The Association Between lncRNA LRRC75A-AS1 and The Clinical Characteristics in Neuroblastoma

Neuroblastoma is the highest mortality rate extracranial soild tumor in childhood. Accumulating evidence indicated that long noncoding RNAs (lncRNAs) are widely expressed in neuroblastoma, and playing an important role in the development and progression. Methods: RNA sequencing was conducted to identify differentially expressed lncRNAs in four Ⅲ phase and four Ⅳ phase tumor tissues of neuroblastoma. RT-qPCR was carried out to validate the result of sequencing. Clinical information was reviewed to analyze the relationship between lncRNA and clinical characteristics. The public da-tabase R2 was used to analyze prognosis. Result: Differentially expressed lncRNAs were identified. LRRC75A-AS1 was the overexpressed lncRNA in Ⅳ phase patients. RT-qPCR was conducted in tumor tissues, confirming the tendency with sequencing. And higher expression of LRRC75A-AS1 was associated with N-MYC (p < 0.001), advanced stage (p = 0.029), Risk group (p = 0.027). Furthermore, LRRC75A-AS1 was correlated with Shimada classification(p = 0.046), LDH level (r = 0.390, p = 0.003), D-Dimer level (r = 0.338, p = 0.012) , and NSE level (r = 0.284, p = 0.05). The neuroblastoma dataset shows that patients with overexpressed LRRC75A-AS1 have a worse prognosis than down-expressed. Conclusion: LRRC75A-AS1 is associated with clinical characteristics of neuroblastoma and may function as a prognostic predictor or a therapeutic target.


Introduction
Neuroblastoma (NB) is a sympathetic embryonic tumor originating from the neural crest of the embryonic sympathetic nervous system,it is the most common extracranial solid tumor in children which accounts for 7-10% of all childhood cancer mortality [1][2][3][4][5]. In order to take tailored treatment approaches for neuroblastoma, pediatric cooperative groups introduce risk factors including clinical stage, age, histologic category, grade of tumor differentiation, MYCN status, DNA ploidy, and 11q exception [6,7]. High-risk neuroblastoma patients often have unfavorable outcomes, with the 5-year overall survival rate less than 50% [6].
The application of genetic difference analysis promote accurate stratification has attracted widespread attention. Therefore, it is of great practical significance and theoretical value to explore effective drug targets and better biomarkers for advanced neuroblastoma.
Long noncoding RNAs (lncRNAs) refer to endogenous RNAs that are longer than 200 nucleotides and lack of specific complete open reading frames (ORFs) and the function of protein-coding [8,9].
Thus they were once considered a part of transcriptional noise, but now have been proved as potential key regulators of promoting or maintaining tumorigenesis and the development of cancer, having clinical potential as prognostic biomarkers for targeted therapeutics and interventions in various cancers [4,10]. Several lines of evidence have shown that lncRNAs have been implicated in initiation and progression of neuroblastoma [5,6], and lncRNAbased prognostic biomarkers have been proposed for tumor stratification and predicting survival. Therefore, deep investigating of the roles and mechanisms of lncRNAs in tumorigenesis provides promises in developing new biomarkers and molecular-targeted therapy. Our research aims to identify lncRNA-based biomarkers DOI: 10.26717/BJSTR.2020.28.004597 21268 that could be used for prognosis prediction and treatment. The purpose of this study was to explore the relationship between lncRNAs and clinicopathological parameters in neuroblastoma patients, to further explore the lncRNAs that lead to the invasion and metastasis of neuroblastoma. In the first, we conducted RNAsequencing to identify differentially expressed lncRNAs in 4 Ⅲ stage and 4 Ⅳ stage patients' tumor tissues of neuroblastoma, and we identified a lncRNA named LRRC75A-AS1 was upregulated lncRNAs in Ⅳ stage neuroblastomas, Futher to explore the relationship between LRRC75A-AS1 and clinical characteristics ,RT-qPCR was carried out to detect 57 cases of neuroblastoma.  c) The quality of tissues was unqualified.

Patient
Clinical features of these patients at diagnosis including age, gender, tumor size, INSS stage, risk group, MYCN status, tumor biomarkers, and metastasis were retrospectively collected. All fresh tissue specimens were preserved in −80℃ until use. R2: Genomics Analysis and Visualization Platform (http://r2.amc.nl) was used to investigate the relationship between lncRNA expression and overall survival with neuroblastoma patients.

Expression Profile Analysis of RAN-Sequencing
The RAN-sequencing was employed to identify neuroblastoma-

RNA Extraction and Real Time qRT-PCR Analysis
Total RNA of specimens was extracted using RNA extraction reagent kit (Bio Teke). RNA concentration and purity were measured

Relations Between Expression Level of LRRC75A-AS1 and Prognosis in Neuroblastoma Patients
Owing    [5,6,11]. Therefore, lncRNA have the potential to serve as novel biomarkers for neuroblastoma diagnosis or prognosis. However, the biological functions of most lncRNAs have yet to be explored. High-risk neuroblastoma patients often have unfavorable outcomes, it is one of the biggest obstacles to improve overall survival of neuroblastoma. Hence, it is urgent to investigate the novel genes and illustrate the molecular mechanisms of neuroblastoma. The main purpose of this study was to investigate the differential expression of LRRC75A-AS1 in neuroblastoma, and to find new prognostic and diagnostic markers for neuroblastoma.
The results of this study show that LRRC75A-AS1 is up-regulated in neuroblastoma.
Emerging reports has revealed that LRRC75A-AS1 was involved in several biological processes through modulation of signaling pathway, Jeong et al. [13] reported that LRRC75A-AS1 can regulate the vascular calcification negatively, and might act as a possible target in the treatment of vascular calcification. Wang et al. [14] found that LRRC75A-AS1 can regulate the expression of tight junction (TJ) proteins through LRRC75A, affecting the inflammatory responses of bovine mammary epithelial cells. Leavey K et al. [15] show that LRRC75A is abnormally expressed in the process of normal villous maturation.
However, little information of the prognostic value and the role of LRRC75A-AS1 in neuroblastoma has been reported.
In the present study, we compare the expression profile of lncRNA between 4 Ⅲ phase and 4 Ⅳ phase tumor tissues of neuroblastoma by using the RNA-sequencing. We found that

Statement of Ethics
This research complies with the guidelines for human studies.
This study approved by the ethics committee of the Children's Hospital of Chongqing Medical University and subjects (or their parents or guardians) have given their written informed consent.

Disclosure Statement
The authors have no conflicts of interest to declare