| Backround:Renal cell carcinoma is one of the most common malignant tumors in urinary system and is a common carcinoma in adult,the morbidity increase rapidly.Clinically,RCC can remain occult for the majority cases course,leading to the fact that about twenty to thirty percent patients present with metastasis when initially diagnosed.Besides,RCC is resistant to radiation and chemotherapy,and many patients who undergo curative surgical resection experience recurrent during subsequent follow-up,which bring great difficulties to the diagnosis and treatment of RCC.Up to now,there are two major factors involved in RCC metastasis: one can promote the metastasis and tumor angiogenesis factors,such as MMPs,CD44,another one is the tumor suppressors such as VHL and PTEN.However,the exact mechanisms need to be further studied.Therefore,it is highly essential to explore new specific and effective factors for diagnosis,prognosis prediction as well as the therapeutic targets of RCC.RSK4 is a member of ribosomal s6 kinase(RSK)family,but its functions remain largely unknown.There are only few studies about the distribution of RSK4 m RNA in human normal tissues.In most studies,RSK4 is considered as a potential tumor suppressing gene,and it is reported that RSK4 expression is reduced in some tumors and RSK4 overexpression could be against the invasion and metastasis of the tumor cells.In our previous study,the expression of RSK4 in RCC is higher than normal kidney,and the overexpression of RSK4 in RCC may lead to invasion and metastasis easily.That is to say,RSK4 may take a crucial role in RCC.According to previous reports,RSK4 may not be the single factor in RCC,while expression of MMP-9 and CD44 are also in high level in RCC.Previous studies showed that with the stimulating of Ras-MEK-ERK signal pathway,MMP-9 can bind with CD44-ICD to take on further regulation.RSK4 was reported to be a downstream factor of Ras-MEK-ERK signal pathway.rp S6,a 70 k D aribosomal protein S6 kinase(p70S6K),is the downstream mediators of m TOR signal pathway,can be phosphorylate in the site of Ser235/236 by RSK family members,while GSK3β,a downstream mediators of Wnt signal pathway,also can be regulated by RSK4.Whether RSK4 could mediate the invasion and metastasis of renal cell carcinoma by rp S6 and GSK3β is also a new direction proposed by this study.Purpose:1.To investigate the expression of RSK4,CD44 and MMP-9 protein in primary renal cell carcinoma(p RCC)and metastatic renal cell carcinoma(m RCC).2.To explore the level of expressions of RSK4,CD44 and MMP-9 protein as well as the association with clinicopathological features and clinical outcome in metastatic renal cell carcinoma(m RCC).3.To preliminarily investigate the function and mechanism of RSK4,CD44 and MMP-9protein in RCC.Methods:1.The expression of RSK4,CD44 and MMP-9 in 52 p RCC samples and 48 m RCC samples were detected by immunohistochemistry.2.The relationship with clinicopathologic features as well as prognosis were analyzed by statistical methods.3.Kaplan–Meier method and Cox hazard regression model were employed to analyze the relationship of RSK4,CD44 and MMP-9 expression and survival in RCC patients.4.Up-and down-regulation of RSK4 were studied by lipofectamine-mediated gene transfection in ACHN cell lines.5.The expression of CD44,MMP-9,rpS6,p-rpS6,GSK3β as well as β-catenin levels in the RSK4 overexpressed ACHN cell lines was studied by western blot.6.The expression of β-catenin in pRCC samples and m RCC samples were detected by immunohistochemistry.Results:1.In the 48 m RCC samples,there were 36(75%,36 /48),33(68.75%,33 /48)and 44(91.7%,44/48)positive for RSK4,CD44 and MMP-9,respectively,while the positive rate in 52 p RCC samples were 23(44.2%,23/52)、18(34.6%,18/52)and 36(69.2%,36/52),respectively.2.Statistical analysis showed that the expression of RSK4,CD44 and MMP-9 in m RCC samples are higher than the p RCC samples(RSK4: P=0.002;MMP-9: P=0.002;CD44:P=0.001).Further analysis showed that expression of the three proteins showed closely related(p=0.008),particularly,the expression of RSK4 and CD44(p=0.019),MMP-9 and CD44(p=0.05)were correlated respectively,while the expression of RSK4 and MMP-9(p=1.00)showed no significance of correlation.Furthermore,the expression of RSK4,CD44 and MMP-9 in m RCC samples weren`t correlated with ages,genders,Fuhrman grades and the metastatic sites(p>0.05).3.The median survival time among 29 m RCC samples are 36m(6-99m),Kaplan–Meier method shows the expression of MMP-9 is related to the length of lifetime after tumor metastasis(p=0.038).4.q RT-PCR and western blot showed while the expression of MMP-9 and CD44 increased in the.RSK4 overexpression cell line.5.Western blot showed due to the overexpression of RSK4 in ACHN cell line,rp S6,p-rp S6,GSK3β as well as β-catenin expression levels increased significantly.6.Statistical analysis verified that,by using immunohistochemistry,m RCC samples were positive for β-catenin in 9(39.1%,9 /23)cases,while the positive rate in p RCC samples were 23(65.7%,23/35).Conclusions:1.The expression of RSK4,CD44 and MMP-9 in m RCC samples were higher than pRCC samples,suggesting that the three are related with the metastasis of renal cell carcinoma.2.There remain a positive relationship among RSK4,CD44 and MMP-9,indicate the potential function in promoting the tumor metastasis.3.In up-and down-regulation of RSK4 ACHN cell lines,the expressions of rpS6,p-rp S6,GSK as well as β-catenin levels are all upregulated.Meanwhile,the expression ofβ-catenin in m RCC samples are higher than p RCC samples,indicating that RSK4 may regulate MMP-9 and CD44 through GSK3β and β-catenin signal pathway in promoting RCC progression. |
PDF Full Text Request