miR-21 and miR-145 cooperation in regulation of colon cancer stem cells.
BACKGROUND
Acquired drug resistance is one of the major reasons for failing cancer therapies. Although the reasons are not fully understood, they may be related to the presence of cancer stem cells (CSCs). We have reported that chemo-resistant (CR) colon cancer cells, highly enriched in CSCs, exhibit a marked up-regulation of miR-21 and that down-regulation of this miR renders the CR cells more susceptible to therapeutic regimens. However, the underlying molecular mechanism is poorly understood. The aim of this investigation is to unravel this mechanism.


METHODS
The levels of miR-145 and miR-21 were manipulated by transfection of mature, antago-miRs or pCMV/miR-145 expression plasmid. Quantitative RT-PCR or/and Western blots were performed to examine the expression of CD44, β-catenin, Sox-2, PDCD4, CK-20 and k-Ras. Colonosphere formation and SCID mice xenograft studies were performed to evaluate the tumorigenic properties of CSC-enriched colon CR cells.


RESULTS
We investigated the role that microRNAs (miRs), specifically miR-21 and miR-145 play in regulating colon CSCs. We found the expression of miR-21 to be greatly increased and miR-145 decreased in CR colon cancer cells that are highly enriched in CSC, indicating a role for these miRNAs in regulating CSCs. In support of this, we found that whereas forced expression of miR-145 in colon cancer cells greatly inhibits CSCs and tumor growth, up-regulation of miR-21 causes an opposite phenomenon. In addition, administration of mature miR-145 or antagomir-21 (anti-sense miR-21) greatly suppresses the growth of colon cancer cell xenografts in SCID mice. This was associated with decreased expression of CD44, β-catenin, Sox-2 and induction of CK-20 indicating that administration of miR-145 or antagomir-21 decreases CSC proliferation and induces differentiation. In vitro studies further demonstrate that miR-21 negatively regulates miR-145 and vice versa. k-Ras appears to play critical role in regulation of this process, as evidenced by the fact that the absence of k-Ras in CR colon cancer cells increases miR-145 expression, suppresses miR-21, and interrupts the negative cooperation between miR-21 and miR-145.


CONCLUSIONS
Our current observations suggest that miR-21, miR-145, and their networks play critical roles in regulating CSCs growth and/or differentiation in the colon cancer and progression of chemo-resistance.
Department of Veterans Affairs Medical Center, 4646 John R, Detroit, MI, 48201, USA. aa5142@wayne.edu.|Departments of Internal Medicine, Wayne State University, Detroit, MI, 48201, USA. aa5142@wayne.edu.
Yingjie
Y
Yu
Department of Veterans Affairs Medical Center, 4646 John R, Detroit, MI, 48201, USA. makkerp@karmanos.org.|Karmanos Cancer Center, Detroit, MI, 48201, USA. makkerp@karmanos.org.|Departments of Internal Medicine, Wayne State University, Detroit, MI, 48201, USA. makkerp@karmanos.org.
Pratima
P
Nangia-Makker
Department of Veterans Affairs Medical Center, 4646 John R, Detroit, MI, 48201, USA. lfarhana@med.wayne.edu.|Departments of Internal Medicine, Wayne State University, Detroit, MI, 48201, USA. lfarhana@med.wayne.edu.
Lulu
L
Farhana
Department of Veterans Affairs Medical Center, 4646 John R, Detroit, MI, 48201, USA. sindhu25raj@gmail.com.
Sindhu
S
G Rajendra
Department of Veterans Affairs Medical Center, 4646 John R, Detroit, MI, 48201, USA. Edi.Levi@va.gov.
Edi
E
Levi
Department of Veterans Affairs Medical Center, 4646 John R, Detroit, MI, 48201, USA. a.majumdar@wayne.edu.|Karmanos Cancer Center, Detroit, MI, 48201, USA. a.majumdar@wayne.edu.|Departments of Internal Medicine, Wayne State University, Detroit, MI, 48201, USA. a.majumdar@wayne.edu.
Adhip P N
AP
Majumdar
D000818:Animals; D014408:Biomarkers, Tumor / Q000378:metabolism; D063646:Carcinogenesis / Q000473:pathology; D002454:Cell Differentiation; D049109:Cell Proliferation; D003110:Colonic Neoplasms / Q000235:genetics* / Q000473:pathology; D015536:Down-Regulation; D019008:Drug Resistance, Neoplasm; D025461:Feedback, Physiological; D045325:HCT116 Cells; D019073:HT29 Cells; D006801:Humans; D016513:Mice, SCID; D035683:MicroRNAs / Q000235:genetics / Q000378:metabolism*; D014411:Neoplastic Stem Cells / Q000378:metabolism* / Q000473:pathology; D018874:Spheroids, Cellular / Q000378:metabolism / Q000473:pathology; D014162:Transfection; D023041:Xenograft Model Antitumor Assays
D014408:Biomarkers, Tumor; C541010:MIRN145 microRNA, human; C524940:MIRN21 microRNA, human; D035683:MicroRNAs