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Adv Biomed Res 2023,  12:186

Study of LncRNA BANCR expression in tumor tissues and adjacent normal tissues in gastric cancer patients

1 Department of Animal Biology, Faculty of Natural Sciences, University of Tabriz, Tabriz, Iran
2 Department of General and Thoracic Surgery, Tabriz University of Medical Sciences, Tabriz, Iran
3 Immunology Research Center, Tabriz University of Medical Sciences, Tabriz, Iran
4 Department of Medical Genetics, School of Medicine, Iran University of Medical Sciences, Tehran, Iran

Date of Submission05-Aug-2022
Date of Acceptance27-May-2023
Date of Web Publication20-Jul-2023

Correspondence Address:
Prof. Reza Safaralizadeh
Department of Animal Biology, Faculty of Natural Sciences, University of Tabriz, Tabriz - 5166616471
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/abr.abr_260_22

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Background: Long non-coding RNAs (lncRNAs) have emerged as crucial regulators in various biological processes, including cancer development and progression. This study aimed to investigate the expression differences of the BRAF-activated non-coding RNA (BANCR) gene in GC tissues compared to adjacent normal tissues. The potential diagnostic significance of BANCR in GC was explored, with the aim of improving diagnostic and therapeutic approaches for this global health burden.
Materials and Methods: Tissue samples from 100 gastric cancer (GC) patients were collected, and BANCR expression was analyzed using quantitative real-time PCR. Correlations between BANCR expression and clinicopathological features were assessed, and its biomarker potential was evaluated.
Results: In individuals diagnosed with GC, the expression of BANCR was notably elevated in tumor tissues compared to adjacent normal tissues (P < 0.0001). However, the analysis of gene expression data did not demonstrate any statistically significant correlation between elevated BANCR expression and clinicopathological features. According to the ROC analysis, BANCR demonstrated an AUC of 0.6733 (P < 0.0001), with a sensitivity of 73% and a specificity of 45%. However, further evaluation is required to determine its potential as a biomarker (CI 95% = 0.5992 to 0.7473).
Conclusions: The observed upregulation of BANCR in GC tissues implies its potential involvement as an oncogenic lncRNA in GC patients. Furthermore, BANCR may serve as a promising biomarker for identification and treatment of GC.

Keywords: BANCR, gastric cancer, H. pylori, LncRNA, RT-PCR, TNM

How to cite this article:
Nikanfar R, Dabbaghi R, Rajabi A, Hashemzadeh S, Baradaran B, Teimourian S, Safaralizadeh R. Study of LncRNA BANCR expression in tumor tissues and adjacent normal tissues in gastric cancer patients. Adv Biomed Res 2023;12:186

How to cite this URL:
Nikanfar R, Dabbaghi R, Rajabi A, Hashemzadeh S, Baradaran B, Teimourian S, Safaralizadeh R. Study of LncRNA BANCR expression in tumor tissues and adjacent normal tissues in gastric cancer patients. Adv Biomed Res [serial online] 2023 [cited 2023 Sep 26];12:186. Available from:

  Introduction Top

In 2020, gastric cancer (GC) became the fifth most frequent cancer, with 1,089,103 new cases reported in 2020, and the fourth leading cause of cancer death, with 768,793 new death cases globally.[1] Despite advances in prediction technology and therapy approaches, most GCs have a poor prognosis, and most GC patients have a short life expectancy due to metastasis and failure to diagnose at the initial stages. Discovering valuable biomarkers can be a significant step forward in primary diagnosis and, therefore, more efficient treatment.[2],[3],[4] Long non-coding RNAs (lncRNAs), with more than 200 base pairs in length, play crucial roles in various biological cellular processes including differentiation, apoptosis, proliferation, and migration by regulation of gene expression.[5] Accordingly, misregulation of lncRNAs has been indicated in many cancers like CRC, lymphoma, breast, and hepatocarcinomatous.[6],[7],[8],[9],[10],[11] BRAF-activated non-coding RNA (BANCR), with four exon transcripts of 693 bp located on nine chromosomes, initially reported by Flockhart et al., has shown significant functions in modifying cell proliferation and migration in malignant melanoma.[12] Additionally, relayed to some other research on BANCR function in other tumors, by promoting cell proliferation, migration, and invasion, BANCR act as an oncogene in some malignancies like endometrial cancer, osteosarcoma, esophageal cancer, hepatocellular carcinoma (HCC), and retinoblastoma.[13],[14],[15],[16],[17] In another way, BANCR levels are downregulated in some cancers. With expression enhanced of BANCR, the tumor growth will suppress, for example, bladder cancer, papillary thyroid carcinoma (PTC), and lung cancer.[18],[19],[20],[21] The primary objective of this research was to assess the BANCR expression level and its utility as a biomarker in tumor tissues of individuals diagnosed with GC. Considering the diagnostic, prognostic, and treatment role of lncRNAs in the GC, the aim of the present study is to explore the potential of BANCR as a marker for personalized therapies in this cohort of patients.

  Materials and Methods Top

Clinical samples collection

In this study, 100 pairs of tumor tissue and 5–7 cm adjacent normal tissue samples of GC patients during surgery were obtained at Imam Reza Hospital in Tabriz under the supervision of a surgeon from November 2020 until the end of February 2020. Various types of gastrectomy were collected and instantly placed in free RNase microtubules, frozen in liquid nitrogen (−196°C), and kept at −80°C in the laboratory until the subsequent RNA isolation step.

RNA extraction and cDNA syntheses

The TRIZOL reagent, following the manufacturer's instructions (Invitrogen), was utilized to extract total RNA from both tumor samples and adjacent normal tissues obtained from patients diagnosed with GC (GC). The nanodrop (Thermo Fisher Scientific, USA) was employed to quantitatively assess the RNA samples, while their quality was evaluated through 1% agarose gel electrophoresis. Subsequently, the RNA specimens were stored at −80°C until further processing, including DNase I treatment and cDNA synthesis. After the RNA extraction process, all samples underwent DNase I treatment (GeneAll, Seoul, Korea) to eliminate any possible DNA contamination. For each DNase I treatment, a 5 μl reaction mixture consisting of the total extracted RNA, 0.5 μl DNase I buffer (10×) and 0.5 μl DNase I enzyme (1 U) was incubated at 37°C for a duration of 30 minutes. Following this, 1 μl of EDTA was introduced to deactivate DNase I. The mixture underwent an incubation process for a period of 10 minutes, with the temperature set at 65°C.

The preparation of complementary DNA (cDNA) was carried out through the utilization of the Takara kit, in compliance with the manufacturer's instructions. In every reaction, a portion of RNA that had been treated with DNase I was combined with a blend comprising 3.5 μl of Master mix Kit TaKaRa and 1.5 μl of DEPC-treated water. Subsequently, the mixture was subjected to incubation at 37°C for 60 minutes, followed by a brief exposure at 85°C for 5 seconds.

Quantitative real-time PCR

The quantitative real-time PCR was conducted using the Light Cycler® 96 Real-Time PCR system (Roche Molecular Systems, Inc., Pleasanton, CA, USA) and the SYBR Green Master Mix (Amplicon, Odense, Denmark). The composition of each reaction quantity of 14 μl, which included 7 μl of SYBR Green Master Mix (2×), 0.6 μl of BANCR and β-actin specific primers (10 μM), 1 μl of cDNA (100 ng/μl), and 5.4 μl of ddH2O. The specific primer sequences employed in qRT-PCR were as follows: LncRNA BANCR primer sequences; Forward: 5'-ACAGGACTCCATGGCAAACG-3' and Reverse: 5'-ATGAAGAAAGCCTGGTGCAGT-3', β-actin primer sequences and Forward: 5'-AGAGCTACGAGCTGCCTGAC-3' and Reverse: 5'-AGCACTGTGTTGGCGTACAG-3'. The thermal cycle protocol consisted of three steps. Firstly, the reaction mixture was subjected to a temperature of 95°C for 10 minutes. Subsequently, the amplification was carried out through 40 cycles, which involved a denaturation step at 95°C for 30 seconds, an annealing step at 60°C for 30 seconds, and an extension step at 72°C for 30 seconds. Finally, the amplification was completed with an extension step at 72°C for 5 minutes (duplicate reactions were performed for each sample). The expression levels of the BANCR gene were normalized to those of β-actin.

Statistical analysis

2−ΔCt ± SEM was computed to compare the amount of BANCR gene expression in tumor tissues against adjacent normal tissues. SPSS statistics and GraphPad Prism software were used for data analysis, and P < 0.05 was considered significant. The Mann-Whitney and one-way ANOVA tests were employed to analyze the data concerning BANCR gene expression and its association with clinicopathological characteristics. Additionally, the receiver operating characteristic (ROC) curve test was utilized to assess the effectiveness of BANSR as a biomarker in GC patients, evaluating sensitivity%, specificity%, and the cutoff score.

  Results Top

LncRNA BANCR expression level in gastric cancer tissues

Normalized expression data was utilized to compute the fold-change (Tumor vs Normal) and P-value. Our results have shown that LncRNA BANCR overexpressed significantly (Fold Change = 1.063208 and SEM = 0.20606, P < 0.0001) in GC samples compared to marginal normal tissues [Table 1] and [Figure 1].
Table 1: Statistical analyses of BANCR gene expression in GC tissues and adjacent normal tissues

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Figure 1: The overexpression of BANCR in tumor tissues compared to marginal tissues in GC patients ****P < 0.0001

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Correlation between LncRNA BANCR and clinicopathological parameters

Our study explored the correlation between high BANCR expression and clinicopathological parameters such as age, gender, tumor size, TNM stage, H. pylori infection, and lymph node metastasis in patients diagnosed with GC. The data revealed no substantial relationship between BANCR overexpression and any of these parameters; tumor size >5 cm (P value = 0.293), lymph node metastasis (P value = 0.147), and H. pylori infection (P value = 0.393), gender (P value = 0.893), age (P value = 0.25), TNM (P value = 0.763), and Lauren assortment (P value = 0.684) [Table 2].
Table 2: Association between BANCR expression and clinicopathological features in GC patient

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ROC curve

The diagnostic value investigation of BANCR revealed that it has an area under the curve (AUC) value of 0.67, sensitivity and specificity percentages of 73% and 45%, respectively, and a cutoff value >0.01045 that requires additional evaluation for its potential as a biomarker (P value <0.0001 and 95% CI = 0.5992 to 0.7473) [Figure 2] and [Table 3].
Figure 2: ROC curve value of BANCR (P < 0.0001)

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Table 3: The statistical assessment of the BANCR biomarker value in GC patients

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  Discussion Top

GC ranks among the top five prevalent cancer types worldwide. Even though therapeutic methods like surgery, chemotherapy, and radiation advance, the survival rate in the whole world has not been too satisfying. For instance, the 5-year survival rate among Iranian patients diagnosed with GC has been recorded to be below 11%.[22],[23],[24] Many investigations have been conducted on the role of lncRNAs in cancer development and management. Detection of GC subtypes based on the determined molecular profile leads to more satisfied and substantial treatment approaches by personalized clinical recognition and identifying biomarkers for screening, prognostic, prompt diagnosis, and surveillance of cancer progression. lncRNAs have considerable potential for cancer diagnosis, prognosis, and treatment due to their specific expression and diverse functions. Furthermore, past procedures for cancer diagnosis were invasive, whereas lncRNAs are obtained from bodily fluids such as blood in a far minimally invasive manner.[25],[26],[27],[28],[29] Many lncRNAs associated with GC have been identified, which play significant regulatory roles in the disease progression.[30],[31] Peng et al. have shown that high expression of the lncRNA GASL1 inhibits tumor growth in GC patients by suppressing the Wnt/-catenin signaling pathway.[32] Moreover, Chen et al. have illustrated that lncRNA HAGLROS knockdown inhibits cell proliferation and metastasis while increasing autophagy through reduced mTOR expression.[33] Dai et al. have proved that in patients with GC, lncRNA UCA1 promotes cell proliferation and prevents apoptosis by interacting with EZH2 and activating the PI3K/AKT pathway.[34] Previous studies on LncRNA BANCR have illustrated that expression of this gene dysregulates in several cancers and, regard of type of cancer, acts as a tumor suppressor or an oncogene.[17],[18],[35] According to a new meta-analysis study on 1240 tumor patients, there was a high correlation between BANCR level with the survival rate of tumor patients, lymph node metastasis, and distant metastasis.[36] As an assassin, several studies have demonstrated that BANCR mainly operates as an epithelial-mesenchymal transition (EMT) accelerator in PTC, breast cancer, and poor prognosis and lymph node metastasis in colorectal cancer (CRC).[37],[38],[39] Yu et al. manipulated an investigation to manufacture BANCR in esophageal squamous cell carcinoma (ESCC) cell lines; the assessment indicated that the overexpression of BANCR promoted the proliferation, migration, and invasion of ESCC cells through activation of the Raf/MEK/ERK signaling pathway.[40] Sadeghpour et al. conducted another investigation on the alteration of BANCR expression in ESCC patients; analysis showed the evaluation of BANCR expression associated closely with tumor differentiation and lymph node metastasis in ESCC patients.[41] Numerous studies have demonstrated that lncRNA BANCR capacity as a tumor promoter in different cancer sorts by acting as a sponge for tumor suppressor miRNAs. In CRC cells, BANCR has been found to sponge miR-203, which mediates its oncogenic effects. The expression of BANCR exhibits a notable increase in both CRC tissues and cell lines. Overexpression of BANCR is associated with the facilitation of cell proliferation, migration, invasion, and the development of resistance to Adriamycin, a chemotherapeutic drug.[42] In addition, BANCR has been shown to sponge miR-338-3p, resulting in the increased expression of IGF1R, which activates the Raf/MEK/ERK pathway, leading to the promotion of cell proliferation, migration, and invasion in ESCC.[43] Similarly, in HCC, BANCR sponges miR-590-5P, which reduces its availability to bind to its target mRNA, oxidized low-density lipoprotein receptor 1 (OLR1), leading to an increase in OLR1 expression. This upregulation of OLR1 expression then contributes to enhanced HCC cell growth and invasion.[44] Furthermore, BANCR promotes melanoma cell growth and invasion by acting as a “sponge” to sequester a tumor-suppressive microRNA called miR-204, leading to the upregulation of Notch2, which is involved in cell growth and differentiation. Collectively, these observations emphasize the significance of BANCR in the advancement of cancer, elucidating its involvement in the regulation of gene expression mediated by miRNAs.[45]

  Conclusion Top

The findings from the qRT-PCR analysis demonstrate a substantial upregulation of BANCR expression in GC tissues in comparison to adjacent normal tissues, suggesting its potential oncogenic role in GC. Nonetheless, no significant association has been observed between high BANCR expression levels and clinicopathological characteristics in patients with GC. Furthermore, BANCR exhibits potential as a biomarker for distinguishing GC tissues from normal tissues, offering a prospect for tailored and precise therapies for GC patients.


The authors take this opportunity to express their gratitude to all the teammates and patients who helped with this experiment.

Financial support and sponsorship


Conflicts of interest

There are no conflicts of interest.

  References Top

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  [Figure 1], [Figure 2]

  [Table 1], [Table 2], [Table 3]


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