Clinicopathological Features' Correlation and Genes’ Expression of NEAT1, lincRNA-ROR and Oct4 in Iranian Patients with Gastric Cancer

Purpose Long non-coding RNAs (LncRNAs) play a critical role in the initiation and development of Gastric Cancer(GC). The aim of this study was to consider the expression of NEAT1, lincRNA-ROR and Oct4 and nally evaluate the correlation between their expression and clinical characteristics in Iranian patients with GC. This cross-sectional study was performed on 41 gastric tumor tissue samples with matched normal adjacent tumor tissues. The RNA level of lncRNA NEAT1 and lincRNA ROR and Oct4 genes were assessed using the quantitative Real-time polymerase chain reaction. B2M was used as an internal control. Also, the relative expression of lncRNA NEAT1 and lincRNA ROR compared Oct4 in GC tissues was evaluated. The 2 −ΔΔCq method was used to determine the expression fold changes.


Introduction
Gastric cancer (GC), a heterogeneous disease with the complicated mechanisms and geographical differences in the prevalence [1,2], is the second common malignancy which is the most important leading cause of mortality among cancers worldwide [3]. In spite of reducing the trend of GC in most parts of the world, the impact of it on public health and burden of the disease is still remained [3,4].
Helicobacter pylori (H. pylori) is a known environmental risk factors of GC that has various signaling pathways which reported to be contributed to the progress and development of GC. The combined effect of Helicobacter pylori infection and various lncRNAs on the increased risk of GC development has been studied [5,6]. Several researches have looked into the relationship between lncRNAs and H. pylori pathogenicity in GC and have suggested that the correlational ndings could be used for early intervention and treatment [7].
In order to better recognize the pathogenesis of GC and detect more actual biomarkers predicting the prognosis of patients, further investigations are required on all aspects of the disease. In recent years, it has been suggested that cancer stem cells (CSCs), known as tumor-initiating cells, play e cient role in tumorigenesis in a variety of human malignancies, including gastric cancer [8,9]. According to a previous functional study, some genes with more attentions are involving in stem cell development such as octamer-binding transcription factor 4 (Oct4) as a major regulator in the progression of tumorigenesis and malignancy [10][11][12][13][14].
In embryonic stem cells, Oct4 has been identi ed as a genetic factor that regulates the transcription, modi cation of chromatin, regulation of long non-coding RNAs (lncRNAs) and microRNAs [15,16]. Oct4 is a key pluripotency programming agent which regulates the expression of lincRNA, the knockdown of lncRNA ROR, that inhibited the proliferation and invasion of gastric cancer stem cells [17,18].
Previous research ndings by Jen et al. suggested evidence of transcriptional regulator of MALAT1 expression in lung cancer by the stemness transcription factor OCT4, that targeted enhancer regions of MALAT1 and activated its expression, therefore leads to proliferation, migration and invasion of cancer cells at in-vitro experiments [17,19]. Also, Jen et al. revealed lung cancer cells that have high expression of NEAT1 and the Oct4-silenced cells re-formed with NEAT1 promoted cell proliferation, migration and invasion [17]. However, our understanding about the role of NEAT1 in the occurrence and development of GC are not fully clear. A number of studies have been found that knock-down of MALAT1 could decreased cell, migration, proliferation and MALAT1 downregulation indicate a reduce expression of genes such as β-catenin, EMT, EZH2, Lin28 and OCT4 [18].
It has been suggested that levels of NEAT1 are involved in the regulation of cell differentiation, proliferation and invasion of gastric cancer [20]. However, the regulation of transcription of lncRNAs by Oct4 in many tumorigenesis cases is still unknown. Up to recent years, many studies on lncRNAs have been focused on the underlying results and mechanisms of lncRNAs and their potential as prognostic and diagnostic markers [5,[21][22][23][24][25][26]. However, little is known about the transcriptional level and their association with other transcription factors such as Oct4 in gastric cancer tissues.
The aim of this study was to evaluate the expression of NEAT1, lincRNA-ROR and Oct4 and their relationship with clinical characteristics in Iranian patients with GC. in this regard, relative expression of lncRNA NEAT1, lincRNA ROR compared with Oct4 in individual samples evaluated too.

Patients
In this designed case-control study, 41 tissue samples of GC with matched normal tissues adjacent to the tumor were prepared from Iranian patients who underwent surgical resection at Imam Reza Hospital, Tehran, Iran, between January 2016 and April 2018. The provided tissue samples transferred to the laboratory in liquid nitrogen immediately following removal through surgery and stored at 80˚C.
Histopathological diagnosis of tissue specimens were con rmed by a pathologist. Detailed clinicopathological parameters including age and sex of enrolled patients, tumor grade, stage and size, history of h pylori infection were recorded according to the unique questionnaire. The tumor stage was determined using American Joint Committee on Cancer Staging Manual (7th edition) [27].

genes' selection
The literature review of effective genes in the progress of patients with GC, resulted in the gene expression of lncRNA NEAT1, lincRNA ROR and Oct4.in this regards the mentioned genes selected for evaluating their correlation in Iranian GC population.

RNA extraction and cDNA synthesis
Total RNA was extracted from the tumor samples of the patients using the Total RNA extraction mini kit (Favorgen, Cat No. FABRK001, Iran). The RNA concentration was quanti ed by a Nanodrop ND-1000 spectrophotometer (Nanodrop Technologies) and its quality was Measured by the A260/A280 and A260/A230 ratio. The concentrations of the samples were normalized and the 1 µg of total RNAs were reverse transcribed to cDNA using the RevertAid RT kit (Thermo Fisher Scienti c, Waltham, Massachusetts, USA). The samples of cDNA stored at -70 ˚C for further evaluations.
Quantitative real-time PCR (qRT-PCR) analysis qRT-PCR was performed using a PCR cycler (Rotor-Gene Q MDx; Qiagen GmbH). cDNA fragments were used as templates to amplify the lncRNAs and Oct4 genes using SYBR® Premix Ex Taq™ (Takara Bio, Inc.), according to the manufacturer's protocol.
The experimental protocol was performed as follows: i) Thermocycling conditions consisted of an initial activation step for 30 sec at 94˚C, 35 cycles at 94˚C for 5 sec and 60˚C for 35 sec; and ii) melting curve analysis.
Primer sequences were designed for all the genes with GeneRunner Software and then the Primer-BLAST (NCBI) was used to check their speci city. Table 1 shows the primer sequences of considered genes. B2M gene used as a normalizer endogenous gene. The 2 − ΔΔCq method was used to determine the expression fold changes (patient vs. normal) receive any preoperative treatment, and those who were undergoing chemotherapy or radiotherapy were eliminated. The more details of demographic characteristics are in Table 2. To explore the role of lncRNA NEAT1, lincRNA ROR and Oct4 in GC, expression levels quanti ed in GC tissues. The differences in the expression levels of NEAT1 in the tumor sample between adjacent normal tissues were not statistically signi cant (p= 0.117) (Fig. 1A). However, gene expression analysis showed signi cant increased difference between lincRNA ROR samples compared to adjacent normal tissues (Mean = 3.337, p< 0.001) ( Figure 1B). Also, there was signi cant upregulation difference of Oct4 level between the samples of GC patients compared with adjacent normal tissues observed (Mean= 4.385, p<0.001) (Fig. 1C).

Lack of associations between the expression of lncRNAs and clinical characteristics
In order to further evaluate the role of lncRNA NEAT1, lincRNA ROR and Oct4 in gastric cancer, the associations between the RNA levels of the gene and several clinicopathological features including tumor stage, grad, size and H. pylori infection, were also investigated.
Relative expression of lncRNA NEAT1, lincRNA ROR and Oct4 in individual samples.
In order to determine association between the expression of the lncRNA NEAT1, lincRNA ROR and the Oct4 gene, the relative expression of these genes was compared in each set of the samples. No signi cant association was observed between the levels of NEAT1comparing occt4 in gastric cancer tissues (R=0.244; P=0.185). In addition, we observed signi cant association between LincRNA ROR and Oct4 (R=0.417; P=0.024) (Fig. 3).

Discussion
With the advances in medicine and life science, still GC remains a worldwide public health concern [28]. So, it is essential to exploring novel effective molecular mechanisms of GC progression for tumorigenesis prevention or improvement survival rate.
Accumulating evidence demonstrates that aberrantly expressed lncRNAs are implicated in GC tumorigenesis, progression and these lncRNAs involved in numerous cell signal pathways and act as either tumor suppressors or oncogenes [24][25][26].
The current study found that lincRNA ROR and Oct4 mRNA level in GC tissues comparing normal adjacent tissues showed signi cant association, but NEAT1 level in GC tissues comparing normal adjacent tissues showed no signi cant association. Also, clinicopathological data comparing with lncRNAs and Oct4 expression levels in GC tissues showed no signi cant association. Another important nding was that signi cant association between the levels of lincRNA ROR expression comparing oct4 mRNA level in gastric cancer tissues.
Oct4 (POU5F1) is an important stem cell transcription factor in the maintenance of self-renewal and are essential in embryogenesis and pluripotency [29]. Increasing evidence over the past decades indicated that the Oct4 is also overexpressed in various tumors stem cells and suggested that Oct4-positive tumor cells have correlation with clinical prognosis, chemo resistance and lymph node metastasis [30]. In addition, post-transcriptional alteration of Oct4 disturbs its activity and further study needs to determine the role of Oct4 in gastric cancer and its clinical relevance, as well as nding correlation with lncRNAs in patients tumor tissue have remained controversial [31]. Additionally, Helicobacter pylori infection, one of the important causes of gastric cancer, has been shown to increase the mRNA level of Oct4 through Wnt/β-catenin signaling pathway in human gastric tumor cells [32].
Shuai Wang (2016) and et al revealed that lincRNA-ROR caused upregulation of Oct4 stemness transcriptional factor. Their data con rmed that lincRNA-ROR was related with core stemness transcriptional factors and the pluripotent state of Gastric CSCs [18]. However, few researches have been conducted the clinical signi cance and biological mechanisms of lincRNA-ROR in gastric cancer. Previously, it has been found that lincRNA-ROR RNA level was signi cantly associated with tumor depth, tumor size, TNM stage, lymph node metastasis and gastric cancer patients' overall survival [33]. Another important recently nding shows that lincRNA-ROR expression levels are positively related with increased multidrug resistance and high level of lincRNA-ROR is a poor prognostic factor for patients with gastric cancer. knockdown of lincRNA-ROR reduced multidrug resistance-associated protein 1 (MRP1) mRNA level and increased apoptosis of drug-resistant gastric tumor cells in response to adriamycin (ADR) and vincristine (VCR) treatment [34].
Previous research by Jayu Jen et al, has indicated that Oct4 interacted by the promoter or enhancer regions of various lncRNAs, Jayu Jen and colleagues con rmed that Oct4 enhancer activities of MALAT1 and potentiated promoter activity of NEAT1 and they suggested that upregulation of Oct4-mediated NEAT1 may play critical roles in embryonic or tumor stemness maintenance in lung cancer cells [17]. In HepG2 cells, the relationship between MALAT1 and Oct4 has been showed that MALAT1 suppression signi cantly decreased the expression levels of transcription factors Oct4, which these outcomes showed that MALAT1 could promote the stem-like properties of liver cancer cells [35].
Some previous studies have shown the important clinical outcome of NEAT1 in gastric cancer [20,[36][37][38][39][40][41][42][43][44][45][46]. In contrast to earlier ndings, no evidence of signi cant NEAT1 overexpression in our study detected. Jing-wei Fu et al found that overexpressed levels of NEAT1 in gastric cancer tissues and cell lines signi cantly increased and associated with clinical stage, lymph node metastasis, distant metastasis and histological type [36]. Farbod Esfandi et al, explore associations of NEAT1 in gastric cancer samples compared with adjacent noncancerous tissues, patients' clinicopathological data and their potential as diagnostic biomarkers. The results of Farbod Esfandi and colleagues study show that signi cant associations between site of primary tumor and relative expression of NEAT1 in cancer samples compared with adjacent noncancerous tissues [42]. meta-analysis by Jian Fang et al, have suggested that High NEAT1 expression is facilitates tumorigenesis of various human cancers and can be used as poor prognosis biomarker in cancer patients [47]. However, most of the studies were assessed by Jian Fang et al, meta-analysis conducted in China; hence, differences may happen between ethnic groups [47].
In conclusion, our data offer the rst suggestion that lincRNA ROR expression correlated with oct4 mRNA level in gastric cancer tissues. However, a further study with more focus on lincRNA ROR molecular mechanisms in association with oct4 mRNA level in gastric cancer tissues and cell lines is therefore suggested.