Overexpression Of miR-136 Research Articles

Urolithin A alleviates respiratory syncytial virus-induced lung infection in neonatal mice by activating miR-136-mediated Sirt1 signaling

To observe the therapeutic effects of urolithin A (UA) on respiratory syncytial virus (RSV)-induced lung infection in neonatal mice and explore the underlying mechanisms. Babl/c mice (5-7 days old) were subjected to nasal instillation of RSV and received intraperitoneal injection of saline or 2.5, 5 and 10 mg/kg UA 2 h after the infection and then once daily for 2 weeks. Bronchoalveolar lavage fluid (BALF) was then collected for detection of inflammatory cells and mediators, and lung pathology was evaluated with HE staining. RSV-infected BEAS-2B cells were treated with 2.5, 5 or 10 µmol/ L UA. Inflammatory factors, cell viability, apoptosis and autophagy were analyzed using ELISA, CCK-8 assay, TUNEL staining, flow cytometry, Western blotting and immunofluorescence staining. The cellular expressions of miR-136 and Sirt1 mRNAs were detected using qRT-PCR. A dual-luciferase reporter system was used to verify the binding between miR-136 and Sirt1. In neonatal Babl/c mice, RSV infection caused obvious lung pathologies, promoted pulmonary cell apoptosis and LC3-Ⅱ/Ⅰ, Beclin-1 and miR-136 expressions, and increased the total cell number, inflammatory cells and factors in the BALF and decreased p62 and Sirt1 expressions. All these changes were alleviated dose-dependently by UA. In BEAS-2B cells, RSV infection significantly increased cell apoptosis, LC3B-positive cells and miR-136 expression and reduced Sirt1 expression (P<0.01), which were dose-dependently attenuated by UA. Dual-luciferase reporter assay confirmed the binding between miR-136 and Sirt1. In RSV-infected BEAS-2B cells with UA treatment, overexpression of miR-136 and Ex527 treatment both significantly increased the inflammatory factors and cell apoptosis but decreased LC3B expression, and these changes were further enhanced by their combined treatment. UA ameliorates RSV-induced lung infection in neonatal mice by activating miR-136-mediated Sirt1 signaling pathway.

LncRNA WWTR1-AS1 upregulates Notch3 through miR-136 to increase cancer cell stemness in cervical squamous cell carcinoma

BackgroundThis Study investigated the role of WWTR1-AS1 in cervical squamous cell carcinoma (CSCC).ResultsWWTR1-AS1 expression was upregulated in CSCC tissues. WWTR1-AS1 was predicted to interact with miR-136, whereas correlation analysis revealed that there was no close correlation between WWTR1-AS1 and miR-136 across CSCC samples. Moreover, WWTR1-AS1 and miR-136 did not regulate the expression of each other. In addition, overexpression of WWTR1-AS1 increased the expression levels of Notch3, which could be targeted by miR-136. Cell stemness analysis indicated that the overexpression of WWTR1-AS1 and Notch3 increased CSCC cell stemness and the capacity of CSCC cell to grow as spheroids. Overexpression of miR-136 decreased CSCC cell stemness and reversed the effects of overexpression of WWTR1-AS1 on Notch3 in CSCC cells.ConclusionTherefore, WWTR1-AS1 may upregulate Notch3 through miR-136 to increase cancer cell stemness in CSCC.

MiR-136 Suppresses the Aggressive Proliferation of Non-Small Cell Lung Cancer Through Restraining Histone Deacetylase 1 (HDAC1) and Phosphorylation of the Janus Kinase 2/Signal Transducer and Activator of Transcription 3 (Jak2/STAT3) Pathway

microRNA-136 can inhibit the proliferating activity of malignant cells and also participate in chemotherapy resistance of colorectal cancer via modulating HDAC1. This study assessed miR-136’s effect on NSCLC cell proliferation and underlying mechanisms. Tumor tissues and paracancerous tissues from NSCLC patients were collected to measure miR-136 and HDAC1 level. Cells were transfected with miR-136-mimics, miR-136-inhibitors or miR-136 mimics+HDAC1-OE followed by analysis of cell viability and apoptosis by CCK-8 method and flow cytometry, phosphorylation of Jak2/STAT3 by western blot. miR-136 was significantly downregulated in tumor tissues and NSCLC cells, accompanied by upregulated HDAC1. miR-136 overexpression suppressed HDAC1 expression, retarded phosphorylation and activation of Jak2/STAT3 signaling, reduced NSCLC cell viability and enhanced apoptosis. In addition, co-transfection of miR-136-mimics and HDAC1-OE reversed the inhibitory effects of miR-136 on NSCLC cells. In conclusion, miR-136 is reduced and HDAC1 is increased in NSCLC and miR-136 overexpression inhibited NSCLC cell proliferation and increased apoptosis possibly through regulating HDAC1/Jak2/STAT3 signal pathway, indicating that miR-136 might be a novel target for the treatment of NSCLC.

The Neuroprotective Effect of miR-136 on Pilocarpine-Induced Temporal Lobe Epilepsy Rats by Inhibiting Wnt/β-Catenin Signaling Pathway

Objective To explore the effect of miR-136 on temporal lobe epilepsy (Ep) and its mechanism of action. Methods 30 male rats were injected intraperitoneally with 30 mg/kg pilocarpine to construct a rat temporal lobe epilepsy model, and they were randomly divided into 5 groups (n = 6 per group): control group, Ep group, agomir NC group, miR-136 agomir group, and miR-136+LiCl group. The brain tissues of the rats were collected 7 days after the treatment. The expression of miR-136 in the hippocampus tissue was detected by qRT-PCR. H&E and Nissl staining were used to observe the histopathological changes and neuron damage in the hippocampus tissue. IL-1β, IL-6, and TNF-α levels in the hippocampus tissue were detected by ELISA. Flow cytometry was used to detect the apoptosis rate in the hippocampus tissue. Western blot was used to detect the expression levels of c-Caspase-3, Bcl-2, β-catenin, Cyclin D1, and c-myc protein in the hippocampus. Results The expression of miR-136 was significantly downregulated in the hippocampus tissue of epileptic rats. After overexpression of miR-136, the number of seizures and the duration of epilepsy in rats were significantly reduced. At the same time, hippocampal tissue damage was improved considerably, and the degree of neuronal damage decreased. Overexpression of miR-136 also significantly reduced the apoptosis rate in the hippocampus tissue and inhibited the levels of inflammatory factors. Meanwhile, miR-136 downregulates the expression of Wnt/β-catenin signaling pathway-related proteins. However, Wnt pathway activator LiCl could destroy the protective effect of miR-136. Conclusion miR-136 could exert its neuroprotective influence on temporal lobe epilepsy rats by inhibiting the Wnt/β-catenin signaling pathway.

Differential Expression of miR-136 in Gestational Diabetes Mellitus Mediates the High-Glucose-Induced Trophoblast Cell Injury through Targeting E2F1.

Background Gestational diabetes mellitus (GDM) seriously affects the health of mothers and infants. The high-glucose-induced inhibition in trophoblast cell viability is an important event in GDM pathogenesis. This study evaluated the expression and clinical significance of miR-136 in GDM patients, and the biological function and related mechanisms of miR-136 in the regulation of trophoblast cell proliferation were explored. Methods The expression of miR-136 in serum and placenta of GDM patients was measured using quantitative Real-Time PCR. Trophoblast cells were stimulated with high-glucose medium to mimic the pathological changes of GDM, and the effect of miR-136 was examined by CCK-8 assay. A luciferase reporter assay was used to confirm the target gene of miR-136, and the relationship of E2F transcription factor 1 (E2F1) with miR-136 in GDM was further analyzed. Results miR-136 expression was significantly elevated in GDM serum and tissue samples. By high-glucose treatment, trophoblast cell proliferation was inhibited and miR-136 expression was promoted. The knockdown of miR-136 could promote the proliferation of trophoblast cells exposed to high glucose, whereas the overexpression of miR-136 could suppress it. In addition, E2F1 was identified as a target gene of miR-136, which could mediate the regulatory effect of miR-136 on trophoblast cell proliferation. Conclusion Collectively, miR-136 expression is increased in both serum and placental tissues in GDM patients, and miR-136 mediates the inhibiting effect of high glucose on trophoblast cell viability by targeting E2F1.

Circ_PUM1 promotes the development of endometrial cancer by targeting the miR-136/NOTCH3 pathway.

Endometrial cancer is one of the most common gynaecological malignancies and the sixth most common cause of cancer‐related death among women. Here, we define the role and molecular mechanism of circ_0000043 (hereafter referred to as circ_PUM1) in the development and progression of endometrial carcinoma. QRT‐PCR was used to detect the expression of circ_PUM1 in normal endometrial tissue and endometrial carcinoma tissues. Changes in cell function and tumorigenicity in nude mice were examined after circ_PUM1 overexpression or knockdown. Bioinformatic analysis and dual‐luciferase reporter assay were used to predict and analyse the miRNAs that circ_PUM1 binds. Gene expression changes were analysed using Western blot. Circ_PUM1 was expressed at significantly higher levels in endometrial cancer tissues than in normal tissues. Up‐regulation of circ_PUM1 promoted the proliferation, migration and invasion of endometrial carcinoma cells. Opposite results were observed with circ_PUM1 knockdown, and the tumorigenic ability of endometrial cancer cells after circ_PUM1 knockdown was reduced compared to control cells. Circ_PUM1 is capable of binding to miR‐136, and up‐regulating its target gene NOTCH3, which can be reversed by overexpression of miR‐136. Circ_PUM1 can compete with miR‐136, leading to up‐regulation of NOTCH3, and thereby promote the development of endometrial cancer.

Silencing of LINC01116 suppresses the development of oral squamous cell carcinoma by up-regulating microRNA-136 to inhibit FN1.

BackgroundOral squamous cell carcinoma (OSCC), one of the most common cancers worldwide with a high mortality rate, is accompanied by poor prognosis, highlighting the significance of early diagnosis and effective treatment. Long non-coding RNAs (lncRNAs) have been linked with the development and progression of various cancers. In this study, aberrantly expressed lncRNA LINC01116, microRNA-136 (miR-136), and fibronectin1 (FN1) were identified in OSCC using a microarray analysis. Therefore, this study aimed to investigate the role of LINC01116/miR-136/FN1 regulatory axis in OSCC.MethodsThe gain-of-function and loss-of-function experiments in vitro were performed to alter the expression of LINC01116 and miR-136 in OSCC cells to elucidate their effects on cellular processes, including epithelial–mesenchymal transition (EMT), viability, invasion, and migration. In addition, the interaction among LINC01116, miR-136, and FN1 was identified. Additionally, the tumorigenicity and lymph node metastasis (LNM) affected by LINC01116 were observed through xenograft tumor in nude mice.ResultsLINC01116 and FN1 were abundant in both OSCC tissues and cells, while miR-136 was poorly expressed. LINC01116 could competitively bind to miR-136, which targets and negatively regulates FN1. Moreover, in response to LINC01116 silencing or miR-136 over-expression, OSCC cells exhibited diminished EMT process and inhibited cell viability, invasion, and migration in vitro, coupling with impaired tumorigenicity and LNM in vivo.ConclusionThe fundamental findings in this study collectively demonstrate that LINC01116 silencing may inhibit the progression of OSCC via the miR-136-mediated FN1 inhibition, highlighting a promising therapeutic strategy for OSCC treatment.

Up-regulation of microRNA-136 induces apoptosis and radiosensitivity of esophageal squamous cell carcinoma cells by inhibiting the expression of MUC1

ObjectiveThis present study is performed to figure out the role of microRNA-136 (miR-136) in radiosensitivity of esophageal squamous cell carcinoma (ESCC) cells through the regulation of MUC1. MethodsSeventy-four ESCC patients were divided into radiotherapy sensitive group and radiotherapy resistance group. Colony formation assay and flow cytometry were used to test the radiosensitivity of radiotherapy resistant strain and parent strain. The expression of miR-136 between radiotherapy resistant strain and parent strain was detected by RT-qPCR, and the expression of miR-136 in Eca109 and TE-1 cells as well as Eca109-R and TE-1-R cells was detected after different doses of X-ray irradiation. Eca109 and TE-1 cells as well as Eca109-R and TE-1-R cells with overexpression of miR-136 or co-overexpression of miR-136 and MUC1 were constructed. Cell proliferation, colony formation and apoptosis was detected by CCK-8 assay, colony formation assay, and flow cytometry, respectively. ResultsThe expression of miR-136 in ESCC tissues was lower and MUC1 mRNA and protein expression was higher than that in adjacent normal tissues. The expression of miR-136 was negatively correlated with the expression of MUC1 mRNA in ESCC. Low expression of miR-136 and high expression of MUC1 were associated with tumor size, lymph node metastasis and distant metastasis. The expression of miR-136 increased while the expression of MUC1 decreased in the radiotherapy sensitive group of ESCC patients relative to the radiotherapy resistant group. The colony formation ability of radiation resistant cell line was stronger than that of parent cell line, and the apoptosis rate showed an opposite trend. Up-regulation of miR-136 reduced the survival rate, suppressed colony formation ability and induced apoptosis of ESCC cells under irradiation, which was reversed by upregulated MUC1. ConclusionThis study demonstrates that up-regulation of miR-136 induces apoptosis and radiosensitivity of ESCC cells by inhibiting the expression of MUC1.

MiR-136 triggers apoptosis in human gastric cancer cells by targeting AEG-1 and BCL2.

Gastric cancer is the second most prevalent cancer across the globe and accounts for about 10% of new cancer cases. It is one among the leading causes of cancer-related deaths around the world. Recently, microRNAs have been identified as important therapeutic targets for the treatment of several cancers owing to their potential to target multiple genes and hinder several biological processes such as proliferation and apoptosis. In the current study, we investigated the potential of miR-136 as therapeutic target for gastric cancer. Total RNA was isolated by RNeasy RNA isolation kit and cDNA was prepared byRevertAid cDNA synthesis kit. The transcript analysis was carried out by quantitative RT-PCR. The transfection of miR-136 mimics or plasmids was carried out by using the Lipofectamine 2000 reagent. Apoptosis was detected by DAPI and acridine orange/ethidium bromide (AO/EB) staining. Protein expression was examined by Western blotting. The results indicated that the expression of miR-136 is significantly downregulated in gastric cancer cells. Transfection and subsequent overexpression of miR-136 in gastric cancer cells significantly promoted apoptosis as evident from DAPI and OA staining. In silico analysis revealed AEG-1 and BCL2 to be the potential targets of miR-136. Therefore, the expression of AEG-1 and BCL2 was determined in untreated control, cisplatin treated control and miR-136 transfected AGS gastric cancer cells. The results revealed that overexpression of miR-136 expression causes significant downregulation of AEG-1 and BCL2 protein expression. Taken together, we conclude that miR-136 promotes apoptosis in gastric cancer cells by targeting AEG-1 and BCL2. Therefore, miR-136 may prove as a potential therapeutic target for the treatment of gastric cancer.

LINC00657 expedites neuropathic pain development by modulating miR-136/ZEB1 axis in a rat model.

Long non-coding RNAs (lncRNAs) are involved in the progression of several diseases. The interactions among lncRNAs, microRNA (miRNAs) or their targeting genes are reported to play crucial roles in the development of diseases. LINC00657 is observed to be upregulated in several cancers. However, the biological role of LINC00657 in neuropathic pain progress is unclear. Hence, in our study, we aimed to investigate the function of LINC00657 in neuropathic pain development. A chronic constriction injury (CCI) rat model was established, and we found that LINC00657 was greatly increased in CCI rats associated with a decrease of miR-136. Inhibition of LINC00657 suppressed neuropathic pain via alleviating mechanical and thermal hyperalgesia. In addition, miR-136 overexpression can also inhibit the neuropathic pain development. MiR-136 was predicted to serve as a miRNA target of LINC00657, and dual-luciferase reporter assay confirmed the correlation between LINC00657 and miR-136. Moreover, we observed that the decrease of LINC00657 was able to inhibit theneuroinflammation of CCI rats by targeting expression of cyclooxygenase-2, tumor necrosis factor-α and interleukin-1βwhile miR-136 inhibitors reversed this phenomenon. Next, by using bioinformatics analysis, ZEB1 was predicted as a direct target of miR-136, and miR-136 could negatively modulate ZEB1 expression. Besides these, ZEB1 was remarkably increased in the CCI rats. Knockdown of ZEB1 can inhibit neuropathic pain development, while miR-136 inhibitors can reverse it. In conclusion, it was implied that LINC00657 can induce the neuropathic pain development via regulating miR-136/ZEB1 axis.

MicroRNA-136 promotes lipopolysaccharide-induced ATDC5 cell injury and inflammatory cytokine expression by targeting myeloid cell leukemia 1.

Osteoarthritis is the most frequent chronic bone and joint diseases in older populations all over the world. Lipopolysaccharide (LPS)-induced murine chondrogenic ATDC5 cell model has been widely used for testing new osteoarthritis therapeutic targets. This study aimed to explore the effects of microRNA-136 (miR-136) on LPS-induced ATDC5 cell injury and inflammatory cytokineexpression, as well as underlying potential mechanism. We found that LPS remarkably inhibited ATDC5 cell viability, induced ATDC5 cell apoptosis, and upregulated the expression of inflammatory cytokines, including interleukin 1β (IL-1β), IL-6, IL-8, and tumor necrosis factor α (TNF-α; P < .01 or < .001). Moreover, LPS obviously upregulated the expression of miR-136 in ATDC5 cells (P < .05). Overexpression of miR-136 markedly exacerbated the LPS-induced ATDC5 cell viability inhibition, cell apoptosis enhancement, and inflammatory cytokine expression (P < .05), and suppression of miR-136 had opposite effects (P < .05). Myeloid cell leukemia 1 (Mcl-1) was a direct target gene of miR-136, which participated in the effect of miR-136 on LPS-induced ATDC5 cell inflammatory injury. Overexpression of Mcl-1 alleviated the LPS-induced inactivation of Wnt/β-catenin and Janus kinase/signal transducer and activator of transcription (JAK/STAT) signaling pathways, while suppression of Mcl-1 had opposite effects. To conclude, this study verified that miR-136 promoted LPS-induced ATDC5 cell injury and inflammatory cytokine expression by targeting Mcl-1, and Mcl-1 was involved in the regulatory effects of LPS on Wnt/β-catenin and JAK/STAT signaling pathways in ATDC5 cells.

MiR-136 Inhibits Malignant Progression of Hepatocellular Carcinoma Cells by Targeting Cyclooxygenase 2.

MicroRNAs (miRNAs) play a vital role in regulating tumor progression. Dysregulated miR-136 expression was linked to the development of various human cancers. In the present study, we investigated the expression and relationship of miR-136 and COX2 in hepatocellular carcinoma (HCC) using relevant experiments, involving CCK-8, Transwell assay, and luciferase reporter assay. We demonstrated that miR-136 expression is obviously decreased in HCC tissues and cells, and negatively correlated with the expression of COX2 mRNA. In vitro assay revealed that overexpression of miR-136 significantly changed the expression of proliferation- and metastasis-related proteins and inhibited the proliferation, migration, and invasion of HepG2 and Hep3B cells. Dual-luciferase reporter assay validated that the 3′-untranslated region (3′-UTR) of COX2 is a direct target of miR-136. Furthermore, COX2 siRNA partially enhanced the miR-136 overexpression-induced inhibitory effects. In conclusion, miR-136 was vital in the regulation of HCC cell proliferation and metastasis by targeting COX2. Thus, our findings provided novel evidence that miR-136 might be recommended as a potential target for the diagnosis and treatment of HCC patients.

Effects of microRNA-136 on melanoma cell proliferation, apoptosis, and epithelial-mesenchymal transition by targetting PMEL through the Wnt signaling pathway.

The study aims to evaluate the effects of miR-136 on the proliferation, apoptosis, and epithelial–mesenchymal transition (EMT) of melanoma cells by targetting premelanosome protein (PMEL) through the Wnt signaling pathway. After establishment of melanoma mouse models, melanoma (model group) and normal tissues (normal group) were collected. Immunohistochemistry was performed to determine PMEL protein concentration. Mouse melanoma cells were assigned into control, blank, negative control (NC), miR-136 mimics, miR-136 inhibitors, siRNA-PMEL, and miR-136 inhibitors + siRNA-PMEL, LiC1 (Wnt signaling pathway activator), and siRNA-PMEL+ LiCl groups. MTT, Scratch test, Transwell assay, and flow cytometry were performed to measure cell proliferation, migration, invasion, and apoptosis. Quantitative real-time PCR (qRT-PCR) and Western blotting were performed to evaluate miR-136, PMEL, β-catenin, Wnt3a, Bcl-2, Bax, Caspase, E-cadherin, and N-cadherin expressions. PMEL is highly expressed in melanoma tissues. MiR-136, Bax, Caspase, and E-cadherin expressions decreased in the model group, whereas PMEL, β-catenin, Bcl-2, Wnt3a, and N-cadherin expressions increased. Bax, Caspase, and E-cadherin expressions increased in the miR-136 mimics and siRNA-PMEL groups, whereas the expressions decreased in the miR-136 inhibitors group and LiC1 group. PMEL, β-catenin, Bcl-2, Wnt3a, and N-cadherin expressions, cell proliferation, migration, and invasion decreased, and the apoptosis rate inceased in the miR-136 mimics and siRNA-PMEL groups; whereas the tendencies were opposite to those in the miR-136 inhibitors group and LiC1 group. In the siRNA-PMEL+ LiCl group, PMEL expression decreased. These findings indicated that overexpression of miR-136 inhibits melanoma cell EMT, proliferation, migration, invasion, and promotes apoptosis by targetting PMEL through down-regulation of the Wnt signaling pathway.

MiR-136 controls neurocytes apoptosis by regulating Tissue Inhibitor of Metalloproteinases-3 in spinal cord ischemic injury

BackgroundSpinal cord ischemia is a serious injury that threatens human health and life. Furthermore, it was widely accepted that miR-136 was mediated in the spinal injury, while whether it regulated neurocytes apoptosis in I/R-induced spinal cord injury remains unclear. MethodsSpinal cord ischemia injury (SCII) rats were induced by clamping the nontraumatic vascular clip on the abdominal aorta. Real-time PCR was conducted to determine the mRNA expression, and western blot was carried out to measure protein expression. TUNEL assay was used to measure cell apoptosis. ResultsMiR-136 was up-regulated, while Tissue Inhibitor of Metalloproteinases-3 (TIMP3) was down-regulated in both SCII rats and hypoxic neurocytes. MiR-136 overexpression protected neurocytes against injury that induced by hypoxia. TIMP3 was the target gene of miR-136. Hypoxia supplementation decreased the expression of miR-136, promoted TIMP3 expression, and urged cell apoptosis, cells transfected with miR-136 mimic reversed the effect that induced by hypoxia, while cells co-transfected with pcDNA-TIMP3 abolished the results that induced by overexpressed miR-136. ConclusionMiR-136 regulated neurocytes apoptosis of SCII by mediating TIMP3.

MicroRNA-136 inhibits colon cancer cell proliferation and invasion through targeting liver receptor homolog-1/Wnt signaling

An increasing number of studies have reported that microRNAs (miRNAs) are involved in the malignant behavior of colon cancer cells through directly targeting multiple tumor suppressors or oncogenes. The expression and role of miR-136 has been reported in several types of human cancer. However, the role of miR-136 in colon cancer remains unclear. In this study, we aimed to investigate the expression and function of miR-136 in colon cancer and the potential underlying mechanism. Here, we found that miR-136 was decreased in colon cancer cell lines and tissues. Overexpression of miR-136 inhibited the proliferation and invasion in SW480 and HCT116 cell lines while suppression of miR-136 exhibited the opposite effect. Liver receptor homolog-1 (LRH-1) was identified as a direct target gene of miR-136. Notably, miR-136 overexpression suppressed LRH-1 expression as well as Wnt signaling in SW480 and HCT116 cell lines. The miR-136 expression level inversely correlated with LRH-1 mRNA expression in colon cancer specimens. Moreover, overexpression of LRH-1 partially reversed the miR-136-induced antitumor effect in SW480 and HCT116 cell lines. Taken together, these findings suggest that miR-136 functions as a negative regulator in colon cancer progression by targeting LRH-1 and that miR-136 downregulation contributes to high expression of LRH-1 and aberrant activation of Wnt signaling, leaving open the possibility that miR-136 may serve as a potential therapeutic target for colon cancer.

Enhanced inflammatory damage by microRNA-136 targeting Klotho expression in HK-2 cells by modulating JAK/STAT pathway.

MiR-136 acts as a tumor suppressor by promoting cell apoptosis and downregulating Bcl-2 in glioma cells. Hence, an attempt has been made to evaluate the role of miR-136 in regulation of inflammatory damage in HK-2 cells. HK-2 cells were cultured and assessed for viability. The cells were then transfected with miR-136 mimic, si- miR-136, si-Klotho, and NC. Dual luciferase test was performed to confirm the target of miR-136 which was assumed to be Klotho. Cell viability, apoptosis, expressions of inflammatory cytokines like TNF-α, IL-1β, IL-6 and IL-8 were assessed in HK-2 cells with overexpressing miR-136 or with knocked down miR-136 activities, following exposure to LPS. LPS induced inflammatory damage decreased cell viability, induced cell apoptosis, and increased the expression of different inflammatory cytokines. It was found that LPS decreased the expression of miR-136. Over-expression of miR-136 inhibited cell viability, enhanced apoptosis, and increased expression of inflammatory cytokines while knockdown of miR-136 showed opposite results with p-values < 0.05. MiR-136 negatively regulated the expression of Klotho with p-value < 0.05. Over-expression of miR-136 inhibited the expression of Klotho and activated JAK/STAT and mTOR signaling pathways and vice versa. Hence, it can be concluded that miR-136 enhances inflammatory damage probably by targeting klotho as has been observed in luciferase assay by inactivation of JAK/STAT and mTOR signaling pathways.

MicroRNA-136 inhibits cancer stem cell activity and enhances the anti-tumor effect of paclitaxel against chemoresistant ovarian cancer cells by targeting Notch3

To identify microRNAs (miRNAs) regulating Notch3 expression in association with paclitaxel resistance, candidate miRNAs targeting Notch3 were predicted using TargetScan. We found that miR-136 directly targets Notch3, and miR-136 was significantly downregulated in OSC tissues relative to normal control tissues, and low expression of miR-136 correlated with poor overall in ovarian cancer patients. Artificial miR-136 overexpression significantly reduced cell viability, proliferation, Cancer stem cell (CSC) spheroid formation, and angiogenesis, and increased apoptosis in paclitaxel-resistant SKpac cells compared with the effects of paclitaxel alone. miR-136 overexpression downregulated cell survival- (survivin, DNA-PK, pS6, S6) and cell cycle- (Cyclin D1, NF-κB) related proteins, and anti-apoptotic proteins (BCL2, and BCL-XL), and upregulated pro-apoptotic proteins (Bim, Bid, and Bax).Taken together, miR-136 targets the Notch3 oncogene and functions as a tumor suppressor. miR-136 overexpression resensitized paclitaxel-resistant ovarian cancer cells and reduced CSC activities, suggesting a promising new target for the treatment of chemoresistant ovarian cancers.

Expression of miR-136 is associated with the primary cisplatin resistance of human epithelial ovarian cancer

MicroRNAs (miRNAs) are involved in regulating the response of cancer cells to various therapeutic interventions, yet their involvement in the chemoresistance of human epithelial ovarian cancer is not fully understood. We found that miR-136 was significantly downregulated in specimens from patients with chemoresistant epithelial ovarian cancer. In the present study, we aimed to clarify the role of miR-136 in regulating the chemoresistance of ovarian cancer. Thirty-four tumor bank specimens and 2 well-established human ovarian cancer cell lines, C13 and OV2008, were used. We found that miR-136 expression was significantly reduced in primary platinum-resistant patients and the ovarian cancer OVC cell line. Enforced expression of miR-136 decreased the chemoresistance to cisplatin in OVC cells through inhibition of cell survival. In addition, we found no association between miR-136 and migration or invasion potential in the ovarian cancer cell lines. However, in the platinum-resistant C13 cell line, the overexpression of miR-136 markedly promoted an apoptotic response to cisplatin. Furthermore, the levels of adducts corrected with their extent of DNA damage/repair, in terms of the percentage of DNA in comet tails, tail length, tail moment (TM), and olive tail moment (OTM), revealed that miR-136 is essential for the repair of cisplatin-induced DNA damage. Our findings suggest that miR-136 may function as an anti-oncogene and deficiency of miR-136 expression in ovarian cancer can induce chemoresistance at least in part by downregulating apoptosis and promoting the repair of cisplatin-induced DNA damage. Thus, miR-136 may provide a biomarker for predicting the chemosensitivity to cisplatin in patients with epithelial ovarian cancer.

MiR-136 modulates glioma cell sensitivity to temozolomide by targeting astrocyte elevated gene-1.

BackgroundRecent studies have linked chemotherapy resistance to the altered expression of microRNAs (miRNAs). Thus, miRNA-based approaches to modulating sensitivity to temozolomide (TMZ) may overcome chemoresistance. The aim of the present study was to investigate whether miR-136 could modulates glioma cell sensitivity to TMZ.MethodsThe proliferation of glioma U251 cell line was evaluated by MTT assay. The expression of astrocyte elevated gene-1 (AEG-1)was detected by real‑time polymerase chain reaction (RT-PCR)and Western blot. The luciferase reporter gene was used to test whether AEG-1 was the target of the miR-136.ResultsThe MTT assay showed that U251 cells with miR-136 overexpression were significantly more sensitive to the therapy of TMZ than control cells. Luciferase assays revealed that miR-136 directly targeted the 3’UTR of AEG-1. qRT–PCR and western blotting analysis found that AEG-1 expression at the mRNA and protein levels decreased in the miR-136 mimic-treatment group relative to control group. Downregulation of AEG-1 expression by siRNAs, U251 cells became more sensitive to the therapy of TMZ. In addition, the enhanced growth-inhibitory effect by the miR-136 mimics transfection was enhanced after the addition of AEG-1 siRNA.ConclusionsThe present study provides the first evidence that miR-136 plays a key role in TMZ resistance by targeting the AEG-1 protein in glioma cell line, suggesting that miR-136 can be used to predict a patient’s response to TMZ therapy as well as serve as a novel potential maker for glioma therapy.Virtual SlidesThe virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/13000_2014_173

Abstract 4180: miRNA-mediated anticancer properties of the PPARα-activator fenofibrate on glioblastoma cell growth and migration.

Abstract Background: Glioblastoma is an aggressive tumor which originates from the glial cells and accounts for nearly 50% of adult brain tumors. These tumors are characterized by rapid proliferation, aggressive metastatic potential, and resistance to radio- and chemotherapy. MicroRNAs (miRNAs) are 22-nucleotides long RNAs that regulate gene expression post-transcriptionally. Fenofibrate is a potent agonist of peroxisome proliferator activated receptor alpha (PPARα) that is used for the treatment of cardiovascular disease and various metabolic disorders. In addition, treatment with Fenofibrate results in the proliferation of peroxisomes and a marked reduction in the growth of Glioblastoma cells. Methods: To evaluate the effects of treatment on miRNA expression, we used TaqMan quantitative real-time PCR assays from Life Technologies (Grand Island, NY). miRNAs were cloned into BLOCK-iT Pol II miR RNAi Expression Vectors (Life Technologies). Cell growth was evaluated using Guava ViaCount Reagent (Guava Technologies, Hayward, CA). Migration of miRNA-expressing cells was assessed 24h after plating on 8 μm membranes (BD Biosciences, San Jose, CA). Results: In this study, we utilized the LN229 Glioblastoma cell line and found that among the miRNAs up-regulated by Fenofibrate treatment, miR-634 and miR-136 targeted CYR61, a protein involved in migration and proliferation of cancer cells. We additionally found that miR-136 was up-regulated by Fenofibrate treatment while miR-155, another miRNA previously shown to target CYR61, was down-regulated by Fenofibrate. All three miRNAs were predicted and validated to directly target the 3’-UTR of Cyr61. Over-expression of miR-136 and miR-634 miRNAs negatively affected proliferation, but not migration, of LN229 cells. Forced expression of miR-155 reduced migration and addition of recombinant Cyr61 reversed this impairment in migration. Investigation of the signaling pathways regulated by miR-634, revealed an increased phosphorylation of p70 S6 kinase (Thr389), suggesting an induction of the mammalian target of rapamycin (mTOR) complex 1 pathway. Induction of the mTOR pathway was dependent on Akt and Erk activation. In miR-634 overexpressing cells, TSC2, a negative regulator of mTOR signaling, was found to be decreased. Further studies on the differential roles of miRs-136, -155, and -634 in controlling cell growth, cell size, and migration will provide important information on the molecular mechanisms involved in the anticancer properties of Fenofibrate. Citation Format: Duane Jeansonne, Marco Pacifici, Adam Lassak, Krzysztof Reiss, Francesca Peruzzi. miRNA-mediated anticancer properties of the PPARα-activator fenofibrate on glioblastoma cell growth and migration. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4180. doi:10.1158/1538-7445.AM2013-4180