MicroRNA-203 leads to G1 phase cell cycle arrest in laryngeal carcinoma cells by directly targeting survivin.

Previous studies have shown that miR-203 acts as a tumor-suppressive microRNA in various cancers, but its roles in laryngeal carcinoma are still contradicted. Here, we found that miR-203 inhibited the growth of laryngeal cancer cells and survivin was a direct target of miR-203. Moreover, silencing of survivin recapitulated the effect of miR-203 on cell cycle progression, whereas overexpression of survivin reversed this effect. Additionally, qRT-PCR showed the reciprocal relationship between miR-203 and survivin in laryngeal cancer tissues. These findings indicate that miR-203 inhibits the proliferation of laryngeal carcinoma cells by directly targeting survivin, suggesting its application in anti-cancer therapeutics.

Inhibiting XIAP expression by RNAi to inhibit proliferation and enhance radiosensitivity in laryngeal cancer cell line.

OBJECTIVES: X-linked inhibitor of apoptosis protein (XIAP) is a novel member of the inhibitors of apoptosis (IAPs) family. The overexpression of XIAP is asscociated with radioresistance of human malignancies. The purpose of the present study was to investigate the effect of shRNA-targeted XIAP on the proliferation, apoptosis and radiosensitivity of human laryngeal carcinoma cells (Hep-2). METHODS: A siRNA expression vector (pSilencer4.1-XIAPshRNA) was constructed and stably transfected into human laryngeal carcinoma cells (Hep-2). The downregulation of XIAP expression was evaluated by RT-PCR and Western blot analyses. Then, we investigated the effect of XIAP-shRNA on the proliferation, cell cycle changes and apoptosis in vitro of Hep-2 cells. Finally, the radiosensitivity of Hep-2 cells was investigated by clonogenic cell survival assay. RESULTS: We established stably transfected cell line (Hep-2/XIAPshRNA) in which the expression of XIAP gene was downregulated. The cell viability of Hep-2/XIAP-RNA cells was obviously decreased compared with that of untransfected Hep-2 cells. Morever, XIAP-shRNA induced cell arrest in the G(0)/G(1) phase of cell cycle by flow cytometry analysis. Results of TUNEL assay indicated that Hep-2 cells stably transfected pSilencer4.1-XIAP-shRNA showed obvious apoptosis characters. Furthermore, the downregulation of XIAP expression could lead to significant radiosensitivity enhancement in laryngeal carcinoma cells. CONCLUSIONS: RNAi-mediated downregulation of XIAP expression can inhibit proliferation, induce apoptosis and diminish the radioresistance of laryngeal carcinoma cells, so combined therapy with XIAP inhibition and radiation may be a potential strategy for the treatment of laryngeal carcinoma.

Suppression of Bcl-xL expression by a novel tumor-specific RNA interference system inhibits proliferation and enhances radiosensitivity in prostatic carcinoma cells.

Bcl-xL, a novel member of anti-apoptotic Bcl-2 family that play important roles in regulating cell survival and apoptosis, is frequently overexpressed in various kinds of human cancers, including prostatic carcinoma. To explore its possibility as a therapeutic target for prostatic carcinoma, we developed a novel tumor-specific RNA interference system by using survivin promoter and employed it to suppress exogenous reporters (LUC and EGFP) and endogenous gene Bcl-xL expression and analyzed its phenotypes. We found that expression of exogenous reporters (LUC and EGFP) was specifically inhibited in tumor cells but not in normal cells. We also observed that the specific inhibition of Bcl-xL in human prostatic carcinoma cells (PC3) strongly suppressed in vitro cell proliferation and in vivo tumorigenicity. We observed significant apoptosis induction and radiosensitivity enhancement in PC3 cells by the RNA interference-mediated suppression of Bcl-xL expression. All these results indicate that inhibition of Bcl-xL expression can result in potent antitumor activity and radiosensitization in human prostatic carcinoma.

Inhibiting proliferation and enhancing chemosensitivity to taxanes in osteosarcoma cells by RNA interference-mediated downregulation of stathmin expression.

Stathmin (Oncoprotein18), a signal transduction regulatory factor, plays an important role in cell division and malignant tumor development. Stathmin is a ubiquitous intracellular phosphoprotein that is overexpressed in a variety of human malignancies, including osteosarcoma. To investigate the potential use of stathmin as a therapeutic target for human osteosarcomas, we employed RNA interference [small interfering RNA (siRNA)] to reduce stathmin expression in human osteosarcoma cell lines and analyzed their phenotypic changes. Results showed that the downregulation of stathmin expression in human osteosarcoma cells significantly inhibited cell proliferation in vitro and tumorigenicity in vivo. The specific downregulation induced cell arrest in the G(2)/M phase of cell cycle and eventually apoptotic cell death. Taxanes are a group of effective chemotherapeutic agents whose activity is mediated through stabilization of the microtubules of the mitotic spindle. In the present study, we also observed a synergistic enhancement of the cytotoxicity effect by combination use of taxanes and RNA interference-mediated stathmin downregulation. All these experimental data indicate that stathmin downregulation can lead to potent antitumor activity and chemosensitizing activity to taxanes in human osteosarcomas.

The therapeutic potential of survivin promoter-driven siRNA on suppressing tumor growth and enhancing radiosensitivity of human cervical carcinoma cells via downregulating hTERT gene expression.

Human telomerase is a ribonucleoprotein complex composed of two subunits, an RNA component (hTR) and a human telomerase reverse transcriptase component (hTERT). The activation of telomerase, a process regulated by the human telomerase reverse transcriptase (hTERT), is a crucial step during cellular immortalization and malignant transformation. hTERT is overexpressed in most malignant cells but undetectable in most normal somatic cells. To explore its possibility as a therapeutic target for human cervical carcinoma, we developed a novel tumor-specific RNA interference system targeting hTERT by using the survivin promoter and investigated the effects of it on the proliferation, apoptosis and radiosensitivity in human cervical carcinoma cells (HeLa). The treatment of HeLa cells by hTERT gene RNAi not only could inhibit the proliferation of human cervical carcinoma cells (HeLa), but also could enhance the radiosensitivity of those cells via downregulation of their mRNA and protein expression. Therefore, survivin promoter-driven siRNA expression vector targeting hTERT may have potential use in radiosensitization therapy with targeted tumor gene silencing effect in human cervical carcinomas.

[Construction of an expression vector under the control of hTERT promoter].

AIM: To construct a luciferase expression vector driven by hTERT(human telomerase reverse transcriptase) core promoter and identify the transcriptional activity of the vector in tumor cells and normal cells. METHODS: hTERT gene core promoter was amplified by PCR using the total genomic DNA from HeLa cells as template. The amplified gene fragment was subsequently cloned into PGL3-basic vector. Then the expression vector was confirmed by restriction enzyme digestion and PCR analysis. The luciferase activity driven by the hTERT gene core promoter was identified after transient transfection of the expression vector into tumor cells and normal cells. RESULTS: The luciferase activity was high in the transfected tumor cells, and very low in transfected normal cells. CONCLUSION: hTERT gene core promoter is tumor-specific and may be useful in gene therapy of tumor.

[Apoptosis of laryngeal carcinoma cell line Hep2 induced by tumor-specific expression of TRAIL gene].

AIM: To explore targeted gene therapy of tumor by using the combination of TRAIL gene with the telomerase promoter. METHODS: TRAIL gene with an IL-2 signal peptide was constructed by PCR and cloned into vector pGL3-181hTERT downstream of hTERT promoter to form an eukaryotic expressing vector. Hep2 cells were transfected by the recombinant vector and apoptosis of the transfected cells was evaluated by trypan-blue exclusion and the agarosegel electrophoresis of DNA. RESULTS: We successfully constructed a recombinant eukaryotic expression vector for TRAIL gene.The expressed product significantly induced the apoptosis of Hep2 cells. CONCLUSION: The recombinant eukaryotic expression vector pGL3-181hTERT/TRAIL was successfully constructed, which provides the possibility for gene therapy of tumor.