A LncRNA Signature To Predict Prognosis Of CRC And The Underlying Mechanism Of GAPLINC Involving In The Development And Progression Of GC

Section I A long noncoding RNA signature to predict prognosis of colorectal cancerAims : Amount of protein coding genes(PCGs) have been reported to have prediction ability of CRC(colorectal cancer) patients’ progress, but few of them achieve clinical significance, therefore, it’s urgent to search for more useful biomarkers of CRC. Increasing evidence suggests long non-coding RNAs(lnc RNAs) are frequently aberrantly expressed in cancers, the study of which might give inspiration for the predition of CRC, however, few related lnc RNA signatures have been established for prediction of cancer prognosis. We aimed to develop a lnc RNA signature to predict prognosis of CRC.Methods : Using a lnc RNA-mining approach, we performed lnc RNA expression profiling in large CRC cohorts from Gene Expression Ominus(GEO), including GSE39582 test series(N=436), internal validation series(N=117); and two independent validation series GSE14333(N=197) and GSE17536(N=145). We were trying to establish a set of lnc RNAs that were significantly correlated with the disease free survival(DFS) in the test series. Based on this six-lnc RNA signature, we established a risk equation and classified the test series patients into high-risk and low-risk subgroups with significantly different DFS. The prognostic value of this sixlnc RNA signature was confirmed in the internal validation series and another two independent CRC sets. Gene set enrichment analysis(GSEA) analysis was used to test the risk score associated pathways. CCK8 assay and Transwell experiments were performed to test the function of three dysregulated lnc RNAs, AK123657, BX648207and BX649059 in CRC cell lines HCT116 and SW1116 after transfection with specific si RNAs.Results: We established a set of six lnc RNAs that were significantly correlated with the disease free survival(DFS) in the GSE39582 test series(N=436). Based on the risk equation: Risk score=(0.07826*AK024680)+(-0.14300*AK123567)+(-0.19355*CR622106) +(-0.00172*BX649059) +(-0.20855*BX648207) +(0.24326*AK026784), the test series patients could be classified into high-risk and low-risk subgroups with significantly different DFS(HR=2.670; P<0.0001). The prognostic value of this six-lnc RNA signature was confirmed in the internal validation series(N=117) and another two independent CRC sets(GSE14333, N=197and GSE17536, N=145). GSEA analysis suggested that risk score positively correlated with several cancer metastasis related pathways. Functional experiments demonstrated three dysregulated lnc RNAs, AK123657, BX648207 and BX649059 were required for efficient invasion and proliferation suppression in CRC cell lines.Conclusions : Our results might provide an efficient tool for clinical prognosis evaluation of CRC.Section II The function and mechanism of GAPLINC involving in the development and progression of gastric cancerAIMS: Growing evidence suggests that cancer lnc RNAs, similar to PCGs, may mediate oncogenic or tumor suppressing effects and may be a new class of cancer biomarkers and therapeutic targets. Therefore, this study aims to identify deregulated lnc RNAs in gastric cancer tissues through Microarray analysis, discuss the potential role of lnc RNAs in predicting patients’ clinical characteristics and progress, and probe the underlying mechanism of lnc RNAs involving in the development of gastric cancer.Methods: To obtain the transcriptional profiles for both lnc RNAs and m RNAs in gastric cancer, paired gastric cancer tissues and normal tissues(N=20) were analyzed using Array Star lnc RNA microarray. We applied a widely used "nearest shrunken centroid method" to classify gastric cancer and normal tissues according to their lnc RNA or m RNAs expression profiles. The trained prediction signature included nine lnc RNAs, wherein we chosen the most upregulated uc002 kmd.1 for further study. We also performed ISH to detect GAPLINC level in 90 gastric cancer tissues and paired noncancerous tissues, and analyze association of GAPLINC expression with the 90 patients’ clinical characteristics. To this end, the MGC803 cells were treated with specific si RNAs for GAPLINC, and the levels of all m RNAs were measured by Affymetrix Human Genome U133 Plus2 microarrays(triple repeats for each condition; data accessible via GEO #GSE51651). The GAPLINC-associated pathways were determined by gene set enrichment analysis(GSEA), which determines whether different pathways(sets of genes) show statistically significant differences between two biologic states. By RNA interference and c DNA transfection we detected the effects of knockeddown and upregulation of GAPLINC expression on cell apoptosis, proliferation, and invasion. Then we predicted the sites on GAPLINC and CD44 3’UTR which binds to mir-211-3p by bioinformatics methods, after constructing and transfecting clones we confirmed the exact binding domain by which mi R-211-3p binds to GAPLINC and CD44 3’UTR through luciferase assay.We established nude mice xenograft model by subcutaneously injecting gastric cancer cells, and then testified the impact ofGAPLINC on the tumor growth by stable knockdown GAPLINC. Moreover,we performed RT-q PCR to measure the GAPLINC、mi RNA level,then RT-q PCR and Western Blot to measure the CD44 expression in the two groups of tumors in nude mice xenograft model.Results: In this study, we identify deregulated lnc RNAs in gastric cancer that are associated with CNV or oncogenic transcription factors.We further tested the transactivation of mutant p53 on one of the bound lnc RNA uc002 kmd.1. Interestingly, lnc RNAs displayed equal predictive power as m RNAs on discriminating cancerous and normal tissue(lowest error rate = 0.196 for both sets). The trained prediction signature included nine lnc RNAs, wherein the most upregulated was uc002 kmd.1(Entriz gene ID: AX721193). We used RT-q PCR to quantify the level of uc001 kmd.1 in 48 normal gastric mucosa and paired gastriccancer mucosa, and confirmed the significant upregulation of uc002 kmd.1 in gastric cancer(P< 0.0001). Furthermore, receiver operating characteristic(ROC) curves were determined to evaluate the sensitivity and specificity of uc002 kmd.1expression in predicting gastric cancer tissues from normal tissues. Notably, uc002 kmd.1 displayed considerable predictive significance, with an area under curve(AUC) of 0.714. Given the cancer-predictive value of this RNA, it is hereafter referred to as GAPLINC(Gastric Adenocarcinoma Predictive Long Intergenic Non-Coding RNA). In addition, by analysising the clinicopathological characteristics of gastric cancer patients, it showed that the expression level of GAPLINC was positively related to the tumor size, metastasis, tumor invading more than 3/5 lymph nodes, AJCC stage(P<0.01). Ectopic expression of GAPLINC resulted in enhanced proliferation and invasiveness. By high throughput microarray screening and GSEA, we found that a considerable part of GAPLINC downstream gene is closely related to “cell migration” pathway. By analyzing the correlation between GAPLINC and m RNAs in the lnc RNA microarray dataset(containing 10 tumors and 10 normal tissues), we found CD44 with the highest correlation coefficient in this pathway(Pearson correlation, R=0.810; P<0.0001). These findings suggest that GAPLINC confers CD44-dependent effects in gastric cancer cells. We cloned the 3’-UTR of CD44 and GAPLINC downstream of a luciferase gene, and cotransfected these reporters with mi R211-3p mimics in gastriccancer cells. As expected, mi R211-3p significantly decreased the luciferase signals of both reporters. GAPLINC confers mi R211-3p dependent effects on CD44 expression.We generated xenograft models by implanting MGC803 GAPLINC-KD(stably knockdown GAPLINC) or the control MGC803 vector cells into nude mice. As a result, suppression of GAPLINC produced a marked decrease in the rate of xenograft subcutaneous tumor growth and CD44 m RNA and protein level, while increase the mi R-211-3p level.Conclusion: Our study suggests that deregulated lnc RNAs in gastric cancer might associate with CNVor oncogenic transcription factors. GAPLINC could be considered as the initial factor which affects the development and progressing of gastric cancer. It regulates the proliferation, invasion and metastasis of gastric cancer both in in vivo histological and in vitro cytological level, which partially depends on the activation of CD44, thus affecting the prognosis of GC patients.