Complementary Binding Sites Research Articles

E2F1-Induced Overexpression of Long Noncoding RNA SBF2-AS1 Promotes Non-Small-Cell Lung Cancer Metastasis Through Regulating miR-362-3p/GRB2 Axis.

Long noncoding RNA SBF2 antisense RNA 1 (lncRNA SBF2-AS1) has been reported to be involved in non-small-cell lung cancer (NSCLC) tumorigenesis. However, the biological role and regulatory mechanism of lncRNA SBF2-AS1 on NSCLC metastasis remain largely unknown. In this study, the expression level and functional role of SBF2-AS1 were investigated in both NSCLC tissues and cell lines. We found that SBF2-AS1 was upregulated in both NSCLC tissues and cell lines. Patients with high levels of SBF2-AS1 have larger tumors, higher malignancy, and poor prognosis. Knockdown of SBF2-AS1 significantly inhibited tumor growth in vivo and cell proliferation, migration, and invasion in vitro. Moreover, bioinformatics analysis, chromatin immunoprecipitation assay, and luciferase reporter assay proved that the upregulation of SBF2-AS1 was mediated by transcription factor E2F1. Further experiments demonstrated that miR-362-3p had complementary binding site with 3'-UTR of SBF2-AS1. Besides, luciferase reporter assay validated that GRB2 was the target protein of miR-362-3p. Rescue experiments showed that SBF2-AS1 silencing inhibited cell invasion and migration, while cotransfection si-SBF2-AS1 and miR-362-3p inhibitor rescued the effect of si-SBF2-AS1. These results demonstrate that E2F1-induced overexpression of SBF2-AS1 promotes the expression of GRB2 by targeting miR-362-3p to facilitate the metastasis of NSCLC.

PD24-06 MOLECULAR ALTERATION OF GABAERGIC PAIN MODULATORY PATHWAY IN THE ROSTRAL VENTROMEDIAL MEDULLA FOLLOWING ZYMOSAN-INDUCED CYSTITIS IN RATS

You have accessJournal of UrologyBladder & Urethra: Anatomy, Physiology & Pharmacology II (PD24)1 Apr 2020PD24-06 MOLECULAR ALTERATION OF GABAERGIC PAIN MODULATORY PATHWAY IN THE ROSTRAL VENTROMEDIAL MEDULLA FOLLOWING ZYMOSAN-INDUCED CYSTITIS IN RATS Bidyut K. Medda*, Sankar Addya, Bhavana Talluri, Faith Hoelzel, Maia Terashvili, Patrick Sanvanson, Reza Shaker, Jyoti N. Sengupta, and Banani Banerjee Bidyut K. Medda*Bidyut K. Medda* More articles by this author , Sankar AddyaSankar Addya More articles by this author , Bhavana TalluriBhavana Talluri More articles by this author , Faith HoelzelFaith Hoelzel More articles by this author , Maia TerashviliMaia Terashvili More articles by this author , Patrick SanvansonPatrick Sanvanson More articles by this author , Reza ShakerReza Shaker More articles by this author , Jyoti N. SenguptaJyoti N. Sengupta More articles by this author , and Banani BanerjeeBanani Banerjee More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000000881.06AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: The rostral ventromedial medulla (RVM) comprises of several subsets of neurons, which send descending projections to the spinal cord to modulate pain. These neurons are the major targets of efferent projections from the periaqueductal gray (PAG), the site of action of opioid-mediated analgesia. The majority of RVM originating descending neurons are GABAergic. Recently, we have documented microRNA (miRNA)-mediated transcriptional deregulation of spinal GABAergic system and visceral hypersensitivity (colon and bladder) in rats with zymosan-induced cystitis. The objective was to study the effect of noxious bladder insult on molecular expression of GABAergic RVM neurons of rats with zymosan-induced cystitis. METHODS: Two groups of Sprague Dawley rats (n=8 /group) were used in this study. Experimental and control groups received transurethral zymosan (1% in saline, 300ul) and saline respectively for 3 days from postnatal (P) days P58 through P60. Rats in both groups were euthanized on P60 and RVM tissues were collected for analysis. Gene expression profiles of experimental and control groups were evaluated using Rat Clariton S transcriptome arrays. miRNA expression profiles were examine using Affymatrix Gene Chips miRNA arrays. Individual gene was further evaluated using immunohistochemistry (IHC) and in situ hybridization (ISH). RESULTS: Intravesical zymosan treatment resulted in 4-fold downregulation of GABAA receptor subunit alpha 6 (GABAAα6) and glycine transporter Gly 2 (p<0.05 vs controls). Fourteen miRNAs demonstrated differential expression in zymosan treated rats (p<0.05 vs controls). The target gene prediction identified two upregulated miRNAs; miR-129-5p and miR-300 with complementary binding sites for 3’UTRs of GABAAα6 gene indicating a possible post-transcriptional deregulation of the RVM GABAergic pathway in zymosan-treated cystitis rats. A significant downregulation of RVM vesicular GABA transporter VGAT expression was observed in cystitis group by ISH (p<0.05 vs controls). The expression profile of GABAAα6 in RVM was validated by IHC. CONCLUSIONS: Cystitis results in miRNA-mediated transcriptional deregulation of GABA receptor and transporter expression in RVM neurons. This finding strongly suggests that the suppression of the RVM inhibitory transmission may contribute towards the development of spinal neuron hyperexcitability and bladder pain. Source of Funding: NIH R01DK099201 © 2020 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 203Issue Supplement 4April 2020Page: e534-e534 Advertisement Copyright & Permissions© 2020 by American Urological Association Education and Research, Inc.MetricsAuthor Information Bidyut K. Medda* More articles by this author Sankar Addya More articles by this author Bhavana Talluri More articles by this author Faith Hoelzel More articles by this author Maia Terashvili More articles by this author Patrick Sanvanson More articles by this author Reza Shaker More articles by this author Jyoti N. Sengupta More articles by this author Banani Banerjee More articles by this author Expand All Advertisement PDF downloadLoading ...

The Modulation of miR-195-TGF-β/Smad3 Pathway on Proliferation, Apoptosis and Invasion of Glioma Cells

Glioma is one of the most common malignant tumors of the central nervous system with the high incidence. The abnormal expression of Smad3 is related to the occurrence, progression, metastasis, and drug resistance of various kinds of tumors. It was reported that the decreased expression of miR-195 was related to the onset of glioma. This study aims to explore whether miR-195 plays an important role in regulating Smad3 and influencing the cell biology of glioma. Bioinformatics analysis was performed to determine the target complementary binding site between miR-195 and Smad3. The expressions of miR-195 and Smad3 mRNA were measured in tumor tissue of patients with glioma.In vitro culture, SHG-44 cells were divided into 2 groups: miR-NC group, miR-195 mimic group. The expression of miR-195 was detected by qRT-PCR, and expression of Smad3 was measured by western blot. Apoptosis was detected by flow detection. Cell proliferation and cell invasion ability was detected by Ed U staining and Transwell experiments respectively. Our result showed that, compared with that of the carcinoma tissue, the expressions of miR-195 and Smad3 in glioma tissue were decreased significantly. Compared with normal glial cells HEB, the expression of miR-195 was decreased significantly, whereas the expression of Smad3 was increased significantly. The transfection of miR-195 mimic significantly reduced the expression Smad3 and p-Smad3, which significantly reduced the ability of cell proliferation, and increased apoptosis. In conclusion, miR195 can target regulate proliferation, apoptosis and invasion of glioma cells through TGF-β /Smad3 pathway.

Long non-coding RNA CCAT1 promotes colorectal cancer cell migration, invasiveness and viability by upregulating VEGF via negative modulation of microRNA-218.

Increasing evidence has demonstrated that long non-coding (lnc) RNA is aberrantly expressed in numerous types of cancer. Colorectal cancer is a common malignancy; however, the role and mechanism underlying the influence of lncRNA-colon cancer associated transcript 1 (CCAT1) in colorectal cancer is yet to be elucidated. The present study revealed that CCAT1 is highly expressed in colorectal cancer tissues. Bioinformatics analysis and a dual-luciferase reporter gene assay indicated that CCAT1 and microRNA (miR)-218 had complementary binding sites. Furthermore, reverse transcription-quantitative PCR revealed that miR-218 was downregulated in colorectal cancer tissues compared with paired adjacent healthy tissues. To investigate the biological effects of CCAT1 on colorectal cancer cells, MTT and Transwell assays were performed. The results revealed that when compared with the control group, CCAT1-short hairpin (sh)RNA significantly inhibited colorectal cancer cell (SW480) viability and decreased migration and invasiveness. In addition, CCAT1-shRNA significantly reduced vascular endothelial growth factor (VEGF) expression in SW480 cells; however, these effects were partially rescued by an miR-218 inhibitor. Furthermore, it was revealed that the CCAT1-plasmid significantly promoted the viability of SW480 cells, increased cell migration and invasiveness, and significantly increased VEGF expression. However, these effects were also partially rescued by with a miR-218 mimic. Taken together, the present results identified that the CCAT1/miR-218 axis serves a key role in the regulation of colorectal cancer progression, which may be used as potential therapeutic target for the treatment of colorectal cancer.

MiR‑200b regulates breast cancer cell proliferation and invasion by targeting radixin

Radixin is an important member of the Ezrin-Radixin-Moesin protein family that is involved in cell invasion, metastasis and movement. microRNA (miR)-200b is a well-studied microRNA associated with the development of multiple tumors. Previous bioinformatics analysis has demonstrated that miR-200b has a complementary binding site in the 3'-untranslated region of radixin mRNA. The present study aimed to investigate the role of miR-200b in regulating radixin expression, cell proliferation and invasion in breast cancer. Breast cancer tissues at different Tumor-Node-Metastasis (TNM) stages were collected; breast tissues from patients with hyperplasia were used as a control. miR-200b and radixin mRNA expression levels were tested by reverse transcription-quantitative PCR. Radixin protein expression was detected by western blotting. The highly metastatic MDA-MB-231 cells were divided into four groups and transfected with a miR-negative control (NC), miR-200b mimic, small interfering (si)RNA-NC or siRNA targeting radixin. Cell invasion was evaluated by Transwell assay and cell proliferation was assessed by 5-ethynyl-2'-deoxyuridine staining. Compared with the control group, radixin mRNA expression was significantly higher in breast cancer tissues and increased with TNM stage. miR-200b expression levels exhibited the opposite trend. Radixin mRNA expression in breast cancer cells was notably higher, whereas miR-200b expression was lower compared with that in normal breast epithelial MCF-10A cells. The expression of radixin was higher, whereas miR-200b was lower in MDA-MB-231 cells compared with that in MCF-7 cells. miR-200b mimic or siRNA-radixin transfection downregulated the expression of radixin in MDA-MB-231 cells and attenuated the invasive and proliferative abilities of these cells. miR-200b-knockdown and radixin overexpression were associated with enhanced cell invasion in breast cancer. In conclusion, miR-200b regulates breast cancer cell proliferation and invasion by targeting radixin expression.

The potential mechanism of miR-130b on promotion of the invasion and metastasis of hepatocellular carcinoma by inhibiting Notch-Dll1

Introduction: This study aimed to elucidate the regulatory role and molecular regulation mechanism of miR-130b gene in the process of invasion and metastasis of hepatocarcinoma, and provide a theoretical basis for seeking of effective prevention and treatment of new targets for hepatocellular carcinoma.Materials and methods: The expression level of miR-130b gene in hepatocarcinoma tissues was determined by qRT-PCR. The biological function and mechanism of miR-130b gene were verified by cell and animal models, and the target gene was verified by double luciferase assay.Results: In the liver cancer tissues of patients with metastasis, the expression level of miR-130b gene was increased, and the difference was significantly significant (p < 0.05). Evaluation of independent risk factors for overall survival showed significant difference (p < 0.01). Up-regulation of miR-130b in MHCC97L– subpopulation cells significantly enhanced the invasion and migration ability, and the difference was statistically significant (p < 0.05). The invasion and migration ability of MHCC97H + subpopulation cells with increased expression of miR-130b was significantly decreased, and the difference was notably significant (p < 0.05). When the expression of miR-130b in MHCC97H + subpopulation cells was inhibited, the expressions of Notch-Dll1 and SOX2, Nanog and E2F3 proteins in transplanted tumor tissues were significantly higher than those in other groups (p < 0.05). When miR-130b in MHCC97L– subpopulation cells was up-regulated, the expressions of Notch-Dll1 and Bcl-2, CCND1, Nanog and MET proteins in transplanted tumor tissues were significantly increased than those in other groups (p < 0.05). The prediction results of bioinformatics data suggest that the target gene of miR-130b may be Notch-Dll1 gene. The experiment of luciferase reporter gene confirmed that miR-130b gene can be inhibited and contains fluorescent reporter gene with complementary binding site, lost activity.Conclusion: The miR-130b gene can inhibit the protein expression of Notch-Dll1, and it can promote the invasion and metastasis of liver cancer cells.

Mannosylated hemagglutinin peptides bind cyanovirin-N independent of disulfide-bonds in complementary binding sites.

Cyanovirin-N (CV-N) has been shown to reveal broad neutralizing activity against human immunodeficiency virus (HIV) and to specifically bind Manα(1→2)Manα units exposed on various glycoproteins of enveloped viruses, such as influenza hemagglutinin (HA) and Ebola glycoprotein. Chemically synthesized dimannosylated HA peptides bound domain-swapped and dimeric CV-N with either four disulfide-bonds (Cys–Cys), or three Cys–Cys bonds and an intact fold of the high-affinity binding site at an equilibrium dissociation constant KD of 10 μM. Cys–Cys mutagenesis with ion-pairing amino-acids glutamic acid and arginine was calculated by in silico structure-based protein design and allowed for recognizing dimannose and dimannosylated peptide binding to low-affinity binding sites (KD ≈ 11 μM for one C58–C73 bond, and binding to dimannosylated peptide). In comparison, binding to HA was achieved based on one ion-pairing C58E–C73R substitution at KD = 275 nM, and KD = 5 μM for two C58E–C73R substitutions. We were utilizing a triazole bioisostere linkage to form the respective mannosylated-derivative on the HA peptide sequence of residues glutamine, glycine, and glutamic acid. Thus, mono- and dimannosylated peptides with N-terminal cysteine facilitated site-specific interactions with HA peptides, mimicking a naturally found N-linked glycosylation site on the HA head domain.

Silencing of long noncoding RNA TUG1 inhibits viability and promotes apoptosis of acute myeloid leukemia cells by targeting microRNA-221-3p/KIT axis.

Acute myeloid leukemia (AML) is a hematological malignancy. This study was attempted to uncover the effects of long noncoding RNA taurine-upregulated gene1 (TUG1) on the viability and apoptosis of AML cells. QRT-PCR was implemented to examine the expression of TUG1, miR-221-3p and KIT in AML. The correlation between TUG1 and clinicopathological features of AML patients was evaluated. The effect of TUG1 on AML cells were studied by RNA interference approach. AML cells were transfected with miR-221-3p mimic and miR-221-3p inhibitor, respectively. Then the viability and apoptosis of AML cells were examined by MTT and flow cytometry assay, respectively. Additionally, dual-luciferase reporter assay was used to confirm the interactions among TUG1, miR-221-3p and KIT. Western blot was applied to analyze protein expression of KIT. The expression of TUG1 and KIT was up-regulated in AML, but miR-221-3p was down-regulated. TUG1 expression had obviously correlation with World Health Organization (WHO) grade in AML patients. The functional experiment stated that TUG1 silencing suppressed the viability and accelerated the apoptosis of AML cells. Moreover, the mechanical experiment demonstrated that TUG1 and KIT were both targeted by miR-221-3p with the complementary binding sites at 3'UTR. Up-regulation of miR-221-3p inhibited the protein expression of KIT. Furthermore, in the feedback experiment, miR-221-3p inhibition or KIT overexpression reversed the repression of tumor behavior induced by TUG1 silencing. TUG1 silencing retarded viability and promoted apoptosis of AML cells via regulating miR-221-3p/KIT axis, providing a potential therapeutic target for AML.

LINC00162 participates in the pathogenesis of diabetic nephropathy via modulating the miR-383/HDAC9 signalling pathway

Diabetic nephropathy (DN) is a common chronic complication of diabetes. In this study, we aimed to explore the potential role of lncRNA LINC-00162 in the pathogenic process of DN. LncRNA microarray analysis, real-time PCR, IHC computational analysis and luciferase assay were performed to explore the regulatory relationship among LINC00162, miR-383 and HDAC9. There was an obvious difference between T2D + DN and T2D − DN patients in their levels of eGRF and albuminuria. A significant difference was observed between T2D + DN and T2D − DN groups in terms of their LINC00162 expression. In particular, LINC00162 and HDAC9 were highly expressed, while miR-383 was lowly expressed in tissues derived from the T2D + DN group compared with those in tissues derived from the T2D − DN group. MiR-383 was able to bind to LINC00162, while HDAC9 was a direct downstream target of miR-383 with a complementary miR-383 binding site located in the 3′ UTR of HDAC9. LINC00162 reduced miR-383 expression and further up-regulated HDAC9 expression, while miR-383 mimics reduced HDAC9 expression under a dose-dependent manner. In summary, we suggested for the first time that down-regulation of LINC00162 was associated with the development of DN in T2D via the up-regulation of miR-383 expression and reduction of HDAC9 expression.

MiR-155 targeting FoxO3a regulates oral cancer cell proliferation, apoptosis, and DDP resistance through targeting FoxO3a.

FoxO3a is a well-defined tumor suppressor gene in the forkhead transcription factor O subfamily (FoxO), and its reduction is related to the occurrence of various tumors. It was found that the expression of miR-155 is abnormally elevated in oral cancer tissues, suggesting that miR-155 may be a tumor-promoting gene for oral cancer. Bioinformatics analysis revealed a targeted complementary binding site between miR-155 and FoxO3a. In this study, we established a cisplatin-resistant cell line of oral cancer to compare the expression of miR-155 and FoxO3a in parental oral cancer cells, and explore whether miR-155 regulates the expression of FoxO3a and affects the proliferation and apoptosis of oral cancer cells. Dual luciferase reporter gene assay validated the targeted regulatory role between miR-155 and FoxO3a. The DDP-resistant oral cancer cell line KB/DDP was established, and the expressions of miR-155 and FoxO3a were compared. The CCK-8 assay was used to calculate the resistance index (RI). KB/DDP cells were divided into miR-NC group and miR-155 inhibitor group. Cell apoptosis was detected by flow cytometry. Cell proliferation ability was detected by EdU staining. There is a targeted regulatory relationship between miR-155 and FoxO3a mRNA. The expression of miR-155 was significantly higher, while the expression of Foxo3a mRNA and protein was obviously lower in KB/DDP cells than that in KB cells. CCK-8 experiments showed that the same concentration of DDP exhibited apparently lower proliferation inhibition in KB/DDP cells than KB cells. Compared with the miR-NC group, transfection of miR-155 inhibitor markedly increased the expression of Foxo3a in KB/DDP cells, which significantly attenuated cell proliferation and enhanced apoptosis in DDP-treated cells. Increased expression of miR-155 and decreased expression of Foxo3a were associated with DDP resistance in oral cancer cells. Inhibition of miR-155 expression upregulated Foxo3a expression, restrained oral cancer cell proliferation, promoted cell apoptosis, and enhanced DDP sensitivity in oral cancer cells.

Role of Multivalent Interactions in Dynamic-Template-Directed Assembly of Conjugated Polymers

Dynamic-template-directed assembly is a promising method to enhance molecular ordering and electronic properties of solution-coated polymer semiconductor thin films over large area. In this work, we establish that multicomponent dynamic templates of complimentary chemistries can promote polymer crystallization through cooperative multivalent interactions. We investigate this phenomenon using a combination of templating substrates including a fluoropolymer, a hydrogen-bonded liquid and an ionic liquid. Template-dependent multiscale morphology is studied by a comprehensive set of characterization techniques to understand how introducing diverse chemical moieties modulates polymer assembly. Our results clearly confirm synergistic effect between components of complimentary chemistries constituting the dynamic template. The relative degree of crystallinity is improved by 50-150% for films deposited on multicomponent dynamic templates compared to their neat constituents. In addition, macroscopic alignment is increased significantly (2 to 5 times) compared to single-component templates. As a result, highly anisotropic charge transport is observed with apparent hole mobilities up to 3.6 cm2V-1s-1. In contrast, such synergistic effect is not observed when using multicomponent dynamic template of comparable chemistries (i.e. ionic liquid and polymerized ionic liquid). We elucidate the origin of this synergistic effect through a combination of attenuated total reflectance Fourier transform infrared spectroscopy and isothermal titration calorimetry. When the dynamic template is comprised of two or more components interacting with complementary binding sites on the conjugated polymer (esp. backbone vs. side chain), the template-polymer interactions is significantly enhanced compared to the sum of single component contributions. These results provide valuable insights on surface-directed conjugated polymer crystallization during large-area solution coating. Template dynamics is rarely studied, and represents a new opportunity for guiding assembly of soft functional matter.

Deregulation of UCA1 expression may be involved in the development of chemoresistance to cisplatin in the treatment of non-small-cell lung cancer via regulating the signaling pathway of microRNA-495/NRF2.

Non-small-cell lung cancer (NSCLC) remains the leading cause of cancer death worldwide. As a platinum-based chemotherapeutic drug, cisplatin has been used for over 30 years in NSCLC treatment while its effects are diminished by drug resistance. Therefore, we aimed to study the potential role of UCA1 in the development of chemoresistance against cisplatin. Real-time polymerase chain reaction, western-blot analysis, and immunofluorescence were used to study the involvement of UCA1, miR-495, and NRF2 in chemoresistance against cisplatin. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay was performed to determine the effect of cisplatin on cell proliferation. Computational analysis and luciferase assay were carried out to explore the interaction among UCA1, miR-495, and NRF2. The cisplatin-R group exhibited lower levels of UCA1 and NRF2 expression but a higher level of miR-495 expression than the cisplatin-S group. The growth rate and half-maximal inhibitory concentration of cellular dipeptidyl peptidase (cisplatinum) of the cisplatin-R group were much higher than those in the cisplatin-S group. MiR-495 contained a complementary binding site of UCA1, and the luciferase activity of wild-type UCA1 was significantly reduced after the transfection of miR-495 mimics. MiR-495 directly targeted the 3'-untranslated region (3'-UTR) of NRF2, and the luciferase activity of wild-type NRF2 3'-UTR was evidently inhibited by miR-495 mimics. Finally, UCA1 and NRF2 expressions in the effective group were much lower than that in the ineffective group, along with a much higher level of miR-495 expression. We suggested for the first time that high expression of UCA1 contributed to the development of chemoresistance to cisplatin through the UCA1/miR-495/NRF2 signaling pathway.

MiR-199 inhibits EMT and invasion of hepatoma cells through inhibition of Snail expression.

MiR-199 expression is associated with liver cancer. Bioinformatics analysis revealed that miR-199 has a complementary binding site to the 3'-UTR region of Snail mRNA. This study investigated whether miR-199 plays a role in regulating Snail expression and affecting epithelial-mesenchymal transition (EMT) and invasion of hepatoma cells. The Dual-Luciferase reporter gene assay validated the targeted regulation between miR-199 and Snail. QRT-PCR was used to detect and compare the expression of miR-199 and Snail mRNA in human normal liver HL7702 cells, low metastatic MHCC97L cells, and high metastatic MHCC97H cells. MHCC97H cells were cultured in vitro and divided into two groups: miR-NC group and the miR-199 mimic group followed by the analysis of the expression of Snail, E-cadherin, and N-cadherin, as well as cell invasion ability by transwell assay. There was a targeted regulatory relationship between miR-199 and Snail mRNA. Compared with HL7702 cells, miR-199 expression was significantly decreased, and Snail expression was significantly increased in MHCC97L and MHCC97H cells, with more changes being observed in high metastatic MHCC97H cells. The transfection of miR-199 mimic significantly downregulated the expression of Snail and N-cadherin in MHCC97H cells, increased E-cadherin expression, inhibited the cell's EMT process, and invasion. The decrease of miR-199 expression plays a role in upregulating the expression of Snail and promoting EMT and invasion of hepatocarcinoma cells. The increase of the expression of miR-199 can inhibit the expression of Snail and inhibit the EMT process and invasion ability of hepatoma cells.

Long non-coding RNA LINC00641 promotes cell growth and migration through modulating miR-378a/ZBTB20 axis in acute myeloid leukemia.

Many researchers have revealed that long noncoding RNAs (lncRNAs) acted as modulators in tumor biology. LncRNA LINC00641 (LINC00641), a newly discovered tumor-related lncRNA, has been reported to act as a modulator in several tumors. Hence, our study aimed to examine the expression and function of LINC00641 in acute myeloid leukemia (AML). The expression pattern of LINC00641 in AML specimens and cell lines was explored using a gene expression profiling interactive analysis (GEPIA) tool and RT-PCR assays. The cell counting kit-8 (CCK-8) assays, transwell migration, and invasion assays were used for the functional study of cell viability, cell migration, and invasion. The influence of LINC00641 on cell cycle and apoptosis was determined using Flow cytometry detection. The regulating associations between LINC00641, miR-378a, and ZBTB20 were investigated in AML cells using the Luciferase reporter assays and RT-PCR assays RESULTS: We found that LINC00641 was highly expressed in AML specimens and cell lines. Functionally, the silence of LINC00641 inhibited the proliferation, migration, invasion, and cell cycle arrest in AML cells while inducing their apoptosis. The results using bioinformatics assays predicted the complementary binding sites within LINC00641 and miR-378a, which was demonstrated by the use of the Luciferase reporter assays. In addition, we also demonstrated that ZBTB20 was a direct target of miR-378a. Moreover, the inhibition of miR-378a could rescue the ZBTB20 protein level decrease induced by LINC00641 knockdown. We firstly identified LINC00641 as a novel AML-related lncRNA whose knockdown inhibited cell proliferation, migration, invasion, and promoted apoptosis by modulating miR-378a/ZBTB20 axis in AML.

Effect of long non-coding RNA colorectal neoplasia differentially expressed targeting microRNA-384 on invasion and migration of lung cancer SPC-A1 cells

Objective To investigate the effect of long non-coding RNA (lncRNA) colorectal neoplasia differentially expressed (CRNDE) targeting microRNA (miRNA, miR)-384 on invasion and migration of lung cancer SPC-A1 cells. Methods Small interfering RNA (siRNA) CRNDE was transfected into lung cancer SPC-A1 cells, and the interference effect was determined by real-time quantitative polymerase chain reaction (Real-time PCR). Cell proliferation was measured by methyl thiazol tetrazolium (MTT) assay, cell invasion and migration were measured by Transwell chamber, and Western blotting was used to detect the expression of matrix metalloproteinase (MMP)-9, MMP-2, E-cadherin and Vimentin in cells. The targeting relationship prediction software predicts that CRNDE binds to miR-384. Luciferase reporting system was used to identify the targeting relationship. In SPC-A1 cells, siRNA CRNDE and miR-384 inhibitors were co-transfected, and the above methods were used to determine the reverse effect of miR-384 inhibitors on the invasion and migration of lung cancer SPC-A1 cells transfected with siRNA CRNDE.Comparison two groups was performed using t test, and comparison three groups was performed with one-way analysis of variance (ANOVA). Results The expression of CRNDE in lung cancer SPC-A1 cells transfected with siRNA CRNDE decreased (1.01±0.12 vs. 0.43±0.06), cell proliferation decreased (0.37±0.05 vs. 0.24±0.02, F=10.467, P<0.05), the number of invasive cells (141.25±15.23 vs. 101.02±9.84, F=9.420, P<0.05) and migrating cells (188.20±16.84 vs. 142.58±11.22, F=8.345, P<0.05) decreased, the expression levels of MMP-9, MMP-2 and Vimentin proteins in cells decreased, and E-cadherin protein level increased. There were complementary binding sites between CRNDE and miR-384. CRNDE targeted and negatively regulated the expression of miR-384. The miR-384 inhibitors reversed the effects of siRNA CRNDE on proliferation (0.26±0.03 vs. 0.35±0.04, t=3.118, P<0.05), migration (139.84±10.57 vs. 176.51±12.45, t=5.276, P<0.05), invasion (99.84±10.02 vs. 134.25±11.25, t=3.890, P<0.05) and expression of MMP-9, MMP-2, E-cadherin and Vimentin in lung cancer cells. Conclusion Down-regulation of lncRNA CRNDE targeting miR-384 inhibits invasion and migration of lung cancer SPC-A1 cells. Key words: Lung cancer; MicroRNA-384; Colorectal neoplasia differentially expressed targeting; Migration; Invasion

MiR-155 affects proliferation and apoptosis of bladder cancer cells by regulating GSK-3β/β-catenin pathway.

GSK-3β negatively regulates Wnt/β-catenin signaling pathway. The abnormal miR-155 expression is associated with bladder cancer. Bioinformatics analysis revealed a complementary binding site between miR-155 and GSK-3β mRNA. This study investigated the role of miR-155 in the proliferation and apoptosis of bladder cancer cells. The dual luciferase reporter gene assay validated the targeted regulation between miR-155 and GSK-3β. Tumor tissues and adjacent tissues were collected from bladder cancer patients and the expression of miR-155 and GSK-3β mRNA was detected by RT-PCR. Bladder cancer cell line BIU-87 cells were cultured in vitro and divided into miR-NC group and miR-155 inhibitor group. The expressions of miR-155, GSK-3β and β-catenin were compared, cell apoptosis was detected by flow cytometry, and cell proliferation was detected by EdU staining. Compared with adjacent tissues, miR-155 expression was significantly increased in bladder cancer tissues, and GSK-3β mRNA expression was significantly decreased. There was a targeted regulatory relationship between miR-155 and GSK-3β. Compared with SV-HUC-1 cells, miR-155 expression in bladder cancer BIU-87 and 5637 cells was significantly increased, and GSK-3β expression was significantly decreased. Transfection of miR-155 inhibitor significantly increased GSK-3β expression in BIU-87 and 5637 cells, decreased β-catenin expression, increased cell apoptosis, and decreased cell proliferation. The increased expression of miR-155 plays a role in reducing the expression of GSK-3β and in promoting the pathogenesis of bladder cancer. Inhibition of miR-155 can up-regulate the expression of GSK-3β, inhibit the activity of Wnt/β-catenin pathway, attenuate proliferation and promote apoptosis of bladder cancer cells.

LncRNA SNHG3 regulates laryngeal carcinoma proliferation and migration by modulating the miR-384/WEE1 axis

LncRNA SNHG3 (SNHG3) is involved in tumor development and progression, but little is known about how SNHG3 functions in laryngeal carcinoma (LC). Real time-PCR (RT-PCR) was used to estimate the expression of SNHG3 in LC tissues and cell lines TU212, TU686, and Hep-2. Cell viability, migration, and invasion were evaluated. Our results showed increased SNHG3 in LC tissues and cell lines. Loss of function of SNHG3 reduced cell viability, migration, and invasion of TU212 and TU686 cells. Western blot analyses demonstrated that the protein levels of MMP2 and MMP9 decreased after SNHG3 silencing. Additionally, bioinformatics software predicted that SNHG3 could sponge miR-384 at the 3′-UTR with complementary binding sites, which was validated by a dual-luciferase reporter assay. RT-PCR analysis revealed that knockdown of SNHG3 upregulated miR-384 expression and that overexpression of miR-384 decreased SNHG3. Furthermore, a dual-luciferase reporter assay showed that miR-384 could bind to the 3′-UTR of WEE1, and inhibition of miR-384 markedly increased WEE1 expression. The mRNA and protein levels of WEE1 were downregulated upon deletion of SNGH3. Suppression of WEE1 partly abolished the tumorigenic migration and invasion potential of the miR-384 inhibitor in migration and invasion. Inhibition of miR-384 partially reversed the biological activities of SNHG3 in TU212 and TU686 cells. Collectively, our results indicate that SNHG3 regulated LC cell migration and invasion via the miR-384/WEE1 axis.

MiR-181b regulates atherosclerotic inflammation and vascular endothelial function through Notch1 signaling pathway.

To explore the influences of micro ribonucleic acid (miR)-181b on the inflammation and vascular endothelial function in atherosclerosis (AS), and its specific molecular regulatory mechanism. 44 apolipoprotein E (ApoE)-/- 7 weeks old male rats were randomly divided into normal diet group (NC group) and AS model group (high-fat diet feeding). Rat aorta was dissected and the serum sample was collected in both groups. The serum levels of inflammatory factors in both groups were detected via enzyme-linked immunosorbent assay (ELISA). The mRNA levels of miR-18b and Notch1 were detected via Reverse Transcription-Polymerase Chain Reaction (RT-PCR). Moreover, aortic endothelial cells were extracted from AS rats. The mir-18b binding target gene was analyzed via bioinformatics and further verified by the Luciferase reporter gene assay. The protein expressions of miR-18b and Notch1 in endothelial cells transfected with miR-181b mimic or inhibitor were detected. Influence of miR-181b on vascular endothelial indexes was also detected. Compared with those in the NC group, the serum levels of interleukin-6 (IL-6), IL-10, IL-13 and tumor necrosis factor-α (TNF-α) in the AS group significantly increased (p<0.05). The mRNA level of miR-18b in AS plaques was significantly lower than that in NC arterial tissues. Conversely, Notch1 level in AS plaques was markedly higher than that in the NC arterial tissues (p<0.05), with the mean difference of 2.12 and 2.82 folds (p<0.05). According to the Pearson correlation analysis, there was a significant negative correlation between mRNA levels of miR-181b and Notch1 in AS tissues (r=-0.65, p=0.014). The bioinformatics analysis showed that there were complementary binding sites between miR-181b and Notch1. The Luciferase reporter gene assay confirmed the presence of direct binding sites between miR-181b and Notch1. Western blotting revealed that the overexpression of miR-181b downregulated Notch1 and hs-CRP, but upregulated BNP (p<0.05). Opposite trends were obtained by miR-181b knockdown (p<0.05). The decline in the miR-181b expression may be an important factor in AS plaque formation and vascular endothelial injury by regulating Notch1.

A dual template imprinted polymer modified electrochemical sensor based on Cu metal organic framework/mesoporous carbon for highly sensitive and selective recognition of rifampicin and isoniazid

A highly sensitive and selective doubly imprinted polymer modified electrochemical sensor based on copper metal organic framework/mesoporous carbon (Cu-MOF/MC) composite was developed for simultaneous determination of Rifampicin (RIF) and Isoniazid (INZ). The molecularly imprinted polymer (MIP) film was formed on the surface of Cu-MOF/MC modified glassy carbon electrode (GCE) by incorporating RIF and INZ as template molecules during electropolymerization of pyrrole, followed by removal of both the template molecules. During this process of sensor fabrication, large numbers of complementary binding sites for template molecules are created on the surface of MIP sensor due to high surface area offered by Cu-MOF/MC composite. Various parameters influencing the performance of MIP sensor such as number of electropolymerization cycles and template: monomer ratio were optimized during the study. Based on the optimized parameters, the electrochemical response of Cu-MOF/MC/MIP/GCE towards determination of RIF and INZ was studied in a wide linear working range of 0.08 μM to 85 μM employing adsorptive stripping differential pulse voltammetry in aqueous as well as real samples. The limit of detection was found to be 0.28 nM and 0.37 nM for RIF and INZ respectively. In addition, the MIP sensor showed good stability, acceptable reproducibility and high selectivity for RIF and INZ over other common interferents. Moreover, the developed sensor was successfully applied for simultaneous determination of RIF and INZ in pharmaceutical formulations, blood serum and urine samples with satisfactory recoveries.

Identification and co-evolution pattern of stem cell regulator miR394s and their targets among diverse plant species

BackgroundMicro RNAs (miRNAs), a class of small non-coding RNAs, have been implicated in various aspects of plant development. miR394 is required for shoot apical meristem organization, stem cell maintenance and abiotic stress responses in Arabidopsis, where it functions by negatively regulating the transcript level of target LEAF CURLING RESPONSIVENESS (LCR), which is an F-box protein-coding gene. The evolutionary conservation of stem cell regulatory miR394-LCR module among plants remains elusive.ResultsOur study has identified 79 miR394 and 43 target sequences across 40 plant species using various homology based search tools and databases, and analysed their co-evolution pattern. We customised an annotation workflow which computationally validates 20 novel miR394s from 14 plant species. Independent phylogenetic trees were reconstructed with precursor MIR394s, mature miR394s, and their target sequences along with complementary miR394 binding sites. The phylogeny revealed that mature sequences of miR394s as well as their targets belonging to the F-box protein encoding gene families, were highly conserved. Though, miR394–3p were complementary to miR394s/miR394–5p, they clustered separately.ConclusionThe existence and separate clustering of miR394–3p and miR394s/miR394–5p indicate their independent regulation. The phylogeny also suggests that miR394s had evolved at the beginning of gymnosperm-angiosperm divergence. Despite strong conservation, some level of sequence variation in miR394s and the complementary binding sites of their targets suggests possible functional diversification of miR394-LCR mediated stem cell regulation in plants.