The Downregulation of miRNA-152 Gene Expression in Colorectal Cancer Patients: A Case-Control Study

MicroRNAs (miRNA or miRs) are Small, endogenous, non-coding RNAs that attach to the 3´-Untranslated Region (UTR of target messenger RNAs (mRNAs) to control gene expression after transcription. This binding either prevents the mRNA from being translated or causes it to degrade [1,2]. MiRNA-152, a member of the miR-148/152 family, is found in the first intron of the COP72 gene on chromosome 17q21.32. A CpG island usually surrounds its promoter region [3]. Aberrant expression of miRNA-152, which can behave as an oncogene or a tumor suppressor based on its activities and patterns of expression, is a frequent feature of many cancer types [3]. Additionally, by downregulating ZEB1/2, SNAI1 and KLF5, it inhibits the Epithelial-Mesenchymal Transition (EMT) [4,5]. By blocking CDC25B and E2F3, it controls the cell cycle [6,7]. Additionally, it improves anti-tumor immunity by inhibiting immunological evasion molecules such as B7-H1 [9] and HLA-G [8]. Furthermore, microRNA-152 targets tumor suppressors such as p27 [10] and PTEN [11] to produce oncogenic effects in prostate cancer and Chronic Myeloid Leukemia (CML). Targeting several oncogenic pathways and compounds, miRNA-152 has tumor-suppressive effects in colorectal cancer. Targeting DNA Methyltransferase (DNMT1) is a crucial strategy that reactivates dormant tumor suppressor genes and lowers global DNA methylation through a negative feedback loop [12]. The biological properties of Colorectal Cancer (CRC) cells are impacted by DNMT1's function in the methylation of microRNA (miR-152-3p); hence, increased DNMT1 may counteract the impact of miR-152-3p overexpression on CRC development and tumor growth [13]. However, in other colorectal diseases, like IBD, where miRNAs modulate inflammatory pathways and impact important biological processes like extracellular matrix dynamics, tight junctions, cellular hemostasis and the microbiota, miR-152 plays a crucial role in the differentiation, maturation and functional control of immune cells. As a result, altered miR-152 expression can influence intestinal barrier integrity, tight junctions and the extracellular matrix and accelerate the course of disease [14]. For the following reasons, this study was created to examine the expression of the microRNA-152 gene in colorectal cancer as opposed to other colorectal diseases: (1) The majority of miRNAs are tumor suppressors (common findings) in CRC tissue and cell lines, miR-152 (particularly miR-152-3p) is downregulated, according to the majority of recent reviews and experimental research; restoring miR-152 inhibits colony formation, causes apoptosis and decreases proliferation. (2) DNA methylation and DNMT1 are important molecular targets. One important and frequently mentioned mechanism is miR-152's direct targeting of DNMT1. Tumor growth is aided by lower miR-152 → increased DNMT1 → hypermethylation and silencing of tumor-suppressor genes (e.g., PTEN/RASSF1A). Restoring miR-152 can reduce DNMT1 activity and demethylate tumour suppressor promoters. (3) Suppresses pathways driving invasion/EMT (Wnt, PI3K/AKT, etc.). miR-152 negatively regulates EMT and metastasis-related signalling (Wnt/β-catenin, PI3K/AKT/mTOR and downstream effectors), lowering migration and invasion in CRC models. (4) Biomarker and therapeutic potential. The objective of this study is to study the microRNA-152 gene expression in colorectal cancer patients compared to colorectal benign disease patients by achieving a molecular study using RT-qPCR.

Patients and Sampling

The Al-Iraqia University Medical College Council has given its approval to the study. The target population comprised 90 samples and was divided into three groups: 30 patients with a confirmed diagnosis of Colorectal Cancer (CRC group) and 30 patients with other Colorectal Diseases (CRD group), including polyps, ulcers, IBD and Hirschsprung disease. The active inflammation and congestion group and the third group was the control group (n = 10), who were healthy individuals. The age of the samples ranged from 16 to 81 years, with a mean of 47.60±14.980 and most of them fell within the age group of 30-60 years old (68.9%). Blood samples were collected from various hospitals (GIT Centre, Oncology Teaching Hospital, Baghdad Teaching Hospital), regardless of gender or age, with an emphasis on elderly patients, from 1st November 2024 to 31 January 2025. During this time, a series of data was collected. These data include: obtaining information from the patients by using the questionnaire (patient data sheet), reviewing patients´ reports (histopathology reports) to know the stage, grade and type of cancer. As well as to know the type of other non-cancerous colorectal disease.

Genetic Analysis

The concentration of miRNAs in blood plasma is in the picomolar range and they make up only 0.01% of total RNA. For miRNA analysis, quantitative PCR (qPCR) techniques are the most often utilized methodologies. Two hundred fifty (250) µL from a blood sample (EDTA tube) was added to a 1.5 ml Eppendorf tube, which contains five hundred (500) µL TRIzol for each patient of two groups (CRC) and (CRD), besides healthy individuals (control groups), then the tube was inverted many times for mixing. For the detection of microRNA-152 in these groups, firstly, the total RNA was isolated from the blood of these groups by using TRIzol and then using RT-qPCR, which was conducted through two phases: in the first phase, we synthesized cDNA from the isolated RNA by using a specific reverse transcriptase enzyme.

The second one involves choosing the cDNA sample from each patient, then using quantitative PCR (qPCR) to amplify and measure the amount of specific cDNA in real time using a fluorescent dye, SYBR^® Green. For each sample, there are two PCR tubes, one for miRNA-152 and the other for the (RNA U6) gene, which is considered a housekeeping gene in this study.

RT-qPCR Protocol:

• Five microliters from each extracted total RNA sample were added to a new PCR tube
• Easy-Script reaction mix that contains dNTPs, buffer and other essential components was added as 10 µL for each sample
• MUL-V Enzyme (reverse transcriptase) was then added to the reaction as 1µl per sample
• One microliter of random primer for the U6 gene, also 1µL specific RT primer for (miR-152) and the volume was completed up to 20 µL by adding nuclease-free water
• This mixture was incubated as shown in the Table 1-3 and Figure 1

Figure 1: The Results were Collected and Analyzed by the Livak Formula

Table 1: The Mixture (Temperature, time and Purpose) of RT-qPCR

Table 2: The RT-qPCR Thermocycling Protocol

Table 3: The Name, the Sequence and the Reference of the Primers that have been used in this Study

Statistical Analysis

Data analysis was conducted using the Statistical Package for the Social Sciences (SPSS) version 26 and STATISTICA version 9. The present analysis utilised analysis of variance (ANOVA) to compare the continuous variables among more than two groups, while a Chi-square test was used to assess association within categorical variables in independent groups.