Aberrant expression of lncRNA Sox2ot is associated with advanced tumor progression and poor prognosis in patients with colorectal cancer

Keqian Zhang Southwest Hospital, Army Medical University Tianqi Mao Southwest Hospital, Army Medical University Zhicheng He Southwest Hospital, Army Medical University Xiaojiao Wu Southwest Hospital, Army Medical University Yu Peng Southwest Hospital, Army Medical University Yanrong Chen Southwest Hospital, Army Medical University Yan Dong Southwest Hospital, Army Medical University Zhihua Ruan Southwest Hospital, Army Medical University Zhe Wang (  usioamk@yeah.net ) Southwest Hospital, Army Medical University https://orcid.org/0000-0001-6065-4143

and functions of Sox2ot in cancers, including lung cancer [17] and hepatocellular carcinoma [18]. Moreover, Liu et al. have shown that lncRNA Sox2ot could promote CRC cell proliferation and motility and knockdown of Sox2ot suppressed cell migration and invasion [19]. However, little is known about the signi cance of Sox2ot expression and its prognosis value in CRC.
In the present study, we investigated the expression level of lncRNA Sox2ot in human CRC tissues, and then explored the association between lncRNA Sox2ot expression and clinicopathological characteristics. In addition we also explored the prognosis value of lncRNA Sox2ot in CRC.

Ethics statement
The study was approved by Ethics Committee of Southwest Hospital, Army Medical University before it was initiated. All patients provided written informed consent to the surgical procedures and gave permission to use resected tissue specimens for research purposes. The procedures of the study were in accordance with the ethical standards of the committee on human experimentation of the institution.

Patients and tissue specimens
We collected 117 paired CRC tissues and adjacent non-cancerous tissues from patients who underwent surgery at Southwest Hospital, Army Medical University. The diagnosis of CRC was con rmed pathologically by two independent experienced pathologists. Besides, we retrospectively reviewed the medical records of 117 patients. All patients were given questionnaires about their symptoms and medical history before taking a physical exam. Patients with a previous history of cancer or who had already received surgery, chemotherapy, or radiotherapy were excluded from this study.
After treatment, postoperative 5-year follow-up constructed at our outpatient department. During the follow-up, the patients were evaluated at the hospital or contacted by telephone or letter every 3 months in the rst 3 years, every 6 months in the forth year and annually thereafter. The following data were collected from the patients for further investigation: general information, preoperative information, details of the surgery, pathology reports, TNM stage, and results of the follow-up. For follow-up purposes, the primary end point was the overall survival (OS), de ned as the time from surgery to mortality due to any cause. Adverse event was de ned as tumor progression or death.
Tumor tissues and adjacent normal tissues from 117 patients were extracted and snap froze in liquid nitrogen. Then all samples were stored at -80℃ until total RNA extraction.

RNA isolation and cDNA synthesis
Total RNA was extracted from all tissues samples with TRIzol ® Reagent (Invitrogen; Carlsbad, CA, USA), following the manufacturer's instructions. The concentration and puri cation of isolated RNA was evaluated with a NanoDrop ® ND-2000 Spectrophotometer (NanoDrop Technologies Inc.; Wilimington, DE, USA), Purity was estimated with the absorbance ratio 260nm/280nm (mean ratio=1.91; range, 1.52-2.37). cDNA was synthesized from RNA with the PrimeScript TM RT reagent Kit (Perfect Real Time; TaKaRa Bio Inc.; Tokyo, Japan) according to the manufacturer's instructions and stored at -80℃ for further use.

Quantitative real-time polymerase chain reaction (qRT-PCR)
The qRT-PCR reaction of lncRNA Sox2ot and GAPDH were performed using LightCycler ® 480 SYBR Green II real-time PCR system (Roche Applied Science) equipped with LightCycler ® 480 software according to manufacturer's instruction. The primers for lncRNA Sox2ot and GAPDH (internal control) were listed in Table 1. The following cycle conditions included: 94℃ 5 min; 30 cycles of denaturation at 94℃ for 30 s, annealing at 60℃ for 30 s and extension at 72℃ for 1 min; nally 72℃ 10 min. When the reactions were nished, expression levels of mRNA were automatically calculated using the number of cycle threshold (CT) and normalized to internal control GAPDH. Fold change in mRNA expression after normalization was calculated using 2 -∆∆CT method.

Statistical analysis
All the statistical analyses were performed with SPSS 19.0 software (SPSS, Inc., Chicago, IL, USA) and GraphPad Prism 5 software (GraphPad Software, Inc., San Diego, La Jolla, CA, USA). All data were presented as mean ± standard deviation (SD). Student's t-test was used to analyze the difference in lncRNA Sox2ot expression between the CRC tissues and adjacent non-cancerous tissues. The correlation between the expression levels of lncRNA Sox2ot and the clinicopathological features of CRC was analyzed using the Chi-square test. Survival curves for the patients were calculated using the Kaplan-Meier method. Prognostic factors were examined by univariate and multivariate analyses using Cox regression model. P<0.05 was considered as statistically signi cant.

Results
LncRNA Sox2ot expression was higher in CRC tissues than adjacent non-cancerous tissues We performed qRT-PCR to measure the expression of lncRNA Sox2ot in CRC tissues and adjacent noncancerous tissues. As shown in Figure 1, the expression level of lncRNA Sox2ot was signi cantly higher in CRC tissues (1.142±0.273) than in the adjacent non-cancerous tissues (0.668±0.281). Besides, there was a signi cant difference between two groups (P<0.001). These results indicated that lncRNA Sox2ot expression was abnormally elevated in CRC patients.

The correlations between lncRNA Sox2ot expression and clinicopathological characteristics of CRC patients
To further investigate whether the expression level of lncRNA Sox2ot was associated with the development of CRC, the relationship between its expression and clinicopathological features was assessed and summarized in Table 2. The results demonstrated that high lncRNA Sox2ot expression was signi cantly associated with N stage (P=0.002), T stage (P=0.001), TNM stage (P=0.008), histological differentiation grade (P=0.002), lymph node involvement (P=0.008) and distant metastasis (P=0.004). Meanwhile, there was no signi cant association between lncRNA Sox2ot expression and age, gender tumor size, tumor histology, primary tumor localization or Karnofsky performance status (all, P>0.05).

LncRNA Sox2ot expression is associated with overall survival in CRC patients
Kaplan-Meier analysis and log-rank test were performed to assess the prognostic value of lncRNA Sox2ot expression in CRC patients. Form the curve, we observed that patients with high expression level of lncRNA Sox2ot had signi cantly shorter survival time than those with low expression level of lncRNA Sox2ot (P<0.001, Figure 2). In addition, we also observed that lncRNA Sox2ot over-expression was an unfavorable prognostic factor in CRC patients (Table 3). Both univariate analysis and multivariate analysis showed that high lncRNA Sox2ot expression (P=0.000, HR=3.783, 95%CI=2.119-6.756) was an independent poor prognostic factor for CRC patients.

Discussion
CRC is a highly heterogeneous disease and the third leading cause of cancer-related death worldwide [20]. Despite recent diagnostic and therapeutic advances have improved the clinical outcomes of CRC patients with early stage, a large fraction of early stage CRC patients still develop recurrence or metastasis. Moreover, there are few reliable markers available to accurately predict metastasis in early stage CRC patients, and individual adjuvant treatment remains a challenge. Therefore, nding new molecular makers for early diagnosis, prognosis and treatment of CRC is of great importance to improve the outcome of this disease.
In recent years, lncRNAs have been increasingly reported to be involved in a number of important events, such as epigenetic regulation, transcriptional regulation, post-transcriptional regulation [21] and some human diseases [22,23]. Emerging evidence showed that lncRNAs may serve as diagnostic or prognostic bio-markers for some cancers [24]. In the present study, we investigated the expression and clinical signi cance of lncRNA Sox2ot in CRC patients. Our results showed that lncRNA Sox2ot expression in CRC tissues was signi cantly higher than that in matched adjacent non-cancerous tissues. The relationships of lncRNA Sox2ot with various clinical features of CRC were analyzed, we found that lncRNA Sox2ot expression was proven to be associated with N stage, T stage, TNM stage, histological differentiation grade, lymph node involvement and distant metastasis, suggesting that lncRNA Sox2ot might be involved in the carcinogenesis of CRC. Furthermore, Kaplan-Meier analysis with the log-rank test indicated that patients with a high level of lncRNA Sox2ot expression had signi cantly shorter overall survival than those with a low level of lncRNA Sox2ot expression. In Cox regression analysis, our results suggested that lncRNA Sox2ot expression level was independent prognostic factors for overall survival of CRC patients. All the results indicated that high lncRNA Sox2ot level was a promising non-invasive bio-marker for prognosis of CRC patients. Moreover the further studies are needed to elucidate the the mechanism of lncRNA Sox2ot in CRC.

Conclusion
In conclusion, our data suggested that lncRNA Sox2ot up-regulation was associated with aggressive progression and poor prognosis in CRC. LncRNA Sox2ot could be used as a new biomarker and a potential therapeutic target for CRC.

Declarations
Ethics approval and consent to participate This study was supported by the Ethics Committee of Southwest Hospital, Army Medical University and also has been carried out in accordance with the World Medical Association Declaration of Helsinki.

Consent for publication
The subjects provided written informed consent for the publication of any associated data and accompanying images.    Figure 1 Relative expression of lncRNA Sox2ot in CRC tissues and adjacent non-cancerous tissues measured by qRT-PCR assay The relative mRNA expression of lncRNA Sox2ot in CRC tissues were signi cantly higher than that in controls. *, P<0.05.