Low expression of hsa_circ_0001811 in gastric cancer and its role in clinical diagnosis

Abstract Background As far as gastric cancer is concerned, there is a lack of specific early diagnostic biomarkers in clinical diagnosis. Circular RNAs (circRNAs) have been found to be stable in gastric cancer tissues and plasma, so they have the potential to become new type of diagnostic biomarkers for gastric cancer. Materials and Methods The hsa_circ_0001811 expressions in gastric cancer tissues and paired non‐cancer tissues, preoperative and postoperative plasma of patients with gastric cancer, and plasma of healthy volunteers were detected using quantitative reverse transcription‐polymerase chain reaction. The receiver operating characteristic (ROC) curves were drawn; and the combined ROC curves were used to analyze their diagnostic values. The correlation between the plasma or tissue hsa_circ_0001811 levels and the clinicopathological factors of gastric cancer was further analyzed. Results Hsa_circ_0001811 was found to be lowly expressed in gastric cancer tissues and plasma from patients with gastric cancer. The area under the ROC curve (AUC) in the gastric cancer tissues and the plasma of patients with gastric cancer was 0.658 and 0.747, respectively. The AUC increased to 0.824 after combination of both. The expression level of hsa_circ_0001811 in gastric cancer tissue correlated with carcinoembryonic antigen (CEA) (P = .0347), tissue differentiation (P = .0138), and lymph node metastasis (P = .0234), while plasma hsa_circ_0001811 level was related to carbohydrate antigen (CA19‐9) (P = .0278), lymph node metastasis (P = .0469), distant metastasis (P = .0384), and age (P = .0085). Conclusions The above results indicate that hsa_circ_0001811 may become a new biomarker for clinical diagnosis of gastric cancer.

particularly important to search for new markers for early diagnosis of gastric cancer. 5 Circular RNAs (circRNAs) are a type of new endogenous RNA produced by non-canonical reverse splicing events. 6 They have no free 5 'and 3' ends and no poly-A tail structure, but form a circular structure with covalent bonds. 7 Therefore, compared with linear RNAs, circRNAs are not easily degraded by exonuclease, can exist stably in vivo, 8 and are highly conservative, indicating that circRNAs have obvious advantages as a new diagnostic biomarker. 9 In addition to stability and conservation, the abundance and tissue-specific expression of circRNA also make it an obvious advantage as a new diagnostic biomarker.
In our previous study, we used circRNA microarray to detect the expression profile of circRNAs in gastric cancer tissues and matched non-cancer tissue samples. 10 hsa_circ_0001811 is one of the significant differently expressed circRNAs between gastric cancer tissues and matched non-cancer tissues. The gene of hsa_circ_0001811 is located at 8q21.11 (chr8:74585342-74601048), and its related gene symbol is staufen double-stranded RNA binding protein 2 (STAU2).

| Clinical specimens and pathological data
In this study, 100 pairs of gastric cancer tissues and matched noncancer tissues, 42 preoperative and postoperative plasma samples of gastric cancer patients, and 42 healthy volunteer plasma samples were obtained from Affiliated People's Hospital of Ningbo University.
All tissue specimens were obtained by experienced surgeons. Tissues were diagnosed by pathology, and the tumor staging is based on the International Cancer Alliance's Tumor-Node-Metastasis (TNM) staging system. The histological grade was evaluated according to the National Comprehensive Cancer Network (NCCN) oncology clinical practice guidelines (V.1.2011). 11 Immediately after obtaining, the tissues were put into RNA-fixer Reagent (Kang Wei) and then stored at −80℃ until use. The patients involved signed an informed consent, and the study was approved by the Ningbo University Medical Ethics Committee.

| Total RNA extraction
When extracting RNA from all tissue samples, the soybean size was taken, crushed and abstracted by TRIzol reagent (Invitrogen), while plasma samples were abstracted by TRIzol LS reagent (Invitrogen).
Then, the concentration and purity of RNA were measured via Smart Spec Plus Spectrophotometer (BioRad).

| Reverse transcription
According to the GoTaq® 2-Step RT-qPCR System (Promega) kit instructions, reverse transcription was performed.

| Statistical analysis
The data statistical analysis was mainly used GraphPad Prism 6 (GraphPad Software) and SPSS 26.0 (SPSS). Chi-squared test was used for clinical-pathological data analysis; paired sample t test was used for analysis hsa_circ_0001811 expression level between gastric cancer tissues and adjacent tissues, and plasma of patients with gastric cancer before and after the operation, while independent sample t test was used for unpaired samples. The experimental data were expressed as mean ± standard deviation. All experiments in this study were repeated three times. When P < .05, the difference was considered as statistically significant.

| Determination of specific primers for hsa_ circ_0001811
PCR products were sequenced by BGI (Shanghai, China). Sanger sequencing results are shown in Figure 1. After comparison with the circBase, the PCR product sequence is consistent with the target cir-cRNA; that is, this primer is a specific primer of hsa_circ_0001811.

| Hsa_circ_0001811 was lowly expressed in gastric cancer tissues and plasma from patients with gastric cancer
We used RT-PCR to detect the expression levels of hsa_circ_0001811 in 100 pairs of gastric cancer tissues and adjacent tissues, and fasting plasma of 42 gastric cancer patients one day before operation and ten days after operation and healthy volunteers. The results showed F I G U R E 2 Expression levels of hsa_circ_0001811 in gastric cancer tissues and plasma. The expression levels of hsa_circ_0001811 were significantly downregulated in gastric cancer tissues and plasma. Larger ΔC t value indicates lower expression. **** P < .0001, A: n = 100, B: n = 42 F I G U R E 3 ROC curve of hsa_ circ_0001811 that compared with paired non-cancer tissues, hsa_circ_0001811 was low expressed in gastric cancer tissues ( Figure 2A); the expression of plasma specimens one day before surgery in gastric cancer patients was lower than those in ten days after surgery and plasma samples of healthy volunteers ( Figure 2B).

| Potential diagnostic value of hsa_circ_0001811 in gastric cancer
The above experimental results prove that the expression of hsa_ circ_0001811 in gastric cancer tissues and adjacent tissues, plasma of patients with gastric cancer, and healthy volunteers are significantly different ( Figure 2). Therefore, we first draw the ROC curve of hsa_circ_0001811 in tissues and plasma and the combination, and obtained the area under ROC curve (AUC), specificity and sensitivity.
As shown in Figure 3, the AUC of hsa_circ_0001811 in gastric cancer tissues was 0.658, while the AUC in plasma was 0.747. When tissues and plasma were combined used, the AUC increased to 0.824, further showing the higher diagnostic value of hsa_circ_0001811.
Then, to further explore its clinical diagnostic value, the relationship between the differential expression of hsa_circ_0001811 in gastric cancer tissues or plasma and the clinical-pathological factors of gastric cancer patients was analyzed. As shown in The bold values indicate statistically significant.
These results indicate that the lower the expression level of hsa_ circ_0001811 in tissues or plasma, the more obvious these indicators involved.

| D ISCUSS I ON
CircRNAs were first identified as faulty spliced transcripts in viruses. 12 CircRNAs can be divided into three types, exon type ecir-cRNAs, 24 intron type ciRNAs, 25 exon-intron type also known as In this study, we focused on hsa_circ_0001811, which belongs to ecircRNAs and is composed of two exons. We first designed its specific primers for hsa_circ_0001811. On this basis, we expanded the tissue sample size to 100 pairs. The results indicated that hsa_circ_0001811 was lowly expressed in gastric cancer tissues compared to adjacent non-cancer tissues ( Figure   The bold values indicate statistically significant. metastasis (Table 1), while plasma hsa_circ_0001811 level was related to CA19-9, lymph node metastasis, and distant metastasis and age (Table 2). These results indicate that it may become a new diagnostic biomarker for gastric cancer.
Because circRNAs are more stable and highly conservative in tissues and plasma than other RNAs, such as long non-coding RNA (ln-cRNA) and miRNA, these characteristics make circRNAs an effective new biomarker for non-invasive diagnosis of gastric cancer.

| CON CLUS ION
In conclusion, in this study, the data we obtained showed that hsa_ circ_0001811 is low expressed in gastric cancer tissues and plasma, and its expression level can be detected by qRT-PCR. These findings indicate that hsa_circ_0001811 may be a new potential biomarker for the diagnosis of gastric cancer.