LncRNA NUTM2A-AS1 silencing inhibits glioma via miR-376a-3p/YAP1 axis.

The lncRNA NUTM2A-AS1 has been shown to be dysregulated in gastric cancer, while the roles in glioma is unclear. The aim of this study was to investigate the roles and potential mechanisms of lncRNA NUTM2A-AS1 in the proliferation and apoptosis of glioma cells. The StarBase software and dual luciferase reporter assay were used to identify the relationship between lncRNA NUTM2A-AS1 and miR-376a-3p, and miR-376a-3p and YAP1. The expression of lncRNA NUTM2A-AS1, miR-376a-3p, and YAP1 in human glioma cell lines was detected by qRT-PCR. MTT and flow cytometry were used to detect the effects of lncRNA NUTM2A-AS1 or miR-376a-3p on the proliferation and apoptosis of U251 and A172 cells, respectively. In addition, changes of Bax and Bcl-2 expression in glioma cells were further verified by western blotting and qRT-PCR. The results showed that the expression of lncRNA NUTM2A-AS1 was elevated in glioma cell lines, while miR-376a-3p was decreased. LncRNA NUTM2A-AS1 was negatively correlated with miR-376a-3p. Silencing of lncRNA NUTM2A-AS1 enhanced the levels of miR-376a-3p, leading to reduced cell proliferation and increased apoptosis in glioma cells. YAP1 was a direct target of miR-376a-3p, and it was negatively regulated by miR-376a-3p in U251 and A172 cells. Further mechanistic studies suggested that miR-376a-3p reduced glioma cell proliferation and increased apoptosis by inhibiting YAP1 expression. In addition, lncRNA NUTM2A-AS1 positively regulated of YAP1 expression in glioma cells. In conclusion, silencing of lncRNA NUTM2A-AS1 inhibited proliferation and induced apoptosis in human glioma cells via the miR-376a-3p/YAP1 axis.

Long Non-Coding RNA NUTM2A-AS1/miR-376a-3p/PRMT5 Axis Promotes Prostate Cancer Progression.

BACKGROUND: In recent years, significant attention has been directed towards long non-coding RNA NUT family member 2A antisense RNA 1 (NUTM2A-AS1) for its oncogenic role in tumours. This study aimed to investigate the functional and molecular mechanisms underlying NUTM2A-AS1 in prostate cancer (PCa). METHODS: NUTM2A-AS1, miR-376a-3p, and protein arginine methyltransferase 5 (PRMT5) levels were assessed in PCa samples and matched non-cancerous prostate samples. The DU145 cell line was conditioned to undergo transfection with relevant plasmids, and a cell counting kit-8 assay was performed to evaluate cell proliferation. A Transwell assay was conducted to analyse cell migration or invasion. Cell apoptosis was assessed using an annexin V-fluorescein isothiocyanate/propidium iodide apoptosis detection kit and flow cytometry. A tumour sphere formation assay was conducted to assess the ability of PCa cells to form tumour spheres. RESULTS: We found elevated expression of NUTM2A-AS1 and PRMT5 and decreased expression of miR-376a-3p in PCa samples. Inhibition of NUTM2A-AS1 or overexpression of miR-376a-3p led to reduced cell proliferation and diminished cancer stem cell-like traits in vitro. NUTM2A-AS1 regulated miR-376a-3p through competitive absorption, thereby modulating PRMT5. Up-regulation of PRMT5 nullified the therapeutic effects of inhibiting NUTM2A-AS1 or overexpressing miR-376a-3p in DU145 cells. CONCLUSIONS: NUTM2A-AS1 promotes cancer stem cell-like traits in PCa cells by targeting PRMT5 through miR-376a-3p. Therefore, these NUTM2A-AS1-based novel insights into tumour therapy hold promise for patients with PCa.

Long Non-Coding RNA NUTM2A-AS1/miR-376a-3p/PRMT5 Axis Promotes Prostate Cancer Progression

Background: In recent years, significant attention has been directed towards long non-coding RNA NUT family member 2A antisense RNA 1 (NUTM2A-AS1) for its oncogenic role in tumours. This study aimed to investigate the functional and molecular mechanisms underlying NUTM2A-AS1 in prostate cancer (PCa). Methods: NUTM2A-AS1, miR-376a-3p, and protein arginine methyltransferase 5 (PRMT5) levels were assessed in PCa samples and matched non-cancerous prostate samples. The DU145 cell line was conditioned to undergo transfection with relevant plasmids, and a cell counting kit-8 assay was performed to evaluate cell proliferation. A Transwell assay was conducted to analyse cell migration or invasion. Cell apoptosis was assessed using an annexin V-fluorescein isothiocyanate/propidium iodide apoptosis detection kit and flow cytometry. A tumour sphere formation assay was conducted to assess the ability of PCa cells to form tumour spheres. Results: We found elevated expression of NUTM2A-AS1 and PRMT5 and decreased expression of miR-376a-3p in PCa samples. Inhibition of NUTM2A-AS1 or overexpression of miR-376a-3p led to reduced cell proliferation and diminished cancer stem cell-like traits in vitro. NUTM2A-AS1 regulated miR-376a-3p through competitive absorption, thereby modulating PRMT5. Up-regulation of PRMT5 nullified the therapeutic effects of inhibiting NUTM2A-AS1 or overexpressing miR-376a-3p in DU145 cells. Conclusions: NUTM2A-AS1 promotes cancer stem cell-like traits in PCa cells by targeting PRMT5 through miR-376a-3p. Therefore, these NUTM2A-AS1-based novel insights into tumour therapy hold promise for patients with PCa. (AU)

MiR-376a-3p increases cell apoptosis in acute myeloid leukemia by targeting MT1X.

Metallothioneins (MTs) are a group of low-molecular weight cysteine-rich proteins that play vital roles in oxidative stress, metal homeostasis, carcinogenesis and drug resistance. However, few studies have analyzed the roles of MTs in acute myeloid leukemia (AML). In this study, we revealed that the expression of metallothionein1X (MT1X), a main isoform of MTs, was highly expressed and acted as a candidate of prognostic indicator in AML patients. In vitro cell function experiments verified that silencing MT1X inhibited the proliferation of AML cells, sensitized cells to doxorubicin, and increased their apoptosis. We also showed that the downregulation of MT1X expression suppressed nuclear factor-κB (NF-κB) signaling by reducing p65, p-IκB-α, and downstream effectors. Elevated p65 and MT1X levels were indicators in AML. Moreover, we revealed that miR-376a-3p had binding sites with 3'-UTR of MT1X, suggesting that MT1X was negatively regulated by miR-376a-3p. Cell functional assay results indicated that miR-376a-3p overexpression significantly inhibited the proliferation, arrested the AML cells in the G0/G1 phase and induced cell apoptosis. The rescue experiments further confirmed that miR-376a-3p could reverse the promotion of MT1X overexpression on the progress of AML cells. Taken together, our results revealed that elevated MT1X expression might be involved in the mechanism underlying AML progression, indicating that the miR-376a/MT1X axis might serve as a promising novel target for the effective treatment of patients with AML.

PSMA3-AS1 induced by transcription factor PAX5 promotes cholangiocarcinoma proliferation, migration and invasion by sponging miR-376a-3p to up-regulate LAMC1.

Long noncoding RNAs (lncRNAs) have been reported to exhibit a crucial regulatory role in tumor progression, including cholangiocarcinoma (CCA). As a promising lncRNA, proteasome 20S subunit alpha 3 antisense RNA 1 (PSMA3-AS1) is involved in development of various tumors. However, the role and function of PSMA3-AS1 in CCA remain unclear. The aim of this study is to examine the expression, function, mechanism, and clinical significance of PSMA3-AS1 in CCA development. By TCGA database analysis, we found that PSMA3-AS1 was overexpressed in CCA. Consistent with the TCGA analysis, PSMA3-AS1 was significantly overexpressed in CCA tissues and cells by RT-qPCR. Upregulated PSMA3-AS1 was related to lymph node invasion, advanced TNM stage and poor survival, and was an independent risk factor of prognosis for CCA patients. Functionally, CCK-8, EdU and colony formation assays confirmed that upregulated PSMA3-AS1 promoted CCA cell proliferation, whereas downregulated PSMA3-AS1 inhibited proliferation. This result was further confirmed by subcutaneous tumor formation in nude mice. Wound healing and transwell assays confirmed that increased PSMA3-AS1 promoted CCA cell migration and invasion, whereas decreased PSMA3-AS1 inhibited these biological phenotypes. In addition, PSMA3-AS1 promoted the EMT process of CCA by downregulating E-cadherin and upregulating N-cadherin and vimentin. Mechanistically, transcription factor PAX5 bound to the promoter region of PSMA3-AS1 and promoted its transcription. Simultaneously, PSMA3-AS1 primarily localized in the cytoplasm could competitively bind miR-376a-3p to upregulate LAMC1, thereby accelerating CCA progression. This study uncovers that PSMA3-AS1 functions as a cancer-promoting gene in CCA, and PAX5/PSMA3-AS1/miR-376a-3p/LAMC1 axis plays a vital role in CCA development.

Long non-coding RNA LINC00488 facilitates thyroid cancer cell progression through miR-376a-3p/PON2.

OBJECTIVE: Long non-coding RNAs (lncRNAs) recently have been identified as influential indicators in a variety of malignancies. The aim of the present study was to identify a functional lncRNA LINC00488 and its effects on thyroid cancer in the view of cell proliferation and apoptosis. METHODS: In order to evaluate the effects of LINC00488 on the cellular process of thyroid cancer, we performed a series of in vitro experiments, including cell counting kit-8 (CCK-8) assay, EdU (5-ethynyl-2'-deoxyuridine) assay, flow cytometry, transwell chamber assay, Western blot and RT-qPCR. The target gene of LINC00488 was then identified by bioinformatics analysis (DIANA and TargetScan). Finally, a series of rescue experiments was conducted to validate the effect of LINC00488 and its target genes on proliferation, migration, invasion and apoptosis of thyroid cancer. RESULTS: Our findings revealed that LINC00488 was highly expressed in thyroid cancer cell lines (BCPAP, BHP5-16, TPC-1 and CGTH-W3) and promoted the proliferation, migration and invasion, while inhibited the apoptosis of thyroid cancer cells (BCPAP and TPC-1). The results of bioinformatics analysis and dual luciferase reporter gene assay showed that LINC00488 could directly bind to miR-376a-3p and down-regulated the expression level of miR-376a-3p. In addition, Paraoxonase-2 (PON2) was a target gene of miR-376a-3p and negatively regulated by miR-376a-3p. Rescue experiment indicated that LINC00488 might enhance PON2 expression by sponging miR-376a-3p in thyroid cancer. CONCLUSION: Taken together, our study revealed that lncRNA LINC00488 acted as an oncogenic gene in the progression of thyroid cancer via regulating miR-376a-3p/PON2 axis, which indicated that LINC00488-miR-376a-3p-PON2 axis could serve as novel biomarkers or potential targets for the treatment of thyroid cancer.

Circular RNA FBXO11 regulates the proliferation and apoptosis of gastric cancer SNU-1 cells through targeting miR-376a-3p/SNRPB axis / 中国肿瘤生物治疗杂志

@#[Abstract] Objective: To investigate the effect of circular RNA FBXO11 (circFBXO11) regulating the miR-376a-3p/SNRPB (small nuclear ribonucleoprotein polypeptides B gene) axis on the proliferation and apoptosis of gastric cancer SNU-1 cells. Methods: Cancer and para-cancerous tissue samples from 30 patients with gastric cancer who underwent surgical resection were surgically resected in the Department of Oncosurgery, the Affiliated Hospital of North China University of Science and Technology from January 2018 to January 2019 were collected. The positive expression rate of SNRPB protein in gastric cancer tissues was detected by Immunohistochemical staining. The expression levels of circFBXO11, miR-376a-3p and SNRPB mRNA in gastric cancer tissues, gastric cancer cell lines (SNU-1, AGS and HS-746T) and gastric mucosal cell line GES1 were detected by qPCR. Dual luciferase reporter gene assay was used to determine the relationship between circFBXO11 and miR-376a-3p as well as between miR-376a-3p and SNRPB. The si-NC, si-circFBXO11, miR-NC, miR-376a-3p, si-SNRPB, si-circFBXO11+anti-miR-NC, si-circFBXO11+anti-miR-376a-3p, si-circFBXO11+pcDNA-NC, si-circFBXO11+pcDNA-SNRPB were transfected into gastric cancer SNU-1 cells, respectively. CCK-8 assay, Flow cytometry and WB assay were used to detect cell proliferation activity, apoptosis rate and protein expressions of SNRPB, cyclin D1 and C-caspase-3, respectively. Results: Compared with para-cancerous tissues, the expression level of circFBXO11 and the positive rate of SNRPB protein in gastric cancer tissues were significantly increased (all P<0.01), while the expression of miR-376a-3p was significantly decreased (P<0.01). Compared with GES1 cells, the expressions of circFBXO11 and SNRPB were significantly increased, while the expression of miR-376a-3p was significantly decreased (all P<0.01) in gastric cancer cells. circFBXO11 negatively regulated miR-376a-3p expression, and miR-376a-3p negatively regulated SNRPB expression. After inhibiting the expression of circFBXO11 or over-expressing miR-376a-3p or suppressing the expression of SNRPB, the proliferation viability of SNU-1 cells was decreased, and the apoptosis rate was increased (P<0.01). Either inhibiting miR-376a-3p or over-expressing SNRPB could partially reverse the effect of circFBXO11 suppression on proliferation and apoptosis of SNU-1 cells (all P<0.01). Conclusion: circFBXO11 is highly expressed in gastric cancer tissues. Inhibiting circFBXO11 inhibits the proliferation and induces apoptosis of gastric cancer cells, and the mechanism is related to the regulation of miR-376A-3p/SNRPB pathway.

LncRNA TTN­AS1 promotes endometrial cancer by sponging miR­376a­3p.

Increasing research has demonstrated that lncRNAs participate in the development of multiple cancer types. However, the role of TTN­AS1 in endometrial cancer (EC) remains unknown. The present study aimed to explore the function of titin­antisense RNA1 (TTN­AS1) in EC progression and the underlying mechanisms. qRT­PCR was performed to assess the TTN­AS1 expression patterns in EC tissues and cell lines. Loss of function experiments were carried out to estimate the effects of TTN­AS1 on EC cell proliferation, migration and invasion. To reveal the underlying mechanisms, informatics tools were used to predict the targets. Rescue experiments were performed to investigate the TTN­AS1­regulated miR­376a­3p/pumilio homolog 2 (PUM2) axis involved. The results of the present study revealed that TTN­AS1 was highly expressed in both EC tissues and cell lines, and TTN­AS1 knockdown inhibited EC cell proliferation, migration and invasion. With respect to the mechanisms, miR­376a­3p was revealed to be targeted by TTN­AS1, and reversed the effects on EC development induced by TTN­AS1. In addition, PUM2 was positively regulated by TTN­AS1, and miR­376a­3p mediated the regulation between them. Furtherly, in vivo experiments confirmed the results. Collectively, TTN­AS1 enhanced EC cell proliferation and metastasis by targeting the miR­376a­3p/PUM2 axis, which may shed light on EC diagnosis and treatment.

Integrated bioinformatics analysis identifies microRNA-376a-3p as a new microRNA biomarker in patient with coronary artery disease.

INTRODUCTION: Coronary artery disease (CAD) is a major global health problem with high incidence and mortality. Despite many advances in treatment, the prognosis of patients with CAD still remains poor. Therefore, this study aimed to identify potential biomarkers and targets associated with the progression of CAD. METHODS: Two gene expression profile datasets (GSE20681 and GSE12288), and two microRNA (miRNA) expression profile datasets (GSE59421 and GSE105449) were downloaded from the Gene Expression Omnibus (GEO) database; R language was used to screen out the differentially expressed genes (DEGs) and differentially expressed miRNAs (DEMs), respectively. In addition, five online bioinformatics tools (miRWalk et al.) were used to identify the target genes of DEMs, and miRNA-gene network was constructed using Cytoscape software. Moreover, CCK-8, flow cytometry assays were used to detect the cell proliferation and apoptosis in human umbilical vein endothelial cells (HUVECs). Meanwhile, the dual luciferase reporter system assay was used to explore the interaction of miR-376a-3p and NRIP1 in HUVECs. RESULTS: In the present study, 150 common DEGs and 5 common DEMs were screened using a Venn diagram in R language. First, a total of 6812 target genes were identified from the overlapping DEMs. Second, 26 overlapping dysregulated genes from 150 overlapping DEGs and 6812 miRNA target genes were identified. Meanwhile, 43 miRNA-gene regulatory pairs were obtained between the 5 common DEMs and 26 dysregulated genes. Downregulation of miR-376a-3p significantly inhibited the proliferation of HUVECs via inducing apoptosis. Moreover, overexpression of miR-376a-3p markedly inhibited the growth of HUVECs via downregulating NRIP1. CONCLUSION: In this study, miR-376a-3p-NRIP1 pair might involve in the progression of CAD, implying that miR-376a-3p may be a therapeutic target for the treatment of CAD.

LncRNA TTN-AS1 sponges miR-376a-3p to promote colorectal cancer progression via upregulating KLF15.

AIMS: Long non-coding RNAs (lncRNAs) play key roles in regulating multiple cancers. TTN-AS1 was reported to function in several human malignancies. However, the biological function of TTN-AS1 in colorectal cancer (CRC) has not been explored. In this study, we aimed to investigate the role and the underlying mechanisms of TTN-AS1 in CRC progression. MAIN METHODS: RT-qPCR was used to detect the expression levels of TTN-AS1, miR-376a-3p and KLF15 in colorectal cancer tissues and cells. CCK-8, colony formation, flow cytometry and transwell assays were performed to determine the cell proliferation, apoptotic rate and invasion ability. Target genes were predicted using bioinformatics methods. si-RNA and miRNA inhibitor were transfected into CRC cells to explore the underlying mechanisms. Tumor xenografts were created to confirm the function of TTN-AS-1 in vivo. KEY FINDINGS: TTN-AS1 upregulation was observed both in CRC tissues and cell lines. Functional investigation showed that knockdown of TTN-AS1 inhibited CRC cell proliferation and invasion, while enhanced cell apoptosis. Bioinformatics analysis identified miR-376a-3p as a target of TTN-AS1. Transfection of miR-376a-3p inhibitor mitigated the alterations induced by TTN-AS1 knockdown. Moreover, TTN-AS1 positively regulated KLF15 via sponging miR-376a-3p. Additionally, these findings were supported by in vivo experiments. SIGNIFICANCE: In conclusions, TTN-AS1 promoted CRC proliferation and invasion through miR-376a-3p/KLF15 axis. Our findings suggested that TTN-AS1 might be a potential therapeutic target in CRC treatment.

Tumor Suppressor Function of miR-127-3p and miR-376a-3p in Osteosarcoma Cells.

Since the introduction of high-dose chemotherapy about 35 years ago, survival rates of osteosarcoma patients have not been significantly improved. New therapeutic strategies replacing or complementing conventional chemotherapy are therefore urgently required. MicroRNAs represent promising targets for such new therapies, as they are involved in the pathology of multiple types of cancer, and aberrant expression of several miRNAs has already been shown in osteosarcoma. In this study, we identified silencing of miR-127-3p and miR-376a-3p in osteosarcoma cell lines and tissues and investigated their role as potential tumor suppressors in vitro and in vivo. Transfection of osteosarcoma cells (n = 6) with miR-127-3p and miR-376a-3p mimics significantly inhibited proliferation and reduced the colony formation capacity of these cells. In contrast, we could not detect any influence of miRNA restoration on cell cycle and apoptosis induction. The effects of candidate miRNA restoration on tumor engraftment and growth in vivo were analyzed using a chicken chorioallantoic membrane (CAM) assay. Cells transfected with mir-127-3p and miR-376a-3p showed reduced tumor take rates and tumor volumes and a significant decrease of the cumulative tumor volumes to 41% and 54% compared to wildtype cells. The observed tumor suppressor function of both analyzed miRNAs indicates these miRNAs as potentially valuable targets for the development of new therapeutic strategies for the treatment of osteosarcoma.

MiR-127 and miR-376a act as tumor suppressors by in vivo targeting of COA1 and PDIA6 in giant cell tumor of bone.

Giant cell tumors of bone (GCTB) are generally benign bone tumors associated with expansive osteolytic defects, a high rate of recurrence and potential malignant transformation. We recently observed silencing of miR-127-3p and miR-376a-3p in GCTB and identified COA1 and PDIA6 as their target genes. Here, we investigate the impact of these microRNAs and their target genes on tumor engraftment and progression of giant cell tumor stromal cells (GCTSC) in vivo by xenotransplantation on the chorioallantoic membrane of chicken eggs. Prior to transplantation, the neoplastic GCTSCs were transfected with miRNA mimics or siRNAs directed against their target genes. Restoration of miR-127-3p and miR-376a-3p reduced the tumor take rate to 17% and 47% compared to 95% in the controls. The tumor volumes were significantly reduced to 29% by both miRNAs. Silencing of COA1 and PDIA6 significantly decreased the tumor volumes to 37.7% and 42.7%, while the tumor take rates remained stable. Our results indicate that re-expression of miR-127-3p and miR-376a-3p induces a strong tumor suppressor effect in GCTSC, which is partially mediated via COA1 and PDIA6.

RNA editing independently occurs at three mir-376a-1 sites and may compromise the stability of the microRNA hairpin.

RNA editing is being recognized as an important post-transcriptional mechanism that may have crucial roles in introducing genetic variation and phenotypic diversity. Despite microRNA editing recurrence, defining its biological relevance is still under extended debate. To better understand microRNA editing function and regulation we performed an exhaustive characterization of the A-to-I site-specific patterns in mir-376a-1, a mammalian microRNA which RNA editing is involved in the regulation of development and in disease. Thorough an integrative approach based on high-throughput small RNA sequencing, Sanger sequencing and computer simulations we explored mir-376a-1 editing in samples from various individuals and primate species including human placenta and macaque, gorilla, chimpanzee and human brain cortex. We observed that mir-376a-1 editing is a common phenomenon in the mature and primary microRNA molecules and it is more frequently detected in brain than in placenta. Primary mir-376a-1 is edited at three positions, -1, +4 and +44. Editing frequency estimations and in silico simulations indicated that editing was not equally recurrent along the three mir-376a-1 sites, nevertheless no epistatic interactions among them were observed. Particularly, the +4 site, located in the seed region of the mature miR-376a-5p, reached the highest editing frequency in all samples. Secondary structure predictions revealed that the +4 position was the one that conferred the highest stability to the mir-376a-1 hairpin. We suggest that molecular stability might partially explain the editing recurrence observed in certain microRNAs and that editing events conferring new functional regulatory roles in particular tissues and species could have been conserved along evolution, as it might be the case of mir-376a-1 in primate brain cortex.

Restoration of miR-127-3p and miR-376a-3p counteracts the neoplastic phenotype of giant cell tumor of bone derived stromal cells by targeting COA1, GLE1 and PDIA6.

Although generally benign, giant cell tumors of bone (GCTB) display an aggressive behavior associated with significant bone destruction and lung metastasis in rare cases. This and the very high recurrence rate observed after surgical resection ranging from 20 to 55% necessitates the development of more effective treatment strategies. To identify valuable therapeutic targets, we screened a previously identified microRNA signature consisting of 23 microRNAs predominantly down-regulated in GCTB. We preselected eight candidate microRNAs and analyzed the impact of their restored expression on the neoplastic phenotype of GCTB stromal cells (GCTSC). A consistent and significant inhibition of cell proliferation, migration, colony formation and spheroid formation could be induced by transfection of primary GCTSC cell lines with miR-127-3p and miR-376a-3p, respectively. Genome wide expression analysis of miR-127-3p and miR-376a-3p transfected cells revealed four novel target genes for each microRNA. Luciferase reporter assays demonstrated direct interactions of miR-127-3p with COA1 and direct interaction of miR-376a-3p with GLE1 and PDIA6, suggesting a pivotal role of these genes in the molecular etiology of GTCB. Interestingly, both microRNAs are located within a chromosomal region frequently silenced in GCTB and many other types of cancers, indicating that these microRNAs and their target genes are valuable therapeutic targets for the treatment of GCTB and possibly other tumor entities.