The Significance Of Detection For Circulating RNA In Diagnosis And Therapy Of Gastric Cancer

Objective To establish a method of detecting low quantity of nucleic acids by nested real-time PCR, with which to determine the circulating RNA in plasma or supernatant of cell culture. To design several culture models in vitro: serum deprivation, hypoxia, low temperature and exposing with antineoplastic agent to analyze the potential factors that enhance circulating RNA release in cancer patients, and moreover, to investigate the significance of diagnosis and therapy of gastric cancer.Methods Circulating RNA was isolated with the combination of Trizol reagent and column method. The nested real-time PCR, based onSYBR green I, was established. Human gastric carcinoma cell line(SGC7901) was cultured under serum deprivation, hypoxia and low temperature conditions in vitro to observe the change of supernatant RNA, through which to analyze the possible factors that affect on the releasing of circulating RNA in tumor patients. SGC cells were also exposed to 5-fluorouracil (5-FU) and Cisplatin (DDP), the concentration of extracellular RNA was measured at 48h and 96h to evaluate the change of circulating RNA in tumor patients after the chemotherapy in vitro. In addition, the expression ofβ-actin gene in cellular and extracellular RNA was determined specifically. Through filtration test and exposure to ribonuclease, the ability of anti-degradation for circulating RNA was observed. Plasma CXCR4 and Bmi-1 mRNA, and CEA and CA19-9 proteins were evaluated in 89 preoperative gastric cancer patients and 42 healthy subjects. Sixty-nine of the cancer patients were also assessed after surgery. The correlation between the diagnostic values of plasma CXCR4 and Bmi-1 expression and the levels of CEAand CA19-9 was investigated.Results Circulating RNA was successfully isolated by the combination of Trizol reagent and column method. The sensitivity of nested real-time PCR is 10^0-1 copies/μl The quantity of extracellular RNA in the supernatant of SGC cells which suffered serum depriving and low temperature descended comparing with the control group while in the hypoxic culture, the extracellular RNA was increased significantly after 48h. When exposing to anti-cancer drugs for 48h, most tumor cells died and after removing the drugs, the extracellular RNA decreased significantly. The gene expression of extracellular RNA was significantly lower than the cellular RNA and was resistant to degradation by RNase. The levels of CXCR4 and Bmi-1 mRNA or CEA and CA19-9 proteins in cancer patients were significantly higher than those in healthy subjects, however there was a decrease in expression after surgery. The levels of CXCR4 and Bmi-1 mRNA had a strong correlation with each other. The expression of the two genes also strongly correlated with the CEA level. The expression of both circulating CXCR4 and Bmi-1 was significantly associated with the age of the patient, and the histological type, clinical stage, extent of cell differentiation, and the metastasis of gastric cancer. The detection of multiple genes has more sensitivity and accuracy than the combination of CEA and CA19-9 for tumor diagnosis.Conclusions Nested real-time PCR is a sensitive method for detecting low quantity of nucleic acids. Hypoxia and metabolic level are two potential factors that contribute to the releasing of circulating RNA in tumor patients. Circulating RNA has the ability of resisting the degradation by RNase. Determination of tumor associated circulating mRNA may provide novel diagnostic and prognostic markers for cancer and has more superiorities than traditional tumor markers.