| BackgroundPancreatic cancer is a malignant digestive tumor with a rapid process, its overall 5-year survival rate is only 5%. About 15%to 20%pancreatic cancer can be resected radically. Thus, the adjuvant chemotherapy plays an important role among multiple therapy measures. Gemcitabine is considered to be the first-line therapy drug for pancreatic cancer. But its therapeutic efficacy is far from satisfactory because the responses to gemcitabine are various.Deoxycytidine kinase (dCK) takes an important part in the metabolism of nucleotide in cells, and it's responsible for the clinical effection of nucleoside analogs. Single nucleotide polymorphism is a DNA sequence variation occurring when a single nucleotide in the genome differs between members of a biological species or paired chromosomes in an individual with a frequency of more than 1%. SNPs in the non-coding and coding region can affect the expression of target gene and the synthesis of target protein. Then the activity and founction of the target protein can be affected. It's been proved that SNPs in the metabolic enzyme of chemotherapy drugs can affect the responses of patients. In recent years, it's reported that the SNPs of dCK gene and the expression level of dCK protein were related to the chemosensitivity of nucleoside analogs.Thus, the aim of this study is to find whether the SNPs of dCK gene or the expression level of dCK protein are associated with the chemosensitivity of gemcitabine in pancreatic cancer cells, and try to investigate the possibility to predict the chemosensitivity of gemcitabine by detecting the specific SNPs of dCK gene and the expression level of dCK protein in pancreatic cancer patients. Objective1. To investigate the relationship between the SNPs of dCK gene and chemosensitivity of gemcitabine in pancreatic cancer cells;2. To investigate the relationship between the expression level of dCK protein and chemosensitivity of gemcitabine in pancreatic cancer cells;3. To investigate the possibility to predict the chemosensitivity of gemcitabine by detecting the specific SNPs of dCK gene and the expression level of dCK protein in pancreatic cancer patients.MethodsIn Part I, the total DNA samples of AsPC-1, BxPC-3, Mia PaCa2, SU86.86, SW1990, T3M4 pancreatic cancer cells were extracted with QIAamp DNA minikits, then seven selected SNPs in the dCK gene were sequenced. The chemosensitivity to gemcitabine were evaluated in vitro with a Cell Counting Kit-8 (CCK-8) test. The relationship between the SNPs of dCK gene and chemosensitivity of gemcitabine was investigated.In Partâ…¡, on the basis of Part I, two groups of pancreatic cancer cells with different chemosensitivity of gemcitabine(sensitive group:BxPC-3, T3M4; resistance group: AsPC-1, SW1990) were chosen, the expression of dCK protein were evaluated with western blot method. The dCK-siRNA reagent was used to knockdown the expression of dCK protein in two pancreatic cancer cells (BxPC-3 and T3M4), then the chemosensitivity of gemcitabine were detected with CCK-8 test. To observe the association between expression of dCK protein and chemosensitivity to gemcitabine in pancreatic cancer cells.In Part III, the A9846G SNP of dCK gene was sequenced in 30 pancreatic cancer patients, and the clinical outcomes were collected. The expression of dCK protein in 43 specimens of pancreatic cancer patients were tested with immunohistochemistry, the clinical features and prognosis were evaluated.Results1. The genotypes of the A9846G and C300T SNPs in the dCK gene were determined in six pancreatic cancer cells. Cells with the 9846AG genotype (BxPC-3 and T3M4) were more sensitive to gemcitabine compared to 9846GG genotype cells (AsPC-1, Mia PaCa2, SU86.86, SW1990) (P< 0.01), cells with the 300CT genotype revealed no significant difference in chemosensitivity to gemcitabine compared to 300CC genotype cells (P> 0.01);2. Pancreatic cancer cells with high expression of dCK protein were more sensitive to gemcitabine than cells with lower expression of dCK protein (P< 0.01). The chemosensitivity to gemcitabine were significantly decreased after dCK-siRNA reagent was transfected into pancreatic cancer cells (P<0.05);3. In 30 pancreatic cancer patients, patients with the AA/AG genotype of A9846G SNP had a better outcome than GG genotype carriers (P=0.057);4. In 43 pancreatic cancer patients, the expression level of dCK protein was significantly associated with the prognosis and patients age (P<0.05).Conclusions1. The A9846G SNP of dCK gene has a significant association with the chemotherapy efficacy of gemcitabine in pancreatic cancer cells;2. There's a positive correlation between the expression level of dCK protein and the chemotherapy efficacy of gemcitabine in pancreatic cancer cells;3. The method of predicting the chemosensitivity of gemcitabine by detecting the specific SNPs of dCK gene and the expression level of dCK protein in pancreatic cancer patients shows a great prospect.
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