Effects Of Skp2 Gene On Growth And Proliferation Of SGC-7901 Gastric Carcinama Cells By Application Of Antisense Oligonucleotides Technique

Objective:In 1978, antisense oligonucleotides technique was first used to inhibit the formation of the Rous sarcoma virus in chicken, and then this technique has been generally accepted as a gene intervention method which has superiority of high specificity and low toxicity. Antisense oligonucleotides are specifically designed to hybridise to the complementary RNA following the Watson-Crick binding rules, and inhibit expression of specific genes at the level of transcription or translation. Molecular biology and genetic engineering are developing so fast that shot nucleotides could be synthesized quickly and simplely. Antisense oligonucleotides technique can be used not only in the research of life science, but also in the treatment of malignant tumour, viral infection, inflammatory diseases and in reducing scar formation. Antisense oligonucleotides technology has entered into clinical trials in some countries whereas just started in China. Whether antisense oligonucleotides can be a new class of drugs for therapy still needs further researches, especially the mechanisms of how it target genes.The key problem of antisense therapy for cancer is how to choose a target gene.The common features of malignant cells are the abnormal functions of some specific genes which control cell proliferation, division or differentiation. So inhibition the translation of abnormal gene can reverse or eliminate the malignant behaviors of proliferation and metastasis in cancer cells. S-phase kinase-associated protain 2(Skp2) was first found in malignant cells, and played an important role in controlling cell proliferation. Skp2 could degradate some cell cycle regulators through ubiquitin-proteasome pathway to control the cell cycle progression. Meanwhile, Skp2 may be an oncogene, it has a high expression level in many malignant carcinomas and is close interrelated with maligant phenotypes and poor prognosis.Gastric cancer is one of the most common gastrointestinal cancers and has the highest mortality rate among all malignant carcinomas. It is noticeable that the prevalence and mortality rates of gastric cancer in China are twice as high as the average of the world. Like other malignant carcinomas, gastric cancer is an acquired genopathy of multi-factor and multi-stage, including activation of proto-oncogene, inactivation of tumor suppressor gene, and abnormal proliferation at the cellular level. As a regulator of cell proliferation and a potential oncogene, Skp2 also has a high expression level and correlate with malignangt phenotypes in gastric carcinomas. So, Skp2 gene plays an important role in carcinogenesis and development of gastric cancer, and may be a target for therapy. In recent years, antisense oligonucleotides technique targeting cancer risk genes has become a prospective therapeutical strategy for gastric cancer. However, cancer treatment research by targeting Skp2 gene has just begun, and research of using Skp2 antisense oligonucleotides in the treatment of gastric cancer has not been reported. Therefore, antisense oligodeoxy-nucleotide (ASODN), which hybridises to the Skp2 mRNA nucleotides sequence, was used in the present study. The functions and mechanisms of Skp2 gene in controlling growth and proliferation of gastric cancer SGC-7901 cells were explored for providing preliminary information about mechanism of cancer treatment targeting Skp2 gene.Methods:According to the experiment design, different treatment methods were performed in three groups: normal control group, nonsense oligodeoxy-nucleotides(NSODN) treated group and antisense oligodeoxy-nucleotides(ASODN) treated group. The concentrations of transfection were 50 nmol/L, 200 nmol/L and 400 nmol/L. The Skp2 oligodeoxy-nucleotides were delivered into SGC-7901 cells using lipofectamine 2000 Reagent. The growth and proliferation of cells were observed with light microscopy, and MTT assay. The cell cycle was detected by flow cytometry. The total RNA of cells was extracted and the expression levels of Skp2 and p27 mRNA were detected by reverse transcription-polymerase chain reaction. The total protein of cells was extracted and the expression levels of Skp2 protein, p27 protein and p53 protein were detected by western-blot.Results:1. After transfected with 200 nmol/L Skp2 ASODN, the growth of SGC-7901 cells was obviously slowed down, unclear cell borders and loose cell junctions were observed, the characteristics of clonal growth in cancer cells were lost.2. 200 nmol/L Skp2 ASODN significantly inhibited the proliferation of gastric cancer cells at 24, 48 and 72 h, p < 0.01. But after transfected with high concentration(400 nmol/L) of oligodeoxy-nucleotides for more than 48 h, both NSODN and ASOND had serious non-antisense inhibition effect, and there was no significant difference between both, p > 0.05.3. The cell cycle of 200 nmol/L NSODN group cells was similar with that of normal control group, but the cell cycle of 200 nmol/L ASODN group cells was changed: cells in the G₀/G₁ phase were decreased slightly, and cells in the S phase were obviously increased compared with that of normal control group, p < 0.05.4. At the concentration of 200 nmol/L, Skp2 mRNA level of NSODN group was notchanged compared with that of normal control group, p > 0.05; but Skp2 mRNA level of ASODN group was obviously lower than that of normal control group, p < 0.05; p27 mRNA levels of NSODN group and ASODN group were not changed compared with that of normal control group, p > 0.05.5. After transfected with 200 nmol/L Skp2 ASODN and Skp2 NSODN, Skp2 protein level of ASODN group was obviously lower than that of normal control group, p < 0.05; both p27 and p53 protein levels of ASODN group were obviously higher than that of normal control group, p < 0.05. As expected, Skp2,p27 and p53 protein levels of NSODN group were not changed compared with that of normal control group, p > 0.05.Conclusion:1. After treatment with Skp2 ASODN, the decrease of both mRNA and protein levels of Skp2 indicated that application of antisense oligonucleotides technique could inhibit expression of target genes effectively in vitro.2. Skp2 antisense oligodeoxy-nucleotides could change phenotype and cell cycle of gastric cancer cells, and the growth and proliferation of cells were inhibited. So it will partly reverse the malignant behaviors of proliferation and metastasis of gastric cancer cells, and promisingly have a certain therapeutic effect for gastric cancer.3. Skp2 antisense oligodeoxy-nucleotides could significantly reduce Skp2 protein level of gastric cancer cells, it makes ubiquitin-proteasome system have an ineffective identification and degradation of negative cell-cycle regulatory protein p27, so high level of p27 protein inhibits the growth and proliferation rates of cells; meanwhile, low level of Skp2 protein interacts DNA replication, leads to high expression level of tumor suppressor gene p53, and may induce apoptosis.