performed to detect the protein level of survivin. Tumor cell growth in vitro and in vivo was assessed by trypan blue exclusion and nude mice experiments. Annexin ¢?/propidium iodide staining followed by flow cytometric analysis and TUNEL assay were used to detect apoptosis in cell culture and in nude mice. We found that RNA interference could efficiently and stably suppress survivin expression in KYSE510 cells. Down-regulation of survivin resulted in significantly inhibition of tumor growth in vitro and in vivo. The mechanism appears to be increased induction of apoptosis. Our results suggest a potential role for the targeting of survivin in the treatments of esophageal carcinoma.

EB1 acts as an oncogene via activating β-catenin/TCF pathway to promote cellular growth and inhibit apoptosis†

Previously we showed that end‐binding protein 1 (EB1) may promote cellular growth by activating β‐catenin/T‐cell factor (TCF) pathway. To further investigate the role of EB1 in regulating cellular growth, we established an EB1‐inducible expression system in which the protein level of EB1 was significantly upregulated upon doxycycline induction. We found that EB1 promoted cellular growth and resulted in a significant increase in colony formation. In addition, EB1 could induce tumor formation in nude mice, activate β‐catenin‐dependent gene expression and upregulate the transcriptional activity of c‐myc. We also showed that EB1 in this manner inhibited apoptosis of 293‐T‐REx cells upon cisplatin and upregulated expression of Bcl‐2, whereas ΔN TCF4, an inhibitor of β‐catenin/TCF pathway, could completely or partially abolish the effects of EB1 on the promotion of cell growth and the inhibition of apoptosis activity. Moreover, knockdown of c‐myc by RNAi could abrogate upregulation of EB1‐dependent induction of Bcl‐2 expression. Overall, EB1 acts as a potential oncogene via activating β‐catenin/TCF pathway to promote cellular growth and inhibit apoptosis.

FRAT1 overexpression leads to aberrant activation of β-catenin/TCF pathway in esophageal squamous cell carcinoma

Esophageal squamous cell carcinoma (ESCC) is an aggressive tumor with a poor prognosis. Although aberrant activation of β‐catenin/T‐cell factor (TCF) pathway has been observed in ESCC, mechanisms underlying this phenomenon remain unknown. Frequently rearranged in advanced T‐cell lymphomas‐1 (FRAT1), overexpressed in some ESCC lines, is a positive regulator of β‐catenin/TCF pathway. However, little is known about the molecular relationship between FRAT1 and β‐catenin/TCF in ESCC. In this study, we analyzed freshly resected ESCC specimens and demonstrated that FRAT1 was overexpressed in approximately 74% of tumor samples compared with matched normal tissue. Overexpression of FRAT1 significantly promoted esophageal cancer cells growth, whereas suppression of FRAT1 level by RNAi markedly inhibited their growth. In addition, FRAT1 overexpression induced the nuclear accumulation of β‐catenin and promoted the transcriptional activity of β‐catenin/TCF. These effects were reversed by coexpression of GSK 3β or ΔN TCF4. Furthermore, accumulation of β‐catenin was correlated with FRAT1 overexpression in ESCC and the basal layer of normal esophageal epithelium. Finally, continued expression of c‐Myc is necessary and sufficient for maintenance of the growth state in cells expressing FRAT1. Taken together, these results support the novel hypothesis that aberrant activation of β‐catenin/TCF pathway in esophageal cancer appears to be due to upstream events such as FRAT1 overexpression, and c‐Myc may be an important element in oncogenesis of human ESCC induced by FRAT1.

Aurora kinase A induces miR-17-92 cluster through regulation of E2F1 transcription factor

Aurora kinase A (AURKA) is an essential mitotic serine/threonine kinase and its abnormal expression is observed in many malignancies, yet the exact role for AURKA in tumorigenesis still remains elusive. Here, through a transcription factor array, we show that the transcription activity of E2F1 was increased by AURKA overexpression. Meanwhile, the E2F1 protein level was found to be upregulated and a correlation between AURKA and E2F1 expression was observed in cancer specimens. Further analysis revealed that AURKA increased E2F1 protein stability by inhibiting proteasome-dependent degradation of this protein. Additionally, a microRNA cluster, miR-17-92, was found to be upregulated upon AURKA overexpression, and this stimulation was largely repressed by E2F1 knockdown. Chromatin immunoprecipitation further demonstrated that AURKA enhanced E2F1 occupancy to the promoter of the miR-17-92 cluster. These data reveal a novel link between AURKA and microRNAs via the regulation of E2F1, providing new clues for understanding the role of AURKA in tumorigenesis.

Krüppel-like factor 4 represses transcription of the survivin gene in esophageal cancer cell lines

Abstract Aberrant expression of survivin has been shown to be regulated at the transcription level in cancer cells. In this study, we demonstrate that there are six putative binding sites of Krüppel-like factor 4 (KLF4) within the 2000-bp region upstream of the transcription start site of the human survivin gene. Luciferase reporter gene assays revealed that survivin promoter activity is repressed upon overexpression of KLF4 in EC9706 cells. A chromatin immunoprecipitation assay indicated that KLF4 indeed binds the survivin promoter in vivo. It specifically binds the site located at position -40 among the six binding sites as determined by electrophoretic mobility shift assay. Ectopic expression of KLF4 decreases the mRNA and protein levels of survivin. Furthermore, overexpression of survivin partially reverses KLF4-induced cell apoptosis. These results indicate that KLF4 is a transcriptional repressor of the human survivin gene in esophageal squamous cancer cells.