| Background: Gastric cancer is one of the most common gastrointestinal malignancies in the world. Some Asian countries such as China, Japan and South Korea are the highest incidence areas of gastric cancer. Every year the number of the new cases of gastric cancer in China is nearly 400,000,which accounts for about 42% of the number of the world's total cases.In 2011,Chinese urban population mortality rate of gastric cancer was 19.66 / 10 million, and the rural mortality rate was about 22.09/10 million.The mortality rate of gastric cancer is located in the second place for the death caused by malignant tumors. Now the surgical treatment of gastric cancer becomes more and more standardized and the drug treatment for gastric cancer has also been further developed.So the survival and quality of life of many gastric cancer patients have been greatly improved.In spite of this,5-year survival rate of gastric cancer patients is still lower than 40%.So it is still the most emphasis and difficulty to fully understand the stomach progress mechanism, and to seek effective method of prevention and cure in gastric cancer.PTEN is a tumor suppressor gene,located on human chromosome l0q23.3.It contains 9 extrons and 8 introns, and is approximately 200 kb long.It encodes the protein which consists of 403 amino acid residues, has phosphatase activity,makes the phosphatidylinosito1-3,4 triphosphate(PIP3) dephosphorylation specifically,inhibits PI3K/Akt signaling pathway resulting in regulating the cell growth,proliferation,apoptosis and survival and inhibiting the cell malignant transformation.Currently PTEN has been recognized as markers for cancer diagnosis and prognosis.Micro RNA is about 19-22 nt and a kind of small non-coding and endogenous short single-stranded RNA,discovered in recent years.It can be complementary bound to the m RNA3'UTR region of target genes and inhibit the translation of target gene's function protein so that it can regulating the espression of target gene at the level of transcription. It has been proved that mi RNA regulates the expression of PTEN,inhibits the translation of PTEN and affects the proliferation of tumor cells in a variety of tumor cells such as glioma, ovarian cancer, breast cancer and bladder cancer.PTENP1 is the pseudogene of PTEN and located on chromosome 9p13.3. Studies have shown that the 3' UTR region of PTENP1 is similar with that of PTEN in a degree of 95% and the region affected by mi RNA in the two genes is identical.Therefore, mi RNA will act on PTEN and PTENP1 simultaneously, making the function of PTEN preserved,which opens up a new path for the treatment of tumor.We cloned the same mi RNA fragments of PTENp1 and PTEN(the PP),constructed the plasmid pc DNA3.1-pp,and compared the differences between gastric cancer cells transfected with the recombinant plasmid,transfected with empty plasmid and untreated in the proliferation,migration and invasion at the cellular level.Then we detected the expression of PTEN after overexpression of PP fragments, providing new ideas for the treatment of tumor.Objective: To construct the plasmid pc DNA3.1-pp used for transient transfection,observe the influence on cell growth, migration and invasion ability after transfecting the plasmid into gastric cancer,study the effect of the expression of PTEN by overexpressd PP fragments and lay a foundation for the further study on the mechanism.Methods: 1. We used the method of PCR,gel recovery,plasmid extraction to obtain fragments,and insert it into the eukaryotic expression vector pc DNA3.1.Then we constructed the recombinant plasmid pc DNA3.1-pp,and the recombinant plasmid was identified by enzyme digestion analysis and sequencing.2.We used liposome to transfect BGC823 cells. The recombinant plasmid pc DNA3.1-pp and pc DNA3.1 empty vector were transfected into the BGC823 cell, and then we used Western blotting to detect the expression of PTEN gene.3.We regarded the BGC823 cells that transfected by recombinant plasmid pc DNA3.1-pp as the research object, and the cells transfected by the empty vector and non-transfected cells as control.By cell counting, scratch test, cell invasion assays we compared the differences in cell growth, migration and invasion ability about three groups.Results: 1. Western blotting was used to detect the expression of PTEN gene which showed that the expression of PTEN protein after transfecting recombinant plasmid pc DNA3.1-pp was significantly higher than that after transfecting empty vector and untreated,and there was no significant difference between empty plasmid group and untreated group in the expression of PTEN.2.The automatic counting is used to record the number of cells divided into three groups in7 days.It can be seen after data processing,the cell number of the group transfected by recombinant plasmid pc DNA3.1-pp was significantly lower than that of empty plasmid group and untreated group, and the difference had statistical significance,but the difference between empty plasmid group and untreated group was not statistically significant.3. Scratch test was used to detect the cell migration ability. The migration rate of the recombinant plasmid cells was significantly lower than the empty plasmid group and untreated group, and the difference had statistical significance, but the difference between empty plasmid group and untreated group was not statistically significant in migration rate.4.Transwell assay was used to detect cell invasion ability.The transmembrane cell number of the recombinant plasmid cells was significantly higher than the empty plasmid group and untreated group, and the difference has statistical significance, but the difference between empty plasmid group and untreated group was not statistically significant.Conclusion: We constructed the BGC823 cells which were transfected transiently by pc DNA3.1-pp recombinant plasmid successfully.When the PP fragment was overexpressed,we found the expression of PTEN increased,and the ability of growth, migration and invasion of gastric cancer cells decreased. |
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