Abstract
Background/Aim: MicroRNAs (miRNAs) are likely to play a significant role in predicting rectal cancer response to chemoradiation therapy and overall cancer prognosis, offering insights that complement other biological tumor markers. This study aimed to conduct miRNA profiling in rectal cancer tissues in patients with good (GR) and bad response (BR) to neoadjuvant chemoradiation therapy (nCRT), followed by the selection of clinically relevant miRNAs. The relationship between selected miRNAs and subsequent disease outcomes and survival prognosis was assessed.
Patients and Methods: Forty patients with locally advanced rectal cancer who received nCRT followed by surgical treatment at the Pauls Stradiņš Clinical University Hospital during the period from 2016 to 2021 were included in the study. Two study groups were created, GR and BR, according to the Dworak tumor regression grading (TRG) system. The identification of 752 miRNAs was conducted in rectal cancer tissues according to the protocol of miRCURY LNA miRNA miRNome PCR Panels. Six up-regulated miRNAs were deemed as clinically significant and subsequently validated in both the BR and GR groups.
Results: MiR-142-5p, miR-182-3p, and miR-99a-3p exhibited statistical significance in the validation procedure. The results showed that BR to nCRT, lower expression of miRNA-142-5p and miR-99a-3p, and higher expression of miR-182-3p were associated with a trend toward worse local recurrence-free survival, distant metastases-free survival, and overall survival.
Conclusion: MiRNAs may potentially serve as clinical biomarkers in the prediction of disease-free survival and overall survival in patients with rectal cancer.
- Rectal cancer
- biomarker
- microRNA
- chemoradiation therapy
- treatment response
- miRNA 142-5p
- miRNA 182-3p
- miRNA 99a-3p
Introduction
Rectal cancer is a highly prevalent type of cancer, accounting for approximately 10-30% of newly diagnosed colorectal cancers (CRC) (1). According to the Globocan Cancer Observatory data of 2022, CRC is the third most commonly diagnosed malignancy and the second leading cause of cancer-related deaths in the world, with approximately 1.9 million new cases and 904,019 deaths per year (2). It is a heterogeneous disease that develops via stepwise accumulation of well-characterized genetic and epigenetic alterations (3). Accurate staging of rectal cancer is crucial as it can influence the choice of treatment strategies (4). If early rectal cancer is managed by surgical resection alone, then more advanced cases demand neoadjuvant (preoperative) combination of chemoradiation therapy to reach the tumor downstaging or downshifting in order to provide safe resection margins and reduce the risk of local recurrence (5). However, responses to this therapy can vary widely among patients, influenced by a multitude of factors including genetic makeup, tumor biology, and the tumor microenvironment (6-8). As a result, we encounter a spectrum of tumor reactions – from non-response to complete response to therapy. Studies show that clinical complete response (cCR) after neoadjuvant chemoradiation therapy (nCRT) can be obtained in 10-40% of cases, and pathological complete response (pCR) is observed in 15-30% of rectal cancer patients (9-10). Patients with a pCR to nCRT have lower rates of local recurrence, improved survival as compared to patients who don’t achieve pCR. The 5-year recurrence-free survival rates are 90.5%, 78.7% and 58.5% for patients with complete, intermediate and poor response (11). Furthermore, patients with pCR after nCRT have improved distant metastatic rates compared to poor responders to nCRT – 7-10.5% and 26-31%, respectively (12, 13).
Understanding the differences of good and bad responders is not just an academic pursuit; it has profound implications for clinical practice. By identifying the underlying mechanisms that drive varied responses, healthcare providers can tailor treatment strategies more effectively, optimizing therapeutic outcomes and minimizing unnecessary side effects.
Currently, several biomarkers have been investigated regarding their effectiveness in the diagnosis and prognosis of rectal cancer. These include carcinoembryonic antigen (CEA) and tissue-based markers such as KRAS mutations, which can indicate prognosis and treatment response. However, the limitations of these markers include variable sensitivity and specificity, as well as the need for invasive procedures to obtain tissue samples (14-16).
MiRNAs represent a promising avenue in rectal cancer, providing a complementary approach to existing biological tumor markers. MiRNAs are small (18–25 nucleotides) single-stranded and non-coding RNAs that down-regulate gene expression at the post-transcriptional level through binding to target mRNAs and triggering their degradation or translational blocking (17). They exert their effects primarily by binding to the 3′ untranslated regions (3′ UTRs) of target messenger RNAs (mRNAs), leading to either mRNA degradation or inhibition of translation. This regulatory function allows miRNAs to fine-tune gene expression, impacting various cellular processes such as proliferation, differentiation, apoptosis, and stress responses. It is believed that up to 30% of human genes are regulated by miRNAs (18-20). Given the great impact of miRNAs on gene expression, it is not surprising that miRNA deregulation contributes to the initiation, progression, and dissemination of any type of human tumor (20-22). They can act as oncogenic miRNAs (onco-miRNAs) or tumor suppressor miRNAs, depending on the function of the targeted mRNA (13). Both the overexpression of specific onco-miRNAs and silencing of tumor suppressor miRNAs have been associated with the tumorigenesis of rectal cancer by inhibiting key components of the main signaling pathways altered in this disease. The overexpression of a miRNA can be due to the amplification of its coding gene or augmented transcription, while miRNA down-regulation can be caused by epigenetic silencing, deletion of its coding gene, or defective biogenesis (18). Understanding the intricate balance between miRNAs and their target oncogenes is crucial for elucidating the molecular mechanisms underlying rectal cancer. This knowledge not only enhances our comprehension of cancer biology but also opens avenues for therapeutic interventions, such as miRNA-based therapies that aim to restore normal regulatory pathways disrupted in cancer (23).
MiRNAs are integral to the regulation of various biological processes in rectal cancer, influencing tumor behavior and the response to treatment through several key mechanisms: (i) regulation of cell proliferation and apoptosis: miRNAs can modulate pathways that control cell cycle progression and apoptosis, two critical processes in cancer development; (ii) impact on cell migration and invasion: miRNAs play a role in the epithelial-to-mesenchymal transition (EMT), a process that enhances the invasive capabilities of cancer cells; (iii) modulation of tumor microenvironment: miRNAs can influence the tumor microenvironment, affecting interactions between cancer cells and surrounding stromal cells. For example, certain miRNAs can regulate the secretion of cytokines and growth factors, modifying immune responses and promoting angiogenesis. This can create a supportive environment for tumor growth and progression; (iv) resistance to chemotherapy and targeted therapies: miRNAs are implicated in the mechanisms of drug resistance in rectal cancer. They can regulate the expression of genes involved in drug metabolism, efflux, and apoptotic pathways; (v) biomarkers for prognosis and treatment response: specific miRNA profiles can serve as prognostic biomarkers, indicating disease progression and therapy outcomes. For example, the expression levels of certain miRNAs have been correlated with patient survival and response to treatment, enabling the identification of patients who may benefit from more aggressive therapies or those who may require alternative treatment strategies (18, 20, 22, 23).
The key advantages of miRNAs as diagnostic tools lie in their ability to reflect the underlying pathophysiological state of the tumor. Specific miRNA signatures have been associated with various stages of rectal cancer, offering potential not only for early detection but also for monitoring disease progression and therapeutic response. Moreover, miRNAs can serve as prognostic indicators, with certain miRNA profiles correlating with patient outcomes, metastasis, and overall survival. Additionally, the multiplex nature of miRNA assays enables the simultaneous assessment of multiple miRNAs, thereby enhancing diagnostic accuracy and providing a more comprehensive understanding of tumor biology. Besides, miRNAs are secreted into bodily fluids with minimal degradation and exhibit high stability during storage. All of these advantages facilitate their use in the clinical setting, and miRNAs are emerging as stable and non-invasive biomarkers regarding diagnosis, staging, and prognosis in rectal cancer management. As research continues to elucidate the roles of miRNAs in rectal cancer, their integration into clinical practice may lead to more personalized and effective management strategies for patients (18, 20, 23).
Although several miRNAs have already been described as potential biomarkers in rectal cancer patients, there is a lack of robust, established markers available in the clinical routine (20, 24, 25). A review by De Palma et al. in 2020, who assessed 61 articles, identified a total of 77 miRNAs that hold predictive value; however, only six miRNAs (let-7f, miR-21, miR-145, miR-622, miR-630, and miR-1183) exhibited significant differences in two or more independent studies (26).
The aim of our study was miRNA profiling in rectal cancer tissue and establishment of their potential association with further prognosis in locally advanced rectal cancer patients.
Patients and Methods
Patient characteristics. A retrospective study was conducted in which patients with morphologically and radiologically verified stage II and III rectal adenocarcinoma, who received nCRT followed by surgical treatment during the period from 2016 to 2021 at Pauls Stradiņš Clinical University Hospital (Riga, Latvia), were selected and were either alive or deceased at the time of the study’s initiation. A signed informed consent form for participation in the study was obtained from each patient or their family member (if the patient had deceased).
Exclusion criteria were as follows: inflammatory bowel disease (Crohn’s disease or ulcerative colitis); known family history suggesting a possible hereditary cancer risk; a history of organ transplantation; concomitant cancer at another site, that could affect survival prognosis; tumor progression (stage IV) during or after nCRT; incomplete nCRT; if a palliative rather than radical surgery had been performed after the nCRT; if no surgery had been performed after nCRT (the patient declined surgery, died due to underlying conditions or comorbidities before surgery, or the patient had achieved a cCR after nCRT); if the patient did not attend adjuvant therapy (if it was indicated) and follow-up visits for dynamic monitoring; if the patient/patient’s relative (if the person was deceased) did not agree to participate in the study.
Tissue samples. Tissue samples were acquired as part of planned treatment at the hospital. The study did not impose an additional burden on the patients. Tissue samples were obtained from the operative material. The study groups were formed based on the post-operative pathomorphosis of the tumor in the radically resected surgical material, using the Dworak classification (Table I), which was assessed by two pathologists (27).
Dworak classification.
Tumor pathomorphosis was evaluated in formalin-fixed, paraffin-embedded (FFPE) tissue samples, which were cut to a thickness of 4 μm using an automatic microtome and then prepared on slides with hematoxylin-eosin (HE) staining.
Study groups. From the initially selected 298 patients, 86 patients were included in the study, meeting the inclusion and exclusion criteria. Patients were divided into groups according to the Dworak classification: Dworak 0-4 patients, Dworak 1-13 patients, Dworak 2-32 patients, Dworak 3-23 patients, Dworak 4-14 patients. For further tissue sample analysis, two groups were formed: the bad response (BR) group, consisting of patients with no tumor response or a minimal response to nCRT (corresponding to Dworak 0 and 1), and the good response (GR) group, consisting of patients with a good response to nCRT (corresponding to Dworak 3). Considering that the Dworak 2 group was the most heterogeneous and exhibited the greatest phenotypic variability, it was not analyzed further. Additionally, Dworak 4 was not evaluated, as no tumor cells were morphologically detected in that group. The formation of the study groups is shown in Figure 1.
Systematization of the tumor samples according to the Dworak classification (tumor regression grade from 0 το 4). For further tissue sample analysis, two groups were formed: Bad response (BR) group (corresponding to Dworak 0 and 1), and good response (GR) group (corresponding to Dworak 3) to nCRT.
Study groups characteristics. A total of 40 patients were included in the study: 17 patients in the BR group and 23 patients in the GR group. The patients were followed up for 3 to 98 months. All patients had morphologically verified rectal adenocarcinoma, and clinical stage evaluation was based on radiological examinations – computed tomography (CT) scans of the abdomen and thorax and pelvic magnetic resonance imaging (MRI). All patients received nCRT and completed the course. Re-staging was performed (pelvic MRI) 6-12 weeks after finishing nCRT. The treatment response was evaluated according to the mrTRG (magnetic resonance imaging tumor regression grade) classification. Radical surgery was performed in all cases. The summary of post-treatment histopathological evaluations, as along with the clinical characteristics of the patients, is summarized in Table II.
Clinical characteristics of the study groups.
Stages of the study. Stage 1. Preparation of FFPE tissue samples. FFPE tissue samples for further analysis from each patient were obtained, including 10 μm sections from the tumor (sample A) and the proximal resection line (sample B). A total of 8 samples were obtained from each tumor and each resection line (16 samples per patient). The tissue samples were stored at −20°C until the next step of the study. The following study workflow is summarized in Figure 2.
The flowchart of the study. Five stages of the study: Stage 1. Acquisition of formalin-fixed, paraffin-embedded (FFPE) tissue samples from bad response (BR) and good response (GR) groups. Samples A – rectal cancer; samples B – proximal resection line. Six samples from GR and BR selected for the next step. Stage 2. Total RNA extraction from selected and all the rest A and B samples. Stage 3. Screening. MiRNA profiling in A and B samples. Stage 4. Selection of target miRNA. Stage 5. Validation. Identification of selected miRNA in all the rest A and B samples.
Stage 2. Isolation of total RNA from FFPE tissue samples. Total RNA (including miRNA) extraction from FFPE tissue samples was performed using the miRNeasy FFPE kit protocol (miRNeasy FFPE Kit for miRNA extraction, QIAGEN, ID: 217504; Venlo, the Netherlands). Total RNA concentration measurements in the samples were performed using fluorometry, following the Qubit™ RNA BR assay kit protocol (Invitrogen from Thermo Fisher Scientific; Catalog No. Q10210, Q10211; Waltham, MA, USA).
Stage 3. Screening (miRNA profiling in selected A and B samples). From each group (BR and GR group), six cases were selected – a total of 12 tumor (A) samples and 12 proximal resection line (B) tissue samples. Reverse transcription reaction to generate complementary DNA (cDNA) was performed using the miRCURY LNA RT kit (QIAGEN, ID: 339340). Reverse transcription polymerase chain reaction (RT-PCR) for miRNA profiling (a total of 752 miRNAs) was conducted in the selected samples using miRCURY LNA miRNA miRNome PCR panels (QIAGEN, ID: 339322). All steps were performed in triplicate (i.e., three technical replicates). All procedures were carried out according to the manufacturer’s protocol, using the ViiA™ 7 Real-Time PCR System (Applied Biosystems from Thermo Fisher Scientific). The obtained data were analyzed using the QIAGEN GeneGlobe online data analysis tool (28).
Stage 4. Selection of target miRNA. From the identified 752 miRNAs, those miRNAs were selected whose expression (fold regulation) was at least 2x higher or lower than the established standard (the miRNA with equal expression levels in all samples (A and B samples, GR and BR group) was considered as the reference), the expression in the tumor was up-regulated and p<0.05. The clinical significance of the selected miRNAs was evaluated by assessing their association with specific genes, using the miRNA online database miRDB (29). Three miRNAs from each group (GR and BR) with the highest p-value were selected and further evaluated in the online program miRTargetLink 2.0 to assess their association with specific genes (30). For normalization, two miRNAs were selected that had a Ct value in RT-PCR >30 and the “fold regulation” value closest to the standard in both groups across all samples.
Stage 5. Validation of selected miRNAs. Selected miRNAs were validated in the rest of the A and B samples [17 patients from GR group (34 samples) and 11 patients from BR group (22 samples)]. cDNA synthesis and RT reaction were performed using the TaqMan™ MicroRNA Reverse Transcription Kit (Applied Biosystems from Thermo Fisher Scientific, ID: 4366596) and primers (Applied Biosystems from Thermo Fisher Scientific; IDs 002681, 002141, 002229, 002248, 002429, 000483, 002642). This was followed by the quantification of the selected miRNAs using the TaqMan™ Small MicroRNA Assay (ID: 4427975; Applied Biosystems from Thermo Fisher Scientific) and TaqMan™ Fast Advanced Master Mix (ID: 4444558; Applied Biosystems from Thermo Fisher Scientific). All steps were performed in triplicate (i.e., three technical replicates). All procedures were carried out according to the manufacturer’s protocol, using the ViiA™ 7 Real-Time PCR System (Applied Biosystems from Thermo Fisher Scientific).
Statistical analysis. The sample size for the first discovery experiment was calculated using the R package “size power” (31). The parameters for the matched two-sample calculation were as follows: the mean number of false positives=1; the anticipated number of un-differentially expressed genes in the experiment=752; power level=0.8; fold change=2; and the anticipated standard deviation of the difference in log-expression between matched treatment and control conditions=0.5. The calculated minimum sample size was 5 per group.
The independent samples t-test was used to compare continuous variables between the two study groups, while the Chi-square test was used for categorical variables.
A t-test, paired t-test, and Mann-Whitney U-test were used for the analysis of verified miRNAs. Local recurrence-free survival (LRFS), distant metastasis-free survival (DMFS), disease-free survival (DFS), and overall survival (OS) were estimated using the Kaplan-Meier method. The log-rank test was used to calculate any significant differences between the groups through univariate analysis. Significance levels were set at p<0.05. DFS (LRFS and DMFS) and OS were considered from the date of surgery to the date of any disease manifestation (local recurrence or metastasis) or patient death. The statistical analysis was carried out using Python version 3.4 and R version 4.4.2.
Ethics approval and consent to participate, and consent for publication. The study was conducted in accordance with the Declaration of Helsinki and approved by the Ethics Committee of Riga Stradiņš University (Nr. 2-PĒK-4/494/2022 on 21.11.2022 and Nr. 2-PĒK-4/120/2023 on 26.01.2023). Informed consent was obtained from all subjects (patients or their family members if the patient had deceased) involved in the study.
Results
Screening of miRNAs in rectal cancer tissue. From the identified 752 miRNAs, there were twenty-five miRNAs from the BR group and thirteen miRNAs from the GR group that met the selection criteria (Table III).
Screening results of miRNA in rectal cancer tissue from the BR and GR group.
Six miRNAs were selected (three from the BR group and three from the GR group) that had the most statistically significant results: miR-665, miR-99a-3p and miR-127-5p from GR group and miR-142-5p, miR-182-3p, and miR-548c-5p from BR group. MiR-151-5p was chosen for normalization, as it showed the most stable expression of fold changes among all tumor samples and was suggested by producer of the kit (QIAGEN).
Validation of selected miRNA in rectal cancer tissue. Subsequently, the validation of the selected miRNAs was performed in other tissue samples from the BR and GR groups using the TaqMan Small RNA Assays protocol (Applied Biosystems from Thermo Fisher; ID 002681, 002141, 002229, 002248, 002429, 000483, 002642). MiRNAs with p<0.05 in all statistical tests were selected as clinically significant. Accordingly, in the BR group, miR-142-5p and miR-182-3p met this criterion, and in the GR group, it was miRNA-99a-3p (Table IV). Three target miRNAs turned out to be false positives – miR-548-5p, miR-665, and miR-127-5p.
The validation results of the selected miRNA in rectal cancer tissue (up-regulated miRNAs with a fold change of at least two times in the BR and GR group).
The string analysis of miRNA target genes showed a relationship between the selected miRNAs and several oncogenes. The string analysis of BR group miRNAs revealed more clinically significant relationships than those of GR group miRNAs (Figure 3 and Figure 4).
Target genes of BR group miRNAs and the interaction between target gene products. FBXW7: F-box and WD repeat domain-containing 7; Becn1: Beclin 1; TP53INP1: tumor protein p53 inducible nuclear protein 1; STAT5B: signal transducer and activator of transcription 5B; NOX4: NADPH oxidase 4; SCO2: synthesis of cytochrome c oxidase 2; PTEN: phosphatase and tensin homolog; RAC1: Ras-related C3 botulinum toxin substrate 1; SMAD3: SMAD family member 3; SOCS1: suppressor of cytokine signaling 1; ZEB1: Zinc finger E-box binding homeobox 1; MYADM: myeloid associated differentiation marker; HIF1A: hypoxia-inducible factor 1 alpha; CLDN1: claudin 1; TGFBR2: transforming growth factor beta receptor 2; TGFB2: transforming growth factor beta 2; NFE2L2: nuclear factor, erythroid 2 like 2; SIRT1: sirtuin 1; BACH2: BTB and CNC homology 2.
Target genes of GR group miRNAs and the interaction between target gene products. The targets of miR-99a-3p are not found in the strongly validated criteria; therefore, there is no interaction analysis. BLVRB: Biliverdin reductase B; CNR2: cannabinoid receptor 2; SPP1: secreted phosphoprotein 1; CD274: cluster of differentiation 274.
The survival rates in BR and GR groups. The following Kaplan-Meier analysis was executed to estimate DFS, LRFS, DMFS, and OS in the BR and GR groups (Figure 5). Afterwards, the population of both study groups was pooled, and the survival parameters (DFS, LRFS, DMFS and OS) were evaluated according to the expression level (lower or higher) of a particular miRNA – miR-142-5p (Figure 6), miR-182-3p (Figure 7), and miR-99a-3p (Figure 8).
Survival results based on clinical responses - Bad response (BR) and good response (GR) to neoadjuvant chemoradiation therapy (nCRT). A) disease-free survival (DFS); B) local recurrence-free survival (LRFS); C) distant metastasis-free survival (DMFS); D) overall survival (OS).
Survival results based on the expression level of miR-142-5p (local recurrence-free survival and distant metastasis-free survival – data not shown). A) disease-free survival (DFS); B) overall survival (OS).
Survival results based on the expression level of miR-182-3p (distant metastasis-free survival – data not shown). A) disease-free survival (DFS); B) local recurrence-free survival (LRFS); C) overall survival (OS).
Survival results based on the expression level of miR-99a-3p (local recurrence-free survival and overall survival–data not shown). A) disease-free survival (DFS); B) distant metastasis-free survival (DMFS).
Although the p-value in none of the groups reached the threshold of statistical confidence (p<0.05), a similar trend was observed in all categories – the estimated DFS, LRFS, DMFS, and OS were worse in the BR group and in cases of lower expression levels of miR-142-5p and miR-99a-3p, and higher expression of miR-182-3p. We assume that statistically significant results were not established due to the limited number of patients in both groups.
Discussion
The role of miRNAs in the behavior of rectal cancer, tumor progression, treatment resistance, and overall patient outcomes has been described in several studies (20, 23, 32, 33). This study focused on miRNA profiling of rectal cancer tissue and verification of clinically relevant ones in association with clinical response to nCRT and survival rates.
We aimed to assess the significance of each miRNA that demonstrated clinical relevance in our study; however, we recognize that miRNAs interact with various factors that may influence their regulation. The importance of miRNA up-regulation versus down-regulation depends on the specific context and the biological pathways involved. Up-regulation of certain miRNAs may be crucial for promoting tumor progression or resistance to therapy, while down-regulation of others may be significant for tumor suppression. Therefore, both up-regulation and down-regulation can play critical roles in various biological processes and disease states, and their importance should be evaluated based on the specific miRNA and the associated conditions (34). In this study, we primarily focused on up-regulated miRNAs, with plans to further analyze down-regulated miRNAs and their potential synergistic interactions.
In our study, it was observed that miR-142-5p was up-regulated in patients with poor response to the nCRT, but its’ lower expression in general was related with worse DFS and OS.
MiR-142-5p has emerged as a significant regulator in various cancers, including rectal cancer, due to its influence on key oncogenes and tumor suppressors (35). MiR-142-5p has been shown to negatively regulate several oncogenes that promote cell proliferation. For instance, it can target genes involved in signaling pathways such as the RAS/RAF/MEK/ERK pathway, which is critical for cell growth and differentiation. By down-regulating these oncogenes, miR-142-5p can inhibit tumor growth and metastasis (36).
Conversely, miR-142-5p may also interact with tumor suppressor genes, leading to altered cancer biology. In some contexts, it can inhibit the expression of tumor suppressor proteins, thereby promoting oncogenic pathways. For example, miR-142-5p has been implicated in the regulation of tumor suppressor genes such as PTEN, which is crucial for controlling cellular growth and apoptosis. The down-regulation of PTEN can contribute to enhanced cell survival and increased resistance to apoptosis (37).
MiR-142-5p has been associated with the development of chemoresistance in rectal cancer through several mechanisms: (i) regulation of drug metabolism and efflux: miR-142-5p can influence the expression of transporters and enzymes involved in drug metabolism. By modulating the levels of these proteins, miR-142-5p can affect the accumulation and efficacy of chemotherapeutic agents, leading to reduced sensitivity to treatment (38); (ii) modulation of apoptotic pathways: by targeting key apoptotic regulators, such as Bcl-2 family members, miR-142-5p can inhibit programmed cell death in response to chemotherapy. This anti-apoptotic effect allows cancer cells to survive despite the presence of cytotoxic agents, contributing to treatment failure (39); (iii) influencing the tumor microenvironment: miR-142-5p may also affect the tumor microenvironment, promoting a supportive niche that enhances resistance to therapy. By modulating cytokine and growth factor secretion, miR-142-5p can influence immune responses and angiogenesis, creating conditions that favor tumor survival during treatment (40).
The role of miR-142-5p as a possible prognostic marker of poor CRC prognosis has been presented in some studies (41-43). The overexpression of miR-142-5p has been associated with cancer in the proximal colorectum, BRAF positive patients, and biological aggressiveness of cancer (6). The study by Kunigenas et al. in 2020 demonstrated that miRNA-142-5p is a diagnostic biomarker of rectal cancer following long-course nCRT. This study showed that miR-142-5p expression levels were significantly increased in rectum tumor tissue samples following long-course nCRT compared to tumor samples collected before the nCRT and compared to adjacent normal rectum tissue. The overexpression of miR-142-5p was observed in patients who received chemotherapy compared to expression levels in the group without the treatment, suggesting that the evaluation of miR-142-5p expression levels could serve as a diagnostic biomarker of cancer therapy (7). Another study of Shi et al. in 2015 demonstrated that expression of miR-142-5p increases during the infusion chemotherapy in stage III CRC, suggesting that miR-124-5p is a potential tumor suppressor in CRC and could serve for evaluation of efficacy of infusion chemotherapy and the progress of CRC (34). Regarding the importance of miR-142-5p in predicting the tumor response, the study of Cervena et al. in 2021 presented the association of miR-142-5p expression levels and therapy response, respectively, the patients who died within a year after the diagnosis, didn’t have benefit from the therapy or had local recurrence, presented significantly lower expression levels in their second plasma sampling compared to the first one (first one taken at the time of diagnosis and the second one – a year after therapy). These findings suggest that miR-142-5p acts as a tumor suppressor. Additionally, miR-142-5p was up-regulated in the presence of 5-FU by SMiR-NBI (the Small Molecule-miRNA Network-Based Inference) model, and this probably means that the effect of miRNA is potentiated in this way (43).
Another miRNA in the study, which was up-regulated in BR group and associated with worse DFS and OS, was miR-182-3p, which belongs to the family of miR-182.
MiR-182-3p has been identified as a significant player in various cancers, including rectal cancer, where it modulates critical pathways involved in tumor progression and chemoresistance. MiR-182-3p can interact with several oncogenes that promote tumor growth and survival. For example, it has been shown to target genes involved in the phosphoinositide 3-kinase (PI3K)/AKT signaling pathway, which is crucial for cell survival and proliferation. By down-regulating these oncogenes, miR-182-3p can inhibit cancer cell growth and reduce invasive potential (44).
MiR-182-3p also influences the expression of tumor suppressor genes. It has been found to down-regulate key tumor suppressors such as PTEN and FOXO1. The inhibition of PTEN, for instance, leads to enhanced AKT signaling, promoting cell survival and proliferation. By suppressing these tumor suppressors, miR-182-3p can facilitate oncogenic processes, contributing to tumor progression (45).
MiR-182-3p is implicated in the development of chemoresistance in rectal cancer through several mechanisms: (i) regulation of drug efflux pumps: miR-182-3p can modulate the expression of drug efflux pumps, such as ATP-binding cassette (ABC) transporters. By increasing the expression of these transporters, miR-182-3p can enhance the ability of cancer cells to expel chemotherapeutic agents, leading to reduced drug accumulation and efficacy (46); (ii) influencing apoptotic pathways: miR-182-3p can affect apoptotic signaling pathways by targeting pro-apoptotic factors. For example, it may down-regulate proteins like BIM, which promote apoptosis, thereby allowing cancer cells to evade cell death in response to chemotherapy (47); (iii) modulating the tumor microenvironment: miR-182-3p may also play a role in shaping the tumor microenvironment, affecting interactions between cancer cells and stromal or immune cells. By influencing cytokine secretion and immune evasion mechanisms, miR-182-3p can create a more favorable environment for tumor survival during treatment (48).
The evidence suggests that miR-182 has an important role as an onco-miRNA in different types of cancers by promoting tumor cell survival and metastasis. It is often up-regulated in various types of tumors and may inhibit apoptosis (45). In particular, it has been demonstrated that miR-182 plays a crucial role in CRC tumorigenesis. Several studies have reported that miR-182 expression is up-regulated in CRC tissues compared to adjacent non-cancerous tissues (49). The up-regulation of miR-182 has been associated with the advanced stage of TNM, i.e., positive regional lymph node status and high depth of tumor invasion, and as well as with local recurrence of the tumor (44, 50). The study of Yang et al. in 2014 suggested that a higher level of miR-182 expression significantly promotes CRC invasion, migration, and cell proliferation in vivo and in vitro (51). A high level of miR-182 has been observed in 5-FU-resistant CRC cell lines. Up-regulation of miR-182 considerably induces drug resistance, proliferation, and colony formation, and causes apoptosis to be reduced in 5-FU-resistant CRC cell lines (52).
Among the miRNAs evaluated, it was observed that miR-99a-3p was up-regulated in the group of GR, and a lower expression level of miR-99a-3p was associated with worse OS and DFS.
MiR-99a-3p has been shown to directly target several oncogenes that promote tumor growth and survival. One of its well-characterized targets is the mTOR (mechanistic target of rapamycin) pathway, a key regulator of cell growth and proliferation. By inhibiting components of the mTOR pathway, miR-99a-3p can reduce cell proliferation and induce apoptosis in cancer cells, countering oncogenic signals (53).
In addition to its effects on oncogenes, miR-99a-3p can also influence tumor suppressor genes. For instance, it has been implicated in the regulation of the tumor suppressor gene PTEN. By down-regulating PTEN, miR-99a-3p can enhance AKT signaling, promoting cell survival and growth. This dual role in targeting oncogenes and tumor suppressors underscores miR-99a-3p’s complex involvement in cancer biology (54).
The role of miR-99a-3p has been associated with various types of cancer; however, the studies revealing its predictive role in CRC are limited. In a study of Pinelo et al. in 2014, expression of miR-99a-3p in stage IV CRC patient blood samples was significantly associated with progression-free survival and OS and therefore was validated as a predictive marker for chemotherapy response (55).
The results obtained from this study, revealed a pronounced trend regarding the significance of certain miRNAs in predicting disease behavior; however, we were unable to achieve statistical significance, which we attribute to several factors. The study had several limiting factors. First, the number of patients included in the study was small due to the selection criteria, as well as the fact that patients with post-treatment pathomorphosis corresponding to Dworak 2 and Dworak 4 were not included. Second, the GR group, whose results were compared to the BR group, could not be considered as a completely favorable response, as it comprises patients whose postoperative morphological examination materials correspond to Dworak 3. A complete favorable response is classified as Dworak 4, where tumor cells are no longer detected, which consequently limits the analysis of this material from a tumor perspective. Lastly, it is essential to note that both groups (GR and BR) still exhibit heterogeneity, which only increases during the analysis process. Considering that the initial selection groups were different, it is worth noting that not only does miRNA expression affect disease outcomes and patient prognosis, but the differing clinical conditions of rectal cancer may also influence variations in miRNA expression. These factors hinder the attainment of statistical significance, and the direct association of miRNA with patient survival prognosis is secondary; however, a certain trend in the association is still observed. MiRNA is a correlating factor to rely on.
The ability to assess miRNA expression profiles not only enhances our understanding of tumor behavior but also offers promising avenues for personalized medicine. By integrating miRNA analysis into clinical practice, patient stratification could be improved, enabling more tailored treatment approaches that consider individual molecular profiles. Additionally, miRNAs could serve as valuable prognostic markers, aiding in the prediction of treatment responses and survival outcomes, as well as detecting cancer progression and determining treatment plans. As research continues to unravel the intricate roles of miRNAs in rectal cancer, future studies should focus on validating these findings in larger, diverse cohorts and exploring the therapeutic potential of targeting miRNAs. Ultimately, incorporating miRNA profiles into clinical decision-making could transform the management of rectal cancer, paving the way for improved prognostic assessments and enhanced patient care.
Acknowledgements
Gratitude to all the medical professionals and patients involved in the study.
Footnotes
Authors’ Contributions
Conceptualization, L.K., E.M., A.G.; methodology, L.K., E.M., Z.D.; software, L.K., M.Š.; validation, Z.D., E.M.; formal analysis, L.K., M.Š., M.N.M.; investigation, L.K., D.Z., I.N., J.N.; data curation, L.K.; writing – original draft preparation, L.K.; writing – review and editing, L.K., Z.D., E.M.; visualization, L.K., M.N.M.; supervision, E.M., Z.D. All Authors have read and agreed to the published version of the manuscript.
Conflicts of Interest
The Authors declare that they have no competing interests related to this work.
Artificial Intelligence (AI) Disclosure
No artificial intelligence (AI) tools, including large language models or machine learning software, were used in the preparation, analysis, or presentation of this manuscript.
- Received June 9, 2025.
- Revision received June 20, 2025.
- Accepted June 30, 2025.
- Copyright © 2025 The Author(s). Published by the International Institute of Anticancer Research.
This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.
References
Jump to section
Related Articles
Cited By...
- No citing articles found.