RNA Binding Protein Immunoprecipitation Assay Research Articles

MicroRNA‑1271‑5p alleviates the malignant development of hepatitis B virus‑mediated liver cancer via binding to AQP5.

Hepatitis B virus (HBV) is a leading cause of liver-related cancer. Progress has been made on the study of microRNA (miRNA or miR) function in HBV-related liver cancer. Hence, the objective of the present study was to determine the role and functional mechanism of miR-1271-5p in HBV-associated liver cancer. miR-1271-5p and aquaporin 5 (AQP5) expression at the mRNA level were measured by reverse transcription-quantitative PCR (RT-qPCR). The levels of hepatitis B e-antigen (HBeAg), hepatitis B surface antigen (HBsAg) and HBV DNA were assessed by ELISA or qPCR. Cell viability, apoptosis, migration and invasion were detected by Cell Counting Kit-8, flow cytometry or Transwell assay. The interaction of miR-1271-5p and AQP5 was predicted by TargetScan, and verified by Dual-luciferase reporter assay and RNA binding protein immunoprecipitation assay. The protein levels of AQP5, Bax, Bcl-2, cleaved-caspase-3 and proliferating cell nuclear antigen were quantified by western blot analysis. Nude mouse tumorigenicity assay was conducted to examine the role of miR-1271-5p in vivo. miR-1271-5p was downregulated, while AQP5 was upregulated in HBV-related liver cancer cells and tissues. Overexpression of miR-1271-5p or AQP5 knockdown inhibited the levels of HBeAg, HBsAg and HBV DNA, blocked cell viability, migration and invasion, and induced apoptosis. AQP5 was confirmed to be a direct target of miR-1271-5p, and miR-1271-5p exerted its role through targeting AQP5. Overexpression of miR-1271-5p impeded tumor growth in vivo by weakening the expression of AQP5. In conclusion, miR-1271-5p blocked the progression of HBV-induced liver cancer by competitively targeting AQP5.

RPL34‐AS1–induced RPL34 inhibits cervical cancer cell tumorigenesis via the MDM2‐P53 pathway

Ribosomal proteins (RPs) are important components of ribosomes and related to the occurrence and development of tumors. However, little is known about the effects of the RP network on cervical cancer (CC). In this study, we screened differentially expressed RPL34 in CC by high‐throughput quantitative proteome assay. We found that RPL34 acted as a tumor suppressor and was downregulated in CC and inhibited the proliferation, migration, and invasion abilities of CC cells. Next, we verified that RPL34 regulated the CC through the MDM2‐P53 pathway by using Act D medicine, MDM2 inhibitor, and a series of western blotting（WB）assays. Moreover, an antisense lncRNA, RPL34‐AS1, regulated the expression of RPL34 and participated in the tumorigenesis of CC. RPL34 can reverse the effect of RPL34‐AS1 in CC cells. Finally, by RNA‐binding protein immunoprecipitation (RIP) assay we found that eukaryotic initiation factor 4A3 (EIF4A3), which binds to RPL34‐AS1, regulated RPL34‐AS1 expression in CC. Therefore, our findings indicate that RPL34‐AS1–induced RPL34 inhibits CC cell proliferation, invasion, and metastasis through modulation of the MDM2‐P53 signaling pathway, which provides a meaningful target for the early diagnosis and treatment of CC.

Circular RNA circMYBPC1 promotes skeletal muscle differentiation by targeting MyHC

Skeletal muscle development is a complex and highly orchestrated biological process mediated by a series of myogenesis regulatory factors. Numerous studies have demonstrated that circular RNAs (circRNAs) are involved in muscle differentiation, but the exact molecular mechanisms involved remain unclear. Here, we analyzed the expression of circRNAs at the adult and embryo development stages of cattle musculus longissimus. A stringent set of 1,318 circRNAs candidates were identified, and we found that 495 circRNAs were differentially expressed between embryonic and adult tissue libraries. We subsequently focused on one of the most downregulated circRNAs (using the adult stage expression as control), and this was named muscle differentiation-associated circular RNA (circMYBPC1). With RNA binding protein immunoprecipitation (RIP) and RNA pull-down assays, circMYBPC1 was identified to promote myoblast differentiation by directly binding miR-23a to relieve its inhibition on myosin heavy chain (MyHC). In addition, RIP assays demonstrated that circMYBPC1 could directly bind MyHC protein. In vivo observations also suggested that circMYBPC1 may stimulate skeletal muscle regeneration after muscle damage. These results revealed that the novel non-coding circRNA circMYBPC1 promotes differentiation of myoblasts and may promote skeletal muscle regeneration. Our results provided a basis for in-depth analysis of the role of circRNA in myogenesis and muscle diseases.

LncRNA LINC01410 Induced by MYC Accelerates Glioma Progression via Sponging miR-506-3p and Modulating NOTCH2 Expression to Motivate Notch Signaling Pathway.

Glioma is a common invasive cancer with unfavorable prognosis in patients. Long non-coding RNAs have been reported to participate in modulating diverse cellular processes. Here, we focused on exploring the role of long intergenic non-protein coding RNA 1410 (LINC01410) in glioma and its underlying mechanism. The expression levels and protein levels of genes were analyzed by quantitative real-time PCR (RT-qPCR) analysis and western blot. Loss-of-function assays were performed to assess the function of LINC01410 in glioma cells. The interactions among MYC, LINC01410, microRNA-506-3p (miR-506-3p) and notch receptor 2 (NOTCH2) were validated through Chromatin immunoprecipitation (ChIP), RNA Binding Protein immunoprecipitation (RIP), RNA pull-down and luciferase reporter assays. Our data supported that LINC01410 was up-regulated in glioma cells. Bioinformatics predictions and the integrated experiments identified that MYC activated LINC01410 transcription and LINC01410 promoted the levels of NOTCH2 through sponging miR-506-3p and further motivated Notch signaling pathway. Rescue assays validated that LINC01410 exerted its influential functions on glioma cell proliferation and apoptosis via enhancing NOTCH2 expression. Our studies identified that LINC01410 accelerates the progression of glioma through acting as a ceRNA for miR-506-3p and elevating NOTCH2 expression to further activate the Notch signaling pathway, which indicated that LINC01410 might act as a novel regulator of glioma progression.

ST8SIA6-AS1 promotes the development of hepatocellular carcinoma cells through miR-338-3p/NONO Axis

BackgroundIncreasing studies have shown a vital fact that long non-coding RNAs (lncRNAs) play a considerable regulatory role in hepatocellular carcinoma (HCC) progression. However, whether ST8 alpha-N-acetyl-neuraminide alpha-2, 8-sialyltransferase 6 antisense RNA 1 (ST8SIA6-AS1) affects the development of HCC is unclear. MethodsThe target genes in HCC cell lines were quantified via utilzing quantitative real-time polymerase chain reaction (RT-qPCR) analysis and western blot. Effects of ST8SIA6-AS1 on proliferative, apoptosis and migratory ability of HCC cells were proved by a series of function experiments. The cellular distribution of ST8SIA6-AS1 was examined through fluorescent in situ hybridization (FISH) assay and subcellular fractionation experiments. RNA pulldown assay was implemented to explore the target of ST8SIA6-AS1. RNA Binding Protein Immunoprecipitation (RIP) and luciferase reporter assays were performed to identify the specific relationships between miR-338-3p and ST8SIA6-AS1/ non-POU domain containing octamer binding (NONO). ResultsThe expression of ST8SIA6-AS1 was apparently elevated in HCC cell. Silenced ST8SIA6-AS1 reduced proliferative, migratory and invasive ability of HCC cells. Moreover, ST8SIA6-AS1 targeted miR-338-3p to modulate the expression of NONO in HCC cells. ConclusionsST8SIA6-AS1 enhances the progression of HCC via miR-338-3p/NONO axis in vitro.

CTCF-silenced miR-137 contributes to EMT and radioresistance in esophageal squamous cell carcinoma

BackgroundEsophageal squamous cell carcinoma (ESCC) is one of the most malignant tumors in gastrointestinal system. MicroRNAs (miRNAs) have been reported to be implicated in cancer development. However, the role of miR-137 has not been fully revealed in ESCC.MethodsQuantitative real-time polymerase chain reaction (qRT-PCR) and western blot analyses were separately used to examine RNA level and protein level. 5-ethynyl-2′-deoxyuridine (EdU) assay, transwell assays and flow cytometry analyses were conducted to assess biological behaviors of ESCC cells. Additionally, the interaction between genes were analyzed via Chromatin Immunoprecipitation (ChIP) assay, RNA Binding Protein Immunoprecipitation (RIP) assay, RNA pull down assay and luciferase reporter assay.ResultsMiR-137 was down-regulated in ESCC cells. Upregulation of miR-137 hindered ESCC cell proliferation, migration, invasion and epithelial mesenchymal transition (EMT). Besides, miR-137 enhanced the sensitivity of ESCC cells to irradiation. Moreover, CCCTC-binding factor (CTCF) inactivated miR-137 transcription in ESCC cells. Furthermore, we revealed enhancer of zeste 2 polycomb repressive complex 2 subunit (EZH2) and paxillin (PXN) as the downstream targets of miR-137. In turn, EZH2 was recruited by CTCF and induced methylation in miR-137 promoter.ConclusionCTCF/Suz12/EZH2 complex-silenced miR-137 facilitates ESCC progression and radioresistance by targeting EZH2 and PXN.

Oncogenic lncRNA ZNF561-AS1 is essential for colorectal cancer proliferation and survival through regulation of miR-26a-3p/miR-128-5p-SRSF6 axis

BackgroundLong non-coding RNAs (lncRNA) are reported to influence colorectal cancer (CRC) progression. Currently, the functions of the lncRNA ZNF561 antisense RNA 1 (ZNF561-AS1) in CRC are unknown.MethodsZNF561-AS1 and SRSF6 expression in CRC patient samples and CRC cell lines was evaluated through TCGA database analysis, western blot along with real-time PCR. SRSF6 expression in CRC cells was also examined upon ZNF561-AS1 depletion or overexpression. Interaction between miR-26a-3p, miR-128-5p, ZNF561-AS1, and SRSF6 was examined by dual luciferase reporter assay, as well as RNA binding protein immunoprecipitation (RIP) assay. Small interfering RNA (siRNA) mediated knockdown experiments were performed to assess the role of ZNF561-AS1 and SRSF6 in the proliferative actives and apoptosis rate of CRC cells. A mouse xenograft model was employed to assess tumor growth upon ZNF561-AS1 knockdown and SRSF6 rescue.ResultsWe find that ZNF561-AS1 and SRSF6 were upregulated in CRC patient tissues. ZNF561-AS1 expression was reduced in tissues from treated CRC patients but upregulated in CRC tissues from relapsed patients. SRSF6 expression was suppressed and enhanced by ZNF561-AS1 depletion and overexpression, respectively. Mechanistically, ZNF561-AS1 regulated SRSF6 expression by sponging miR-26a-3p and miR-128-5p. ZNF561-AS1-miR-26a-3p/miR-128-5p-SRSF6 axis was required for CRC proliferation and survival. ZNF561-AS1 knockdown suppressed CRC cell proliferation and triggered apoptosis. ZNF561-AS1 depletion suppressed the growth of tumors in a model of a nude mouse xenograft. Similar observations were made upon SRSF6 depletion. SRSF6 overexpression reversed the inhibitory activities of ZNF561-AS1 in vivo, as well as in vitro.ConclusionIn summary, we find that ZNF561-AS1 promotes CRC progression via the miR-26a-3p/miR-128-5p-SRSF6 axis. This study reveals new perspectives into the role of ZNF561-AS1 in CRC.

LncRNA NKX2-1-AS1 promotes tumor progression and angiogenesis via upregulation of SERPINE1 expression and activation of the VEGFR-2 signaling pathway in gastric cancer.

Long noncoding RNAs (lncRNAs) can compete with endogenous RNAs to modulate the gene expression and contribute to oncogenesis and tumor metastasis. lncRNA NKX2‐1‐AS1 (NKX2‐1 antisense RNA 1) plays a pivotal role in cancer progression and metastasis; however, the contribution of aberrant expression of NKX2‐1‐AS1 and the mechanism by which it functions as a competing endogenous RNA (ceRNA) in gastric cancer (GC) remains elusive. NKX2‐1‐AS1 expression was detected in paired tumor and nontumor tissues of 178 GC patients by quantitative reverse transcription PCR (qRT‐PCR). Using loss‐of‐function and gain‐of‐function experiments, the biological functions of NKX2‐1‐AS1 were evaluated both in vitro and in vivo. Further, to assess that NKX2‐1‐AS1 regulates angiogenic processes, tube formation and co‐culture assays were performed. RNA binding protein immunoprecipitation (RIP) assay, a dual‐luciferase reporter assay, quantitative PCR, Western blot, and fluorescence in situ hybridization (FISH) assays were performed to determine the potential molecular mechanism underlying this ceRNA. The results indicated that NKX2‐1‐AS1 expression was upregulated in GC cell lines and tumor tissues. Overexpression of NKX2‐1‐AS1 was significantly associated with tumor progression and enhanced angiogenesis. Functionally, NKX2‐1‐AS1 overexpression promoted GC cell proliferation, metastasis, invasion, and angiogenesis, while NKX2‐1‐AS1 knockdown restored these effects, both in vitro and in vivo. RIP and dual‐luciferase assays revealed that the microRNA miR‐145‐5p is a direct target of NKX2‐1‐AS1 and that NKX2‐1‐AS1 serves as a ceRNA to sponge miRNA and regulate angiogenesis in GC. Moreover, serpin family E member 1 (SERPINE1) is an explicit target for miR‐145‐5p; besides, the NKX2‐1‐AS1/miR‐145‐5p axis induces the translation of SERPINE1, thus activating the VEGFR‐2 signaling pathway to promote tumor progression and angiogenesis. NKX2‐1‐AS1 overexpression is associated with enhanced tumor cell proliferation, angiogenesis, and poor prognosis in GC. Collectively, NKX2‐1‐AS1 functions as a ceRNA to miR‐145‐5p and promotes tumor progression and angiogenesis by activating the VEGFR‐2 signaling pathway via SERPINE1.

MiR-27a-3p/miR-27b-3p Promotes Neurofibromatosis Type 1 via Targeting of NF1.

The dysregulation of microRNAs (miRNAs) is a crucial molecular signature of disease development. The potential implication of miRNAs in neurofibromatosis type 1 (NF1) remains poorly investigated. The expression levels of miR-27a-3p, miR-27b-3p, and neurofibromin 1 (NF1) were detected by real-time quantitative polymerase chain reaction (RT-qPCR) analysis. The functional roles of miR-27a-3p and miR-27b-3p in NF1 were explored by CCK8 (Cell Counting Kit-8), 5-ethynyl-2'-deoxyuridine (EdU), terminal deoxynucleoitidyl transferase dUTP nick-end labeling (TUNEL), and transwell assays. Luciferase reporter, RNA pull-down, and RNA binding protein immunoprecipitation (RIP) assays were employed to study the probable target relationship between miRNA and messenger RNA (mRNA). MiR-27b-3p and miR-27a-3p were upregulated in dermal and plexiform human Schwann cells (HSC) from NF1 neurofibromas as well as cell lines of malignant peripheral nerve sheath tumors (MPNSTs). MiR-27a-3p/miR-27b-3p mimics promoted the proliferative, migratory, and invasive ability of dermal HSC and MPNST cell ST88-14, while inhibiting the apoptotic capacity. MiR-27a-3p/miR-27b-3p inhibitors elicited the opposite impacts on the above cellular behaviors in dermal HSC and ST88-14. Intriguingly, NF1 was revealed to be the target of both miR-27a-3p and miR-27b-3p, and was negatively modulated by them. MiR-27a-3p/miR-27b-3p upregulation suppressed the expression of NF1 in dermal HSC and ST88-14. Furthermore, NF1 depletion counterbalanced the functional alteration induced by miR-27a-3p/miR-27b-3p inhibition. Our study suggests that both miR-27b-3p and miR-27a-3p are involved in upstream molecular activity responsible for the depletion of NF1, representing promising targets for therapeutic application in NF1.

Propofol suppresses cell viability, cell cycle progression and motility and induces cell apoptosis of ovarian cancer cells through suppressing MEK/ERK signaling via targeting circVPS13C/miR-145 axis

BackgroundPropofol is a kind of common intravenous anaesthetic agent that plays an anti-tumor role in a variety of cancers, including ovarian cancer. However, the working mechanism of Propofol in ovarian cancer needs further exploration.MethodsThe viability and metastasis of ovarian cancer cells were assessed by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and transwell assays. Flow cytometry was used to evaluate the cell cycle and apoptosis. Quantitative real-time polymerase chain reaction (qRT-PCR) was used to examine the abundance of circular RNA vacuolar protein sorting 13 homolog C (circVPS13C) and microRNA-145 (miR-145). The target relationship between miR-145 and circVPS13C was predicted by circinteractome database and verified by dual-luciferase reporter assay, RNA-binding protein immunoprecipitation (RIP) assay and RNA-pull down assay. Western blot assay was used to detect the levels of phosphorylated extracellular regulated MAP kinase (p-ERK), ERK, p-MAP kinse-ERK kinase (p-MEK) and MEK, in ovarian cancer cells.ResultsPropofol treatment suppressed the viability, cell cycle and motility and elevated the apoptosis rate of ovarian cancer cells. Propofol up-regulated miR-145 in a dose-dependent manner. Propofol exerted an anti-tumor role partly through up-regulating miR-145. MiR-145 was a direct target of circVPS13C. Propofol suppressed the progression of ovarian cancer through up-regulating miR-145 via suppressing circVPS13C. Propofol functioned through circVPS13C/miR-145/MEK/ERK signaling in ovarian cancer cells.ConclusionPropofol suppressed the proliferation, cell cycle, migration and invasion and induced the apoptosis of ovarian cancer cells through circVPS13C/miR-145/MEK/ERK signaling in vitro.

LINC00466 Impacts Cell Proliferation, Metastasis and Sensitivity to Temozolomide of Glioma by Sponging miR-137 to Regulate PPP1R14B Expression.

PurposeLINC00466 is a newfound long non-coding RNA (lncRNA) that has been rarely explored in cancers. However, the specific role and molecular mechanism of LINC00466 in glioma remain to be further elucidated.MethodsBioinformatic analysis was used to screen differentially expressed genes. Quantitative real-time PCR (qRT-PCR) was used to determine the expression of LINC00466, microRNA-137 (miR-137) and protein phosphatase 1 regulatory subunit 14B (PPP1R14B). Dual-luciferase reporter gene assay and RNA binding protein Immunoprecipitation (RIP) assays were employed to verify the binding relationship among LINC00466, miR-137 and PPP1R14B. The sensitivity of glioma cells to temozolomide (TMZ) was measured by cell counting kit-8 (CCK8) assay. The xenograft nude models were used to test the effects of LINC00466 on glioma tumor growth in vivo.ResultsHighly expressed LINC00466 and PPP1R14B and lowly expressed miR-137 were eventually revealed in glioma tissues. Overexpression of LINC00466 could promote proliferation, metastasis and drug sensitivity to TMZ of glioma cells. LINC00466 could bind to miR-137, and up-regulation of miR-137 could attenuate the enhancing effects caused by LINC00466 overexpression. We took a further step and found that miR-137 could bind to PPP1R14B. Besides, LINC00466 could function as a sponge to miR-137 to regulate PPP1R14B. In addition, overexpression of LINC00466 could promote tumor growth in vivo.ConclusionThese findings validate LINC00466 could restrain the miR-137 expression to up-regulate PPP1R14B and therefore promote proliferation, metastasis and resistance to TMZ of glioma.

LncRNA MCTP1-AS1 Regulates EMT Process in Endometrial Cancer by Targeting the miR-650/SMAD7 Axis.

BackgroundLong noncoding RNAs (lncRNAs) play critical roles in the pathogenesis of several diseases, especially some kinds of cancer. This study aimed to investigate the expression of MTCP1-AS1 and its effects on endometrial cancer (EC).MethodsMTCP1-AS1 expression level was determined in human EC tissues and cell lines by qRT-PCR. The role of MTCP1-AS1 on EC cell proliferation, migration, invasion and epithelial to mesenchymal transition (EMT) was detected by CCK8, wound-healing assay, transwell assay and Western blot, respectively. Moreover, luciferase reporter assay and RNA-binding protein immunoprecipitation (RIP) assay were performed to verify the targeting relationship between miR-650, MCTP1-AS1 and SMAD7 in EC cells.ResultsOur data showed that MCTP1-AS1 expression was downregulated in EC tissues and cell lines. Overexpression of MCTP1-AS1 inhibited cell proliferation, migration, invasion and EMT process of EC cells. Moreover, MCTP1-AS1 was proved to be the target of miR-650 and reversely correlated with its expression. In addition, MCTP1-AS1 reversed the effect of miR-650 on the EC cells, which might be associated with the role of SMAD7. Moreover, Western blot showed siRNA-SMAD7 transfection could rescue the repressed TGF-β/SMAD pathway induced by MCTP1-AS1 in EC cells.ConclusionTaken together, these data suggested that lncRNA MCTP1-AS1 inhibited cell proliferation, migration, invasion and EMT process of EC cells via targeting the miR-650/SMAD7 axis and it has the potential to be explored as a therapeutic target for the treatment of EC in the future.

Exosomal Long Non-coding RNA HOTTIP Increases Resistance of Colorectal Cancer Cells to Mitomycin via Impairing MiR-214-Mediated Degradation of KPNA3.

It has been reported that long non-coding RNA HOXA distal transcript antisense RNA (lncRNA HOTTIP) functions as a tumor promoter in colorectal cancer (CRC). Hence, we paid attention to exploring whether exosomes could carry lncRNA HOTTIP to affect the mitomycin resistance in CRC and to identify the underlying mechanisms. High expression of HOTTIP was detected in mitomycin-resistant CRC cells. Inhibition of HOTTIP reduced the mitomycin resistance. In the co-culture system of mitomycin-resistant cells or their derived exosomes with CRC cells, the HOTTIP was found to be transferred into the parental cells via extracellular vesicles (EVs) secreted from mitomycin-resistant cells and to contribute to the mitomycin resistance. Based on the bioinformatics databases, possible interaction network of HOTTIP, microRNA-214 (miR-214) and Karyopherin subunit alpha 3 (KPNA3) in CRC was predicted, which was further analyzed by dual-luciferase reporter, RNA binding protein immunoprecipitation and RNA pull-down assays. As HOTTIP down-regulated miR-214 to elevate the KPNA3 expression, HOTTIP enhanced the mitomycin resistance through impairing miR-214-dependent inhibition of KPNA3. Finally, HOTTIP was suggested as an independent factor predicting mitomycin response in patients with CRC. Those data together confirmed the promotive effects of EV-carried HOTTIP on the mitomycin resistance, while targeting HOTTIP might be a promising strategy overcoming drug resistance in CRC.

LINC00461 facilitates HNSCC development and reduces chemosensitivity by impairing miR-195-mediated inhibition of HOXA10

Homeobox A10 (HOXA10) has been regarded to serve as an oncogene in head and neck squamous cell carcinoma (HNSCC). This study was intended to explore the interaction among the long intergenic noncoding RNA 00461 (LINC00461), microRNA (miR)-195, and HOXA10, and to investigate its role in epithelial-mesenchymal transition (EMT) and chemoresistance in HNSCC. The effects of LINC00461, miR-195, and HOXA10 on the EMT and chemoresistance of HNSCC cells were analyzed by comprehensive analysis of gain- and loss-of-function techniques. The intimate relationships among LINC00461, miR-195, and HOXA10 were investigated by several procedures such as RNA-binding protein immunoprecipitation, RNA pull-down, and dual-luciferase reporter assays. A xenotransplantation tumor model in nude mice was established for the assessment of the tumorigenic ability of the cells in vivo. Our findings indicated that LINC00461 was highly expressed in HNSCC and its overexpression induced EMT and precipitated the chemoresistance of HNSCC cells to cisplatin. The LINC00461 could bind to miR-195 while miR-195 targeted HOXA10 independently. Moreover, LINC00461 impaired miR-195-mediated inhibition of HOXA10 to induce EMT and increase the chemoresistance in HNSCC. Tumor weight and volume were reduced by lentivirus-mediated elevation of miR-195 by inhibition of HOXA10, which could be annulled by LINC00461 overexpression. LINC00461 downregulates the expression of miR-195 to subsequently upregulate the expression of HOXA10, thereby promoting EMT and enhancing chemoresistance in HNSCC.

Silencing of long noncoding RNA MYLK-AS1 suppresses nephroblastoma via down-regulation of CCNE1 through transcription factor TCF7L2.

Nephroblastoma, a pediatric kidney cancer, caused by pluripotent embryonic renal precursors. Long noncoding RNAs (lncRNAs) are commonly abnormal expressed in many cancers. In the present study, we fousced on one newly discrovered lncRNA, MYLK Antisense RNA 1 (MYLK-AS1), and its functional role in proliferation and cycle distribution of nephroblastoma cells. Micorarray-based analysis revealed the highly expressed Cyclin E1 (CCNE1) and MYLK-AS1 in nephroblastoma. After nephroblastoma tissue sample collection, RT-qPCR confirmed the upregulated expression of MYLK-AS1 and CCNE1 in nephroblastoma tissues and cells. Kaplan-Meier curve exhibited that patients with elevated CCNE1 had lower overall survival rate in follow-up study. RNA binding protein immunoprecipitation, chromatin immunoprecipitation, and dual-luciferase reporter gene assay were employed to determine the relationship among MYLK-AS1, TCF7L2, and CCNE1, which validated that transcription factor 7-like 2 (TCF7L2) could specifically bind to MYLK-AS1 and TCF7L2 could positively promote CCNE1. After gain- and loss-of function assays, the conclusion that silencing of MYLK-AS1 could inhibit expression of CCNE1 through the transcription factor TCF7L2 to regulate the cell proliferation and cell cycle distribution of nephroblastoma cells was obtained. Subsequently, the subcutaneous tumor formation ability of nephroblastoma cell in nude mice was observed and the silencing of MYLK-AS1 exerts suppressive role in the tumorigenic ability of nephroblastoma cells in vivo. Taken together, MYLK-AS1 constitutes a promising biomarker for the early detection and treatment of nephroblastoma.

LncRNA BCYRN1-induced autophagy enhances asparaginase resistance in extranodal NK/T-cell lymphoma.

Background: Asparaginase (ASP) is the cornerstone drug in the treatment of extranodal NK/T-cell lymphoma (ENKTCL), and the mechanisms of resistance to ASP remain largely unknown. Long non-coding RNAs play important roles in chemotherapy resistance in various cancers. However, the expression of BCYRN1 and its role in ENKTCL still remain unidentified.Methods: Lentivirus-mediated BCYRN1 overexpression and knockdown were performed in SNK-6 cells. Cell autophagy was analyzed by adenovirus expressing GFP-LC3B fusion protein. RNA pull-down and RNA Binding Protein Immunoprecipitation Assay were performed to investigate the relationship between BCYRN1 and p53. Western blot analysis was performed to assess the effect of BCYRN1 on different autophagy pathways. Finally, in vivo xenograft tumor model was constructed to analyze the effect of BCYRN1 on tumor growth and ASP resistance.Results: BCYRN1 was overexpressed in ENKTCL than normal NK cells, and patients with higher expression had significantly inferior progression-free survival (PFS). The IC50 value of ASP was significantly increased in BCYRN1-overexpressed SNK-6 cells and BCYRN1 overexpression could resist the inhibitory effect of ASP on proliferation. ASP could induce concurrent apoptosis and autophagy in ENKTCL, and the latter process was enhanced by overexpression of BCYRN1, mainly through affecting both PI3K/AKT/mTOR and p53/mTOR pathways. BCYRN1 could induce the degradation of p53 via ubiquitination, thus resulting in enhancement of autophagy and ASP resistance, which could be reversed by drug-induced autophagy inhibition. The effect of BCYRN1 on tumor growth and autophagy were confirmed in vivo xenograft model.Conclusions: It was found that BCYRN1 was a valuable prognostic biomarker in ENKTCL. BCYRN1 could promote resistance to ASP by inducing autophagy, which could be reversed by inhibition of autophagy. Our findings highlight the feasibility of combining autophagy inhibition and ASP in the treatment of ENKTCL.

Downregulation of lncRNA HCP5 has inhibitory effects on gastric cancer cells by regulating DDX21 expression

LncRNA HCP5 has been confirmed to play crucial roles in many types of cancers. However, the role of lncRNA HCP5 in regulating the occurrence and development of gastric cancer (GC) remains unknown. In the current study, we aimed to investigate the precise effects of lncRNA HCP5 on cell proliferation, migration and invasion and molecular mechanisms in gastric cancer. Using RT-qPCR analysis, we found that lncRNA HCP5 was differentially expressed in GC cell lines. CCK-8, wound healing and transwell assay indicated that the proliferation, migration and invasion of gastric cancer cells were inhibited by downregulation of lncRNA HCP5 and lncRNA HCP5 overexpression exhibited the opposite effects in gastric cancer cells. Mechanistically, RNA binding protein immunoprecipitation and dual luciferase reporter assay confirmed the interaction between lncRNA HCP5 and DDX21. The effects of lncRNA HCP5 overexpression the proliferation, migration and invasion of GC cells were partly rescued by DDX21 silencing. Taken together, downregulation of lncRNA HCP5 exerted inhibitory effects on GC cell proliferation, migration and invasion through modulation of DDX21 expression, demonstrating the function of lncRNA HCP5 and DDX21 in GC progression.

Long Noncoding-RNA Component of Mitochondrial RNA Processing Endoribonuclease Promotes Carcinogenesis in Triple-Negative Breast Cancer Cells via the Competing Endogenous RNA Mechanism.

PurposeTriple-negative breast cancer (TNBC) is a subtype of breast cancer. Increasing evidence supports that dysregulation of long noncoding RNAs (lncRNAs) plays a vital role in cancer progression. RNA component of mitochondrial RNA processing endoribonuclease (RMRP), a lncRNA, is characterized as a tumor-propeller in some cancers, but its mechanism in TNBC remains poorly understood. This study aimed to determine whether and how RMRP functions in TNBC.MethodsCell proliferation was determined by cell counting kit-8 (CCK-8) and colony formation assays and cell apoptosis by flow cytometry analysis and terminal deoxynucleotidyl transferase-mediated nick end labeling (TUNEL) assay. Cell migration and invasion were determined by transwell assays. RNA-binding protein immunoprecipitation (RIP), luciferase reporter, and RNA pulldown assays were implemented to assess the interaction of RMRP with other molecules in TNBC cells.ResultsRMRP expression was elevated in TNBC cells. RMRP knockdown repressed cell proliferation, migration, and invasion, but induced apoptosis in TNBC. In addition, RMRP was found to target microRNA-766-5p (miR-766-5p) in TNBC cells. Silencing miR-766-5p enhanced cell viability and decreased apoptosis, whereas miR-766-5p overexpression had opposite effects. Furthermore, miR-766-5p was found to bind to yes-associated protein 1 (YAP1). Moreover, miR-766-5p inhibition reversed the repressive effect of RMRP knockdown on the malignant progression of TNBC.ConclusionThe present study manifested that RMRP promotes the growth, migration, and invasion of TNBC cells via the miR-766-5p/YAP1 axis. These findings provide novel perspectives for TNBC treatment.

Exosomal circular RNA circ_0074673 regulates the proliferation, migration, and angiogenesis of human umbilical vein endothelial cells via the microRNA-1200/MEOX2 axis

ABSTRACT Circular RNAs (circRNAs) are implicated in the pathogenesis of gestational diabetes mellitus (GDM). The aim of this study was to investigate the roles and molecular mechanism underlying the effects of circ_0074673 in GDM. Exosomal morphology was visualized by transmission electron microscopy (TEM), while exosomal size and concentration were determined by nanoparticle tracking analysis (NTA). The expression of CD9 and CD63 was measured by western blotting. The levels of circ_0074673, miR-1200 and mesenchyme homeobox 2 (MEOX2) were determined by quantitative real-time polymerase chain reaction (qPCR). Cellular proliferation, migration, and angiogenesis were measured by Cell Counting Kit-8 (CCK-8), transwell, and tube formation assays, respectively. The binding relationship between circ_0074673 or MEOX2 and miR-1200 was evaluated by luciferase reporter assay, RNA-binding protein immunoprecipitation (RIP) assay and RNA-pull-down assay. The results showed that exosomal size and concentration were greater in the umbilical cord blood of patients with GDM than in that of the healthy controls. The expression of circ_0074673 was upregulated in exosomes from GDM and in human umbilical vein endothelial cells (HUVECs) co-cultured with exosomes. High glucose (HG) treatment suppressed cellular proliferation, migration, and angiogenesis. Circ_0074673 knockdown enhanced the proliferation, migration, and angiogenesis of HG treated HUVECs (HG-HUVECs). As circ_0074673 and MEOX2 directly bind to miR-1200, circ_0074673 silencing promoted the biological functions of HG-HUVECs by sponging miR-1200 and further targeting MEOX2. Altogether, the loss of exosomal circ_0074673 facilitated the proliferation, migration, and angiogenesis of HG-HUVECs via the miR-1200/MEOX2 axis, suggesting that circ_0074673 is a potential therapeutic target for GDM.

LINC01224 Promotes Colorectal Cancer Progression by Sponging miR-2467.

IntroductionColorectal cancer (CRC) is one of the most common human cancers and a leading cause of cancer-related death. Accumulating evidence has confirmed that long non-coding RNA (lncRNA) plays crucial roles in CRC development.MethodsqRT-PCR was performed to examine the expressions of LINC01224 and miR-2467. CCK-8 assay, colony formation assay and transwell invasion assay were used to examine the progression of breast cancer cells. Luciferase and RNA-binding protein immunoprecipitation (RIP) assay were applied to verify the binding site. Correlation analysis of miR-2467 and LINC01224 expression in lung cancer tissues was shown. Pancreatic cancer cells growth in vivo was evaluated using xenograft tumor assay.ResultsLINC01224 expression was observed to be up-regulated in CRC tissues and cell lines. Functional studies suggested that LINC01224 silence inhibited CRC cells proliferation and invasion of CRC cells, while co-transfection with a miR-2467 inhibitor reversed these biological effects. Luciferase reporter assays illustrated that LINC01224 regulated miR-2467 directly, and RNA-binding protein immunoprecipitation (RIP) further confirmed that the suppression of LINC01224 by miR-2467 was in an RISC-dependent manner. Finally, LINC01224 silence inhibited the growth CRC cells in vivo.ConclusionIn conclusion, our findings showed that LINC01224 promoted CRC progression through sponging miR-2467. LINC01224 may serve as a potential diagnostic biomarker and therapeutic target for CRC patients.