Circular RNA mitochondrial translation optimization 1 correlates with less lymph node metastasis, longer disease‐free survival, and higher chemotherapy sensitivity in gastric cancer

Abstract Objective Circular‐mitochondrial translation optimization 1 (circ‐MTO1) inhibits the progression of gastric cancer by regulating the growth, apoptosis, and invasion of tumor cells. However, its clinical potential as a biomarker for gastric cancer remains to be further evaluated. This study aimed to assess circ‐MTO1 expression and its correlation with clinical features and prognosis in gastric cancer patients, as well as the effect of circ‐MTO1 on the sensitivity to chemotherapy in gastric cancer cells. Methods Circ‐MTO1 in tumor and adjacent tissues of 97 gastric cancer patients undergoing resection was examined by reverse transcription‐quantitative polymerase chain reaction. HGC‐27 and NCI‐N87 cells transfected by circ‐MOT1 overexpression plasmid (OE‐circ‐MOT1) and negative control (OE‐NC) were treated with 0‒6.4 μM oxaliplatin. Relative cell viability was detected using Cell Counting Kit‐8. Results Circ‐MTO1 was insufficiently expressed in gastric tumor tissue (median (interquartile range): 0.403 (0.288‒0.518)) compared with adjacent tissue (median (interquartile range): 1.000 (0.715‒1.524)) (p < 0.001). Besides, tumor circ‐MTO1 was correlated with less lymph node metastasis (p = 0.014) and low TNM stage (p = 0.039), while was not correlated with demographic features or other clinical characteristics (all p > 0.05). Furthermore, tumor circ‐MTO1 high expression was independently correlated with prolonged disease‐free survival (DFS) (p = 0.013, adjusted hazard ratio (95% confidential interval): 0.314 (0.126‒0.782)), but was not correlated with overall survival (p > 0.05). Lastly, in gastric cancer cells, OE‐circ‐MTO1 apparently decreased relative cell viabilities at oxaliplatin concentrations of 0.4, 0.8, 1.6, and 3.2 μM (all p < 0.05). Conclusion Circ‐MTO1 correlates with less lymph node metastasis, prolonged DFS, and improved chemotherapy sensitivity in gastric cancer.


| INTRODUC TI ON
Gastric cancer, originating from gastric mucosa epithelium, is a worldwide health burden since it is one of the top leading causes of cancer-related mortality in humans 1,2 with over 950,000 newly diagnosed cases around the world annually (approximately 60% of new cases in east Asia such as China, Japan, and Korea). 3 Over the past decades, progresses on the screening and diagnosis of gastric cancer bring an increased number of early-stage gastric cancer patients. 4,5 These early-stage patients, together with intermediate patients with gastric cancer, are recommended to be treated with gastric resection. [6][7][8] Besides, neoadjuvant or adjuvant treatments such as chemoradiotherapy are supposed to be applied to creating conditions for surgical resection. 9,10 In addition, a huge number of studies are devoted to making contributions to the treatment for advanced gastric patients including chemotherapy, targeted therapy, and immunotherapy. [11][12][13][14] In spite of these, patients still commonly experience dissatisfied survivals. 15 Therefore, finding biomarkers for monitoring the progression and prognosis of gastric cancer is important for more accurate treatment and better prognosis.
This study aimed to assess the expression of circ-MTO1, its correlation with clinical characteristics, and prognosis in postoperative patients with gastric cancer, as well as the effect of circ-MTO1 on oxaliplatin sensitivity in gastric cancer cell lines.

| Patients
Ninety-seven patients with gastric cancer who underwent surgical resection in our hospital from January 2016 to December 2019 were retrospectively included in this study. All patients were screened out from the electronic medical record system (EMRS), according to the following criteria: (i) pathologically confirmed as gastric cancer; (ii) received surgical resection without neoadjuvant therapy; (iii) had freshly frozen specimens including tumor and adjacent tissue; (iv) had complete clinicopathological information and follow-up records; and (v) without other primary cancers or malignant diseases.
Approval was obtained from the Ethical Inspection Committee, and the informed consent was signed by patients or their relatives.

| Data acquisition
Clinical features were collected from the EMRS, including demographics, smoke and drink status, complications, helicobacter pylori infection status, tumor location, pathological grade, tumor size, clinical T stage, clinical N stage, clinical TNM stage, and adjuvant treatment (chemotherapy with XELOX regimen or S-1 monotherapy, with or without radiotherapy). In addition, follow-up information of patients was also reviewed to abstract the main time points for estimation of progression-free survival (PFS) and overall survival (OS).

| Circ-MTO1 determination
The freshly frozen tumor and adjacent tissues of patients were acquired from the sample library of the hospital, which were available for reverse transcriptase quantitative polymerase chain reaction (RT-qPCR). Briefly, total RNA was extracted from tumor or adjacent tissues by TRIzol™ Reagent (Thermo Fisher Scientific), followed by the removal of linear RNA with RNase R (Epicentre), and reverse transcription into cDNA using PrimeScript™ RT reagent Kit (Perfect Real Time) (Takara). Subsequently, qPCR was conducted by TB Green™ Fast qPCR Mix (Takara) to quantify the circ-MTO1 expressions, which was calculated by 2 −ΔΔCt method with glyceraldehyde-

3-phosphate dehydrogenase (GAPDH) as an internal reference.
Primers for PCR were designed according to previous studies. 22,23

| Cell culture
The gastric cancer cell lines including HGC-27 and NCI-N87 were purchased from Cell Blank of Chinese Academy of Sciences (Shanghai). The cells were all cultured in the 90% RPMI1640 medium (Gibco) with 10% Fetal Bovine Serum (Gibco) and were maintained in at 37℃ in a humidified atmosphere of 5% CO 2 .
Meanwhile, the cells without transfection were used as blank controls.
After transfection, all cells including OE-circ-MTO1, OE-NC, and blank cells were treated with oxaliplatin (Sigma-Aldrich) at the following concentrations: 0, 0.2, 0.4, 0.8, 1.6, 3.2, and 6.4 μM. After incubation for 48 h, the relative cell viability in each cell group was detected using Cell Counting Kit-8 (Beyotime) as per the instruction of the kit.

| Circ-MTO1 expression in tumor tissue and adjacent tissue
Circ-MTO1 expression was obviously lower in tumor tissue than in adjacent tissue of gastric cancer patients (p < 0.001). The median (IQR) value of circ-MTO1 expression in tumor tissue was 0.403 (0.288-0.518), while that in adjacent tissue was 1.000 (0.715-1.524) (Figure 1).

| Correlation of tumor circ-MTO1 with accumulating DFS and OS
According to the median of circ-MTO1 expression in gastric tumor tissues (Figure 1), tumor circ-MTO1 expression was divided by high (over 0.403) and low (below 0.403) expressions. In gastric cancer patients, tumor circ-MTO1 high expression was associated with better accumulating DFS (p = 0.027) ( Figure 3A). In terms of accumulating OS, no correlation of tumor circ-MTO1 with OS was found (p > 0.05) ( Figure 3B).  Figure 4B).

| DISCUSS ION
The main findings were listed as follows: 1) Circ-MTO1 was down- CircRNAs, widely presenting in transcriptomes, play vital roles in the regulation of various biological activities. 25 Circ-MTO1 is one of the commonly studied circRNAs, which acts as a cancer-suppressor gene involved in a variety of cancers. [19][20][21][22] circ-MTO1 suppresses the growth and invasion of gastric, colorectal, and prostate cancer cells. [19][20][21][22] Moreover, it is insufficiently expressed in hepatocellular tumor tissues. 21 In this study, circ-MTO1 was down-regulated in gastric tumor tissue compared with adjacent tissue, which could be explained by that overexpression of circ-MTO1 inhibited the proliferation of gastric cancer cells via multiple mechanisms such as regulating miR-199a-3p/PAWR axis, miR-3200-5p/PEBP1 axis, miR-9/p21 axis, and Wnt/β-catenin signaling pathway. 19,[21][22][23] The viability of gastric cancer cells was decreased, leading to its down-regulation in gastric tumor tissue compared with adjacent tissues.
Circ-MTO1 is clinically correlated with tumor characteristics. 19,20 For example, a study shows that insufficient circ-MTO1 expression is associated with advanced lymph node metastasis and TNM stage in colorectal cancer patients. 19 Another study exhibits that circ-MTO1 is associated with decreased pathological T stage and N stage in patients with prostate cancer. 20   Furthermore, no correlation of circ-MTO1 with OS in gastric cancer patients was observed in this study. This was attributed by that the number of death events was too small, leading to the low statistical power. Further study with long follow-up duration was supposed to be conducted in the future.
It could not be ignored that there were a few limitations in this study. Firstly, the sample size was relatively small, which might lead to poor statistical power. Further study with a larger sample size was supposed to be performed. Secondly, specific mechanism about how circ-MTO1 regulated gastric cancer progression was still unclear and was required to be extensively explored in the future. Lastly, since our study was a cohort study, selection bias and confounding factors such as diversified resection treatments and tumor status might exist.
Conclusively, circ-MTO1 correlates with less lymph node metastasis, prolonged DFS, and increased chemotherapy sensitivity in gastric cancer. This study provides evidences for selecting optimized treatment strategies and improving prognosis for gastric cancer patients.

ACK N OWLED G EM ENTS
None.

CO N FLI C T O F I NTE R E S T
No potential conflict of interest was reported by the authors.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available in this article.