| Non-coding RNA(ncRNA) is a kind of non-protein coding and gene expression regulating RNA,which plays an important in physiological processes. With the development and application of high throughput biochip and sequencing technique, a large number of significant differential expressed ncRNAs were observed in CRC cancer tissue and cell line, and the ncRNAs were considered as "oncogenes"or "tumor suppressors" in its development and progression. Functional genetic polymorphism in ncRNA may affect CRC risk, drug efficacy and clinical prognosis by affecting its expression or alternation of RNA secondary structure. And circulating significant differential expressed and stable lncRNAs are candidate clinical applied biomarkers for CRC.For this, we screened CRC relative ncRNAs and their functional SNPs by means of literature retrieving and bioinformatics, identified eligible patients, collected clinical data and performed 3 years following-up to investigate the role the SNPs and circulating lncRNA in CRC risk assessment, chemotherapeutic drug efficacy judgment, prognostic prediction and diagnosis and their possible mechanism using MassArray platform, qRT-PCR, common PCR, agarose gel electrophoresis, DNA sequencing, luciferase reporter gene assay, cell culture, transmission and scanning electron microscope observation, nanosight, western blot and ELISA detection.In the first section, we screened and selected 24 functional SNPs (13 SNPs in 13 miRNAs and 11 SNPs in 8 lncRNAs) which were rarely reported in CRC), detected the genotype in a total number of 1358 CRC patients and 1079 healthy individuals using MassArray genotyping platform and investigated the association between these SNPs and susceptibility to CRC,5-Fu based chemotherapeutic drug efficacy and clinical 3 years’prognosis. Our results showed that CCAT2 rs10808556 were positively associated with susceptibility to CRC in co-dominant (CC vs.TT) and recessive (CC vs. TT+TC) models, miR-608 rs4919510 GG genotype and RPL34-AS1 rs11731416 GC、CC. GC+CC genotypes and C allele were significantly negative associated with risk of CRC, rs4919510 and rs11731416 could play a cumulative effect to decrease CRC risk. MiR-126 rs4636297 and HOTAIR rs2366152, PCAT1 rs1551513 and CCAT2 rs10808556 as well as RPL34-AS1 rs11731416 and miR-373 rs3859501 were associated with DCR, ORR as well as DCR and ORR, respectively, in 276 CRC patients undergoing 5-Fu based chemotherapeutic therapy. And only CCAT2 rs10808556 CC genotype was observed to significantly associated with poor RFS and 3 years’ OS in comparison with TT or TT+TC genotype carrier. There was a linkage disequilibrium between rs10808556 and rs6983267 which was identified by GWAS(R2=0.848).Meanwhile, the region of the two loci presented an enhancer activity, and allele C of rs10808556 and allele G of rs6983267 as well as two of them could significantly increase promoter activity of luciferase reported gene in luciferase reported gene assay.Bioinformatical prediction showed that SP1 and SP2 could bind to rs10808556 and allele T of the locus could significantly decrease the binding ability between them. The genotypes of rs10808556 were significantly associated with expression of CCAT2 and CARlo5. These data illustrated that rs10808556 may affect the binding of transcript factor SP1 and SP2, induce differential enhance activity and lead to aberrant expression of CCAT2 and CARLo5, consequently, contributing to CRC carcinogenesis, resistance of 5-Fu based chemotherapeutic drug and poor prognosis.In the second section, we screened 18 lncRNAs which played roles in CRC development and progression by literature retrieving, PCAT1, CCAT2, UCA1, lncRNA-P21 and MALAT1 were significantly differential expressed in small sample size of screening stage and PCAT1, CCAT2, UCA1 and MALAT1 were validated in 236 CRC preoperative and 213 healthy individual serum samples. The diagnostic efficacy of PCAT1, CCAT2 and UCA1 was higher than 0.700, and only PCAT1 could stable existence in serum, plasma, hemolysis, jaundice, fatty serum sample, and without inference of temperature, strong acid and alkali, freeze-thaw, digestion of DNase and RNase and different time placed at room temperature. The optimal cut-off value of PCAT1 was 1.615 for CRC diagnosis, its AUC, sensitivity, specificity, accuracy, NPV, PPV and Youden index were 0.779,77.54%,64.32%,71.27%,72.11%, 70.66% and 41.86%, and its diagnostic value was superior to CEA or CA199, the combined diagnosis of PCAT1+CEA and PCAT1+CEA+CA199 were superior to single circulating PCAT1, however there was no significantly difference between them, circulating PC ATI was significantly decreased in patients with one month post-operation in comparison with pre-operation and its diagnostic value for CRC was confirmed in 50 CRC pre-operative serum samples and 50 healthy serum samples, indicating that circulating PCAT1 could be considered as a biomarker for clinical CRC diagnosis as well as efficacy judgment of post-operation. PC ATI was significantly higher expressed in CRC tissue, HT29 and HCT116 cell lines, and circulating PC ATI expression in pre-operated serum and 48h cell culture solution was significantly higher than post-operated serum and 24h cell culture solution, and thre was no difference in two groups’ blood samples, and its expression wasn’t associated with count of blood red cell, white cell, lymphocyte, monocyte, neutriphil and platelet. We confirmed that exosome was presented in serum sample using transmission scanning electron microscope, nanosight, western blot detection, and higher PCAT1 expression was also examined in CRC exosome sample in comparison with CRC exosome-free serum sample and exosome from healthy individuals, but no difference was examined in exosome-free serum between two groups as well as exosome sample and exosome-free serum sample from healthy individuals, but there was a positive linear correlation between serum exosome concentration and circulating PC ATI expression in CRC patients. Meanwhile, PCAT1 was detected in CRC cancer tissue, cell liens, blood, plasma, exosome, exosome-free serum, and the 87bp PCAT1 was originated from approximately a length of 320bp PCAT1 fragment in serum exosome.In summary, we got the results as following:1. miR-608 rs4919510 and RPL34-AS1 rs11741516 might be protective genetic loci for CRC.2. MiR-126 rs4636297, miR-373 rs3859501, HOTAIR rs2366152, PCAT1 rs1551513, RPL34-AS1 rs11731416, CCAT2 rs10808556 could affect clinical efficacy of 5-FU based chemotherapeutic drug.3. CCAT2 rs10808556 could affect the binding of transcript factor SP1 and SP2, mediated different enhancer activity and induced abberant expression of CCAT2 and CaRLo5 to promote CRC carcinogenesis, decrease efficacy of 5-FU based chemotherapeutic drug and poor survival, its CC genotype could be used as a clinical biomarker for CRC risk assessment, efficacy judgment of 5-FU based chemotherapeutic drug and prediction of prognosis.4. Circulating differential expressed PCAT1 was originated from eoxosme particle which was released by CRC cell, and sample fragment of PCAT1 was protected by exosome and was stable in serum without interfering of hemolysis, fatty, jaundice, temperature, strong acid and alkali, DNase or RNase and different placed time in room temperature.5. The cut-off value of circulating PCAT1 was 1.615 for CRC diagnosis, and its AUC, sensitivity, specificity, accuracy, NPV, PPV and youden index were 0.779,77.54%, 64.32%,71.27%,72.11%,70.66% and 41.86%. Circulating PCAT1 and CEA combined diagnosis could increase its diagnostic efficacy for CRC, and it could be considered as a biomarker for CRC diagnosis and post-operated efficacy judgment. |
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