LncRNA HCP5 : A Potential Biomarker for Diagnosing Gastric Cancer

Abstract

Background: It has been reported that long non-coding RNAs (lncRNAs) can be regarded as a biomarker and had particular clinical significance for early screening and gastric cancer (GC) diagnosis. Therefore, this study aimed to investigate whether serum HCP5 could be a new diagnostic biomarker.

Methods: Filtered out the HCP5 from the GEO database. The specificity of HCP5 was verified by real-time fluorescence quantitative PCR (qRT-PCR), and then the stability of HCP5 was verified by room temperature storage and repeated freeze-thaw experiments. Meanwhile, the accuracy of HCP5 was verified by agarose gel electrophoresis (AGE) and Sanger sequencing. Simultaneously, the expression level of serum HCP5 was detected by qRT-PCR in 98 patients with primary gastric cancer, 21 gastritis patients, 82 healthy donors, and multiple cancer types. Then, the methodology analysis was carried on. Moreover, receiver operating characteristic (ROC) was used to evaluate its diagnostic efficiency.

Results: qRT-PCR method had good repeatability and stability in detecting HCP5. The expression level of HCP5 in the serum of gastric cancer patients was remarkably higher than that of healthy controls, and it could distinguish gastritis patients from healthy donors. Besides, the expression of HCP5 was increased dramatically in MKN-45 and MGC-803. The FISH assay showed that HCP5 was mainly distributed in the cytoplasm of MKN-45 and BGC-823 cells. When HCP5 was combined with existing tumor markers, the diagnostic efficiency of HCP5 was the best, and the combined diagnosis of carcinoembryonic antigen (CEA), carbohydrate antigen199 (CA199), and HCP5 can significantly improve the diagnostic sensitivity. Besides, compared with the expression levels of thyroid cancer (THCA), colorectal cancer (CRC), and breast cancer (BRCA), serum HCP5 in gastric cancer was the most specific. Moreover, the high expression of serum HCP5 was related to differentiation, lymph node metastasis, and nerve invasion. The term of serum HCP5 after the operation was significantly lower than that of patients with primary gastric cancer.

Conclusion: Serum HCP5 can be used as a potential biomarker of non-invasive fluid biopsy, which had a unique value in the early diagnosis, development, and prognosis of gastric cancer.

Keywords: GC; HCP5; biomarker; diagnosis; gastric cancer; gastritis.

Figure 1

Methodology evaluation of HCP5 in GC serum samples. (A, B) Standard curves of serum HCP5 and 18S in a ten-fold serial dilution to show the linearity. (C, D) Stability of HCP5 under room temperature incubation time or multiple freeze-thaw cycles. Data were presented as raw Ct value (n=3). (E) The specificity of PCR products by the melting curve. (F) The validation of PCR products by the agarose gel electrophoresis. (G) The product sequence was verified by Sanger sequencing.

Figure 2

Concentrations of serum HCP5, CEA, and CA199 in GC cases and diagnostic efficacy. (A) Detection of serum HCP5 in GC patients (n=98), healthy donors (n=82). (B) Detection of serum HCP5 in GC (n=98) and gastritis (n=21) patients. (C) The concentrations of serum CEA. (D) The concentrations of serum CA199. ^*P < 0.05, ^**P < 0.01, ^****P < 0.001, NS means no statistically difference. (E) The ROC curve of serum HCP5 in GC cases. (F) The ROC curve of serum CEA in GC cases. (G) The ROC curve of serum CA199 in GC cases. (H) The diagnostic efficacy of the combined diagnosis.

Figure 3

The origin of serum HCP5 in GC cases. (A) The expression level of HCP5 in 20 pairs of GC tissues. (B) HCP5 was secreted into the culture medium by MKN-45 and MGC-803 cells in a time-dependent manner. *P < 0.05, **P < 0.01, ****P < 0.001. (C) The FISH assay of HCP5 in MKN-45 and BGC-823 cells. Scale bars: 20μm.

Figure 4

The specificity of serum HCP5 in GC and differentiating from gastritis and healthy donors. (A) Detection of serum HCP5 in 19 thyroid cancer patients. (B) Detection of serum HCP5 in 20 colorectal cancer patients. (C) Detection of serum HCP5 in 17 breast cancer patients. (D) The expression levels of HCP5 in gastritis patients (n=21) and healthy donors (n=21). (E) The concentrations of CEA in gastritis patients (n=21) and healthy donors (n=21). (F) The concentrations of CA199 in gastritis patients (n=21) and healthy donors (n=21). *P < 0.05, **P < 0.01, NS means no statistically difference.

Figure 5

Serum HCP5 in monitoring tumor dynamics in GC patients. (A) Detection of serum HCP5 expression in GC pre-operation patients (n=98), post-operation patients (n=46) and recurrence patients (n=57). (B) Altered expression of serum HCP5 in 15 paired samples preoperatively and postoperatively. (C) The survival curve of patients with GC. ^*P < 0.05, ^**P < 0.01, ^****P < 0.001, NS means no statistically difference.