PLCE1 Influences The Occurrence And Development Of Gastric Cancer And Its Drug Resistance

PLCE1(phospholipase C epsilon-1,PLCE1)is a multifunctional phospholipase,it can act as not only a second messenger for its common catalytic properties shared by the members of phospholipase family,which can hydrolyze PIP2 to IP3 and DAG,but also an oncogene because of its Ras related domain and Guanine Exchange Factor domain.GWAS has launched the discussion about the correlations between the single nucleotide polymorphisms of PLCE1 and diseases since 2010.Our previous studies have confirmed the relationship between the PLCE1 rs2274223 and rs3765524 with the risk of gastric cancer.It has also been found by motifscan that PLCE1 should have interactions with DNA-PK,which is a key molecule involved in the progress of non-homologous end joining when DNA double-strands are damaged.This study has been designed in the following four parts to reveal the relationship between PLCE1 gene polymorphisms and its expression level and related functions as well as the influences caused by PLCE1 on biologic behaviors of gastric cancer cells during the occurrence and development of gastric cancer and to confirm whether PLCE1 is involved in the gastric cancer chemotherapy drug resistance or not and found itsmolecular mechanisms by the way.Part 1: PLCE1 rs3765524 and rs2274223 influence its gene expression.Objective: This study aimed to find the influences on PLCE1 expression caused by PLCE1 rs3765524 C>T and rs2274223 A>G.Methods: DNA sequencing technology was used to genotype PLCE1 rs3765524 and rs2274223 for a variety of gastric cancer cell lines.RT-PCR and WB were used to detect the PLCE1 expression level in different gastric cancer cell lines.Overlapping extension PCR and two-pointmutation technology were used to build restructuring PLCE1 eukaryotic expression vector,which were PLCE1 Minor and PLCE1 Major.Gene transfection technology was used to evaluate the influences on PLCE1 expression caused by its SNPS.Results:There were PLCE1 rs3765524 and rs2274223 polymorphisms in different gastric cancer cell lines.Trs3765524-Grs2274223(PLCE1 Minor)and Crs3765524-Ars2274223(PLCE1Major)were often linked together in gastric cancer cell lines.The different PLCE1 expression level in different gastric cancer cell lines was related to PLCE1 genotype but not to the differentiation level.PLCE1 expression level is higher in gastric cancer cell lines with PLCE1 Minor than PLCE1 Major.PLCE1 cDNA was cloned and found to be a novel transcript variant and then,it was used to build restructuring PLCE1 Minor and PLCE1 Major eukaryotic expression vector successfully.By transfection,it was found that the transient or stable expression level of mRNA and protein of PLCE1 Minor were significantly higher than that of PLCE1 Major in HEK-293 T or SGC-7901 cell line respectively.Conclusion: There were different PLCE1 genotypes on rs3765524 and rs2274223 in different gastric cancer cell lines.It was found that PLCE1 expression level is obviously related to its genotypes,but not to differentiation.Last but not least,PLCE1 Minor can obviously improve the expression level of PLCE1.Part 2: PLCE1 affects biology behaviors of gastric cancer cells.Objective: This study aimed to appraise the influences on gastric cancer cell proliferation,abilities of plate clone formation,migration and invasion and also the effect of cell apoptosis caused by PLCE1.Methods: MTS,plate clone formation,transwell,flow cytometry technique,and some other assays were used.Results: Bytransfection,it was found that PLCE1 Minor and PLCE1 Major could promote gastric cancer cell proliferation,invasion and migration and also enhance the colony-forming ability,but inhabit its apoptosis compared with the control of empty vector.What’s more,PLCE1 Minor acted more strongly than PLCE1 Major.Conclusion: PLCE1 overexpression could promote gastric cancer cell proliferation,clone formation,invasion and metastasis,but inhibit the apoptosis of them,and the promotion is positively correlated to PLCE1 expression level.Part 3: PLCE1 induced chemotherapy drug resistance of gastric cancer cell lines.Objective: This study aimed to reveal that PLCE1 should induce chemotherapy drug resistance of gastric cancer cell lines.Methods: MTS assay,IC50 calculated by Probit method,flow cytometry technology,shRNA interference and gene transfection were used.Results: Different gastric cancer cell lines with different PLCE1 expression level had different sensitivity to various chemotherapy drugs.For example,the IC50 of chemotherapy drugs such as HCPT,DOXO and 5-FU on various gastric cancer cell lines with different PLCE1 expression level was positively related to its PLCE1 expression level,in which the HCPT was the most typical.Further study found that the IC50 of HCPT on four different gastric cancer cell lines increases with their PLCE1 expression level.When PLCE1 was knocked down,the IC50 was reduced,but the apoptosis was increased.On the other hand,when PLCE1 was transfected into gastric cancer cells,the results were reversals of what caused by PLCE1 knocked down,and in which,PLCE1 Minor was more powerful.Conclusion:High expression of PLCE1 could survive gastric cancer cells from chemotherapy drugs such as HCPT,by enhancing its IC50 and reducing the apoptosis at the same time.Part 4: The molecular mechanisms of drug resistance caused by PLCE1 in gastric cancer cells.Objective: This study aimed to illustrate molecular mechanisms of drug resistance caused by PLCE1.Methods: SGC-7901 gastric cancer cell line was induced to resistant to HCPT.RT-PCR and WB were used to detect relative gene expression.Transwell assay was used to appraise the cell migration and invasion ability Immunoprecipitation assay was used to detect the interaction between two different kinds of protein.Immunofluorescence assay was used to test the Golgi dispersion areas.Results: SGC-7901 cell line was successfully induced to resistant to HCPT and it was named SGC-7901/ HCPT with the resistance index of 6.8 + /-0.3.It was found that PLCE1 expression level was enhanced in SGC-7901/ HCPT than in SGC-7901 cell line and the migration and invasion abilities of the former were also enhanced.It was also found that the expression level of PLCE1 and DNA-PKcs would increase after treated by HCPT for a while.Meanwhile,the expression level of snail,fanscin1,vimentin and slug increased and at the same time the expression of Ecadherin level decreased.It was first found that there was consistency on the expression level of PLCE1 and DAN-PKcs and also the interaction between PLCE1 and DNA-PKcs by Immunoprecipitation assay.It was found by Immunofluorescence assay that PLCE1 could promote dispersal of Golgi in gastric cancer cells caused by HCPT.The inhibitor of DNA-PK enhanced the cytotoxicity of HCPT.Conclusion:High PLCE1 expression level was associated with gastric cancer cells resistance to HCPT,which might be involved in increased migration and invasion abilities,overexpression of DNA-PKcs and the reorganization and dispersal of Golgi caused by PLCE1.