LncRNA-KCNQ1OT1 Promotes the Odontoblastic Differentiation of Dental Pulp Stem Cells via Regulating miR-153-3p/RUNX2 Axis

Background and Objective: Long non-coding RNAs (LncRNAs) play a key role in the odontoblastic differentiation. This study aimed to explore the role of LncRNA-KCNQ1OT1 in the odontoblastic differentiation of human dental pulp stem cells (DPSCs) and its possible mechanism. Methods: The expression of LncRNA-KCNQ1OT1, miR-153-3p, RUNX2 in the odontoblastic differentiation was detected by qRT-PCR. Interaction between LncRNA-KCNQ1OT1 and miR-153-3p and interaction between miR-153-3p and RUNX2 were detected by dual-luciferase assay. The cell viability of DPSCs was detected by cell counting kit-8 (CCK-8), and the effect of LncRNA-KCNQ1OT1 and miR-153-3p on the odontoblastic differentiation of DPSCs was observed by alizarin red staining, alkaline phosphatase (ALP) activity assay and Western blot for RUNX2, DSPP, DMP-1. Results: During odontoblastic differentiation of DPSCs, the expression of LncRNA-KCNQ1OT1 increased, miR-153-3p expression decreased, and RUNX2 expression increased. Dual-luciferase assay showed that LncRNA-KCNQ1OT1 sponges miR-153-3p and miR-153-3p targets on RUNX2. After LncRNA-KCNQ1OT1 and miR-153-3p expressions of DPSCs were changed, the cell viability was not notably changed, but the odontoblastic differentiation was notably changed which was confirmed with alizarin red staining, ALP activity and Western blot for RUNX2, DSPP, DMP-1. Conclusion: LncRNA-KCNQ1OT1 promotes the odontoblastic differentiation of DPSCs via regulating miR-153-3p/RUNX2 axis, which may provide a therapeutic clue for odontogenesis.

LncRNA-KCNQ1OT1 Promotes the Odontoblastic Differentiation of Dental Pulp Stem Cells Via Regulating miR-153-3p/RUNX2 Axis

Background and Objective: Long non-coding RNAs (LncRNAs) play a key role in the odontoblastic differentiation. This study aimed to explore the role of LncRNA-KCNQ1OT1 in the odontoblastic differentiation of human dental pulp stem cells (DPSCs) and its possible mechanism. Methods: The expression of LncRNA-KCNQ1OT1, miR-153-3p, RUNX2 in the odontoblastic differentiation was detected by qRT-PCR. Interaction between LncRNA-KCNQ1OT1 and miR-153-3p and interaction between miR-153-3p and RUNX2 were detected by dual-luciferase assay. The cell viability of DPSCs was detected by cell counting kit-8 (CCK-8), and the effect of LncRNA-KCNQ1OT1 and miR-153-3p on the odontoblastic differentiation of DPSCs was observed by alizarin red staining, alkaline phosphatase (ALP) activity assay and Western blot for RUNX2, DSPP, DMP-1.Results: During odontoblastic differentiation of DPSCs, the expression of LncRNA-KCNQ1OT1 increased, miR-153-3p expression decreased, and RUNX2 expression increased. Dual-luciferase assay showed that LncRNA-KCNQ1OT1 sponges miR-153-3p and miR-153-3p targets on RUNX2. After LncRNA-KCNQ1OT1 and miR-153-3p expressions of DPSCs were changed, the cell viability was not notably changed, but the odontoblastic differentiation was notably changed which was confirmed with alizarin red staining, ALP activity and Western blot for RUNX2, DSPP, DMP-1.Conclusion: LncRNA-KCNQ1OT1 promotes the odontoblastic differentiation of DPSCs via regulating miR-153-3p/RUNX2 axis, which may provide a therapeutic clue for odontogenesis.

MicroRNA-5100 Modulates Lung Cancer Cell Proliferation and Apoptosis via Inhibiting X-Linked Inhibitor of Apoptosis Protein (XIAP) Expression

This study assesses the miR-5100 expression and its function in human lung cancer. The expression of miR-5100 was analyzed by miScript miRNA method. Cancer cells were transfected with miR-5100 mimics (miR-5100), miR-5100 inhibitors (ASO-miR-5100), XIAP inhibitors (si-XIAP), negative controls (NC) followed by analysis of cell proliferation by MTT and apoptosis by flow cytometry, the expression of XIAP related proteins by Western blot. miR-5100’ target was predicted by bioinformatics website and verified by dual luciferase assay. Finally, a xenogeneic tumor inhibition model was established to detect tumor progression after treatments. Lung cancer cells and tissues exhibited significantly reduced miR-5100 level. Dual luciferase assay showed that miR-5100 bound XIAP 3′-UTR and reduced XIAP mRNA and protein level. Further, miR-5100 inhibited cell proliferation, increased apoptosis and the expression of cleaved-capsase-3 and cleaved-capsase-9, the XIAP downstream factor. Finally, miR-5100 inhibited tumor growth, decreased cellular proliferation and promoted apoptosis, accompanied by reduced XIAP expression in vivo. miR-5100 inhibits lung cancer cell proliferation and enhances apoptosis through inhibiting XIAP expression in vitro and in vivo.

LncRNA TCL6 Contributes to CSC-like Properties via Modulating TP53 in HCC

BackgroundHepatocellular carcinoma (HCC) is the most common malignant tumors, accounting for most of the adult primary liver cancer. Herein, we aimed to analyze the expression of long non-coding RNA-T cell leukemia/lymphoma 6 (lncRNA-TCL6) in HCC and elucidate its mechanism involved in the HCC progression. Methodse performed RNA extraction and quantitative real-time polymerase chain reaction assays, spheroid formation assays, flow cytometry and western blot assays to assess the effect of TCL6 on the liver CSCs marker CD133 expression rate, sphere-forming ability of liver stem cells, and the relationship between TCL6 expression and stem cell factor (TP53, P21, CD44, KLF4, OCT4, Nanog, and Sox2). In addition, we used a dual luciferase assay to verify the relationship between miR-106a-5p and TP53.ResultsKnockdown of TCL6 expression significantly improved the CD133 expression rate and the liver stem cells sphere-forming ability in HCC, while TCL6 overexpression in HCC showed the opposite effect. Knockdown of TCL6 upregulated the KLF4mRNA expression, while TCL6 overexpression in HCC inhibited the TP53 and CDKN1A expression. Western blot assays showed that TCL6 expression was positively correlated with TP53 and P21, while negatively correlated with stem cell factor. Dual luciferase assay showed that TP53 was a target of miR-106a-5p.ConclusionResults suggested that reprogramming-related TCL6 may be a novel tumor suppressor gene in HCC, which inhibits the self-renewal of liver CSCs, in part by promoting the TP53 expression.

Inhibition of miR-96-5p May Reduce Aβ 42/Aβ40 Ratio via Regulating ATP-Binding Cassette A1

Background: Imbalance between amyloid-β (Aβ) production and clearance results in Aβ accumulation. Regulating Aβ levels is still a hot point in the research of Alzheimer’s disease (AD). Objective: To identify the differential expression of ATP-binding cassette A1 (ABCA1) and its upstream microRNA (miRNA) in AD models, and to explore their relationships with Aβ levels. Methods: Quantitative real-time polymerase chain reaction (qRT-PCR) and western blotting were performed to determine the expression of ABCA1 in 5xFAD mice, SH-SY5Y cells treated with Aβ oligomers and SH-SY5YAβPP695 cells (AD models). TargetScan was used to predict the upstream miRNAs for ABCA1. Dual-luciferase assay was conducted to identify the regulation of the miRNA on ABCA1. qRT-PCR was used to measure the expression of miRNA in AD models. Finally, enzyme-linked immunosorbent assays were performed to detect Aβ 42 and Aβ40 levels. Results: The expression of ABCA1 was significantly down regulated in AD models at both mRNA and protein levels. Dual-luciferase assay showed that miR-96-5p could regulate the expression of ABCA1 through binding to the 3 untranslated region of ABCA1. The level of miR-96-5p was significantly elevated in AD models. The expression of ABCA1 was enhanced while Aβ 42 levels and Aβ 42/Aβ 40 ratios were reduced in SH-SY5Y A βPP695 cells after treated with miR-96-5p inhibitor. Conclusion: The current study found that miR-96-5p is the upstream miRNA for ABCA1. Suppression of miR-96-5p in AD models could reduce Aβ 42/Aβ 40 ratios via up regulating the expression of ABCA1, indicating that miR-96-5p plays an important role in regulating the content of Aβ.

miR-133a-3p Functions as a Tumor Suppressor in Colorectal Cancer by Targeting PFKFB3

Background Recent studies reveal that PFKFB3 plays an important role in tumorigenesis and tumor progression. Our study aims to identify an novel microRNA which can suppress the expression of PFKFB3 and to provide a potential target for tumor therapy. Methods Bioinformatics methods were implemented to explore the expression and clinical significance of PFKFB3 and miR-133a-3p in colorectal cancer (CRC). qRT-PCR was performed to detect PFKFB3, miR-133a-3p, KI67 and MMP9 mRNA expression, while western bot was carried out for the detection of protein expression of PFKFB3, miR-133a-3p, KI67 and MMP9. Bioinformatics analysis was used to predict the binding sites of miR-133a-3p on PFKFB3 3’UTR, while dual-luciferase assay was conducted to validate their binding relationship. CCK-8 assay, KI67 detection, Transwell assay and MMP9 detection were employed to measure CRC cell proliferative and invasive abilities. Results PFKFB3 expression is up-regulated in colorectal cancer, and is significantly associated with poor prognosis. Silencing PFKFB3 could inhibit the proliferation and invasion of colorectal cancer cells. miR-133a-3p is down regulated in colorectal cancer, which has diagnostic value for colorectal cancer. Dual luciferase assay confirmed that PFKFB3 was the direct acting site of miR-133a-3p. Overexpression of miR-133a-3p could significantly reduce the expression of PFKFB3 and inhibit the effect of PFKFB3 on the proliferation and invasion of colorectal cancer cells. Conclusions Our study suggested that miR-133a-3p functions as a novel tumor suppressor in colorectal cancer by targeting PFKFB3.

LncRNA AFAP1-AS1 Promotes the Progression of Colorectal Cancer through miR-195-5p and WISP1

Objective. Colorectal cancer (CRC) is the most common cancer. But, the molecular mechanisms of CRC progression are not fully understood. This study was conducted to explore how the long noncoding RNA actin filament-associated protein 1-antisense RNA1 (lncRNA AFAP1-AS1) participates in CRC progression through the regulation of microRNA-195-5p (miR-195-5p) and wingless-type inducible signaling pathway protein-1 (WISP1). Methods. The expressions of AFAP1-AS1, miR-195-5p, and WISP1 were detected by RT-qPCR or western blot. A dual-luciferase assay confirmed the target relationship of AFAP1-AS1, miR-195-5p, and WISP1. Colony formation, wound-healing, and Transwell assays were used to detect the growth, migration, and invasion abilities of cells, respectively. Results. AFAP1-AS1 and WISP1 expressions were notably increased, and miR-195-5p expression was markedly reduced in CRC. The dual-luciferase assay verified that AFAP1-AS1 could bind to miR-195-5p. AFAP1-AS1 knockdown could inhibit the malignant behavior of CRC cells. miR-195-5p could target and regulate WISP1 expression. Overexpression of WISP1 or miR-195-5p inhibition reversed the inhibition effect of AFAP1-AS1 knockdown on the biological activity of CRC cells. Conclusions. AFAP1-AS1 knockdown may inhibit the proliferation, migration, and invasion of CRC cells through the miR-195-5p/WISP1 axis.

miR-223-5p Suppresses OTX1 to Mediate Malignant Progression of Lung Squamous Cell Carcinoma Cells

Background. Lung squamous cell carcinoma (LUSC) features high morbidity and mortality as a worldwide malignant tumor. This study mainly explored a miR-223-5p-dependent mechanism that affected proliferation, invasion, and migration of LUSC cells. Methods. Expression data of mature miRNAs and sequencing data of total RNA of LUSC were downloaded from TCGA database. Differentially expressed mRNAs were obtained. Function of miR-223-5p in LUSC cells was detected by assays like qRT-PCR, MTT, wound healing assay, Western blot, and Transwell assay. Western blot was performed to analyze the relationship between OTX1 and JAK/STAT signaling pathways. Dual-luciferase assay detected the relationship between miR-223-5p and OTX1. The way how miR-223-5p regulated LUSC cell biological functions via OTX1 was further explored. Results. It was noted that miR-223-5p expression in LUSC tissue and cells was significantly reduced. Overexpression of miR-223-5p negatively regulated the proliferation, invasion, and migration of LUSC cells. The downstream target gene OTX1 was detected to be notably elevated in LUSC cells. A negative correlation between OTX1 and miR-223-5p was also found. As analyzed by GSEA, OTX1 was significantly enriched in the JAK/STAT signaling pathway and activated the pathway. Dual-luciferase assay demonstrated that OTX1 was a direct molecular target of miR-223-5p in LUSC cells. Rescue experiment verified that miR-223-5p regulated the malignant phenotypes of LUSC cells by pairing with OTX1. Conclusion. This study indicated that miR-223-5p was lowly expressed in LUSC cells. The impact of miR-223-5p on cell proliferation, invasion, and migration was realized by targeting OTX1. It is likely that miR-223-5p can be a novel target for LUSC treatment, which provides new ideas for future LUSC treatment.

MiR-133a-3p Functions as a Tumor Suppressor in Colorectal Cancer by Targeting PFKFB3

Background: Recent studies reveal that PFKFB3 plays an important role in tumorigenesis and tumor progression. Our study aims to identify an novel microRNA which can suppress the expression of PFKFB3 and to provide a potential target for tumor therapy.Methods: Bioinformatics methods were implemented to explore the expression and clinical significance of PFKFB3 and miR-133a-3p in colorectal cancer (CRC). qRT-PCR was performed to detect PFKFB3, miR-133a-3p, KI67 and MMP9 mRNA expression, while western bot was carried out for the detection of protein expression of PFKFB3, miR-133a-3p, KI67 and MMP9. Bioinformatics analysis was used to predict the binding sites of miR-133a-3p on PFKFB3 3’UTR, while dual-luciferase assay was conducted to validate their binding relationship. CCK-8 assay, KI67 detection, Transwell assay and MMP9 detection were employed to measure CRC cell proliferative and invasive abilities. Results: PFKFB3 is up-regulated in CRC and significantly associated with poor prognosis. Overexpressed PFKFB3 promotes CRC cell proliferation and invasion. miR-133a-3p is down-regulated and has diagnostic value in CRC. Dual-luciferase assay confirmed that there was a binding relationship between miR-133a-3p and PFKFB3.Overexpressed miR-133a-3p remarkably reduced PFKFB3 expression in CRC cells, weakened the promoting effect of PFKFB3 on cell proliferation and invasion.Conclusions: Our study suggests that miR-133a-3p functions as a novel tumor suppressor in colorectal cancer by targeting PFKFB3.

Long Noncoding RNA NEAT1 Promotes Tumorigenesis in H. Pylori Gastric Cancer By Sponging miR-30a To Regulate COX-2/BCL9 Pathway

Background: Helicobacter pylori (H. pylori) is a carcinogenic factor for gastric cancer. Our previous study demonstrated that H. pylori decreased the expression of microRNA(miRNA)-30a to promote the tumorigenesis in gastric cancer. However, the upstream regulatory mechanism of miR-30a hasn’t well-elucidated. In this study, we found the long non‑coding RNA (lncRNA) NEAT1 may sponge miR-30a to regulate COX-2/BCL9 pathway. Methods: The expression of NEAT1 was detected in gastric cancer tissues and tumour adjacent tissues by fluorescence in-situ hybridization(FISH) analysis and RT-qPCR. LncRNA-miRNA interaction networks were constructed using the RNAhybird and Starbase v.2.0. and then validated using dual-luciferase assay. The effects of NEAT1 dysregulation on the proliferative, migratory and invasive abilities of H. pylori filtrate infected gastric cancer cells were observed by cell counting kit-8 (CCK-8), colony formation, wound healing test and transwell assays. Western blot and RT-qPCR were performed to detect protein and RNA expression. The Immunohistochemistry(IHC) was carried out to analyze the location and expression of COX-2 and BCL9Results: FISH and RT-qPCR demonstrated that the expression of NEAT1 was up-regulated in gastric cancer tissues, especially in H. pylori gastric cancer tissues, and the expression of NEAT1 is negatively correlated with miR-30a(miR-30a-3p, miR-30a-5p). The proliferation, migration and invasion of H. pylori filtrate infected gastric cancer cells could be largely enhanced by the up-regulation of NEAT1, while the downregulation of NEAT1 decreased these abilities, and miR-30a could reverse the effect of NEAT1 on these abilities. Dual-luciferase assay identified that NEAT1 directly targeted miR-30a(miR-30a-3p, miR-30a-5p).Due to miR-30a(miR-30a-3p, miR-30a-5p) negatively regulated the expression of downstream COX-2 and BCL9, NEAT1 was identified to indirectly upregulate the expression of COX-2 and BCL9.IHC showed that the expression of COX-2 and BCL9 were increased in H. pylori gastric cancer tissues.Conclusion: The study demonstrated that lncRNA NEAT1 may act as a promoter oftumorigenesis in H. pylori gastric cancer, by sponging miR-30a(miR-30a-3p, miR-30a-5p) to regulate COX-2/BCL9 pathway.