Dual-luciferase Reporter Gene Experiment Research Articles

GATA3 Exerts Distinct Transcriptional Functions to Regulate Radiation Resistance in A549 and H1299 Cells

Background Radiation resistance of lung cancer cells is a vital factor affecting the curative effect of lung cancer. Transcription factor GATA3 is involved in cell proliferation, invasion, and migration and is significantly expressed in a variety of malignancies. However, the molecular mechanism governing GATA3 regulation in lung cancer cells' radiation resistance is unknown. Methods Radiation-resistant cell models (A549-RR and H1299-RR) were made using fractionated high-dose irradiation. Use clone formation, CCK-8, F-actin staining, cell cycle detection, and other experiments to verify whether the model is successfully constructed. Cells were transiently transfected with knockdown or overexpression plasmid. To explore the relationship between GATA3/H3K4me3 and target genes, we used ChIP-qPCR, ChIP-seq, and dual luciferase reporter gene experiments. Xenograft tumor models were used to evaluate the effect of GATA3 depletion on the tumorigenic behavior of lung cancer cells. Results We report that transcription factors GATA3 and H3K4me3 coactivate NRP1 gene transcription when A549 cells develop radiation resistance. However, the mechanism of radiation resistance in H1299 cells is that GATA3 acts as a transcription inhibitor. The decrease of GATA3 will promote the increase of NRP1 transcription, in which H3K4me3 does not play a leading role. Conclusions GATA3, an upstream transcriptional regulator of NRP1 gene, regulates the radioresistance of A549 and H1299 cells by opposite mechanisms, which provides a new target for radiotherapy of lung cancer.

Circ_0000467 modulates malignant characteristics of colorectal cancer via sponging miR-651-5p and up-regulating DNMT3B

Circular RNAs (circRNAs) are widely expressed in cancer tissues and participate in modulating the progression of malignant tumors, playing a pro- or anti-cancer role. This work is conducted to probe the precise role of circ_0000467 in colorectal cancer (CRC) and its regulatory mechanism. The differentially expressed circRNAs in CRC tissues and paracancerous tissues were screened by bioinformatics analysis. The expression levels of circ_0000467, miR-651-5p and DNA methyltransferases 3B (DNMT3B) mRNA in CRC tissues and cells were detected by qRT-PCR. circ_0000467 knockdown cell model was constructed to investigate the effects of circ_0000467 on CRC cell growth, migration and invasion by CCK-8 and Transwell experiments. Western blot was performed to examine DNMT3B protein expression in CRC cells. Dual-luciferase reporter gene experiment was executed to validate the targeting relationship between circ_0000467 and miR-651-5p, miR-651-5p and DNMT3B. Circ_0000467 expression and DNMT3B mRNA expression were increased and miR-651-5p expression was down-regulated in CRC tissues and cell lines. Knockdown of circ_0000467 repressed CRC cell growth, migration and invasion. Dual-luciferase reporter gene experiments validated that miR-651-5p was a direct target of circ_0000467 and miR-651-5p could specifically bind with DNMT3B 3’UTR. Functional compensation experiments showed that the regulatory effect of circ_0000467 on CRC cells’ behaviors could be partially counteracted by miR-651-5p. Circ_0000467 may enhance the growth and metastasis of CRC cells by targeting miR-651-5p and up-regulating DNMT3B expression. Circ_0000467 may be a potential diagnostic biomarker and therapeutic target for CRC.

Circ_0001955 promotes the progression of non-small cell lung cancer via miR-769-5p/EGFR axis

ABSTRACT To elaborate on the role of circular RNA 0001955 (circ_0001955) on the proliferation and apoptosis of non-small cell lung cancer (NSCLC) cells and its underlying mechanism. Circ_0001955 expression in NSCLC was screened out through bioinformatics analysis based on GEO database. Circ_0001955, microRNA-769-5p (miR-769-5p), and epidermal growth factor receptor (EGFR) expression in NSCLC tissues and cell lines was examined using quantitative real-time PCR (qRT-PCR) and Western blot. Cell proliferation and apoptosis were examined using the CCK-8 method, BrdU experiment and flow cytometry analysis, respectively. Bioinformatics prediction, dual-luciferase reporter gene experiment and RNA immunoprecipitation (RIP) experiments were applied to validate the targeting relationship between miR-769-5p and circ_0001955 and the 3’ UTR of EGFR. Pearson’s correlation analysis was employed to validate the correlations among them. Circ_0001955 expression was up-regulated in NSCLC tissues and cell lines, and its overexpression was strongly associated with increased tumor TNM stage and lymph node metastasis. Circ_0001955 overexpression enhanced the proliferation and restrained the apoptosis in NSCLC cells, whereas knocking down circ_0001955 exerted the opposite effects. Circ_0001955 directly targeted miR-769-5p and negatively regulated its expression. EGFR, a target gene of miR-769-5p, could be indirectly and positively regulated by circ_0001955. Correlation analysis indicated that circ_0001955 was negatively correlated with miR-769-5p expression, while circ_0001955 was positively correlated with EGFR expression. Circ_0001955 facilitates the proliferation and represses the apoptosis of NSCLC cells by modulating miR-769-5p/EGFR axis.

Bone Marrow Mesenchymal Stem Cells (BMSCs)-Triggered Up-Regulation of miR-198 Impedes the Aggressive Migration and Invasion of Cervical Cancer Cells

As a subset of RNAs without protein-coding function, short non-coding RNAs (microRNAs) are reported to contribute to the progress of multiple disorders. Nevertheless, the precise function of miR-198 in human cervical cancer is still an open question. RNA sequencing between cervical cancer cell lines and normal cervical epithelial cells identified CCAR1 to be highly expressed in cervical cancer. Cells were transfected with si-CCAR1 followed by analysis of cell behaviors using clonogenic assay and transwell migrating assay. The binding of miR-198 and CCAR1 was verified by a dual-luciferase reporter gene experiment. CCAR1 was highly expressed in cervical cancer tissues and cell lines and associated with tumor staging. Knockdown of CCAR1 restrained the malignant phenotypes of cervical cancer cells. CCAR1 was a target of miR198. Co-culture with BMSCs upregulated miR-198 expression, resulting in impediment of the aggressive phenotypes of cervical cancer cells, which was mediated by suppression of CCAR1 and release of inflammatory factors. In conclusion, CCAR1 level is increased in cervical cancer tissues or cell lines. Co-culture of BMSCs can facilitate the proliferating, migrating and invading activities of cervical cancer cells but reduce the release of inflammatory factors which is possibly through manipulating the axis of miR-198/CCAR1.

1468-P: Effect of Kcnq1ot1 on Influencing the Function of Pancreatic ß-Cells Targeting miR-92a-1-5p

Objective: To study the role and mechanism of Kcnq1ot1 by targeting to regulate miR-92a-1-5p to effect insulin secretion in pancreatic β cells. Methods: The qRT-PCR was used to detect the expression of Kcnq1ot1 in different tissues of C57BL/6J mice, Min6 with PA intervention, islets of T2D model mice and the serum of T2D patients. The biological information website was used to predict that Kcnq1ot1 had complementary binding sites with miR-92a-1-5p, which was verified by the luciferase reporting gene system. The si-Kcnq1ot1, miR-92a-1-5p mimics, miR-92a-1-5p inhibitors and their respective controls were respectively transfected into Min6, and the effect of Ins1, Ins2, Insr, Nkx6.1, Irs1 and Ngn3 were detected by qRT-PCR, the Western blot method was used to detect the expression of insulin and Insr, and the GSIS experiment was used to detect its effect on insulin secretion. Results: The expression of Kcnq1ot1 in pancreas was significantly higher than that in other tissues. Compared with the control group, the expression of Kcnq1ot1 in T2D patients' serum, Min6 after PA treatment and the islets of T2D model mice were lower. And the expression of Kcnq1ot1 in serum is negatively correlated with FBG or HbA1c. Low expression of Kcnq1ot1 led to remarkable reduction of insulin secretion in Min6 cells. Down-regulation of Kcnq1ot1 or overexpression of miR-92a-1-5p reduced the mRNA expression levels of Ins1, Ins2, Insr, Nkx6.1, Irs1 and Ngn3, and the protein levels of insulin and Insr. We screened miR-92a-1-5p as targets of Kcnq1ot1, and their interaction was validated with dual luciferase reporter gene experiment. After knocking down Kcnq1ot1, the expression of miR-92a-1-5p was found to increase. Further rescue experiments showed that down-regulation of miR-92a-1-5p could reverse the effect of low Kcnq1ot1 expression on the function of pancreatic β-cells. Conclusion: Kcnq1ot1 may regulate the function of pancreatic β cells by targeting to negatively regulate miR-92a-1-5p. Disclosure Y.Chen: None. Y.Li: None. W.Xu: None. B.Lin: None. X.Mai: None. Y.Peng: None. D.Chen: None. Y.Su: None. W.Li: n/a. Funding Science & Technology Project of Guangzhou（202102010175）

Hsa_circ_0006427 Suppresses Multiplication, Migration and Invasion of Non-Small Cell Lung Cancer Cells through miR-346/VGLL4 Pathway.

Objective Circular RNAs (circRNAs) are identified as key modulators in cancer biology. Nonetheless, the role of circ_0006427 in non-small cell lung cancer (NSCLC) and its modulatory mechanism are undefined. This study aimed to investigate the potential function and mechanism of circ_0006427 in NSCLC. Materials and Methods In this experimental study, circ_0006427, miR-346 and vestigial like family member 4 (VGLL4) mRNA expressions were analyzed in NSCLC tissues and cells, using quantitative reverse transcription polymerase chain reaction (qRT-PCR). Multiplication, migration and invasion of NSCLC cells were examined using the CCK-8 method and Transwell experiment, respectively. Dual-luciferase reporter gene experiments were conducted to identify the paring relationship between circ_0006427 and miR-346. Western blot was employed to determine expressions of VGLL4 and epithelial-mesenchymal transition (EMT) markers on protein levels. Immuno-histochemistry (IHC) method was adopted to assess VGLL4 protein expression in NSCLC tissues. Results Circ_0006427 expression was down-regulated in NSCLC tissues and cells, and circ_0006427 suppressed multiplication, migration, invasion and EMT of NSCLC cells. miR-346 expression was upregulated in NSCLC tissues and cells, and miR-346 worked as a target of circ_0006427. VGLL4 was down-regulated in NSCLC tissues and cells, and knockdown of VGLL4 accelerated multiplication, migration, invasion and EMT of NSCLC cells. Circ_0006427 enhanced VGLL4 expression by competitively binding with miR-346. ConclusionCirc_0006427/miR-346/VGLL4 axis regulated NSCLC progression.

MiR-221 Regulates Suppressors of Cytokine Signaling 3-Janus Kinase 2/Signal Transducer and Activator of Transcription 3 (SOCS3-JAK2/STAT3) Pathway and Affects Thyroid Cancer Cell Proliferation and Apoptosis

Objective: Suppressors of cytokine signaling 3 (SOCS3) negatively regulates JAK-STAT signaling. Bioinformatics analysis showed a targeted relationship between miR-221 and SOCS3 mRNA 3′-UTR. This study investigated whether miR-221 regulates SOCS3 expression and affects thyroid cancer cells. Methods: Dual-luciferase reporter gene experiments verified the relationship between miR-221 and SOCS3. The tumor tissues and adjacent tissues of patients with thyroid cancer were collected to detect miR-221 and SOCS3 level. Thyroid cancer cell line KTC-1 cells were assigned into miR-NC group and miR-221 inhibitor group followed by analysis of SOCS3, p-JAK2, and p-STAT3 level by Real-time PCR, cell apoptosis and cell proliferation by flow cytometry and cell invasion by Transwell assay. Results: Compared with adjacent tissues, miR-221 level in tumor tissues was increased, and SCOS3 mRNA level was decreased. There was a targeted relationship between miR-221 and SOCS3 mRNA. MiR-221 level in KTC-1 and TPC-1 cells was increased, while SOCS3 mRNA level was decreased. MiR-221 inhibitor can significantly upregulate SOCS3 mRNA and protein in KTC-1 cells, reduce the expression of p-JAK2, p-STAT3 protein, increase cell apoptosis, and reduce cell proliferation and invasion. Conclusion: The increased miR-221 and decreased SOCS3 expression are related to thyroid cancer pathogenesis. MiR-221 can inhibit the expression of SOCS3, affect JAK-STAT signaling activity, and regulate the proliferation and apoptosis of thyroid cancer cells.

Circ_0000514 promotes the malignant biological behaviors of non-small cell lung cancer cells by modulating miR-330-5p and HMGA2

BackgroundCircular RNA 0000514 (circ_0000514) has a modulatory function in the progression of several cancers, but its role and mechanism in non-small cell lung cancer (NSCLC) are obscure. MethodsThe relative expressions of circ_0000514, microRNA-330–5p (miR-330–5p) and high mobility group AT-hook 2 (HMGA2) mRNA in 67 cases of NSCLC and adjacent lung tissues were examined by quantitative real-time polymerase chain reaction (qRT-PCR). The changes in cell phenotypes were examined by cell counting kit-8 (CCK-8), 5-Ethynyl-2′-deoxyuridine (EdU), Transwell and flow cytometry assays, respectively. Bioinformatics analysis, dual-luciferase reporter gene experiment and RNA immunoprecipitation (RIP) experiment were adopted to predict and validate the relationship between circ_0000514 and miR-330–5p, and miR-330–5p and HMGA2 3′UTR, respectively. In addition, Western blot was performed to examine the effects of circ_0000514 overexpression or knockdown on HMGA2 protein expression. ResultsCirc_0000514 expression was up-regulated in NSCLC tissues. Circ_0000514 significantly facilitated the growth, cell cycle progression, metastatic potential, and repressed the apoptosis of NSCLC cells. Circ_0000514 was identified as a molecular sponge to decoy miR-330–5p, and HMGA2 was proven to be a target gene of miR-330–5p. Moreover, circ_0000514 positively regulated HMGA2 expression, and its biological effect was dependent on miR-330–5p/HMGA2 axis. ConclusionCirc_0000514 is an oncogenic circRNA in NSCLC progression, and it modulates miR-330–5p/HMGA2 axis.

Cancer-suppressing miR-520-3p gene inhibits proliferation, migration, and invasion of gastric cancer cells through targeted regulation of KLF7

Gastric cancer (GC) is among the most common malignant tumors. Numerous studies have reported that microRNAs (miRNAs) play significant roles in carcinogenesis and treatment. An miRNA, miR-520-3p, has been identified as a cancer-suppressing gene in several cancers. However, the role and underlying mechanism of miR-520-3p regulation of GC remain unknown. In this study, the expression levels of miR-520-3p in cancer tissues of patients with GC -and in adjacent normal tissues, gastric cancer cell lines, and human normal gastricepithelial cells- were detected by qRT-PCR. RNA interference was performed in GC cell lines. After the corresponding treatment, the cells were characterized in vitro or in vivo to evaluate their molecular function. CCK-8, cell colony formation, and a Transwell assay were used to detect cell proliferation rate, viability, and invasion ability. A dual-luciferase reporter gene experiment was used to explore the potential molecular mechanisms of miR-520-3p. The results showed that the expression of miR-520-3p was significantly downregulated in GC tissues and cells, and upregulation of miR-520-3p could inhibit the proliferation, vitality, and invasion of GC cells both in vivo and in vitro. The expression of Kruppel-like factor 7 (KLF7) was greatly upregulated in GC tissues. MiR-520-3p can adsorbKLF7 in GC cells, and KLF7 can reverse the inhibitory effect of miR-520-3p overexpression on the proliferation of GC cells. This study revealed that miR-520-3p plays a significant role in inhibiting the proliferation, invasion, and migration of GC cells by targeting KLF7. These data demonstrate that miR-520-3p may serve as a novel prognostic biomarker and a potential therapeutic target for GC.

Involvement of MiRNA-211-5p and Arhgap11a Interaction During Osteogenic Differentiation of MC3T3-E1 Cells.

ObjectiveMicroRNAs (miRNAs) are well-recognized for their abilities to regulate gene expression post-transcriptionally in plants and animals. Recently, miRNA-messenger RNA (mRNA) regulatory relationships have been confirmed during biological processes, including osteogenic differentiation. This study aimed to find out more candidate miRNA-mRNA pairs involved in the osteogenic differentiation of MC3T3-E1 cells.MethodsAn MC3T3-E1-based microarray dataset (accessioned as GSE46400) downloaded from the Gene Expression Omnibus included MC3T3-E1 cells with or without 14-day osteoblast differentiation osteoblast induction. Multiple miRNA-mRNA prediction databases were searched by differentially expressed genes (DEGs) to obtain pairs of a miRNA-DEG regulatory network. The MC3T3-E1 cells were cultured and incubated in the osteogenic differentiation medium for 14 days. The expressions of candidate miRNAs and mRNAs were determined by real-time quantitative PCR(RT-qPCR) in MC3T3-E1 cells. The miRNA-mRNA interactions were verified by dual-luciferase reporter gene assays and experiments using mimics miRNA or their inhibitors.ResultsWe identified 715 upregulated DEGs and 603 downregulated DEGs between MC3T3-E1 cells with and without osteoblast induction by analyzing the raw data of the GSE46400 dataset. There were 7 overlapped miRNA-mRNA pairs identified during osteogenic differentiation of MC3T3-E1 cells, including mmu-miR-204-5p-Arhgap11a, mmu-miR-211-5p-Arhgap11a, mmu-miR-24-3p-H2afx, mmu-miR-3470b-Chek2, mmu-miR-3470b-Dlgap5, mmu-miR-466b-3p-Chek1, and mmu-miR-466c-3p-Chek1. The Arhgap11a, H2afx, Chek2, Dlgap5, and Chek1 were hub genes downregulated in MC3T3-E1 cells after osteogenic differentiation, verified by RT-qPCR results. The RT-qPCR also determined declined expressions of miR-204-5p and miR-24-3p concomitant with elevated expressions of miR-211-5p, miR-3470b, miR-466b-3p, and miR-466c-3p in the MC3T3-E1 cells, with osteoblast induction compared with undifferentiated MC3T3-E1 cells. Dual-luciferase reporter gene assays demonstrated Arhgap11a as the target of miR-211-5p. MiR-211-5p upregulation by its mimic increased Arhgap11a expression in MC3T3-E1 cells.ConclusionOur study characterizes miR-211-5p targeting Arhgap11a promotes the osteogenic differentiation of MC3T3-E1 cells, which provides novel targets to promote the osteogenesis process during bone repair.

Effect of Carvacrol on the Biological Behavior of Leukemia Cells and Its Mechanism

To explore the effect of carvacrol on the biological behavior of leukemia cells and its regulation to circ-0008717/miR-217 molecular axis. Human acute lymphoblastic leukemia cells Molt-4 were cultured in vitro, and different concentrations of carvacrol were added to the cells. si-NC and si-circ-0008717 were transfected into Molt-4 cells (si-NC group, si-circ-0008717 group). pcDNA, pcDNA-circ-0008717, anti-miR-NC, anti-miR-217 were transfected into Molt-4 cells and then added to carvacrol-treated cells (carvacrol+pcDNA group, carvacrol+pcDNA-circ-0008717 group, carvacrol+anti-miR-NC group, carvacrol+anti-miR-217 group). MTT, plate clone formation experiment, and flow cytometry were used to detect the viability of the cell, colony formation number, and apoptosis rate of cells, respectively. The RT-qPCR method was used to detect the expression levels of circ-0008717 and miR-217. The dual luciferase reporter gene experiment was used to detect the targeting relationship between circ-0008717 and miR-217. After carvacrol treatment, the cell viability decreased significantly (r=-0.9405), expression level of circ-0008717 decreased (r=-0.9117), colonies formed number decreased (r=-0.9256), while the cell apoptosis rate increased (r= 0.8464), and the expression level of miR-217 increased (r=0.9468). Compared with the si-NC group, the expression level of miR-217 in si-circ-0008717 group increased (P＜0.001), the cell apoptosis rate increased (P＜0.001), while cell viability decreased (P＜0001), the number of colonies formed decreased (P＜0.001). Compared with the carvacrol+pcDNA group, the cell viability of the carvacrol+pcDNA-circ-0008717 group increased (P＜0.001), the number of colonies formed increased (P＜0.001), while the cell apoptosis rate decreased (P＜0.001). circ-0008717 could target miR-217. The cell viability of the carvacrol+anti-miR-217 group increased (P＜0.001), and the number of colonies formed increased (P＜0.001), while the cell apoptosis rate decreased (P＜0001) as compared with the carvacrol+anti-miR-NC group. Carvacrol can promote the expression of miR-217 by down-regulating the expression of circ-0008717, thereby reducing the proliferation and cloning ability of leukemia cells and promoting cell apoptosis.

Circular RNA circ_0004277 Inhibits Acute Myeloid Leukemia Progression Through MicroRNA-134-5p / Single stranded DNA binding protein 2

ABSTRACT Circular RNAs (circRNAs) are crucial non-coding RNAs in the process of tumorigenesis. Nevertheless, the biological function of circ_0004277 in acute myeloid leukemia (AML) is blurred. Microarray data of circRNAs were utilized to evaluate circRNAs’ differential expression in AML. Quantitative real-time polymerase chain reaction (qRT-PCR) was executed to determine circ_0004277 and microRNA-134-5p (miR-134-5p) expression levels. The growth, migration and invasion of AML cells were tested by the cell counting kit-8 and Transwell experiment. Dual-luciferase reporter gene experiment, RNA immunoprecipitation (RIP) experiment and RNA pull-down experiment were executed to determine the targeting relationship between circ_0004277 and miR-134-5p. Western blot assay was used to detect single stranded DNA binding protein 2 (SSBP2) expression. We observed that circ_0004277 was down-regulated in AML, while miR-134-5p was up-regulated. Functionally, circ_0004277 overexpression or inhibition of miR-134-5p remarkably suppressed AML cell viability, migration and invasion. Furthermore, miR-134-5p served as a direct downstream target of circ_0004277 and SSBP2 was identified as a target of miR-134-5p. Compensation experiments showed that miR-134-5p mimics abolished the biological function of circ_0004277 on malignant phenotypes of AML cells. Collectively, circ_0004277 impedes AML development by adsorbing miR-134-5p and up-regulating SSBP2.

MiR-16 Inhibits Extracellular Signal-Regulated Kinases (ERK) Mitogen-Activated Protein Kinases (MAPK) Signaling to Affect Epithelial-Mesenchymal Transition (EMT) and Invasion of Glioma Cells

Abnormal MEK1 expression is associated with tumor cell EMT, invasion and metastasis. Decreased miR-16 level is associated with glioma. Bioinformatics analysis showed a relationship between miR-16 and MEK1. This study assessed whether miR-16 regulates MEK1 expression and affects glioma cell EMT and invasion. The tumor tissues and adjacent glioma tissues were collected to measure miR-16 and MEK1 mRNA. The dual luciferase assay validated the relation of miR-16 with MEK1. U251 cells were cultured and assigned into NC group and mimic group, followed by analysis of cell biological behaviors, and MEK1, p-ERK1/2, E-cadherin, N-Cadherin expression. Compared with adjacent tissues, miR-16 expression was significantly decreased and MEK1 was elevated in glioma tissues. Compared with HEB, miR-16 in glioma U251 and SHG44 cells was decreased and MEK1 was increased. Dual luciferase reporter gene experiments confirmed the relation of miR-16 with MEK1. Transfection of miR-16 mimic significantly down-regulated MEK1, p-ERK1/2 and N-cadherin in U251 cells, upregulated E-cadherin, inhibited cell proliferation, promoted apoptosis, and attenuated EMT and invasion of glioma cells. In conclusion, decreased miR-16 expression and increased MEK1 expression is related to glioma pathogenesis. Overexpression of miR-16 can inhibit MEK1 expression, ERK/MAPK signaling, glioma cell proliferation, promote apoptosis, and attenuate EMT and invasion.

The Circular RNA circSKA3 Facilitates the Malignant Biological Behaviors of Medulloblastoma via miR-520h/CDK6 Pathway.

Circular RNAs (circRNAs) are reported to participate in the development of diverse human malignancies. This work investigated the mechanism of circSKA3 in modulating medulloblastoma progression. A total of 15 cases of medulloblastoma were collected in this work. Daoy cells were used to construct cell models. The expression level of circSKA3, microRNA-520h (miR-520h), and cyclin-dependent kinase 6 (CDK6) mRNA in tissues or cells was examined by quantitative real-time polymerase chain reaction (qRT-PCR). Western blot was employed to detect CDK6 protein expression. CCK-8 experiment, Transwell assay, and flow cytometry were applied to detect the regulatory effects of circSKA3 on cell proliferation, migration, invasion, and cell cycle. Dual-luciferase reporter gene experiment was executed to determine the relationship between circSKA3 and miR-520h, and between miR-520h and CDK6. circSKA3 was remarkably up-modulated in medulloblastoma tissues. CircSKA3 depletion markedly suppressed Daoy cell viability, migration, invasion, and cell cycle progression. CircSKA3 overexpression induced the opposite effects. circSKA3 could decoyed miR-520h, which targeted the 3' UTR of CDK6. circSKA3 expression in medulloblastoma tissues was negatively correlated with miR-520h expression and positively correlated with CDK6 expression. "Rescue" experiments revealed that miR-520h down-modulation or CDK6 overexpression remarkably counteracted the inhibitory effect of circSKA3 knockdown on Daoy cells. circSKA3 facilitates medulloblastoma progression through miR-520h/CDK6.

Necdin, one of the important pathway proteins in the regulation of osteosarcoma progression by microRNA-200c

ABSTRACT MicroRNA-200c (miR-200c) generally acts as a tumor suppressor in multiple cancer types and a promising therapeutic target in tumorigenesis. However, only a few studies have explained the role of miR-200c in the development of osteosarcoma (OS). In this study, we investigated the role of miR-200c in OS progression and identified the regulatory pathway protein NDN involved in inhibiting the occurrence and development of OS. Firstly, we found that miR-200c is downregulated in OS cells and tissues. As well, in vitro and in vivo experiments showed that upregulating miR-200c inhibits the proliferation, invasion, metastasis of Saos-2 cells, promotes the apoptosis of Saos-2 cells and suppresses tumor growth in mice, indicating miR-200c plays a major role in regulating the OS progression. Furthermore, bioinformatics analysis showed that an anti-tumor protein, necdin (NDN), might be a potential target by miR-200c. To verify this hypothesis, we measured the expression level of NDN in OS cells and tissues and found NDN is downregulated, suggesting NDN is functional in OS progression. Moreover, we found that the expression levels of NDN and miR-200c in in vivo and in vitro experiments were positively correlated. However, the results of dual-luciferase reporter gene experiment showed miR-200c does not directly act on the 3ʹ untranslated region (UTR) of NDN gene, indicating that NDN might be an important pathway protein which regulates OS progression in the presence of miR-200c. Therefore, miR-200c/NDN could be potential targets for developing effective treatment against OS.

Circ_0064288 acts as an oncogene of hepatocellular carcinoma cells by inhibiting miR-335-5p expression and promoting ROCK1 expression

BackgroundReportedly, circular RNA (circRNA) is a key modulator in the development of human malignancies. This work is aimed to probe the expression pattern, biological effects and mechanism of circ_0064288 on hepatocellular carcinoma (HCC) progression.MethodsThe differentially expressed circRNA was screened by analyzing the expression profiles of circRNAs in HCC tissues and normal tissues. Quantitative real-time polymerase chain reaction (qRT-PCR) was performed to examine the expression of circ_0064288, miR-335-5p and Rho associated coiled-coil containing protein kinase 1 (ROCK1) mRNA in HCC specimens. After circ_0064288 was overexpressed or knocked down in HCC cells, cell growth was detected by the CCK-8 experiment, and cell migration was evaluated using Transwell experiment and scratch healing experiment. The targeting relationship between miR-335-5p and circ_0064288 and ROCK1 mRNA was predicted and verified using bioinformatic analysis and dual-luciferase reporter gene experiments, respectively. Western blot was executed to examine ROCK1 protein expression in HCC cells.ResultsCirc_0064288 and ROCK1 expression was up-modulated in HCC, while miR-335-5p was down-modulated. High circ_0064288 expression was associated with shorter survival time of HCC patients. It was also revealed that circ_0064288 overexpression remarkably enhanced HCC cell growth and migration, while knockdown of circ_0064288 induced opposite effects. Additionally, circ_0064288 could competitively bind with miR-335-5p thereby up-modulate ROCK1 expression. MiR-335-5p overexpression partly counteracted the effect of circ_0064288 overexpression on HCC cells.ConclusionCirc_0064288 facilitates HCC cell growth and migration by modulating the miR-335-5p/ROCK1 axis.

CCNB1, Negatively Regulated by miR-559, Promotes the Proliferation, Migration, and Invasion of Ovarian Carcinoma Cells.

Cyclin B1 (CCNB1) is regarded as an oncogene in multiple tumors. This work aims to investigate the expression, function, and related mechanisms of CCNB1 in ovarian carcinoma (OC). Three microarray datasets (GSE14407, GSE18520, and GSE54388) were obtained from the Gene Expression Omnibus (GEO) database and screened for differentially expressed genes (DEGs) of OC tissues and normal ovarian tissues. CCNB1 expression in OC tissues and paracancerous tissues was detected by immunohistochemistry. Kaplan-Meier plotter database was utilized to analyze the correlation between CCNB1 expression and the prognosis of OC patients. After the loss-of-function and gain-of-function cell models were established, cell counting kit-8 (CCK-8), bromo-deoxyuridine (BrdU), and transwell experiments were employed to examine the proliferation, migration, and invasion of OC cells, respectively. The targeting relationship between miR-559 and CCNB1 was verified using the dual-luciferase reporter gene experiment. The expressions of CCNB1 mRNA and miR-559 were detected by quantitative reverse transcription-polymerase chain reaction (qRT-PCR). Western blot was used to quantify the protein expression of CCNB1. In addition, xenograft nude mouse models were established to examine the effects of CCNB1 on lung metastasis in vivo. CCNB1 expression was markedly increased in OC tissues and cell lines. The overall survival, progression-free survival, and post-progression survival of OC patients with high CCNB1 expression were significantly shorter. OC cell proliferation, migration, and invasion were enhanced by CCNB1 overexpression while CCNB1 knockdown led to opposite effects. MiR-559 expression was remarkably reduced in OC tissues and cell lines, and miR-559 markedly suppressed the malignant characteristics of OC cells. Besides, miR-559 directly targeted the 3' UTR of CCNB1 mRNA and reduced CCNB1 expression at both the mRNA and protein levels. Overexpression of CCNB1 accelerated lung metastasis of OC cells in vivo. CCNB1, of which expression is modulated by miR-559, facilitates proliferation, migration, and invasion of OC cells, therefore, working as a potential therapeutic target of OC. This work provides new insights into the clinical diagnosis and treatment of OC.

Long noncoding RNA LINC01426 promotes the progression of lung adenocarcinoma via regulating miRNA-125a-5p/ casein kinase 2 alpha 1 axis

ABSTRACT Although long noncoding RNAs (lncRNAs) in lung adenocarcinoma (LUAD) have been increasingly studied, LINC01426 has not been fully investigated in LUAD. The GEPIA database revealed that LINC01426 was upregulated in LUAD tissues. In our study, we further verified the significantly high expression of LINC01426 in LUAD tissues and cell lines. We also analyzed the LINC01426 expression level and LUAD clinical features and found that high LINC01426 expression was associated with tumor diameter; tumor, node, and metastases (TNM) staging; lymph node metastasis (LNM); and overall survival (OS) rate of LUAD patients. In vitro experiments revealed that suppression of LINC01426 could repress the proliferation, migration and invasion of LUAD cells. Then, the bioinformatic analysis revealed that there were binding domains between miR-125a-5p and the 3′-UTR of LINC01426. As revealed by dual-luciferase reporter gene experiment and RNA Binding Protein Immunoprecipitation (RIP) assay, miR-125a-5p could bind to LINC01426. Additionally, the results of qRT-PCR and Pearson’s analysis respectively revealed that miR-125a-5p was slightly expressed in LUAD and its expression was negatively correlated with LINC01426. Moreover, casein kinase 2 alpha 1 (CSNK2A1) was predicted to bind to miR-125a-5p. CSNK2A1 expression was remarkably high in LUAD tissues, negatively associated with miR-125a-5p, and positively correlated with LINC01426. Subsequently, our results showed that CSNK2A1 enhanced the malignant progression of LUAD cells. Overall, our study revealed that LINC01426 might regulate the malignant phenotype of LUAD via the miR-125a-5p/CSNK2A1 axis.

Hsa_circ_0000418 promotes the progression of glioma by regulating microRNA-409-3p / pyruvate dehydrogenase kinase 1 axis

ABSTRACT Glioma is the commonest intracranial malignancy, and circRNAs are important regulatory factors which are implicated in the development of glioma. Nonetheless, the role of circRNAs in glioma is largely unknown. The research is performed to elaborate on the biological role of has_circ_0000418 (circ_0000418) in glioma progression and its potential molecular mechanism. The differentially expressed circRNAs in glioblastoma patient derived cells and neural progenitor cells were analyzed based on the microarray data of GSE146463. Additionally, qRT-PCR and Western blot experiments were conducted to measure the expression of circ_0000418, microRNA-409-3p (miR-409-3p) and pyruvate dehydrogenase kinase 1 (PDK1) in glioma tissues/cells. Cell growth and cell cycle distribution were monitored using CCK-8 assay, BrdU assay and flow cytometry. Bioinformatics prediction, dual-luciferase reporter gene experiment and RIP assay were conducted to verify the targeting relationship between circ_0000418 and miR-409-3p, miR-409-3p and PDK1 3ʹUTR. In this work, we observed that, circ_0000418 expression level was significantly up-regulated in glioma tissues and cell lines. Circ_0000418 overexpression facilitated glioma cell growth and accelerated cell cycle progression, while knockdown of circ_0000418 produced the opposite effects. Circ_0000418 specifically combined with miR-409-3p, and circ_0000418 negatively modulated the expression of miR-409-3p. PDK1 acted as a target gene of miR-409-3p, and PDK1 could be positively and indirectly modulated by circ_0000418 in glioma cells. In summary, circ_0000418 enhances glioma cell growth and accelerates cell cycle progression by regulating miR-409-3p/PDK1 axis.

Circular RNA hsa_circ_0083756 promotes intervertebral disc degeneration by sponging miR-558 and regulating TREM1 expression.

ObjectivesIntervertebral disc degeneration (IVDD) is a leading cause of low back pain. Circular RNAs (circRNAs) have been demonstrated to exert vital functions in IVDD. However, the role and mechanism of hsa_circ_0083756 in the development of IVDD remain unclear.Materials and methodsRT‐qPCR was performed to detect expressions of hsa_circ_0083756, miR‐558 and TREM1 in nucleus pulposus (NP) tissues and cells. CCK8 assay, flow cytometry, TUNEL assay, RT‐qPCR and WB were used to clarify the roles of hsa_circ_0083756 in NP cells proliferation and extracellular matrix (ECM) formation. Bioinformatics analyses, dual‐luciferase reporter gene experiment, RNA immunoprecipitation (RIP) assay and FISH assay were performed to predict and verify the targeting relationship between hsa_circ_0083756 and miR‐558, as well as that between miR‐558 and TREM1. Ultimately, the effect of hsa_circ_0083756 on IVDD was tested through anterior disc‐puncture IVDD animal model in rats.Resultshsa_circ_0083756 was upregulated in degenerative NP tissues and cells. In vitro loss‐of‐function and gain‐of‐function studies suggested that hsa_circ_0083756 knockdown promoted, whereas hsa_circ_0083756 overexpression inhibited NP cells proliferation and ECM formation. Mechanistically, hsa_circ_0083756 acted as a sponge of miR‐558 and subsequently promoted the expression of TREM1. Furthermore, in vivo study indicated that silencing of hsa_circ_0083756 could alleviate IVDD in rats.Conclusionshsa_circ_0083756 promoted IVDD via targeting the miR‐558/TREM1 axis, and hsa_circ_0083756 may serve as a potential therapeutic target for the treatment of IVDD.