Roles Of MiR-148a/-148b/-152 In Colorectal Cancer

ObjectiveMicroRNA(miRNA) is a class of endogenous, non-coding 21-25 nucleotides RNA, which was discovered in recent years. MiRNAs are transcribed as primary transcripts (pri-miRNA). An 70 nt precursor called the pre-miRNA, which is cleaved by Dicer to generate an 22nt mature miRNA, is excised out from the pri-miRNA by RNase-â…¢.The mature miRNA then gets assembled into the effector complexes. The "seed region", which is the core sequence that encompasses the first 2-7 nucleotides at the 5'portion of miRNA, is essential for the specific suppression of targe mRNA. In 1993,the first miRNA-lin-4 was discovered. Since then, increasing numbers of studies showed at least 500 miRNAs aberrantly expressed in different types of cancers, and showed that miRNAs were involved in the regulation of the proliferation, differentiation and apoptosis. Therefore, miRNAs were deemed to play a crucial role in the initiation and progression of human cancers, which could regulate many oncogenes and tumor suppressor genes.Colorectal cancer is one of the most common malignancies in the world. Altered expression of miRNAs have been reported in colorectal cancer, and may play a critical part in carcinogenesis. MiR-143 and miR-145, which were discovered in the early study, played important roles in the initiation and progression of colorectal cancer. Let-7,which was also found aberrant expression in colorectal cancer, was associated with not only carcinogenesis but also development of cancer. With more and more miRNAs were discovered in colorectal cancer, miRNAs might be involved in the diagnosis and therapy of colorectal cancer.In the present study, we detected the expression of miR-148a, miR-148b and miR-152 relative to their non-tumorous control in colorectal cancer tissues and cell lines using quantity real-time PCR. We studied the function of miR-148b in the colorectal cancer cell lines and detected preliminarily the role of miR-148b in the initiation and progression of cancer. 1. Expression of miR-148b in the colorectal cancer tissuesWe detected the relative expression of miR-148b in colorectal cancer tissues of 101 patients compared to their matched non-tumor adjacent tissues(NATs). Therefore, the value of the relative expression ratio< 1.0 was considered as low-expression in cancer relative to the non-tumorous control. We analyzed the association between miR-148b expression and clinicopathological characteristics.2. The expression of miR-148b in colorectal cancer and the effects on colorectal cancer cell proliferationWe detected the expression of miR-148b in colorectal cancer cell lines, HCT-116 and HT-29, by quantity real-time PCR. MiR-148b was over-expressed in colorectal cancer cell lines by transfecting miR-148b mimics. MTS assay was used to investigate the effect of miR-148b on the proliferation of colorectal cancer cell lines.3. Expression of miR-148a and miR-152 in the colorectal cancer tissues and cell linesWe detected the expression of miR-148a and miR-152 in the colorectal cancer tissues and cell lines(HCT-116 and SW-620) by quantity real-time PCR. We analyzed the association between these two miRNAs expression and clinicopathologic characteristics.Results1. Expression of miR-148b in the colorectal cancer tissues(1) We found low-expression of miR-148b in colorectal cancer tissues relative to their NATs. Among 101 patients with colorectal cancer,84 (83%) cases showed low-expression of miR-148b (p<0.001). The median fold change was 0.16. Low expression of miR-148b was associated with increased tumor size (p<0.001) and advanced pT stage (p=0.012).(2) The products of PCR were confirmed by TA cloning and sequencing assay.2. The expression of miR-148b in colorectal cancer cell lines and the effects on colorectal cancer cell proliferation As there is no normal colorectal epithelial cell, we randomly selected 3 NATs as control. The results of quantity real-time PCR showed that low-expression of miR-148b in the colorectal cancer cell lines (HT-29[p<0.001] and HCT-116[p<0.001]) at different levels between them. After transfection with miR-148b mimics and negative control(NC), we found up-regulation of miR-148b in the experience group comparing to NC group. As the results of MTS assay showed, up-regulation of miR-148b suppressed the proliferation of HT-29 from 48 hours(p<0.05) and HCT-116 from 72 hours(p<0.05).3. Expression of miR-148a and miR-152 in the colorectal cancer tissues and cell lines(1) We found low-expression of miR-148a and miR-152 in colorectal cancer tissues relative to their NATs.69 (68%) cases showed low-expression of miR-148a (p<0.001) and 80 (79%) cases showed low-expression of miR-152 (p<.001) in cancer tissues.The median fold change was 0.36 and 0.11, respectively. Low expression of miR-148a and miR-152 was associated with increased tumor size (p=0.018 and p=0.004, respectively) and advanced pT stage (p=0.023 and p=0.002, respectively).(2) As there is no normal colorectal epithelial cell, we randomly selected 3 NATs as control. The results of quantity real-time PCR showed that low-expression of miR-148a and miR-152 in the colorectal cancer cell lines (HCT-116[all p<0.001] and SW-620[all p<0.001]) at different levels among them.(3) A strong correlation among the expression of miR-148a, miR-148b and miR-152 in colorectal cancer tissues(p<0.001).(4) The products of PCR were confirmed by TA cloning and sequencing assay.Conclusions1. MiR-148b was down-regulated in colorectal cancer tissues relative to their NATs. Low expression miR-148b was associated with increased tumor size and advanced pT stage.2. MiR-148b was down-regulated in colorectal cancer cell lines (HCT-116 and HT-29) relative to non-tumorous control.3. Up-regulation of miR-148b suppressed the proliferation of colorectal cancer cells in MTS assay.4. MiR-148a and miR-152 were down-regulated in colorectal cancer tissues relative to their NATs. Low expression miR-148a and miR-152 was associated with increased tumor size and advanced pT stage.5. MiR-148a and miR-152 were down-regulated in colorectal cancer cell lines (HCT-116 and SW-620) relative to non-tumorous control.6. A strong correlation among the expression of miR-148a, miR-148b and miR-152 in colorectal cancer tissues.