The Biological Significance Of CCL21/CCR7 Axis In The Invasion And Metastases Of Colorectal Carcinoma

PartⅠ:Expression of CCR7 and MMP-9 in Colorectal Carcinomaand its Clinical SignificanceObjective:To investigate the expressions of C-C chemokine receptor(CCR7)andmatrix metalloproteinases-9(MMP-9),to explore their correlation to clinical pathologicalfeatures of colorectal carcinoma,and the relationship between expressions of CCR7 andMMP-9.Methods:The expressions of CCR7 and MMP-9 proteins in 65 cases of colorectalcarcinoma were detected by SABC immunohistochemistry,the expressions of CCR7 in 4strains of colorectal carcinoma cell line examined by RT-PCR and Western blot.Results:41 of the 65 colorectal carcinoma specimens(63.1%)were positive in CCR7,the expression of CCR7 was correlated with invasion depth and lymph node metastasis ofcolorectal carcinoma(P＜0.05).MMP-9 expression was detected in 75.4%(49/65)ofcolorectal carcinoma specimens,the expression of MMP-9 was assiociated with invasiondepth and lymph node metastasis of colorectal carcinoma(P＜0.05).Significant relevancewas found between the expression of MMP-9 and CCR7(P＜0.05).Conclusion:The expression of the chemokine receptor CCR7 and MMP-9 incolorectal carcinoma tissues may be associated with its progression and metastasis.Molecular targets of CCR7 and MMP-9 may also be important factors in the metastasis ofcolorectal carcinoma. PartⅡ:Construction and Identification of pGC-silencer-CRM30CCR7 short hairpin RNA Expression VectorsObjective:To construct CCR7 shRNA eukaryotic expression vectors to transfect intoSW480 cells in order to further study the silencing effects of the vector on the targetinggene CCR7.Methods:The shRNA oligonucleotides targeting for CCR7 gene were synthesized andcloned into pGC-silencer-CRM30 to generate shRNA eukaryotic expression vectors.Therecombinants were named pGC-silencer-CRM30_CCR7A,_CCR7B and _CCR7C.shRNAexpression plasmids were identificated by sequencing.The eukaryotic expression vectorswere transfected into SW480 cells.The green fluorescent protein(GFP)was detected byfluorescence microscope,and the silencing effects of the recombinant vectors weredetermined by RT-PCR.Procedures were optimized to maximize the silencing effect.Two-weeks after addition of G-418,clones stablly expressing pGC-silencer-CRM30_CCR7C were individually selected and expanded.Results:The recombinant sequence identified by sequencing was the same as thetargeting one.In SW480 cells transfected with the recombinant vectors,the expression ofgreen fluorescent protein(GFP)was detected,the silencing effect of pGC-silencer-CRM30_CCR7C was more evident than other recombinant vectors.After screening withG418,CCR7-silencing stable SW480 cells were obtained,and named CCR7-shRNASW480.Conclusion:shRNA recombinant was established successfully by RNAi techniqueand transfected into SW480 cells.The expression of CCR7 was completely knocked downin SW480 cells. PartⅢ:Effect of CCL21/CCR7 on Invasion ofColorectal Carcinoma cell line SW480Objective:To investigate the role of CCL21/CCR7 in invasion of colorectalcarcinoma cell line SW480.Methods:parental SW480 and CCR7-shRNA cell SW480 were exposed to varyingconcentrations of CCL21(10,100 ng/ml)for 48 h.The invasive ability was detected byWound healing assay and Transwell assay,the expression of Snail mRNA was evaluated byRT-PCR,the expressions of MMP-9 and E-cadherin were determined by Western Blot.Results:CCL21 drove more parental SW480 cell migrated into the gap than controlgroup at same time-points after inducing the lesion.The counts of parental SW480penetrating through membrane in 100ng/ml group were 113±7,significantly more than48±4 in control group(P＜0.05).The expression of MMP-9 in 100ng/ml group was0.83±0.02,significantly higher than 0.38±0.01 in control group(P＜0.05),the expression ofE-cadherin was significantly lowered after exposure to CCL21(P＜0.05),the expression ofSnail mRNA was significantly up-regulated(P＜0.05);these functions could be blocked bytransfecting the CCR-shRNA.Conclusion:CCL21 increased the invasive ability in SW480 cells,induced theMMP-9 expression and activity,and enhanced the survival capacity of SW480 cells. PartⅣ:Effects of CCL21/CCR7 on the Survival andApoptosis of Colorectal Carcinoma line SW480Objective:To investigate the role of CCL21/CCR7 in survival of colon cancer cellline SW480 at suboptimal circumstance and explore the mechanism.Methods:The growth curve of CCR7-shRNA SW480 was drwaed by using MTTmethod,the cell cycle was detected by flow cytometry.Parental SW480 cells werepre-incubated with CCL21 for 2h before exposure to VP-16(20 ng/mL),the cellproliferation was detected by MTT assay,cell apoptosis by flow cytometry and Hoechst33258 staining;the expression of Bcl-2/Bax was examined by Western blot.Results:CCL21 alone did not promote the proliferation,but pre-incubation withCCL21 confer SW480 resistance to death induced by VP-16,inhibition rate reduced from68.3% to 47.4% with treatment of 100ng/mL CCL21;consistently,apoptosis decreasedfrom 65.2% to 48.7% after exposure to 100ng/ml CCL21.The expression of Bcl-2 wassignificantly elevated,and the expression of Bax was significantly decreased with treatmentwith CCL21,the effect was blocked by Transfecting SW480 cell with CCR-shRNA.Conclusion:CCL21 enhanced the invasive ability in SW480 cells,induced MMP-9expression,and promoted the survival of SW480 cells under the suboptimal circumstance.