Development of NRSE containing Tubb3 promoter-based cancer gene therapy

• Main Authors: Yin Rei Huang, 黃穎蕾
• Other Authors: K. Y. Chong
• Format: Others
• Published: 2013
• Online Access: http://ndltd.ncl.edu.tw/handle/42540665815396713735

碩士 === 長庚大學 === 醫學生物技術暨檢驗學系 === 101 === βIII-tubulin (TUBB3) is a microtubule element which expressed exclusively in neurons and a specific marker for neurons in nervous tissue. The sequence analysis of promoter of TUBB3 revealed the presence of a conserved motif, neuron restrictive silencer element (NRSE), which is known to bind with the neuron-restrictive silencing factor (NRSF). NRSF bind to NRSE exerted repression to the transcription of TUBB3 during neuronal undifferentiated stage. Study also demonstrated that NRSE dsRNA appeared at an early stage of neurogenesis, competing with NRSF for bind to NRSE of Tubb3 promoter, and produced a derepression of transcriptional activity. Recent studies indicated that TUBB3 expression level was associated with an ascending histological grade of malignancy and poor prognosis. Furthermore, NRSF has been identified a negative regulator of tumor suppressor gene expression to the cancers, reduced expression of NRSF in the certain types of cancers resulted in tumor suppress genes derepression and cancer progression. Several studies reported that the expression of the truncated splice variant, NRSF4 worksed as a dominant-negative regulator and NRSF4 competing with NRSF bind to NRSE of the regulated genes, NRSF no longer associated with repressor proteins resulted in an increment of the regulated genes. Therefore, we hypothesize NRSF4 or NRSE dsRNA may take parted in the regulation of TUBB3 expression in TUBB3 positive cancer cells. To develop NRSE containing Tubb3 promoter-based cancer gene therapy, we studied: (1) To evaluate the expression of TUBB3 and NRSF in human normal and cancer cell lines, (2) To develop a NRSE containing Tubb3 promoter based cancer gene therapy. Our results shown that gene expression of TUBB3 and NRSF exhibited different patterns in human normal cell lines, cancer cell lines and multi-drug resistant cancer cell lines. A remarkably higher level of TUBB3 expression was found in multi-drug resistant cell lines including MES-SA/Dx5 and MCF-7/ADR when compared with their parental cell lines including MES-SA and MCF-7. Furthermore, we also found that other regulators including NRSE dsRNA and NRSF4 higher expression in MES-SA/Dx5. These findings suggested that NRSE dsRNA and/or NRSF4 may participate the regulation of NRSF-regulated gene in MES-SA/Dx5. Moreover, we have constructed a cMET shRNA expression mediated apoptosis system by a NRSE containing TUBB3 promoter (NRSE-Tubb3-shCMET system).When transfected of NRSE-Tubb3-shCMET system into the tested cell lines, we found that the viability was decreased in the transfected cancer cell lines with TUBB3 and cMET positive in a dose dependent manner, but not in the normal cell lines and cancer cell lines with TUBB3 and cMET negative. In addition, intratumoral infection with Lentiviral vector carrying NRSE-Tubb3-shCMET into MES-SA/Dx5 cells, triggered apoptosis was correlated to suppressed tumorigenicity in vivo. In conclusion, our results demonstrated that the possible application of NRSE containing Tubb3 promoter based cancer gene therapy in treatment of TUBB3 positive cancer.