Expression Level And Role Of DCN In Proliferation And Metastasis In Renal Cell Carcinoma

Background and ObjectiveRenal cancer is one of the common genitourinary carcinomas and its high mortality rate harm to human healthy.Renal cell carcinoma(RCC),the most common subtype of renal cancer,accounts for over 80%of renal carcinoma and the morbidity is increasing year by year.Despite of recent advances in therapy,postoperative recurrence and drug resistance are two important factors which affect the prognosis of renal cell carcinoma.Extracellular matrix(ECM)is macromolecule synthesized by cells and secreted into extracellular space.ECM distributed on the cell surface or intercellular space composed by fibrillar collagens,nonfibrillar collagens,elastic fibers and non collagen glycoprotein(such as,proteoglycan and hyaluronic acid).When tumor metastasis occurs,ECM is the first protective screen.As a functional component of the extracellular matrix,DCN is involved in inhibiting the tumor develpoment,including affecting tumor cell proliferation,decreasing malignant characters,regulating tumor angiogenesis and reducing tumor metastasis.Nonetheless,there are few limited reports have shown the relationship between DCN and RCC.Herein,we not only search for DCN mRNA expression using massive microarray data in OncomineTM(Compendia Bioscience,Ann Arbor,MI),and also examined the expression profile of DCN protein in RCC tissues and adjacent normal kidney tissue by IHC.Moreover,the relationship between DCN protein expression level and clinicopathological parameters of RCC patients was analyzed.Then through ectopic expression analyses and animal model,the roles of DCN in proliferation,migration,invasion and the underline mechanism for decorin expression were further explored.Methods and Materials1.Though public database Oncomine,we explored the expression level of DCN mRNA in RCC.Meanwhile,we also screened several types of RCC specimen and detected the DCN protein expression level in RCC.Furthermore,the relationship between DCN protein expression level and clinicopathological parameters of RCC patients was analyzed.2.We searched the expression level of DCN mRNA in different human tumor cells and focus on DCN mRNA expression level in RCC cell lines.qRT-PCR detected the DCN mRNA level in 786-0 and OS-RC-2 cell lines after 5-azac treatment.The full-length CDS sequence of DCN was constructed into pEGFP-Nl vector plasmid to obtain the pEGFP-N1-DCN plasmid,and then transfected into RCC cell lines.The cell proliferation,cell cycle,migration and invasion of RCC cell lines were analysed by MTT,flow cytometry,clone forming and Transwell.What's more,the expression levels of p21 and E-cadherin protein were examined by western blot.3.6 week-male BALB/c mice(14)were used for the tumor formation assay.7 mice/group and 2 x 106 suspended cells/each mouse were injected into the right axilla.Tumor growth was measured by calipers every 5 days and v=a2b/2.After 27 days,the tumors were removed from all of the animals and the weight of each tumor was examined and finally entered into the statistical analysis.More over,qRT-PCR and IHC were used to examine the DCN mRNA and protein level in tumor.The tumor tissues were also under HE staining and Ki67 IHC staining.Results1.The expression of DCN in RCC tissues is much lower than in adjacent normal kidney:we examined the Oncomine for analysis and visualization of DCN mRNA in RCC and normal tissues.DCN mRNA levels were significantly lower in RCC.However,No statistical differences of DCN mRNA levels between RCC and normal.IHC was performed to determine the expression of DCN protein in 94 RCC tissues and adjacent normal tissues.We found stain for DCN was positive in nuclear of cancer cells and weaker positive in tumor stroma.But positive stain of DCN was multiply in normal kidney tissues,it could be positive in nuclear,cytomembrane and stroma.The expression level of DCN protein in tumor stroma is significantly lower than in normal kidney stroma,though cancer cells and normal cells showed no statistical differences.Examination of the correlation between DCN protein expression and clinical pathological features showed that decreased decorin expression was correlated with tumor size(>4cm),while no association between DCN expression and other clinicopathological factors.2.DCN protein could inhibit the proliferation and metastasis of RCC cell lines:DCN protein was upregulated 2.7 and 2.3-fold in 786-0 and OS-RC-2,respectively,through pEGFP-N1-DCN transfection.The result of MTT assay showed that since 24h in 786-0 and 48h in OS-RC-2,the cell proliferation rates were both decreased in pEGFP-N1-DCN group,compared to the pEGFP-N1 group.The tablet clone forming experiments had showed the same result that over-expression DCN protein suppress the ability of cell clone formation.The cell cycle analysis showed that,compared to pEGFP-N1 group,786-0 transfected with pEGFP-N1-DCN had an obvious cell cycle arrest at the G1 phase,and a decreased S phase.The western blot showed an increase of p21 protein as well as DCN protein over-expression.The antitumous effect of DCN might be the modulation of cancer cell cycle,result in cell proliferation suppression.The transwell assays showed that the migratory and invasive capacity of cells transfected with pEGFP-N1-DCN were both reduced in 786-0 and OS-RC-2,as compared to pEGFP-N1 group.And the E-cadherin protein was increased when DCN over-expression.3.DCN protein could inhibit tumor formation and growth:the tumor formation rate and tumor size of pEGFP-N1-DCN group in nude mice was reduced than pEGFP-N1 group.The expression level of DCN mRNA and protein was higher in pEGFP-N1-DCN group,as compared to pEGFP-N1 group.Meanwhile,IHC showed stronger positive staining of Ki67 in pEGFP-N1 group.ConclusionIt is the first time to investigate the expression level and role of DCN in renal cell carcinoma.1.The expression level of DCN mRNA in RCC cancer tissues is lower than in adjacent normal kidney tissues.DCN protein in tumor stroma is significantly decreased,as compared to normal kidney stroma,and decreased decorin expression in stroma was correlated with tumor size.2.The suppression in proliferation and metastasis of DCN is linked to the regulation of p21 and E-cadherin by DCN.3.DCN protein could inhibit tumor formation and growth in nude mice.