The Applied Research On Rac1 MRNA Quantification And Its Relation With The Biological Behavior Of Gastric Carcinomas

The disordered infiltrating growth at the original site and distant metastasis of the Gastric carcinoma led to disease progression, which will be out of control and deprive the lives of patients. Tumor invasion and metastasis is related to many factors, such as extracellular molecules, cell adhesion molecules, proteases, angiogenic factors, cytokines and growth factors, as well as the potential cell signal transduction pathway components such as: Rac1 and so on.The whole name of Rac1 is Ras-related C3 botulinum toxin substrate 1, and its promoter is rich in GC base pairs, with the characteristics of housekeeping gene, encoding a GTP enzyme (GTPase) activity of guanylate (GTP)-binding protein with the molecular weight around for the 20-25kD.Rac1 is regulated by the guanine nucleotide exchange factor (GEF), and GTPase activating protein (GAP). When Rac1 is combined with GTP, it shows activation status; with GDP, inactivation status.When the GTP hydrolysis, Rac1 can activate the downstream effect factors such as IQGAP,IRS P53,WAVE,PAK,JNK,MLK2/3and so on, thus giving rise to a series of cell responses including cell motility, cell-cell and cell-matrix adhesions, cytoskeletal rearrangements, cell apoptosis, gene transcription, and superoxide production ,a variety of physiological processes.Rac1 shows a low level of expression or non-expression in human normal tissues, but shows high expression in breast cancer, lung cancer, colon carcinoma, ovarian cancer and other malignant tumor cells. And its expression level is closely related to the degree of differentiation, invasion and metastasis capacity of tumor cells.The invasion and metastasis of tumor cell includes cell-cell and cell-matrix adhesions changes, cell matrix hydrolysis, actin cytoskeletal rearrangements and cell polarity, cell motility changes et al. Rac1 regulates these processes through varieties of signaling pathways. It has been taken much attention to the role of Rac1, which affects the genesis, development, invasion and metastasis of the tumor cells.Rac1 will probably become an effective molecular target for the treatment of tumor. This experiment used the real-time fluorescence quantitative PCR (RTFQ-PCR) TaqMan probe technology,and detected the Rac1mRNA level in 52 cases of gastric carcinomas and 12 cases of benign tissues. The relation between Racl quantification and the biological behavior of gastric carcinoma was analyzed. This experiment is to lay foundation for carring out tumor metastasis-related genes detection in the clinical laboratory. TaqMan probe 5' end is with fluorescent material FAM, 3' end is with fluorescent quenching substance TAMRA.When the integrity of the probe, the 3' end fluorescent quenching materials constraint 5' end fluorescent substances, fluorescences can not be detected. When the TaqMan probe is decomposed, the 5' end fluorescent materials will be free and send out fluorescences.During the PCR annealing process, the probe hybridisms with the template. During the extension process, it degradates with Taq enzyme 5'-3' exonuclease activity, and the free fluorescent materials send out fluorescences.Amplification of a DNA chain of each, there is the formation of a fluorescent molecule.The fluorescence signal strengths will be monitored by the Real-Time PCR Detection System in real time, eventually reaching the purpose to quantitate the unknown templates.The results of this study showed that: Rac1mRNA was expressed in either gastric carcinoma tissues or benign lesions.The positive rates were respectively 100% (52/52) and 33.3% (4/12). Four benign lesions with positive expression were the serious ulcers or inflammation specimens.The magnitude of RaclmRNA expression Md(QR) in the gastric carcinoma tissues and benign lesions were as follows: 7.41×10(54.01×106), 0(3.55×103). The positive rate and magnitude of Rac1mRNA expression in gastric carcinoma were higher than benign lesions(P<0.01).The gastric carcinomas were grouped according to the patients'gender, age, primary tumor site, tumor size, differentiation, invasion depth, lymph node metastasis and clinical stages. The results showed that:the magnitude of RaclmRNA expression had nothing to do with the patients'gender, age and tumor size,and inter-group comparison was no significant difference (P> 0.05); But in the degree of tumor differentiation, depth of invasion, lymph node metastasis, clinical stage grouping, the difference between groups was statistically significant (P <0.05).With the lower degree of differentiation of gastric carcinoma, to deepen the depth of invasion, lymph node metastasis happened, TNM staging increased, there is increasing trend of Racl expression. According to the level of RaclmRNA in the gastric carcinoma tissues and benign gastric disease tissues, the best critical point for RTFQ-PCR Racl level testing whether the gastric tissue samples benign or malignant is 1.73×104copies(5μgRNA),which was determined by ROC curvet, and its detection sensitivity and specificity were 88.5% and 100%. The consistency test statistic Kappa=0.742,P<0.05。 According to the level of RaclmRNA in the gastric cancer lymph node metastasis group and no metastasis group, the best critical point for RTFQ-PCR Racl level testing whether the incidence of lymph node metastasis of gastric cancer is 7.49×105copies(5μgRNA), which was determined by ROC curvet, and its detection sensitivity and specificity were 57.9% and 78.6%. The consistency test statistic Kappa=0.282,P<0.05。Conclusions: The positive rate and value of Racl expression in the gastric carcinoma tissues were significantly higher than the benign lesions. So the abnormal expression of Racl may be associated with the occurrence of gastric carcinoma, which may be one of the reference targets to identify benign or malignant gastric lesions. The value of Racl was positively correlated with invasion and metastasis of gastric carcinoma, and it may become the reference targets to evalue the incidence of lymph node metastasis of gastric carcinoma.