Correlation between expression levels of lncRNA FER1L4 and RB1 in patients with colorectal cancer

Colorectal cancer (CRC) is a major life-threatening malignancy. Studies demonstrated the lncRNA fer-1 like family member 4 (FER1L4) was downregulated in different cancers and its expression was positively correlated with the retinoblastoma 1 (RB1) mRNA in a competing endogenous RNAs network. We investigated expression levels of FER1L4 and RB1 in patients with colorectal cancer. 50 paired colorectal tumors and non-tumor marginal tissues, 30 paired adenomatous colorectal polyps (ACPs) and matched adjacent normal tissues were obtained from the patients. Total RNA was extracted from the samples and cDNAs were synthesized. Their expression was quantified by qRT-PCR. Correlation between FER1L4 and RB1 expression levels was analyzed by Pearson correlation test. Finally, ROC curve analysis was used to evaluate their biomarker potency. We observed significant downregulation of FER1L4, but upregulation of RB1 in the colorectal tumors compared with non-tumor and the polyp tissues. However, RB1 expression was positively correlated with FER1L4 expression both in the tumor and polyp samples. ROC curve analysis showed both FER1L4 and RB1 expression levels could discriminate tumor from non-tumor and tumor from polyp samples. None of the clinicopathological characteristics of patients were associated with FER1L4 or RB1 expression levels. Despite the downregulation of FER1L4 and upregulation of RB1 in tumors compared with non-tumor tissues, the expression of RB1 was positively correlated with the expression of FER1L4 in the colorectal tumor as well as in the polyp tissues. FER1L4 expression level might be considered as a potential biomarker for colorectal cancer development.


Introduction
Colorectal cancer (CRC) as a gastrointestinal malignancy is the third most common cancer and also the second leading cause of cancer-related deaths in the world [1,2]. In recent decades, the incidence rate of CRC is increasing due to lifestyle changes including dietary habits, obesity, smoking, inflammatory bowel disease, diabetes mellitus, and lack of physical activity [2][3][4]. Distant metastasis and recurrence of disease are the cause of most CRC-related deaths [5]. Therefore, it is essential to identify novel biomarkers for early stages and to understand the molecular mechanisms of CRC to discover new therapeutic target [5,6].
Long non-coding RNAs (lncRNAs) are a class of RNAs with a length longer than 200 nucleotides and less than 1 Mb, but they have little ability to code protein [7,8]. LncRNAs participate in many biological functions, including cell proliferation, apoptosis, metastasis, and gene expression regulation; thus, they could play crucial roles in cancer biology [8][9][10][11]. Different cancer types have been reported to be associated with aberrant expression of lncRNAs and the related dysregulation of mRNAs [5,8,12]. In colorectal cancer, some lncRNAs have been demonstrated to play a role as a tumor suppressor or oncogene, implying their potential as a diagnostic marker or therapy target for CRC [13][14][15].
Fer-1-like family member 4 (FER1L4) is a long noncoding RNA that is located on chromosome 20 [16]. This lncRNA has been confirmed to be involved in tumorigenesis and tumor development [17]. LncRNA FER1L4 expression has been reported to decrease in various cancers, including gastric cancer [16], colorectal cancer [6], osteosarcoma [18], endometrial carcinoma [19], 1 3 and hepatocellular carcinoma [20]. However, there is little published data about the involvement of FER1L4 in colorectal cancer [6]. On the other hand, the retinoblastoma gene, RB1, acts as a tumor suppressor gene in different tumor types, but its expression has been reported to increase in colorectal cancer [21][22][23][24][25][26][27][28]. The lncRNA FER1L4 and RB1 cooperate in a competing endogenous RNAs (ceRNAs) network involving cancer biology, where, the expression of FER1L4 positively correlates with the RB1 expression. This study aimed to evaluate FER1L4 and RB1 expression levels in colorectal tumor versus paired marginal non-tumor tissues as well as in adenomatous colorectal polyps (ACPs) versus their matched adjacent normal tissues. Finally, the correlation between FER1L4 and RB1 expression levels in colorectal cancer was further explored.

Study subjects
A total of 160 specimens, including 50 colorectal tumors, 50 corresponding adjacent non-tumorous tissues, 30 adenomatous colorectal polyps, and 30 polyps' matched adjacent normal tissues were obtained from the Shahid Mahallati Hospital (Tabriz, Iran). The study was approved by the Research Ethics Committee of the University of Tabriz (Tabriz, Iran) and written consents were collected from all patients. All specimens were confirmed by a pathologist as polyp or tumor and included in the study. Exclusion criteria were receiving of clinical treatments such as radiotherapy, chemotherapy, or immunotherapy by the patients before surgery or colonoscopy. Clinicopathological features of patients were reported in Tables 1 and 2.

RNA extraction, cDNA synthesis, and expression analysis
RNX-plus Reagent (SinaClone, Iran) was used to isolate total RNA from tissues according to the manufacturer's instructions. The quality and quantity of isolated RNAs were measured using agarose gel electrophoresis and Nan-oDrop® ND-1000 UV-Vis Spectrophotometer (Thermo Fisher). A total of 1000 ng RNA was reverse transcribed into cDNA in a volume of 10 μl using PrimeScript RT Reagent kit (TaKaRa) following the manufacturer's protocol. Primers were; RNU6 Forward: 5′-CTC GCT TCG GCA GCA CAT -3′ and Reverse: 5ʹ-GGA ACG CTT CAC GAA TTT GC-3ʹ,

Statistical analysis
Data were analyzed using GenEx 7, SPSS v25, and Graph-Pad Prism v8 software programs. Kolmogorov-Smirnov, one-way analysis of variance test, Kruskal-Wallis, independent samples T test, and Mann-Whitney U test were used as appropriate. The Kolmogorov-Smirnov test was used to determine the normality of the data. Expression levels in tumor and polyp tissues and their margins were assessed by ANOVA test. The clinicopathological features in the two groups by independent samples T test and Mann-Whitney U test were evaluated in normal and abnormal data, respectively. Non-normal data in more than two groups were analyzed first by the Kruskal-Wallis test and then ANOVA test. A receiver operating characteristic (ROC) curve was established to evaluate the diagnostic value. Also, the Pearson test was used to investigate the correlation between FER1L4 and RB1. The data were presented as the mean ± SD and P < 0.05 was considered statistically significant.

Study subjects
Clinicopathological characteristics of the study subjects are shown in Tables 1 and 2

LncRNA FER1L4 is downregulated in colorectal cancer
We analyzed the expression level of FER1L4 in 50 paired CRC tumor and non-tumor tissues as well as in 30 paired ACPs and their adjacent normal tissues by qRT-PCR. The results indicated that the expression level of lncRNA FER1L4 was significantly reduced in the colorectal cancer tissues compared with their matched adjacent non-tumor tissues (P = 0.0027, Fold change: 0.268), however, there was no significant difference between its expression in ACPs and their matched adjacent normal tissues (P = 0.99, Fold change: 0.930). Comparing its expression between the tumor and polyp tissues revealed a significantly decreased expression in tumor tissues (P = 0.00029, Fold change: 0.159) (Fig. 1a). Additionally, ROC curve suggested a significant diagnostic ability for FER1L4 in CRC patients. The results demonstrated that the area under the ROC curve (AUC) was 0.764 (99% confidence interval 0.5993-0.9310; P = 0.0003) for the paired tumor versus marginal tissues, and 0.907 (99% confidence interval 0.7716-1.0000; P < 0.0001) for the tumor versus polyp tissues (Fig. 2). The association of FER1L4 expression level in CRC, as well as in polyp tissues, with the clinicopathological features of the patients including gender, age, smoking status, size, Fig. 1 Relative expression of FER1L4 and RB1 were quantified by qRT-PCR. a FER1L4 expression level in tumors was significantly lower than in their adjacent non-tumor tissues, but there was no significant difference between adenomatous colorectal polyps and their adjacent normal tissues (P = 0.99). Interestingly, as figure shows a significant decrease was detected in tumor group compared with the polyp group. b RB1 expression level in tumors was significantly higher than in their adjacent non-tumor tissues, but there was no significant difference between adenomatous colorectal polyps and their adjacent normal tissues (P > 0.05). As figure shows a significant upregulation was detected in tumor group compared with the polyp group. *P < 0.05, **P < 0.01, ***P < 0.001 location, histological grade, and TNM stage, was further analyzed. The results showed no association between its expression level neither in the CRC nor in the polyp tissues with the clinicopathological features (Tables 1 and 2).

RB1 is upregulated in colorectal cancer
The RB1 expression level was also compared between tumor and non-tumor as well as between polyp and their adjacent normal tissues. As shown in Fig. 1b, the expression level of RB1 was significantly higher in the tumor tissues compared with the corresponding adjacent non-tumor tissues (P = 0.00065, Fold change: 3.678), however, there was no significant difference between polyps and matched normal tissues (P = 0.90, Fold change: 1.317). Comparing RB1 expression levels between tumor and polyp tissues revealed that its expression was significantly increased in tumor tissues (P = 0.01343, Fold change: 3.067).
ROC curve analysis identified an area under the curve (AUC) of 0.766 (99% confidence interval 0.6202-0.9117; P < 0.0001) for tumor versus non-tumor and an AUC of 0.708 for tumor versus polyp analyzes (99% confidence interval 0.5388-0.8779; P = 0.0040) (Fig. 2). Furthermore, the association between clinicopathological characteristics of the patients and the RB1 expression level was analyzed. As shown in Tables 1 and 2, none of the clinicopathological characteristics showed a significant association with its expression in the tumor or polyp groups.

RB1 expression positively correlates with FER1L4 expression
Correlation between FER1L4 and RB1 expression levels both in tumor samples and in polyp samples was investigated. We observed a statistically significant positive correlation between FER1L4 and RB1 expression levels in tumor (R = 0.407, P = 0.023) and in polyp samples (R = 0.518, P = 0.033) (Fig. 3).

Discussion
Over 1.8 million patients are diagnosed annually with colorectal cancer [1]. So, identification of the pathogenic molecular pathways and determination of therapeutic targets are needed. It has been recognized that mutations within the noncoding genome and deregulation of noncoding RNAs play critical roles in human diseases [14]. LncRNA, as a novel molecular target, impacts cancer pathogenesis [29]. As an example, LncRNA FER1L4 has been confirmed to retard tumor progression [6]. It is downregulated in several cancers, including gastric cancer [16], osteosarcoma [18], endometrial carcinoma [19], hepatocellular carcinoma [30], esophageal squamous cell carcinoma [31], and lung cancer [32]. However, its expression in glioma is upregulated [17]. Furthermore, the expression level of FER1L4 significantly associates with lymph node metastasis, distant metastasis, and TNM stage of diverse cancers [6,16,18,19]. Previous experimental studies determined that FER1L4 could suppress proliferation, migration, and invasion but induce apoptosis [6,20,[31][32][33][34]. FER1L4 functions as a ceRNA to regulate the expression of PTEN through miR-106a-5p and miR-18a-5p in gastric cancer and Osteosarcoma [10,35]. It also interacts with RB1 which is mediated by miR-106a-5p. Both RB1 and FER1L4 are targets of miR-106a-5p. It was reported that knockdown of FER1L4 by siRNA could decrease both FER1L4 and RB1 levels in gastric cancer. FER1L4 knockdown elevates miR-106a-5p level that provides more chance to bind to other targets, such as RB1 and PTEN mRNAs [36].
To reveal if the lncRNA FER1L4 deregulates in CRC, here we analyzed its expression level in 50 pairs of colorectal tumors and their non-tumor marginal tissues as well as in 30 pairs of adenomatous colorectal polyps and their adjacent normal tissues. Furthermore, the expression level of RB1 as a ceRNA of FER1L4 was also investigated. The results showed that FER1L4 was significantly downregulated in the colorectal tumor tissues, but there was no significant difference between the polyps and their adjacent normal tissues (Fig. 1a). Consistent with these findings, Yue et al. observed that FER1L4 significantly downregulated in colorectal tumors compared with the marginal non-tumor tissues [6]. Interestingly, as Fig. 1a shows, we observed a significant difference between FER1L4 expression in the colorectal tumor and polyp tissues. This observation implies that deregulation of FER1L4 might have occurred after the transformation of adenomatous polyps to colorectal tumors. Furthermore, ROC curve analysis confirmed that FER1L4 expression level can significantly discriminate colorectal tumors from non-tumor tissues, as well as from adenomatous polyps, which implies its potential as a biomarker for colorectal cancer development. Although Yue et al. reported association of FER1L4  [6] we did not observe any significant association with the clinicopathological features. These different observations may result from common study limitations such as heterogeneity of subjects, genetic background of the study population, limited number of samples used in different studies, and sampling errors. Furthermore, multifactorial nature of cancer with diverse drivers and molecular signaling pathways implies that every tumor might have a unique pathogenesis.
RB1 is one of the first described tumor suppressor genes in a wide variety of human cancers, but its involvement and function in colorectal cancer are largely controversial. Some researchers reported its downregulation [37,38], but, others observed its upregulation in CRC [21][22][23][24][26][27][28]. Here we also observed significant upregulation of RB1 in the colorectal tumor compared with their matched marginal nontumor tissues. However, its expression was not significantly different between polyps and their matched normal tissues. As stated above, miR-106a-5p targets the RB1 mRNA and regulates its function in a ceRNA network. Sponging miR-106a-5p by the lncRNA FER1L4 improves RB1 level and its appropriate tumor suppressor function [36]. As a general role, this ceRNA interaction network suggests that downregulation of FER1L4 should result in the downregulation of RB1 mRNA, implying a positive correlation between their expression levels. Despite downregulation of FER1L4 and upregulation of RB1 that we and some other researchers [21][22][23][24][25][26][27][28]37] observed in colorectal tumors compared with their corresponding non-tumor tissues, a positive correlation between their expressions levels was identified when their expressions were analyzed just in tumor or polyp tissues in our study. This observation was consistent with their molecular relationship in the ceRNA network. Probably, the RB1 upregulation which was observed by us and some other researchers might be explained by its increased gene copy number and infrequent allelic loss at the RB1 locus in colorectal cancer [21,[39][40][41]. Lai et al. observed allelic imbalance (AI) of RB1 in colorectal carcinomas and suggested that AI was indicative of allelic loss [26]. So in this situation, the remaining allele could increase RB1 expression by dosage compensation or homeostatic mechanisms, because mRNA and protein of RB1were observed in all samples with AI [26]. Furthermore, RB1 mRNA, Rb protein, and Rb Phosphorylation were reported to be increased in colorectal carcinoma compared with paired normal colonic tissue [23-25, 27, 28]. RB1 commonly performs its tumor suppressor role by restraining E2F transcription factors, but this inhibition is abolished by phosphorylation of the Rb protein. Rb phosphorylation as an oncogenic driver leads to increased proliferation and decreased apoptosis in CRC [22,23].
LncRNAs have been revealed to associate with all major types of malignancies. They involve in tumorigenesis and tumor metastasis by impacting gene expression at both the transcriptional and translational levels [42]. Importantly, having critical roles in the regulation of antigen presentation, they allow tumor cells to escape immune surveillance [43]. Everincreasing number of lncRNAs entering to the clinical trials [44] provides promised implications of them for the future cancer diagnosis, prognosis and therapy. In the present study, our results suggested that FER1L4 and RB1 might be considered as potential molecular biomarkers for diagnosis and progression of colorectal cancer, however, further studies are needed to confirm these findings.

Conclusions
In summary, we observed significant downregulation of lncRNA FER1L4 but overexpression of RB1 in colorectal cancer. However, their expression was positively correlated both in the tumor and polyp samples. Neither FER1L4 nor RB1 expression levels were associated with the clinicopathological characteristics of the patients. However, both FER1L4 and RB1 expression levels could discriminate colorectal tumor from non-tumor tissues, as well as from the adenomatous polyps, highlighting their potential as a biomarker for colorectal cancer development.