Screening And Functional Studies Of Long Noncoding RNA Associated With Colorectal Cancer

Objective:Screening and verification lnc RNA and m RNA expression profiles a ssociated with colorectal cancer and conducting preliminary bioinformatic analys is of differentially expressed m RNAs.Methods:1.lncRNA microarray hybridization and data extraction:Randomly selected five cases of colorectal cancer tissues, extracting RNA,passing quality testing, synthetic c DNA, purification tag, passing testing of the labeling efficiency, conducting microarray hybridization, scanning, image-digital conversion, data normalization, set the fold change≥2 for the screening criteri a, geting lnc RNA and m RNA expression profiles.2.q RT-PCR verifying the reliability of microarray results:Selection six lnc RNAs with large fold-changes and in the relative positions of loci for intronic antisense, natural antisense or bidirectional（the two were up-regulated lnc RNAs: ENST00000421055, ENST00000441270;the four were do wnregulated lnc RNAs: uc003 hws.1, ENST00000565575, ENST00000563660, N R₀40058） were SYGB Green I q RT-PCR detected, and used the GAPDH as i nternal reference.3. GO analysis of differentially expressed m RNA GO classify into biological processes、cellular components and molecular f unctions based on Gene Ontology（www.geneontology.org）.A significant degree of GO functional pathways by P value indicates that the more significant funct ional ways with the smaller P value,（P <0.05 was statistically significant）.4. KEGG analysis of differentially expressed m RNA KEGG analysis based on the KEGG database（Kyoto Encyclopedia of Gen es and Genomes, http://www.genome.jp/kegg）, A significant degree of KEGG bi ological pathways by P value indicates that the more significant biological path ways with the smaller P value,（P <0.05 was statistically significant）.Result:1.lnc RNA and m RNA expression profiles:The differential expression data of lnc RNAs was 11724（4908 lnc RNAs w ere up, 6816 lnc RNAs were down）,the differential expression data of m RNAs was 7339（3860 m RNAs were up, 3479 m RNAs were down）.2.q RT-PCR verifying the reliability of microarray results:The curves of GAPDH, ENST00000421055, ENST00000441270, uc003 hws.1, ENST00000565575, ENST00000563660, NR₀40058 showed specificity of P CR amplification, using 2-△△CTmethod to calculate the expression of ENST00000421055, ENST00000441270, uc003 hws.1, ENST00000565575, ENST000005 63660, NR₀40058 in five pairs of colorectal cancer tissues,the changes consi stent with the microarray results.3.3. GO analysis of differentially expressed m RNA The result of GO analysis showed that m RNAs differentially expression m ainly enriched in twenty Cell Biologys、twenty cell components and twenty mo lecular functions, the highest enrichment degree of biological process in upregu lated m RNAs was the mitotic cell cycle, containing 210 differentially expressed m RNAs;the highest enrichment degree of cell component in upregulated m RN As was the cytosol, containing 508 differentially expressed m RNAs;the highest enrichment degree of molecular functiont in upregulated m RNAs was the pro tein binding, containing 1386 differentially expressed m RNAs;the highest enric hment degree of biological process in downregulated m RNAs was the multicell ular organismal process, containing 962 differentially expressed m RNAs;the hi ghest enrichment degree of cell component in downregulated m RNAs was the extracellular region, containing 421 differentially expressed m RNAs;the highest enrichment degree of molecular functiont in downregulated m RNAs was the a ctin binding, containing 81 differentially expressed m RNAs.4. KEGG analysis of differentially expressed m RNA The result of KEGG analysis showed that m RNAs differentially expressed mainly enriched in the twenty signaling pathways. The highest enrichment degr ee of biological pathway in upregulated m RNAs was the Cell cycle- Homo sapiens（human）, containing 124 differentially expressed m RNAs.The highest en richment degree of biological pathway in downregulated m RNAs was the Syste mic lupus erythematosus- Homo sapiens（human）, containing 137 differentially expressed m RNAs.Conclusion:1.Getting lnc RNAs and m RNAs expression profiles associated with colorectal cancer Using lnc RNA Microarray screened the lnc RNAs and m RNAs expression p rofiling associated with colorectal cancer, including 11,724 lnc RNAs and 7339 m RNAs.2.q RT-PCR verifying the reliability of microarray results Using SYGB Green I q RT-PCR detected the expression of these lnc RNAs in five cases of colorectal cancer tissues, the result was consistent with the m icroarray results and confirmed the reliability of microarray results.3.GO and KEGG analysis of differentially expressed m RNAs The result of GO analysis showed that m RNAs differentially expression m ainly enriched in twenty Cell Biologys、twenty cell components and twenty mo lecular functions, The result of KEGG analysis showed that m RNAs differential ly expression mainly enriched in twenty signaling pathways, Through research colorectal cancer related m RNAs, it was hopeful to find more lnc RNAs as pot ential diagnostic markers and therapeutic targets.