Introduction: MicroRNAs have emerged as post-transcriptional regulators that are critically involved intumorigenesis. This study was designed to explore the effect of miRNA 133b on the proliferation and expressionof TBPL1 in colon cancer cells.
Methods: Human colon cancer SW-620 cells and human colon adenocarcinomaHT-29 cells were cultured. MiRNA 133b mimcs, miRNA 133b inhibitors, siRNA for TBPL1 and scrambled controlwere synthesized and transfected into cells. MiR-133b levels in cells and CRC tumor tissue was measured byreal-time PCR. TBPL1 mRNA was detected by RT-PCR. Cell proliferation was studied with MTT assay. Westernblotting was applied to detect TBPL1 protein levels. Luciferase assays were conducted using a pGL3-promotervector cloned with full length of 3’UTR of human TBPL1 or 3’UTR with mutant sequence of miR-133b targetsite in order to confirm if the putative binding site is responsible for the negative regulation of TBPL1 by miR-133b.
Results: Real time PCR results showed that miRNA 133b was lower in CRC tissue than that in adjacenttissue. After miR-133b transfection, its level was elevated till 48h, accompanied by lower proliferation in bothSW-620 and HT-29 cells. According to that listed in http://www.targetscan.org, the 3’-UTR of TBPL1 mRNA(NM_004865) contains one putative binding site of miR-133b. This site was confirmed to be responsible for thenegative regulation by miR-133b with luciferase assay. Further, Western blotting and immunohistochemistryboth indicated a higher TBPL1 protein expression level in CRC tissue. Finally, a siRNA for TBPL1 transfectionobviously slowed down the cell proliferation in both SW-620 and HT-29 cells.
Conclusion: MiR-133b might actas a tumor suppressor and negatively regulate TBPL1 in CRC.