Downregulation of miR‐26 promotes invasion and metastasis via targeting interleukin‐22 in cutaneous T‐cell lymphoma

It has been reported that certain microRNAs (miRNA) are associated with the pathogenesis of lymphoma. We have previously demonstrated that histone deacetylase inhibitors restore tumor‐suppressive miRNAs, such as miR‐16, miR‐29, miR‐150, and miR‐26, in advanced cutaneous T‐cell lymphoma (CTCL). Among these, the function of miR‐26 remains unclear. In this study, we aimed to reveal the function of miR‐26 in CTCL oncogenesis. First, we confirmed that the miR‐26 family was markedly dysregulated in CTCL cell lines and primary samples. In vivo analysis using miR‐26a‐transduced CTCL cells injected into immunodeficient NOG mice demonstrated the significant prolonged survival of the mice, suggesting that the miRNA had a tumor‐suppressive function. We performed gene expression assays and identified 12 candidate miR‐26 targets, namely RGS13, FAM71F1, OAF, SNX21, CDH2, PTPLB, IL22, DNAJB5, CASZ1, CACNA1C, MYH10, and CNR1. Among these, IL22 was the most likely candidate target because the IL‐22–STAT3–CCL20–CCR6 cascade is associated with tumor invasion and metastasis of advanced CTCL. In vitro analysis of IL22 and IL22RA knockdown and miR‐26 transduction demonstrated inhibited CTCL cell migration. In particular, IL22 knockdown induced cell apoptosis. Finally, we conducted in vivo inoculation analysis of mice injected with shIL22‐transfected CTCL cells, and found no tumor invasion or metastasis in the inoculated mice, although the control mice showed multiple tumor invasions and metastases. These results, along with our previous data, demonstrated that miR‐26 is a tumor suppressor that is associated with tumor invasion and the metastasis of advanced CTCL by regulating the IL‐22–STAT3–CCL20 cascade. Therefore, a IL‐22‐targeting therapy could be a novel therapeutic strategy for advanced CTCL.     

microRNA‐99a‐5p induces cellular senescence in gemcitabine‐resistant bladder cancer by targeting SMARCD1

Patients with advanced bladder cancer are generally treated with a combination of chemotherapeutics, including gemcitabine, but the effect is limited due to acquisition of drug resistance. Thus, in this study, we investigated the mechanism of gemcitabine resistance. First, gemcitabine‐resistant cells were established and resistance confirmed in vitro and in vivo. Small RNA sequencing analyses were performed to search for miRNAs involved in gemcitabine resistance. miR‐99a‐5p, selected as a candidate miRNA, was downregulated compared to its parental cells. In gain‐of‐function studies, miR‐99a‐5p inhibited cell viabilities and restored sensitivity to gemcitabine. RNA sequencing analysis was performed to find the target gene of miR‐99a‐5p. SMARCD1 was selected as a candidate gene. Dual‐luciferase reporter assays showed that miR‐99a‐5p directly regulated SMARCD1. Loss‐of‐function studies conducted with si‐RNAs revealed suppression of cell functions and restoration of gemcitabine sensitivity. miR‐99a‐5p overexpression and SMARCD1 knockdown also suppressed gemcitabine‐resistant cells in vivo. Furthermore, β‐galactosidase staining showed that miR‐99a‐5p induction and SMARCD1 suppression contributed to cellular senescence. In summary, tumor‐suppressive miR‐99a‐5p induced cellular senescence in gemcitabine‐resistant bladder cancer cells by targeting SMARCD1.     

Long non‐coding RNA RACGAP1P promotes breast cancer invasion and metastasis via miR‐345‐5p/RACGAP1‐mediated mitochondrial fission

Long non‐coding RNAs (lncRNAs) are emerging as key molecules in various cancers, yet their potential roles in the pathogenesis of breast cancer are not fully understood. Herein, using microarray analysis, we revealed that the lncRNA RACGAP1P, the pseudogene of Rac GTPase activating protein 1 (RACGAP1), was up‐regulated in breast cancer tissues. Its high expression was confirmed in 25 pairs of breast cancer tissues and 8 breast cell lines by qRT‐PCR. Subsequently, we found that RACGAP1P expression was positively correlated with lymph node metastasis, distant metastasis, TNM stage, and shorter survival time in 102 breast cancer patients. Then, in vitro and in vivo experiments were designed to investigate the biological function and regulatory mechanism of RACGAP1P in breast cancer cell lines. Overexpression of RACGAP1P in MDA‐MB‐231 and MCF7 breast cell lines increased their invasive ability and enhanced their mitochondrial fission. Conversely, inhibition of mitochondrial fission by Mdivi‐1 could reduce the invasive ability of RACGAP1P‐overexpressing cell lines. Furthermore, the promotion of mitochondrial fission by RACGAP1P depended on its competitive binding with miR‐345‐5p against its parental gene RACGAP1, leading to the activation of dynamin‐related protein 1 (Drp1). In conclusion, lncRNA RACGAP1P promotes breast cancer invasion and metastasis via miR‐345‐5p/RACGAP1 pathway‐mediated mitochondrial fission.

Abbreviations

CDS

coding sequence

ceRNAs

competitive endogenous RNAs

Drp1

dynamin‐related protein 1

FFPE

formalin‐fixed paraffin‐embedded

lncRNAs

long non‐coding RNAs

miRNAs

microRNAs

RACGAP1

Rac GTPase activating protein 1

TCGA

The Cancer Genome Atlas

     

Homocysteine induces podocyte apoptosis by regulating miR‐1929‐5p expression through c‐Myc,DNMT1 and EZH2

Chronic kidney disease (CKD) is a common and complex disease in kidneys which has been associated with an increased risk of renal cell carcinoma. Elevated homocysteine (Hcy) levels are known to influence the development and progression of CKD by regulating podocyte injury and apoptosis. To investigate the molecular mechanisms triggered in podocytes by Hcy, we used cbs^+/− mice and observed that higher Hcy levels increased the apoptosis rate of podocytes with accompanying glomerular damage. Hcy‐induced podocyte injury and apoptosis in cbs^+/− mice was regulated by inhibition of microRNA (miR)‐1929‐5p expression. Overexpression of miR‐1929‐5p in podocytes inhibited apoptosis by upregulating Bcl‐2. Furthermore, the expression of miR‐1929‐5p was regulated by epigenetic modifications of its promoter. Hcy upregulated DNA methyltransferase 1 (DNMT1) and enhancer of zeste homolog 2 (EZH2) levels, resulting in increased DNA methylation and H3K27me3 levels on the miR‐1929‐5p promoter. Additionally, we observed that c‐Myc recruited DNMT1 and EZH2 to the miR‐1929‐5p promoter and suppressed the expression of miR‐1929‐5p. In summary, we demonstrated that Hcy promotes podocyte apoptosis through the regulation of the epigenetic modifiers DNMT1 and EZH2, which are recruited by c‐Myc to the promoter of miR‐1929‐5p to silence miR‐1929‐5p expression.     

The lncRNA ‘UCA1’ modulates the response to chemotherapy of ovarian cancer through direct binding to miR‐27a‐5p and control of UBE2N levels

Ovarian cancer (OC) is the leading cause of death in patients with gynecologic cancers. Due to late diagnosis and resistance to chemotherapy, the 5‐year survival rate in patients with OC is below 40%. We observed that UCA1, a lncRNA previously reported to play an oncogenic role in several malignancies, is overexpressed in the chemoresistant OC cell line OAW42‐R compared to their chemotherapy‐sensitive counterpart OAW42. Additionally, UCA1 overexpression was related to poor prognosis in two independent patient cohorts. Currently, the molecular mechanisms through which UCA1 acts in OC are poorly understood. We demonstrated that downregulation of the short isoform of UCA1 sensitized OC cells to cisplatin and that UCA1 acted as competing endogenous RNA to miR‐27a‐5p. Upon UCA1 downregulation, miR‐27a‐5p downregulated its direct target UBE2N leading to the upregulation of BIM, a proapoptotic protein of the Bcl2 family. The upregulation of BIM is the event responsible for the sensitization of OC cells to cisplatin. In order to model response to therapy in patients with OC, we used several patient‐derived organoid cultures, a model faithfully mimicking patient’s response to therapy. Inhibition of UBE2N sensitized patient‐derived organoids to platinum salts. In conclusion, response to treatment in patients with OC is regulated by the UCA1/miR‐27a‐5p/UBE2N axis, where UBE2N inhibition could potentially represent a novel therapeutic strategy to counter chemoresistance in OC.     

Concomitant overexpression of mir‐182‐5p and mir‐182‐3p raises the possibility of IL‐17–producing Treg formation in breast cancer by targeting CD3d,ITK, FOXO1, and NFATs: A meta‐analysis and experimental study

T cells are polarized toward regulatory T cells (Tregs) in tumor microenvironment by the shuttling of microRNAs that target T cell–activating signaling pathways. We evaluated the expression of the miR‐182 cluster (miR‐96, 182, and 183) in peripheral blood mononuclear cells (PBMCs) of patients with breast cancer (BC), and T cell polarization by the expression of FOXO1, NFATs, ITK, TCR/CD3 complex, and IL‐2/IL‐2RA. Twenty‐six microRNAs overexpressed in tumor tissues and sera of these patients were extracted by a meta‐analysis. Then, the expression of the miR‐182 cluster was investigated in PBMCs and sera of these patients and correlated with their targets in PBMCs. Finally, miR‐182 was cloned into Jurkat cells to evaluate its effects on T cell polarization. FOXO1, CD3d, ITK, NFATc3, NFATc4, and IL‐2RA were targeted by miR‐182, due to which their expression decreased in PBMCs of patients. Although IL‐6, IL‐17, and TGF‐β increased after miR‐182 transduction, IL‐2 dramatically decreased. We revealed CD4⁺FOXP3⁺ T cell differentiation in the miR‐182–transduced group. Although miR‐182 has inhibitory effects on T cells by the inhibition of FOXO1, TCR/CD3 complex, NFATs, and IL‐2/IL‐2RA signaling pathways, it increases FOXP3, TGF‐β, and IL‐17 expression to possibly drive T cell deviation toward the transitional state of IL‐17–producing Tregs and Treg formation in the end.     

Downregulation of miR‐326 and its host gene β‐arrestin1 induces pro‐survival activity of E2F1 and promotes medulloblastoma growth

Persistent mortality rates of medulloblastoma (MB) and severe side effects of the current therapies require the definition of the molecular mechanisms that contribute to tumor progression. Using cultured MB cancer stem cells and xenograft tumors generated in mice, we show that low expression of miR‐326 and its host gene β‐arrestin1 (ARRB1) promotes tumor growth enhancing the E2F1 pro‐survival function. Our models revealed that miR‐326 and ARRB1 are controlled by a bivalent domain, since the H3K27me3 repressive mark is found at their regulatory region together with the activation‐associated H3K4me3 mark. High levels of EZH2, a feature of MB, are responsible for the presence of H3K27me3. Ectopic expression of miR‐326 and ARRB1 provides hints into how their low levels regulate E2F1 activity. MiR‐326 targets E2F1 mRNA, thereby reducing its protein levels; ARRB1, triggering E2F1 acetylation, reverses its function into pro‐apoptotic activity. Similar to miR‐326 and ARRB1 overexpression, we also show that EZH2 inhibition restores miR‐326/ARRB1 expression, limiting E2F1 pro‐proliferative activity. Our results reveal a new regulatory molecular axis critical for MB progression.

Abbreviations

ARRB1

β‐arrestin1

BTC

bulk tumor cell

CSCs

cancer stem cells

EZH2

enhancer of zeste homolog 2

GCP

granule cell progenitors

MB

medulloblastoma

OFC

oncosphere‐forming cell

     

Circ_0008039 supports breast cancer cell proliferation,migration, invasion,and glycolysis by regulating the miR‐140‐3p/SKA2 axis

Circular RNAs (circRNAs) have been shown to modulate gene expression and participate in the development of multiple malignancies. The purpose of this study was to investigate the role of circ_0008039 in breast cancer (BC). The expression of circ_0008039, miR‐140‐3p, and spindle and kinetochore‐associated protein 2 (SKA2) was detected by qRT‐PCR. Cell viability, colony formation, migration, and invasion were evaluated using methylthiazolyldiphenyl‐tetrazolium bromide (MTT) assay, colony formation assay, and transwell assay, respectively. Glucose consumption and lactate production were measured using commercial kits. Protein levels of hexokinase II (HK2) and SKA2 were determined by western blot. The interaction between miR‐140‐3p and circ_0008039 or SKA2 was verified by dual‐luciferase reporter assay. Finally, a mouse xenograft model was established to investigate the roles of circ_0008039 in BC in vivo. We found that circ_0008039 and SKA2 were upregulated in BC tissues and cells, while miR‐140‐3p was downregulated. Knockdown of circ_0008039 suppressed BC cell proliferation, migration, invasion, and glycolysis. Moreover, miR‐140‐3p could bind to circ_0008039 and its inhibition reversed the inhibitory effect of circ_0008039 interference on proliferation, migration, invasion, and glycolysis in BC cells. SKA2 was verified as a direct target of miR‐140‐3p and its overexpression partially inhibited the suppressive effect of miR‐140‐3p restoration in BC cells. Additionally, circ_0008039 positively regulated SKA2 expression by sponging miR‐140‐3p. Consistently, silencing circ_0008039 restrained tumor growth via increasing miR‐140‐3p and decreasing SKA2. In conclusion, circ_0008039 downregulation suppressed BC cell proliferation, migration, invasion, and glycolysis partially through regulating the miR‐140‐3p/SKA2 axis, providing an important theoretical basis for treatment of BC.

Abbreviations

ANOVA

analysis of variance

BC

breast cancer

circRNAs

circular RNAs

DMSO

dimethyl sulfoxide

ECAR

extracellular acidification rate

ECL

enhanced chemiluminescence

FBS

fetal bovine serum

HK2

hexokinase II

MEGM

mammary epithelial growth medium

miR‐140‐3p

microRNA‐140‐3p

MTT

methylthiazolyldiphenyl‐tetrazolium bromide

PBS

phosphate‐buffered saline

PRKAR1B

protein kinase A regulatory subunit R1‐beta

SD

standard ± deviation

SKA2

spindle and kinetochore‐associated protein 2

     

The miR‐19b‐3p‐MAP2K3‐STAT3 feedback loop regulates cell proliferation and invasion in esophageal squamous cell carcinoma

Esophageal squamous cell carcinoma (ESCC) is one of the most refractory malignancies worldwide. Mitogen‐activated protein kinase 3 (MAP2K3) has a contradictory role in tumor progression, and the function and expression patterns of MAP2K3 in ESCC remain to be determined. We found that MAP2K3 expression to be downregulated in ESCC, and MAP2K3 downregulation correlated with clinically poor survival. MAP2K3 inhibited ESCC cell proliferation and invasion in vitro and in vivo. MAP2K3 suppressed STAT3 expression and activation. Mechanistically, MAPSK3 interacted with MDM2 to promote STAT3 degradation via the ubiquitin–proteasome pathway. Furthermore, exosomal miR‐19b‐3p derived from the plasma of patients with ESCC could suppress MAP2K3 expression to promote ESCC tumorigenesis. STAT3 was found to bind to the MIR19B promoter and increased the expression of miR‐19b‐3p in ESCC cells. In summary, our results demonstrated that the miR‐19b‐3p‐MAP2K3‐STAT3 feedback loop regulates ESCC tumorigenesis and elucidates the potential of therapeutically targeting this pathway in ESCC.     

circ_0006089 promotes gastric cancer growth,metastasis, glycolysis,and angiogenesis by regulating miR‐361‐3p/TGFB1

Circular RNA (circRNA) participates in a variety of pathophysiological processes, including the development of gastric cancer (GC). However, the role of circ_0006089 in GC progression and its underlying molecular mechanism need to be further revealed. Quantitative real‐time PCR was utilized for detecting circ_0006089, microRNA (miR)‐361‐3p and transforming growth factor‐β1 (TGFB1) expression. The interaction between miR‐361‐3p and circ_0006089 or TGFB1 was confirmed using a dual‐luciferase reporter assay and an RNA immunoprecipitation (RIP) assay. Cell proliferation, metastasis, apoptosis, and angiogenesis were determined using colony formation assay, EdU assay, transwell assay, flow cytometry, and tube formation assay. Cell glycolysis was evaluated by detecting glucose consumption, lactate production, and ATP levels. In addition, western blot (WB) analysis was used to measure protein expression. Xenograft tumor models were used to assess the effect of circ_0006089 knockdown on GC tumorigenesis. circ_0006089 had been found to be upregulated in GC tissues and cells, and it could act as an miR‐361‐3p sponge. circ_0006089 knockdown suppressed GC proliferation, metastasis, glycolysis, angiogenesis, and increased apoptosis, while this effect could be revoked by miR‐361‐3p inhibitor. TGFB1 was targeted by miR‐361‐3p, and its overexpression reversed the effects of miR‐361‐3p on GC cell function. Also, circ_0006089 promoted TGFB1 expression via sponging miR‐361‐3p. Animal experiments showed that silenced circ_0006089 inhibited GC tumorigenesis through the miR‐361‐3p/TGFB1 pathway. Our results revealed that the circ_0006089/miR‐361‐3p/TGFB1 axis contributed to GC progression, confirming that circ_0006089 might be a potential therapeutic target for GC.     

Circulating exosomal miR‐363‐5p inhibits lymph node metastasis by downregulating PDGFB and serves as a potential noninvasive biomarker for breast cancer

Sentinel lymph node (LN) biopsy is currently the standard procedure for clinical LN‐negative breast cancer (BC) patients but it is prone to false‐negative results and complications. Thus, an accurate noninvasive approach for LN staging is urgently needed in clinical practice. Here, circulating exosomal microRNA (miRNA) expression profiles in peripheral blood from BC patients and age‐matched healthy women were obtained and analyzed. We identified an exosomal miRNA, miR‐363‐5p, that was significantly downregulated in exosomes from plasma of BC patients with LN metastasis which exhibited a consistent decreasing trend in tissue samples from multiple independent datasets. Plasma exosomal miR‐363‐5p achieved high diagnostic performance in distinguishing LN‐positive patients from LN‐negative patients. The high miR‐363‐5p expression level was significantly correlated with improved overall survival. Functional assays demonstrated that exosomal miR‐363‐5p modulates platelet‐derived growth factor (PDGF) signaling activity by targeting PDGFB to inhibit cell proliferation and migration. Our study revealed, for the first time, plasma exosomal miR‐363‐5p plays a tumor suppressor role in BC and has the potential for noninvasive LN staging and prognosis prediction of BC.     

CircPCBP2 promotes the stemness and chemoresistance of DLBCL via targeting miR‐33a/b to disinhibit PD‐L1

Diffuse large B‐cell lymphoma (DLBCL) is the most common lymphoid malignancy with a high relapse rate of up to 40%. The prognosis of the disease needs improvement and requires a understanding of its molecular mechanism. We investigated the mechanisms of DLBCL development and its sensitivity to chemotherapy by focusing on circPCBP2/miR‐33a/b/PD‐L1 axis. Human DLBCL specimens and cultured cancer cell lines were used. Features of circPCBP2 were systematically characterized through Sanger sequencing, Actinomycin D, RNase R treatment, and FISH. The expression levels of circPCBP2, miR‐33a/b, PD‐L1, stemness‐related markers, ERK/AKT and JAK2/STAT3 signaling were measured using qRT‐PCR, western blotting, and immunohistochemistry. Stemness of DLBCL cells was assessed through spheroid formation assay and flow cytometry. Cell viability and apoptosis upon cyclophosphamide, doxorubicin, vincristine, and prednisone (CHOP) treatment were determined using MTT assay and flow cytometry, respectively. Interactions of circPCBP2‐miR‐33a/b and miR‐33a/b‐PD‐L1 were validated using dual luciferase activity assay and RNA‐RIP. Nude mouse xenograft model was used to assess the function of circPCBP2 in DLBCL growth in vivo. circPCBP2 was upregulated in human DLBCL specimens and cultured DLBCL cells while miR‐33a/b was reduced. Knockdown of circPCBP2 or miR‐33a/b overexpression inhibited the stemness of DLBCL cells and promoted cancer cell apoptosis upon CHOP treatment. circPCBP2 directly bound with miR‐33a/b while miR‐33a/b targeted PD‐L1 3’‐UTR. circPCBP2 disinhibited PD‐L1 signaling via sponging miR‐33a/b. miR‐33a/b inhibitor and activating PD‐L1 reversed the effects of circPCBP2 knockdown and miR‐33a/b mimics, respectively. circPBCP2 knockdown restrained DLBCL growth in vivo and potentiated the anti‐tumor effects of CHOP. In conclusion, circPCBP2 enhances DLBCL cell stemness but suppresses its sensitivity to CHOP via sponging miR‐33a/b to disinhibit PD‐L1 expression. circPCBP2/miR‐33a/b/PD‐L1 axis could serve as a diagnosis marker or therapeutic target for DLBCL.     

Promotion of tumorigenesis by miR‐1260b–targeting CASP8: Potential diagnostic and prognostic marker for breast cancer

MicroRNAs are reported as promising biomarkers for the diagnosis and treatment of breast cancer. miR‐1260b is identified as a tumor‐associated noncoding microRNA in other cancers, although the role of miR‐1260b and its clinical relevance in breast cancer remain unclear. In this study, miR‐1260b as a potential prognostic biomarker was observed by univariate and multivariate Cox regression analyses in 102 breast tumor tissues. The tumorigenic role of miR‐1260b in terms of proliferation, apoptosis, and migration of breast cancer cells was investigated using gain‐ and loss‐of‐function assays in vitro. Additionally, the potential early diagnosis and treatment monitoring marker of miR‐1260b was validated in 129 plasma samples. We found that high miR‐1260b expression was markedly associated with bulky tumor size, advanced stage, and lymph node invasion. Particularly, the high–miR‐1260b‐expression group showed shorter overall survival than the low–miR‐1260b‐expression group. The inhibition of oncogenic miR‐1260b induced apoptosis and decreased migration and invasion of MDA‐MB‐231 cells. CASP8 was revealed as a direct target gene of miR‐1260b, which is closely related to apoptosis. Furthermore, miR‐1260b expression levels in plasma were significantly higher in patients with breast cancer than in healthy controls. The patients who tested positive for miR‐1260b showed 16.3‐ and 18.2‐fold higher risks in the early stage and locally advanced stage, respectively, compared with healthy controls, and the risk was decreased 6.2‐fold after neoadjuvant chemotherapy. Taken together, miR‐1260b may be a potential novel diagnostic, prognostic, and therapeutic target in breast cancer.     

HPV+ HNSCC‐derived exosomal miR‐9‐5p inhibits TGF‐β signaling‐mediated fibroblast phenotypic transformation through NOX4

Human papillomavirus (HPV) is a significant risk factor for head and neck squamous cell carcinoma (HNSCC). HPV⁺ HNSCC patients have a higher survival rate, which may be related to its unique tumor microenvironment. Exosomes are emerging as a communication tool between tumor cells and the tumor microenvironment, including cancer‐associated fibroblasts (CAFs). In this study, 111 clinical samples tissues and public sequencing data were analyzed. Our study found fewer CAFs infiltrated in HPV⁺ HNSCC, and poor CAF infiltration level was associated with a good prognosis. HPV⁺ HNSCC cell‐derived exosomes can significantly reduce the phenotypic transformation of fibroblasts. miR‐9‐5p, as a miRNA enriched in HPV⁺ HNSCC cell‐derived exosomes, can be transferred to fibroblasts. miR‐9‐5p mimic transfection decreased the expression of NOX4 and the level of intracellular reactive oxygen species (ROS), which inhibited the transforming growth factor beta 1(TGF‐β1)‐induced increase of αSMA levels. Therefore, these results indicated that HPV⁺ HNSCC‐derived exosomal miR‐9‐5p inhibits TGF‐β signaling‐mediated fibroblast phenotypic transformation through NOX4, which is related to the excellent prognosis of HPV patients.