Proliferation Of 786-O Cells Research Articles

The mechanism of action and experimental verification of Gan-song Yin on renal clear cell carcinoma based on network pharmacology and bioinformatics

BackgroundGan-song Yin (GSY) is originated from the scripture “Gan-song Pills”, a medical work of the Ningxia ethnic minorities, and its treatment of kidney diseases has good results. Its method of treating Renal clear cell carcinoma (KIRC) is still unknown, nevertheless.MethodsFirstly, utilizing a network pharmacology strategy to screen GSY for active components and targets and looking up KIRC-related targets in GeneCards and GEO databases. Secondly, protein interaction networks were constructed and analyzed for GO and KEGG enrichment. Molecular docking was then performed and clinical and other correlations of the network pharmacology results were analyzed using bioinformatic analysis methods. Finally, we performed in vitro cellular experiments with 786-O cells and ACHN cells to validate the results of network pharmacology and bioinformatic analysis.ResultsWith the help of network pharmacological analysis, six hub targets were eliminated. Bioinformatics study revealed that the hub targets has clinically significant clinical guiding importance. The results showed that GSY inhibited the proliferation of 786-O cells and ACHN cells, induced cell apoptosis, blocked cell cycle, and reduced cell colony formation ability. qRT-PCR results showed that GSY promoted the expression of ALB and CASP3 genes, and inhibited the expression of EGFR, JUN, MYC and VEGFA genes. Western blot results showed that GSY could promote the expression of ALB and CASP3 protein, and inhibit the expression of EGFR, JUN, MYC and VEGFA protein.ConclusionsNetwork pharmacology and bioinformatics analysis showed that GSY could act on multiple targets through a variety of components to achieve the effect of treating KIRC. In this study, we confirmed that GSY inhibits KIRC by regulating the expression of core targets through in vitro cellular experiments, thus providing a reference for subsequent related studies.

Immune-pyroptosis-related genes predict the prognosis of kidney renal clear cell carcinoma

BackgroundKidney renal clear cell carcinoma (KIRC) is a common cancer of the adult urological system. Recent developments in tumor immunology and pyroptosis biology have provided new directions for kidney cancer treatment. Therefore, there is an urgent need to identify potential targets and prognostic biomarkers for the combination of immunotherapy and pyroptosis-targeted therapy. MethodsThe expression of immune-pyroptosis-related differentially expressed genes (IPR-DEGs) between KIRC and healthy tissues was examined using the Gene Expression Omnibus datasets. The GSE168845 dataset was selected for subsequent analyses. Data of 1793 human immune-related genes were downloaded from the ImmPort database (https://www.immport.org./home), while those of 33 pyroptosis-related genes were extracted from previous reviews. The independent prognostic value of IPR-DEGs was determined using differential expression, prognostic, and univariate and multivariate Cox regression analyses. The GSE53757 dataset was used to further verify the GSDMB and PYCARD levels. In our cohorts, the association among DEGs and clinicopathological features and overall survival was analyzed. The least absolute shrinkage and selection operator Cox regression model was established to evaluate the correlation of IPR-DEGs with the immune score, immune checkpoint gene expression, and one-class logistic regression (OCLR) score. KIRC cells and clinical tissue samples were subjected to quantitative real-time polymerase chain reaction to examine the GSDMB and PYCARD mRNA levels. The GSDMB and PYCARD levels in a healthy kidney cell line (HK-2 cells) and two KIRC cell lines (786-O and Caki-1 cells) were verified. The tissue levels of GSDMB and PYCARD were evaluated using immunohistochemical analysis. GSDMB and PYCARD were knocked down in 786-O cells using short-interfering RNA. Cell proliferation was examined using the cell counting kit-8 assay. Cell migration was measured by transwell migration assays ResultsGSDMB and PYCARD were determined to be IPR-DEGs with independent prognostic values. A risk prognostic model based on GSDMB and PYCARD was successfully established. In the GSE53757 dataset, the GSDMB and PYCARD levels in KIRC tissues were significantly higher than those in healthy tissues. The GSDMB and PYCARD expression was related to T stage and OS in our cohort. The GSDMB and PYCARD levels were significantly correlated with the immune score, immune checkpoint gene expression, and OCLR score. The results of experimental studies were consistent with those of bioinformatics analysis. The GSDMB and PYCARD levels in KIRC cells were significantly upregulated when compared with those in healthy kidney cells. Consistently, GSDMB and PYCARD in KIRC tissues were significantly upregulated when compared with those in adjacent healthy kidney tissues. GSDMB and PYCARD knockdown significantly decreased 786-O cell proliferation (p < 0.05). Transwell migration result reflects that silencing GSDMB and PYCARD inhibited 786-O cell migration (p < 0.05) . ConclusionsGSDMB and PYCARD are potential targets and effective prognostic biomarkers for the combination of immunotherapy and pyroptosis-targeted therapy in KIRC.

Effects of p-cresol, a uremic toxin, on cancer cells.

Though p-cresol exists at a low concentration in the blood, it accumulates in various organs of uremic patients. Previous research has shown that the p-cresol promoted bladder cancer cell invasion and migration. This study aims to see if p-cresol had similar effects on kidney cancer cells and liver cancer cells. For 48 hours, 786-O human renal cancer cells and HepG2 human liver cancer cells were treated with p-cresol at concentrations of 0, 10, 20, 40, and 70 µM. The effects of p-cresol on cell viability, apoptosis, migration, and invasion were then analyzed using the CCK-8, TUNEL, and Transwell migration/invasion assays, respectively. P-cresol at 0 to 70 µM for 48 hours had no significant toxic effects on 786-O cells or HepG2 cells. We chose 40 µM p-cresol for 48 hours for the following experiment. The viability and proliferation of 786-O cells and HepG2 cells were unaffected after 48 hours of treatment, with 40 µM p-cresol. However, 40 µM p-cresol for 48 hours promoted HepG2 cell migration and invasion but did not have the same effect on the 786-O cell line. P-cresol may be responsible for HepG2 cells' malignant biological behavior. Because the liver is the primary site of p-cresol metabolism, it is important to study the responses of cancer cells in the liver to p-cresol.

MiR-184-5p inhibits cell proliferation, invasion and predicts prognosis of clear cell renal cell carcinoma by targeting NUS1 dehydrodolichyl diphosphate synthase subunit: Results from large-scale comprehensive identification and validation.

Clear cell renal cell carcinoma (ccRCC) has become a common malignant cancer with increasing incidence rate and high recurrence risk in genitourinary oncology around the world. Recently, miRNAs were identified to affect pathogenesis, development, molecular functions, and prognosis of ccRCC. In this study, microRNA-184-5p (miR-184-5p) was identified from three independent ccRCC cohorts and was determined as a significantly distinct prognostic biomarker. Relative miR-184-5p expression was found in A-498 and 786-O ccRCC cells compared with HK-2 cells. After ccRCC cells were transfected with miR-184-5p mimics or inhibitor, biological abilities of miR-184-5p in tumor cell proliferation, cycle, apoptosis and invasion were determined. Additionally, we confirmed the direct relationship between miR-184-5p and NUS1 dehydrodolichyl diphosphate synthase subunit (NUS1) by using the Luciferase reporter and rescue assays. These results indicated that the expression level of miR-184-5p in human ccRCC cells and tissues was reduced, and the up-regulation of miR-184-5p regulated A-498 and 786-O cell proliferation, invasion and apoptosis by directly targeting NUS1. These findings may provide new theoretical targets for treatment strategies and drug development of ccRCC.

CX3CL1 in the red bone marrow promotes renal cell carcinoma to metastasize to the spine by involving the Src-related pathway.

Spinal metastasis (SM) frequently occurs in renal cell carcinoma (RCC) patients. Our preliminary work showed that CX3CL1 plays a positive role in SM. The objective of the present study was to verify whether CX3CL1 activates the downstream pathway by binding to CX3CR1 in RCC cells, ultimately promoting RCC to metastasize to the spine. The expression of CX3CL1 and CX3CR1 in tissue samples was detected by immunohistochemistry and western blotting. ELISA was used to quantify the concentration of CX3CL1 in the serum. The expression level of CX3CR1 in RCC cell lines was also detected. The CellTiter-Glo assay and flow cytometry were used to analyze cell viability and apoptosis of RCC cells. Transwell and wound healing assay were used to analyze the effect of CX3CL1 on the invasion and migration ability of RCC cells. Specific inhibitors were used to interfere with key molecules in the signaling pathway to further explore the signal transduction in RCC cells after CX3CL1 stimulation. The expression of CX3CR1 in SM from RCC was higher than that in limb bone metastases. Among the five RCC cell lines, 786O cells expressed the highest level of CX3CR1. CX3CL1 neither inhibited the proliferation of 786O cells nor promoted the apoptosis of 786O cells. However, it promoted the migration and invasion of RCC cells. After CX3CL1 stimulation, Src and Focal adhesion kinase (FAK) phosphorylation levels increased in RCC cells. Bosutinib and PF-00562271 inhibited Src/FAK phosphorylation and cell motility and invasion triggered by CX3CL1 stimulation. CX3CL1 in the red bone marrow of spinal cancellous bone enhances migration and invasion abilities of RCC cells, thereby promoting RCC metastasize to the spine. The migration and invasion of RCC cells activated by CX3CL1 are at least partially dependent on Src/FAK activation.

Effect of ginsenoside Rg3 on proliferation and apoptosis of 786-0 cells and AktmTORSTAT3 signaling in renal carcinoma

Surgery is currently the major approach treating kidney cancer, which is insensitive to chemo-/radio-therapy. However, due to the limitation of current diagnostic technique, certain number of patients has lost the opportunity of surgery. Therefore the searching for novel effective treatment approach is of critical importance. Ginsenoside Rg3 can inhibit proliferation and induce apoptosis of various tumor cells. It effect in kidney cancer, however, is still lacked. This study thus treated kidney cancer cell line 786-O with ginsenoside Rg3 to illustrate its effect on cell proliferation and apoptosis, and investigated possible mechanism.Gradient concentrations of ginsenoside Rg3 was used to treat 786-O cells, whose proliferation was measured by CCK8 assay. The apoptotic rate was quantified using flow cytometry. Western blotting was used to measure protein expression levels of p-Akt, p-mTOR and p-STAT3. Ginsenoside Rg3 significantly inhibited proliferation of kidney cancer cell 786-O in a dose- and time-dependent manner: higher concentration and longer treatment time caused more significant inhibition on cell proliferation. Similar patterns also occurred for the induction for cell apoptosis by ginsenoside Rg3. The expression levels of p-Akt, p-mTOR and p-STAT3, however, were decreased with higher dosages of ginsenoside Rg3, and were negatively correlated with apoptotic ratio. Ginsenoside Rg3 can exert anti-tumor role via inducing apoptosis and inhibiting proliferation of 786-O cells, possibly via the blocking of Akt/mTOR/STAT3 pathway.

Investigation on the antitumor effects of paeonol against renal cell carcinoma based on network pharmacology and experimental validation

Ethnopharmacological relevanceRenal cell carcinoma (RCC) is the most common cancer of the urinary system, the current treatments for RCC are unsatisfactory. Paeonol is the main pharmacologically active ingredient of the traditional Chinese medicine (TCM) moutan cortex (Paeonia suffruticosa Andrews) and Paeonia albiflora Pall, and has been used in TCM to treat various diseases including cancer. However, the underlying therapeutic mechanisms of paeonol in RCC have not been investigated yet. Aim of the studyThis study aimed to explore the potential antitumor effects and mechanisms of paeonol on RCC based on network pharmacology and experimental validation. Materials and methodsNetwork pharmacological analysis was performed to predict the potential targets and mechanism of paeonol against RCC. The antitumor effects and the priority targets of paeonol against RCC were further assessed by in vitro experiments. Results104 intersection targets shared by paeonol and RCC were collected, 15 hub genes were obtained, among these genes, VEGFA expression was higher in RCC, and the higher expression of IL-6 or lower expression of AKT1, JUN, MAPK1, and MAPK8 were correlated to the shorter overall survival (OS) in RCC patients. GO and KEGG analyses suggested that the genes were mainly enriched in the positive regulation of cell death and apoptosis pathway. In vitro experiments showed that paeonol inhibited 786-O cell proliferation, migration, invasion, and promoted apoptosis. When 786-O cells were treated with paeonol, the expression of Bax increased while Bcl-2 and VEGFA decreased. ConclusionThe present study demonstrated that paeonol might play an essential role in RCC by regulating cell proliferation, apoptosis, metastasis, and invasion through the Bcl-2/Bax signaling pathway and VEGFA, providing a theoretical and experimental scientific basis for future investigations of the antitumor effects of paeonol against RCC.

Fatty Acid Synthase Correlates With Prognosis-Related Abdominal Adipose Distribution and Metabolic Disorders of Clear Cell Renal Cell Carcinoma.

Purpose: Lipid metabolism reprogramming is a major pathway in tumor evolution. This study investigated fatty acid synthase (FASN) mRNA expression in anthropometric adipose tissue and elucidated the prognostic value and potential mechanism of clear cell renal cell carcinoma (ccRCC).Materials and Methods: Transcription profiles were obtained from 533 ccRCC samples in The Cancer Genome Atlas (TCGA) cohorts. Real-time quantitative PCR (RT-qPCR) and immunohistochemistry were performed to detect FASN expression in 380 paired ccRCC and normal tissues from the Fudan University Shanghai Cancer Center (FUSCC). Visceral adipose tissue (VAT) and subcutaneous adipose tissue were at the level of the umbilicus as measured by magnetic resonance imaging (MRI). Non-targeted metabolomics and in vitro experiments were used to reveal the biological functions of FASN.Results: Increased FASN expression was significantly relevant to advanced T, N, and American Joint Committee on Cancer (AJCC) stages (p < 0.01) and significantly correlated to poor progression-free survival (PFS) and overall survival (OS) of 913 ccRCC patients in FUSCC and TCGA cohorts. Pearson's correlation coefficient indicated that FASN amplification was positively correlated to VAT% (r = 0.772, p < 0.001), which significantly correlated to poor PFS (HR = 2.066, p = 0.028) and OS (HR = 2.773, p = 0.023) in the FUSCC cohort. Transient inhibition or overexpression of FASN significantly regulated A498 and 786O cell proliferation and migration by regulating epithelial–mesenchymal transition. Inhibition of FASN led to a higher apoptotic rate and decreased lipid droplet formation compared with normal control in ccRCC cells. Non-targeted metabolomics showed that decreased de novo lipogenesis might be required to sustain an elevation of glycolytic activity in 786O cells by regulating galactinol, dl-lactate, N-acetylaspartylglutamate, and sucrose, thereby participating in carcinogenesis and progression of ccRCC.Conclusion: This study demonstrated that FASN expression is positively related to aggressive cell proliferation, migration, apoptosis, and lipid droplet formation and regulates metabolic disorders of the ccRCC microenvironment. Additionally, elevated FASN mRNA expression is significantly correlated to the abdominal obesity distribution, especially VAT%, which is a significant predictor of a poor prognosis for ccRCC patients.

Bidirect effects from cisplatin combine with rosmarinic acid (RA) or hot water extracts of Glechoma hederacea (HWG) on renal cancer cells

BackgroundCisplatin (CDDP) is a chemotherapeutic drug which also causes adverse side effects. Glechoma hederacea is a traditional Chinese herb belonging to the Labiatae family and has many biological activities. Our previous study indicated that rosmarinic acid (RA) was the most abundant phytochemical in G. hederacea. However, the antioxidant or anti-inflammatory effects of the combined treatment of G. hederacea, RA and CDDP on human renal cell carcinoma (RCC) 786-O cells have not been clearly demonstrated. We aimed to investigate the bioefficacy of hot water extracts of G. hederacea (HWG) and RA in inhibiting RCC 786-O cell activity and its synergism with CDDP against metastatic renal cancer cell.MethodsBioactivities of the combination treatment of HWG, RA, HWG/CDDP and RA/CDDP were assessed using the MTT assay and transwell migration, and the crude extract/compound efficacy was evaluated using wound healing migration assays, flow cytometry and western blotting.ResultsOur study indicates that CDDP inhibits 786-O cell proliferation and migration and HWG and RA protect against these effects. On the other hand, HWG and RA demonstrate a low cytotoxic effect in human renal proximal tubular epithelial cell line -2 (HK-2 cells). Cell cycle analysis found that HWG/CDDP and RA/CDDP combined treatment exerted cytotoxicity by inducing G2/M arrest and apoptosis. RA in combined with CDDP significantly inhibiting the expression of p-FAK (Tyr 925) in RCC 786-O cells in vitro.ConclusionWe propose that the inhibition of RA on RCC 786-O cell invasion and migration may partly occur through the downregulation of FAK phosphorylation. The HWG/CDDP and RA/CDDP combined treatments may be effective strategies for intervention of RCC 786-O cell activity.

G250 Antigen-Targeting Drug-Loaded Nanobubbles Combined with Ultrasound Targeted Nanobubble Destruction: A Potential Novel Treatment for Renal Cell Carcinoma.

PurposeWe intended to design G250 antigen-targeting temsirolimus-loaded nanobubbles (G250-TNBs) based on the targeted drug delivery system and to combine G250-TNBs with ultrasound targeted nanobubble destruction (UTND) to achieve a synergistic treatment for renal cell carcinoma (RCC).MethodsThe filming-rehydration method was combined with mechanical shock and electrostatic interactions to prepare temsirolimus-loaded nanobubbles (TNBs). G250-TNBs were prepared by attaching anti-G250 nanobodies to the surface of TNBs using the biotin-streptavidin-bridge method. The ability of G250-TNBs to target the G250 antigen of RCC cells and the synergistic efficacy of G250-TNBs and UTND in the treatment of RCC were assessed.ResultsThe average diameter of the prepared G250-TNBs was 368.7 ± 43.4 nm, the encapsulation efficiency was 68.59% ± 5.43%, and the loading efficiency was 5.23% ± 0.91%. In vitro experiments showed that the affinity of G250-TNBs to the human RCC 786-O cells was significantly higher than that of TNBs (P <0.05), and the inhibitory effect on 786-O cell proliferation and the induction of 786-O cell apoptosis was significantly enhanced in the group treated with G250-TNBs and UTND (G250-TNBs+ UTND group) compared with the other groups (P <0.05). In a nude mouse xenograft model, compared with TNBs, G250-TNBs could target the transplanted tumors and thus significantly enhance the ultrasound imaging of the tumors. Compared with all other groups, the G250-TNBs+UTND group exhibited a significantly lower tumor volume, a higher tumor growth inhibition rate, and a higher apoptosis index (P <0.05).ConclusionThe combined G250-TNBs and UTND treatment can deliver anti-tumor drugs to local areas of RCC, increase the local effective drug concentration, and enhance anti-tumor efficacy, thus providing a potential novel method for targeted therapy of RCC.

Oleic acid promotes cell invasion through an integrin-linked kinase signaling pathway in renal cell carcinoma

The change of fatty acid composition has been regarded as an indicator of altered lipid metabolism during human tumourigenesis, but the details are still unclear. We have previously demonstrated a monounsaturated fatty acid (MUFA) named oleic acid (OA) was involved in renal cell carcinoma (RCC) cell growth, as an extracellular signaling molecule to regulate 786-O cell proliferation via the integrin-linked kinase (ILK) pathway. In this study, we further observe the effects of OA on cell invasion of RCC and the potential mechanism by which OA worked was determined. The transwell invasion assay showed OA increased cell invasion of RCC in a dose-dependent manner. Western blotting results indicated ILK, COX-2, and MMP-9 proteins were involved for their high expressions and these effects were reversed when down-regulating the expression of ILK by special siRNA. The MMPs inhibitor GM6001 could weaken the abilities of OA on RCC cells invasion. These results suggested MUFA indeed affected cell invasion of RCC, which was depended by the regulation of ILK pathway.

Effects of parthenolide on proliferation inhibiting and cell cycle of human renal cancer 786-O cells

Objective To explore the effects of parthenolide on growth inhibition, cell aoptosis and molecular mechanism of human 786-O kidney cancer cells. Methods The inhibitory effect of parthenolide on human786-O cells was determined by MTT assay.The cell apoptosis and the cell cycle were analyzed by flow cytometry.Bax and the gene change of Bax and Bcl-2 were analyzed by fluorescence quantitative PCR, Bax and Bcl-2 family proteins were analyzed by Westerm blot. Results Parthenolide inhibited the proliferation of 786-O cells in a dose-dependent and time-dependent manner.As showed by flow cytometry, parthenolide treatment of the 786-O cells led to significant G1-phase cell cycle arrest in a dose-dependent manner and a decrease in cell population in the S-phase, whereas the population of cells in G2/M phase did not chang significantly.Flow cytometIy also showed a dose-dependent increase in 786-O cells apoptosis by parthenolide treatment.Westem blot analysis indicated a down-regulation of phosphorylated CyclinDl in a dose-dependent manner in 786-O cells by parthenolide treatment.In the gene and protein level, parthenolide treatment to 786-O cell lines resulted a significant reduction in Bcl-2 expression but an increase in Bax protein in a dose-dependent manner. Conclusions Parthenolide treatment can 1ead to a significant dose-dependent and time-dependent inhibition in the growth and an increase in apoptosis of human 786-O Kidney cancer cells.The mechanism of action of parthenolide may be related to inhibition of CyclinDl activation and regulation of Bcl-2 and Bax expression in human 786-O Kidney cancer cells. Key words: Kidney Neoplasms; Tanacetum parthenium; Cell Proliferation; Apoptosis; Cell Cycle

Scavenger receptor BI promotes cytoplasmic accumulation of lipoproteins in clear-cell renal cell carcinoma

Clear-cell renal cell carcinomas (ccRCCs) are characterized by inactivation of the von Hippel-Lindau (VHL) gene and intracellular lipid accumulation by unknown pathomechanisms. The immunochemical analysis of 356 RCCs revealed high abundance of apoA-I and apoB, as well as scavenger receptor BI (SR-BI) in the ccRCC subtype. Given the characteristic loss of VHL function in ccRCC, we used VHL-defective and VHL-proficient cells to study the potential influence of VHL on lipoprotein uptake. VHL-defective patient-derived ccRCC cells and cell lines (786O and RCC4) showed enhanced uptake as well as less resecretion and degradation of radio-iodinated HDL and LDL (125I-HDL and 125I-LDL, respectively) compared with the VHL-proficient cells. The ccRCC cells showed enhanced vascular endothelial growth factor (VEGF) and SR-BI expression compared with normal kidney epithelial cells. Uptake of 125I-HDL and 125I-LDL by patient-derived normal kidney epithelial cells as well as the VHL-reexpressing ccRCC cell lines, 786-O-VHL and RCC4-O-VHL cells, was strongly enhanced by VEGF treatment. The knockdown of the VEGF coreceptor, neuropilin-1 (NRP1), as well as blocking of SR-BI significantly reduced the uptake of lipoproteins into ccRCC cells in vitro. LDL stimulated proliferation of 786-O cells more potently than 786-O-VHL cells in a NRP1- and SR-BI-dependent manner. In conclusion, enhanced lipoprotein uptake due to increased activities of VEGF/NRP1 and SR-BI promotes lipid accumulation and proliferation of VHL-defective ccRCC cells.

Expression and significance of Cystatin-C in clear cell renal cell carcinoma

PurposeCystatin-C (Cys-C) has been studied as a valuable prognostic indicator in several malignancies. The goal of this study is to explore the expression and prognostic significance of Cys-C in clear cell renal cell carcinoma (CCRCC). MethodsImmunohistochemistry and western blot assays were performed to evaluate the level of Cys-C expression in CCRCC tissue. Expression levels of Cys-C in CCRCC tissue samples in relation to clinicopathological characteristics of the tumors were assessed. Their prognostic significance was analyzed by univariate and multivariate analyses. In addition, the expression of Cys-C in 786-O cell lines was inhibited by using CRISPR/Case9 and the effects of Cys-C knockout on 786-O cells in vitro were evaluated using MTT method, colony formation assay, cell cycle assay, and cell migration and invasive assay. ResultsThe expression level of Cys-C was lower in CCRCC tissues (n = 253) than in paired adjacent non-cancerous tissues (n = 164) by immunohistochemistry (P < 0.001). Among the 253 patients, the results showed that patients with low Cys-C expression level in cancer tissue has longer overall survival (OS) than that with high Cys-C level. Furthermore, knockout of Cys-C in 786-O cell line has ability to suppress cell proliferation, induce G0/G1 phase arrest, inhibited cell invasion, decreased phosphorylation of ERK1/2, STAT-3 and enhanced phosphorylated JNK expression. ConclusionsA decrease in serum Cys-C is a favorable prognostic indicator for CCRCC patients. Inhibition of Cys-C suppressed RCC 786-O cell proliferation and invasion. These results indicated that Cys-C could serve as an ideal prognostic biomarker in patients with CCRCC.

Molecular Recognition and In-Vitro-Targeted Inhibition of Renal Cell Carcinoma Using a DNA Aptamer

Renal cell carcinoma (RCC) is the most common malignant tumor of the urinary system, and it has a high frequency of local invasion and distant metastasis. Although multiple advances have been made in the diagnosis and therapy of RCC, the vast majority of patients with metastatic RCC remain incurable. In this study, an aptamer named SW-4 against RCC 786-O cells was identified from a known sequence pool. The identified aptamer exhibited high binding affinity for target cells with dissociation constants in the nanomolar range. Binding analysis revealed that SW-4 only bound to RCC 786-O cells, but not HEK293T cells or human proximal tubular HK-2 cells, indicating that SW-4 has excellent binding selectivity. By sequence optimization, the 26-nt truncated SW-4b demonstrated improved binding affinity, and it was internalized into target cells via caveolae-mediated endocytosis in a temperature-dependent manner. Furthermore, fluorescence imaging confirmed that SW-4b accumulated at tumor sites in 786-O xenograft nude mice models and specifically recognized clinical RCC tissues. Meanwhile, SW-4b inhibited proliferation of 786-O cells by arresting cell cycle progression at the S phase. Taken together, these results indicate that SW-4b is a potential candidate for development into a novel tool for diagnosis and targeted therapy of RCC.

Effect of up-regulating the expression of microRNA-613 on biological behavior of human renal cell carcinoma cells

Objective To observe the effect of microRNA-613 (miR-613) on the biological behavior of human renal cell carcinoma cells and explore its molecular mechanism. Methods The synthesized miR-613 mimics and miR-NC were respectively transferred into 786-O cells by Lipofectamine 3000 and divided into experimental group and control group. Real time quantitative polymerase chain reaction (qPCR) was used to detect the expression of miR-613 in cells of two groups to verify the transfection success. The proliferation of 786-O cells was detected by methyl thiazolyl tetrazolium (MTT) assay and the migration of 786-O cells was detected by Transwell migration assay. Bioinformatics predicts the target gene of miR-613. Dual luciferase reporter assay validates the targeted relationship between miR-613 and target genes, and their binding sites. qPCR and Western blot were used to detect the expression of target genes. Results qPCR test results showed that the expression of miR-613 in the experimental group (16.22±1.08) was significantly higher than that in the control group (1.06±0.20), with statistically significant difference (P<0.01). The results of MTT showed that the cells in the experimental group showed a decrease in absorbance value at the d-th point after transfection (P<0.05). Transwell cell migration test results showed that the cells in the control group and experimental group migrated from the upper chamber of Transwell to the lower chamber of the cells were respectively (95.55±17.88) and (199.10±22.74), with statistically significant difference (P<0.05). Bioinformatics predicts that the target gene of miR-613 is Cortactin (CTTN). Dual luciferase reporter assay confirmed that miR-613 can target CTTN gene (P<0.05). miR-613 can significantly inhibit the CTTN gene expression in renal cell carcinoma cells (P<0.01). Conclusions Up-regulation of miR-613 expression can inhibit the proliferation and migration of renal cell carcinoma cells, and its possible molecular mechanism is that miR-613 inhibits the expression of CTTN gene. Key words: Carcinoma, renal cell/PP; MicroRNAs; Cell proliferation; Cell migration assays

MiR-200c-3p inhibits cell migration and invasion of clear cell renal cell carcinoma via regulating SLC6A1

In this study, we investigated the mechanism of miR-200c-3p and SLC6A1 in regulating cell activity of clear cell renal cell carcinoma (CCRCC). The mRNA and miRNA expressions of tissue specimens were analyzed by CapitalBio Corporation (Beijing, China). The expression of SLC6A1 in CCRCC cells was examined through qRT-PCR and western blot. The migration and invasion ability of 786-O cells was testified by transwell assay after transfected. 786-O cell proliferation ability was detected by MTT assay. Dual luciferase reporter assay verified the association between SLC6A1 and miR-200c-3p. SLC6A1 was high expressed and miR-200c-3p was low expressed in CCRCC tissues and cells. Besides, lower SLC6A1 expression indicated longer survival time and higher survival rate. MiR-200c-3p could directly target at SLC6A1 and reduce its expression. MiR-200c-3p inhibited the proliferation, migration and invasion in 786-O cells by down-regulating SLC6A1 expression. The results suggested that the miR-200c-3p served as a suppressor for CCRCC via down-regulating SLC6A1.

MiR-502-mediated histone methyltransferase SET8 expression is associated with clear cell renal cell carcinoma risk.

Genetic variants may affect the interactions between microRNAs (miRNAs/miRs) and their target genes by modulating their binding affinity or by creating, or destroying a miRNA-binding site. SET domain containing (lysine methyltransferase) 8 (SET8) is the sole lysine methyltransferase that catalyzes the monomethylation of histone H4 lysine 20, and is associated with tumor growth, invasion and metastasis. In the present study, the rs16917496 polymorphism within the miR-502 binding site of the SET8 mRNA 3′ untranslated region (3′UTR) in patients with clear cell renal cell carcinoma (ccRCC) and healthy controls was genotyped. The SET8 CC genotype was associated with a decreased ccRCC risk compared with the CT [P=0.003; odds ratio (OR)=0.318; 95% confidence interval (CI), 0.146–0.691], TT (P=0.011; OR=0.402; 95% CI, 0.197–0.819) and CT+TT (P=0.004; OR=0.370; 95% CI, 0.186–0.736) genotypes. The SET8 CC genotype was associated with reduced SET8 expression based on immunostaining of ccRCC tissue. Low SET8 protein levels were negatively associated with tumor-node-metastasis staging in patients with ccRCC according to the size of tumor and lymph node metastases. SET8-knockdown inhibited renal carcinoma 786-O cell proliferation, migration and invasion. c-Myc and matrix metalloproteinase-7 mRNA expression were downregulated upon SET8-knockdown in renal carcinoma 786-O cells. These data indicated that SET8 may be a functional tumor promoter and that its activation, which is partially regulated by changing the miR-502 and SET8 3′UTR binding affinity, may serve an important role in ccRCC development.

MicroRNA-302c-3p inhibits renal cell carcinoma cell proliferation by targeting Grb2-associated binding 2 (Gab2).

The expression and biological function of Grb2-associated binding 2 (Gab2) in renal cell carcinoma (RCC) cells was tested here. We showed that Gab2 expression was significantly elevated in human RCC tissues and RCC cells. It was correlated with over-activation of Akt and downregulation of microRNA-302c-3p (“miR-302c-3p”), a putative Gab2-targeting microRNA. Knockdown of Gab2 inhibited Akt activation and 786-O RCC cell proliferation. Reversely, forced over-expression of Gab2 led to Akt hyper-activation to facilitate 786-O cell proliferation. Exogenous expression of miR-302c caused Gab2 downregulation, Akt inhibition and 786-O cell proliferation inhibition. On the other hand, miR-302c-3p depletion by expressing its anti-sense (“antagomiR-302c”) led to Gab2 upregulation, Akt activation and increased 786-O cell proliferation. Significantly, miR-302c-3p failed to affect the proliferation of 786-O cells with shRNA-depleted Gab2. Together, we suggest that miR-302c-3p depletion in human RCC cells leads to Gab2 over-expression, Akt hyper-activation and cell proliferation.

Expression of SET8 in renal cell carcinoma and the effects of SET8 silencing on proliferation and migration of 786-O cells

Objective To investigate the expression of lysine methyltransferase SET8 protein in the clear cell carcinoma of kidney and the effects of silencing SET8 gene by shRNA on proliferation and migration of human renal cell carcinoma cell line 786-O in vitro. Methods Immunohistochemistry(IHC) was employed to detect the expression of SET8 in 156 tissue samples of clear cell renal cell carcinoma in the study between December, 2006 and December, 2011.Four pairs of SET8-shRNA were designed and synthesized. Transfection was performed with cationic lipid vectors(LipofectamineTM2000). The transfection efficiency was observed by fluorescence confocal microscopy. The effecive shRNA sequences were screened by Western blot. After transfection with effective shRNA sequences, 786-O cells were divided into 3 groups: (1)SET8-shRNA transfection; (2)negative control transfection; (3)blank control.The assays of methyl thiazolyl tetrazolium(MTT), colony formation and scratch wound were applied to examine variations in cell proliferation, colony formation and migration. Results The positive rate of SET8 expression was 98.7%(154/156), and its expression was closely associated with TNM staging, the size of tumor and lymph node metastasis(all P<0.05). The transfection efficiency was around 70% with a concentration of SET8-shRNA 2μg/L and lipofectamine 2000 4μl/well. SET8-shRNA2 was chosen as the effective sequences with a suppression ratio up to 64%. At 48h post-transfection, the expression of SET8 protein was significantly lower in SET8-shRNA2 group than that in negative control transfection group and blank control group(t=-38.174, P<0.01; t=-58.766, P<0.01). While SET8 gene was silenced by shRNA in SET8-shRNA transfection group, the proliferation, colony formation and migration ability were decreased markedly. Conclusion SET8 protein plays a significant role in the carcinogenesis and progression of renal cell carcinoma. SET8-shRNA can effectively inhibit the expression of SET8 protein in 786-O cells and thus suppress malignant phenotypes of RCC. Key words: Lysine methyltransferaseSET8; Clear cell renal cell carcinoma; 786-O cell; Cell proliferation; Colony formation; Migration