Pancreatic cancer is one of the most aggressive cancers, which is usually diagnosed in an advanced state for which there are few or no effective therapies [
1]. One of the major hallmarks of pancreatic cancer is its extensive local tumor invasion and early systemic dissemination [
2]. Over the past two decades, numerous efforts have been made in improving treatment and survival of pancreatic cancer patients but the outcome has been disappointing [
3]. Emerging evidence suggest that the resistance could in fact be due to the enriched existence of tumor initiating cells, also classified as cancer stem-like cells in a tumor mass. The CSCs have the capacity of self-renewaland the potential to regenerate into all types of differentiated cells giving rise to heterogeneous tumor cell populations in a tumor mass, which contributes to tumor aggressiveness [
4,
5]. The existence of CSCs or cancer stem-like cells in a tumor mass is believed to be responsible for tumor recurrence because of their intrinsic and extrinsic drug-resistance characteristics [
3].
It has recently become clear that EMT is associated with drug resistance and cancer cell metastasis [
7]. During this process, the expression of E-cadherin is down-regulated, which is a transmembrane protein essential for the stable adherens junctions, and the expression of the mesenchymal molecules vimentin, fibronectin, and/or N-cadherin are up-regulated [
8,
9]. In pancreatic cancer cells, EMT is also reported to be a crucial step for tumor cell migration and invasion [
10]. Recent studies have demonstrated that EMT plays a great role not only in tumor metastasis, but also in tumor recurrence that is believed to be tightly linked with the biology of cancer stem-like cells or cancer-initiating cells. However, the mechanisms by which EMT cells generate the stem-like cells remain to be elucidated [
11‐
13]. Importantly, emerging evidence implicated the critical role of microRNAs because they are key regulatory molecules in biological and pathologic processes including EMT [
7]. MicroRNAs are small and non-coding RNA molecules that can regulate gene expression by interacting with multiple mRNAs and inducing either degradation of mRNA or inhibition of their translation to functional proteins [
7,
14]. Members of the miR-200 family are downregulated in human cancer cells and tumors due to aberrant epigenetic gene silencing and play a critical role in the suppression of EMT, tumor cell adhesion, migration, invasion and metastasis, by targeting and repressing the expression of key mRNAs that are involved in EMT (ZEB1 and ZEB2), and participates in a signalling network with the E-cadherin transcriptional repressors ZEB1/deltaEF1 and ZEB2/SIP1, and TGF-β2 that is postulated to facilitate maintenance of stable epithelial or mesenchymal states but also allow reversible switching between these states in response to EMT effectors (such as TGF-β) [
15,
16]. In ovarian and breast cancer, low expression of miRNA-200 plays important roles in cancer metastasis [
14,
17,
18]. MiR-200 changed the tumor environment, inhibiting the process of EMT and metastasis [
19,
20]. These findings hypothesizes that the expression of miR-200 in pancreatic cancer cell is correlated with stemness, EMT and metastasis.
Since miR-200 is associated with EMT, which is believed to be associated with cancer stem cells or cancer stem-like cells, we investigated the effects of miR-200 family on pancreatic CSC functions in this study. We identified a highly tumorigenic subpopulation of pancreatic cancer cells expressing the cell surface markers CD24, CD44 and ESA in pancreatic adenocarcinoma cell line PANC-1. And CD24+CD44+ESA+ cells in PANC-1 were sorted by BD FACS Aria II for further study. Then, we analysed the miR-200 family and transcription factors Oct4 and Nanog expression in CSCs of pancreatic cancer cell line PANC-1, and determined their relationships with EMT markers and repressors of E-cadherin transcription. In order to study the role of miR-200a for EMT in CSCs, miR-200a mimic was transformed into CSCs. In addition, invasion and metastasis were determined in CSCs and transformed CSCs.