Twenty samples from 10 patients, including 6 men and 4 women, were used to perform qRT-PCR. The age at diagnosis ranged from 43 to 79 years. Primary tumors were observed in the colon of 3 patients and in the rectum of 7 patients. The histological subtypes pure adenocarcinoma and mucinous adenocarcinoma were found in 9 and 1 patients, respectively. The staging of patients following the American Classification Joint Committee on Cancer showed 2 patients classified as stage I, 3 patients as stage II, and 5 patients as stage III.
From the miRNA–TF–gene networks, we found that miR-20a might be playing the most important miRNA role for CRC. Therefore, we performed qRT-PCR to examine the expression of miR-20a and TFs that interacted with miR-20a, including PPARA, PPARD, PPARG, and EPAS1 in CRC. The relative expression of miR-20a was 1.84 ± 0.73 folds upregulated in tumor tissues versus the adjacent nontumor tissues (P < .01). The relative expression levels of PPARA, PPARG, and EPAS1 were 0.36 ± 0.15 (P < .01), 0.26 ± 0.18 (P < .01), and 0.48 ± 0.28 (P < .05) folds downregulated in tumor tissues versus adjacent nontumor tissues, respectively. The relative expression of PPARD was 0.87 ± 0.57 folds downregulated in tumor tissues versus the adjacent nontumor tissues, but this was not statistically significant. Because the most enriched KEGG pathway of the target genes was the PI3K-Akt signaling pathway, we chose the main PI3K-Akt signaling pathway-related elements from the genes in the miRNA–TF–gene networks, including phosphatase and tensin homolog deleted on chromosome 10 (PTEN) and B-cell lymphoma 2-associated agonist of cell death (BAD), to perform qRT-PCR verification. The relative expression levels of PTEN and BAD were 0.50 ± 0.48 and 0.25 ± 0.36 folds downregulated in tumor tissues versus adjacent nontumor tissues, respectively (Fig. 8).
CRC is a digestive tract tumor with a relatively high incidence rate among various malignant tumors. In the past several decades, numerous studies have been conducted to understand the causes and underlying mechanisms of CRC occurrence and development; however, the incidence and mortality of CRC remain very high globally. This might be because studies often focus on a single genetic effect. However, cancer genomics is complex, with underlying networks involving a variety of factors such as noncoding RNAs, coding genes, and TFs. The overall aim of the current study was to explore the correlation among miRNAs, TFs, and target genes in CRC.
Initially, we integrated 3 cohorts of profile datasets of individuals from 3 different geographical regions (China, Italy, and the USA) and applied bioinformatics analysis to identify 14 commonly altered DEMs. By predicting the targets of the DEMs and the associated cancer-related TFs, we finally identified 5 miRNAs (miR-145, miR-497, miR-30a, miR-31, and miR-20a) that were considered to regulate CRC proliferation through TFs. By consulting the literature, we found that all the 5 miRNAs are related to the generation and proliferation of malignant tumors.[25–29] Among the 5 miRNAs, miR-20a was considered to be the most important miRNA in CRC because it interacted with 4 TFs (PPARA, PPARD, PPARG, and EPAS1), while the others interacted with only 1 TF. MiR-20a, a member of the miR-17–92 cluster, has been shown to function as an oncomir in CRC. A study performed by Cheng et al demonstrated that miR-20a was upregulated in CRC and that it promoted CRC invasion and metastasis by downregulating Smad4. Xu et al reported that miR-20a enhances the epithelial-to-mesenchymal transition of CRC cells by modulating matrix metalloproteinases. In the present study, on the basis of miRNA microarray data and qRT-PCR verification, we demonstrated that miR-20a was upregulated in CRC. The results were consistent with several studies. However, the result of qRT-PCR was not exactly the same as that of the microarray. In the qRT-PCR test, the relative expression of miR-20a was 1.84 ± 0.73 folds upregulated in CRC tissues compared to normal tissues, which was less than 2 folds. The strength of microarray-based techniques lies in their ability to quantify large numbers of miRNAs simultaneously in a single experiment. However, the specificity of microarray-based miRNA tests is lower than that of qRT-PCR tests. Therefore, we believe that the results of qRT-PCR are more reliable. Of course, we cannot exclude the errors in the results of qRT-PCR tests, which could partly be due to the small sample number. Fan et al reported that miR-20a overexpression suppresses the expression levels of PPARG in bone marrow stem cells, indicating that PPARG might be the direct target of miR-20a. The result of this study is consistent with the putative target of miR-20a in our study. Besides, in the present study, we found that the PI3K/Akt signaling pathway was the most significantly enriched pathway in the miRNA–TF–gene networks. This finding was similar to the report of Jiang et al, which demonstrated that miR-20a suppresses multiple myeloma progression by modulating the PTEN/PI3K/Akt signaling pathway.
TFs are the final players of signal transduction cascades, which often begin with extracellular ligand-binding events, followed by signal integration and processing, ultimately resulting in the initiation or repression of target gene transcription. MiRNAs and TFs combine together in a functional network to alter gene expression in cancer.
The PI3K/Akt pathway is an important intracellular signal transduction pathway to control the progression of tumor cells, including apoptosis, transcription, translation, metabolism, and angiogenesis. During malignant transformation, various genetic alterations may occur in any of the PI3K pathway components, such as the receptor tyrosine kinase genes EGFR, HER2, KIT, PTEN, PIK3CA, and AKT. By KEGG analysis in the present study, we found that the PI3K/Akt signaling pathway was the most significantly enriched pathway in the miRNA–TF–gene networks. Simultaneously, we found 2 important genes in the miRNA–TF–gene networks: PTEN and BAD. As a negative regulator of PI3K/Akt signaling, PTEN is closely correlated with the carcinogenesis, progression, and prognosis of CRC. A study by Hsu demonstrated that the expression of PTEN in CRC was lower than that in normal colon mucosa, and negative expression of PTEN was correlated with tumor size and poor prognosis in CRC. As an important downstream target of PI3K/Akt signaling, BAD can promote apoptosis by binding B-cell lymphoma 2 and inhibiting its function. In the present study, the expression levels of PTEN and BAD were lower in CRC tumor tissues compared with adjacent nontumor tissues, as confirmed by qRT-PCR, suggesting that PTEN and BAD might play an inhibitory role in CRC. Nevertheless, further research needs to explore the underlying mechanisms in more detail.
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