Serum miR-34a as a potential biomarker for diagnosis of inflammatory bowel diseases in Egyptian patients

IBD is a multifactorial disease. Although dysbiosis of commensal bacteria and breakdown of the intestinal barrier are considered as major pathological mechanisms in the development of IBD, other important factors such as genetic aberrations also contribute to its development. Our results revealed that serum miR-34a RQ values were significantly lower and serum MACF1 RQ values were significantly higher in IBD patients compared to healthy controls. In addition, serum miR-34a in relation to pathological activity and disease severity in the IBD group revealed a significant difference (p>0.05). Serum miR-34a RQ and serum MACF1 RQ value-based biomarker panels can act as a potential biomarker for IBD diagnosis and prognosis.


Background
Ulcerative colitis (UC) and Crohn's disease (CD) are two chronic, relapsing, and remitting inflammatory bowel diseases (IBD) that have no definitive medication treatment and can cause severe long-term morbidity [1]. Only the colon is affected by UC, which is mostly limited to the mucosal and, to a lesser extent, submucosal compartments. It could include all layers of the intestine, and this CD could also contain various digestive system components, ranging from the oral cavity to the anus [2]. These diseases have long been regarded as a problem in Western civilization, and Western lifestyles have played a significant role in disease development [3,4]. It is also increasingly being seen as an emergent worldwide illness [5]. Irritable bowel disease is linked to a significant increase in the risk of colorectal cancer (CRC), especially after years of active disease [6,7]. MACF1 (also known as macrophin 1 or trabeculin-alpha) is a human actincrosslinking protein that is encoded by MACF1, which is found on chromosome 1p34 and spans over 270 kb [8]. It is encoded by MACF1, which has at least 102 exons and spans over 270 kb. MACF1 appears to serve an important role in organisms. The potential role of MACF1 was investigated using MACF1 knockout animals or conditional knockout in specific organs. The findings revealed that MACF1 is required for embryonic development as well as the maintenance of the neural system, bone, colon, cardiomyocyte function, and skin integrity [9]. MACF1 has also been linked to a variety of malignancies, including breast cancer in women, colon cancer, and liver cancer [10,11]. It is also linked to lung cancer, renal cell carcinoma, and some tumors like glioblastoma [12]. Through the Wnt/catenin pathway, MACF1 1 is involved in colon cancer cell proliferation and progression. As a result, we anticipated that inhibiting MACF1

Open Access
The Egyptian Journal of Internal Medicine might slow the evolution of CRC. Using MIDB [13], bioinformatic investigation indicated that MACF1 is a target of miR-34a-5p. MicroRNAs (miRs) are 22-nucleotide noncoding RNAs that regulate the amounts of numerous proteins in basic biological processes [14]. The miR-34 family is made up of miRs, which are encoded by two separate genes: miR-34a is transcriptionally independent, whereas miR-34b and miR-34c share a first copy linked in the human genome on chromosome 1, miR-34a, and miR-34b and miR-34c homologs on chromosome 11. In vertebrates, the miR-34 family is highly conserved [15]. Several microRNAs have also been discovered as important regulators of inflammation resolution during the healing process [16][17][18]. The first aim of this work is to identify a novel molecular signature and a selected target gene involved in IBD with high accuracy to detect IBD through in silico data analysis. The second aim is to characterize the expression of serum non-coding RNA profile and the associated target gene to evaluate their usefulness as diagnostic biomarkers.

Methods
This case-control study was conducted on 51 subjects subdivided into two groups: group 1: a total of 37 inflammatory bowel disease patients; group 2: control group: a total of 14 normal healthy subjects. Patients who complain from any other inflammatory diseases were excluded from the study. Patients were selected from within the National Tropical Hepatology and Tropical Medicine Research Institute (NHTMRI), and their consent to conduct this study was obtained.

Sample collection and processing
The venous blood (5 mL) was withdrawn from each subject under complete aseptic conditions and was left to clot for 30 min. The serum was separated by centrifugation at 4000gf or 15 min. All samples were kept at −80°C until use.

Relative expression of miR-34a and MACF1 using quantitative real-time polymerase chain reaction miRNA expression analysis
The quantification of miR-34a levels was performed using the SYBR-green fluorescent-based primer assay (hsa-miR-34) primer assay, and the Hs_SNORD_68 primer assay was used as a housekeeper gene for normalization.
The qPCR was performed in the 5-plex Rotor Gene PCR System (Qiagen, Hilden, Germany). The 20-ul reaction mixture/reaction consist of 2x QuantiTectsyber green PCR mastermix, 10x miscript universal primer, 2μL primer assay, and 50pg-3ng cDNA. Both targets were amplified in duplicates for each sample. The thermal protocol consists 15 min for HotStarTaq DNA polymerase   [19].

Gene expression analysis for mRNA
The quantification of MACF1 gene expression levels was amplified from cDNA using a QuantiTectqPCR Assay for Human genes; [Hs_ MACF primer assays (Qiagen, Germany). The ACTB_1_SG QuantiTect Primer Assay cat no: 249900 was used as a housekeeper gene. The PCR amplification was conducted using the QuantiTect SYBER Green master mix. The 20-ul reaction mixture/ reaction consist of a 10-μl syber green PCR mastermix, 2-ul primer assay (10μM), RNAase H 2 O, and cDNA. The thermal protocol consists 10 min for HotStarTaq DNA polymerase activation at 95°C followed by 40 cycles of denaturation at 94°C for 15 min, primer annealing for 30 s at 55°C, and extension at 70°C for 30 s. The 2∆∆Ct method was conducted for the analysis of MACF1 gene expression levels, using ACTB as an endogenous reference control for normalization purposes [20].

Statistical analysis
All analyses were done using the Statistical Package for the Social Sciences (SPSS software version 20, Chicago, IL, USA) on a personal computer. Mann-Whitney and Kruskal-Wallis tests were used for statistical comparison of the non-parametric data variables between groups. Chi-square analysis was used to find out the relation between various qualitative data. Variables were crosstabulated in all possible combinations against each other. The correlation coefficients (r) were calculated using the Spearman's correlation. Receiver operating characteristic (ROC) curve determined the best value that gave maximum sensitivity and specificity.

Demographic, clinicopathological, and laboratory parameters of study subjects
Different demographic and laboratory parameters of subjects of the two groups are shown in Table 1. Moreover,  the characteristics of IBD patients were investigated including pathological activity and assessment of disease severity by Mayo score.

Serum miR-34a and MACF1 RQvalues in inflammatory bowel disease patients
Results showed that serum miR-34a RQ values were significantly lower and serum MACF1 RQ values were significantly higher in IBD patients compared to healthy controls (p<0.01, Table 2). ROC curve was done to determine the best cutoff values for miR-34a and MACF1 discriminating the IBD group from healthy control group (Figs. 1 and 2 ) ( Table 3). The positivity rates of serum miR-34a and MACF1 were estimated among the groups of the study (p<0.01, Table 2). A correlation analysis was performed between the two groups regarding miR-34a, MACF1 RQ values, and hemoglobin level (p<0.05, Table 4). In addition, serum miR-34a in relation to pathological activity and disease severity in the IBD group revealed a significant difference (p>0.05, Table 5).

Discussion
Although the Mayo score is used to diagnose and characterize IBD, coloscopy is one of its most essential characteristics. The discovery of valid non-invasive diagnostic biomarkers is required because sigmoidoscopy and colonoscopy are criticized for their high costs and worsening  of inflammatory bowel disease. For the first time, we used serum to investigate a network of genetic and epigenetic indicators in IBD (has-miR-34a, MACF1 mRNA). We believe that this strategy has a better likelihood of success than the more traditional single-marker approach. MiR-34a is a tumor suppressor gene that also has an antiproliferative function. It is found in the second exon of the 33-kb transcript and is considered a tumor suppressor gene as well as has antiproliferative activity [21,22]. However, it has a role in the inflammatory process and prevents epithelial to mesenchymal transition which is one of the risk factors for a progression of CRC [23].
Females are more likely to develop immune-mediated illnesses. Other immune-mediated illnesses, such as sarcoidosis, type 1 diabetes, and IBD, show substantially less gender-specific abnormalities [24,25]. In agreement with the current study that revealed no significant difference in sex between IBD patients and the healthy group (p>0.05, Table 1). Consistent with our study, El-Daly et al [26]. reveal suppression of miR-34a in four stages of colitis-related carcinogenesis, and there was a dependent decrease in miR-34a expression associated with a tumor stage. Also, HOU et al. [27] indicated that miR 34a 3p expression is reduced in FLS from RA patients. Our study revealed a significant difference in serum miR-34a expression in relation to pathological activity and disease severity in the IBD group. In addition, there is a significant positive correlation between miR-34a and HB level in IBD patient [28]. In K562 cells, a steady level of miR-34a increased the level of fetal HB as previously observed by Yanlei Ma et al. [29]. Our study showed MACF1 1RQ values were significantly higher in IBD patients compared to healthy controls (p<0.01, Table 2). Since ROC curve analysis reveals miR-34a ( Fig. 1 ) values had higher sensitivity (97.3%, 100%) and lower specificity (85.7%, 1%), it could be used to confirm the diagnosis rather than decline it (good positive test). The study limitations include the following: it was performed at a single center in Egypt with a relatively limited sample size. Moreover, in vitro functional analysis is needed to elucidate the biological mechanisms of RNA-RNA cross-talk in IBD is still needed which is currently running in our center.