Serum exosomal long noncoding RNA CRNDE as a prognostic biomarker for hepatocellular carcinoma

Abstract Background Accumulating evidence has shown that long noncoding RNA (lncRNA) CRNDE functions as an oncogene in many cancer types. However, its clinical value has not yet been explored in hepatocellular carcinoma (HCC). Methods A total of 166 patients with HCC and 100 healthy volunteers were enrolled in this study. The expression levels of serum exosomal lncRNA CRNDE were detected in patients with HCC and controls by quantitative real‐time PCR (qRT‐PCR). Results The serum exosomal lncRNA CRNDE expression levels were significantly increased in patients with HCC compared with normal controls. High serum exosomal lncRNA CRNDE expression was significantly associated with tumor size, tumor differentiation, and TNM stage. Receiver operating characteristic (ROC) analysis revealed that an area under the ROC curve (AUC) of 0.839, with a sensitivity and specificity of 69.3% and 85.0%. In addition, the overall survival (OS) and disease‐free survival (DFS) were significantly longer in patients with lower serum exosomal lncRNA CRNDE expression compared to those with higher CRNDE expression. Moreover, HCC patients with cirrhosis had worse OS and DFS than those without cirrhosis. Univariate and multivariate analyses indicated that high serum exosomal lncRNA CRNDE expression was an independent indicator of poor prognosis. Conclusion Taken together, serum exosomal lncRNA CRNDE might serve as a potential biomarker for HCC diagnosis and prognosis.


| INTRODUC TI ON
In 2018, liver cancer is one of the most common cancers in China with about 3,68,960 cancer-related deaths and has become a major public health problem in the country. 1 The majority of liver cancer (75%-80%) is hepatocellular carcinoma (HCC). 2 Though great improvements in treatment strategies have been made in the past decades, the long-term survival rate of patients with HCC remains very unfavorable mainly because of late diagnosis, cancer recurrence, and tumor metastasis. 3,4 Treatment of surgical resection is usually used for early-stage HCC. However, most patients are diagnosed at advanced stages due to the lack of reliable markers for early detection. 5 Therefore, to improve the outcome of patients with HCC, the identification of novel biomarkers for predicting the recurrence and prognosis of this disease is urgently required.
Long noncoding RNAs (lncRNAs) are a class of transcripts longer than 200 nucleotides and are involved in a variety of cellular processes including cell proliferation, apoptosis, and differentiation. [6][7][8] LncRNAs actively participate in the initiation and progression of different cancer types including HCC. For instance, TGLC15 was significantly upregulated in HCC, and TGLC15 overexpression predicted unfavorable clinical outcome. Mechanistically, increased TGLC15 promoted the oncogenic activities of HCC cells in vitro and in vivo through interacting with SOX4. 9 Similarly, a novel lncRNA termed HLNC1 was demonstrated to promote tumorigenesis of HCC by interacting with USP49. 10 Exosomes are membrane vesicles with a size range of 30-150 nm and can be found in serum, plasma, urine, saliva, and breast milk. Exosomes contain various types of nucleic acids, such as miRNAs, proteins, and lncRNAs. 11,12 lncRNAs in serum exosomes can be stably detected and emerged as candidate biomarkers for the detection of HCC.
The colorectal neoplasia differentially expressed (CRNDE) gene locates on chromosome 16, and has been reported as an oncogene in several malignancies, such as colorectal cancer, 13 nonsmall cell lung cancer, 14 cervical cancer, 15 pancreatic cancer, 16 and papillary thyroid cancer. 17 However, to the best of our knowledge, the clinical significance of lncRNA CRNDE in serum exosomes of patients with HCC has not been analyzed. In this study, the ln-cRNA CRNDE expression levels in exosomes isolated from the serum samples of patients with HCC and healthy volunteers were detected. The application significance of serum exosomal lncRNA CRNDE as a biomarker for the detection and prognosis of HCC was assessed.

| Ethical approval
Signed informed consents were collected from all participants prior to the recruitment. The study protocol was approved by the Ethics Committee of Shulan International Medical College.

| Exosomal isolation
After blood was drawn from patients with HCC and healthy volunteers, serum was immediately isolated by centrifugation at 2000 g for 10 min at room temperature and then stored at −80°C until further use. Exosomes were isolated from serum using ExoQuick Exosome Precipitation Solution (System Biosciences). Briefly, serum was centri-

| Quantitative real-time PCR
The total RNA was extracted from the serum using an miRNeasy Serum/Plasma kit (Qiagen). In the RNA isolation step, 2 μl synthetic The relative expression levels of serum exosomal CRNDE were normalized against cel-miR-39 using the 2 -ΔΔCt method.

| Statistical analysis
All statistical calculations were performed using GraphPad Prism 9.0 p values less than 0.05 were considered statistically significant.

| Relationship between serum exosomal lncRNA CRNDE expression and clinical parameters
According to the median serum exosomal lncRNA CRNDE expres-

| The diagnostic efficiency of serum exosomal lncRNA CRNDE for HCC
Receiver operating characteristic curve analysis was performed to analyze the efficiency of serum exosomal lncRNA CRNDE as a diagnostic indicator for HCC detection. Figure 2 demonstrated that the sensitivity was 69.3% and the specificity was 85.0% with an AUC of 0.839 (95% CI = 0.794-0.885), indicating that serum exosomal lncRNA CRNDE could well identify HCC cases from normal controls.

| Relationship between serum exosomal lncRNA CRNDE expression and HCC prognosis
The Kaplan-Meier method was used to plot OS and DFS according to the serum exosomal lncRNA CRNDE levels. Patients with HCC in high serum exosomal lncRNA CRNDE expression group had significantly shorter OS (p = 0.0073, Figure 3A) and DFS (p = 0.0217, Figure 3B). Then, OS and DFS curves of patients with HCC stratified by cirrhosis were also plotted. The subgroup of patients with cirrhosis had significant shorter OS (p = 0.0070, Figure 3C) and DFS (p = 0.0024, Figure 3D) compared to those without cirrhosis.
To assess whether serum exosomal lncRNA CRNDE might serve as an independent prognostic marker for OS and DFS in HCC, the effects of serum exosomal lncRNA CRNDE expression levels and prognosis of patients was evaluated. As shown in LncRNA CRNDE upregulation was also found in colorectal cancer (CRC) tissues and associated with poor clinical variables.

| CON CLUS IONS
In conclusion, the study highlighted the potential role of serum exosomal lncRNA CRNDE for the diagnosis and prognosis of HCC. The results demonstrated that serum exosomal lncRNA CRNDE could serve as a novel predictive biomarker for the prognosis of HCC. The limitation of the study is the relatively small sample size, and future studies with a larger sample size will be required.

ACK N OWLED G EM ENTS
The study was supported by National S&T Major Project of China (No. 2018ZX10301201).

CO N FLI C T O F I NTE R E S T
No competing interests.

I N FO R M E D CO N S E NT
Signed informed consent was collected from all participants prior to the recruitment.

DATA AVA I L A B I L I T Y S TAT E M E N T
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.