The Effect Of Calcium Transport Protein Gene RNA Interference Combination With Cisplatin Treatment On Gastrointestinal Tumor

The gastrointestinal tract tumor is very common in our country. It is very difficult to diagnose it in early stage. Chemotherapy and radiotherapy are the main therapies in late stage. The tumor cell can be killed by these drugs, but meanwhile the normal tissue cell also killed by these drugs. This means all the drugs have a lot of side effect, and their efficacy is very low. So it is urge to find new therapy to cure the gastrointestinal tract tumor.The cisplatin is the common drug to treat tumors in clinical. The main target of cisplatin is DNA. It can combine with the N7 atom of guanine and adenine in tumor cell's DNA, then form the intra- or inter-compounds, and finally to block the DNA duplication. On the other hand, the cisplatin can combine with mitochondria DNA to form comoles compound, then the mitochondria can not repair the nucleotide. But the drug resistance will appear when the cisplatin was used in longterm. On the other hand, the cisplatin has a lot of side effects, such as the nephrotoxicity and bone marrow depression. These limit its application in clinical.The calcium channel maybe a potential target for the tumor treatment because the calcium can regulate the cell growth and cell death. CaT1 represent a new family of Ca2+ transporter/channels with structural homology to the capsaicin receptor, VR1 and was cloned from human intestine in 1999. It is also distally related to the TRP ion channels. CaT1 is rather specific for calcium. Apical calcium entry into epithelial cells is mainly by CaT1. Ca2+ is involved in cell differentiation and proliferation, as well as apoptosis. If CaT1 expression is linked to cell proliferation and/or apoptosis, altered CaT1 function might have significant implications for tumour growth. Thus, inhibition of CaT1 might be a therapeutic strategy to prevent uncontrolled growth of cancers of epithelial origin.RNA interference (RNAi) is the process of sequence-specific posttranscriptional gene silencing triggered by double-stranded RNAs (dsRNAs) homologous to the silenced gene. Whether artificial synthetic or Small RNA (siRNAs) transcripted from plasmids can target complementary mRNAs for degradation RNAi. RNAi has the characteristic of specific, high efficacy and no toxicity. So it is a good method for tumor therapy. There is no report about using siRNA-CaT1 to study the proliferation, differentiation and apoptosis in malignant tumor.Objective: To study the effect of calcium transport protein gene RNA interference combination with cisplatin treatment on gastrointestinal tumor. Methods(1)Using human CaT1(TRPV6) gene sequences from GenBank, selected suitable target site, synthesized oligonucleotides as DNA template encoding siRNA-CaT1, annealed and ligated into pSilencerTMneo 3.1-H1 siRNA expression vector to construct plasmid pSH1Si-CaT1, then amplified in E. coli. The recombinant pSlincer 3.1-H1 plasmids were identified by restriction endonuclease cutting and DNA sequencing and then transfected into Human Gastric Carcinoma Cell Line BGC-823. The expression of CaT1 mRNA and protein was examined by RT-PCR and Westernblot.(2)MTT assay was used to observe the cell growth inhibition of BGC-823 treated with CaT1-RNAi, DPP and the combination agent.(3)RT-PCR,Westernblot were used to observe the change of the expression of Akt,Bax,Bcl-2,Caspase-3,IκB and NF-κBp65. Results and discussion(1) The calcium transport protein1(CaT1) gene-specific small interfering RNA(siRNA) expression vectors was successfully constructed, and can inhibit the expression of CaT1 gene when transfected into BGC-823 cell.(2) MTT assay showed that siRNA-CaT1 and DPP can significantly inhibit BGC-823 growth, and the inhibition rate of cells treated with combination agents was higher than that of siRNA-CaT1 group and DPP group.The inhibition efficacy of siRNA-CaT1 combination with DPP 0.625μg/ml is equal to DDP 2.5μg/ml. So it suggested that siRNA-CaT1 can decrease the dosage of DDP, then lessen the side effect of DDP.(3)The RT-PCR and Westernblot data showed that the expression of Bax is increasing gradually and the expression of Bcl-2 is decreasing gradually in BGC-823 cell treated with siRNA-CaT1 and DPP group. So the ratio of Bax/Bcl-2 is increasing gradually. The efficacy is more higher in BGC-823 cell treated with siRNA-CaT1 combination with DPP group. The expression of Caspase-3 is also increasing in BGC-823 cell treated with siRNA-CaT1 group, DPP group and siRNA-CaT1 combination with DPP group. It suggested that apoptosis is involved in the BGC-823 cell death induced by DPP and siRNA-CaT1. This is the main reason that the survival rate is decreasing in BGC-823 cell treated with siRNA-CaT1 group and DPP group. The Akt is activated by calcium concentration in cytoplasm. When the expression of CaT1 gene is inhibited by RNAi technology, the calcium concentration in cytoplasm is decreasing rapidly and inhibit the expression of Akt at the same time and then affect the survival rate in BGC-823 cell treated with siRNA-CaT1 group and DPP group.(4)The RT-PCR and Westernblot data showed that the expression of NF-κB is increasing through inhibiting the expression of IκB in BGC-823 cell treated with siRNA-CaT1 and DPP group. The efficacy is more higher in BGC-823 cell treated with siRNA-CaT1 combination with DPP group. It suggested that the high expression of NF-κBp65 is also involved in cell apoptosis induced by siRNA-CaT1 and DPP group. This is another reason that the survival rate is decreasing in BGC-823 cell treated with siRNA-CaT1 group and DPP group.Conclusion: On the base of siRNA-CaT1 plasmid construction, siRNA-CaT1 can inhibit the human gastric cancer cell line BGC-823 growth and have synergistic effect with DPP. The mechanism involves that the combination agent can inhibit the Akt expression. On the other hand it can inhibit IκB expression, then increase NFκBp65, increase the ratio of bax/bcl-2 and promote the caspase-3 and other apoptosis related genes expression.