The Study Of MiR-144 Suppressing Cells Proliferation And Migration In Colorectal Cancer By Targeting NRAS

OBJECTIVE:Colorectal cancer is a type of malignant cancer that has become particularly prevalent worldwide.There are 1.8 million newly diagnosed patients with CRC and 0.88 million deaths due to CRC worldwide in 2018.Although CRC has been intensively investigated at the molecular level,the precise mechanisms that participate in CRC initiation and development remain complicated and obscure.NRAS is the archetypical member of the RAS superfamily and has been recently extensively investigated as an oncogene.A mutation in the NRAS gene leads to the abnormal proliferation and metastasis of cells in CRC.However,the exact function of NRAS and its regulation in CRC should be elucidated because of fewer related studies.MicroRNAs(consisting of 19-23 nucleotides)are a class of conserved noncoding RNA molecules,mainly post-transcriptionally regulating gene expression.Researches indicated most miRNAs are considered oncomiRs or tumor-suppressive miRNAs based on their target mRNA(s)and/or overall effects on tumor development.MiR-144 has been reported to impede cell multiplication and promotes cell apoptosis in prostate cancer by targeting CEP55.However,studies of miR-144 function in CRC are limited.The research aimed to investigate the expression status,specific roles of miR-144 and NRAS in CRC.Moreover,it should be verified that there existed the target correlation between miR-144 and NRAS.METHODS:CRC and adjacent noncancerous tissues were obtained randomly from 13 patients who underwent surgical resection of CRC in general surgery department of our hospital.All the specimens were confirmed by pathologic diagnosis.The expressions of miR-144 and NRAS in CRC tumor tissues and noncancerous tissues were detected by western blot,qRT-PCR.Then,miR-144 mimics and inhibitors were transfected into the CRC cell lines(SW480,LoVo,Caco2)to up-regulate and reduce the expression of miR-144 respectively,while NRAS vector and siRNA were used to transfect into SW480 cells to increase and down-regulate the expression of NRAS respectively.Online software Targetscan and luciferase reporter analysis were utilized to predict and verify the direct interaction between miR-144 and NRAS.207nt sequence of NRAS 3'-UTR containing miR-144 binding site was inserted into pMIR-REPORT plasmid to design and synthesize wild-type luciferase plasmid.6nt of the above sequence was changed and then inserted into the same vector to design and synthesize mutant luciferase plasmid.NRAS overexpression vector The CRC cell migration and invasion in different groups were examined by CCK8,EdU and transwell assays.Besides,tumor xenografts model were established in mice to observe regulatory effects of miR-144 and NRAS on the growth of CRC in vivo.RESULTS:The expression of miR-144 was significantly suppressed in CRC tissues with compared to adjacent noncancerous tissues,and the difference was statistically significant(P<0.001).It was investigated that the expression level of NRAS was consistently higher in CRC tissues than matched noncancerous tissues(P<0.001).Moreover,there existed negative correlation between miR-144 level and NRAS protein level in CRC(R=-0.6624).Targetscan software predicted miR-144 regulate NRAS as a potential candidate.The result of luciferase reporter analysis showed the luciferase signal of wild-type plasmid was weaker than mutant plasmid.It was confirmed miR-144 combine straightly with NRAS 3'UTR.MiR-144 levels in SW480,LoVo and Caco2 cells transfected with mimics were 70,25 and 35 times compared to the control group respectively(P<0.001),while miR-144 levels in above cells transfected with inhibitor were 0.13,0.25 and 0.4 times compared to the control group respectively(P<0.001).NRAS protein level was significantly reduced after miR-144 overexpression in above cells,while the level was elevated when miR-144 was down-regulated.Futhermore,vitro experiments demonstrated that miR-144 inhibited SW480 cell proliferation and migration by targeting NRAS.After sacrificing the mice,we observed a markedly decreased tumor weight in miR-144 overexpression group compared with the control group(p<0.001),whereas NRAS overexpression increased tumor weights(p<0.001).Vivo experiments indicated that miR-144 inhibits xenograft tumors growth by silencing NRAS.CONCLUSION:MiR-144 suppresses CRC cellproliferation and migration by targeting NRAS.Besides,miR-144 could inhibit xenograft tumors growth by repressing NRAS expression in vivo.In summary,this research identified a novel miR-144-NRAS axis in CRC that could promote the research and treatment of CRC.