Feng-Qiang Yang

Upregulation of long non-coding RNA MALAT1 correlates with tumor progression and poor prognosis in clear cell renal cell carcinoma

Long noncoding RNAs (lncRNAs) have been investigated as a new class of regulators of cellular processes, such as cell growth, apoptosis, and carcinogenesis. LncRNA metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) has recently been identified to be involved in tumorigenesis of several cancers such as lung cancer, pancreatic cancer, and cervical cancer. However, the role of lncRNA MALAT1 in clear cell renal cell carcinoma (ccRCC) remains unclear. Expression levels of lncRNA MALAT1 in ccRCC tissues and renal cancer cell lines were evaluated by quantitative real-time PCR (qRT-PCR), and its association with overall survival of patients was analyzed by statistical analysis. Small interfering RNA (siRNA) was used to suppress MALAT1 expression in renal cancer cells. In vitro assays were conducted to further explore its role in tumor progression. The expression level of MALAT1 was higher in ccRCC tissues and renal cancer cells compared to adjacent non-tumor tissues and normal human proximal tubule epithelial cells HK-2. The ccRCC patients with higher MALAT1 expression had an advanced clinical features and a shorter overall survival time than those with lower MALAT1 expression. And multivariate analysis showed that the status of MALAT1 expression was an independent predictor of overall survival in ccRCC. Additionally, our data indicated that knockdown expression of MALAT1 decreased renal cancer cell proliferation, migration, and invasion. Our data suggested that lncRNA MALAT1 was a novel molecule involved in ccRCC progression, which provided a potential prognostic biomarker and therapeutic target.

MiR-506 Is Down-Regulated in Clear Cell Renal Cell Carcinoma and Inhibits Cell Growth and Metastasis via Targeting FLOT1

Background Some microRNAs (miRNAs) are abnormally expressed in cancer and contribute to tumorigenesis. In the present study, we investigated the role of miR-506 in clear cell renal cell carcinoma (ccRCC). Methods miR-506 expression was detected in renal cancer cell lines 786-O, ACHN, Caki-1, and Caki-2 and ccRCC specimens by quantitative real-time-PCR. We assessed the association of miR-506 expression with pathology and prognosis in ccRCC patients. We over-expressed and knocked-down miR-506 expression in two renal cancer cell lines, 786-O and ACHN, and assessed the impact on cell proliferation, migration and invasion. A luciferase reporter assay was conducted to confirm the target gene of miR-506 in renal cancer cell lines. Results miR-506 was significantly down-regulated in renal cancer cell lines and ccRCC specimens. Low miR-506 expression in ccRCC specimens was associated with an advanced clinical stage and poor prognosis. miR-506 expression was an independent prognostic marker of overall ccRCC patient survival in a multivariate analysis. Over-expression of miR-506 in renal cancer cells decreased cell growth and metastasis, In contrast, down-regulation of miR-506 expression promoted renal cancer cell growth and metastasis. FLOT1, a potential target gene of miR-506, was inversely correlated with miR-506 expression in ccRCC tissues. Consistent with the effect of miR-506, knockdown of FLOT1 by siRNA inhibited cell malignant behaviors. Rescue of FLOT1 expression partially restored the effects of miR-506. Conclusions miR-506 exerts its anti-cancer function by directly targeting FLOT1 in renal cancer, indicating a potential novel therapeutic role in renal cancer treatment.

VPA inhibits renal cancer cell migration by targeting HDAC2 and down-regulating HIF-1α.

Cell migration plays major roles in human renal cancer-related death, but the molecular mechanisms remain unclear. Valproic acid (VPA) is a broad-spectrum inhibitor of class I and II histone deacetylases and shows great anticancer activity in a variety of human cancers. In this study, we found that VPA significantly inhibited cell migration but not proliferation of human renal cancer ACHN cells. Mechanistic studies found that VPA significantly inhibited the expression of HIF-1α. Knockdown of HIF-1α could obviously inhibited cell migration, while over-expression of HIF-1α markedly rescued the inhibition of VPA on cell migration. Further studies found that knockdown of HDAC2 completely mimicked the effects of VPA on HIF-1α and cell migration, and over-expression of HIF-1α could also rescue the effects of HDAC2 knockdown on cell migration. Collectively, these results indicated that the potential of specific inhibition of HDAC2 by small molecular chemicals may lead to future therapeutic agents in human renal cancer treatment.

Up-regulation of flotillin-2 is associated with renal cell carcinoma progression

Flotillin-2 (FLOT2) is a highly conserved protein isolated from caveolae/lipid raft domains that tether growth factor receptors linked to signal transduction pathway. FLOT2 has recently been identified to be involved in tumorigenesis of several cancers such as breast cancer, melanoma, and gastric cancer. However, the role of FLOT2 in renal cell carcinoma (RCC) remains unclear. The expression levels of FLOT2 in RCC patients and renal cancer cell lines were evaluated by quantitative real-time PCR (qRT-PCR) and Western blot. FLOT2 protein expression was also analyzed in archived paraffin-embedded RCC tissues using immunohistochemistry (IHC), and its association with overall survival of patients was analyzed by statistical analysis. Small-interfering RNA (siRNA) was used to suppress FLOT2 expression in RCC cell lines. In vitro assays were performed to further explore its role in tumor progression. The expression level of FLOT2 was higher in RCC tissues and cell lines than in corresponding adjacent normal tissues and normal human proximal tubule epithelial cell line HK-2. IHC analysis revealed high expression levels of FLOT2 in RCC specimens. The RCC patients with higher FLOT2 expression had an advanced clinical stage and poorer prognosis than those with lower FLOT2 expression. FLOT2 expression was an independent prognostic marker of overall RCC patient survival in a multivariate analysis. In vitro assays indicated that knockdown of FLOT2 reduced cell proliferation, migration, and invasion. Our data suggest that FLOT2 is a novel molecule involved in RCC progression, which provide a potential prognostic biomarker and therapeutic target.

Valproic acid upregulates NKG2D ligand expression and enhances susceptibility of human renal carcinoma cells to NK cell-mediated cytotoxicity

Introduction We aimed to investigate the effect of valproic acid (VPA) on NKG2D ligand expression in human renal carcinoma cell lines and to investigate the mechanisms. Material and methods Different concentrations of VPA from 0.5 mM to 8.0 mM were applied to 786-O and ACHN cell lines, respectively. Cell viability after treatment with VPA was determined by flow cytometry (FCM). Real-time PCR and FCM were used to detect the changes of mRNA and protein level of NKG2D ligands (MICA/B and ULBPs) in the two cell lines treated with 4 mM VPA. The cytotoxicity assay and CD107a mobilization assay were carried out to detect the cytotoxicity changes of NK cells against renal carcinoma cell lines after the same treatment. Results Valproic acid can efficiently upregulate MICA/B, ULBP1 and ULBP2 expression in the renal carcinoma cell lines at the mRNA and protein level (p < 0.05). 786-O and ACHN cells treated with VPA were more susceptible to killing by NK cells than untreated cells and the enhanced cytotoxicity of NK cells was blocked by the pretreatment of NK cells with anti-NKG2D monoclonal antibodies (p < 0.05). Conclusions Valproic acid can clearly induce the expression of NKG2D ligands of renal carcinoma cell lines, thereby enhancing the cytotoxicity of NK cells against renal carcinoma cell lines.

Epigallocatechin gallate attenuates interstitial cystitis in human bladder urothelium cells by modulating purinergic receptors

BACKGROUND Epigallocatechin gallate (EGCG) has exhibited antitumor properties against bladder cancer. However, its effects in interstitial cystitis (IC) have not been investigated. METHODS Here, we performed repeated cystoscopy and re-biopsy of bladder mucosa before and after intravesical irrigation of EGCG in eight patients diagnosed with IC based on clinical and histopathologic assessments. Six normal bladder tissue samples were obtained from age-, race-, and sex-matched asymptomatic control subjects. IC symptom index was used to compare the therapeutic effect in IC patients. Patient-derived bladder epithelial cells were cultured and cell stretch experiments and ATP assays were performed. The expression of purinergic receptors X1, X2, and X3, and Y1, Y2, and Y11, in biopsied samples was detected by Western blotting and real-time polymerase chain reaction, respectively. Moreover, the expression of inducible NO synthase, phosphorylated Akt, and phosphorylated NF-κB was also assessed. RESULTS All EGCG-treated patients demonstrated different extents of remission of symptoms. We found a significant upregulation in P2X1, P2X2, and P2X3 receptor proteins and P2Y1, P2Y2, and P2Y11 receptor transcripts in IC patients. However, EGCG therapy attenuated the expression of all purinergic receptors. In addition, EGCG demonstrated prominent antioxidative and antiinflammatory effects via inhibition of the upregulation of iNOS and phosphorylated NF-κB. Furthermore, the stretch-activated release of ATP in cultured bladder urothelial cells was greater in cells derived from IC patients, compared with those from the control patients, but EGCG, at all concentrations tested, effectively abolished the increase in ATP release from stretched IC patient-derived cells. CONCLUSIONS Our study suggests that inhibition of the expression of purinergic receptors and ATP release in urothelial cells by EGCG supports further development of EGCG as a novel therapeutic option for IC.

Matrix metallopeptidase 2 (MMP2) media la eliminación de MHC class I polypeptide-related sequence A (MICA) en el cáncer de células renales

INTRODUCTION The MHC class i chain-related molecule A (MICA) is a ligand for the natural killer group 2, member D (NKG2D) immunoreceptor activation. The engagement of tumor cell surface MICA to NKG2D stimulates the NK and T cell antitumor immunity. Shedding of MICA by tumor cells facilitates tumor immune evasion, which might partially contribute to tumor progression. MATERIAL AND METHODS Inmunohistochemistry was performed on both normal and neoplastic renal tissue. Human renal carcinoma cell lines 786-0 and ACHIN were transfected and target sequences to silence human MMP2 by shRNA expression were established. The degree of MICA shedding was measured and quantitative real-time PCR and Western-blot analysis were performed. RESULTS The membrane type matrix metalloproteinase 2 (MMP2) mediated the MICA shedding, which was blocked by suppression of MMP2 expression. Concomitantly, MMP2 over-expression enhanced the MICA shedding, indicating that MMP2 was involved in the renal cell carcinoma-associated proteolytic release of soluble MICA (sMICA), which facilitated the tumor immune escape. CONCLUSIONS These findings suggested that MMP2 might be a new potential target for tumor immune therapy. Elucidation of the mechanisms by which tumors shed MICA could be of a great importance for cancer treatment in order to reinforce the NK and T cell antitumor immunity.

HOXA11-AS promotes the growth and invasion of renal cancer by sponging miR-146b-5p to upregulate MMP16 expression: YANG et al.

Recently, increasing studies showed that long noncoding RNAs (lncRNAs) play critical roles in tumor progression. However, the function and underlying mechanism of HOMEOBOX A11 antisense RNA (HOXA11‐AS) on renal cancer remain unclear. In the current study, our data showed that the expression of HOXA11‐AS was significantly upregulated in clear cell renal cell carcinoma (ccRCC) tissues and cell lines. High HOXA11‐AS expression was associated with the advanced clinical stage, tumor stage, and lymph node metastasis. Function assays showed that HOXA11‐AS inhibition significantly suppressed renal cancer cells growth, invasion, and ETM phenotype. In addition, underlying mechanism revealed that HOXA11‐AS could act as a competing endogenous RNA (ceRNA) that repressed miR‐146b‐5p expression, which regulated its downstream target MMP16 in renal cancer. Taken together, our findings suggested that HOXA11‐AS could promote renal cancer cells growth and invasion by modulating miR‐146b‐5p–MMP16 axis. Thus, our findings suggested that HOXA11‐AS could serve as potential therapeutic target for the treatment of renal cancer.

Inhibitory effect of valproate on proliferation of human kidney carcinoma ACHN cells and its mechanism

Objective: To investigate the effects of VPA (valproate) on proliferation, cell cycle distribution and apoptosis of human kidney carcinoma ACHN cells and the possible underlying mechanisms. Methods: The effect of VPA on the proliferation of ACHN cells was examined by CCK-8 (cell counting kit-8) assay. Flow cytometry was used to analyze the cell cycle distribution and apoptosis of ACHN cells exposed to VPA. The mRNA expressions of cyclin E1, P21WAF1, Bcl-2 and Bax were detected by real-time fluorescence quantitative-PCR. Results: Incubation with various concentrations of VPA for 48 h resulted in a significant inhibition of proliferation of ACHN cells with an IC50 (50% inhibitory concentration) value of 4.21 mmol/L. After treatment with VPA, the cell cycle was arrested obviously at G0/G1 phase and the apoptotic rate was significantly increased as compared with the control group. After treatment with 4 mmol/L VPA for 48 h, the levels of P21WAF1 and Bax mRNAs were both significantly increased, and at the same time, the levels of cyclin E1 and Bcl-2 mRNAs were obviously decreased. Conclusion: VPA can inhibit the proliferation of kidney carcinoma ACHN cells by inducing cell-cycle arrest and apoptosis. DOI:10.3781/j.issn.1000-7431.2013.03.004

GATA-3 es subregulado en pacientes con carcinoma renal de células claras

BACKGROUND GATA-3 is a transcription factor involved in human growth and development. Recent studies found its association with breast cancer, however, its expression profile in renal cell carcinoma (RCC) has not been investigated. MATERIAL AND METHOD The study included 35 patients submitted to radical nephrectomy with confirmed pathological diagnosis of RCC. Normal control kidney tissues were obtained from 25 living kidney donors and tissues were biopsied before implantation. The majority of RCC samples were diagnosed as clear cell renal cell carcinoma (94.3%) except for 1 case of papillary RCC and 1 case of collecting duct carcinoma. GATA-3 expression was evaluated by quantitative PCR and Western blotting (WB) in RCC samples and normal kidneys respectively, immunohistochemical staining was performed as well. Meanwhile, the GATA-3 expression in two cancer cell lines (786-O, ACHN) and normal kidney epithelial cells (HK-2) was detected by PCR and WB. In addition, renal cancer cells and HK-2 cells were cultivated and detected by confocal microscopy for the exact intra-cellular localization of GATA-3. RESULTS Data showed a significant down-regulation of GATA-3 expression present in neoplastic tissues compared with normal tissues; similarly, GATA-3 was significantly attenuated in all renal cancer cell lines compared with normal HK-2 cells. Confocal displayed a strong cytoplasmic immno-fluorescence activity of GATA-3 with peri-nuclear zone in HK-2, whereas the intensity in cancer cells was markedly weaker than that of HK-2. CONCLUSIONS In summary, our present study clarifies that the aberrant expression profile of GATA-3 in human RCC is possibly involved with tumorigenesis, and the complicated mechanism is worthy of further investigation.