The Mechanism Of Micro-374 In The Formation And Migration Of The Tumor Cell Transformed From Mesenchymal Stem Cell

Objective In the previous study, we established the tumor cell line K3 transformed from r BM-MSC in vivo successfully. Besides, mi RNAs have great roles in the progression of cancer, so we investigated the key mi RNAs regulating the transformation of r BM-MSC and clarified the mechanism.Methods We employed microarrays to screen the differently expressed mi RNAs between r BM-MSC and K3. The most representative ones were selected and verified by q RT-PCR and corresponding mi RNA mimics/inhibitors were transfected into r BM-MSC. After detecting the transfection efficiency, cell growth curve and the colony forming assay were used to detect the cell proliferation. We observed the migrated cell numbers and the expression of E-cadherin, N-cadherin to assess migration ability of transfected cell.Additionally, K3 cells were also transfected with corresponding inhibitors and mimics to further identify the function of those selected mi RNAs in cell proliferation and migration. According to those selected key mi RNAs, we predicted the downstream m RNA by mi RGen, Target Scan and Pic Tar softwares and confirmed signal pathway by western-blot.Results Microarrays and q RT-PCR analysis showed a higher expression level of mi R-374 and lower expession level of mi R-199 a, mi R-145, mi R-34 a,mi R-214, mi R-350 in K3 than those in r BM-MSC. After transfection of mi R-374 mimics, the colony numbers and cellular proportion in S-period were increased, suggesting an enhanced proliferative capacity of r BM-MSC after mi R-374 upregulation. In addition, mi R-374 upregulation also elevated the migrated cell numbers of r BM-MSCs. Immunofluorescence staining showed a reduced E-cadherin expression and an increased N-cadherin expression. These results collectively suggested that mi R-374 could promote the r BM-MSC migration. The knockdown of mi R-374 in K3 leaded to an impaired cell proliferation and migration capacity, which were identified by counting the migrated cell numbers and analyzing the expression quantity of E-cadherin,N-cadherin. Next, Wnt5 a was selected and verified as the targetgene of mi R-374. Western-blot indicated the downsream protein Ca MK Ⅱ and PKC significantly reduced when mi R-374 over-expression.Conclusions mi R-374 was upregulated during the period of r BM-MSC transformed to K3, gain and loss function assay suggested mi R-374 promoted cell proliferation and migration. Wnt5 a as targetgene of mi R-374 affected the expression of Ca MKⅡ and PKC to regulate the calcium signal transduction pathways.