Angiotensin II directly impairs adipogenic differentiation of human preadipose cells

microRNAs (miRNAs) are small non-coding RNAs that have been suggested to play an essential role in tumorigenesis. Reduced expression of miR-338 has been reported in several types of cancers; however, the role of miR-338 in oral squamous cell carcinoma (OSCC) has not been elucidated. In this study, we demonstrated that miR-338 was dramatically downregulated in OSCC tissues and cell lines. Overexpression of miR-338 significantly inhibited proliferation, colony formation, migration, and invasion of OSCC cells. In addition, neuropilin1 (NRP1) was identified as a target of miR-338 in OSCC cells and inversely correlated with miR-338 in OSCC tissues. Furthermore, restoration of NRP1 attenuated the tumor-suppressive effects of miR-338. Taken together, miR-338 might inhibit growth and metastasis of OSCC cells by targeting NRP1.     

MiR‐133a‐3p Inhibits Oral Squamous Cell Carcinoma (OSCC) Proliferation and Invasion by Suppressing COL1A1

The aim of our study was to investigate the effects of miR‐133a‐3p on human oral squamous cell carcinoma (OSCC) cells by regulating gene COL1A1. OSCC tissues, adjacent tongue epithelial tissues, the immortalized oral epithelial cell line HIOEC, and OSCC cell lines (CAL‐27, TCA‐8113, SCC‐4, SCC‐9, and SCC‐15) were used in this research. Quantitative real‐time PCR (RT‐qPCR) was employed to determine the expression of miR‐133a‐3p and COL1A1. Dual luciferase reporter gene assay and Western blot were applied to verify the binding relationship between miR‐133a‐3p and COL1A1. Functional assays were also conducted in this study, including CCK‐8 assay, colony formation assay, flow cytometry analysis as well as Transwell assay. MiR‐133a‐3p was found low‐expressed both in OSCC tissues and cells lines compared with normal tissues and cell line, respectively, whereas COL1A1 was just the opposite. The over‐expression of miR‐133a‐3p or the down‐regulation of COL1A1 suppressed the proliferation, invasion, and mitosis of OSCC cells, whereas simultaneous down‐regulation of miR‐133a‐3p and up‐regulation of COL1A1 led to no significant alteration of cell activities. MiR‐133a‐3p could inhibit the proliferation and migration of OSCC cells through directly targeting COL1A1 and reducing its expression. J. Cell. Biochem. 119: 338–346, 2018. © 2017 Wiley Periodicals, Inc.     

Systematic evaluation of microRNA processing patterns in tissues, cell lines, and tumors

Very little is known regarding regulation of microRNA (miRNA) biogenesis in normal tissues, tumors, and cell lines. Here, we profiled the expression of 225 precursor and mature miRNAs using real-time PCR and compared the expression levels to determine the processing patterns. RNA from 22 different human tissues, 37 human cancer cell lines, and 16 pancreas and liver tissues/tumors was profiled. The relationship between precursor and mature miRNA expression fell into the following four categories: (1) a direct correlation exists between the precursor and mature miRNA expression in all cells/tissues studied; (2) direct correlation of the precursor and mature miRNA exists, yet the expression is restricted to specific cell lines or tissues; (3) there is detectable expression of mature miRNA in certain cells and tissues while the precursor is expressed in all or most cells/tissues; or (4) both precursor and mature miRNA are not expressed. Pearson correlation between the precursor and mature miRNA expression was closer to one for the tissues but was closer to zero for the cell lines, suggesting that processing of precursor miRNAs is reduced in cancer cell lines. By using Northern blotting, we show that many of these miRNAs (e.g., miR-31, miR-105 and miR-128a) are processed to the precursor, but in situ hybridization analysis demonstrates that these miRNA precursors are retained in the nucleus. We provide a database of the levels of precursor and mature miRNA in a variety of cell types. Our data demonstrate that a large number of miRNAs are transcribed but are not processed to the mature miRNA.     

Circle RNA hsa_circRNA_100290 serves as a ceRNA for miR-378a to regulate oral squamous cell carcinoma cells growth via Glucose transporter-1 (GLUT1) and glycolysis

Aerobic glycolysis (the Warburg effect) is a robust metabolic hallmark of most tumors, including oral squamous cell carcinoma (OSCC). Glucose transporter 1 (GLUT1), a major glucose transporter regulating the glucose uptake, is upregulated in OSCC and participated in the cell glycolysis of OSCC. The deregulation and function of noncoding RNAs in cancers have been widely reported. Reportedly, hsa_circular RNA (circRNA)_100290 (circ_SLC30A7) is significantly upregulated (fold change = 6.91, p < 0.0000001) in OSCC. According to online tools prediction (miRWalk, miRanda, and Targetscan), miR-378a could simultaneously target circRNA_100290 and GLUT1. Herein, the expression of circRNA_100290 and GLUT1 remarkably increased in oral tumor tissue specimens and cells. In OSCC cell lines, cell proliferation and glycolysis could be remarkably downregulated by circRNA_100290 silence, which could be rescued by GLUT1 overexpression. Conversely, miR-378a expression could be remarkably inhibited in tumor tissue specimens and cells. The effect of miR-378a overexpression on OSCC cells was similar to those of circRNA_100290 silence. miR-378a directly bound to circRNA_100290 and GLUT1 3′-untranslated region, circRNA_100290 could remarkably relieve miR-378a-induced inhibition on GLUT1 via acting as a competing endogenous RNA (ceRNA). miR-378a inhibition remarkably attenuated the effect of circRNA_100290 silence on cell proliferation and glycolysis in OSCC cell lines. In summary, circRNA_100290 serves as a ceRNA to counteract miR-378a-mediated GLUT1 suppression, thus promoting glycolysis and cell proliferation in OSCC. We provide a reliable experimental basis for understanding the mechanism of cell growth and glycolysis deregulation in OSCC.     

CircKRT1 drives tumor progression and immune evasion in oral squamous cell carcinoma by sponging miR-495-3p to regulate PDL1 expression

Regulatory functions of circRNAs by targeting the micro RNA (miRNA)/mRNA axis have been increasingly found in oral squamous cell carcinoma (OSCC). CircRNA keratin 1 (CircKRT1) and miR-495-3p were dysregulated in OSCC. Programmed death ligand 1 (PDL1) was an important immunotherapeutic molecule in OSCC. Our objective was to explore whether circKRT1 could regulate cancer progression and immune evasion in OSCC by affecting the miR-495-3p/PDL1 axis. RNA expression was examined by quantitative real-time polymerase chain reaction. All protein levels were detected by western blot. OSCC cell growth was assessed by CCK-8 and colony formation assays. Cell migratory and invasive abilities were evaluated by transwell assay. CD8⁺ T-cell cytotoxicity was determined via lactate dehydrogenase assay. CD8⁺ T-cell percentage and apoptosis were analyzed by flow cytometry. Target screening was performed by Veen Diagram and RNA pull-down assay. Target binding was verified using dual-luciferase reporter and RNA immunoprecipitation assays. A xenograft in mice was conducted for in vivo experiment. CircKRT1 and PDL1 were highly expressed in OSCC tissues and cells. CircKRT1 knockdown repressed OSCC cell growth, migration, invasion, epithelial–mesenchymal transition, and CD8⁺ T-cell apoptosis, but enhanced CD8⁺ T cytotoxicity and percentage. The inhibitory effects of circKRT1 downregulation on OSCC progression and immune evasion were related to PDL1 expression inhibition. CircKRT1 sponged miR-495-3p and miR-495-3p targeted PDL1. OSCC progression and immune evasion were regulated by circKRT1 via the miR-495-3p/PDL1 axis. CircKRT1 also facilitated OSCC progression in vivo by regulating miR-495-3p and PDL1. This study clarified that circKRT1 worked as a miR-495-3p sponge to regulate PDL1, consequently affecting cancer progression and immune evasion in OSCC.     

MicroRNA alterations and associated aberrant DNA methylation patterns across multiple sample types in oral squamous cell carcinoma

Background

MicroRNA (miRNA) expression is broadly altered in cancer, but few studies have investigated miRNA deregulation in oral squamous cell carcinoma (OSCC). Epigenetic mechanisms are involved in the regulation of >30 miRNA genes in a range of tissues, and we aimed to investigate this further in OSCC.

Methods

TaqMan® qRT-PCR arrays and individual assays were used to profile miRNA expression in a panel of 25 tumors with matched adjacent tissues from patients with OSCC, and 8 control paired oral stroma and epithelium from healthy volunteers. Associated DNA methylation changes of candidate epigenetically deregulated miRNA genes were measured in the same samples using the MassArray® mass spectrometry platform. MiRNA expression and DNA methylation changes were also investigated in FACS sorted CD44^high oral cancer stem cells from primary tumor samples (CSCs), and in oral rinse and saliva from 15 OSCC patients and 7 healthy volunteers.

Results

MiRNA expression patterns were consistent in healthy oral epithelium and stroma, but broadly altered in both tumor and adjacent tissue from OSCC patients. MiR-375 is repressed and miR-127 activated in OSCC, and we confirm previous reports of miR-137 hypermethylation in oral cancer. The miR-200 s/miR-205 were epigenetically activated in tumors vs normal tissues, but repressed in the absence of DNA hypermethylation specifically in CD44^high oral CSCs. Aberrant miR-375 and miR-200a expression and miR-200c-141 methylation could be detected in and distinguish OSCC patient oral rinse and saliva from healthy volunteers, suggesting a potential clinical application for OSCC specific miRNA signatures in oral fluids.

Conclusions

MiRNA expression and DNA methylation changes are a common event in OSCC, and we suggest miR-375, miR-127, miR-137, the miR-200 family and miR-205 as promising candidates for future investigations. Although overall activated in OSCC, miR-200/miR-205 suppression in oral CSCs indicate that cell specific silencing of these miRNAs may drive tumor expansion and progression.     

miR-613 inhibits cell migration and invasion by downregulating Daam1 in triple-negative breast cancer

Dishevelled-associated activator of morphogenesis 1 (Daam1) is a formin protein and participates in regulating cell migration of triple-negative breast cancer (TNBC) cells. The specific miRNA targeting Daam1 and mediating cell migration and invasion remains obscure. This experiment investigated the suppressive role of miR-613 in TNBC cells. The luciferase activity of Daam1 3′-untranslated region (3′-UTR) based reporters constructed in HEK-293T and MCF-7 cells suggested that Daam1 was the target gene of miR-613. Overexpressed miR-613 reduced the protein level of Daam1, weakened RhoA activity, and retarded the cell migration, cell invasion and colony formation of TNBC cells. Overexpression of Daam1 or RhoA rescued cell migration and invasion in miR-613-overexpressed TNBC cells, but failed to reverse colony formation. MiR-613 was significantly downregulated in breast cancer tissues compared with that in adjacent normal tissues. This downregulation in TNBC tissues and lymphnode metastatic breast cancer tissues was more obvious than that in non-TNBC tissues and non-metastatic cancer tissues, respectively. MiR-613 weakens the resistance of TNBC cells against paclitaxel rather than adriamycin, cyclophosphamide, docetaxel, and kaempferol. Taken together, miR-613 is involved in cell migration and invasion of TNBC cells via targeting Daam1/RhoA signaling pathway.     

miR-21 inhibitor sensitizes human OSCC cells to cisplatin

miR-21 as a tumor oncogenic molecule has been reported. However, whether miR-21 can affect the sensitivity of oral squamous cell carcinoma (OSCC) cells to cisplatin remain unclear. The aim of this study is to evaluate the roles of miR-21 in the sensitivity of OSCC cells to cisplatin. RT-PCR assay was performed to detect the expression of miR-21 in 10 pairs of OSCC and noncancerous tissue samples. Then As-miR-21 oligonucleotides were used to down the miR-21 expression. Finally, the effects of miR-21 downregulation the sensitivity of OSCC cells (CA-27) to cisplatin in vitro were also detected. The level of miR-21 expression in OSCC tissues was significantly higher than that in corresponding noncancerous tissues. Down the expression of miR-21 could significantly inhibit growth and induce apoptosis of CA-27 cells. Moreover, downregulation of miR-21 could sensitize CA-27 cells to cisplatin possibly by increasing cisplatin induced apoptosis. This study demonstrated that combination of cisplatin application with miR-21 downregulation might be a potential strategy for the treatment of human OSCC.     

The effects of MOTILIPERM on cisplatin induced testicular toxicity in Sprague–Dawley rats

Genome-wide miRNA expression profile has identified microRNA (miR)-96 as one of upregulated miRNAs in clinical bladder cancer (BC) tissues compared to normal bladder tissues. The aim of this study was to confirm the expression pattern of miR-96 in BC tissues and to investigate its involvement in carcinogenesis. Quantitative real-time PCR was performed to detect the expression levels of miR-96 in 60 BC and 40 normal control tissues. Bioinformatics prediction combined with luciferase reporter assay were used to verify whether the cyclin-dependent kinase inhibitor CDKN1A was a potential target gene of miR-96. Cell counting kit-8 and apoptosis assays were further performed to evaluate the effects of miR-96-CDKN1A axis on cell proliferation and apoptosis of BC cell lines. We validated that miR-96 was significantly increased in both human BC tissues and cell lines. According to the data of miRTarBase, CDKN1A might be a candidate target gene of miR-96. In addition, luciferase reporter and Western blot assays respectively demonstrated that miR-96 could bind to the putative seed region in CDKN1A mRNA 3′UTR, and significantly reduce the expression level of CDKN1A protein. Moreover, we found that the inhibition of miR-96 expression remarkably decreased cell proliferation and promoted cell apoptosis of BC cell lines, which was consistent with the findings observed following the introduction of CDKN1A cDNA without 3′UTR restored miR-96. Our data reveal that miR-96 may function as an onco-miRNA in BC. Upregulation of miR-96 may contribute to aggressive malignancy partly through suppressing CDKN1A protein expression in BC cells.     

miR-30a attenuates drug sensitivity to 5-FU by modulating cell proliferation possibly by downregulating cyclin E2 in oral squamous cell carcinoma

We aimed to determine the functional role of the miRNA, which affects drug sensitivity to 5-FU in oral squamous cell carcinoma (OSCC), using two types of 5-FU-resistant and parental OSCC cell lines. MiRNA microarray data showed that miR-30a was significantly upregulated in two resistant cell lines. Therefore, we investigated the effects and molecular mechanism of miR-30a on 5-FU sensitivity. Stable overexpression of miR-30a in parental OSCC cells decreased cell proliferation and attenuated drug sensitivity to 5-FU. Cell cycle analysis indicated that miR-30a overexpression increased the proportion of G1 phase cells and decreased the proportion of S phase cells. MiR-30a knockdown using siRNA reversed the effects of miR-30a overexpression. DNA microarray analysis using miR-30a-overexpressing cell lines and a TargetScan database search showed that cyclin E2 (CCNE2) is a target of miR-30a. A luciferase reporter assay confirmed that a miR-30a mimic interacted with the specific binding site in the 3' UTR of CCNE2. CCNE2 knockdown with siRNA in OSCC cells yielded decreased drug sensitivity to 5-FU, similar to miR-30a overexpressing cells. These findings suggest that miR-30a in OSCC may be a novel biomarker of 5-FU-resistant tumors, as well as a therapeutic target for combating resistance.     

Downregulation of NEAT1 reverses the radioactive iodine resistance of papillary thyroid carcinoma cell via miR-101-3p/FN1/PI3K-AKT signaling pathway

Considering the resistance of papillary thyroid cancer (PTC) ¹³¹I therapy, this study was designed to find a solution at molecular respect. By probing into lncRNA-NEAT1/miR-101-3p/FN1 axis and PI3K/AKT signaling pathway, this study provided a potential target for PTC therapy. ¹³¹I-resistant cell lines were established by continuous treatment with median-lethal ¹³¹I. Bioinformatic analysis was applied to filtrate possible lncRNA/miRNA/mRNA and related signaling pathway. Luciferase reporter assay was employed in the verification of the targeting relationship between lncRNA and miRNA as well as miRNA and mRNA. MTT assay and flow cytometry assay were performed to observe the impact of NEAT1/miR-101-3p/FN1 on cell viability and apoptosis in radioactivity iodine (RAI)-resistant PTC cell lines, respectively. Western blot and qRT-PCR were conducted to measure the expression of proteins and mRNAs in RAI-resistant PTC tissues and cells. Meanwhile, endogenous PTC mice model were constructed, in order to verify the relation between NEAT1 and RAI-resistance in vivo. NEAT1 was over-expressed in RAI-resistant PTC tissues and cell lines and could resist RAI by accelerating proliferation accompanied by suppressing apoptosis. It indicated that overexpressed NEAT1 restrained the damage of RAI to tumor in both macroscopic and microcosmic. Besides, NEAT1/miR-101-3p exhibited a negative correlation by directly targeting each other. The expression of FN1, an overexpressed downstream protein in RAI-resistance PTC tissues, could be tuned down by miR-101-3p, while the decrease could be restored by NEAT1. In conclusion, both in vitro and in vivo, NEAT1 suppression could inhibit ¹³¹I resistance of PTC by upregulating miR-101-3p/FN1 expression and inactivated PI3K/AKT signaling pathway both in vitro and in vivo.     

Downregulation of miR-33b promotes non-small cell lung cancer cell growth through reprogramming glucose metabolism miR-33b regulates non-small cell lung cancer cell growth

Glucose metabolism is a common target for cancer regulation and microRNAs (miRNAs) are important regulators of this process. Here we aim to investigate a tumor-suppressing miRNA, miR-33b, in regulating the glucose metabolism of non-small cell lung cancer (NSCLC). In our study, quantitative real-time polymerase chain reaction (qRT-PCR) showed that miR-33b was downregulated in NSCLC tissues and cell lines, which was correlated with increased cell proliferation and colony formation. Overexpression of miR-33b through miR-33b mimics transfection suppressed NSCLC proliferation, colony formation, and induced cell-cycle arrest and apoptosis. Meanwhile, miR-33b overexpression inhibited glucose metabolism in NSCLC cells. Luciferase reporter assay confirmed that miR-33b directly binds to the 3′-untranslated region of lactate dehydrogenase A (LDHA). qRT-PCR and Western blot analysis showed that miR-33b downregulated the expression of LDHA. Moreover, introducing LDHA mRNA into cells over-expressing miR-33b attenuated the inhibitory effect of miR-33b on the growth and glucose metabolism in NSCLC cells. Taken together, these results confirm that miR-33b is an anti-oncogenic miRNA, which inhibits NSCLC cell growth by targeting LDHA through reprogramming glucose metabolism.     

Antisense inhibition of human miRNAs and indications for an involvement of miRNA in cell growth and apoptosis

Of the over 200 identified mammalian microRNAs (miRNAs), only a few have known biological activity. To gain a better understanding of the role that miRNAs play in specific cellular pathways, we utilized antisense molecules to inhibit miRNA activity. We used miRNA inhibitors targeting miR-23, 21, 15a, 16 and 19a to test efficacy of antisense molecules in reducing miRNA activity on reporter genes bearing miRNA-binding sites. The miRNA inhibitors de-repressed reporter gene activity when a miRNA-binding site was cloned into its 3′-untranslated region. We employed a library of miRNA inhibitors to screen for miRNA involved in cell growth and apoptosis. In HeLa cells, we found that inhibition of miR-95, 124, 125, 133, 134, 144, 150, 152, 187, 190, 191, 192, 193, 204, 211, 218, 220, 296 and 299 caused a decrease in cell growth and that inhibition of miR-21 and miR-24 had a profound increase in cell growth. On the other hand, inhibition of miR-7, 19a, 23, 24, 134, 140, 150, 192 and 193 down-regulated cell growth, and miR-107, 132, 155, 181, 191, 194, 203, 215 and 301 increased cell growth in lung carcinoma cells, A549. We also identified miRNA that when inhibited increased the level of apoptosis (miR-1d, 7, 148, 204, 210, 216 and 296) and one miRNA that decreased apoptosis (miR-214) in HeLa cells. From these screens, we conclude that miRNA-mediated regulation has a complexity of cellular outcomes and that miRNAs can be mediators of regulation of cell growth and apoptosis pathways.     

Specificity of miR-378a-5p targeting rodent fibronectin

One criterion for microRNA identification is based on their conservation across species, and prediction of miRNA targets by empirical approaches using computational analysis relies on the presence of conservative mRNA 3′UTR. Because most miRNA target sites identified are highly conserved across different species, it is not clear whether miRNA targeting is species-specific. To predict miRNA targeting, we aligned all available fibronectin 3′UTRs and observed significant conservation of all 20 species. Twelve miRNAs were predicted to target most fibronectin 3′UTRs, but rodent fibronectin showed potential binding sites specific for five different miRNAs. One of them, the miR-378a-5p, contained a complete matching seed-region for all rodent fibronectin, which could not be found in any other species. We designed experiments to test whether the species-specific targeting possessed biological function and found that expression of miR-378a-5p decreased cancer cell proliferation, migration, and invasion, resulting in inhibition of tumor growth. Silencing fibronectin expression produced similar effects as miR-378a-5p, while transfection with a construct targeting miR-378-5p produced opposite results. Tumor formation assay showed that enhanced expression of fibronectin in the stromal tissues as a background environment suppressed tumor growth, while increased fibronectin expression inside the tumor cells promoted tumor growth. This was likely due to the different signaling direction, either inside-out or outside-in signal. Our results demonstrated that species-specific targeting by miRNA could also exert functional effects. Thus, one layer of regulation has been added to the complex network of miRNA signaling.     

Differential expression of miR-195 in esophageal squamous cell carcinoma and miR-195 expression inhibits tumor cell proliferation and invasion by targeting of Cdc42

MicroRNAs (miRNA) have played an important role in carcinogenesis. In this study, Agilent miRNA microarray was used to identify differentially expressed miRNAs in esophageal squamous cell carcinoma (ESCC) tissues and miR-195 was downregulated in ESCC compared with normal esophageal tissues. Moreover, Cdc42 was confirmed as target gene of miR-195. Ectopic expression of miR-195 in ESCC cells significantly downregulated Cdc42 by directly binding its 3′ untranslated regions, and induced G1 cell cycle arrest, leading to a significant decrease in cell growth, migration, and invasion in vitro. Therefore, our findings demonstrated that miR-195 may act as a tumor suppressor in ESCC by targeting Cdc42.     

microRNA-1297 involves in the progression of oral squamous cell carcinoma through PTEN

Background

Oral squamous cell carcinoma (OSCC) oncogenic mechanisms still remain elusive. Herein, we proposed to understand the biological role of a newly discovered OSCC miRNA.

miR-29a-3p enhances the radiosensitivity of oral squamous cell carcinoma cells by inhibiting ADAM12

Oral squamous cell carcinoma (OSCC) is the most common malignant tumor in the head and neck, and radiotherapy is the main approach for this disease, while irradiation resistance is a huge challenge that influences radiosensitivity. This study aims to determine the role and function of miR-29a-3p and ADAM12 in the radiosensitivity of OSCC cells. The expression pattern of ADAM12 in OSCC cells was searched in TCGA database. The binding of miR-29a-3p and ADAM12 was predicted by Starbase and verified using dual luciferase reporter gene assay. The RNA or protein expressions of miR-29a-3p and ADAM12 were measured by RT-qPCR or western blot. OSCC cell lines were treated by various γ-ray irradiation dosages before the alteration on miR-29a-3p expression and on the cell viability, proliferation, migration and cell apoptosis was detected. ADAM12 was highly expressed in OSCC cells, whose expression in resistant cells was positively correlated with irradiation dosage. Overexpression of ADAM12 in OSCC cells lead to increased cell proliferation and migration ability as well as inhibited cell apoptosis. miRNAs potentially binding ADAM12 in PITA, microT, miRmap and targetscan were screened, among which miR-29a-3p had the maximum differential expression levels in OSCC cells determined by RT-qPCR. Overexpression of miR-29a-3p resulted in suppressed cell viability, proliferation, migration ability and increased cell apoptosis, while this expression pattern can be partially counteracted by ADAM12 overexpression in OSCC cells. miR-29a-3p through targeting and inhibiting AMDM12 enhances the radiosensitivity of OSCC cells.Key words: miR-29a, ADAM12, oral squamous cell carcinoma, radio-resistance, cell viability