1.MiR-204 Mediated PAR₂ Activation-induced Tumor Cell Migration 2.The Correlation Of Tyrosine And Threonine Kinase Expression And The Prognosis Of Colorectal Cancer

Colorectal cancer is the third common malignant tumor in the world.The progress of surgical operation,radiation therapy and chemotherapy drastically improve treatment of colorectal cancer.However,there are still 70%-80%CRC patients that died of recurrence or distal metastasis of colorectal cancer.The high metastasis rate hinders the colorectal cancer treatment,as well as high recurrence rate and drug-resistance.Proteinase activated receptors-2(PAR2)is a seven transmembrane spanning domain G-protein coupled receptors and affects the process of inflammation and cancer development.Most recently,we found that activation of PAR2 promotes cell migration through the downregulation of miR-125b and its downstream target gene Gab2.Therefore,we aimed to study(1)whether miR-204 mediated PAR2 activation-induced tumor cell migration and the mechanisms underlied;(2)whether tyrosine and threonine protein kinase(TTK)is a potential prognosis factor of CRC.(The first part)Firstly,we found that PAR2 activating peptide decreased the miR-204 expression,while stable knock down of P AR2 significantly increased miR-204 expression in different CRC cells.In addition,overexpression or inhibition of miR-204 dramatically suppressed or promoted cell migration respectively.It was important that over-expressing miR-204 blocked cell migration that was induced by PAR2 activating peptide.Therefore,it seems like that PAR2 regulated the expression of miR-204,and the latter was closely related to the cell migration.Furthermore,we predicted the target genes of miR-204 by the combination of miRNAs database(such as Targetscan and MIRDB)with PAR2-related transcriptome.Four genes(MYO10\ACSL4\USP47\NUAK1)were predicted as target genes,and were confirmed by different methods.It revealed that MYO10 and ACSL4 were regulated by both miR-204 and PAR2 signaling.Furthermore,we demonstrated that activation of PAR2 promotes cell migration through the downregulation of miR-204 and its downstream target MYO10 and ACLS4.Besides that,we noticed that three miRNAs(miR-204/miR-205/miR-34a)regulated by PAR2 likely target the 3’UTR region of ACSL4.However,there was no significantly synergistic effect on regulating ACSL4 expression.(The second part)Tyrosine and Threonine Kinase is a dual specificity serine/threonine and tyrosine protein kinase,and plays an important role in centrosome duplication and chromosome attachment during mitosis.Colitis-associated colon cancer model was induced by azoxymethane(AOM)and dextran sulfate sodium(DSS)in mice,and the transcriptome chip of mouse colons indicated that TTK is related with the occurrence of CRC.The study verified that the mRNA and protein expression of TTK were gradually elevated during the development of colon cancer in the first place.In addition,The expression of TTK at both mRNA and protein levels were significantly higher than adjacent normal tissues in 24 pairs CRC clinical samples.Furthermore,the high level of TTK is related with the poor prognosis of CRC patients(GSE17536).In summary,our data indicated that the activation of PAR2 promotes cell migration through the downregulation of miR-204 and its downstream target MYO10 and ACLS4.In addition,elevated expression of TTK is related to colonic carcinogenesis both in mouse model and human CRC specimens.TTK might be a potential prognosis factor in CRC patients.