ABSTRACT
Objective:To investigate the expression of MEK/ERK signaling pathways in renal cell car-cinoma with bone metastasis,and to analyze the differences of expressions of VEGFR-2,MEK,ERK on the primary and metastasis tissue and its mechanism.Methods:The tissue samples were obtained from 7 renal cell carcinoma patients kindly provided by Department of Urology,Peking University People’s Hos-pital from January 1,2009 to January 1,2010.The expression of MEK/ERK signaling pathways was de-tected in the 7 renal cell carcinoma patients`primary and matched metastatic tissues with ICH,The anti-body concentrations were 1 ∶200,1 ∶25,and 1 ∶250,respectively.The mutation of the twentieth exon of the PDGFRA gene,the second exon of the K-ras gene,the fifteenth exon of the Brafgene and the se-cond exon of the MEK1 gene were detected with PCR.Results:The expression intensities of VEGFR-2, MEK,and ERK were measured by H-score [intensity (1,2,3,or 4)multiplied by the distribution (%)].VEGFR-2,MEK,and ERK expressions were divided into 3 groups according to the positive dis-tribution of the tumor cells:1,0 -5%;2,6% -50%;and 3,>50%,To assess intratumor heteroge-neity,three distinct microscopic fields (×200)from each specimen were used to evaluate the expres-sions,Subsequently,the scores were averaged to obtain a single concatenated score for each tissue. VEGFR-2,MEK,and ERK expressions were assessed by 2 independent pathologists who were blinded to the clinicopathological data.The data were expressed as the mean value of the triplicate experiments.The expressions of MEK,and ERK were higher in the metastatic tissues than in the matched RCC tissues (6.10 ±4.10 vs.1.33 ±0.51,P =0.015;9.10 ±2.24 vs.4.43 ±2.84,P =0.021 )while the ex-pression of VEGFR-2 was not different between the primary and metastatic tissues (P =0.901).No mu-tation was detected on the twentieth exon of the PDGFRA gene,the second exon of the K-ras gene,the fifteenth exon of the Brafgene and the second exon of the MEK1 gene.Conclusion:MEK/ERK signa-ling pathways may play an important role in the metastasis and the resistance of sunitinib in RCC patients with bone metastasis.
ABSTRACT
Objective:To investigate the change of biological characteristics after stable knockdown of CKLF-like MARVEL transmembrane domain containing 3 (CMTM3)expression in PC3 by lentivirus shRNA and to reveal new therapeutic targets.Methods:The research includes two groups:sh393 is the experimental group in which CMTM3 is knocked down in PC3 cell line;shN is the control group in which CMTM3 is negatively knocked down.The expression of CMTM3 was detected by Western blot.The mi-gration ability of PC3 after stable knockdown was detected by Transwell and Wound healing assay.The invasion ability of PC3 was detected by Matrigel assay.Results were obtained from at least three indivi-dual experiments.Results:The expression of CMTM3 in sh393 group is significant lower than shN group (0.004 0 ±0.000 4 vs.0.490 0 ±0.055 7,P <0.001)detected by Western blot.It also had statistical significance in Matrigel assays (248.6 ±4.5 vs.113.0 ±3.3),Transwell (203.6 ±1.9 vs.103.0 ± 1.2)and Wound healing assays (95.0 ±2.9 vs.33.0 ±1.5)that knockdown of CMTM3 promoted mi-gration,and invasion of PC3 cells in vitro (P <0.001).Conclusion:Negative correlation exists between the stable knockdown of CMTM3 and change of biological characteristics in PC3 cells,and knocking down CMTM3 affects migration,and invasion ability in PC3 cells.