Serum exosomal miR-210 as a potential biomarker for clear cell renal cell carcinoma

Exosomal microRNAs (miRNAs) are suggested to reflect molecular changes occurring in their cells of origin and are potential indicators in the early detection of cancers. This study aimed to determine whether certain exosomal miRNAs from tumor tissue can be used as noninvasive biomarkers for clear cell renal cell carcinoma (ccRCC). Based on ccRCC miRNA expression profiles and the literature, we selected six miRNAs (miR-210, miR-224, miR-452, miR-155, miR-21, and miR-34a) and analyzed their expression in tissues, sera, and serum exosomes through quantitative real-time polymerase chain reaction in hypoxia-induced (with CoCl₂) renal cell lines. miR-210, miR-224, miR-452, miR-155, and miR-21 were upregulated in tumor tissues compared with normal tissues. Serum miR-210 and miR-155 levels were higher in patients with ccRCC than in healthy controls (HCs). Furthermore, only exosomal miR-210 was significantly upregulated in patients with ccRCC than in HCs. Moreover, receiver operating characteristic (ROC) curve analysis revealed an area under the ROC curve of 0.8779 (95% confidence interval, 0.7987-0.9571) and a sensitivity and specificity of 82.5% and 80.0%, respectively. Moreover, exosomal miR-210 was upregulated at an advanced stage, and Fuhrman grade and metastasis decreased significantly one month after surgery. Acute hypoxia exposure activates miR-210 and release of exosomes with upregulated miR-210 in both normal and tumor RCC cell lines and interferes with vacuole membrane protein 1 mRNA expression, especially in the metastatic ccRCC cell line. In conclusion, Serum exosomal miR-210 originating from tumor tissue has potential as a novel noninvasive biomarker for the detection and prognosis of ccRCC.     

Circular RNA circDVL1 inhibits clear cell renal cell carcinoma progression through the miR-412-3p/PCDH7 axis

Clear cell renal cell carcinoma (ccRCC) is a primary kidney cancer with high aggressive phenotype and extremely poor prognosis. Accumulating evidence suggests that circular RNAs (circRNAs) play pivotal roles in the occurrence and development of various human cancers. However, the expression, clinical significance and regulatory role of circRNAs in ccRCC remain largely unclear. Here we report that circDVL1 to be reduced in the serums and tissues from ccRCC patients, and to negatively correlate with ccRCC malignant features. Overexpression of circDVL1 inhibits proliferation, induces G1/S arrest, triggers apoptosis, and reduces migration and invasion in different ccRCC cells in vitro. Correspondingly, circDVL1 overexpression suppresses ccRCC tumorigenicity in a mouse xenograft model. Mechanistically, circDVL1 serves as a sponge for oncogenic miR-412-3p, thereby preventing miR-412-3p-mediated repression of its target protocadherin 7 (PCDH7) in ccRCC cells. Collectively, our results demonstrate that circDVL1 exerts tumor-suppressive function during ccRCC progression through circDVL1/miR-412-3p/PCDH7 axis, and suggest that circDVL1 could be a novel diagnostic and prognositc marker and therapeutic target for ccRCC.     

miR-34a-5p对三阴性乳腺癌细胞的影响及相关机制研究*

目的: 探讨miR-34a-5p在三阴性乳腺癌(triple negative breast cancer,TNBC)中的表达,分析miR-34a-5p对TNBC细胞增殖、凋亡、迁移的作用,对TNBC荷瘤小鼠肿瘤生长的影响以及在TNBC中对B7-H1表达的影响。方法: 利用RT-qPCR、Western blot分析TNBC细胞中miR-34a-5p、B7-H1的表达,并利用Kaplan-Meier分析二者的表达与TNBC患者的生存关系;将miR-34a-5p转染TNBC细胞,通过CCK-8、流式细胞术及划痕实验检测miR-34a-5p对TNBC细胞增殖、凋亡、迁移的影响;利用RT-qPCR、Western blot检测miR-34a-5p、B7-H1表达水平的变化,双荧光素酶基因报告验证miR-34a-5p与B7-H1的相互作用;利用RT-qPCR、Western blot、IHC检测miR-34a-5p对MDA-MB-231荷瘤小鼠miR-34a、B7-H1表达的影响。结果: TNBC细胞中miR-34a-5p呈低表达,B7-H1呈高表达,二者均与TNBC患者的不良预后有关,差距具有统计学意义(P<0.01);miR-34a-5p抑制TNBC细胞增殖、侵袭,促进细胞凋亡,并且在TNBC细胞中靶向抑制B7-H1;miR-34a-5p agomir在体内抑制MDA-MB-231成瘤裸鼠的肿瘤生长和B7-H1表达。结论: miR-34a-5p在TNBC发生、发展中发挥着重要作用,靶向miR-34a-5p/B7-H1可能成为TNBC患者新的分子治疗策略。     

Down-Regulated miR-30a in Clear Cell Renal Cell Carcinoma Correlated with Tumor Hematogenous Metastasis by Targeting Angiogenesis-Specific DLL4

Background

Endothelial DLL4 plays an important role in controlling of tumor angiogenesis, which is required for tumor invasive growth and metastasis. However, the regulation of DLL4 in clear cell renal cell carcinoma (ccRCC) has not yet been systematically elucidated.

Methodology

We performed bioinformatical analysis to explore miRNAs targeting DLL4. miR-30a was selected as a representative to validate its functional association in endothelial cell. Then, the expressions of DLL4 and mature miR-30a from 90 cases of ccRCC and 28 cases of nonmatched adjacent non-tumor tissues were measured by quantitative real-time PCR. Finally, the expression of miR-30a was correlated with DLL4 expression, tumor features (metastatic condition and microvessel density), and patient metastasis-free survival. The univariate and multivariate analyses were performed to select the risk factors associated with hematogenous metastasis, respectively.

Principal Findings

miR-30a negatively regulated DLL4 and inhibited the proliferation and migration of endothelial cells. DLL4 was up-regulated in ccRCC and further increased in hematogenous metastatic cases, while miR-30a was down-regulated in tumor tissues and further decreased in hematogenous metastatic ccRCC (student t test, all p<0.05). Additionally, expression of miR-30a was inversely correlated with expression of DLL4 and microvessel density (linear correlation analysis, both p<0.05). Low-level miR-30a also indicated a higher probability of developing metastasis (log-rank test, p = 0.010). Most importantly, miR-30a expression was an independent predictor of ccRCC hematogenous metastasis by the univariate analysis and binary logistic regression model (both p<0.05).

Conclusions

Down-regulated miR-30a in ccRCC was associated with tumor hematogenous metastasis through increasing microvessel density by targeting angiogenesis-specific DLL4.     

B7-H4 expression associates with cancer progression and predicts patient’s survival in human esophageal squamous cell carcinoma

A retrospective cohort study including 112 patients suffering from esophageal squamous cell carcinoma (ESCC) was performed to investigate the expression of B7-H4 in ESCC and determine its association with patient’s clinicopathological parameters and survival. Expression levels of B7-H4 on tumor cells and densities of tumor infiltrating lymphocytes (TILs) in the surgical specimens of ESCC tissues were characterized using immunohistochemical assays. Uni- and multivariate analyses were performed to evaluate the prognostic value of B7-H4 expression levels and densities of TILs in tumor sections. Positive B7-H4 immunostaining was observed in 107 of 112 (95.5%) of ESCC tissue sections. We further divided all patients into two major subgroups, a lower B7-H4 expression group with 46 patients and a higher B7-H4 expression group with 66 patients. We found that expression levels of B7-H4 on tumor cells were significantly correlated with patient’s gender (P = 0.0288), distant metastasis (P = 0.0500), and TNM stage (P = 0.0258). Moreover, tumor cell B7-H4 expression was inversely correlated with densities of CD3⁺ T cells in tumor nest (P = 0.0424) and CD8⁺ T cells in tumor stroma (P = 0.0229). The overall survival rate of the patients with higher B7-H4 expression was significantly worse than that of the patients with lower B7-H4 expression (P = 0.0105, Hazard Ratio: 1.854, 95%CI:1.152–2.902). Markers of cell-mediated immune responses such as CD3, CD8, and T-bet were associated with better patient survival. The present study demonstrated that B7-H4 expression in human ESCC is associated with cancer progression, reduced tumor immunosurveillance and worse patient outcomes. B7-H4 can serve as a novel prognostic predictor for human ESCC and a potential target for the immune therapy against this malignancy.     

Overexpression of B7-H1 (PD-L1) significantly associates with tumor grade and postoperative prognosis in human urothelial cancers

PURPOSE: The programmed death-1 (PD-1)/B7-H1 (also called PD-L1) pathway negatively regulates T cell activation and has been suggested to play an important role in regulating antitumor host immunity. To investigate the clinical significance of B7-H1 expression to the tumor grade and postoperative prognosis of patients with urothelial cancer, we analyzed the relationship between B7-H1 expression and various clinicopathological features and postoperative prognosis. EXPERIMENTAL DESIGN: Sixty-five urothelial cancer cases were examined. B7-H1 expression in tumors and the numbers and phenotypes of tumor-infiltrating lymphocytes were evaluated by immunohistochemistry and flow cytometry. RESULTS: A substantial expression of B7-H1 was observed in all urothelial cancers investigated. Tumor specimens from patients with higher WHO grade or primary tumor classifications showed significantly higher percentages of tumor-associated B7-H1. Tumor-associated B7-H1 expression was significantly associated with a high frequency of postoperative recurrence and poor survival rate. Furthermore, multivariate analysis indicated that tumor-associated B7-H1 was more significant prognostic factor than WHO grade. CONCLUSIONS: Our results demonstrate that the aberrant expression of B7-H1 in urothelial cancer is associated with aggressive tumors, suggesting a regulatory role of tumor-associated B7-H1 in antitumor immunity. Therefore, the manipulation of tumor-associated B7-H1 may become a beneficial target for immunotherapy in human urothelial cancer.     

Impact of miR-21, miR-126 and miR-221 as Prognostic Factors of Clear Cell Renal Cell Carcinoma with Tumor Thrombus of the Inferior Vena Cava

Clear cell renal cell carcinoma (ccRCC) characterized by a tumor thrombus (TT) extending into the inferior vena cava (IVC) generally indicates poor prognosis. Nevertheless, the risk for tumor recurrence after nephrectomy and thrombectomy varies. An applicable and accurate prediction system to select ccRCC patients with TT of the IVC (ccRCC/TT) at high risk after nephrectomy is urgently needed, but has not been established up to now. To our knowledge, a possible role of microRNAs (miRs) for the development of ccRCC/TT or their impact as prognostic markers in ccRCC/TT has not been explored yet. Therefore, we analyzed the expression of the previously described onco-miRs miR-200c, miR-210, miR-126, miR-221, let-7b, miR-21, miR-143 and miR-141 in a study collective of 74 ccRCC patients. Using the expression profiles of these eight miRs we developed classification systems that accurately differentiate ccRCC from non-cancerous renal tissue and ccRCC/TT from tumors without TT. In the subgroup of 37 ccRCC/TT cases we found that miR-21, miR-126, and miR-221 predicted cancer related death (CRD) accurately and independently from other clinico-pathological features. Furthermore, a combined risk score based on the expression of miR-21, miR-126 and miR-221 was developed and showed high sensitivity and specificity to predict cancer specific survival (CSS) in ccRCC/TT. Using the combined risk score we were able to classify ccRCC/TT patients correctly into high and low risk cases. The risk stratification by the combined risk score (CRS) will benefit from further cohort validation and might have potential for clinical application as a molecular prediction system to identify high- risk ccRCC/TT patients.     

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.     

Identification of a three-miRNA signature as a novel potential prognostic biomarker in patients with clear cell renal cell carcinoma

Current studies suggest that some microRNAs (miRNAs) are associated with prognosis in clear cell renal cell carcinoma (ccRCC). In this paper, we aimed to identify a miRNAs signature to improve prognostic prediction for ccRCC patients. Using ccRCC RNA-Seq data of The Cancer Genome Atlas (TCGA) database, we identified 177 differentially expressed miRNAs between ccRCC and paracancerous tissue. Then all the ccRCC tumor samples were divided into training set and validation set randomly. Three-miRNA signature including miR130b, miR-18a, and miR-223 were constructed by the least absolute shrinkage and selection operator (LASSO) Cox regression model in training set. According to optimal cut-off value of three-miRNA signature risk score, all the patients could be classified into high-risk group and low-risk group significantly. Survival of patients was significantly different between two groups (hazard ratio, 5.58, 95% confidence interval, 3.17-9.80; P < 0.0001), and three-miRNA signature performed favorably prognostic and predictive accuracy. The results were further validated in the validation set and total set. Multivariate Cox regression analyses and subgroup analyses showed that three-miRNA signature was an independent prognostic factor. Two nomograms that integrated three-miRNA signature and three clinicopathological risk factors were constructed to predict overall survival and disease-free survival after surgery for ccRCC patients. Functional enrichment analysis showed the possible roles of three-miRNA signature in some cancer-associated biological processes and pathways. In conclusion, we developed a novel three-miRNA signature that performed reliable prognostic for patient survival with ccRCC, it might facilitate ccRCC patients counseling and individualize management.     

Circ_0101692 knockdown retards the development of clear cell renal cell carcinoma through miR-384/FN1 pathway

PurposeCircular RNA_0101692 (circ_0101692) is overexpressed in clear cell renal cell carcinoma (ccRCC) by microarray analyses. However, its function and action mechanism in ccRCC tumorigenesis is still elusive.MethodsWestern blotting and qRT-PCR were executed to assess the circ_0101692, miR-384 and FN1 expression in ccRCC cells and tissues. Target relationships among them were determined via dual luciferase reporter and/or RNA immunoprecipitation assays. Cell proliferation was evaluated by CCK-8 assay. Caspase-3 activity assay was utilized to analyze cell apoptosis. To find out whether ccRCC cells might migrate, a transwell assay was performed. To assess the effects of circ_0101692 on tumor development in vivo, a mouse xenograft model was used.ResultsHigh expression of circ_0101692 and FN1, and decreased miR-384 were determined in ccRCC. Cell growth, migration and viability were decreased whereas cell apoptosis was stimulated when circ_0101692 was knockdown. miR-384 inhibitor transfection attenuated the inhibiting impacts of circ_0101692 silencing on ccRCC cell progression. FN1 deletion further inverted the cancer-promoting effect of miR-384 downregulation on cell viability and migration. In addition, circ_0101692 could sponge miR-384 to relieve the inhibition of miR-384 on FN1 in ccRCC.ConclusionsCirc_0101692 targeted miR-384/FN1 axis to facilitate cell proliferation, migration and repress apoptosis, thereby accelerating the development of ccRCC. This points out that circ_0101692/miR-384/FN1 axis might be a prospective target implemented for the future treatment of ccRCC.     

miR-219-5p targets TBXT and inhibits breast cancer cell EMT and cell migration and invasion

miR-219-5p has been reported to act as either a tumor suppressor or a tumor promoter in different cancers by targeting different genes. In the present study, we demonstrated that miR-219-5p negatively regulated the expression of TBXT, a known epithelial–mesenchymal transition (EMT) inducer, by directly binding to TBXT 3′-untranslated region. As a result of its inhibition on TBXT expression, miR-219-5p suppressed EMT and cell migration and invasion in breast cancer cells. The re-introduction of TBXT in miR-219-5p overexpressing cells decreased the inhibitory effects of miR-219 on EMT and cell migration and invasion. Moreover, miR-219-5p decreased breast cancer stem cell (CSC) marker genes expression and reduced the mammosphere forming capability of cells. Overall, our study highlighted that TBXT is a novel target of miR-219-5p. By suppressing TBXT, miR-219-5p plays an important role in EMT and cell migration and invasion of breast cancer cells.     

LncRNA GABPB1‑IT1 inhibits the tumorigenesis of renal cancer via the miR-21/PTEN axis

Long noncoding RNA (lncRNA) (GABPB1‑IT1) has been reported to be downregulated in lung cancer, while its expression and function in other cancers are unknown. In this study, the expression levels of GABPB1‑IT1 in tissue samples from 62 ccRCC patients were measured by performing RT-qPCR. Potential base pairing formed between GABPB1‑IT1 and miR-21 was explored using the online program IntaRNA 2.0 and further confirmed by Dual-luciferase activity assay and RNA pulldown assay. The role of GABPB1‑IT1 and miR-21 in regulating the expression of PTEN was evaluated by RT-qPCR and Western blot. The role of GABPB1‑IT1, miR-21, and PTEN in regulating the proliferation of Caki-2 cells was explored by CCK-8 assay. It was observed that GABPB1‑IT1 was downregulated in ccRCC and predicted poor survival. GABPB1‑IT1 directly interacted with miR-21, while it did not regulate the expression of each other. Moreover, upregulation of PTEN, which is a target of miR-21, was observed in ccRCC cells with overexpression of GABPB1‑IT1. Overexpression of GABPB1‑IT1 and PTEN decreased the proliferation rates of ccRCC cells. In addition, overexpression of GABPB1‑IT1 reduced the enhancing effects of miR-21 on cell proliferation. Therefore, GABPB1‑IT1 may upregulate PTEN by sponging miR-21 in ccRCC to inhibit cancer cell proliferation. Our study characterized a novel GABPB1‑IT1/miR-21/PTEN axis in ccRCC.     

Long non-coding RNA TUG1/microRNA-187-3p/TESC axis modulates progression of pituitary adenoma via regulating the NF-κB signaling pathway

The molecule mechanisms of long non-coding RNAs (lncRNAs) and microRNAs (miRNAs) in human diseases have been broadly studied recently, therefore, our research aimed to assess the effect of lncRNA taurine upregulated gene 1 (TUG1)/miR-187-3p/tescalcin (TESC) axis in pituitary adenoma (PA) by regulating the nuclear factor-kappa B (NF-κB) signaling pathway. We observed that TUG1 was upregulated in PA tissues and was associated with invasion, knosp grade and tumor size. TUG1 particularly bound to miR-187-3p. TUG1 knockdown inhibited cell proliferation, invasion, migration, and epithelial–mesenchymal transition, promoted apoptosis, and regulated the expression of NF-κB p65 and inhibitor of κB (IκB)-α in PA cells lines in vitro, and also inhibited tumor growth in vivo, and these effects were reversed by miR-187-3p reduction. Similarly, miR-187-3p elevation inhibited PA cell malignant behaviors and modulated the expression of NF-κB p65 and IκB-α in PA cells, and reduced in vivo tumor growth as well. TUG1 inhibition downregulated TESC, which was targeted by miR-187-3p. In conclusion, this study suggests that TUG1 sponges miR-187-3p to affect PA development by elevating TESC and regulating the NF-κB signaling pathway.Subject terms: Cell biology, Diseases     

Clinical significance and regulation of the costimulatory molecule B7-H3 in human colorectal carcinoma

B7-H3, a member of the B7-family molecules, plays an important role in adaptive immune responses, and was shown to either promote or inhibit T-cell responses in various experimental systems. B7-H3 was expressed in some human cancers and correlated with poor outcome of cancer patients. However, its exact role in cancer is not known. In the present study, we studied the expression of B7-H3 in the pathologic specimens of 102 patients treated for colorectal carcinoma (CRC) by immunohistochemistry. Strong B7-H3 expression was found in cancer tissues from 54.3% CRC patients, while minimal expression was found in adjacent normal colorectal tissues. Higher B7-H3 expression in tumor positively correlated with a more advanced tumor grade. In addition, consistent with a role of B7-H3 in suppressing tumor immune surveillance, the expression of B7-H3 in cancer cells negatively correlated with the intensity of tumor infiltrating T lymphocytes in both tumor nest and tumor stroma. Furthermore, we found that the level of soluble B7-H3 in sera from CRC patients was higher than healthy donors. TNF-α, an important cancer-promoting inflammatory molecule, was subsequently found to significantly increase the release of soluble B7-H3 in colon cancer cell lines. Therefore, our data suggest that both soluble and membranous B7-H3 proteins are involved in colon cancer progression and evasion of cancer immune surveillance.     

Hsa_circ_0003596 enhances the development of cell renal clear cell carcinoma through the miR-502-5p/IGF1/PI3K/AKT axis

Accumulating research findings have shown that circular RNAs (circRNAs) play an indispensable role in tumorigenesis and tumor progression. The current study aimed to explore the role and modulatory mechanism of hsa_circ_0003596 in clear cell renal cell carcinoma (ccRCC). Quantitative real-time polymerase chain reaction was adopted to detect the expression of hsa_circ_0003596 in ccRCC tissue and cell lines. 5-Ethynyl-2′-deoxyuridine, cell counting kit 8 and the colony formation assay were utilized to assess the proliferation potential of the ccRCC cells. Transwell along with wound healing assays were adopted to quantify infiltration coupled with the migration potential of the cells. The current research study found that the circRNA hsa_circ_0003596 was overexpressed in ccRCC tissue and cell lines. Further, result showed that hsa_circ_0003596 was associated with distant metastasis of renal cancer. Notably, the knockdown of hsa_circ_0003596 can lower the proliferation, infiltration and migration potential of ccRCC cells. The results of in vivo experiments found that the reduction of hsa_circ_0003596 significantly hampered the growth of tumors in mice. In addition, it was evident that hsa_circ_0003596 acts as a “molecular sponge” for miR-502-5p to upregulate the expression of the microRNA-502-5p (miR-502-5p) target insulin-like growth factor 1 (IGF1R). Furthermore, it was found that the phosphatidylinositol 3-kinase (PI3K)/AKT signaling was the downstream cascade of hsa_circ_0003596/miR-502-5p/IGF1R cascade, which is partly responsible for the cancer-promoting effect. Overall, the results of the present study showed that hsa_circ_0003596 facilitated the proliferation, infiltration and migration of ccRCC through the miR-502-5p/IGF1R/PI3K/AKT axis. Therefore, it was evident that hsa_circ_0003596 can serve as a possible biomarker and therapeutic target against ccRCC.     

Phosphoinositide-dependent kinase 1–associated glycolysis is regulated by miR-409-3p in clear cell renal cell carcinoma

Clear cell renal cell carcinoma (ccRCC) is the most popular kidney cancer in adults. Metabolic shift toward aerobic glycolysis is a fundamental factor for ccRCC therapy. MicroRNAs (miRNAs) are thought to be important regulators in ccRCC development and progression. Phosphoinositide-dependent kinase 1 (PDK1) is required for metabolic activation; however, the role of PDK1-induced glycolytic metabolism regulated by miRNAs is unclear in ccRCC. So, the purpose of the current study is to elucidate the underlying mechanism in ccRCC cell metabolism mediated by PDK1. Our results revealed that miR-409-3p inhibited glycolysis by regulating PDK1 expression in ccRCC cells. We also found that miR-409-3p was regulated by hypoxia. Our results indicated that PDK1 facilitated ccRCC cell glycolysis, regulated by miR-409-3p in hypoxia.