Chang Zhou

A miR-200b/200c/429-Binding Site Polymorphism in the 3′ Untranslated Region of the AP-2α Gene Is Associated with Cisplatin Resistance

The transcription factor AP-2α functions as a tumor suppressor by regulating various genes that are involved in cell proliferation and apoptosis. Chemotherapeutic drugs including cisplatin induce post-transcriptionally endogenous AP-2α, which contributes to chemosensitivity by enhancing therapy-induced apoptosis. microRNAs (miRNAs) miR-200b, miR-200c and miR-429 (miR-200b/200c/429) are up-regulated in endometrial and esophageal cancers, and their overexpression correlates with resistance to cisplatin treatment. Using computational programs, we predicted that the 3′ untranslated region (UTR) of AP-2α gene contains a potential miRNA response element (MRE) for the miR-200b/200c/429 family, and the single nucleotide polymorphism (SNP) site rs1045385 (A or C allele) resided within the predicted MRE. Luciferase assays and Western blot analysis demonstrated that the miR-200b/200c/429 family recognized the MRE in the 3′ UTR of AP-2α gene and negatively regulated the expression of endogenous AP-2α proteins. SNP rs1045385 A>C variation enhanced AP-2α expression by disrupting the binding of the miR-200b/200c/429 family to the 3′ UTR of AP-2α. The effects of the two polymorphic variants on cisplatin sensitivity were determined by clonogenic assay. The overexpression of AP-2α with mutant 3′ UTR (C allele) in the endometrial cancer cell line HEC-1A, which has high levels of endogenous miR-200b/200c/429 and low levels of AP-2α protein, significantly increased cisplatin sensitivity, but overexpression of A allele of AP-2α has no significant effects, compared with mock transfection. We concluded that miR-200b/200c/429 induced cisplatin resistance by repressing AP-2α expression in endometrial cancer cells. The SNP (rs1045385) A>C variation decreased the binding of miR-200b/200c/429 to the 3′ UTR of AP-2α, which upregulated AP-2α protein expression and increased cisplatin sensitivity. Our results suggest that SNP (rs1045385) may be a potential prognostic marker for cisplatin treatment.

microRNA-372 maintains oncogene characteristics by targeting TNFAIP1 and affects NFκB signaling in human gastric carcinoma cells

Aberrant microRNA (miRNA) expression has been investigated in gastric cancer, which is one of the most common malignancies. However, the roles of miRNAs in gastric cancer remain largely unknown. In the present study, we found that microRNA-372 (miR-372) directly targets tumor necrosis factor, α-induced protein 1 (TNFAIP1), and is involved in the regulation of the NFκB signaling pathway. Furthermore, overexpression of TNFAIP1 induced changes in AGS cells similar to those in AGS cells treated with miR-372-ASO. Collectively, these findings demonstrate an oncogenic role for miR-372 in controlling cell growth and apoptosis through downregulation of TNFAIP1. This novel molecular basis provides new insights into the etiology of gastric cancer.

Identification of a novel human cancer/testis gene MAEL that is regulated by DNA methylation

The mouse maelstrom (MAEL) gene has been found to be expressed in male germ cells and to play a role in spermatogenesis. Here, we cloned the human MAEL gene by digital differential display and found that, among human tissues, MAEL is only expressed in the testis, but it is also expressed in various cancer cell lines. The transcription start site of the MAEL gene is 74-bp upstream of the start codon. The region from −216 to +150 is the basal promoter of the MAEL gene, and a CpG island (−295 to +148) is located in this region. Treatment with the demethylating agent 5′-Aza-2-Deoxycytidine significantly upregulated MAEL expression. These results suggest that MAEL is a novel cancer/testis-associated gene and is regulated by DNA methylation.

MicroRNA-373 is upregulated and targets TNFAIP1 in human gastric cancer, contributing to tumorigenesis.

The role of microRNAs (miRNAs) in regulating gene expression is currently an area of intense interest. Previous studies have shown that miRNA-372 plays crucial roles in gastric tumorigenesis by targeting the mRNA of tumor necrosis factor, α-induced protein 1 (TNFAIP1). The present study showed that miR-373 is upregulated in gastric adenocarcinoma tissue and gastric carcinoma cell lines when compared to normal gastric tissues. The overexpression of miR-373 in the gastric cancer cells increased cell proliferation. A bioinformatics search revealed a conserved target site within the 3′ untranslated region (UTR) of TNFAIP1, an immediate-early response gene of the endothelium induced by TNF-α. The overexpression of miR-373 caused the suppression of a luciferase reporter containing the TNFAIP1 3′UTR in the HEK293 cells and reduced the levels of TNFAIP1 protein in the AGS cells. The mRNA levels of TNFAIP1 in the gastric cancer and normal gastric tissues were negatively correlated with the expression levels of miR-373 in these tissues. Moreover, the knockdown of TNFAIP1 had a similar effect to the overexpression of miR-373. The overexpression of TNFAIP1 may partly rescue the inhibition of proliferation caused by the inhibitor, miR-373-ASO. Taken together, these findings demonstrate an oncogenic role for miR-373, similar to that of miR-372, in controlling cell growth through the downregulation of TNFAIP1.

Functional characterization of the promoter region of human TNFAIP1 gene

Tumor necrosis factor, alpha-induced protein 1 (TNFAIP1) is an immediate-early response gene of endothelium induced by TNF alpha. However, little is really known concerning the TNFAIP1 expression regulation. To better understand how TNFAIP1 expression is regulated, we functionally characterized the promoter region of human TNFAIP1 gene. Deletion mutation analysis, gel electrophoretic mobility shift, and site-directed mutagenesis assays allowed the identification of one functional Sp1-binding site within the human TNFAIP1 core promoter region. Moreover, chromatin immunoprecipitation analysis indicated that Sp1 was associated in vivo with the TNFAIP1 promoter. Further, Sp1 overexpression enhanced TNFAIP1 promoter activity. These findings suggest that Sp1 is implicated in the control of basal TNFAIP1 gene expression. Accordingly, Sp1 is supposed to be involved in the elevation of TNFAIP1 in response to TNF alpha induction, and thus participate in inflammation-associated angiogenesis.

Identification and characterization of the novel protein CCDC106 that interacts with p53 and promotes its degradation

MINT‐7681212: p53 (uniprotkb:P04637) and CCDC106 (uniprotkb:Q9BWC9) colocalise (MI:0403) by fluorescence microscopy (MI:0416)

Human intersectin 2 (ITSN2) binds to Eps8 protein and enhances its degradation.

Participates in actin remodeling through Rac and receptor endocytosis via Rab5. Here, we used yeast two-hybrid system with Eps8 as bait to screen a human brain cDNA library. ITSN2 was identified as the novel binding factor of Eps8. The interaction between ITSN2 and Eps8 was demonstrated by the in vivo co-immunoprecipitation and colocalization assays and the in vitro GST pull-down assays. Furthermore, we mapped the interaction domains to the region between amino acids 260-306 of Eps8 and the coiled-coil domain of ITSN2. In addition, protein stability assays and immunofluorescence analysis showed ITSN2 overexpression induced the degradation of Eps8 proteins, which was markedly alleviated with the lysosome inhibitor NH4Cl treatment. Taken together, our results suggested ITSN2 interacts with Eps8 and stimulates the degradation of Eps8 proteins.

Curcumin inhibits AP-2γ-induced apoptosis in the human malignant testicular germ cells in vitro

Aim:To investigate the effects of curcumin on proliferation and apoptosis in testicular cancer cells in vitro and to investigate its molecular mechanisms of action.Methods:NTera-2 human malignant testicular germ cell line and F9 mouse teratocarcinoma stem cell line were used. The anti-proliferative effect was examined using MTT and colony formation assays. Hoechst 33258 staining, TUNEL and Annexin V-FITC/PI staining assays were used to analyze cell apoptosis. Protein expression was examined with Western blot analysis and immunocytochemical staining.Results:Curcumin (5, 10 and 15 μmol/L) inhibited the viability of NTera-2 cells in dose- and time-dependent manners. Curcumin significantly inhibited the colony formation in both NTera-2 and F9 cells. Curcumin dose-dependently induced apoptosis of NTera-2 cells by reducing FasL expression and Bcl-2-to-Bax ratio, and activating caspase-9, -8 and -3. Furthermore, curcumin dose-dependently reduced the expression of AP transcription factor AP-2γ in NTera-2 cells, whereas the pretreatment with the proteasome inhibitor MG132 blocked both the curcumin-induced reduction of AP-2γ and antiproliferative effect. Curcumin inhibited ErbB2 expression, and decreased the phosphorylation of Akt and ERK in NTera-2 cells.Conclusion:Curcumin induces apoptosis and inhibits proliferation in NTera-2 cells via the inhibition of AP-2γ-mediated downstream cell survival signaling pathways.

Caudatin targets TNFAIP1/NF-κB and cytochrome c/caspase signaling to suppress tumor progression in human uterine cancer

Caudatin, a C-21 steroidal glyco-side isolated from Chinese herbs, has a long history of use for the treatment of multiple diseases, including cancers. However, the precise mechanisms of actions of caudatin in human uterine cancer cells remain unclear. In this study, we investigated the molecular mechanisms by which caudatin inhibits cell growth in human cervical carcinoma cell line (HeLa) and endometrial carcinoma cell line (HEC-1A). Treatment with caudatin promoted cell morphology change, inhibited cell proliferation, colony formation, migration and spheroid formation, and induced cell apoptosis. Our results showed that the expression of tumor necrosis factor; α-induced protein 1 (TNFAIP1) was downregulated in uterine cancer cells and tissues compared to paired adjacent non-tumor uterine tissues. Further molecular mechanism study showed that caudatin can directly regulate TNFAIP1 expression in a concentration-dependent manner and also associated with the downregulation of NF-κB and upregulation of BAX/BcL-2 ratio and caspase-3. Moreover, we found that overexpression of TNFAIP1 inhibits the growth and invasion, and induces apoptosis in uterine cancer cells through inhibition of the NF-κB pathway, suggesting that TNFAIP1 may act as a potential therapeutic target for the treatment of cancer. We found that caudatin inhibited tumorigenicity and upregulated TNFAIP1 in vivo. Taken together, caudatin impacts on cell proliferation, migration and apoptosis of uterine cancer cells by regulating several carcinogenesis-related processes, including a novel mechanism involving the targeting of TNFAIP1/NF-κB signaling. Our findings provide new insights into understanding the anticancer mechanisms of caudatin in human uterine cancer therapy.

The Regulation of Downstream Target Gene AP-2γ by Transcription Factor OCT-4

In recent years,OCT-4 and AP-2γ have been widely used as clinical markers of testicular germ cell tumors.Transcription factor OCT-4,which plays an important role in embryonic development,gives full play to a variety of biological functions in different developmental periods and differentiations.The effect of OCT-4 is realized through the regulation of the target genes.In this study,we found OCT-4 binding sites within the sequence of AP-2γ promoter region by a variety of bionemerics.AP-2γ,a novel target gene of OCT-4,was identified by chromatin immunoprecipitation(ChIP)-PCR.A combination of sequence analysis,reporter gene assays,Western blot,immunofluorescence assay and mouse cryptorchidism model experiment further confirmed that AP-2γ was the target gene of OCT-4.OCT-4 inhibited the transcriptive activity of AP-2γ.The expression of AP-2γ gene was confirmed to be significantly altered by silencing or overexpression of OCT-4.This new discovery is conductive to study the malignant process of germ cell tumors at the molecular level.