Selective blockade of DCAMKL-1 results in tumor growth arrest by a Let-7a MicroRNA-dependent mechanism.
OBJECTIVE
MicroRNAs (miRNAs) are non-coding RNAs that regulate gene expression. The tumor suppressor miRNA let-7a has been reported to be inhibited posttranscriptionally in embryonic stem cells and in human cancers. Microtubule-associated kinase DCAMKL-1 is a putative intestinal stem cell marker that is expressed in Apc(Min/+) adenomas. We investigated the role of DCAMKL-1 on expression of let-7a miRNA and the oncogene c-Myc and in tumorigenesis.


METHODS
Human tissue microassay slides were immunostained for DCAMKL-1. HCT116 and SW480 cells were transfected with DCAMKL-1 small interfering RNA (siRNA) (si-DCAMKL-1) and analyzed for DCAMKL-1, c-Myc (using immunoblot and real-time reverse-transcription polymerase chain reaction [RT-PCR]), and pri-let-7a miRNA (using real-time RT-PCR) levels. A liposomal preparation of si-DCAMKL-1 was administered into HCT116 xenografts in nude mice, and tumor volumes were measured. A luciferase reporter assay, with a plasmid containing a let-7a-binding site at the 3' untranslated region, was utilized to measure let-7a in cell lines. Cells were isolated from normal mouse intestine using DCAMKL-1 and fluorescence-activated cell sorting (FACS) and subjected to pri-let-7a miRNA analysis.


RESULTS
Expression of DCAMKL-1 was increased in human colorectal cancers. siRNA-mediated blockade of DCAMKL-1 resulted in H tumor xenograft growth arrest, increased pri-let-7a miRNA, a corresponding decrease in luciferase activity, and decreased expression of the oncogene c-Myc. DCAMKL-1(+) cells isolated by FACS demonstrated a significant decrease in pri-let-7a miRNA, compared with more differentiated cells.


CONCLUSIONS
DCAMKL-1 is a negative regulator of let-7a miRNA biogenesis in intestinal stem and colorectal cancer cells; it could represent a novel target for anti-cancer stem cell-based strategies.
Sureban
Sripathi M
SM
Department of Medicine, Digestive Diseases and Nutrition Section, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma, USA.
May
Randal
R
Ramalingam
Satish
S
Subramaniam
Dharmalingam
D
Natarajan
Gopalan
G
Anant
Shrikant
S
Houchen
Courtney W
CW
D000704:Analysis of Variance; D000818:Animals; D015153:Blotting, Western; D045744:Cell Line, Tumor; D015179:Colorectal Neoplasms / Q000235:genetics* / Q000503:physiopathology; D004195:Disease Models, Animal; D005260:Female; D005434:Flow Cytometry; D015972:Gene Expression Regulation, Neoplastic; D045325:HCT116 Cells; D006801:Humans; D007150:Immunohistochemistry; D052118:Lysosomal-Associated Membrane Protein 1 / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D008819:Mice, Nude; D035683:MicroRNAs / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism; D011336:Probability; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D011897:Random Allocation; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012680:Sensitivity and Specificity; D014183:Transplantation, Heterologous; D047368:Tumor Burden / Q000235:genetics
D052118:Lysosomal-Associated Membrane Protein 1; D035683:MicroRNAs; D012333:RNA, Messenger; C487931:mirnlet7 microRNA, human