| Gastric cancer is one of the most common tumors.Symptoms usually appear late in the disease course and most patients are diagnosed at an advanced stage,which delays treatment.Apropriate treatment plan and prolonged survival time are essential for studies on gastric cancer.Nowadays,the most common treatment for gastric cancer is still surgery and chemotherapy,while relapse and metastasis often happen for advanced stage patients.As the development of molecular-pathology,people could know more about the nature of the disease with genomic,trancriptomic,and epidemic information.And targeted therapy has got good performance to prolong survival time with trastuzumab targeting HER-2 and ramucirumab targeting VEGFR-2,which highlights the importance of molecular-pathology study and the advantage of personalized therapy.Searching for new targeting point is still central to gastric cancer because of more urgent need.Clinicalpathological characters are output of inner molecular alteration in gastric cancer,searching for new target point need a comprehensive uderstanding of molecular pathological features including genomic,transcriptomic,and post-transcriptomic alterations.mi RNA,methylation,and copy number all have impact on gastric cancer formation and development because of their regulation on gene expression.Now,there have more studies of single omics studies on gastric cancer,little is about multidimensional data and their interactions.Combining multi-dimensional data to dig up pathways and key genes is needed.Subsequently,expression and biology experiments on key genes will support the exploration of drug target.Objectives:Using bioinformatics methods to study on multi-dimensional data of gastric cancer,aiming at constructing gene-gene interation network and distinguishingimportant signaling pathways and key genes.Exploring basic biological function and downstream target of key genes in gastric cancer.Methods:1)The data of gastric cancer comes from TCGA,stored in Broad GDAC Firehose database.Differential analysis of m RNA,mi RNA,and methylation were performed using R software limma package.Copy number alteration was distinguished on GISTIC2.0.Subsequently,we got regulating relasionship between mi RNA and m RNA from Starbase database,and distinguished those differentially expressed genes with alteration on regulatory factor.2)Combining these regulatory factors to construct interactive network based on protein-protein information provided by STRING.Using DAVID online tool and network parameters to distinguish important signal pathways and key genes,seperately.Doing literature searching analysis for those key genes.3)Using q PCR and Western-blot experiment to detect expression of NR3C1 and PARK2 in human gastric cancer cell lines and normal gastric mucosal cell line;Analyzing correlation between the expression of PARK2 and gastric cancer clinic-pathological characters using TCGA data.4)Using lentiviral infection to construct PARK2 overexpressed cells in MGC80-3 and BGC-823 gastric cancer cell lines;evaluating biological function of PARK2 in gastric cancer through CCK-8 cell vialility experiment,cell cycle checking experiment and cell apoptosis detection experiment.5)Filtering out the targets of PARK2 from the network;analyzing biological process of there genes and their expression correlation with PARK2.Using q PCR and Western-blot experiment to check the expression alteration of possible targets in PARK2 overexpressed cells.Results:1)A total of 3602 differentially expressed genes,251 differentially expressed mi RNAs,604 differentially methylated gene sites,52 altered chromosomal regions locating 2137 genes were identified.Three groups of candidate genes controlled by different regulatory mechanisms were screened out consisting of 212,87,and 113 altered genes.2)Interactive networks under regulatory factors were constructed consisting of415,228,and 233 genes,respectively,all of which were enriched in cell cycleassociated pathways(cell cycle,P53 signaling,DNA replication),viral carcinogenesis,HTLV-1 infection,and progesterone mediated oocyte maturation pathways.Nine hub genes(SRC,KAT2 B,NR3C1,CDK6,MCM2,PRKDC,BLM,CCNE1,PARK2)were identified that were presumed to be key genes;seven of these were shown to be implicated in gastric cancer through searching literatures.3)The m RNA expression of NR3C1 was not decreased,while m RNA and protein expression of PARK2 were both decreased in gastric cancer cell lies.Clinical correlation analysis showed that the expression of PARK2 was associated with grade and invasion depth.4)We constructed PARK2 overexpressed MGC80-3 and BGC-823 cell lines.Overexpressed PARK2 cells showed less cell viability,G1 and S phase block,and more cell apoptosis.5)21 genes were detected as targets for PARK2,of which CCNE1,CCNA1,and PCNA were on cell cycle pathway.CCNE1 and PCNA were negatively correlated with PARK2,while only CCNE1 showed decreased expression in PARK2 overexpressed cells.Conclusion:1)Cell cycle associated pathways,viral carcinogenesis,HTLV-1 infection,and progesterone-mediated oocyte maturation are important signaling pathways in gastric cancer;2)SRC、 NR3C1、KAT2B、 CDK6、 MCM2、 PRKDC、 BLM、 CCNE1、and PARK2 are key genes in gastric cancer;3)PARK2 showed reduced copy number and decreased m RNA expression in gastric cancer,and its overexpression lead to decreased cell viability,blocked cell cycle G1 and S phase,and more cell apoptosis;4)CCNE is a PARK2 downstream target,which contributes to the function of cell cycle block. |
