Circulating Long Non-Coding RNAs LINC00324 and LOC100507053 as Potential Liquid Biopsy Markers for Esophageal Squamous Cell Carcinoma: A Pilot Study

Abstract

Background: Despite the availability of advanced technology to detect and treat esophageal squamous cell carcinoma (ESCC), the 5-year survival rate of ESCC patients is still meager. Recently, long non-coding RNAs (lncRNAs) have emerged as essential players in the diagnosis and prognosis of various cancers.

Objective: This pilot study focused on identifying circulating lncRNAs as liquid biopsy markers for the ESCC.

Methodology: We performed next-generation sequencing (NGS) to profile circulating lncRNAs in ESCC and healthy individuals' blood samples. The expression of the top five upregulated and top five downregulated lncRNAs were validated through quantitative real-time PCR (qRT-PCR), including samples used for the NGS. Later, we explored the diagnostic/prognostic potential of lncRNAs and their impact on the clinicopathological parameters of patients. To unravel the molecular target and pathways of identified lncRNAs, we utilized various bioinformatics tools such as lncRnome, RAID v2.0, Starbase, miRDB, TargetScan, Gene Ontology, and KEGG pathways.

Results: Through NGS profiling, we obtained 159 upregulated, 137 downregulated, and 188 neutral lncRNAs in ESCC blood samples compared to healthy individuals. Among dysregulated lncRNAs, we observed LINC00324 significantly upregulated (2.11-fold; p-value = 0.0032) and LOC100507053 significantly downregulated (2.22-fold; p-value = 0.0001) in ESCC patients. Furthermore, we found LINC00324 and LOC100507053 could discriminate ESCC cancer patients' from non-cancer individuals with higher accuracy of Area Under the ROC Curve (AUC) = 0.627 and 0.668, respectively. The Kaplan-Meier and log-rank analysis revealed higher expression levels of LINC00324 and lower expression levels of LOC100507053 well correlated with the poor prognosis of ESCC patients with a Hazard ratio of LINC00324 = 2.48 (95% CI: 1.055 to 5.835) and Hazard ratio of LOC100507053 = 4.75 (95% CI: 2.098 to 10.76)]. Moreover, we also observed lncRNAs expression well correlated with the age (>50 years), gender (Female), alcohol, tobacco, and hot beverages consumers. Using bioinformatics tools, we saw miR-493-5p as the direct molecular target of LINC00324 and interacted with the MAPK signaling pathway in ESCC pathogenesis.

Conclusion: This pilot study suggests that circulating LINC00324 and LOC100507053 can be used as a liquid biopsy marker of ESCC; however, multicentric studies are still warranted.

Keywords: ESCC; LncRNA; biomarkers; esophageal squamous cell carcinoma; long non-coding RNA; next generation sequencing.

Figure 1

Summary of differentially expressed lncRNAs identified in esophageal squamous cell carcinoma (ESCC) patients’ blood samples. (A) Venn diagram showing the distribution of differentially expressed lncRNAs in ESCC. (B) Heatmap showing the list of differentially expressed lncRNAs according to their Log2 Fold change (p-value < 0.05). (C) Top five upregulated lncRNAs in ESCC. (D) Top five downregulated lncRNAs in ESCC. (E) Relative expression of LINC00324 using TCGA database (F) Normalized expression of LINC00324 (ΔCt values). (G) ROC curve to demonstrate the diagnostic potential of LINC00324. (H) Relative expression of LINC00324 using TCGA database. (I) Normalized expression of LOC100507053 (ΔCt values). (J) ROC curve to demonstrate the diagnostic potential of LOC100507053. Experiments were performed in triplicates for at least three independent times. Data are presented as the mean ± SEM. *represents p<0.05, **represents p<0.01 and calculated using a Mann Whitney test. ΔCt value was used to show relative expression of LINC00324 and LOC100507053 using ΔCt = Ct(Mean Ct value of LncRNA target - Mean Ct value of GAPDH). Small ΔCt value indicates higher expression while large ΔCt value indicates lower expression.

Figure 2

Association of LINC00324 expression with ESCC patients’ lifestyle status and clinicopathological characteristics compared to healthy individuals. (A) Age of ESCC patient. (B) Gender of ESCC patients. (C) Tobacco smoking status of ESCC patients. (D) Alcoholic status of ESCC patients. (E) Consumption of hot beverages status. (F) Histopathological grading of the ESCC patients. (G) TNM staging of the ESCC patients. A Scatter plot with Bar graphs represents the normalized expression of LINC00324 in ESCC patients compared to healthy individuals. The data are expressed as mean ± SEM where *represents p-value < 0.05, **represents p-value < 0.01, ***represents p-value < 0.001, and ****represents p-value < 0.0001 calculated using unpaired (Mann-Whitney test), paired t-tests and Chi-square test. (H) Kaplan-Meier survival plot represents the relationship between the levels of LINC00324 expression and survival percentage of ESCC patients [TNM: tumor node metastasis].

Figure 3

Association of LOC100507053 expression with ESCC patients’ lifestyle status and clinicopathological characteristics compared to healthy individuals. (A) Age of ESCC patient. (B) Gender of ESCC patients. (C) Tobacco smoking status of ESCC patients. (D) Alcoholic status of ESCC patients. (E) Consumption of hot beverages status. (F). Histopathological grading of the ESCC patients. (G) TNM staging of the ESCC patients. A scattered plot with Bar graphs represents the normalized expression of LOC100507053 in ESCC patients compared to healthy individuals. The data are expressed as mean ± SEM where *represents p-value < 0.05, **represents p-value < 0.01, ***represents p-value < 0.001, and ****represents p-value < 0.0001 calculated using unpaired (Mann-Whitney test), paired t-tests and Chi-square test. (H) Kaplan-Meier survival plot represents the relationship between the levels of LOC100507053 expression and survival percentage of ESCC patients.

Figure 4

In silico target prediction of LINC00324 and LOC100507053 using online databases. (A) hsa-miR-493-5p predicted as miRNA target of LINC00324 from Starbase, lncRnome, and RAID v2.0 databases. (B) 399 mRNA targets of hsa-miR-493-5p predicted from Starbase, miRdb, and TargetScan databases. (C) 365 mRNA targets of LOC100507053 predicted from the RAID v2.0 database. (D–G) Gene ontology (GO) biological, cellular, biological processes and KEGG pathway of predicted target has-miR-493-5p of LINC00324..

Figure 5

Co-expression network of LINC00324 and LOC100507053. (A) A total of nine lncRNAs and twelve mRNAs interacted with LINC00324. (B) A total of three lncRNAs and eighteen mRNAs interacted with LOC100507053.