MicroRNA-338-3p Suppresses Colorectal Carcinoma Cell Invasion And Migration

PartⅠExpression differences of microRNA-338-3p between different colorectal carcinoma cell lines.Objective:Recent studies found that miRNAs are involved in the regulation of tumor cell invasion and metastasis. Some reports show that miR-338-3p is involved in the regulation of occurrence and development in several kinds of tumor and associated with tumor malignant biological behavior. Although the previous studies indicated the down-regulation of miR-338-3p in colorectal carcinoma (CRC), but the expression difference in miR-338-3p in CRC cell lines is unclear. In this study, we detected miR-338-3p expression of different malignancy CRC cell lines, in order to find out the relationship between miR-338-3p and malignant biological behavior of CRC.Methods:Five kinds of human colorectal cancer cell lines (LOVO, HT-29, HCT116, SW620, SW480) and human umbilical vein endothelial cells (HUVEC), preserved by Department of Pathology, Southern Medical University, were cultured conventionly. The gene sequence was searched in the miRbase database, using Primer-Express2.0to design primers and probes. The forward primer of MiR-338-3p was5’-ACA CTC CAG TGG GTC CAG CAT CAG TGA TTT T-3’, its reverse primer was5’-CTC AAC TGG TGT CGT GGA-3’,the amplified fragment was72bp long. The forward primer of internal control U6was5’-CTC GCT TCG GCA GCA CA-3’, its reverse primer was5’-CTC GCT TCG GCA GCA CA-3’, the amplified fragment was94bp long. Total RNA was extracted in each group by Trizol, and was dissolved in DEPC water. RNA concentration was measured by BioPhotometer plus.5S rRNA,18S rRNA and28s rRNA bands of total RNA were analysed by agarose gel electrophoresis, and three bands of complete can prove that total RNA extraction was relatively complete. MiR-338-3p expression was detected by real-time RT-PCR. Take U6as internal control, and its copy number as the calibration baseline. Use the SW620value as the correction. The exact content of miR-338-3p in each group was calculated according to the formula:target gene expression=2-ΔΔCT.All data in the experiment was presented as average±standard deviation (x±s). The data were test by One-way-ANOVA. All the statistical analysis were performed with SPSS13.0statistic software (SPSS Inc, Chicago, IL, USA), and P<0.05were identified statistically significant difference.Results:Compared with HUVEC, the expression level of miR-338-3p in five kinds of CRC cell lines were down-regulated (P<0.001). The expression levels of miR-338-3p among these five kinds of CRC cell lines were significantly deferent (P<0.05).Conclusion:MiR-338-3p expressions were down-regulated in CRC cell lines compared with normal tissue, indicating that miR-338-3p might play an important role in tumor occurrence and development process. In each kind of CRC cell lines, miR-338-3p also presented significantly differences, suggesting that miR-338-3p was associated with tumor malignant biological behavior. PartⅡMicroRNA-338-3p suppresses invasion and migration of colorectal cancer cells by down-regulating SMO.Objective:In the first part, we found that miR-338-3p, which has been detected in human CRC cell lines, were differentially expressed, and presumably associated with malignant biological behavior of tumor. But the mechanism between its imbalance expression and CRC invasion and metastasis is still unknow. So the aim of this study was to explore the effects of miR-338-3p on the invasion and migration ability of CRC cells and its mechanisms.Methods:By reading literature and the detection results of the first part of this research, we selected human CRC cell lines SW620and SW480, which collect from the same parental sample, to perform the next experiment. The cells were preserved by Department of Pathology, Southern Medical University. The miR-338-3p target gene was predicted by TargetScan and miRanda. The gene sequences were searched from the miRbase database, using the software Primer-Express2.0to design primers and probes. The forward primer of SMO mRNA was5’-CAT TAC CTT CAG CTG CCA CT-3’, its reverse primer was5’-CTT TGG CTC ATC GTC ACT CT-3’, and the amplified fragment was292bp long. The forward primer of internal control β-actin was5’-TGG ATC AGC AAG CAG GAG TA-31, its reverse primer was5’-TCG GCC ACA TTG TGA ACT TT-3’,and the amplified fragment was275bp long. Human CRC cell lines SW620and SW480were cultured conventionally. After2-3generation’s normal transmission, the exponential growth phase cells were cultured in24well plates, and the experiment started at about80%cell fusion. Cells were transfected with50nM miR-338-3p precursor (pre-miR-338-3p) or miR-338-3p specific inhibitor (anti-miR-338-3p) by LipofectamineTM RNAiMAX, and real-time quantitative PCR (Real-time RT-PCR) was used to detect miR-338-3p expressions. SMO mRNA was analyzed by semi-quantitative RT-PCR, using (3-actin as internal control. SMO protein was analyzed by Western-blot, using β-actin as internal control. Transwell invasion assay and migration assay were used to detect vitro invasion and migration ability. The data within two groups were tested by t test, and data among groups were tested by One-way-ANOVA. All the statistical analyses were analysed with SPSS13.0statistic software (SPSS Inc, Chicago, IL, USA) and P<0.05were identified to be statistically significantly difference.Results:An incomplete complementary binding site between Hsa-miR-338-3p and SMO mRNA3’-UTR was predicated by bioinformatics websites TargetScan and Miranda. The results of real-time RT-PCR showed that, the expression of miR-338-3p in SW620was lower than that in SW480(P<0.001). And the results of Western-blot showed that SMO expression in SW620was significantly higher than that in SW480(P<0.05). By transfection of pre-miR-338-3p into SW620cells, miR-338-3p expression was up-regulated, SMO protein was down-regulated, and the invasion and migration abilities were decreased (P<0.05). On the contrary, miR-338-3p expression was decreased in SW480cells with anti-miR-338-3p transfection, SMO protein was increased, and the invasion and migration abilities were enhanced (P<0.05). But semi quantitative RT-PCR results presented that SMO mRNA expression had no statistical difference with or without transfection.Conclusion:Enhancement of miR-338-3p expression down-regulated expression of SMO in higher invasive and migratory CRC cell line SW620, and decreased tumor cell invasiveness and migratoryness by transfected pre-miR-338-3p. In contrast, anti-miR-338-3p increased tumor invasiveness and migratoryness by up-regulating levels of SMO with inhibitation of miR-338-3p in lower invasive and migratory CRC cell lines SW480. These present that miR-338-3p suppress invation and migration of CRC by down-regulating SMO. Base on the invariableness of SMO mRNA expression among all groups and the bioinformatics software prediction results, miR-338-p may down-regulate SMO expression by post-transcriptional gene silencing and then inhibits the activation of Sonic Hedgehog signaling pathway to suppress invation and migration, and this effect may possibly cause by incomplete complementary binding between miR-338-3p and SMO mRNA3’-untranslated region (3’-UTR). Together, these results suggest that miR-338-3p would be a tumor suppressor in CRC.