MiR-192 Targets C-X-C Motif Chemokine Ligand 2 And Inhibits Invasion And Migration In Human Colorectal Cancer

Objectives 1 To investigate the expression difference of miR-192 in colorectal cancer cells HCT116 and HT29 and evaluate the influence of miR-192 on the cell proliferation,invasion,migration,colony formation and spheroid of colorectal cancer by up-regulating miR-192 and blocking its function.2 To investigate the candidate target gene of miR-192 and molecular mechanism for its regulation.3 To further study the expression of miR-192 and its target gene in colorectal cancer as well as the correlation between its target gene and clinical characteristics.Methods 1 Detect the expression of miR-192 and CXCL2 mRNA in CRC tissues,adjacent nontumor tissues,cell lines HCT116 and HT29 using real-time PCR.2 CCK-8 and colony formation were used to evaluate the influence of miR-192 on CRC cells growth.3 Transwell assay was employed to evaluate the invasion capacity of miR-192 on CRC cells.4 The influence of miR-192 on migration ability of colorectal cancer cell was assessed using wound healing assay.5 Cell spheroid assay was used to evaluate the effect of miR-192 on self renewing of CRC stem cell.6 miRDB and Target Scan were used to predict the targets of miR-192.The wild-type and mutant miR-192 binding sequence at the 3’-UTR of human CXCL2 were cloned into p MIR-REPORT luciferase vector.The recombinant p MIR-REPORT luciferase vectors were transfected into HCT116 after miR-192 was up-regulated,and then luciferase reporter assay and Western blot were used to confirm the candidate target of miR-192.7 Western blot was performed to detect the expression of CXCL2 in CRC tissue and adjacent nontumor tissue;ELISA assay was performed to detect the expression of CXCL2 in sera of CRC,healthy control and nontumor disease control,and the correlation between serum CXCL2 expression and clinic pathological characteristics of CRC was analyzed.Serum levels of carcinoembryonic antigen(CEA)and carbohydrate antigen 19-9(CA19-9)in all subjects were measured using immunoluminometric assay.Results 1 The relative expression level of miR-192 in CRC tissues was significantly lower than that in the adjacent normal tissues(P<0.01),but the relative expression level of CXCL2 m RNA in CRC tissues was significantly higher than that in the adjacent normal tissues(P<0.01).The inverse correlation was showed in the expression levels of miR-192 and CXCL2 m RNA.The expression of miR-192 in HCT116 was significantly lower than that in HT29(P<0.01).2 CCK-8 cell growth assay showed that the OD450 value of HCT116 cell in miR-192 overexpressed group was significantly lower than that in the control group(P<0.05);The OD450 value of HT29 cell in zip-miR-192 overexpressed group was significantly higher than that in control group(P<0.05).Colony formation assay showed that the colony numbers of HCT116 in miR-192 overexpressed group was significantly lower than that in control group(P<0.05);the colony numbers of HT29 in zipmiR-192 overexpressed group was significantly higher than that in control group(P<0.05).3 Transwell assay showed that,compared with control group,the number of invading and migration HCT116 cells in miR-192 overexpressed group was significantly decreased,and the percentage of invasion was also significantly decreased(P<0.05);the number of invading and migration HT29 cells in zip-miR-192 overexpressed group was significantly increased,and the percentage of invasion was also significantly increased(P<0.05).4 Wound-healing assay showed that,compared with control group,the ability of the HCT116 cells to migrate into the wound area wassignificantly slower in the miR-192 overexpressed group,and wounds heal slower;the ability of the HT29 cells to migrate into the wound area was significantly faster in the zipmiR-192 overexpressed group,and wounds heal faster.5 Spheroid assay showed that,the ratio of HCT116 spheroid was significantly decreased in miR-192 overexpressed group compared with that in control group(P<0.05);the ratio of HT29 spheroid was significantly increased in zip-192 overexpressed group compared with that in control group.6 Luciferase report and Western blot showed that the CXCL2 acts as the target gene of miR-192.7 WB show that the CXCL2 protein expression in CRC tissues was higher than that in matched adjacent normal tissues.ELISA results showed that the serum CXCL2 levels were much higher in patients with colorectal cancer than those in healthy controls(P<0.01)and other disease controls.Serum CXCL2 levels in colorectal cancer were correlated positively with tumor-node-metastasis staging(P<0.01)and the depth of infiltration(P<0.05),but not with the histological grade,tumor embolus,lymph node metastasis,gross pathologic tumor type,or patient gender.The sensitivity and specificity of the assay for serum CXCL2 were 56.1%(69/123)and 95.31%(203/213),respectively.The diagnosis sensitivity was 22.2(4/18)for I stage and 66.7%(26/39)for II stage when combined the data of CXCL2 with CEA and CA19-9.The area under the ROC curve constructed with CXCL2(0.834)was larger than that constructed with CEA(0.739)or CA19-9(0.676)for discriminating colorectal cancer from matched controls.Conclusions 1 The expression of miR-192 is lower in CRC tissues,while the expression of CXCL2 is higher in CRC tissues.There is an inverse correlation between the levels of miR-192 and CXCL2 in CRC tissues.The expression of miR-192 is significantly lower in HCT116 cell with higher invasive capacity than that in HT29 cell with low invasive capacity,while the expression of CXCL2 is inverse in the two cell lines.2 miR-192 negatively regulates the expression of CXCL2.3 The up-regulation of miR-192 expression in HCT116 cell can effectively inhibit cell growth,invasion,migration,colony formation and spheroid.The fuction-blocking of miR-192 in HT29 cell can effectively enhance cell growth,invasion,migration,colony formationand spheroid.4 The abnormal increase of CXCL2 expression in CRC tissues indicated that CXCL2 may play a role in tumorigenesis and development.The concentration of CXCL2 in CRC sera indicated that the serum CXCL2 level could be a useful biomarker for colorectal cancer diagnosis.