| Breast cancer has become the number one killer among women cancers. Known and suspected causes for breast cancer include high estrogen level and low progestin level. Metabolic disorder of steroid hormone promotes the development of some kinds of breast cancer, but the involved regulation mechanism remains unclear. Human tissue kallikreins, a new subgroup of serine proteases, involve in the carcinogenesis of steroid hormone dependent tumor. Kallikrein 6(KLK6), a member of the kallikrein gene family, encodes a secreted serine protease (hK6) with predicted trypsin-like enzymatic activity. It had been shown KLK6 expression level changed in breast cancer, which suggested KLK6 may be involved in carcinogenesis. But the role of KLK6 remains unknown. This study attempted to observe and identify K.LK.6 gene regulation mechanisms by estrogen siRNA, and K.LK6 gene function and its role in breast cancer development. The study was designed as follows:1. To develop a real-time quantitative RT-PCR method for detection of K.LK6 mRNA.2. To detect KLK6 mRNA levels in 33 breast cancer tissues and 25 normal breast tissues using real-time quantitative RT-PCR and analyze the relationship between KLK6 mRNA levels and clinicopathological variables such as tumor metastasis, estrogen receptor, progestin receptor and CerbB-2 gene expression.3. To study the regulation of KLK.6 gene expression by 17-p-estradiol and tamoxifen in human breast cancer cell line MCF-7, and its effect on cell cycle.4. To study the influence of KLK6 gene expression on cell proliferation and apoptosis by interfering its expression in MCF-7 breast cancer cell line with the small interfering RNA (siRNA) in vitro.5. To observe the effect of KLK6 gene silencing on tumor growth following intra-tumoral injection of siRNA or siRNA expression plasmid in nude mice with MCF-7 breast cancer.Results were as follows:1. Using SYBR Green I as report dye and GAPDH as inner reference, a real-time quantitative RT-PCR method was established. The amplification efficiencies for KLK6 and GAPDH of the real-time quantitative RT-PCR method were 0.99 and 0.98, respectively; inter-coefficient of variation were 2.0% and 0.8%, respectively; intra—coefficient of variation were 3.2% and 3.9%, respectively. The DNA sequencing and melting curve analysis of gene amplification product showed that the method for KLK6 mRNA quantification with SYBR Green I is simple, specific, reproductive and reliable, and can be used to study KLK6 expression.2. The levels of KLK6 mRNA were significantly higher in breast cancer tissues (0.4327±0.2348) than in normal tissues (0.2194±0.1154, P<0.01); KLK6 expression levels were obviously lower in cancer with node metastasis (0.3096±0.1l92) than no-metastasis (0.5527±0.2615, P<0.01). KLK6 expression levels were obviously lower in estrogen receptor positive tumor (0.2811±0.1164) than in estrogen receptor negative tumor (0.5097±0.2521, P<0.01). No correlation was found between KLK6 mRNA expression and progestin receptor and CerbB-2 gene expression (P>0.05).3. 17-P-estradioI inhibited KLK6 and hK6 expression in MCF-7 cells and...
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