| Objective: To elucidate the suppressive role of ING4 in EMT and metastasis of colorectal cancer(CRC)and its molecular mechanism.Methods:(1)A total of 82 pairs of paraffin-embedded or snap-frozen CRC tumor and adjacent non-tumor tissues were obtained from randomly selected 82 patients who have undergone surgery for CRC.The clinicopathological characteristics of these patients were collected based upon medical history.The expression of ING4 in human CRC tumor and adjacent non-tumor clinical tissues was analyzed by immunohistochemistry and Western blot,respectively.The correlation between the expression of ING4 in CRC tumor tissues and clinicopathological parameters features of CRC patients was analyzed.(2)The total proteins derived from a panel of human CRC cell lines such as HT29,LS174 T,SW480,SW620 and Lo Vo as well as a colorectal mucous epithelial cell line NCM460(used as a normal control)were extracted.The expression of ING4 in CRC cells was detected by Western blot.The relationship between the expression of ING4 in CRC cells and metastatic capability of CRC cells was assessed.(3)The p Lenti6.3/ING4/IRES/GFP expressing human ING4 or blank control p Lenti6.3/IRES/GFP lentiviral plasmid was cotransfected into 293 T human embryonic kidney cells with helper packaging plasmids including p LP1,p LP2 and VSVG by Lipofectamine 2000,respectively.The lentivirus expressing ING4(LV-ING4)or blank lentivirus(LV)was consequently generated and the titre was then determined.(4)A highly metastatic CRC cell line SW620 that expressed the lowest level of ING4 was infected with LV-ING4 or LV(control)at a multiplicity of infection(MOI)of 50 and selected with drug Blasticidin S(BSD),leading to the generation of SW620-ING4 and SW620-mock(control)CRC cells.A lowly metastatic CRC cell line SW480 that expressed the highest level of ING4 was infected with LV-sh ING4 or LV-shcontrol(control)and selected with drug Puromycin(Puro),resulting in formation of SW480-sh ING4 and SW480-shcontrol(control)CRC cells.The transgene efficiency in SW620 CRC cells was analyzed according to GFP reporter by fluorescence microscopy and flow cytometry.The overexpression or knockdown efficiency of ING4 in SW620 or SW480 CRC cells was determined by q RT-PCR and Western blot analysis.(5)The effect of lentivirus-mediated ING4 overexpression or silence on CRC cell migration,invasion and metastasis was evaluated by in vitro Transwell migration and invasion assays and by in vivo intravenous(i.v.)injection of CRC cells through murine tail vein in an athymic BALB/c nude mouse model.(6)The effect of ING4 on expression of EMT-inducing transcription factor(EMT-TF)(Snail1)and EMT-associated markers(E-cadherin,N-cadherin and Vimentin)was analyzed by gene expression microarray,q RT-PCR and Western blot,respectively.(7)To wonder whether Snail1 knockdown would attenuate ING4 deficiency-induced EMT and metastasis in CRC cells,the ING4-silenced SW480-sh ING4 CRC cells were transfected with Snail1 si RNA and then subjected to Snail1 knockdown function studies.(8)The expression of Snail1,E-cadherin,N-cadherin and Vimentin in human CRC tumor tissues was further analyzed by immunohistochemistry.The clinical correlation of ING4 with Snail1,E-cadherin,N-cadherin or Vimentin was analyzed.(9)To delineate a possible mechanism for ING4-induced downregulation of Snail1,the effect of ING4 on expression of ING4-elicited upregulation of miRNAs(miR-940,miR-1228-3p and miR-1825)potentially targeting Snail1 in CRC cells was analyzed by miRNA expression microarray and q RT-PCR,respectively.(10)The wild-type(wt)and mutant(mut)Snail1-3'UTR dual luciferase reporter plasmids were constructed and then cotransfected with miR-940 mimics,miR-1228-3p mimics or miRNA mimics negative control(NC)into 293 T cells.The luciferase activity was detected to validate whether Snail1 is a target of miR-940 and miR-1228-3p.Results:(1)We found the expression of ING4 in human CRC tumor tissues was significantly lower than that in adjacent non-tumor tissues,which was strongly associated with the tumor size,the presence of lymph node metastasis and the TNM stage.(2)Compared with normal colorectal mucous epithelial cells,the expression of ING4 in human CRC cells was downregulated.Moreover,the expression level of ING4 in highly metastatic CRC cells(SW620 and LoVo)was much lower than that in lowly metastatic CRC cells(HT29,LS174 T and SW480).(3)The ING4-overexpressed SW620-ING4 vs SW620-mock,and the ING4-silenced SW480-sh ING4 vs SW480-shcontrol CRC stable cell lines were successfully established.(4)The lentivirus-mediated ING4 overexpression remarkably suppressed in vitro migration and invasion ability of SW620 CRC cells as well as its in vivo metastasis capability to lung in BALB/c nude mice,whereas knockdown of ING4 significantly promoted these processes.(5)ING4 significantly downregulated EMT-TF Snail1 and mesenchymal markers such as N-cadherin and Vimentin as well as upregulated epithelial marker E-cadherin in SW620 CRC cells,while ING4 silence had an opposite effect in SW480 CRC cells.(6)Knockdown of Snail1 was capable of reversing EMT phenotype and attenuating metastatic ability in SW480-sh ING4 CRC cells,demonstrating that ING4 deficiency-triggered upregulation of Snail1 was an important molecular mechanism for ING4 deficiency-induced EMT and metastasis in CRC.(7)ING4showed a negative correlation with the expression of Snail1,N-cadherin and Vimentin as well as a positive correlation with the expression of E-cadherin in CRC tumor tissues.(8)ING4 obviously upregulated Snail1-targeted miR-940 and miR-1228-3p in SW620 CRC cells,whereas silence of ING4 suppressed their expression in SW480 CRC cells.Snail1-3'UTR dual luciferase reporter assay showed that Snail1 was a functional target of miR-940 and miR-1228-3p.These data indicated that ING4 downregulated Snail1 in CRC cells very possibly via upregulating miR-940 and miR-1228-3p.Conclusions: ING4 deficiency-induced elevation of Snail1 is critical for ING4deficiency-induced promotion of EMT and metastasis in CRC.ING4 markedly suppresses expression of Snail1 in CRC cells,which may be an important molecular mechanism for ING4-mediated inhibition of EMT and metastasis in CRC.ING4 inhibits EMT and metastasis in CRC via repression of Snail1 very probably through a miR-940/miR-1228-3p-Snail1 pathway. |
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