Renal Carcinoma Tissues Research Articles

MiR-182 affects renal cancer cell proliferation, apoptosis, and invasion by regulating PI3K/AKT/mTOR signaling pathway.

PI3K/AKT/mTOR signaling pathway plays a crucial role in tumorigenesis and development. It was shown that mTOR overexpression was associated with the pathogenesis of renal cancer. Down-regulation of MiR-182 was found in renal carcinoma tissue. This study thus aims to investigate the influence of miR-182 in regulating mTOR expression and renal carcinoma cell proliferation, invasion, and apoptosis. The targeted regulatory relationship between miR-182 and mTOR was tested by dual luciferase assay. Renal carcinoma tissue and benign renal tissue were collected to detect miR-182 and mTOR expressions. MiR-182, mTOR, p-mTOR, and Survivin levels were compared between HK-2 and A498 cells. Renal carcinoma A498 cells were divided into four groups, including miR-NC, anti-miR-182 mimic, si-NC, and si-mTOR groups. Cell apoptosis and proliferation were evaluated by flow cytometry. Cell invasion was determined by transwell assay. Bioinformatics analysis revealed the complementary relationship between miR-182 and the 3'-UTR of mTOR mRNA. The level of miR-182 was significantly reduced, while mTOR expression was upregulated in renal carcinoma tissue compared with that in benign lesion, which was associated with TNM stage. MiR-182 expression was markedly declined, whereas mTOR, p-mTOR, and Survivin levels were apparently upregulated in A498 cells compared with that in HK-2 cells. The treatment of miR-182 mimic or si-mTOR transfection significantly downregulated mTOR, p-mTOR, and Survivin expressions, restrained cell proliferation and invasion, and enhanced cell apoptosis. The decreasing level of miR-182 plays a role in enhancing mTOR expression and promoting renal carcinoma pathogenesis. Overexpression of miR-182 inhibited mTOR expression and weakened cell proliferation and invasion, which provides leads to the future therapy of renal cancer.

MiR-155 affects renal carcinoma cell proliferation, invasion and apoptosis through regulating GSK-3β/β-catenin signaling pathway.

Glycogen Synthase Kinase-3β (GSK-3β) negatively regulates Wnt/β-catenin signaling pathway through degrading β-catenin protein. It plays an inhibitory role in various tumors, while the influence in the pathogenesis of renal carcinoma has not been elucidated. MicroRNA-155 (MiR-155) was found to be upregulated in renal carcinoma tissue. Bioinformatics analysis revealed the complementary binding site between miR-155 and 3'-UTR of GSK-3β. This study investigated the influence of miR-155 in regulating GSK-3β expression, Wnt/β-catenin signaling pathway activity, and renal carcinoma cell proliferation, invasion, and apoptosis. The targeted regulatory relationship between miR-155 and GSK-3β were tested by dual luciferase assay. Renal carcinoma tissue and benign renal tissue were collected to detect miR-155 and GSK-3β expressions. MiR-155, GSK-3β, and β-catenin levels were compared between HK-2 and 786-O cells. Renal carcinoma 786-O cells were cultured in vitro and divided into four groups, including miR-NC, anti-miR-155, pIRES2-blank, and pIRES2-GSK-3β groups. Cell apoptosis was evaluated by flow cytometry. Cell invasion was determined by transwell assay. Cell proliferation was assessed by EdU staining. MiR-155 targeted regulated GSK-3β expression. MiR-155 and β-catenin expressions were significantly increased, while GSK-3β level was significantly declined in renal carcinoma tissue compared with benign renal tissue. MiR-155 and β-catenin expressions were significantly elevated, whereas GSK-3β level was significantly downregulated in 786-O cells compared with HK-2 cells. Anti-miR-155 or pIRES2-GSK-3β transfection significantly up-regulated GSK-3β expression, attenuated β-catenin level, restrained cell proliferation and invasion, and enhanced cell apoptosis. MiR-155 promoted renal carcinoma pathogenesis. Inhibition of miR-155 increased GSK-3β expression, attenuated Wnt/β-catenin signaling pathway, weakened proliferation and invasion, and facilitated apoptosis in renal carcinoma cells.

Association between the expression of inhibitor of growth family member 4 and the progression of clear cell renal carcinoma.

Inhibitor of growth family member 4 (ING4) is a candidate tumor suppressor that serves important roles in tumor growth and angiogenesis. In the present study ING4 expression was assessed in clear cell renal carcinoma (CCRC) tissues and its association with the progression of CCRC was determined. The expression of ING4 in 125 patients with CCRC was analyzed using reverse transcription-quantitative polymerase chain reaction (RT-qPCR), western blot analysis and immunohistochemical methods. A total of 40 adjacent normal renal tissues were used as control samples. The results identified that ING4 expression was positive in 100% of normal renal tissues, but in only 82.3% CCRC samples. RT-qPCR and western blotting results demonstrated that expression levels of ING4 mRNA and protein were significantly decreased in CCRC compared with in normal tissues (P<0.0001). ING4 expression was not associated with sex, age or tumor volume (P>0.05), but was associated with the nuclear grade of renal cancer (P<0.0001), the clinical stage of CCRC (P<0.0001) and lymphatic metastasis (P<0.0001). The results of the present study indicated that dysregulation of ING4 expression may be involved in the initiation and progression of CCRC.

Formation and Determination of Endogenous Methylated Nucleotides in Mammals by Chemical Labeling Coupled with Mass Spectrometry Analysis.

5-Methylcytosine (5-mC) is an important epigenetic mark that plays critical roles in a variety of cellular processes. To properly exert physiological functions, the distribution of 5-mC needs to be tightly controlled in both DNA and RNA. In addition to methyltransferase-mediated DNA and RNA methylation, premethylated nucleotides can be potentially incorporated into DNA and RNA during replication and transcription. To exclude the premodified nucleotides into DNA and RNA, endogenous 5-methyl-2'-deoxycytidine monophosphate (5-Me-dCMP) generated from nucleic acids metabolism can be enzymatically deaminated to thymidine monophosphate (TMP). Therefore, previous studies failed to detect 5-Me-dCMP or 5-methylcytidine monophosphate (5-Me-CMP) in cells. In the current study, we established a method by chemical labeling coupled with liquid chromatography-electrospray ionization mass spectrometry (LC-ESI-MS/MS) for sensitive and simultaneous determination of 10 nucleotides, including 5-Me-dCMP and 5-Me-CMP. As N,N-dimethyl-p-phenylenediamine (DMPA) was utilized for labeling, the detection sensitivities of nucleotides increased by 88-372-fold due to the introduction of a tertiary amino group and a hydrophobic moiety from DMPA. Using this method, we found that endogenous 5-Me-dCMP and 5-Me-CMP widely existed in cultured human cells, human tissues, and human urinary samples. The presence of endogenous 5-Me-dCMP and 5-Me-CMP indicates that deaminases may not fully deaminate these methylated nucleotides. Consequently, the remaining premethylated nucleosides could be converted to nucleoside triphosphates as building blocks for DNA and RNA synthesis. Furthermore, we found that the contents of 5-Me-dCMP and 5-Me-CMP exhibited significant decreases in renal carcinoma tissues and urine samples of lymphoma patients compared to their controls, probably due to more reutilization of methylated nucleotides in DNA and RNA synthesis. This study is, to the best of our knowledge, the first report for detecting endogenous 5-Me-dCMP and 5-Me-CMP in mammals. The detectable endogenous methylated nucleotides indicate the potential deleterious effects of premodified nucleotides on aberrant gene regulation in cancers.

Rce1 expression in renal cell carcinoma and its regulatory effect on 786-O cell apoptosis through endoplasmic reticulum stress.

Ras and a-factor-converting enzyme 1 (Rce1) is located in the endoplasmic reticulum (ER) and is thought to be responsible for endoproteolytic processing of the vast majority of CAAX proteins. Endoplasmic reticulum stress (ERS) plays an important role in renal cell carcinoma (RCC); however, the expression and role of Rce1 in RCC have not been extensively studied. We aimed to investigate the expression of Rce1 in RCC tissues and its molecular mechanism in ERS-induced apoptosis in RCC 786-O cells. We first used western blotting, quantitative reverse transcriptase-polymerase chain reaction, and immunohistochemistry to detect the Rce1 expression in renal carcinoma tissues and paracancerous tissues. It was found that Rce1 expression was upregulated in RCC tissues, and its positive expression level was strongly associated with clinicopathologic features. Next, we detected the expression of Rce1 in human embryonic kidney cell line HEK293 and human renal carcinoma cell lines 786-O, ACHN, and A498. Higher expression of Rce1 was found in human renal carcinoma cell lines, especially in 786-O cells. Knockdown of Rce1 in 786-O cells increased apoptosis and inhibited proliferation (P < 0.05). Moreover, downregulation of Rce1 upregulated the expression of the pro-apoptotic protein Bax, but downregulated the expression of the anti-apoptotic protein Bcl-2. Further studies showed that downregulation of Rce1 also affected the expression of ERS factors. In conclusion, our results indicated that Rce1 plays a key role in RCC. Low expression of Rce1 might indirectly increase apoptosis and inhibit proliferation of renal carcinoma cells through ERS.

Expression and clinical significance of HMGA2 in renal carcinoma

Objective: To detect the high mobility group A2 (HMGA2) expression in renal carcinoma, and to explore the relationship with clinicopathological features and its significance for prognosis. Methods: 50 renal carcinoma specimens, 50 corresponding adjacent normal kidney tissue samples, and 40 benign renal tumor specimens were used in this study. The expressions of HMGA2 mRNA and protein were detected by RT-PCR, Western blot and immunohistochemical assays, and its relationship with clinicopathological features and prognosis in the renal carcinoma patients was analyzed. Results: The RT-PCR results showed that the relative expression levels of HMGA2 mRNA in the renal carcinoma, benign renal tumor tissues, and adjacent normal renal tissues were 0.84±0.23, 0.19± 0.06 and 0.08±0.04, respectively, and the expression in renal carcinoma tissue was significantly higher than those of the other 2 groups (P<0.01). The Western blot results showed that the relative expression levels of HMGA2 protein in the renal carcinoma, benign renal tumor tissues, and adjacent normal renal tissues were 0.91±0.24, 0.12±0.04 and 0.03±0.01, respectively, and the expression in renal carcinoma tissue was significantly higher than those of the other 2 groups (P<0.01). Immunohistochemical results showed that the expression of HMGA2 protein exhibited brown and tan granular, which mainly distributed in the cell nuclei. Among the 50 cases of renal carcinoma, 34 cases exhibited positive expression, with a positive rate of 68.0%. Among the 40 cases of benign tumor tissues, 3 cases had positive expression, with a positive rate of 7.5%, while among the 50 cases of adjacent normal renal tissues, there was only 1 case exhibiting positive expression of HMGA2 protein, with a positive rate of 2.0%. The protein expression of HMGA2 was significantly higher in the renal carcinoma than in the benign tumors and normal renal tissues (P=0.004). There was no statistically significant difference in the association of HMGA2 protein expressions with age, sex, tumor size and histological type (P>0.05), while significant difference did exist in the association with different statuses of TNM staging and lymph node metastasis (P<0.05). The median time to progression (TTP) in 34 HMGA2 protein-positive patients was (22.36±1.48) months and that of 16 HMGA2 protein-negative patients was (34.55±1.87) months (P<0.05). Conclusions: HMGA2 plays an important role in the tumorigenesis and development of renal carcinoma, and may be used as an important predictor for estimating the prognosis of renal carcinoma. HMGA2 might become a new diagnostic and prognostic marker for renal carcinoma.

Expression and clinical significance of Apollon in renal carcinoma.

Apollon, namely baculoviral inhibitor of apoptosis proteins (IAP) repeat containing 6, is an unusually large member of the IAP family, and may be important in oncogenesis. The aim of the present study was to assess the association between renal carcinoma (RC) and Apollon expression, and to highlight the link between Apollon expression and the occurrence, development and prognosis of RC. Apollon expression was detected by immunohistochemistry, western blotting and reverse transcription-quantitative polymerase chain reaction in RC tissues, adjacent non-cancerous tissues and paired normal tissues, respectively, in order to analyze the association between Apollon expression and clinicopathological features of RC. Kaplan-Meier survival estimate was used to assess the prognostic significance. It was observed that Apollon expression was higher in carcinoma tissues than in adjacent non-cancerous tissues and normal control tissues at the protein and messenger RNA level (P<0.001). There was a significant difference in T-stage (P=0.006), nodal involvement (P=0.007) and tumor-node-metastasis-stage (P=0.035) in patients categorized according to different Apollon expression levels. A prognostic significance of Apollon was also identified by the Kaplan-Meier method. The results of the present study indicate that Apollon expression is associated with the biological characteristics of renal cancer, and is potentially a valuable predictor and novel target for RC.

Expression and relationship between hepaCAM protein and multidrug resistance protein in renal carcinoma

Objective To investigate the expression and relationship between hepatocyte cell adhesion molecule (hepaCAM) protein and some multidrug resistance proteins in renal carcinoma tissue. Methods Expressions of hepaCAM, multidrug resistance associated protein (MRP), P-glycoprotein (P-gp), lung resistance protein (LRP), and topoisomerase Ⅱ (TOPO Ⅱ) protein were detected by immunohistochemistry in different areas of human renal cell carcinoma tissues and their relationships were analyzed. Results In the peripheral zone of renal tumor, hepaCAM, MRP, P-gp and LRP protein were showed positive expression. In the central region of the renal tumor, the expressions of hepaCAM, P-gp and LRP were negative or weakly positive, while the expressions of MRP and TOPO Ⅱ protein were positive. The expressions of MRP and TOPO Ⅱ protein in the central region of tumor were stronger than those in the peripheral zone of tumor (31.23±5.67 vs. 23.89±4.56; 45.66±2.34 vs. 5.23±0.66), with statistically significant differences (t=-6.20, P=0.00; t=-100.16, P=0.00). While the expressions of other proteins (hepaCAM, P-gp and LRP) in the central region of tumor were weaker than those in the peripheral zone of tumor (3.21±1.12 vs. 27.25±2.23; 2.34±0.33 vs. 51.23±3.45; 4.22±1.78 vs. 44.23±1.45), with statistically significant differences (t=60.87, P=0.00; t=90.35, P=0.00; t=107.18, P=0.00). Correlation analysis showed that the expression of hepaCAM protein in the central region of renal carcinoma was related with the expression of MRP protein (r=0.94, P=0.01), but it was not related with the expressions of P-gp, LRP and TOPO Ⅱ protein (r=0.22, P=0.44; r=0.14, P=0.80; r=0.34, P=0.07). Conclusion The expression of hepaCAM protein in renal carcinoma may be related to tumor drug-resistance. Key words: Kidney neoplasms; Cell adhesion molecules; Multidrug resistance-associated proteins

Expression of septin-9 in human renal cell carcinoma and clinical significance

Objective To study the expression of septin-9 protein and gene in renal cell carcinoma (RCC) and normal renal tissues and reveal the role of septin-9 gene in the process of angiogenesis in renal tumors. Methods The clinicopathologic data of 60 cases of RCC freshly excised in our hospital were retrospectively analyzed. The expression of septin-9 protein and mRNA was detected by Western blotting and real-time quantitative polymerase chain reaction (Real-time PCR). Tumor microvessel density (MVD) in CD105-labeled RCC was measured. The correlation between the expression of septin-9 in RCC and MVD was analyzed. Results (1) RCC tissues and adjacent cancer tissues both had the expression of septin-9 gene and protein. The gene expression of septin-9 in different pathological grades of RCC was 1.258±0.372, 1.339±0.408, 1.502±0.476, 1.689±0.511, and the protein expression was 1.325±0.315, 1.597±0.376, 1.634±0.513, 1.805±0.572, which was significantly higher than that in tumor adjacent tissues and normal tissue (P<0.05). With the increases in pathological grades of RCC, the expression of septin-9 gene and protein was up-regulated. (2) The positive expression rate of CD105 in RCC tissue was 76.67% (46/60), and 13.33% (2/15) in cancer adjacent tissue with the difference being statistically significant (P<0.05). The MVD in the cancer adjacent tissue was 12.38±2.05. MVD in different pathological grades was 21.59±4.39, 29.84±7.43, 38.61±6.27, and 52.18±6.22 respectively. The MVD was increased with the increases in the pathological grades. Conclusion The expression of septin-9 played an important role in the development of human RCC probably by interfering with the tumor microvascular formation. Key words: Septin-9; CD105; Microvessel density; Renal cell carcinoma

Mitochondrial Sirt3 supports cell proliferation by regulating glutamine-dependent oxidation in renal cell carcinoma

Clear cell renal carcinoma (RCC), the most common malignancy arising in the adult kidney, exhibits increased aerobic glycolysis and low mitochondrial respiration due to von Hippel-Lindau gene defects and constitutive hypoxia-inducible factor-α expression. Sirt3 is a major mitochondrial deacetylase that mediates various types of energy metabolism. However, the role of Sirt3 as a tumor suppressor or oncogene in cancer depends on cell types. We show increased Sirt3 expression in the mitochondrial fraction of human RCC tissues. Sirt3 depletion by lentiviral short-hairpin RNA, as well as the stable expression of the inactive mutant of Sirt3, inhibited cell proliferation and tumor growth in xenograft nude mice, respectively. Furthermore, mitochondrial pyruvate, which was used for oxidation in RCC, might be derived from glutamine, but not from glucose and cytosolic pyruvate, due to depletion of mitochondrial pyruvate carrier and the relatively high expression of malic enzyme 2. Depletion of Sirt3 suppressed glutamate dehydrogenase activity, leading to impaired mitochondrial oxygen consumption. Our findings suggest that Sirt3 plays a tumor-progressive role in human RCC by regulating glutamine-derived mitochondrial respiration, particularly in cells where mitochondrial usage of cytosolic pyruvate is severely compromised.

Downregulation of microRNA-206 suppresses clear cell renal carcinoma proliferation and invasion by targeting vascular endothelial growth factor A.

MicroRNA-206 (miR-206) has been discovered to have anticancer properties in different tissues. However, the role of miR-206 on renal carcinoma is still ambiguous. In the present study, we investigated the role of miR-206 on the development of renal carcinoma. The results indicated that miR-206 was significantly downregulated in 69 clear cell renal carcinoma (ccRCC) tissues and low-level of miR-206 related to shorter metastasis-free survival time for patients with ccRCC. The results indicated that vascular endothelial growth factor A (VEGFA) was a direct target of miR-206 in renal cancer cells. Further studies revealed that upregulation of miR-206 inhibited renal cancer cell proliferation, invasion and migration, suggesting that miR-206 functioned as a tumor suppressor. RNA interference targeting VEGFA mRNA could mimic the upregulation of miR-206 functions, and also suppressed tumor formation in vivo in nude mice. These results suggest that miR-206 plays an important role in ccRCC tumorigenesis by targeting VEGFA.

MYSM-1 suppresses migration and invasion in renal carcinoma through inhibiting epithelial-mesenchymal transition.

Renal cell carcinoma (RCC) is the most common malignant renal tumor and is prone to metastasis. However, the molecular variation and mechanism underlying renal cell carcinoma metastasis remains largely unknown. In our previous study, it was found that MYSM-1 was significantly downregulated in renal cell carcinoma tissues as compared with normal renal tissues without metastasis, using proteomics approach. Therefore, we hypothesized that MYSM-1 may suppress the metastasis of renal cell carcinoma in light of paucity of data regarding MYSM-1 in the cancers. In the present study, to confirm the expression status of MYSM-1 in renal cell carcinoma, immunohistochemistry with renal carcinoma tissue microarray was performed. It was shown that MYSM-1 was remarkably decreased in renal carcinoma tissues compared with paired normal control tissues; and that low expression of MYSM-1 was significantly associated with poor overall prognosis and metastasis. To investigate the biological roles of MYSM-1 in vitro in renal carcinoma cell lines, both knockdown using siRNA and over-expression were carried out. It was found that MYSM-1 could suppress the proliferation, migration, and invasion of renal carcinoma cells. In addition, we found that MYSM-1 could inhibit the epithelial-mesenchymal transition. Together, our results demonstrate that MYSM-1 could suppress the metastasis of renal carcinoma cells may be through inhibiting the epithelial-mesenchymal transition (EMT) process.

DACH1 inhibits cyclin D1 expression, cellular proliferation and tumor growth of renal cancer cells.

BackgroundRenal cell carcinoma (RCC) is a complex with diverse biological characteristics and distinct molecular signature. New target therapies to molecules that drive RCC initiation and progression have achieved promising responses in some patients, but the total effective rate is still far from satisfaction. Dachshund (DACH1) network is a key signaling pathway for kidney development and has recently been identified as a tumor suppressor in several cancer types. However, its role in renal cell carcinoma has not been fully investigated.MethodsImmunohistochemical staining for DACH1, PCNA and cyclin D1 was performed on human renal tissue microaraays and correlation with clinic-pathological characteristics was analyzed. In vitro proliferation, apoptosis and in vivo tumor growth were evaluated on human renal cancer cell lines with decitabine treatment or ectopic expression of DACH1. Downstream targets and potential molecular mechanism were investigated through western blot, immunoprecipitation and reporter gene assays.ResultsExpression of DACH1 was significantly decreased in human renal carcinoma tissue. DACH1 protein abundance was inversely correlated with the expression of PCNA and cyclin D1, tumor grade, and TNM stage. Restoration of DACH1 function in renal clear cell cancer cells inhibited in vitro cellular proliferation, S phase progression, clone formation, and in vivo tumor growth. In mechanism, DACH1 repressed cyclin D1 transcription through association with AP-1 protein.ConclusionOur results indicated that DACH1 was a novel molecular marker of RCC and it attributed to the malignant behavior of renal cancer cells. Re-activation of DACH1 may represent a potential therapeutic strategy.

AB80. A novel protein is lower expressed in renal cell carcinoma.

Engrailed-2 (EN2) has been identified as a candidate oncogene in breast cancer and prostate cancer. It is usually recognized as a mainly nuclear staining in the cells. However, recent studies showed a cytoplasmic staining occurred in prostate cancer, bladder cancer and clear cell renal cell carcinoma. The inconsistency makes us confused. To clarify the localization and expression of EN2 in renal cell carcinoma, anti-EN2 antibody (ab28731) and anti-EN2 antibody (MAB2600) were used for immunohistochemistry (IHC) respectively. Interestingly, we found that EN2 detected by ab28731 was mainly presented in cytoplasm while EN2 detected by MAB2600 was mainly presented in nucleus. To further investigate the different patterns observed above, lysates from full-length EN2 over expression in HEK293T cells were used to identify which antibody the EN2 molecule bound by western blot. Results showed ab28731 did not react with the lysates. For this reason, the novel specific protein detected by ab28731 was not the EN2 molecule and was named nonEN2. Then using the renal carcinoma tissue microarray and renal tissues, we found that the protein expression levels of nonEN2 in kidney tumor tissues was significantly lower than that in kidney normal tissues (p<0.05), so was in renal cell lines. Taken together, nonEN2 is lower expressed and may play an important role in renal cell carcinoma.

A Novel Protein Is Lower Expressed in Renal Cell Carcinoma

Engrailed-2 (EN2) has been identified as a candidate oncogene in breast cancer and prostate cancer. It is usually recognized as a mainly nuclear staining in the cells. However, recent studies showed a cytoplasmic staining occurred in prostate cancer, bladder cancer and clear cell renal cell carcinoma. The inconsistency makes us confused. To clarify the localization and expression of EN2 in renal cell carcinoma, anti-EN2 antibody (ab28731) and anti-EN2 antibody (MAB2600) were used for immunohistochemistry (IHC) respectively. Interestingly, we found that EN2 detected by ab28731 was mainly presented in cytoplasm while EN2 detected by MAB2600 was mainly presented in nucleus. To further investigate the different patterns observed above, lysates from full-length EN2 over expression in HEK293T cells were used to identify which antibody the EN2 molecule bound by western blot. Results showed ab28731 did not react with the lysates. For this reason, the novel specific protein detected by ab28731 was not the EN2 molecule and was named nonEN2. Then using the renal carcinoma tissue microarray and renal tissues, we found that the protein expression levels of nonEN2 in kidney tumor tissues was significantly lower than that in kidney normal tissues (p < 0.05), so was in renal cell lines. Taken together, nonEN2 is lower expressed and may play an important role in renal cell carcinoma.

Cadherin-11 in Renal Cell Carcinoma Bone Metastasis

Bone is one of the common sites of metastases from renal cell carcinoma (RCC), however the mechanism by which RCC preferentially metastasize to bone is poorly understood. Homing/retention of RCC cells to bone and subsequent proliferation are necessary steps for RCC cells to colonize bone. To explore possible mechanisms by which these processes occur, we used an in vivo metastasis model in which 786-O RCC cells were injected into SCID mice intracardially, and organotropic cell lines from bone, liver, and lymph node were selected. The expression of molecules affecting cell adhesion, angiogenesis, and osteolysis were then examined in these selected cells. Cadherin-11, a mesenchymal cadherin mainly expressed in osteoblasts, was significantly increased on the cell surface in bone metastasis-derived 786-O cells (Bo-786-O) compared to parental, liver, or lymph node-derived cells. In contrast, the homing receptor CXCR4 was equivalently expressed in cells derived from all organs. No significant difference was observed in the expression of angiogenic factors, including HIF-1α, VEGF, angiopoeitin-1, Tie2, c-MET, and osteolytic factors, including PTHrP, IL-6 and RANKL. While the parental and Bo-786-O cells have similar proliferation rates, Bo-786-O cells showed an increase in migration compared to the parental 786-O cells. Knockdown of Cadherin-11 using shRNA reduced the rate of migration in Bo-786-O cells, suggesting that Cadherin-11 contributes to the increased migration observed in bone-derived cells. Immunohistochemical analysis of cadherin-11 expression in a human renal carcinoma tissue array showed that the number of human specimens with positive cadherin-11 activity was significantly higher in tumors that metastasized to bone than that in primary tumors. Together, these results suggest that Cadherin-11 may play a role in RCC bone metastasis.

Proliferation inhibition and the underlying molecular mechanisms of microRNA-30d in renal carcinoma cells

To investigate the inhibitory effects of microRNA-30d (miR-30d) on renal carcinoma cell proliferation and the underlying molecular mechanisms, miR-30d expression in renal cell carcinoma (RCC) specimens was analyzed by quantitative polymerase chain reaction (qPCR). The inhibition of the proliferation of miR-30d on renal carcinoma cells (ACHN cell line) was analyzed by MTT and colony formation assays. The effects of miR-30d on cyclin E2 expression were detected by the luciferase activity of the reporter gene. In addition, the effects of miR-30d on endogenous cyclin E2 expression at the RNA and protein levels were investigated by qPCR and western blot analysis, respectively. Cell cycles were analyzed by flow cytometry. The results showed the following: i) Expression of miR-30d was significantly downregulated in renal carcinoma tissues compared with paraneoplastic tissues; ii) overexpression of miR-30d inhibited renal carcinoma cell proliferation and colony formation; iii) miR-30d inhibited cyclin E2 3′ untranslated region-mediated reporter gene expression; and iv) overexpression of miR-30d downregulated endogenous cyclin E2 expression and blocked the cell cycle at the G1 phase. In conclusion, miR-30d functions as a tumor suppressor gene in RCC and inhibits renal carcinoma cell proliferation. Cell cycle regulatory factor cyclin E2 is a target gene of miR-30d. miR-30d inhibits renal carcinoma cell proliferation via the regulation of cyclin E2 expression at the post-transcriptional level.

Downregulation of tumstatin expression by overexpression of ornithine decarboxylase

Tumor angiogenesis, a pivotal process for cancer growth and metastasis, requires both upregulation of pro-angiogenic molecules and downregulation of anti-angiogenic molecules. Anti-angiogenesis therapy represents a promising way for cancer treatment. Tumstatin, a novel endogenous angiogenesis inhibitor, inhibits endothelial cell proliferation, pathological angiogenesis and tumor growth. Ornithine decarboxylase (ODC), overexpressed in various cancers, is associated with cell transformation, tumor invasion and angiogenesis. We found that the expression of tumstatin was suppressed in ODC-overexpressing human cancer cells and renal carcinoma tissues. We presumed that ODC overexpression may downregulate the expression of tumstatin. To be able to test this hypothesis, we generated HEK293 cells that overexpress ODC (ODC transfectants) and characterized the following experimental groups: PBS-treated group, mock transfectants, ODC transfectants, ODC transfectants transfected with pcDNA-ODCr (an antisense ODC-expressing plasmid) group and putrescine-treated group. The effect of ODC overexpression on tumstatin expression was examined by reverse transcriptase-polymerase chain reaction (RT-PCR), western blot analysis and dual luciferase reporter assay. ODC-overexpressing cells and putrescine-treated cells showed suppressed tumstatin mRNA and protein expression, and decreased tumstatin gene promoter activity. Thus, ODC overexpression suppresses the expression of tumstatin, which may provide fundamental evidence for the combination of anti-angiogenic therapy and conventional therapy for cancer treatment.

ALS2CL gene expression in clear-cell renal cell carcinoma and the implication

Objective To study the differential expression of ALS2CL in clear cell renal cell carcinoma (ccRCC) and normal kidney tissue and the implication.Methods Real-time quantitative polymerase chain reaction (Real-time PCR),immunohistochemistry (IPH) and Western blotting were applied to examine the expression status of ALS2CL in 26 ccRCC tumor and adjacent normal kidney tissue samples as well as two kinds of ccRCC-derived cell lines (786/O,SN12-PM6).Results As compared with normal control group,the two ccRCC-derived cell lines (786/O,SN12-PM6) and 65.38％ (17/26) of ccRCC alls showed an obvious down-regulated expression of ALS2CL mRNA.Under the examination of ALS2CL protein by IPH,73.08％ (19/26) of ccRCC specimens were negative or weakly positive.Western blotting also showed a highly down-regulated rate-53.85％ (14/26) in ccRCC samples,and the low expression of ALS2CL was also found in 786/O and SN12-PM6 cells.All of them showed statistically significant difference between ccRCC group and corresponding normal control group (P ＜ 0.05).Conclusion The absence or downregulated expression of ALS2CL may be closely related to the occurrence of ccRCC. Key words: Clear cell renal cell carcinoma; Gene expression; Tumor suppressor genes

Regulatory T cells, dendritic cells and neutrophils in patients with renal cell carcinoma

We evaluated dendritic cells (DC), regulatory T lymphocytes (Treg) and neutrophils in 37 patients with newly diagnosed renal cell carcinoma (RCC) in the tumor and peripheral blood (PB) and correlated these parameters with tumor staging (early-T1, 2, late-T3, 4 and metastatic disease). The number of myeloid and plasmacytoid DC in blood of RCC patients was higher than in healthy controls. The percentage of myeloid dendritic cells (mDC) from CD45+ cells in tumors was higher in comparison with peripheral blood irrespective of disease stage. Higher percentage of these cells expressed a maturation marker in the periphery in the early stage (CD83 expressing cells). The number of plasmacytoid dendritic cells (pDC) in PB was similar in both early and late stage groups, but the early group displayed a significantly higher percentage of pDC in tumor cell suspension. Neutrophil counts in the peripheral blood of RCC patients were higher than in healthy controls, but the counts in both tumor stage groups were similar. The proportion of neutrophils from CD45+ cells was higher in late stage tumors. Higher percentage of Treg from CD4+ cells was detected in renal carcinoma tissue in comparison to PB with no difference between stages of the disease. Our results reflect the complex interplay between various cells of the immune system and the tumor microenvironment. Activation of dendritic cell subpopulations at early stages of the disease course is counterbalanced by the early appearance of T regulatory cells both in the periphery and tumor tissue. Later stages are characterized by the accumulation of neutrophils in the tumor. Appropriate timing of anticancer strategies, especially immunotherapy, should take these dynamics of the immune response in RCC patients into account.