Complementary Binding Sites Research Articles

Circular RNA hsa_circ_0004543 Aggravates Cervical Cancer Development by Sponging MicroRNA hsa-miR-217 to Upregulate Hypoxia-Inducible Factor.

Cervical cancer (CC) is the 4th principal source of cancer death in females with 604,000 new patients and 342,000 deaths in 2020 worldwide. It has been extensively shown that circRNAs are involved in regulating CC development. Nevertheless, the function and mechanisms of hsa_circ_0004543 in regulating CC need to be clearly elucidated. Herein, hsa_circ_0004543 expressions were compared between 40 paired paracancerous and cancerous specimens from CC patients and between 6 CC cell lines and a normal human cervical epithelial cell line based on qRT-PCR. Potential complementary binding sites between hsa-miR-217 and hsa_circ_0004543 were predicted using the interactome, while binding sites for the hypoxia-inducible factor-1a (HIF-1a) were predicted by TargetScan. The function and mechanism of hsa_circ_0004543 in the development of CC were estimated by silencing hsa_circ_0004543 with/without hsa-miR-217 or HIF-1a overexpression. The association between gene expressions was evaluated with Pearson's correlation analysis. Molecular mechanisms were explored by ribonucleic acid (RNA) pulldown, dual-luciferase activity, and rescue experimental assays. Our results revealed that the hsa_circ_0004543 expression was considerably increased in CC tissues and cells. Its silencing repressed proliferation and metastasis, while it increased apoptosis of CC cells. The investigation of the mechanism showed that hsa-miR-217 silencing or HIF-1a overexpression rescued hsa_circ_0004543, and silencing inhibited malignant phenotypes of CC cells. hsa_circ_0004543 upregulated the HIF-1α expression by sponging hsa-miR-217 in CC development. Therefore, the hsa_circ_0004543 functioned as a competing endogenous RNA (ceRNA) of hsa-miR-217 to increase CC oncogenesis and metastasis by the upregulation of the HIF-1α expression. Consequently, targeting the hsa_circ_0004543/hsa-miR-217/HIF-1α axis might be a potential treatment approach for CC.

The Apis mellifera alpha 5 nicotinic acetylcholine receptor subunit expresses as a homomeric receptor that is sensitive to serotonin

Insect nicotinic acetylcholine receptors (nAChRs) are molecular targets of highly effective insecticides such as neonicotinoids. Functional expression of these receptors provides useful insights into their functional and pharmacological properties. Here, we report that the α5 nAChR subunit of the honey bee, Apis mellifera, functionally expresses in Xenopus laevis oocytes, which is the first time a homomeric insect nAChR has been robustly expressed in a heterologous system without the need for chaperone proteins. Using two-electrode voltage-clamp electrophysiology we show that the α5 receptor has low sensitivity to acetylcholine with an EC50 of 2.37 mM. However, serotonin acts as an agonist with a considerably lower EC50 at 119 μM that is also more efficacious than acetylcholine in activating the receptor. Molecular modelling indicates that residues in the complementary binding site may be involved in the selectivity towards serotonin. This is the first report of a ligand-gated ion channel activated by serotonin from an insect and phylogenetic analysis shows that the α5 subunit of A. mellifera and other non-Dipteran insects, including pest species, belong to a distinct subgroup of subunits, which may represent targets for the development of novel classes of insecticides.

Down-regulation of lncRNA TTTY15 targeting miR-4500 to inhibit the biological characteristics of A172 glioma cells

To explore the effect and mechanism of down-regulating lncRNA TTTY15 targeting miR-4500 on the proliferation, apoptosis, migration and invasion of A172 glioma cells. The difference in TTTY15 expression between the glioma cells and tissue was determined with a qRT-PCR method. Complementary binding sites of TTTY15 and miR-4500 were predicted with Starbase software, and the targeting relationship was validated with a luciferase reporter system. A172 glioma cells were divided into Control, si-NC (transfected with control siRNA), si-TTTY15 (transfected with TTTY15 siRNA), si-TTTY15+Anti-miR-NC (co-transfected with TTTY15 siRNA and inhibitor control) and si-TTTY15+Anti-miR-4500 (co-transfected with TTTY15 siRNA and miR-4500 inhibitor) groups. Proliferation, apoptosis, migration and invasion, and the expression of Bax, Bcl-2, MMP-2 and MMP-9 proteins of the A172 glioma cells were respectively detected with CCK-8, flow cytometry, Transwell chamber and Western blotting assays. The expression of TTTY15 in glioma cells and glioma tissues have both increased. The expression levels of TTTY15 and miR-4500 in glioma tissues were inversely correlated. TTTY15 and miR-4500 are mutually targeted. Compared with those of the Control and si-NC groups, the glioma cells in the si-TTTY15 group showed increased level of miR-4500, decreased survival rate, increased apoptosis rate, enhanced cell migration and invasion, increased expression of Bax protein, and decreased expression of Bcl-2, MMP-2 and MMP-9 proteins (P<0.05). Compared with those of the si-TTTY15+Anti-miR-NC group, the A172 glioma cells in the si-TTTY15+Anti-miR-4500 group showed decreased level of miR-4500, increased cell survival rate, decreased apoptosis rate, enhanced cell migration and invasion, decreased expression of Bax protein, and increased expression of Bcl-2, MMP-2, and MMP-9 proteins (P<0.05). Down-regulating TTTY15 targeting miR-4500 can inhibit the proliferation, migration, invasion and induce apoptosis of the A172 glioma cells.

Molecular mechanism of non-coding RNA targeting zinc finger binding protein 1 and cervical cancer cells suppression

The main aim of the study is to explore the mechanism of microrNA-145-3p targeting ZEB1 on migration and invasion in cervical cancer cells. In this study, cervical cancer cell line C33A was selected and transfected. After transfection of C33A cells, the expression level of Mir-145-3p was measured by QT-PCR. Human cervical cancer cell line (C33A) was cultured in RPMI 1640 medium and cell transfection experiment was performed. The related targets of Mir-145-3p, and a complementary binding site was found between the 3′UTR terminal of ZEB1 and Mir-145-3p was performed using a Targetscan biological software. The luciferase reporter system was used to determine the targeting relationship between Mir-145-3p and complementary binding site. The expression of e-cadherin and vimentin proteins was detected by western blot analysis. After 48 h, the expression level of Mir-145-3p in mimics group was significantly increased, the inhibitor group was significantly down-regulated and was statistically significant (P < 0.05). The clone formation, migration and invasion ability and vimentin expression in the MIMics group were significantly decreased than NC group. However, the apoptosis rate and e-cadherin protein expression were significantly increased than control group. In the inhibitor group, the cell cloning, cell migration, invasion and vimentin protein expression were significantly increased, while the cell apoptosis rate and e-cadherin protein expression were significantly decreased (P < 0.05). Analysis of luciferase expression genes revealed that mirNa-145-3p and ZEB1 have targeted regulatory relationship. Compared with Mir-NC, there was no significant difference between Mir-145-3p MUT and Mir-NC (P > 0.05), and luciferase activity of Mir-145-3p WT was significantly down-regulated (P > 0.05). Overexpression of microrNA-145-3p downregulated ZEB1 expression and inhibited proliferation, migration, invasion, and epithelial-mesenchymal transformation of cervical cancer cells, and promoted cell apoptosis.

Peripheral Blood Levels of miR-448 and SIRT1 in Patients with Deep Venous Thrombosis and Their Relationship.

The goal of this study was to investigate the changes in peripheral blood levels of miR-448 and silent information regulator 1 (SIRT1) in patients with deep venous thrombosis (DVT) and to analyze their relationship. A total of 112 patients treated from January 2019 to June 2020 were divided into DVT group (n = 40) and non-DVT group (n = 72). Fasting venous blood was extracted to separate serum and peripheral blood mononuclear cells (PBMCs). Enzyme-linked immunosorbent assay (ELISA) was employed to measure serum SIRT1 protein, and qPCR was utilized to detect miR-448 expression in PBMCs. The clinical data, serum indicators, and expressions of SIRT1 and miR-448 were compared, and the correlations of miR-448 and SIRT1 with DVT were analyzed using a multivariate Cox regression model. TargetScan Release 7.1 was used to predict the possibility of binding sites between miR-448 and SIRT1 mRNA 3'-untranslated region (3'-UTR), and dual-luciferase reporter assay was used to determine the targeting of miR-448 and SIRT1. HeLa cells were divided into overexpression, inhibition, and blank control groups. The cells were harvested 24 hours after transfection, followed by detection of SIRT1 mRNA expression by qPCR and measurement of supernatant SIRT1 protein expression by ELISA. Serum SIRT1 protein level was lower and miR-448 expression in PBMCs was higher in DVT group than those in non-DVT group (p < 0.05). DVT group had a larger number of patients with vascular diseases and history of venous thrombosis than that of non-DVT group (p < 0.05). miR-448 was an independent risk factor for postoperative DVT, and SIRT1 was a protective factor (p < 0.01). There were potential complementary base binding sites between miR-448 and SIRT1 mRNA 3'-UTR. Dual-luciferase reporter assay verified the targeted regulation between miR-448 and SIRT1. HeLa cell SIRT1 mRNA expression and supernatant SIRT1 protein expression were lower in overexpression group while higher in inhibition group than those in blank control group (p < 0.05). Serum SIRT1 protein level decreases while miR-448 expression in PBMCs increases in patients with DVT, and miR-448 inhibits SIRT1 expression through binding to SIRT1 mRNA 3'-UTR with complementary bases, thus inducing inflammatory response to participate in the formation of DVT. Targeting miR-448 to regulate cytokine expression may become an effective target and approach for the treatment of DVT. miR-488 combined with SIRT1 has a high predictive value for the occurrence of DVT.

Downregulation of microRNA-512-3p enhances the viability and suppresses the apoptosis of vascular endothelial cells, alleviates autophagy and endoplasmic reticulum stress as well as represses atherosclerotic lesions in atherosclerosis by adjusting spliced/unspliced ratio of X-box binding protein 1 (XBP-1S/XBP-1U)

ABSTRACT AS is an important pathological basis of cardiovascular disease. miRNAs are involved in almost all steps of AS, including the injury and dysfunction of endothelial cells and vascular smooth muscle cells. This work elucidated the biological functions of miR-512-3p in AS and probed into the underlying molecular mechanism. In the present work, ox-LDL-treated HUVECs served as the in vitro model of AS and ApoE-/- mice were nourished with a high-fat diet to establish an in vivo model of AS. Proliferation, apoptosis, and migration of HUVECs were evaluated by CCK-8, TUNEL staining, Western blot, and transwell assays. Immunofluorescence examined LC3 expression and levels of autophagy-related and ER stress-related proteins were determined by Western blot assay. In addition, starBase predicted the complementary binding sites of XBP-1 to miR-512-3p and luciferase reporter assay confirmed the interaction between miR-512-3p and XBP-1. Moreover, H&E staining was employed to evaluate atherosclerotic lesions in AS model mice. Results revealed that ox-LDL treatment decreased the proliferative and migrative activities and promoted the apoptosis of HUVECs as well as induced autophagy and ER stress, which were abrogated by miR-512-3p silencing. Importantly, ox-LDL treatment elevated miR-512-3p expression and XBP-1 was a direct target of miR-512-3p. Mechanistically, knockdown of miR-512-3p enhanced the viability, suppressed the apoptosis, and promoted the migration of ox-LDL-treated HUVECs, alleviated atherosclerotic lesions in AS model mice as well as repressed autophagy and ER stress by targeting XBP-1 to manipulate the ratio of XBP-1S/XBP-1 U.

CircFOXM1 promotes proliferation and metastasis of hepatocellular carcinoma via regulating miR-1179/SPAG5 axis

Hepatocellular carcinoma (HCC) predominantly occurs in patients with chronic liver disease, accounting for 70–90% of all liver cancer cases. The role of circFOXM1/miR-1179/SPAG5 axis in HCC has not been reported. This study aimed to explore the regulatory mechanism of circFOXM1 in HCC proliferation and metastasis. RNA polymerase inhibitor actinomycin D and RNase R exonuclease were used to identify circFOXM1 in HCC cells. The qRT-PCR was used to detect circFOXM1 expression. Specific siRNA for circFOXM1 was designed, and the sequence of circFOXM1 was inserted in pLCDH-ciR to overexpress circFOXM. Cell proliferation was detected by CCK8 in vitro, by tumor volume and tumor weight of HCC xenograft in vivo. Cell migration was detected by transwell test. Binding status of circFOXM1 with miR-1179 was detected by luciferase reporter gene assay. Rescue experiments were applied to identify the oncogenic mechanism of circFOXM1 in HCC cells. Actinomycin D assay confirmed the cyclization of circFOXM1. RNase R treatment showed that circFOXM1 was not affected by RNase R exonuclease. CCK8 assay, tumor volume and tumor weight showed that circFOXM1 effectively promoted HCC cell proliferation. Transwell assay showed that circFOXM effectively promoted migration and invasion abilities of HCC cells. Luciferase reporter gene activity assay showed that miR-1179 had complementary binding sites with circFOXM1 and SPAG5. CircFOXM1 silencing inhibited malignant phenotypes in HCC cells were partly rescued by either miR-1179 silencing or SPAG5 overexpression. CircFOXM1 promoted HCC cell proliferation and metastasis by regulating miR-1179/SPAG5 axis.

SARS-CoV-2-Impedimetric Biosensor: Virus-Imprinted Chips for Early and Rapid Diagnosis.

Due to the current global SARS-CoV-2 pandemic, rapid and accurate diagnostic tools are needed to prevent the spread of COVID-19 across the globe. An electrochemical sensing platform was constructed using CNTs/WO3-screen printed electrodes for imprinting the complete virus particles (SARS-CoV-2 particles) within the polymeric matrix to create viral complementary binding sites. The sensor provided high selectivity toward the target virus over other tested human corona and influenza respiratory interference viruses. The sensitivity performance of the sensor chips was evaluated using different viral concentrations, while the limits of detection and quantification were 57 and 175 pg/mL, respectively. Reaching this satisfied low detection limit (almost 27-fold more sensitive than the RT-PCR), the sensor was applied in clinical specimens obtained from SARS-CoV-2 suspected cases. Thus, dealing directly with clinical samples on the chip could be provided as a portable device for instantaneous and simple point of care in hospitals, airports, and hotspots.

Long noncoding RNA KIF9-AS1 promotes cell apoptosis by targeting the microRNA-148a-3p/suppressor of cytokine signaling axis in inflammatory bowel disease.

ObjectiveInflammatory bowel disease (IBD) is a chronic intestinal disease. This study was attempted to investigate the effects of long noncoding RNA KIF9-AS1 (KIF9-AS1) on the development of IBD and its underlying mechanism of action.MethodsQuantitative real time PCR (qRT-PCR) was implemented to examine the expression of KIF9-AS1 and microRNA-148a-3p (miR-148a-3p). The IBD mouse model was induced by dextran sulfate sodium (DSS). The body weight, disease activity index (DAI) score, colon length and histological injury were used to evaluate the colon injury. The levels of proinflammatory cytokines were measured by ELISA. In vitro, IBD was simulated by DSS treatment in colonic cells. Then the apoptosis of colonic cells was detected by flow cytometry assay. Furthermore, a dual-luciferase reporter assay was used to demonstrate the interactions among KIF9-AS1, miR-148a-3p and suppressor of cytokine signaling (SOCS3).ResultsKIF9-AS1 expression was upregulated in IBD patients, DSS-induced IBD mice and DSS-induced colonic cells, whereas miR-148a-3p expression was downregulated. KIF9-AS1 silencing attenuated the apoptosis of DSS-induced colonic cells in vitro and alleviated colon injury and inflammation in DSS-induced IBD mice in vivo. Additionally, the mechanical experiment confirmed that KIF9-AS1 and SOCS3 were both targeted by miR-148a-3p with the complementary binding sites at 3′UTR. Moreover, miR-148a-3p inhibition or SOCS3 overexpression reversed the suppressive effect of KIF9-AS1 silencing on the apoptosis of DSS-induced colonic cells.ConclusionKIF9-AS1 silencing hampered the colon injury and inflammation in DSS-induced IBD mice in vivo, and restrained the apoptosis of DSS-induced colonic cells by regulating the miR-148a-3p/SOCS3 axis in vitro, providing a new therapeutic target for IBD.

Berberine Potentiates Insulin Secretion and Prevents β-cell Dysfunction Through the miR-204/SIRT1 Signaling Pathway.

Pancreatic β-cell dysfunction is a key link during the progression of type 2 diabetes (T2DM), and SIRT1 participates in the regulation of various physiological activities of islet β-cells. However, as a key link in signal transduction, it is not clear how SIRT1 is regulated. By TargetScan prediction, we found that miR-204, which is enriched in islets, has highly complementary binding sites with SIRT1. Therefore, we speculate that miR-204 may be the upstream regulatory target of SIRT1 in islets and thus participate in the occurrence of β-cell dysfunction. In this study, we explored the association between miR-204 and β-cell dysfunction, the therapeutic effects of berberine (BBR) on β-cell function and the possible mechanisms. We found that miR-204 increased and SIRT1 mRNA and protein levels decreased significantly in islets both in vivo and in vitro. MIN6 cells induced by palmitic acid exhibited increased apoptosis, and the accumulation of insulin and ATP in the supernatant decreased. Importantly, palmitic acid treatment combined with miR-204 silencing showed opposite changes. MiR-204 overexpression in MIN6 cells increased apoptosis and decreased insulin and ATP production and SIRT1 expression. SIRT1 overexpression reversed the damage to β-cells caused by miR-204. The BBR treatment effectively improved insulin synthesis, reduced miR-204 levels, and increased SIRT1 expression in islet tissue in diabetic mice. Overexpression of miR-204 reversed the protective effect of BBR on apoptosis and insulin secretion in MIN6 cells. Our study identifies a novel correlation between miR-204 and β-cell dysfunction in T2DM and shows that administration of BBR leads to remission of β-cell dysfunction by regulating the miR-204/SIRT1 pathway.

Long non-coding RNA miR155HG silencing restrains ovarian cancer progression by targeting the microRNA-155-5p/tyrosinase-related protein 1 axis.

Ovarian cancer (OC) is the third commonest gynecological malignancy worldwide. The long non-coding (lnc)RNA microRNA (miR)155HG functions as an oncogene in different human cancers. However, the function and molecular mechanism of miR155HG in OC remain elusive. The present study indicated that the expression levels of miR155HG and tyrosinase-related protein 1 (TYRP1) were significantly increased, whereas that of miR155-5p was decreased in OC tissues and cells, as detected by real-time quantitative polymerase chain reaction. It was demonstrated that knockdown of miR155HG markedly inhibited OC cell viability, migration and invasion while promoting apoptosis, as indicated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, wound healing, Transwell and western blot assays. Mechanistically, it was revealed that miR155HG and TYRP1 were both targeted by miR-155-5p with complementary binding sites in the 3' untranslated region. A dual-luciferase reporter assay was used to confirm the targeting relationship between miR155HG, miR-155-5p and TYRP1. In addition, the interaction between miR155HG and miR-155-5p was further demonstrated by radioimmunoprecipitation and pull-down assays. In addition, feedback approaches determined that miR-155-5p inhibition or TYRP1 overexpression markedly reversed the inhibitory effects of miR155HG knockdown on OC cell viability, migration and invasion as well as weakened the promotive effect of miR155HG knockdown on OC cell apoptosis. Thus, miR155HG silencing inhibited the malignant biological behavior of OC cells by targeting the miR-155-5p/TYRP1 axis. The present study provides novel insights into the underlying mechanism of OC progression.

Administration of simvastatin halts progression of cirrhosis via up-regulating expression of miR-34a and interleukin-10 in rats

IntroductionSimvastatin (SIM) treatment has been found to be able to reduce the expression of miR-34a, and we found that interleukin-10 (IL-10) is a potential target gene of miR-34a by searching the online microRNA (miRNA) database. Furthermore, it has been shown that IL10 up-regulation could halt the progression of cirrhosis. The objective of this study was to explore the underlying mechanism of Simvastatin/miR-34a/IL-10 involved in HBV associated cirrhosis.Material and methodsReal-time PCR, western-blot analysis, immunohistochemistry, computational analysis, luciferase assay was carried out to explore the underlying mechanism of miR-34a involved in HBV associated cirrhosis.ResultsSIM treatment dose-dependently decreased the levels of miR-34a while increasing the levels of IL-10 mRNA and protein. Levels of IL-10 mRNA and protein were remarkably decreased, while miR-34a mRNA level and active caspase-3 protein level was apparently increased in Cirrhosis group compared with sham group. Accordingly, SIM treatment obstructed the dysregulated miR-34a expression and IL-10 expression in cirrhosis animals. By performing computational analysis, we identified that a complementary binding site of miR-34a was located in IL-10 3’ untranslated region (3’UTR), and miR-34a reduced luciferase activity of wild-type IL-10 3’UTR.ConclusionsOur data also suggested that SIM may become a new therapeutic strategy for HBV-associated cirrhosis via targeting the miR-34a/IL-10 axis.

MicroRNA-184 enhances the sensitivity of pheochromocytoma-12 cells to doxorubicin by targeting ADAM22

Pheochromocytoma (PCC) is a catecholamine-producing and neuroendocrine tumor with the 5-year overall survival of advanced stage PCC lower than 40%. Increasing evidence has shown that aberrant expression of microRNAs (miRNAs) plays important roles in the development and chemotherapy resistance of cancers including PCC. The tumor-suppressive function of miR-184 has been identified in several types of cancers. The aim of this study is to explore the function and the underlying mechanism of miR-184 in the chemo-resistance of PCC. miR-184 expression was significantly lower in doxorubicin (Dox)-resistant pheochromocytoma-12 (PC-12) cells and PCC patients. Consistently, in vitro analysis showed that overexpression of miR-184 obviously improved the sensitivity of PC-12/Dox cells, while knockdown of miR-184 sensitised PC-12/Dox cells to chemotherapeutics. To further understand the possible functional mechanism of miR184 in the chemo-resistance of PCC, the targets of miR-184 were predicted. The results of miRDB database suggested A disintegrin and metalloproteinase 22 (ADAM22) carrying the potential complementary binding sites of miR-184 within its 3′-untranslated region (UTR). Further experiments confirmed that miR-184 bound the 3′-UTR of ADAM22 mRNA and down-regulated the expression of ADAM22 in PC-12/Dox cells. Moreover, ADAM22 was overexpressed in Dox-resistant PC-12 cells and PCC patients. Additionally, overexpression of ADAM22 attenuated miR-184-mediated chemo-sensitivity of PC-12/Dox cells. miR-184 played a role in the chemo-sensitivity of PC-12/Dox cells at least partially via negatively regulating ADAM22. These results suggested miR-184 as a possible novel target to attenuate the chemo-resistance of PCC.

Long Noncoding RNA FGD5-AS1 Knockdown Decrease Viability, Migration, and Invasion of Non-Small Cell Lung Cancer (NSCLC) Cells by Regulating the MicroRNA-944/MACC1 Axis.

Objective:Long noncoding RNA FGD5 antisense RNA 1 (FGD5-AS1) participates in the regulation of non-small cell lung cancer (NSCLC) progression, but the underlying mechanisms are not fully revealed. This study aimed to determine the regulatory mechanism of FGD5-AS1 on the viability, migration, and invasion of NSCLC cells.Methods:QRT-PCR was performed to measure the expression of FGD5-AS1, microRNA-944 (miR-944), and MACC1 in NSCLC. The correlation between FGD5-AS1 and clinicopathological features of NSCLC patients was analyzed. The viability of NSCLC cells were detected using MTT assay, and the migration and invasion were measured by transwell assay. Additionally, dual-luciferase reporter assay was used to demonstrate the interactions among FGD5-AS1, miR-944, and MACC1. Furthermore, exosomes were isolated from NSCLC cells and identified by transmission electron microscopy (TEM) and western blot. Then, the macrophages treated with exosomes were co-cultured with NSCLC cells to assess the effect of exosomes containing lower FGD5-AS1 level on NSCLC.Results:The expression of FGD5-AS1 and MACC1 was increased in NSCLC, but miR-944 expression was decreased. FGD5-AS1 expression had significantly correlation with TNM stage and metastasis in NSCLC patients. FGD5-AS1 knockdown decreased the viability, migration, and invasion of NSCLC cells. Additionally, FGD5-AS1 and MACC1 were both targeted by miR-944 with the complementary binding sites at 3’ UTR. In the feedback experiments, miR-944 inhibition or MACC1 overexpression reversed the reduction effect of FGD5-AS1 knockdown on the tumorigenesis of NSCLC. Moreover, silencing of FGD5-AS1 suppressed macrophages M2 polarization, and eliminated the promoting effects of exosomes mediated macrophages on NSCLC cell migration and invasion.Conclusions:FGD5-AS1 knockdown attenuated viability, migration, and invasion of NSCLC cells by regulating the miR-944/MACC1 axis, providing a new therapeutic target for NSCLC.

THAP9-AS1 Promotes Tumorigenesis and Reduces ROS Generation through the JAK2/STAT3 Signaling Pathway by Increasing SOCS3 Promoter Methylation in Osteosarcoma.

Increasing studies have demonstrated that dysfunction of long noncoding RNAs (lncRNAs) plays critical roles in the development of human cancers. THAP9-AS1 has been reported to be dysregulated and associated with tumor progression in some cancers. However, the function and mechanism of THAP9-AS1 in osteosarcoma (OS) remain unclear. In the present study, we found that the expression of THAP9-AS1 was significantly upregulated in OS tissues and associated with the advanced stage of tumors and poor prognosis of patients. Blast comparison results showed that the SOCS3 promoter region and THAP9-AS1 had base complementary pairing binding sites. The interactions between THAP9-AS1, DNA methyltransferases (DNMTs), and SOCS3 were assessed by RIP and ChIP assays. The results of methylation-specific PCR (MSP) and bisulfite sequencing PCR (BSP) validated that THAP9-AS1 enhanced the methylation level of the SOCS3 promoter. The mRNA levels of SOCS3 in OS cells could be reversed by the demethylation agent 5-aza-2′-deoxycytidine. The mRNA expression of SOCS3 was downregulated in OS tissues and negatively correlated with THAP9-AS1 expression in tumors. Moreover, the western blot and immunofluorescence (IF) assay data showed that THAP9-AS1 activated the JAK2/STAT3 signaling pathway by upregulating p-JAK2 and p-STAT3 and the nuclear translocation of p-STAT3. Functionally, ectopic expression of THAP9-AS1 promoted cell proliferation, migration, and invasion and inhibited apoptosis, and this phenomenon could be reversed by SOCS3. Introduction of the JAK/STAT inhibitor AG490 partially abolished the stimulative effect of THAP9-AS1 on cellular processes. In addition, THAP9-AS1 decreased oxidative stress by reducing reactive oxygen species (ROS) and enhancing the mitochondrial membrane potential of OS cells via the SOCS3/JAK2/STAT3 pathway. Stable overexpression of THAP9-AS1 contributed to tumor growth and metastasis in vivo. In total, our findings suggested that upregulation of THAP9-AS1 might recruit DNMTs to epigenetically inhibit SOCS3, thereby activating the JAK2/STAT3 signaling pathway and oncogenesis of OS. These results provide novel insights for the understanding of OS progression.

Long non-coding RNA KCNQ1OT1 facilitates the progression of cervical cancer and tumor growth through modulating miR-296-5p/HYOU1 axis

ABSTRACT Literature reports that lncRNA KCNQ1OT1 is markedly up-regulated in cervical cancer (CC) tissues and cell lines, and KCNQ1OT1 can promote the proliferation and metastasis of CC cells. This current work was designed to investigate the molecular mechanism underlying the participation of KCNQ1OT1 in CC progression. Herein, RT-qPCR was utilized for determining the levels of KCNQ1OT1, miR-296-5p and HYOU1 in clinical tumor tissue specimens and CC cell lines. Then, starBase predicted the complementary binding sites of KCNQ1OT1 and miR-296-5p or miR-296-5p and HYOU1. Dual-luciferase reporter assay/RIP assay validated the interplays among KCNQ1OT1/miR-296-5p/HYOU1. In addition, CCK-8, wound healing and transwell assays were employed to assess the proliferative, migrative and invasive properties of CC cells. Moreover, nude mice xenograft model was established by subcutaneously injection with SiHa cells in order to validate the precise functions of KCNQ1OT1/miR-296-5p/HYOU1 axis in CC in vivo. Besides, Immunohistochemical staining examined Ki-67 expression in xenograft tumors and western blotting analysis detected expressions of MMP2/9 and Wnt/β-catenin signaling pathway in CC cells and xenograft tumors. Elevated KCNQ1OT1 and HYOU1 as well as reduced miR-296-5p were observed in clinical tumor tissue specimens and CC cell lines. Results revealed that upregulation of miR-296-5p counteracted the enhancing effects of overexpressed KCNQ1OT1 on the proliferative, migrative and invasive abilities of CC cells. Additionally, HYOU1 overexpression abolished the suppressing effects of silenced KCNQ1OT1 on the malignant behaviors of CC cells and tumor growth. To conclude, KCNQ1OT1 could aggravate the malignant behaviors of CC and facilitate tumor growth through modulating miR-296-5p/HYOU1 axis.

MiR-409-3p inhibits the proliferation and migration of human ovarian cancer cells by targeting Rab10

Ovarian cancer is a leading cause of gynecological cancer-related mortality. It has been reported that miR-409-3p is involved in the proliferation and migration of cancer cells. However, the role of miR-409-3p in ovarian cancer has not been well studied. The present study aimed to investigate the functional role of miR-409-3p in the pathogenesis of ovarian cancer, and its potential mechanism. It was found that the expression levels of miR-409-3p in 6 ovarian cancer tissues were upregulated. Through proliferation, migration and colony formation assays, it was revealed that overexpression of miR-409-3p inhibited the proliferation and migration of ovarian cancer cells. It was predicted from bioinformatics assays that the complementary binding sites were within miR-409-3p and Rab10. It was also demonstrated that the downregulation of the expression of Rab10 reversed the miR-409-3p downregulation-induced abnormal proliferation of ovarian cancer cells. These results suggest that miR-409-3p expression can be used as a predictive marker for the prognosis of ovarian cancer. Thus, the miR-409-3p/Rab10 axis may be a novel therapeutic target for ovarian cancer.

LncRNA LINC01535 upregulates BMP2 expression levels to promote osteogenic differentiation via sponging miR‑3619‑5p.

Osteoporosis is a debilitating skeletal disease that causes bones to collapse and is accompanied by a high risk of bone fracture. It was previously demonstrated that the osteogenic differentiation of human bone marrow‑derived mesenchymal stem cells (hBMSCs) serves an important role in the process of human bone formation. Accumulating research has indicated that long non‑coding RNAs (lncRNAs) participate in hBMSC osteogenic differentiation. For example, LINC01535 was reported to serve as a carcinogenic factor in cervical cancer; however, its latent function and molecular mechanism in the osteogenesis of hBMSCs remain to be investigated. The present study showed that the expression levels of LINC01535 were upregulated upon increasing osteogenic differentiation time. In addition, the inhibition of LINC01535 inhibited hBMSC proliferation and osteogenic differentiation and promoted cell apoptosis. Using bioinformatics analysis, LINC01535 was discovered to have complementary binding sites for microRNA (miR)‑3619‑5p, and further experiments demonstrated that LINC01535 functioned as a sponge of miR‑3619‑5p. Additionally, bone morphogenetic protein2 (BMP2) was confirmed to be a target of miR‑3619‑5p. The results revealed that LINC01535 regulated the expression levels of BMP2 via sponging miR‑3619‑5p. In conclusion, the findings of the present study suggested that LINC01535 may accelerate the osteogenic process of hBMSCs via targeting the miR‑3619‑5p/BMP2 axis, which may offer an innovative therapeutic method for osteoporosis.

Long noncoding RNA LINC00858 promotes the proliferation, migration and invasion of gastric cancer cells via the miR-363-3p/FOXP4 axis.

Long non-coding RNA (lncRNA) LINC00858 has been found to exert oncogenic activity in several types of cancers, except gastric cancer (GC). In the present study, we aimed to explore the potential role of LINC00858 in GC and the underlying molecular mechanism. The expression patterns of LINC00858 were determined using qRT-PCR in GC samples and cell lines. Cell proliferation was examined utilizing CCK-8 assay. Cell migration and invasion were evaluated using transwell assays. We used the bioinformatics software StarBase and TargetScan to predict lncRNA-miRNA and miRNA-mRNA interactions. Our findings revealed that LINC00858 expression was markedly upregulated in GC tissues and cell lines. Loss-of-function experiments demonstrated that LINC00858 silencing inhibited the proliferation, migration and invasion of GC cells. Bioinformatics analysis showed that there were several complementary binding sites between LINC00858 and microRNA (miR)-363-3p, and further Luciferase reporter assay confirmed the interaction between LINC00858 and miR-363-3p. In addition, forkhead box P4 protein (FOXP4) was found to be a target gene of miR-363-3p in GC cells. FOXP4 overexpression reversed the inhibitory effects of miR-363-3p mimics on cell proliferation, migration and invasion of BGC-823 cells. Collectively, LINC00858 acted as an oncogene in GC via regulating miR-363-3p/FOXP4 axis, which indicated that LINC00858 might be a novel therapeutic target for the treatment of GC.

Suppression of circular RNA circDHCR24 alleviates aortic smooth muscle cell proliferation and migration by targeting miR-149-5p/MMP9 axis

Circular RNAs (circRNAs) are involved in many courses of atherosclerosis and coronary artery disease (CHD). However, the role and effect of circRNAs in vascular restenosis after PCI remains unclear. Human aortic vascular smooth muscle cell (HA-VSMC) was cultured and stimulated with PDGF-BB. The expression profile of circRNAs in HA-VSMCs was screened using microarray analysis. A total 257 aberrantly expressed circRNAs were screened with 2 fold change. Has_circ_0113656 (also called circDHCR24) was validated by qRT-PCR to be significantly up-regulated in PDGF-BB induced HA-VSMCs. CircDHCR24 silencing obviously inhibited the proliferation, migration and phenotypic switch. Moreover, bioinformatics analysis predicted that miR-149-5p had complementary binding sites in 3′-UTR of circDHCR24. Luciferase reporter assay and RIP assay further verified the circDHCR24 acts as a spong for miR-149-5p in HA-VSMCs. Besides, bioinformatics analysis, luciferase reporter assay and RIP assay proved MMP9 was a directly target of miR-149-5p. Finally, cells were transfected with si-circDHCR24 with or without miR-149 inhibitor, and the results showed that co-transfection si-circDHCR24 and miR-149 inhibitor reversed the effect of si-circDHCR24 on cell proliferation, migration and phenotypic switch. Taken together, our study suggested for the first time that the knockdown of circDHCR24 alleviates HA-VSMCs proliferation, migration and phenotypic switching, thereby preventing vascular restenosis.