CircCYP24A1 hampered malignant phenotype of renal cancer carcinoma through modulating CMTM-4 expression via sponging miR-421

Renal cell carcinoma (RCC) is a lethal urinary malignancy. Circular RNAs (circRNAs) contribute to the malignant phenotype and progression of several types of human cancers, including RCC. In this study, we identified relatively low hsa_circ_0060927 (circCYP24A1) expression in RCC tissue through high-throughput sequencing and RT–qPCR. Fluorescence in situ hybridization (FISH) was used to validate the expression and subcellular localization of circCYP24A1 in RCC tissues. CCK-8, Transwell, EdU, and wound-healing assays indicated that circCYP24A1 overexpression inhibited the proliferation, invasion, and migration of RCC cells. Dual-luciferase reporter, RNA immunoprecipitation (RIP), FISH, and RNA-pulldown assays verified that circCYP24A1 inhibited RCC progression by sponging miR-421, thus inducing CMTM-4 expression. Xenograft assays and metastasis models further indicated that circCYP24A1 significantly inhibited the metastasis and proliferation of RCC cells in vivo. Taken together, circCYP24A1 is a prognosis-related circRNA in RCC that functions through the circCYP24A1/miR-421/CMTM-4 axis to modulate RCC progression.Subject terms: Renal cell carcinoma, Cancer metabolism     

SNHG12 promotes carcinogenesis of human renal cell cancer via functioning as a competing endogenous RNA and sponging miR-30a-3p

Small nucleolar RNA host gene 12 (SNHG12) has been indicated in the tumorigenesis of various human cancers, including clear cell renal cell carcinoma (ccRCC). However, the underlying mechanisms of SNHG12 driving progression of ccRCC remain incompletely understood. In the present study, we discovered that SNHG12 is up-regulated in ccRCC and that overexpression of SNHG12 predicted poor clinical outcome of ccRCC patients. SNHG12 knockdown notably inhibited proliferation and migration of RCC cells. Furthermore, we discovered that miR-30a-3p, a putative ccRCC inhibitor, was competitively sponged by SNHG12. Via the crosstalk network, SNHG12 was capable of up-regulating multiple target genes of miR-30a-3p, namely, RUNX2, WNT2 and IGF-1R, which have been identified to facilitate tumorigenesis of ccRCC. Taken together, our present study suggested a novel ceRNA network, in which SNHG12 could promote the malignancy of ccRCC although competitively binding with miR-30a-3p and consequently release the expression of its downstream cancer-related genes.     

KIF20B promotes the progression of clear cell renal cell carcinoma by stimulating cell proliferation

Renal cell carcinoma (RCC) is a common urinary system cancer with high morbidity and mortality rate. Clear cell renal cell carcinoma (ccRCC) is a highly aggressive and common type of RCC. More and effective therapeutic targets are badly needed for the treatment of ccRCC. Kinesin family protein (KIF)20B, also named M-phase phosphoprotein 1, was reported as a microtubule-associated, plus-end-directed kinesin. KIF20B was involved in multiple cellular processes such as cytokinesis. Multiple studies indicated the oncogenic role for KIF20B in several types of tumors, including breast cancer and bladder cancer. However, the possible role of KIF20B in the progression of renal carcinoma is still unknown. Herein, our study demonstrated that KIF20B was relatively highly expressed in ccRCC tissues. In addition, KIF20B was inversely related to the clinical features including tumor size and T stage. We further found that inhibition of the KIF20B expression by a specific short hairpin RNA obviously reduces proliferation of ccRCC cells both in vitro and in vivo. Our study reveals the involvement of KIF20B in ccRCC progression. Generally, KIF20B is a promising novel therapeutic for the treatment of clear cell RCC.     

Circular RNA circPPP6R3 upregulates CD44 to promote the progression of clear cell renal cell carcinoma via sponging miR-1238-3p

Circular RNAs (circRNAs) are a type of covalently closed circular-formed RNAs and play crucial roles in the oncogenesis and progression of various human cancers. Here we identified a novel circRNA, circPPP6R3, to be highly expressed both in clear cell renal cell carcinoma (ccRCC) tissues and cell lines based on analyzing high-throughput sequencing data and qRT-PCR analysis. Highly expressed circPPP6R3 was positively correlated with higher histological grade, T stage, and M stage as well as advanced clinical stage of ccRCC patients. Functionally, knockdown of circPPP6R3 attenuated the proliferation, migration, and invasion of ccRCC cells whereas overexpression had the reverse effects. Mechanistically, the biotin-labeled pull-down assay and dual-luciferase reporter assay revealed that circPPP6R3 directly interacted with miR-1238-3p. miR-1238-3p inhibitors had a rescue effect on the proliferative and metastatic capacities by knockdown of circPPP6R3. Moreover, RNA-sequencing analysis and dual-luciferase reporter assay indicated that circPPP6R3 upregulated CD44, a cell-surface glycoprotein contributed to the cell adhesion and metastasis, via sponging to miR-1238-3p. Further investigation revealed that MMP9 and Vimentin were regulated by CD44 in ccRCC. Our study thus provided evidence that the regulatory network involving circPPP6R3/miR-1238-3p/CD44 axis might provide promising biomarkers as well as a therapeutic approach for ccRCC.Subject terms: Tumour biomarkers, Renal cell carcinoma, Epithelial-mesenchymal transition, Non-coding RNAs     

Circ_RPL23A acts as a miR-1233 sponge to suppress the progression of clear cell renal cell carcinoma by promoting ACAT2

Clear cell renal cell carcinoma (ccRCC) is a prevalent urological carcinoma with high metastatic risk. Circular RNAs (circRNAs) have been identified as effective diagnostic and therapeutic biomarkers for ccRCC. This research aims to disclose the effect and regulatory mechanism of circRNA ribosomal protein L23a (circ_RPL23A) in ccRCC. We performed quantitative real-time polymerase chain reaction (qRT-PCR) to examine circ_RPL23A, microRNA-1233 (miR-1233) and acetyl-coenzyme A acetyltransferase 2 (ACAT2). Cell cycle progression, apoptosis, cell viability, invasion and migration, which were respectively conducted by using flow cytometry, 3-(4, 5-dimethylthiazol-2-y1)-2, 5-diphenyl tetrazolium bromide (MTT), transwell assays. The levels of ACAT2 protein and cell cycle proteins, proliferation-associated protein, and epithelial-mesenchymal transition (EMT) associated proteins were measured by western blot. Target relationship was analyzed via dual-luciferase reporter assay and RNA pull down assay. The animal model was used to study how circ_RPL23A affects in vivo. Circ_RPL23A was lower expressed in ccRCC tissues and cells. The elevated circ_RPL23A suppressed cell cycle progression, proliferation, migration and invasion but promoted apoptosis in ccRCC cells. MiR-1233 was a target of circ_RPL23A and direct targeted to ACAT2. Besides, circ_RPL23A exerted its anti-tumor effect by sponging miR-1233, and then relieved the inhibition effect of miR-1233 on ACAT2. Overexpression of circ_RPL23A also curbed ccRCC tumor growth in vivo. Circ_RPL23A inhibited ccRCC progression by upregulating ACAT2 expression by competitively binding miR-1233, which might provide an in-depth cognition for ccRCC pathogenesis and circ_RPL23A might be a promising biomarker in ccRCC diagnosis and treatment.

     

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.     

Circular RNA circABCC4 regulates lung adenocarcinoma progression via miR-3186-3p/TNRC6B axis

Lung adenocarcinoma (LUAD), a general kind of bronchogenic malignancy globally, is depicted as one of the most critical factors affecting human health severely. Featured with loop structure, circular RNA (circRNA) has been described as an essential regulator of multiple human malignancies. Nevertheless, knowledge concerning the regulatory function of circRNA in LUAD progression remains limited. Identified as a novel circRNA, circABCC4 has not been studied in LUAD as yet. This is the first time to probe into the underlying role of circABCC4 in LUAD. In this study, a notably elevated expression of circABCC4 was found in LUAD tissues and cells. Besides, circABCC4 is verified to be characterized with a circular structure in LUAD. Functional assays elucidated that knockdown of circABCC4 significantly impaired LUAD cell proliferation, migration as well as accelerated cell apoptosis. Molecular mechanism experiments later revealed that circABCC4 could bind with miR-3186-3p and miR-3186-3p was a tumor suppressor in LUAD. Moreover, TNRC6B was validated to combine with miR-3186-3p, and its expression was respectively negatively and positively regulated by miR-3186-3p and circABCC4 in LUAD. Final rescue experiments further delineated that TNRC6B upregulation partially restored circABCC4 downregulation-mediated effect on LUAD progression. In sum, circABCC4 regulates LUAD progression via miR-3186-3p/TNRC6B axis.     

Circular RNA circ-EGLN3 promotes renal cell carcinoma proliferation and aggressiveness via miR-1299-mediated IRF7 activation

Renal cell carcinoma (RCC) is a highly lethal cancer with increasing incidence worldwide. The purpose of the present study was to investigate the functions and molecular mechanisms of circular RNA (circRNA), circ-EGLN3, in RCC progression. The expression levels of circ-EGLN3 were assessed by a quantitative real-time polymerase chain reaction. Kaplan-Meier analysis was applied to uncover the prognostic role of circ-EGLN3 in patients with RCC. Cell viability was analyzed using cell counting kit-8 and cell apoptosis was assessed using flow cytometric experiment. Cell migratory and invasive abilities were determined by wound scratch and transwell experiments. Subcellular distribution detection was utilized to investigate the location of circ-EGLN3. Dual-luciferase reporter test was utilized for identifying the molecular mechanism of circ-EGLN3. The results indicated that circ-EGLN3 was elevated in RCC tissues and cell lines and predicted unfavorable prognosis for the patients with RCC. Silenced circ-EGLN3 hindered cell proliferation, migration, and invasion but facilitated apoptosis of RCC cells. Ectopic expressed circ-EGLN3 induced the opposite effects mentioned above. Mechanistically, circ-EGLN3 was mainly located at the cytoplasm. Circ-EGLN3 acted as a competing endogenous RNA (ceRNA) to enhance the IRF7 level via sponging miR-1299. Moreover, circ-EGLN3 mediated elevation of IRF7 is responsible for RCC cell proliferation and aggressiveness. Collectively, our study suggested that circ-EGLN3 knockdown suppressed RCC progression through acting as a ceRNA to regulate the IRF7 expression by targeting miR-1299. Circ-EGLN3 might be a potential therapeutic target for RCC management.     

Up-regulated circBACH2 contributes to cell proliferation,invasion, and migration of triple-negative breast cancer

An increasing amount of evidence has proven the vital role of circular RNAs (circRNAs) in cancer progression. However, there remains a dearth of knowledge on the function of circRNAs in triple-negative breast cancer (TNBC). Utilizing a circRNA microarray dataset, four circRNAs were identified to be abnormally expressed in TNBC. Among them, circBACH2 was most significantly elevated in TNBC cancerous tissues and its high expression was positively correlated to the malignant progression of TNBC patients. In normal human mammary gland cell line, the overexpression of circBACH2 facilitated epithelial to mesenchymal transition and cell proliferation. In TNBC cell lines, circBACH2 knockdown suppressed the malignant progression of TNBC cells. Mechanistically, circBACH2 sponged miR-186-5p and miR-548c-3p, thus releasing the C-X-C chemokine receptor type 4 (CXCR4) expression. The interference of miR-186-5p/miR-548c-3p efficiently promoted the cell proliferation, migration, and invasion suppressed by circBACH2 knockdown in the TNBC cell lines. Finally, circBACH2 knockdown repressed the growth and lung metastasis of TNBC xenografts in nude mice. In summary, circBACH2 functions as an oncogenic circRNA in TNBC through a novel miR-186-5p/miR-548c-3p/CXCR4 axis.Subject terms: Cancer, Cell biology     

CircRNA fibroblast growth factor receptor 3 promotes tumor progression in non-small cell lung cancer by regulating Galectin-1-AKT/ERK1/2 signaling

The dysregulation of circular RNA (circRNA) expression is involved in the progression of several cancers, including non-small cell lung cancer (NSCLC). However, the role and underlying molecular mechanisms of circRNA FGFR3 (circFGFR3) in NSCLC progression remains unknown. Here, we used quantitative real-time polymerase chain reaction to validate that circFGFR3 expression was higher in NSCLC tissues than in the paratumor tissues. Furthermore, our study indicated that the forced circFGFR3 expression promoted NSCLC cell invasion and proliferation. Mechanistically, we found that circFGFR3 promoted NSCLC cell invasion and proliferation via competitively combining with miR-22-3p to facilitate the galectin-1 (Gal-1), p-AKT, and p-ERK1/2 expressions. Clinically, we revealed that the high circFGFR3 expression correlates with the poor clinical outcomes in patients with NSCLC. Together, these data provide mechanistic insights into the circFGFR3-mediated regulation of both the AKT and ERK1/2 signaling pathways by sponging miR-22-3p and increasing Gal-1 expression.     

Pseudogene HSPB1P1 contributes to renal cell carcinoma proliferation and metastasis by targeting miR-296-5p to regulate HMGA1 expression

With the aid of next-generation sequencing technology, pseudogenes have been widely recognized as functional regulators in the development and progression of certain diseases, especially cancer. Our present study aimed to investigate the functions and molecular mechanisms of HSPB1-associated protein 1 pseudogene 1 (HSPB1P1) in renal cell carcinoma (RCC). HSPB1P1 expression at the mRNA levels was determined by quantitative real-time polymerase chain reaction, and its clinical significance was assessed. Cell viability was detected by Cell Counting Kit-8 assay. Cell migration and invasion were detected by transwell assays. The location of HSPB1P1 in RCC cells was detected by subcellular distribution analysis. The direct relationship between HSPB1P1 and miR-296-5p/HMGA1 axis was verified by dual-luciferase reporter assay and RNA immunoprecipitation assay. Our results identify the elevated expression of HSPB1P1 in RCC tissues and cell lines, which predicted advanced progression and poor prognosis in patients with RCC. Knockdown of HSPB1P1 suppressed cell proliferation, migration, and invasion, and reversed epithelial–mesenchymal transition process in RCC. HSPB1P1 was mostly enriched in the cytoplasm and functioned as a miRNA sponge for miR-296-5p and then regulated high-mobility group A1 expression. In conclusion, our study indicated that HSPB1P1 contributed to RCC progression by targeting the miR-296-5p/HMGA1 axis, and should be considered as a promising biomarker and therapeutic target for clinical applications.     

microRNA-505 negatively regulates HMGB1 to suppress cell proliferation in renal cell carcinoma

microRNAs have been recognized to regulate a wide range of biology of renal cell carcinoma (RCC). Although miR-505 has been reported to play as a suppressor in several human tumors, the physiological function of miR-505 in RCC still remain unknown. Therefore, the role of miR-505 and relevant regulatory mechanisms were investigated in RCC in this study. Quantitative real-time polymerase chain reaction was conducted to detect the expression of miR-505 and high mobility group box 1 (HMGB1) in both RCC tissues and cell lines. Immunohistochemical staining was used to assess the correlation between HMGB1 expression and PCNA expression in RCC tissues. Subsequently, the effects of miR-505 on proliferation were determined in vitro using cell counting kit-8 proliferation assays and 5-ethynyl-2′-deoxyuridine incorporation. The molecular mechanism underlying the relevance between miR-505 and HMGB1 was confirmed by luciferase assay. Xenograft tumor formation was used to reflect the proliferative capacity of miR-505 in vivo experiments. Overall, a relatively lower miR-505 and higher HMGB1 expression in RCC specimens and cell lines were found. HMGB1 was verified as a direct target of miR-505 by luciferase assay. In vitro, overexpression of miR-505 negatively regulates HMGB1 to suppress the proliferation in Caki-1; meanwhile, knock-down of miR-505 negatively regulates HMGB1 to promote the proliferation in 769P. In addition, in vivo overexpression of miR-505 could inhibit tumor cell proliferation in RCC by xenograft tumor formation. Therefore, miR-505, as a tumor suppressor, negatively regulated HMGB1 to suppress the proliferation in RCC, and might serve as a novel therapeutic target for RCC clinical treatment.     

Oncofetal H19 RNA promotes tumor metastasis

The oncofetal H19 gene transcribes a long non-coding RNA(lncRNA) that is essential for tumor growth. Here we found that numerous established inducers of epithelial to mesenchymal transition(EMT) also induced H19/miR-675 expression. Both TGF-β and hypoxia concomitantly induced H19 and miR-675 with the induction of EMT markers. We identified the PI3K/AKT pathway mediating the inductions of Slug, H19 RNA and miR-675 in response to TGF-β treatment, while Slug induction depended on H19 RNA. In the EMT induced multidrug resistance model, H19 level was also induced. In a mouse breast cancer model, H19 expression was tightly correlated with metastatic potential. In patients, we detected high H19 expression in all common metastatic sites tested, regardless of tumor primary origin. H19 RNA suppressed the expression of E-cadherin protein. H19 up-regulated Slug expression concomitant with the suppression of E-cadherin protein through a mechanism that involved miR-675. Slug also up-regulated H19 expression and activated its promoter. Altogether, these results may support the existence of a positive feedback loop between Slug and H19/miR-675, that regulates E-cadherin expression. H19 RNA enhanced the invasive potential of cancer cells in vitro and enhanced tumor metastasis in vivo. Additionally, H19 knockdown attenuated the scattering and tumorigenic effects of HGF/SF. Our results present novel mechanistic insights into a critical role for H19 RNA in tumor progression and indicate a previously unknown link between H19/miR-675, Slug and E-cadherin in the regulation of cancer cell EMT programs.     

HAO2 inhibits malignancy of clear cell renal cell carcinoma by promoting lipid catabolic process

Hydroxy acid oxidase 2 (HAO2) has been reported to inhibit tumor progression through metabolic pathway. The current study was designed to evaluate the prognostic significance and probable mechanism of HAO2 in patients with clear cell renal cell carcinoma (ccRCC). The study screened The Cancer Genome Atlas Kidney Clear Cell Carcinoma (TCGA-KIRC) database for patients with ccRCC having complete clinical information and HAO2 expression. Low HAO2 was associated with shorter overall survival (OS) and shorter disease-free survival (DFS). Gene set enrichment analysis (GSEA) showed HAO2 was associated with neutral lipid catabolic process, metabolic process, lipid oxidation, epithelial–mesenchymal transition (EMT), and Kirsten rat sarcoma viral oncogene signaling (KRAS). Western blot analysis and immunohistochemistry analysis checked HAO2 expression in ccRCC cancer tissues, normal tissues, and renal cancer cell lines. HAO2 was downregulated in ccRCC cancer tissues and ccRCC cell lines when compared with their control group. Overexpression of HAO2 by plasmid promoted lipid catabolic process, eliminated lipid accumulation, inhibited KRAS expression, controlled the proliferation, migration, and invasion activity of ccRCC tumor cells. Our results indicated that HAO2 inhibits malignancy ccRCC by promoting lipid catabolic process, HAO2 could be an effective molecular marker and treatment for ccRCC.