The Clinical Significance Of LncRNA CRNDE-h Expression In Colorectal Cancer

Part 1 Increased expression of the long non-coding RNA CRNDE-h indicates a poor prognosis in colorectal cancer, and is positively correlated with IRX5 mRNA expressionObjective:The long non-coding RNA (lncRNA) CRNDE-h plays important roles in the early stages of human development and cancer progression. We investigated the expression and clinical significance of lncRNA CRNDE-h in colorectal cancer (CRC).Methods:The expression level of lncRNA CRNDE-h was analyzed in 142 CRC tissues and 142 paired adjacent non-tumorous tissues, along with 21 inflammatory bowel disease (IBD),69 hyperplastic polyp, and 73 colorectal adenoma samples, using quantitative real-time polymerase chain reaction. The association between lncRNA CRNDE-h and IRX5 mRNA was examined in the same 142 CRC tissues.Results:We found that lncRNA CRNDE-h level was elevated in the CRC and adenoma groups compared with the other groups (all at P＜0.001). In CRC. upregulation of lncRNA CRNDE-h was significantly correlated with large tumor size. positive regional lymph node metastasis, and distant metastasis (all at P<0.05). Area under the curve (AUC) for lncRNA CRNDE-h showed diagnostic capability for distinguishing CRC from other groups. Patients with CRC with high lncRNA CRNDE-h expression level had poorer overall survival than those with low lncRNA CRNDE-h expression (log-rank test, P＜0.001). Further multivariable Cox regression analysis suggested that increased expression of lncRNA CRNDE-h was an independent prognostic indicator for CRC (hazard ratio [HR]= 2.173; 95% CI. 1.282-3.684, P=0.004). Furthermore, lncRNA CRNDE-h expression was positively correlated with IRX5 mRNA in CRC tissues.Conclusions:Our data offer convincing evidence for the first time that lncRNA CRNDE-h is associated with adverse clinical characteristics and poor prognosis, which suggests that it might play an important role in CRC development and progression and might have clinical potential as a useful prognostic predictor.Part 2 Detection of exosomal long noncoding RNA CRNDE-h in serum and its potential diagnostic and prognostic value for CRCObjective:Cancer-secreted long non-coding RNAs (lncRNAs) are emerging mediators of cancer-host crosstalk. The aim of this study was to clarify the clinical significance of the levels of lncRNA CRNDE-h in exosomes extracted from the serum of patients with colorectal cancer (CRC).Methods:The study was divided into four parts:(1) The exosome isolated and lncRNA detected method for accurately and reproducibly measures CRC-related exosomal CRNDE-h in serum were optimized in preliminary pilot stage.(2) The stability of exosomal CRNDE-h was systematically evaluated in human serum. The exosome was subjected to harsh circumstances including incubation at room temperature, store at-80℃, treated with RNase A, multiple freeze-thaw cycles, and strong acid-base treatment. The same procedure was carried out in isolated nucleic acid, which acted as a control for this assay. And quantitative real-time PCR analysis was performed with Power SYBR Green. Results were normalized to endogenous controls.(3) The origin of exosomal CRNDE-h was explorated in vitro and in vivo. Six human colorectal cell lines (CCC-HIE-2, HCT116, SW480, SW620, HT-29 and LoVo) were selected and the exosomal CRNDE-h expression level was examined in the cell culture medium which was collected after 1day,2 days,3 days to show that the exosomal lncRNA CRNDE-h secreted from CRC cell lines. BALB/c nude mouse xenograft model system was used and the serum exosomal CRNDE-h expression level was evaluated in the blood sample which was collected once the tumor models were well established. RT-qPCR was used to measure the lncRNA CRNDE-h expression in serum samples and paired CRC tissues, and then correlation of the levels of lncRNA CRNDE-h between the two groups was analyzed. The serum exosomal CRNDE-h expression level was evaluated in CRC patients (n=20) before (pre-operation) and 14 days after (14 days post-operation) surgical removal of the tumor to demonstrate that serum exosomal CRNDE-h is derived from colorectal tumors.(4) The diagnostic and prognostic value of exosomal CRNDE-h for CRC was validated in an independent cohort of 468 patients. The expression level of lncRNA CRNDE-h was analyzed in 148 CRC patients, along with 80 normal colonoscopy,80 inflammatory bowel disease (IBD),80 hyperplastic polyp, and 80 colorectal adenoma samples, using quantitative real-time polymerase chain reaction.Results:(1) Exosome in the serum exhibitedround vesicular membranes that ranged from 40 nm to 100 nm. In addition, CD63 which was the protein marker onthe surface of exosomesand Hsp 70 which was the protein marker in the exosomes was used to confirm the evaluation of exosome by Western blot. (2)The average of threshold cycles for lncRNA CRNDE-h obtained using the same amount of RNA. The Ct value for the whole serum samples was lower than the supernatant and higher than the exosome samples. The exosome membrane protected lncRNA CRNDE-h when exosome samples were left under harsh conditions including multiple freeze-thaw cycles, incubation at room temperature, incubation at-80 ℃, acid-base treatment, and incubation of RNase A. (3) exosomal CRNDE-h enter cell culture medium at levels can be measurable, and exosomal CRNDE-h derived from human CRC xenografts enter the circulation, which can remarkably distinguish xenografted mouse from controls. Spearman’s rank correlation analysis showed that there was a significant correlationbetween exosomal CRNDE-h amplification and lncRNA CRNDE-h expression in matched CRC tissue samples (r=0.570, P< 0.001), which was consistent with our previous hypothesis. Serum levels of exosome lncRNA CRNDE-h were significantly declined 14d after surgical treatment compared with before surgery. (4) The expression levels of exosomal CRNDE-h were significantly higher in colorectal cancer compared with normal colonoscopy, IBD, hyperplastic polyp, and adenoma (all at P< 0.001, respectively). The high expression of CRNDE-h was significantly correlated with regional lymph nodes metastasis (P=0.019) and distant metastasis (P=0.003). At a cut-off value of 0.020, the optimal sensitivity and specificity were 70.3% and 94.4% in identifying patients with colorectal cancer with an AUC value of 0.892 (95% CI:0.860-0.918). Patients with CRC with high exosomal CRNDE-h expression level had poorer overall survival than those with low exosomal CRNDE-h expression (log-rank test, P＜0.001). Further multivariable Cox regression analysis suggested that increased expression of lncRNA CRNDE-h was an independent prognostic indicator for CRC (P=0.004).Conclusions:Detection of lncRNA CRNDE-h in exosome shed a light on utilizing exosomal CRNDE-h as a noninvasive serum-based biomarker for diagnosis and prognosis of CRC.