Study Of Relationship Between Gastric Cancer Lymphnode Metastasis And MicroRNA Expression With Its Target Genes

In this study of gastric cancer, we compare the primary gastric cancer and corresponding lymph node metastases in microRNA expression profiling to find the differential expression of microRNA. Meanwhile, we detect the lymph node metastasis predicting capbility of differential expression microRNA. Finally, adoption of bioinformatics predicts differential expression microRNA target genes to find out the candidate target genes suitable for follow up research.Part One:The establishment of tumor tissue purification and RNA quality control systemObjective:To establish the standard process of tumor tissue purification by laser microdissection and RNA extraction and quality control system. Methods:Detect normal gastric mucosa RNA in vitro degradation by UV spectrophotometer, agarose gel electrophoresis, real-time PCR. Select five cases of primary gastric cancer and corresponding lymph node metastases, purify the tumor tissue by laser microdissection and test RNA quality by UV spectrophotometer, agarose gel electrophoresis and Agilent2100 bio-analyzer. Results:According to the time in vitro, six groups were set in 0′,15′,30′,60′,120′,240′, respectively. No obvious RNA degradation was found by UV spectrophotometry and agarose gel electrophoresis.The real-time PCR detection found expression of miR-21, U6,18S, and GAPDH at 0′and 240′were no significant difference. UV spectrophotometer and agarose gel electrophoresis shown the existence of total RNA degradation in laser microdissected samples. Agilent2100 bio-analyzer indicated total RNA degradation, but no significant degradation of the microRNA. Conclusions:Within the in vitro time of 240′, the normal gastric mucosa had no significant degradation of total RNA. The degradation of total RNA does not indicate the degradation of microRNA. Laser microdissection derived total RNA quality and quantity meets the follow-up experiments'requirements.Part two:Microarray analysis of microRNA expression and validation in a gastric cancer cohortObjective:Search for different expressing microRNA between gastric cancer primary tumor and corresponding lymph node metastasis. Study the differential expressed microRNAs in a gastric cancer cohort. Methods:The use of microRNA microarray tested five cases of primary gastric cancer and corresponding lymph node metastases to find differential expression of microRNA. The use of stem-loop RT-PCR detected differential expressed microRNAs in a cohort with 33 cases of gastric cancer, and found the correlation between differential expressed microRNAs and lymph node metastasis. Results:Microarray had found five significant differential expressed microRNAs including miR-24-1*, miR-510, miR-1284 which were down-expressed in metastases, and miR-10a, miR-1259 which were up-expressed in metastases. Stem-loop RT-PCR found miR-10a expression is significantly correlated with metastasis, but the expression level is not correlated with the amount of metastatic lymph nodes. Conclusions:we have found five significant differential expressed microRNAs including miR-10a, miR-24-1*, miR-510, miR-1259, miR-1284. miR-10a expression is significantly correlated with metastasis, but the expression level is not correlated with the amount of metastatic lymph nodes.Part Three:Targets prediction of differential expression microRNAsObjective:Predict the target genes of differential expressed microRNAs to provide the candidates of further research. Methods:The use of TargetScan to predict the target genes of miR-10a,miR-24-1*,miR-510 respectively. Search for published dates from gastric cancer gene expression profiling or proteomics studies to improve the prediction efficiency. Results:We have found 186,438,62 genes of miR-10a, miR-24-1*, miR-510 by TargetScan respectively. Combined the published date, we further filter out CDK6 and TPM4 as miR-10a's target gene, PURA as miR-24-1*'s and EGR1 as mi-510's. Conclusions:we filter out CDK6 and TPM4 as miR-10a's target gene, PURA as miR-24-1*'s and EGR1 as mi-510's by bioinformatics.Part four:SummaryLaser microdissection derived total RNA quality and quantity meets the follow-up experiments'requirements. five significant differential expressed microRNAs were found in microarray analysis, including miR-10a, miR-24-1*, miR-510, miR-1259, miR-1284. miR-10a expression is significantly correlated with metastasis, but the expression level is not correlated with the amount of metastatic lymph nodes. CDK6 and TPM4 is found to be miR-10a's target gene by bioinformatics, so as PURA to miR-24-1* and EGR1 to mi-510. These four genes can be candidates in following research.