It is becoming clear that deregulated expression of miRNAs is connected to pathogenesis of many cancer types. In this study, we analysed expression profile of 667 miRNAs in 8 tissues of patients with CRC and paired non-tumoural tissues. Using unsupervised hierarchical clustering, we gained a set of 42 miRNAs that showed significantly different expression between tumoural tissue and adjacent normal mucosa. Four of them (miR-135b, miR-766, miR-183* and miR-135b*) were significantly overexpressed in tumour tissues, and they are supposed to function as oncogenes, although their specific role in pathogenesis of CRC is not known. Nevertheless, miR-183 is one in a cluster of three related miRNAs; this cluster also includes miR-182 and miR-96. It is situated on chromosome 7q32.2  and was described to be overexpressed in many other types of cancer, including lung cancer , breast cancer , colon cancer  and prostate cancer . These miRNAs were also up-regulated in our study, but they did not meet the criterion P < 0.0005. Furthermore, Nagel et al. proved that miR-135b binds 3′ UTR of the APC gene, which leads to accumulation of free β-catenin in cells. The other identified miRNAs were down-regulated in CRC tissues, which is in accordance with previous observations that miRNA levels are globally decreased in human cancers . Several of them were already described in connection with CRC in previous studies (miR-192, miR-215, miR-26b, miR-143, miR-145, miR-16 , miR-139-5p , miR-138 , miR-451 , miR-378, miR-378* , miR-133b , miR-144* , miR-195 , miR-194 , miR-30c, miR-26a , miR-342-3p  and miR-101 ), whereas remaining were identified in this study for the first time (miR-190, miR-422a, miR-375, miR-376, miR-486-5p, miR-598, miR-99a, miR-636, miR-100, miR-411, miR-30b, miR-30e, miR-30e*, miR-30a*, miR-192a*, miR-127-3p, miR-200b, miR-140-5p and miR-186). It seems that miR-30 family is important in CRC, because most of its members were down-regulated in tumour tissue. Xi et al.  described decreased expression of these miRNAs in CRC cell lines with TP53 gene deletion. MiR-143 and miR-145 are tumour suppressive miRNAs that are very repeatedly described as associated with CRC. Among their target molecules, there are oncogenes APC , ERK, RAF , EGFR , MYC , MAPK, CCND2 and transcription factors FOS, YES and FLI . Boominathan  showed that expression of miR-145 depends on correct function of p53. This important molecule is also connected with other miRNAs – miR-192, miR-194 and miR-215- that belong to the same family and affect cell cycle arrest through p21 accumulation . Lower expression of miR-195 is associated with lymph node positivity and shorter survival of patients with CRC. It is supposed that this miRNA can bind anti-apoptotic protein BLC-2 and thus promote apoptosis . Similarly, miR-133b is able to regulate some proteins from the BCL-2 family . Moreover, among important targets of this miRNA, there are oncogenes KRAS and MAP3K1  supporting miR-133b function as an important tumour suppressor. Decreased levels of miR-101 are connected with enhanced function of COX-2 (cyclooxygenase 2), oversized oxidation of arachidonic acid and subsequently deregulated activation of prostaglandins, which leads to increased proliferation of cancer cells . Although the number of research groups interested in miRNAs functioning in cancer in last years dramatically increased, new pathogenic miRNAs are still being discovered and further studies are necessary to understand the modus operandi of this complex regulatory network in CRC pathogenesis.
Therefore, we chose five miRNAs (miR-215, miR-375, miR-378, miR-422a and miR-135b) for further validation and in vitro analyses to find out what is their role in CRC. We used new collection of 125 paired-samples of tumour tissue and adjacent non-tumoural mucosa to confirm our results from miRNA profiling. We proved that miR-215, miR-375, miR-378 and miR-422a are significantly down-regulated, whereas miR-135b is up-regulated in tumour tissue (P < 0.0001). Furthermore, we analysed the correlation between the miRNA expression levels and clinical–pathological features of the CRC patients (see Table 1). It was found that lower expression of miR-215 and miR-422a is associated with advanced stages of the disease. Interestingly, expression of miR-135b was the highest in stage I, which is in accordance with previous studies describing the role of this miRNA in early stages of CRC by targeting APC and activating Wnt signalling . Importantly, we also observed the relationship between lymph node positivity and the expression of miR-215, miR-378, miR-422a and miR-135b. This fact indicates a possible role of these miRNAs in migration of colon cancer cells, which was also analysed in this study. Moreover, expression of these miRNAs could contribute to better differentiation between clinical stages II and III, which is currently based on examination of lymph node positivity. Unfortunately, this approach is not precise enough, because about 25% of patients with lymph node involvement are not detected due to insufficient number of examined lymph nodes. Subsequently, these patients do not receive adjuvant systemic therapy increasing probability of relapse. Finally, we detected increased expression of miR-135b in samples of patients with higher pre-operative serum levels of CAE and CA19-9. This is in agreement with previous observations describing higher levels of miR-135b also in plasma of CRC patients, indicating its potential usage as circulating biomarker in CRC .
To explore the function of validated miRNAs in pathogenesis of CRC, DLD-1 and HCT-116 colon cancer cells were transfected with particular miRNA precursors or inhibitors. The functional analyses of miR-215 showed that overexpression of this miRNA leads to cell cycle arrest and enhanced apoptosis of HCT–116 cells carrying wt-p53, but no effect was observed in the case of DLD–1 cells containing mut-p53. These results are in accordance with previous studies that describe direct connection between miR-215 function and p53 status. Braun et al. found out not only p53-responsive induction of miR-215 but also direct feedback of this miRNA on the activity of p53, which results in activation of apoptosis and cell cycle arrest in cells with wild-type p53, but not in cells with mutated p53. Moreover, we detected significantly decreased migration of cells transfected with pre-miR-215, supporting our observation that expression of this miRNA is lower in primary tumours with lymph node positivity. Karaayvaz et al. () observed prognostic potential of miR-215 in the small cohort of 34 CRC patients of II and III clinical stages. Expression levels of miR-215 were decreased in tumours, but, interestingly, higher levels of miR-215 were associated with worse survival (P = 0.025). Differences in miR-215 levels between tumour of clinical stages II and III were not evaluated . These results together indicate that miR-215 is important in pathogenesis of CRC and could be used not only as a new biomarker of the disease but also as a potential therapeutic target for prevention of metastases.
The analyses of other miRNAs revealed that higher expression of miR-378, miR-375 and miR-422a is associated with accumulation of cells in G1 phase in HCT-116 cells. Concerning DLD-1 cells, we did not observe any significant effect on cell cycle and apoptosis. Therefore, we suppose that these miRNAs function in a similar way as miR-215, although the precise mechanism is not known and further identification of target molecules will be necessary. Using MTT assay, we observed a decreased viability of both DLD-1 and HCT-116 cells 48 hrs after transfection with pre-miR-375 or anti-miR-135b. Tsukamoto et al.  described that miR-375 inhibits expression of PDK1 (phosphoinositide-dependent kinase-1) and anti-apoptotic protein 14–3–3zeta by binding to its 3′ UTR, and its ectopic expression markedly reduced viability of gastric cancer cells.
In conclusion, we proved that miR-215, miR-375, miR-378 and miR-422a evince significant tumour suppressive properties, whereas miR-135b functions as an oncogene. Our observations from analysis of clinical CRC samples indicate potential usage of validated miRNAs as biomarkers, and our functional screening suggests that some of them, mainly miR-215, represent potentially important targets for novel therapeutic strategies in CRC. However, exact molecular mechanisms of these miRNAs functioning could not be fully understood as their target molecules have not been experimentally validated.