Introduction

Colorectal cancer (CRC) is one of the most common and lethal disorders around the world [1] and is known as the third most common cancer as well as the fourth most deadly tumor worldwide [2]. It is responsible for 8.5% of all cancer deaths worldwide, emphasizing the importance of developing early diagnostic biomarkers [3].

Long noncoding RNA (LncRNA) is a group of genetic sequences that do not contribute to protein synthesis but can affect cellular activities such as chromatin remodeling, cell cycle advancement, and neoplasm formation [4].

LncRNA H19 was the first lncRNA discovered, with a 2.3 kb polyadenylated, spliced, and capped non-coding RNA encoded by its gene. It's on chromosome 11p15.5 in humans [5]. LncRNA H19 has been linked to a variety of cancers [6]. Anomalies in lncRNA H19 expression have been observed in a variety of cancers, implying that H19 plays an important role in cancer progression [7]. The H19 gene's single nucleotide polymorphisms (SNPs) may impact the gene's expression and function. Chu et al. [8] reported the association between three H19 polymorphisms (rs2839698, rs217727, and rs2107425) and cancer susceptibility. H19 also promotes epithelial-to-mesenchymal transition (EMT), a critical stage in cancer metastasis, by acting as miRNA sponges and inhibiting the functions of related miRNA, according to Zhou et al. [9].

miR-200a, miR-200b, miR-200c, miR-141, and miR-429 are the five members of the miR-200 family. The miR-200 family is involved in cell transformation and tumorigenesis, cancer metastasis, tumor growth, angiogenesis, invasion, migration, and tumor cell survival within the circulation. miR-200 can be a potential diagnostic and prognostic tool for patients with cancer [10]. In recent years, single nucleotide polymorphisms (SNPs) of candidate genes have become the focal point of various studies on the genetic risk of cancer incidence; the expression of the host lncRNA may vary depending on the genetic variations of the non-coding RNA gene. However, there have been few investigations into the relationship between SNPs in lncRNAs and colon cancer risk. Xue et al. [11] reported that HOTAIR SNPs contributes to the susceptible to colorectal cancer. Also, Li et al. [12] stated that H19 rs2839689 increases the susceptibilities to colorectal cancer in the Chinese population. Moreover, Fu et al. [13] reported that SNP rs12982687 of UCA1 provide high susceptibility of CRC. Thus, the current study aimed to scrutinize the biochemical contribution of LncRNA H-19 rs2107425 to the susceptibility of CRC and the potential relationship between miR-200a and LncRNA H-19 in patients with colorectal cancer.

Subjects and Methods

The present ongoing study included 100 unrelated participants were analyzed, the age of all participants ranged from 29 to 62 years. The participants were divided into two main groups: Group I contained healthy controls and Group II consisted of individuals diagnosed with colorectal cancer (CRC), according to the colonoscopic examination and signs of CRC like significant unexplained weight loss and unexplained anemia; screening for colorectal cancer, and metastasis. The healthy participants were age, gender, and ethnicity matched to colorectal cancer patients.

Informed written consent was received from all the participants enrolled during this study. Characteristics of all enrolled participants, including age, gender, and family history, were collected. The study was performed with the approval of the Al-Kasr Al-Ainy Hospital's ethics committee at Cairo University, Egypt, and in accordance with the Helsinki Declaration (2008).

Regarding, All study participants underwent a thorough history and clinical examination, including routine laboratory tests like hemoglobin level, white blood cell count, Platelets, liver function profile, kidney function tests, and carcinoembryonic antigen (CEA) assay. Full colonoscopy; and imaging with abdominal ultrasound and computed tomography to stage CRC subjects. Patients who had previously received chemo- or radiation for CRC, were diagnosed with inflammatory bowel disease (IBD), had cancer at the other site at the time of selection, or had a history of recurrent malignancies were also ruled out.

All enrolled participants' venous blood samples (10 mL) were withdrawn and centrifuged at 4000 r/min for 10 min in serum separator tubes. Sera were stored at -80 °C until they were analyzed.

The QIAamp DNA Minikit (Qiagen, Valencia, CA) was used to extract genomic DNA from whole blood samples from all subjects according to the manufacturer's guidelines. The yield was determined using NanoDrop2000 (Thermo Scientific, USA). Genotyping was performed using real-time PCR with the TaqMan allelic discrimination assay using predesigned primer/probe sets for rs2107425 (C/T) (Applied Biosystems, USA). DNA amplification was performed in a 25 μl volume containing 12.5 μl TaqMan master mix, 1.25 μl primer/probe, 1 μl DNA, and 10.25 μl H2O. Real-time PCR was performed employing a Rotor gene Q System (Qiagen) with the subsequent conditions: 95 °C for 10 min, followed by 45 cycles at 92 °C for 15 s and 60 °C for 90 s. Fluorescence was measured at the end of every cycle and therefore the endpoint.

Total RNA was extracted from serum by the miRNeasy extraction kit (Qiagen, Valencia, CA) using QIAzol lysis reagent according to the manufacturer’s instructions.

Reverse transcription (RT) was applied to 60 ng of total RNA in a final volume of 20 µ l RT reactions using the RT2 first strand kit (Qiagen, Valencia, CA) in accordance with the manufacturer's guidelines. Before performing real-time PCR, the RT products were diluted in 50 μl RNAase-free water. Serum expression levels of the lncRNA H19 and miR-200a were evaluated using GAPDH, and SNORD68 as interior controls, using customized primers and the Maxima SYBR Green PCR kit (Thermo, USA) in line with the manufacturer’s protocol.

The specificity of the target PCR product was confirmed by visualizing one peak within the melting curve analysis. qRT-PCR data analysis was accomplished based on the 2−ΔΔct comparative expression method [14].

Statistical Analysis

The statistical software SPSS 21.0 was used to analyse the data (SPSS Inc., Chicago, IL, USA). The data is presented as a mean with standard error. One-way ANOVA has been used for descriptive statistics. The chi-square test was applied to compare the genotype frequencies for rs2107425. Hard-Weinberg equilibrium applied using an online calculator [15]. Pearson correlations coefficient test were used to test relation between clinical and laboratory variables. P < 0.05 was considered statistically significant.

Results

The present ongoing study included 100 unrelated participants were analyzed. The participants were divided into 70 CRC patients and 30 healthy controls. Male CRC patients represented 61.43%, while female CRC patients represented 38.57%. Male healthy control participants constituted 46.67%, while female controls constituted 53.33%. According to the radiological and colonoscopy findings performed on the CRC patients, tumor sites in the cecum and ascending colon represented 20% of the cases, transverse and flexures represented 25.7%, descending and rectosigmoid tumor sites represented 41.4%, and rectal represented12.8%, as shown in Table 1.

Table 1 Demographic data and clinical-pathological features of the studied groups
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Regarding biochemical and hematologic variables, there were a significant alterations in hemoglobin and total leukocyte count in CRC patients compared to the healthy control group (P < 0.05). Meanwhile, a significant alteration (P < 0.05) in liver enzymes (ALT, AST), total bilirubin, and albumin levels were observed in CRC patients compared with those in healthy controls. Moreover, CRC patients showed a significant elevation in creatinine and CEA levels as compared to the healthy control group. Additionally, this study shows a remarkable, statistically significant fold change elevation in miR-200a levels in CRC patients compared to healthy participants (mean fold change 6.66, P < 0.05), as shown in Table 2. Here also, LncH19 shows a highly remarkable fold change elevation in its expression level in the CRC patients compared to healthy subjects (mean fold change 9, P < 0.05). Moreover, the expression levels of LncRNA H-19 and miR-200a were significantly higher in participants with stage III than those in stage II (P < 0.05). Moreover, the other biochemical variables studied were numerically differed between stages II and III but didn’t reach statistical significance (P > 0.05) (Table 2).

Table 2 Biochemical comparison between healthy control and CRC patients regarding laboratory investigations (mean ± SE)
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In this study, The patients with CRC had a statistically significantly lower distribution of the CC genotype than the healthy control participants (35.7% and 66.7%, respectively), a statistically higher distribution of the CT genotype than the healthy control participants (45.7% and 30%, respectively), and a statistically higher distribution of the TT genotype than the healthy control participants (18.6% and 3%, respectively) (χ2 = 9.22, P < 0.05) (Table 3). Moreover, the patients with CRC had a statistically higher distribution of T allele frequency (41.4% and 18.3%, respectively) and a lower percentage of C allele frequency than the healthy control participants (58.6% and 81.7%, respectively) (χ2 = 9.92, P < 0.05) (Table 3). Additionally, the average fold expression of miR-200a and LncRNA H19 for CT and TT genotypes of the rs2107425polymorphism was statistically significantly higher for CT and TT genotypes than for CC genotypes (p < 0.05) (Table 4).

Table 3 Genotype and allele frequency of LncH19 rs2107425(C/T) gene single nucleotide polymorphism (SNP) between healthy control and CRC subjects
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Table 4 Expression fold of miR-200a and H19 regarding CRC patients(mean ± SE)
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To assess the relationship between LncRNA H-19 and miR-200a with patients’ clinical and laboratory variables, Pearson correlation analysis was conducted. LncRNA H-19 and miR-200a positively correlated with distant metastasis, stage of the tumor, WBC count, ALT, AST, total bilirubin, and CEA, and negatively with levels of hemoglobin and albumin. Additionally, a significant positive correlation was observed between the expression levels of LncRNA H-19 and miR-200a among studied participants (Table 5).

Table 5 Correlation of serum H-19 and miR-200a expression levels with clinical and biochemical analysis of studied participants
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Discussion

Recent advancements in colorectal cancer diagnosis and therapy have improved health satisfaction; screening for CRC during an early stage, when it has been curable, can minimize both the disease's incidence and mortality [16]. Hence, there is a crucial need for noninvasive biomarkers to complement and improve the diagnosis and prognosis of colorectal cancer. This study explored the biochemical contribution of LncRNA H-19 rs2107425 to the susceptibility of colorectal cancer and the potential relationship between miR-200a and LncRNA H-19 in patients with colorectal cancer. Recently, the single nucleotide polymorphisms (SNPs) of lncRNA genes have been widely confirmed to regulate the expression and function of lncRNAs, ultimately resulting in tumor susceptibility and a poor prognosis [17]. The LncRNA H19 has been widely renowned for its aberrant expression profile and role in carcinogenesis, and it is suggested to be a novel biomarker for cancer diagnosis[18].

Zhang et al. [19] and Ratti et al. [20], reported that the elevation of differential expression of lncRNA and miRNA are associated with an increased susceptibility of cancer formation, inflammation, and neurological diseases.

LncRNA H-19 expression level were up-regulated with a remarkable increase in patients with CRC compared with healthy controls. These findings are consistent with the results of [21,22,23], which reported that patients with CRC exhibited a noticeable elevation in LncRNA H-19 expression compared with healthy controls; and considered LncRNA H-19 as a novel CRC prognostic oncogenic biomarker. Additionally, the loss of imprinting of the gene has been suggested as a cause of H19 overexpression in CRC [18].

The miR-200a differential expression fold was assessed in patients with CRC and compared with that in healthy participants. All participants with CRC showed elevated miR-200a expression with a significant difference. Moreover, the differential expression fold of LncRNA H-19 and miR-200a were lower in participants with CRC with stage II than that in those with stage III, it were significant. This implies that the differential expression of LncRNA H-19 and miR-200a might be a biomarkers for predicting the progress of colorectal cancer.

Our findings support the study by Chen et al. [23], and previous studies of Yang et al. [24], which reported the impact of miR-200a and its oncogenic roles in tumor growth and metastasis. Additionally, Our findings have coincided with Carter et al. [25], who showed that miR-200a expression is frequently up-regulated in colorectal cancer samples compared to control ones.

GWAS has identified multiple independent single nucleotide polymorphisms (SNPs) associated with colorectal cancer risk. Previous studies has found links and relationships between cancer susceptibility and the three most prevalent SNPs in LncRNA H-19 (rs2839698 G > A, rs217727 G > A, and rs2107425 C > T) [12, 26, 27].

In the current study, we evaluate the association between H19 rs2107425 polymorphism and CRC risk. To our knowledge, no previous research has looked into the link between rs2107425 in H19 and colorectal cancer risk in an Egyptian population. Our current study found that there was a remarkable association between H19 rs2107425 polymorphism and CRC, and the T allele of rs2107425 was significantly higher in CRC than in healthy control participants. Also, we found that the CC genotype has a higher percentage of controls compared to CRC participants. Both the mutant and heterozygote genotypes were noticed with increased frequency in CRC patients than in healthy control participants.

These findings agreed with the findings obtained by [27], who reported that T allele carriers have a significantly increased risk of developing lung cancer and that the functional SNP rs2107425 in H19 is highly likely to be involved in lung cancer development. In contrast, Chu et al. [8] reported that in the population studied, the H19 rs2107425 polymorphism was not linked to colon cancer susceptibility, and the frequency of the rs2107425 polymorphism in colon cancer cases and controls did not differ significantly. Verhaegh et al. [28], reported that H19 genetic polymorphisms (rs2839698 and rs2107425) might be associated with the susceptibility of bladder cancer in European Caucasians. Similarly, Yin et al. [29], observed that rs2107425 polymorphism may be able to influence the risk of lung cancer in Chinese female never smokers. All of these studies suggested that genetic variants of LncRNA H-19 may have a significant impact on many tumor susceptibilities.

Additionally, this study found a significant positive correlation between serum LncRNA H-19 and miR-200a, suggesting their concomitant expression in CRC. Additionally, we found a remarkable correlations between ncRNAs (LncRNA H-19 and miR-200a) and tumor stages, and distant metastases. Our data were consistent with previous studies [21, 30,31,32], which reported that LncRNA H-19 and miR-200a were associated with distant metastases in cancers. Similarly, Becker et al. [33] reported that the over expression of miR-200a enhance the malignant transformation and acts as an oncomiR rather than a tumor suppressor by cooperating with an oncogene in malignant cell transformation.

To the best of our knowledge, This study is the first to detect the relationship between LncRNA H-19 rs2107425 to the susceptibility of colorectal cancer and the correlation between miR-200a and LncRNA H-19 in colorectal cancer. However, this study has some limitations, like the sample size, the selection of a single SNP for lncH19 (rs2107425), and the study of miR-200a of the miR-200 family. Therefore, further large-scale studies and clinical trials are needed. Nevertheless, our results implicate rs2107425 as genetic markers of CRC susceptibility that correlate with LncRNA H-19 and miR-200a expression, respectively. Serum LncRNA H, with rs2107425 and serum miR-200a, could predict the risk of CRC diagnosis among non-CRC groups, implications for stages of CRC screening, genetic counseling, and hold promise for large-scale application.