Effect Of MiR-146b-3p On The Proliferation Activitiy Of Pancreatic Cancer Stem Cells And Its Mechanism

Objective： To observe the effect of miR-146b-3p on the proliferation activity ofpancreatic cancer stem cells （P-CSCs） by up-regulating the expression level ofmiR-146b-3p in vitro and to investigate the possible mechanism.Methods： We compared the different miRNAs expression profiles of P-CSCs andnon-P-CSCs by Microarrays. The expression level of these different miRNAs in pancreaticcancer and adjacent non-tumorous tissues was examined by utilizing qPCR assay.miR-146b-3p level was up-regulated by transfecting miR-146b-3p precursor in CSC^highMIA PaCa-2and CSClowBxPC-3cells, the changes of the cell proliferation, apoptosis andcell cycle were detected by CFSE corporation assay, Annexin â…¤/FITC apoptosis assay orPI corporation assay respectively; the changes of the expression levels of apoptosis-relatedproteins and cycle-related proteins were measured; the changes of the mRNA and proteinexpression levels of Hedgehog alternative pathway, MAP3K10â†'DYRK2â†'Gli2, weredetected to clarify whether MAP3K10was post-transcriptional regulated by miR-146b-3p.By using PCR assay and site mutagenesis, we amplified the3′untranslated regions （3′UTR）of human MAP3K10DNA, cloned the DNA fragments into luciferase reporter geneplasmids pGL3-Promoter and obtained pGL3-MAP3K10and pGL3-MAP3K-mut plasmids; then investigated whether miR-146b-3p targets MAP3K10directly by using dual-luciferasereporter assay.Results： We obtained the special miRNAs expression profile of P-CSCs, includingmiR-146b-3p as the most highly downregulated miRNA. Seven different miRNAs of theprofile, including miR-146b-3p, were poorly-expressed in pancreatic cancer tissuescompared with paired adjacent non-tumorous tissues. For CSC^highMIA PaCa-2, the cellproliferation activity decreased, cell apoptosis rate increased, percentage of cells in G0/G1phase increased and S phase decreased, anti-apoptosis protein Bcl-2decreased andpro-apoptosis protein Bax increased, cycle-related protein P27increased, by up-regulatingthe expression level of miR-146b-3p; but no notable change was ovserved in CSClowBxPC-3. The mRNA levels of Hedgehog alternative pathway, MAP3K10â†'DYRK2â†'Gli2,didn’t change notablely; the protein levels of MAP3K10and Gli2decreased, but increasedin DYRK2. The relative luciferase activity （RLA） of group transfected with reporterpGL-3-MAP3K10and miR-146b-3p precursor declined remarkablely compared with othercontrol groups.Conclusions： The expression level of miR-146b-3p was down-regulated in P-CSCsand pancreatic cancer tissues. With up-regulation of miR-146b-3p expression level, theproliferation activity of P-CSCs decreased and the possible mechanism may be related tothe dysregulation of Hedgehog signaling pathway by miR-146b-3p via post-transcriptionalregulation of MAP3K10.