The Expression Of ASPP1 And ASPP2 MRNA In Tissue And Peripheral Blood Of Patients With NSCLC

Objectives: To establish a real-time RT-PCR assay to measure the mRNA expression levels of ASPP1 and ASPP2 in tumor tissue and peripheral blood. The assay is used to search the variation of the ASPP1 and ASPP2 mRNA of patients with Non-small cell lung cancer, and find the relationship between the expression of ASPP1, ASPP2 mRNA and therapeutic effect of patients with NSCLC.Methods: 1.The reaction of real-time RT-PCR assay was confirmed after we designed and synthesized the specific primers according to the sequence. 2. The standard preparations were produced, including ASPP1, ASPP2 andβ-Actin. Plasmid DNA of ASPP1 was constructed as its standard substance. We can take advantage of the RNA extracted from the cultured HL-60 as the standard ASPP2 andβ-Actin substance. 3. The total RNA was extracted from the tumor tissues and the monocytes. The monocytes were isolated from the whole blood of the patients with NSCLC and normal control. The RNA specimens were detected about the content, purity and integrity. 4. The correction value and the relative magnitude were calculated after the mRNA expression levels of ASPP1 and ASPP2 had been detected by RQ-RT-PCR. And then we analyze the results by the statistic method. 5. We monitor the target gene mRNA levels and observe the therapeutic efficacy in the whole blood of the patients with NSCLC. According to this, we research the relations between the level of ASPP1 and ASPP2 and the therapeutic efficacy and explore the sensitivities to the chemotherapy.Result: 1.The primers are specific and the nonspecific reaction does not appear in the whole reactive progression. 2. The satisfactory amplification curve and standard curve were obtained in the quantitative standard substance and a single melting peak appears in the melting curve. The results are stable and repeated. 3. The RNA specimen is enriched concentration and integrity. 4. The correction values are significantly different between lung tissues and whole bloods respectively(P﹤0.05). The two group correction values are also different in the whole bloods of the normal control and patient(P﹤0.05). 5. The ASPP1 and ASPP2 mRNA levels are correlated to the tumor markers, such as CYFRA21-1 and CEA(P﹤0.05). 6. The different patients express the distinct ASPP1 and ASPP2 and they appear different sensitivity to the chemotherapy.Discussion: The standard substances are selected successfully and can be applied to the quantitative test. It is a sensitive, specific and stable method to analysis the ASPP1 and ASPP2 mRNA by RQ-RT-PCR. The results indicate that the ASPP1 and ASPP2 mRNA expression levels are different in the whole blood and the lung tissue. When the NSCLC patients are compared with the normal control, the descent expressions of ASPP1 and ASPP2 imply the ASPP1 and ASPP2 are correlated to the tumor development. The ASPP1 and ASPP2 mRNA levels in the whole blood can reflect the facts of NSCLC patients in the research. The results show it is sensitive to the chemotherapy in the patients with high level ASPP1and ASPP2, so it is useful for monitoring the chemotherapy effect in patients with NSCLC by ASPP1 and ASPP2 mRNA expression in peripheral blood.