title: miRNA-338-3p suppresses cell growth of human colorectal carcinoma by targeting smoothened.
abstract: OBJECTIVE
  To investigate the regulative effect of miRNA-338-3p (miR-338-3p) on cell growth in colorectal carcinoma (CRC).
  
  
  METHODS
  The lentiviral vector pLV-THM-miR-338-3p and pLV-THM-miR-338-3p-inhibitor were constructed. The recombinant viral vector encoding the pre-miR-338-3p or miR-338-3p-inhibitor and the two packaging plasmids psPAX2 and pMD2.G were cotransfected into human embryonic kidney 293T cells to package lentivirus. The supernatant containing the lentivirus particles was harvested to determine the viral titer, and this supernatant was then used to transduce CRC-derived cell line, SW-620. Flow cytometry was utilized for sorting the green fluorescent protein (GFP)⁺ cells to establish the SW-620 cell line stably expressing pre-miR-338-3p or miR-338-3p-inhibitor. Moreover, the expression of miR-338-3p was determined by real-time reverse transcriptase polymerase chain reaction, and Western blotting was used to detect the expression of the smoothened (SMO, the possible target of miR-338-3p) protein in SW-620 cells. Furthermore, the status of CRC cell proliferation and apoptosis were detected by 3-(4,5-dimethyl-2 thiazoyl)-2,5-diphenyl-2H-tetrazolium bromide assay and flow cytometry, respectively.
  
  
  RESULTS
  Restriction enzyme digestion and DNA sequencing demonstrated that the lentiviral vector pLV-THM-miR-338-3p and pLV-THM-miR-338-3p-inhibitor were constructed successfully. GFP was expressed after the SW-620 cells were transduced by the lentivirus. Expression of miR-338-3p in SW-620 cells transduced with the lentivirus pLV-THM-miR-338-3p was significantly increased (relative expression 3.91 ± 0.51 vs 2.36 ± 0.44, P < 0.01). Furthermore, overexpression of miR-338-3p inhibited the expression of SMO protein in SW-620 cells, which showed obviously suppressed proliferation ability [cellular proliferation inhibition rate (CPIR) 61.9% ± 5.2% vs 41.6% ± 4.8%, P < 0.01]. Expression of miR-338-3p in SW-620 cells transduced with the lentivirus pLV-THM-miR-338-3p-inhibitor was significantly decreased (relative expression 0.92 ± 0.29 vs 2.36 ± 0.44, P < 0.01). Moreover, the downregulated expression of miR-338-3p caused upregulated expression of the SMO protein in SW-620 cells, which showed significantly enhanced proliferation ability (CPIR 19.2% ± 3.8% vs 41.6% ± 4.8%, P < 0.01). However, anti-SMO-siRNA largely, but not completely, reversed the effects induced by blockage of miR-338-3p, suggesting that the regulative effect of miR-338-3p on CRC cell growth was indeed mediated by SMO.
  
  
  CONCLUSIONS
  miR-338-3p could suppress CRC growth by inhibiting SMO protein expression.
authors.affiliation: Department of General Surgery, Nanfang Hospital of Southern Medical University, Guangzhou 510515, Guangdong Province, China. sunkai9602@sina.com
authors.forename: Kai
authors.initials: K
authors.lastname: Sun
authors.forename: Hai-Jun
authors.initials: HJ
authors.lastname: Deng
authors.forename: Shang-Tong
authors.initials: ST
authors.lastname: Lei
authors.forename: Jing-Qing
authors.initials: JQ
authors.lastname: Dong
authors.forename: Guo-Xin
authors.initials: GX
authors.lastname: Li
mesh_terms: D017209:Apoptosis; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D015179:Colorectal Neoplasms / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D005822:Genetic Vectors; D057809:HEK293 Cells; D006801:Humans; D016086:Lentivirus / Q000235:genetics; D035683:MicroRNAs / Q000378:metabolism*; D034622:RNA Interference; D043562:Receptors, G-Protein-Coupled / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction; D000072086:Smoothened Receptor; D014161:Transduction, Genetic; D014162:Transfection; D015854:Up-Regulation
chemical_list: C532050:MIRN338 microRNA, human; D035683:MicroRNAs; D043562:Receptors, G-Protein-Coupled; C491023:SMO protein, human; D000072086:Smoothened Receptor
keywords: Colorectal carcinoma; Hsa-miRNA-338-3p; Lentivirus; Smoothened