Study Of P33～(ING1) And P21 MRNA Expression Levels In HNE1 Cells Based On Molecular Beacon

As one of the gene diseases that threaten human being health, malignant cancer has been studied extensively during past years. A considerable amount of data on molecular biology indicates that carcinogenesis process notably accompanied by mRNA expression level change of tumor-associated genes. So to establish a quick, accurate and sensitive method for detecting mRNA expression level is essential. Based on molecular beacon, the thesis has developed application systems to detect mRNA expression level of tumor suppressor gene p21 and p33ING1 in total cellular RNA quantitatively and quickly. The thesis is composed of three major parts: In the first part, a new method for detecting p21mRNA in trace total RNA samples was developed based on molecular beacon (MB). p21 MB was synthesized for the determination of a well-known p53 activating tumor suppressor gene p21 mRNA in p33ING1 overexpression nasopharyngeal carcinoma( NPC) cell line. Furthermore, The effect of p33ING1 gene transfection and chemical drug Fluorouracil( 5-FU ) treatment on p21 mRNA expression was quantitatively measured combining with p21/cDNA standard curve. The data that consistent with results obtained from experiment employing traditional reverse transcription polymerase chain reaction (RT-PCR) method showed that p21 mRNA expression level could be enhanced not only through p33ING1 plasmid transfection but also by 5-FU treatment. The copies of p21mRNA were between 1.92×109 and 8.30×1010/μg. The proposed method is suitable for cancer diagnosis and drug evaluation. In the second part, the p33ING1mRNA expression level in NPC cells with plasmid transfection or not were studied based on MB quantitative detection assay. The reliability of MB detection results was further proved from protein level by molecular biology method. Results suggested that p33ING1 could inhibit cell proliferation not only by inducing the expression of proteins on p53 pathway but also by restraining the expression of proteins on STAT3 pathway. In the last part, p33ING1 mRNA level in cellular total RNA was detected quantitatively based on MB assay in order to conduct the fundamental investigation of the effect of 5-FU on HNE1 cells. During the operation, the effects of 5-FU concentrations and treatment time in HNE1 cells and HNE1 cells transfected by p33ING1 were measured in vitro. Traditional molecular biochemistry methods were applied to explore whether p33ING1 could enhance 5-FU induced apoptosis in HNE1 cells and its molecular mechanism. The results were as follows: p33ING1 mRNA expression level in tumor cells was enhanced not only by the 5-FU concentration but also by lapse of time. The MTT results also proved that high expression of p33ING1 mRNA could increase cell's sensitivity to chemical drug 5-FU. The detection method based on molecular beacon can be used to provide useful evidence quickly and quantitatively for clinical individual therapy and new chemical drugs development.