Construction of circRNA-mediated ceRNA network and immunoassay for investigating pathogenesis of COPD.

Background: The chronic respiratory condition known as chronic obstructive pulmonary disease (COPD) was one of the main causes of death and disability worldwide. This study aimed to explore and elucidate new targets and molecular mechanisms of COPD by constructing competitive endogenous RNA (ceRNA) networks. Methods: GSE38974 and GSE106986 were used to select DEGs in COPD samples and normal samples. Cytoscape software was used to construct and present protein-protein interaction (PPI) network, mRNA-miRNA co-expression network and ceRNA network. The CIBERSORT algorithm and the Lasso model were used to screen the immune infiltrating cells and hub genes associated with COPD, and the correlation between them was analyzed. COPD cell models were constructed in vitro and the expression level of ceRNA network factors mediated by hub gene was detected by reverse transcription-quantitative polymerase chain reaction (RT-qPCR). Results: In this study, 852 differentially expressed genes were screened in the GSE38974 dataset, including 439 upregulated genes and 413 downregulated genes. Gene clustering analysis of PPI network results was performed using the Minimum Common Tumor Data Element (MCODE) in Cytoscape, and seven hub genes were screened using five algorithms in cytoHubba. CCL20 was verified as an important hub gene based on mRNA-miRNA co-expression network, GSE106986 database validation and the analysis of ROC curve results. Finally, we successfully constructed the circDTL-hsa-miR-330-3p-CCL20 network by Cytoscape. Immune infiltration analysis suggested that CCL20 can co-regulate immune cell migration and infiltration through chemokines CCL7 and CXCL3. In vitro experiments, the expression of circDTL and CCL20 was increased, while the expression of hsa-miR-330-3p was decreased in the COPD cell model. Conclusion: By constructing the circDTL-hsa-miR-330-3p-CCL20 network, this study contributes to a better understanding of the molecular mechanism of COPD development, which also provides important clues for the development of new therapeutic strategies and drug targets.

[Qingshen Granules alleviates renal fibrosis in mice by regulating exosomes, miR-330-3p, and CREBBP expression].

OBJECTIVE: To explore the effects of Qingshen Granules (QSG) on adenine-induced renal fibrosis in mice and in uric acid (UA)-stimulated NRK-49F cells and its mechanism for regulating exosomes, miR-330-3p and CREBBP. METHODS: A mouse model of adenine-induced renal fibrosis were treated daily with QSG at 8.0 g·kg-1·d-1 via gavage for 12 weeks. An adenoassociated virus vector was injected into the tail vein, and renal tissues of the mice were collected for analyzing exosomal marker proteins CD9, Hsp70, and TSG101 and expressions of Col-III, α-SMA, FN, and E-cad using Western blotting and immunofluorescence and for observing pathological changes using HE and Masson staining. In the cell experiment, NRK-49F cells were stimulated with uric acid (400 µmol/L) followed by treatment with QSG-medicated serum from SD rats, and the changes in expressions of the exosomal markers and Col-III, α-SMA, FN, and E-cad were analyzed. Dual luciferase reporter assay was employed to examine the targeting relationship between miR-330-3p and CREBBP, whose expressions were detected by RT-qPCR and Western blotting in treated NRK-49F cells. RESULTS: The mouse models of adenine-induced renal fibrosis showed significantly increased levels of CD9, Hsp70, and TSG101, which were decreased by treatment with QSG. The expressions of Col-III, α-SMA, and FN increased and Ecad decreased in the mouse models but these changes were reversed by QSG treatment. QSG treatment obviously alleviated renal fibrosis in the mouse models. Intravenous injection of adeno-associated viral vector obviously inhibited miR-330-3p, increased CREBBP levels, and reduced fibrosis in the mouse models. Dual luciferase assay confirmed CREBBP as a target of miR-330-3p, which was consistent with the results of the cell experiments. CONCLUSION: QSG inhibits renal fibrosis in mice by regulating the exosomes, reducing miR-330-3p levels, and increasing CREBBP expression.

circRNA-0015004 act as a ceRNA to promote RCC2 expression in hepatocellular carcinoma.

Although circular RNAs (circRNA) have been demonstrated to modulate tumor initiation and progression, their roles in the proliferation of hepatocellular carcinoma (HCC) are still poorly understood. Based on the analysis of GEO data (GSE12174), hsa-circRNA-0015004 (circ-0015004) was screened and validated in 80 sets of HCC specimens. Subcellular fractionation analysis was designed to determine the cellular location of circ-0015004. Colony formation and cell counting kit-8 were performed to investigate the role of circ-0015004 in HCC. Dual-luciferase reporter gene assays, RNA immunoprecipitation and chromatin immunoprecipitation were employed to verify the interaction among circ-0015004, miR-330-3p and regulator of chromatin condensation 2 (RCC2). The expression level of circ-0015004 was significantly upregulated in HCC cell lines and HCC tissues. HCC patients with higher circ-0015004 levels displayed shorter overall survival, and higher tumor size and TNM stage. Moreover, knockdown of circ-0015004 significantly reduced HCC cell proliferation in vitro and inhibited the growth of HCC in nude mice. Mechanistic studies revealed that circ-0015004 could upregulate the expression of RCC2 by sponging miR-330-3p, thereby promoting HCC cell proliferation. Furthermore, we identified that Ying Yang 1 (YY1) could function as an important regulator of circ-0015004 transcription. This study systematically demonstrated the novel regulatory signaling of circ-0015004/miR-330-3p/RCC2 axis in promoting HCC progression, providing insight into HCC diagnosis and treatment from bench to clinic.

LncRNA GABPB1-AS1 is a potential target for the diagnosis of prostate cancer.

BACKGROUND: Prostate cancer is an adverse tumor that occurs in the male reproductive system. The symptoms of patients in the early stage are not obvious and are generally difficult to detect. AIM: The aim of this study was to determine the regulation of lncRNA GABPB1-AS1 (GABPB1-AS1) on prostate cancer progression and explore the diagnostic potential of GABPB1-AS1. METHODS: The contents of serum GABPB1-AS1 and miR-330-3p were examined by RT-qPCR assay. The functions of silencing GABPB1-AS1 and miR-330-3p inhibitor in prostate cancer cells were determined using transfection assay, CCK-8 assay and Transwell assay. The target of GABPB1-AS1 was predicted and verified at the molecular level by bioinformatics and luciferase reporter gene assay. The function of GABPB1-AS1 in prostate cancer diagnosis was evaluated via ROC method. RESULTS: GABPB1-AS1 was upregulated in prostate cancer serum, which was associated with patients' Gleason score and TNM stage. Mechanistically, GABPB1-AS1 directly targeted miR-330-3p, and there was a negative correlation between them. Reduced levels of GABPB1-AS1 in cells after knockdown of GABPB1-AS1 resulted in decreased prostate cancer cell growth and activity, and these inhibitory effects were repaired by miR-330-3p inhibitor. CONCLUSION: The present study confirmed that GABPB1-AS1 was overexpressed in prostate cancer, and its sponge miR-330-3p may be an effective target for timely diagnosis of prostate cancer.

Bta-miR-330 promotes bovine intramuscular pre-adipocytes adipogenesis via targeting SESN3 to activate the Akt-mTOR signaling pathway.

Consumers are more inclined to choose beef with a high intramuscular fat content (IMF), which regulated by lots of factors. It is very significant to find a miRNA that plays a key role in the accumulation of IMF. In our study, we found that bta-miR-330 was highly expressed in Japanese black cattle and differentially expressed at intramuscular pre-adipocytes differentiation processes. Furthermore, we transfected the bta-miR-330 mimic & inhibitor in intramuscular pre-adipocytes. The results showed that bta-miR-330 inhibits the proliferation but promotes the adipogenesis of intramuscular pre-adipocytes. Subsequently, our study showed that bta-miR-330 binds to SESN3, which inhibits the adipogenesis of intramuscular pre-adipocytes. Moreover, we established the mechanism that bta-miR-330 promotes the adipogenesis of intramuscular pre-adipocytes by targeting SESN3 to activate the Akt-mTOR signaling pathway. Overall, our results revealed that bta-miR-330-SESN3-Akt-mTOR axis plays an important role in adipogenesis of intramuscular pre-adipocytes, which provides a molecular basis for increasing IMF content in beef cattle.

MicroRNA-330-5p Mediates the TDRG1-Regulated Myocardial Inflammation and Apoptosis after Myocardial Infarction by Inhibiting MAPK1.

Acute myocardial infarction (AMI) is a cardiovascular illness with the highest disability and mortality rates worldwide. This study aimed to estimate the mechanism of TDRG1 in myocardial damage.qRT-PCR was used to study the levels of TDRG1. After establishing hypoxia/reoxygenation (H/R) model, the inflammation was assessed by qRT-PCR, oxidation was detected by commercial kits, and apoptosis was estimated by qRT-PCR and flow cytometry. The luciferase intensity and RNA immunoprecipitation assay were detected for the identification of target relationship. The functional enrichment was unveiled by GO and Kyoto Encyclopedia of Genes and Genomes (KEGG). The protein interaction was conducted for screening key genes.The expression of TDRG1 was elevated and negatively correlated with miR-330-5p in the serum AMI patients. TDRG1/miR-330-5p axis regulated inflammation, oxidation, and viability and apoptosis of HL-1 cells induced by H/R. GO and KEGG analyses indicate that 76 overlapping targets of miR-330-5p were primarily involved in focal adhesion, calmodulin binding, and ErbB and Rap1 signaling pathways. MAPK1 was the top key gene and was a target gene of miR-330-5p.TDRG1/miR-330-5p axis could participate in the regulation of apoptosis and inflammation of H/R-induced cardiomyocytes.

Impacts of TP53TG1 in cancer-associated fibroblasts-derived exosomes on epithelial-mesenchymal transition capacity of colorectal carcinoma cells by targeting miR-330-3p.

Objective: This research aims at clarifying the action and mechanisms of action of TP53TG1 in cancer-associated fibroblasts (CAF)-derived exosomes (EXs) on colorectal carcinoma (CRC) cells. Methods: CAF and CAF-EXs isolated from CRC tissues were incubated with CRC SW480 cells to determine alterations in biological behavior, epithelial-mesenchymal transition (EMT) capacity, and TP53TG1 and miR-330-3p expression. In addition, a dual luciferase reporter (DLR) assay was conducted to verify the connection between TP53TG1 and miR-330-3p, and the impacts of the two genes on CRC cells were analyzed. Results: CRC-CAF-EXs extracted from CRC tissues were successfully identified and were able to promote SW480 multiplication, invasiveness, migration, and EMT ability while inhibiting apoptosis (P < 0.05). In addition, TP53TG1 increased and miR-330-3p decreased in SW480 when cultured with CRC-CAF-EXs (P < 0.05). The DLR assay identified notably reduced fluorescence activity of TP53TG1-WT after transfection with miR-330-3p-mimics (P < 0.05). Furthermore, SW480 cell multiplication, invasiveness and migration were found to be enhanced and the apoptosis decreased after up-regulating TP53TG1, while suppressing TP53TG1 and up-regulating miR-330-3p contributed to quite the opposite effect (P < 0.05). Moreover, by elevating TP53TG1 and miR-330-3p simultaneously, we found a cell activity similar to the NC group (P > 0.05). Conclusion: By targeting miR-330-3p, TP53TG1 in CRC-CAF-EXs can enhance CRC cell activity and EMT capacity and inhibit apoptosis.

Effects of miR-330 restoration on pancreatic cancer cells oncogenesis.

BACKGROUND: Inappropriate expressions of various miRNAs have reported in different human malignancies. Evidence suggested that miR-330 may play as both onco-miR and/or tumor suppressor-miR in different cancers. In the present study, we evaluated effects of miR-330 on proliferation and migration of pancreatic cancer (PC) cells as well as underlying molecular mechanisms. DESIGN: The expression of miR-330 was evaluated in clinical tissue samples of patients with PC. Transfection of the PC cells (PANC-1) by miR-330 was conducted by pCMV vector. The cancer-related genes expression was investigated in mRNA and protein level following transfection of the PC cells. Furthermore, the PC cells viability, invasion, migration, mitochondrial membrane potential, apoptosis, autophagy, and cell cycle profile were investigated after transfection by miR-330. RESULTS: The results indicated that expression of miR-330 downregulated in patients with PC. Stable increase of miR-330 expression after transfection in PC cells reduces viability, mitochondrial membrane potential, invasion, and migration. Further assessments demonstrated that upregulation of miR-330 increases apoptosis and autophagy percentage in the PC cells. Moreover, a cell cycle arrest was observed in G1, Sub-G1, and S phases following transfection of the PC cells. These findings can be explained by modified mRNA and protein expression of apoptosis- and metastasis-related genes. CONCLUSION: Our study suggested that miR-330 acts as a tumor suppressor in PC cells, and revealed that upregulation of miR-330 may provide an effective therapeutic approach for overcoming progression and metastasis in patients with PC.

Biogated mesoporous silica nanoagents for inhibition of cell migration and combined cancer therapy.

Migration is an initial step in tumor expansion and metastasis; suppressing cellular migration is beneficial to cancer therapy. Herein, we designed a novel biogated nanoagents that integrated the migration inhibitory factor into the mesoporous silica nanoparticle (MSN) drug delivery nanosystem to realize cell migratory inhibition and synergistic treatment. Antisense oligonucleotides (Anti) of microRNA-330-3p, which is positively related with cancer cell proliferation, migration, invasion, and angiogenesis, not only acted as the locker for blocking drugs but also acted as the inhibitory factor for suppressing migration via gene therapy. Synergistic with gene therapy, the biogated nanoagents (termed as MSNs-Gef-Anti) could achieve on-demand drug release based on the intracellular stimulus-recognition and effectively kill tumor cells. Experimental results synchronously demonstrated that the migration suppression ability of MSNs-Gef-Anti nanoagents (nearly 30%) significantly contributed to cancer therapy, and the lethality rate of the non-small-cell lung cancer was up to 70%. This strategy opens avenues for realizing efficacious cancer therapy and should provide an innovative way for pursuing the rational design of advanced nano-therapeutic platforms with the combination of cancer cell migratory inhibition.

The lncRNA-MALAT1/miR-330-3p Axis Promotes Growth and Metastasis of Gastric Cancer Through Regulation of Vascular Endothelial Growth Factor A: A Genetic and Cell Evaluation Study

SUMMARY: Overexpression of metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) in various tumor tissues and cell lines was found to promote tumor cell proliferation, migration, and invasion. However, the role of MALAT1 in gastric cancer (GC) is still unclear. We aimed to investigate the correlation between long-chain non-coding RNAs (lncRNAs), MALAT1, MicroRNAs (miRNA) and vascular endothelial growth factor A (VEGFA) in gastric cancer and to disclose underlying mechanism. The correlation between MALAT1 levels and clinical features was analyzed by bioinformatics data and human samples. The expression of MALAT1 was down regulated in AGS cells to detect the cell proliferation, migration, and invasion characteristics, as well as the effects on signal pathways. Furthermore, we validated the role of MALAT1/miR-330-3p axis in GC by dual luciferase reporter gene assays. Expression of MALAT1 was higher in cancer tissues than in para-cancerous tissues. The high MALAT1 level predicted malignancy and worse prognosis. Down-regulation of MALAT1 expression in AGS cells inhibited cell proliferation, migration, and invasion by targeting VEGFA. By dual luciferase reporter gene assay and miR-330-3p inhibitor treatment, we demonstrate that MALAT1 sponged miR-330-3p in GC, leading to VEGFA upregulation and activation of the mTOR signaling pathway. The MALAT1/miR-330-3p axis regulates VEGFA through the mTOR signaling pathway and promotes the growth and metastasis of gastric cancer.

Se descubrió que la sobreexpresión del transcrito 1 de adenocarcinoma de pulmón asociado a metástasis (MALAT1) en varios tejidos tumorales y líneas celulares promueve la proliferación, migración e invasión de células tumorales. Sin embargo, el papel de MALAT1 en el cáncer gástrico (CG) aún no está claro. Nuestro objetivo fue investigar la correlación entre los ARN no codificantes de cadena larga (lncRNA), MALAT1, los microARN (miARN) y el factor de crecimiento endotelial vascular A (VEGFA) en el cáncer gástrico y revelar el mecanismo subyacente. La correlación entre los niveles de MALAT1 y las características clínicas se analizó mediante datos bioinformáticos y muestras humanas. La expresión de MALAT1 se reguló negativamente en las células AGS para detectar las características de proliferación, migración e invasión celular, así como los efectos sobre las vías de señales. Además, validamos el papel del eje MALAT1/miR- 330-3p en GC mediante ensayos de genes indicadores de luciferasa dual. La expresión de MALAT1 fue mayor en tejidos cancerosos que en tejidos paracancerosos. El alto nivel de MALAT1 predijo malignidad y peor pronóstico. La regulación negativa de la expresión de MALAT1 en células AGS inhibió la proliferación, migración e invasión celular al apuntar a VEGFA. Mediante un ensayo de gen indicador de luciferasa dual y un tratamiento con inhibidor de miR-330-3p, demostramos que MALAT1 esponjaba miR-330-3p en GC, lo que lleva a la regulación positiva de VEGFA y la activación de la vía de señalización mTOR. El eje MALAT1/miR-330-3p regula VEGFA a través de la vía de señalización mTOR y promueve el crecimiento y la metástasis del cáncer gástrico.

CircITGA7 regulates malignant phenotypes in bladder cancer cells via targeting miR-330-3p/KLF10 axis.

Bladder cancer (BCa) is one of the common malignancies. Circular RNAs (circRNAs) play regulatory roles in cancer progression. CircITGA7 is a circRNA generated from several exons of ITGA7. The potential role of circITGA7 in BCa remains unknown and needs to be explored. Quantitative real time polymerase chain reaction (qRT-PCR) was used to assess circITGA7 and miR-330-3p expression in BCa tissues and cell lines. Kaplan-Meier analysis was used to evaluate the overall survival of these BCa patients. The biological function of circITGA7 was examined by overexpression of circITGA7 using CCK-8, EdU, wound-healing, and Transwell assays. Xenograft assay was performed to further validate the in vitro results. To explore the mechanism of circITGA7, luciferase reporter, RNA pull-down, fluorescence in situ hybridization (FISH) assays were employed to examine the binding interaction among circITGA7, miR-330-3p and kruppel-like factor 10 (KLF10). Western blot was used to study the protein levels of KLF10.CircITGA7 was downregulated in BCa tissues and cell lines and indicated longer overall survival. Moreover, circITGA7 restricted cell proliferation, migration and invasion of BCa through negatively regulating miR-330-3p. The in vivo model showed that circITGA7 influenced the tumor growth. Besides, the overexpression of miR-330-3p promoted cell progression by directly targeting KLF10. Mechanistically, circITGA7 inhibited BCa progression by activating KLF10 via targeting miR-330-3p.CircITGA7 alleviates BCa cell progression via circITGA7/hsa-miR-330-3p/KLF10 axis, which may provide novel therapeutic targets for BCa.

miR-330-5p Suppress Cell Growth and Invasion via Disrupting HSF4-mediated MACC1/STAT3 Pathway in Colorectal Cancer.

BACKGROUND: Recently, miRNAs are demonstrated to restrain mRNA translation through novel pattern with bind complementary sites in the coding sequence (CDS). Heat Shock Transcription Factor 4 (HSF4) has been newly described as a tumor-associated transcription factor. Therefore, the present study intends to explore miRNAs that bind CDS region of HSF4, and identify the function of their interactions in the malignant biological behavior of colorectal cancer (CRC). METHODS: Prognostic value of HSF4 and correlation between HSF4 and MACC1 expression were estimated via bioinformatics with the Cancer Genome Atlas (TCGA) data. HSF4 and downstream MACC1/STAT3 signaling cascade was characterized by immunoblotting. To characterize the effects of miR-330-5p and HSF4 on the malignant phenotype of CRC cells by functional experiments. The binding activity of miR-330-5p to coding sequence (CDS) of HSF4 was identified using DIANA-microT-CDS algorithm and dual-luciferase reporter assay. RESULTS: HSF4 was aberrantly overexpressed and associated with poor outcomes of CRC patients. Overexpression of HSF4 was correlated with Tumor Node Metastasis stage, and positively regulated malignant behaviors such as growth, migration, invasion of CRC cells. Moreover, miR-330-5p suppressed CRC cell growth, colony formation, migration and invasive. Interestingly, miR-330-5p recognized complementary sites within the HSF4 CDS region to reduce HSF4 expression. In rescue experiments, restoration of HSF4 expression functionally alleviated miR-330-5p-induced inhibition of cell growth, colon formation, invasion, and wound healing of CRC cells. HSF4 was associated positively with the well-known oncogenic factor MACC1 in TCGA cohort CRC samples, and knockdown of HSF4 resulted in downregulation of MACC1. In mechanism, MACC1 was suppressed upon miR-330-5p-induced downregulation of HSF4, leading to inactivation of phosphorylation of downstream STAT3. CONCLUSION: miR-330-5p suppresses tumors by directly inhibiting HSF4 to negatively modify activity of MACC1/STAT3 pathway.

Long non-coding RNA MEG3 acts as a suppressor in breast cancer by regulating miR-330/CNN1.

BACKGROUND: The current study aimed to investigate the molecular mechanism of long non-coding RNA (lncRNA) MEG3 in the development of breast cancer. METHODS: The regulating relationships among lncRNA MEG3, miRNA-330 and CNN1 were predicted by bioinformatics analysis of breast cancer samples in the Cancer Genome Atlas database. The differential expression of lncRNA MEG3, miRNA-330 and CNN1 was first validated in breast cancer tissues and cells. The effects of lncRNA MEG3 on breast cancer malignant properties were evaluated by manipulating its expression in MCF-7 and BT-474 cells. Rescue experiments, dual-luciferase assays, and RNA immunoprecipitation (RIP) experiments were further used to validate the relationships among lncRNA MEG3, miRNA-330 and CNN1. RESULTS: Bioinformatics analysis showed that lncRNA MEGs and CNN1 were significantly downregulated in breast cancer tissues, while miR-330 was upregulated. These differential expressions were further validated in our cohort of breast cancer samples. High expression levels of lncRNA MEG3 and CNN1 as well as low expression of miR-330 were significantly associated with favorable overall survival. Overexpression of lncRNA MEG3 significantly inhibited cell viability, migration and invasion, decreased cells in S stage and promoted cell apoptosis. Dual-luciferase reporter gene assay and RIP experiments showed that lncRNA MEG3 could directly bind to miR-330. Moreover, miR-330 mimics on the basis of lncRNA MEG3 overexpression ameliorated the tumor-suppressing effects of lncRNA MEG3 in breast cancer malignant properties by decreasing CNN1 expression. CONCLUSION: Our study indicated lncRNA MEG3 is a breast cancer suppressor by regulating miR-330/CNN1 axis.

Circ_0004140 promotes lung adenocarcinoma progression by upregulating NOVA2 via sponging miR-330-5p.

BACKGROUND: Circular RNAs (circRNAs) play a significant role in the tumorigenesis and progression of diverse human cancers, including lung adenocarcinoma. A previous study suggested that circ_0004140 expression was increased in lung adenocarcinoma cells. However, the molecular mechanism of circRNA circ_0004140 involved in lung adenocarcinoma is poorly defined. METHODS: Circ_0004140, microRNA-330-5p (miR-330-5p), and NOVA alternative splicing regulator 2 (NOVA2) expression were determined by real-time quantitative polymerase chain reaction (RT-qPCR). Cell proliferation, apoptosis, migration, invasion, and angiogenesis ability were assessed using 5-ethynyl-2'-deoxyuridine (EdU), flow cytometry, wound healing, transwell, and capillary-like network formation assays. Proliferating cell nuclear antigen (PCNA), cyclin D1, and NOVA2 protein levels were detected using Western blot assay. The interaction between miR-330-5p and circ_0004140 or NOVA2 was verified by dual-luciferase reporter assay. Xenograft tumor model was utilized to assess the role of circ_0004140 in tumor growth in vivo. RESULTS: Circ_0004140 was upregulated in lung adenocarcinoma tissues and cell lines. Circ_0004140 silencing suppressed cell proliferation, migration, invasion and tube formation ability, and triggered the apoptosis of lung adenocarcinoma cells. Circ_0004140 acted as a molecular sponge for miR-330-5p, and miR-330-5p silencing largely reversed circ_0004140 knockdown-induced effects in lung adenocarcinoma cells. NOVA2 was a target of miR-330-5p, and NOVA2 overexpression might largely overturn miR-330-5p overexpression-induced influences in lung adenocarcinoma cells. Circ_0004140 upregulated NOVA2 expression via sponging miR-330-5p in lung adenocarcinoma cells. Circ_0004140 silencing restrained xenograft tumor growth in vivo. CONCLUSION: Circ_0004140 knockdown might suppress the malignant biological behaviors of lung adenocarcinoma cells via miR-330-5p-dependent regulation of NOVA2.

METTL3-mediated m6A modification of circRNF220 modulates miR-330-5p/survivin axis to promote osteosarcoma progression.

BACKGROUND: Circular RNAs (circRNAs) play a crucial role in regulating various physiological processes. However, the precise regulatory mechanisms of circRNF220s in osteosarcoma (OS) are not well understood. METHODS: The abundances of circRNF220, miR-330-5p, and survivin were determined using qRT-PCR. To assess the m6A accumulation in circRNF220, a methylated RNA immunoprecipitation (Me-RIP) assay was conducted. Cellular multiplication, motility, and invasion were examined using the cell Counting Kit-8 (CCK-8), EdU, colony formation, Transwell, and wound-healing assays. The binding relationships were measured through RNA immunoprecipitation (RIP) and luciferase reporter assays. In vivo functionality was assessed using xenograft models. RESULTS: CircRNF220 was identified as being overexpressed in both OS cells and tissues. In vitro experiments demonstrated that silencing circRNF220 impeded the proliferation, invasion, and motility of OS cells. Similarly, in vivo studies confirmed that downregulating circRNF220 inhibited the growth of OS. Further mechanistic investigations unveiled that METTL3-modulated circRNF220 regulated the progression of OS by upregulating survivin expression through acting as a sponge for miR-330-5p. CONCLUSION: The modulation of METTL3-regulated circRNF220 has been found to promote the progression of OS by modulating the miR-330-5p/survivin axis. This novel finding suggests a potentially unique approach to managing OS.

METTL3 Promotes the Growth and Invasion of Melanoma Cells by Regulating the lncRNA SNHG3/miR-330-5p Axis.

Accumulating evidence indicates that m6A methyltransferase 3 (METTL3) plays a pivotal role in different malignancies including melanoma. However, the function and underlying mechanisms by which METTL3 contributes to the tumorigenesis of melanoma remain undocumented. The association of METTL3 and long noncoding RNA (lncRNA) small nucleolar RNA host gene 3 (SNHG3) with clinicopathological characteristics and prognosis in patients with melanoma was analyzed by real-time quantitative polymerase chain reaction (RT-qPCR), Western blotting, and The Cancer Genome Atlas data sets. The role of METTL3 in melanoma cells was assessed by in vitro and in vivo experiments. The m6A dot blot, methylated RNA immunoprecipitation (MeRIP), and RT-qPCR were used to verify METTL3-mediated m6A modification of lncRNA SNHG3. The effect of METTL3 on lncRNA SNHG3 was determined by luciferase gene reporter assay, RT-qPCR, and Western blotting. We found that METTL3 was upregulated in melanoma tissue samples and associated with poor survival in patients with melanoma. Knockdown of METTL3 suppressed the growth and invasion of melanoma cells in vitro and in vivo, whereas restored expression of METTL3 promoted these effects. Mechanistic investigations showed that knockdown of METTL3 reduced SNHG3 m6A levels and its messenger ribonucleic acid (mRNA) expression levels. SNHG3 could act as a sponge of microRNA (miR)-330-5p to upregulate the expression of CCHC-type zinc finger nucleic acid binding protein (CNBP). SNHG3 overexpression reversed METTL3-knockdown-caused antitumor effects, miR-330-5p upregulation and CNBP downregulation. SNHG3 had a positive correlation with METTL3 expression but a negative correlation with miR-330-5p expression in melanoma tissue samples. In conclusion, our findings demonstrated that METTL3-mediated m6A modification of lncRNA SNHG3 promoted the growth and invasion of melanoma cells by regulating the miR-330-5p/CNBP axis.

SNHG3/miR-330-5p/HSD11B1 Alleviates Myocardial Ischemia-reperfusion Injury by Regulating the ERK/p38 Signaling Pathway.

BACKGROUND: Studies have found that microRNAs (miRNAs) participate in the pathogenesis of myocardial ischemia-reperfusion injury (MIRI). miR-330-5p alleviated cerebral IR injury and regulated myocardial damage. However, the mechanism of the effect of miR-330-5p on MIRI needs to be further studied. OBJECTIVE: The study aimed to explore the role and mechanism of miR-330-5p in MIRI. METHODS: The oxygen-glucose deprivation reperfusion (OGD/R) model was constructed in cardiomyocytes to simulate MIRI in vitro. QRT-PCR was used for the detection of gene expression. ELISA was used for evaluation of the levels of aldehyde dehydrogenase 2 family member (ALDH2), 4-hydroxynonenal (4-HNE), and malondialdehyde (MDA). Flow cytometry was used to evaluate apoptosis. Western blot was employed for protein determination. Bioinformatic analysis was performed for predicting the targets of miR-330-5p. RESULTS: miR-330-5p was found to be down-regulated in MIRI-induced cardiomyocytes (Model group). miR-330-5p mimic enhanced ALDH2 activity, inhibited apoptosis, and suppressed 4-HNE and MDA of MIRI-induced cardiomyocytes. miR-330-5p inhibited ERK expression while increasing the p38 expression. Bioinformatic analysis showed hydroxysteroid 11-beta dehydrogenase 1 (HSD11B1) to be a target of miR-330-5p. HSD11B1 expression was inhibited by miR-330-5p mimic while increased by miR-330-5p inhibitor in MIRI-induced cardiomyocytes. HSD11B1 overexpression reversed the effect of miR-330-5p on ALDH2, 4-HNE, MDA, apoptosis, and ERK/p38 signaling pathway. Furthermore, lncRNA small nucleolar RNA host gene 3 (SNHG3) was the upstream lncRNA of miR-330-5p. SNHG3 decreased miR-330-5p expression and increased HSD11B1 expression. CONCLUSION: SNHG3/miR-330-5p alleviated MIRI in vitro by targeting HSD11B1 to regulate the ERK/p38 signaling pathway.

Circ-PDZD8 promotes cell growth and glutamine metabolism in non-small cell lung cancer by enriching LARP1 via sequestering miR-330-5p.

BACKGROUND: The deregulation of circular RNA (circRNA) is widely reported in carcinogenesis. The purpose of this study was to investigate the role of circRNA-PDZ domain containing 8 (circ-PDZD8) in non-small cell lung cancer (NSCLC) progression. METHODS: The histological structure of tissues was identified by hematoxylin-eosin (HE) staining analysis. The expression levels of circ-PDZD8, miR-330-5p and la ribonucleoprotein 1 (LARP1) mRNA were ascertained by qPCR. Cell counting kit-8, colony formation, flow cytometry, and transwell assays were employed for functional analysis. Glutamine metabolism was monitored by glutamine consumption, alpha ketoglutarate (α-KG) level and adenosine triphosphate (ATP) level. A xenograft model was established to ascertain the role of circ-PDZD8 in vivo. The putative binding relationships were verified by dual-luciferase and RIP studies. RESULTS: Circ-PDZD8 expression was highly increased in NSCLC. Circ-PDZD8 knockdown inhibited cell growth, migratory capacity, invasiveness and glutamine metabolism but enhanced cell apoptosis in NSCLC cells. Circ-PDZD8 blocked miR-330-5p expression, and miR-330-5p inhibition overturned the effects of circ-PDZD8 absence. LARP1 targeted by miR-330-5p, and miR-330-5p upregulation-impaired cell growth, motility and glutamine metabolism were recovered by LARP1 overexpression. Circ-PDZD8 knockdown was also shown to impede solid tumor growth. CONCLUSION: Circ-PDZD8 promotes NSCLC cell growth and glutamine metabolism by increasing LARP1 via competitively targeting miR-330-5p.

NONHSAT021545/miR-330-3p/EREG: A Cooperative Axis in Breast Cancer Prognosis and Treatment.

Lymphatic metastasis is the most common form in breast cancer (BC) progression. Previously, we observed that lnc045874, a most conservative homology of Homo Sapiens NONHSAT021545 (lnc021545), miR-330-3p, and EREG may have some effects in mouse hepatocarcinoma cell lines with different lymphatic metastasis potentials. Through data from TCGA and GEO database analysis, we speculated that miR-330-3p might be a tumor promoter, while EREG could be a tumor suppressor in BC. MiR-330-3p was upregulated, while lnc021545 and EREG were downregulated in 50 BC tissues. MiR-330-3p advanced the metastatic behaviors of BC cells, whereas lnc021545 and EREG resulted in the opposite effects. The three molecules' expressions were correlated respectively and showed that miR-330-3p targeted lnc021545 and EREG to affect their expressions. Lnc021545/miR-330-3p axis affected BC metastasis by regulating EREG in epithelial-to-mesenchymal transition. In 50 BC patients, these three molecules and their cooperation are associated with aggressive tumor phenotypes, patient outcomes, and trastuzumab therapy. We finally discovered that lnc021545, miR-330-3p, and EREG formed a multi-gene co-regulation system that affected the metastasis of BC and the cooperation reflects the synergistic effects of the three molecules, recommending that their cooperation may provide a more accurate index for anti-metastasis therapeutic and prognostic evaluation of BC.

miR-330-3p alleviates the progression of atherosclerosis by downregulating AQP9.

Atherosclerosis (AS) is the main cause of cardiovascular diseases. However, the role of AQP9 in AS is not well understood. In the present study, we predicted that miR-330-3p might regulate AQP9 in AS through bioinformatics analysis, and we established AS model using ApoE-/- mouse (C57BL/6) with high-fat diet (HFD). Hematoxylin and eosin (H&E) and Oil red O staining were used to determine atherosclerotic lesions. CCK8 and Ethyny1-2-deoxyuridine (EdU) assays were used to investigate human umbilical vein endothelial cells (HUVECs) proliferation after treatment with 100 µg/mL ox-LDL. Wound scratch healing and transwell assays were used to measure the cell invasion and migration ability. Flow cytometry assay was used to determine apoptosis and cell cycle. A dual-luciferase reporter assay was performed to investigate the binding of miR-330-3p and AQP9. We identified that the expression of miR-330-3p in AS mice model decreased while the expression level of AQP9 increased. miR-330-3p overexpression or down-regulation of AQP9 could reduce cell apoptosis, promote cell proliferation, and migration after ox-LDL treatment. Dual-luciferase reporter assay result presented that AQP9 was directly inhibited by miR-330-3p. These results suggest that miR-330-3p inhibits AS by regulating AQP9. miR-330-3p/AQP9 axis may be a new therapeutic target for AS.