| ObjectiveTo investigate the correlation between the of cytoskeletal regulator diaphanous related formin 3(DIAPH3)expression in colorectal cancer(CRC)and the clinical pathological parameters of CRC patients.To further investigate the effect of DIAPH3 expression on the invasion and metastasis capability in CRC cell line and its mechanism.Methods:1.Immunohistochemistry was applied to detect DIAPH3 expression in 118 colorectal cancer tissues and paired normal adjacent mucosal tissues and analyzed the correlation between the expression level of DIAPH3 in colorectal cancer and the pathological characteristics of patients.Besides,the effects of DIAPH3 expression on the prognosis of CRC patients were analyzed with The Cancer Genome Atlas(TCGA)database.2.Western blot was used to detect the difference of DIAPH3 expression in CRC cell lines and normal colonic mucosal cell and high DIAPH3 expression CRC cell lines were selected to inhibit the expression of DIAPH3 by lentivirus transfection.qPCR and Western Blot were used to verify the transfection efficiency,and selected CRC cell lines with the most effective transfected fragments were selected to identify the effects of DIAPH3 expression on proliferation and migration ability3.The transfected cells were divided into control group si-NC and transfection group si-DIAPH3.CCK-8,Clone formation,Transwell and wound healing assay were applied for detecting the effect of DIAPH3 expression on the proliferation and migration ability.4.Western blot was performed to verify the effect of DIAPH3 expression on EGFR/Src signal pathway,a key pathway for proliferation and metastasis in CRC,and further verify its effects by using DIAPH3 functional activator Intramimic-01(IMM-01)and EGFR pathway inhibitor gefitinibResults1.Statistically significant correlation analysis indicated that the expression level of DIAPH3 protein in CRC tissues was associated with local invasion,distant metastasis and clinical stage(P<0.05).Besides,TCGA Analysis of survival data from websites https://www.proteinatlas.org and http://gepia.cancer-pku.cn/recommended that low expression of DIAPH3 might be related to worse overall survival in CRC.2.Western blot and immunohistochemistry were used to determine the protein expression level of DIAPH3 in colorectal cancer,the paired normal colon,and colorectal cell lines(SW480,HCT116,HCT8,KM12,RKO,and LoVo).Results from Western blot and their gray value indicated that DIAPH3 was downregulated in CRC,compared with their paired normal tissues and normal colon cell NCM460(P<0.05).3.SW480 and KM 12 cell lines were selected for transfection to silence DIAPH3.Western blot and RT-qPCR assays showed that fragment sh-1 reduced the DIAPH3 expression of both protein and mRNA the most,compared to the empty vector sh-nc.Next,CCK-8,Clone formation,Transwell and wound healing assay indicated that the proliferation and migration ability were enhanced compared with the control group,and the differences were statistically significant(P<0.05)4.Western blot showed that inhibition of DIAPH3 could stimulate EGFR/Src pathway.Moreover,FH1-FH2(code domain of DIAPH3)targeted activator IMM-01 could reverse the activity of EGFR/Src pathway.The difference was statistically significant(P<0.05).ConclusionDecreased expression of DIAPH3 promotes progression of colorectal cancer and indicates poor prognosis.Stable DIAPH3 knockdown could activate the EGFR/Src pathway to promote the proliferation and migration of colorectal cancer. |
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