The objectives of the present study were to identify the aberrant expression of microRNA (miRNA) in colorectal carcinoma (CRC) tissues from published miRNA profiling studies and to investigate the effects of the identified miRNA inhibitor and mimic miR-96-5p on CRC cell migration and invasion. The altered expression of the regulators of cytoskeleton mRNA in miR-96-5p inhibitor-transfected cells was determined. The miR-96-5p expression level in five CRC cell lines, HCT11, CaCo2, HT29, SW480 and SW620, and 26 archived paraffin-embedded CRC tissues were also investigated by reverse-transcriptase quantitative polymerase chain reaction (RT-qPCR). Cell viability in response to the miR-96-5p inhibitor and mimic transfections was determined by an MTT assay. A Matrigel invasion assay was conducted to select the invasive subpopulation designated SW480-7, by using the parental cell line SW480. The effects of miR-96-5p mimic-or inhibitor-transfected SW480-7 cells on cell migration and invasion were evaluated using the Transwell and Matrigel assays, and the change in expression of the regulators of cytoskeleton mRNAs was identified by Cytoskeleton Regulators RT 2-Profiler PCR array followed by validation with RT-qPCR. CRC tissues exhibited a significant increase in miR-96-5p expression, compared with their matched normal adjacent tissues, indicating an oncogenic role for miR-96-5p. The results demonstrated that the miR-96-5p inhibitor decreased the migration of SW480-7 cells, but had no effect on invasion. This may be due to the promotion of cell invasion by Matrigel, which counteracts the blockade of cell invasion by the miR-96-5p inhibitor. The miR-96-5p mimic enhanced SW480-7 cell migration and invasion, as expected. It was determined that there was a >2.5 fold increase in the expression of genes involved in cytoskeleton regulation, myosin light chain kinase 2, pleckstrin homology like domain family B member 2, cyclin A1, IQ motif containing GTPase activating protein 2, Brain-specific angiogenesisinhibitor 1-associated protein 2 and microtubule-actin crosslinking factor 1, in miR-96-5p inhibitor-transfected cells, indicating that they are negative regulators of cell migration. In conclusion, the miR-96-5p inhibitor blocked cell migration but not invasion, and the latter may be due to the counteraction of Matrigel, which has been demonstrated to stimulate cell invasion.
The objective of this study was to determine the effect of miR‑29a‑3p inhibitor on the migration and invasion of colorectal cancer cell lines (CRC) and the underlying molecular mechanisms. miR‑29a‑3p was detected using reverse transcription-quantitative polymerase chain reaction (RT‑qPCR) in the CRC cell lines HCT11, CaCo2, HT29, SW480 and SW620. An invasive subpopulation designated SW480‑7 was derived from the parental cell line, detected by Transwell and Transwell Matrigel assays. Cytoskeleton Regulators RT2 profiler PCR array and western blot analysis were utilized to identify the alterations in expression of downstream mRNAs. siRNA against CDC42BPA was transfected into SW480‑7 and effects on cell migration and invasion were investigated. Data obtained showed that miR‑29a‑3p was detected in these five CRC cell lines. miR‑29a‑3p inhibitor had no effect on viability but stimulated cell migration and invasion of SW480‑7 cells. In contrast, miR‑29a‑3p mimic suppressed cell migration and invasion. TargetScan miRBD and DIANA were employed to identify the potential direct target genes of miR‑29a‑3p in the Cytoskeleton Regulators RT2-Profiler PCR array. Cytoskeleton Regulators RT2-Profiler PCR array data showed that 3 out of the 5 predicted targets genes, CDC42BPA (2.33-fold), BAIAP2 (1.79-fold) and TIAM1 (1.77-fold), in the array were upregulated by miR‑29a‑3p. A significant increase in expression IQGAP2, PHLDB2, SSH1 mRNAs and downregulation of PAK1 mRNA was also detected with miR‑29a‑3p inhibition. Increase in CDC42BPA, SSH1 and IQGAP2 mRNA expression correlated with increased protein level in miR‑29a‑3p transfected SW-480-7 cells. Silencing of CDC42BPA (an enhancer of cell motility) partially abolished miR‑29a‑3p inhibitor-induced stimulation of cell migration and invasion. miR‑29a‑3p expression in stage II and III CRC is relatively lower than that of stage I CRC. However, the data need to be interpreted with caution due to the small sample size. In conclusion, inhibition of miR‑29a‑3p stimulates SW480‑7 cell migration and invasion and downstream expression IQGAP2, PHLDB2, SSH1 mRNAs are upregulated whilst PAK1 mRNA is downregulated. Silencing of CDC42BPA expression partially reduces miR29a‑3p inhibitor-induced migration and invasion of SW480‑7 cells.
Interleukin-17 (IL-17) is a pro-inflammatory cytokine found in various cancers. Current evidence indicates that IL-17 plays a vital role in tumour initiation and progression in colorectal carcinoma (CRC) via binding with its receptor, IL-17RA. However, the association between clinicopathological features and presence of IL-17 and IL-17RA protein in primary CRC tissues remains unclear. This study also investigates the difference between the presence of IL17 and IL-17RA in the paired tumour tissues versus adjacent normal tissues. The presence of IL-17RA and IL-17 protein in primary CRC tissues was determined by immunohistochemistry. Associations between clinicopathological features and IL-17RA and IL-17 immunoreactivity, were analyzed by χ2 tests. We found that both IL-17RA (p = 0.001) and IL-17 (p = 0.025) in tumour cells of primary CRC tissues was significantly lower as compared to adjacent normal tissue. Positive immunoreactivity for IL-17RA and IL-17 were detected in 51.0% and 16.8% of tumour tissues, respectively. Furthermore, negative immunoreactivity of IL-17R was significantly associated with advanced stage according to TNM classifier (p = 0.027), high grade of tumour (p = 0.019), increased depth of tumour invasion (p = 0.023) and vascular invasion (p = 0.039). Positive IL-17 immunoreactivity was associated with advanced stage (p = 0.008) and lymph node metastasis (p = 0.008). Thus, this study suggests that the loss of IL-17RA expression occurs as tumour progresses and this may predict the aggressiveness of tumour whilst expression of IL-17 promotes tumour progression and lymph node metastasis. Thus, loss of IL-17RA could be a useful prognostic biomarker for tumour progression in CRC patients.
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