scholarly journals MicroRNA-29c Increases the Chemosensitivity of Pancreatic Cancer Cells by Inhibiting USP22 Mediated Autophagy

2018 ◽  
Vol 47 (2) ◽  
pp. 747-758 ◽  
Author(s):  
Limin Huang ◽  
Chaoquan Hu ◽  
Hui Cao ◽  
Xiaoliang Wu ◽  
Rongpin Wang ◽  
...  
Keyword(s):  
Pancreatic Cancer ◽  
Flow Cytometry ◽  
Annexin V ◽  
Luciferase Reporter ◽  
Pancreatic Cancer Cells ◽  
Reporter Assays ◽  
Direct Target ◽  
Induced Apoptosis

Background/Aims: Pancreatic cancer (PC) is an aggressive malignancy with a poor survival rate. Despite advances in the treatment of PC, the efficacy of therapy is limited by the development of chemoresistance. Here, we examined the role of microRNA-29c (miR-29c) and the involvement of autophagy and apoptosis in the chemoresistance of PC cells in vivo and in vitro. Methods: We employed qRT-PCR, western blot and immunofluorescence to examine the expression level of miR-29c, USP22 and autophagy relative protein. In addition, we used MTT assay to detect cell proliferation and transwell assay to measure migration and invasiveness. The apoptosis was determined using annexin V-FITC/PI apoptosis detection kit by flow cytometry. Luciferase reporter assays confirmed the relationship between USP22 and miR-29c. Results: miR-29c overexpression in the PC cell line PANC-1 enhanced the effect of gemcitabine on decreasing cell viability and inducing apoptosis and inhibited autophagy, as shown by western blotting, immunofluorescence staining, colony formation assays, and flow cytometry. Ubiquitin specific peptidase (USP)-22, a deubiquitinating enzyme known to induce autophagy and promote PC cell survival, was identified as a direct target of miR-29c. USP22 knockdown experiments indicated that USP22 suppresses gemcitabine-induced apoptosis by promoting autophagy, thereby increasing the chemoresistance of PC cells. Luciferase reporter assays confirmed that USP22 is a direct target of miR-29c. A xenograft mouse model demonstrated that miR-29c increases the chemosensitivity of PC in vivo by downregulating USP22, leading to the inhibition of autophagy and induction of apoptosis. Conclusions: Taken together, these findings reveal a potential mechanism underlying the chemoresistance of PC cells mediated by the regulation of USP22-mediated autophagy by miR-29c, suggesting potential targets and therapeutic strategies in PC.

scholarly journals Tumor Suppressive Role of miR-342-5p in Human Chondrosarcoma Cells and 3D Organoids

2021 ◽  
Vol 22 (11) ◽  
pp. 5590
Author(s):  
Clément Veys ◽  
Abderrahim Benmoussa ◽  
Romain Contentin ◽  
Amandine Duchemin ◽  
Emilie Brotin ◽  
...  
Keyword(s):  
Luciferase Reporter ◽  
Functional Screening ◽  
Western Blots ◽  
Egfr Expression ◽  
Reporter Assays ◽  
Direct Target ◽  
Induced Apoptosis ◽  
First Time

Chondrosarcomas are malignant bone tumors. Their abundant cartilage-like extracellular matrix and their hypoxic microenvironment contribute to their resistance to chemotherapy and radiotherapy, and no effective therapy is currently available. MicroRNAs (miRNAs) may be an interesting alternative in the development of therapeutic options. Here, for the first time in chondrosarcoma cells, we carried out high-throughput functional screening using impedancemetry, and identified five miRNAs with potential antiproliferative or chemosensitive effects on SW1353 chondrosarcoma cells. The cytotoxic effects of miR-342-5p and miR-491-5p were confirmed on three chondrosarcoma cell lines, using functional validation under normoxia and hypoxia. Both miRNAs induced apoptosis and miR-342-5p also induced autophagy. Western blots and luciferase reporter assays identified for the first time Bcl-2 as a direct target of miR-342-5p, and also Bcl-xL as a direct target of both miR-342-5p and miR-491-5p in chondrosarcoma cells. MiR-491-5p also inhibited EGFR expression. Finally, only miR-342-5p induced cell death on a relevant 3D chondrosarcoma organoid model under hypoxia that mimics the in vivo microenvironment. Altogether, our results revealed the tumor suppressive activity of miR-342-5p, and to a lesser extent of miR-491-5p, on chondrosarcoma lines. Through this study, we also confirmed the potential of Bcl-2 family members as therapeutic targets in chondrosarcomas.

scholarly journals Aurora-a confers radioresistance in human hepatocellular carcinoma by activating NF-κB signaling pathway

BMC Cancer ◽  
2019 ◽  
Vol 19 (1) ◽  
Author(s):  
Ze-Tian Shen ◽  
Ying Chen ◽  
Gui-Chun Huang ◽  
Xi-Xu Zhu ◽  
Rui Wang ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Western Blot ◽  
Luciferase Reporter ◽  
Aurora A ◽  
Western Blot Assay ◽  
Induced Apoptosis ◽  
Mtt Assays ◽  
Hcc Cells

Abstract Background Radiotherapy failure is a significant clinical challenge due to the development of resistance in the course of treatment. Therefore, it is necessary to further study the radiation resistance mechanism of HCC. In our early study, we have showed that the expression of Aurora-A mRNA was upregulated in HCC tissue samples or cells, and Aurora-A promoted the malignant phenotype of HCC cells. However, the effect of Aurora-A on the development of HCC radioresistance is not well known. Methods In this study, colony formation assay, MTT assays, flow cytometry assays, RT-PCR assays, Western blot, and tumor xenografts experiments were used to identify Aurora-A promotes the radioresistance of HCC cells by decreasing IR-induced apoptosis in vitro and in vivo. Dual-luciferase reporter assay, MTT assays, flow cytometry assays, and Western blot assay were performed to show the interactions of Aurora-A and NF-κB. Results We established radioresistance HCC cell lines (HepG2-R) and found that Aurora-A was significantly upregulated in those radioresistant HCC cells in comparison with their parental HCC cells. Knockdown of Aurora-A increased radiosensitivity of radioresistant HCC cells both in vivo and in vitro by enhancing irradiation-induced apoptosis, while upregulation of Aurora-A decreased radiosensitivity by reducing irradiation-induced apoptosis of parental cells. In addition, we have showed that Aurora-A could promote the expression of nuclear IkappaB-alpha (IκBα) protein while enhancing the activity of NF-kappaB (κB), thereby promoted expression of NF-κB pathway downstream effectors, including proteins (Mcl-1, Bcl-2, PARP, and caspase-3), all of which are associated with apoptosis. Conclusions Aurora-A reduces radiotherapy-induced apoptosis by activating NF-κB signaling, thereby contributing to HCC radioresistance. Our results provided the first evidence that Aurora-A was essential for radioresistance in HCC and targeting this molecular would be a potential strategy for radiosensitization in HCC.

scholarly journals Restore of miR-541 resensitizes pancreatic cancer cells to gemcitabine-induced apoptosis through suppression of HAX-1

2020 ◽  
Author(s):  
Hong Liu ◽  
Xuemei Gan ◽  
Jun Zhang ◽  
Xingdiao Zhang ◽  
Jie Xiong ◽  
...  
Keyword(s):  
Pancreatic Cancer ◽  
Cancer Cells ◽  
Luciferase Reporter ◽  
Pancreatic Cancer Cells ◽  
Reporter Assays ◽  
Potential Strategy ◽  
Induced Apoptosis ◽  
Mtt Assays

Abstract Background: MiR-541 acts as a tumor suppressor in some cancers. However, the role of miR-541 in regulating the chemosensitivity to cancer cells is still unclear. The aim of this study is to explore the effect of miR-541 on chemoresistance of pancreatic cancer (PCa) cells to gemcitabine-induced apoptosis.Methods: Gemcitabine-resistant Panc-1 and Capan-2 PCa cell lines (Panc-1/R and Capan-2/R) were established through long term exposure to gemcitabine. Effect of miR-541 on changing the sensitivity of Panc-1/R and Capan-2/R to gemcitabine-induced cytotoxicity was evaluated by MTT assays. Regulation of miR-541 on HAX-1 was confirmed by bioinformatics, western blot analysis and luciferase reporter assays. Cell apoptosis and mitochondrial membrane potential (MMP) was measured by flow cytometry analysis.Results: Comparison with Panc-1 and Capan-2, downregulation of miR-541 was observed in Panc-1/R and Capan-2/R cells. Overexpression of miR-541 was found to increase the cytotoxicity of gemcitabine to Panc-1/R and Capan-2/R cells. However, transfection with HAX-1 plasmid can abolish the effect of miR-541 on gemcitabine-induced cytotoxicity against Panc-1/R and Capan-2/R.Conclusion: Downregulation of miR-541 is responsible for development of gemcitabine resistance in PCa. Overexpression of miR-541 may represent a potential strategy to reverse the chemoresistance of PCa.

scholarly journals circZMYM2 Competed Endogenously with miR-335-5p to Regulate JMJD2C in Pancreatic Cancer

2018 ◽  
Vol 51 (5) ◽  
pp. 2224-2236 ◽  
Author(s):  
Yong An ◽  
Huihua Cai ◽  
Yue Zhang ◽  
Shengyong Liu ◽  
Yunfei Duan ◽  
...  
Keyword(s):  
Pancreatic Cancer ◽  
Cell Proliferation ◽  
Tumor Growth ◽  
Underlying Mechanism ◽  
Luciferase Reporter ◽  
Tissue Samples ◽  
Paired Normal Tissue ◽  
Reporter Assays ◽  
Induced Apoptosis

Background/Aims: We aimed to study the involvement of circZMYM2 (hsa_circ_0099999) in pancreatic cancer (PC) cell proliferation, apoptosis and invasion and to figured out the underlying mechanism of circZMYM2 regulating miR-335-5p and JMJD2C. Methods: CircRNA differential expressions in twenty PC samples and paired normal tissue samples were analyzed using Arraystar Human CircRNA microarray V1. CircZMYM2 expression level was determined via qRT-PCR. The effects of circZMYM2 inhibition and overexpression on cell proliferation, cell apoptosis and cell invasion were investigated by CCK-8 assays, Flow cytometry assays and Transwell assays. An animal experiment on nude mice was put forward to test the influence of circZMYM2 knockdown on tumor growth. The relationship between circZMYM2, miR-335 and JMJD2C was verified by RNA pull down, dual-luciferase reporter assays and rescue experiment. The effect of circZMYM2 and miR-335-5p on the expression of JMJD2C protein was detected by western blot. Results: CircZMYM2 overexpression was observed in both PC tissues and cells. Knockdown of circZMYM2 inhibited proliferation, induced apoptosis, and weakened invasion ability of cancer cells. Tumor growth was restrained in vivo. CircZMYM2 repressed the expression of its target miR-335-5p. MiR-335-5p attenuated pancreatic cancer development via inhibition of JMJD2C. Conclusion: Our study demonstrated that circZMYM2 promoted PC progression. CircZMYM2 had a sponge effect on miR-335-5p and modulated the downstream oncogene JMJD2C.

scholarly journals METTL3 Promotes Pancreatic Tumor Progression Through Regulating the miR-196a/CPEB3 Axis

2020 ◽  
Author(s):  
Pingping Ge ◽  
Dong Fan ◽  
Lei He ◽  
Qiong Wu ◽  
Jin Sun ◽  
...  
Keyword(s):  
Pancreatic Cancer ◽  
Tumor Progression ◽  
Cancer Cells ◽  
Pancreatic Tumor ◽  
Luciferase Reporter ◽  
Pancreatic Cancer Cells ◽  
And Migration ◽  
Proliferation And Migration

Abstract Background: Methyltransferase-like 3(METTL3)-mediated N6-methyladenosine (m6A) modification has been reported to regulate microRNAs maturation. Here, the study was designed to investigate the regulatory effect of m6A-dependent miRNA maturation on pancreatic cancer progression which is still limited before.Results: We found that METTL3 significantly upregulated in the pancreatic tumor tissues. Overexpression of METTL3 promoted cancer cell proliferation and migration in vitro and tumor progression in vivo. METTL3-mediated m6A modification facilitated miR-196a maturation in pancreatic cancer cells, and miR-196a increased the proliferation and migration of cancer cells in vitro. Luciferase reporter assay verified that cytoplasmic polyadenylation element binding protein 3 (CPEB3) was a direct target gene of miR-196a. In vivo studies proved that overexpression of miR-196a inhibited the anti-tumor effect of knockdown of METTL3, and overexpression of CPEB3 inhibited the miR-196a-enhanced tumor progression. Conclusions: We identified that METTL3 was upregulated in pancreatic cancer, leading to the upregulation of miR-196a, resulting in the downregulation of CPEB3, which promoted the pancreatic tumor progression. We first demonstrated that CPEB3 was a tumor suppressor gene in pancreatic cancer, and the METTL3 regulated miR-196a/CPEB3 axis may be a therapeutic target for pancreatic cancer therapy.

scholarly journals Investigation of the role of the miR17-92 cluster in BMP9-induced osteoblast lineage commitment

2021 ◽  
Vol 16 (1) ◽  
Author(s):  
Yunyuan Zhang ◽  
Xuran Jing ◽  
Zhongzhu Li ◽  
Qingwu Tian ◽  
Qing Wang ◽  
...  
Keyword(s):  
Osteogenic Differentiation ◽  
Osteoblastic Differentiation ◽  
Luciferase Reporter ◽  
Morphogenetic Protein ◽  
Bone Morphogenetic Protein 9 ◽  
Reporter Assays ◽  
Direct Target

Abstract Background Bone morphogenetic protein 9 (BMP9) has been identified as a crucial inducer of osteoblastic differentiation in mesenchymal stem cells (MSCs). Although microRNAs (miRNAs) are known to play a role in MSC osteogenesis, the mechanisms of action of miRNAs in BMP9-induced osteoblastic differentiation remain poorly understood. Methods In this study, we investigate the possible role of the miR17-92 cluster in the BMP9-induced osteogenic differentiation of MSCs by using both in vitro and in vivo bone formation assays. Results The results show that miR-17, a member of the miR17-92 cluster, significantly impairs BMP9-induced osteogenic differentiation. This impairment is effectively rescued by a miR-17 sponge, an antagomiR sequence against miR-17. Using TargetScan and the 3′-untranslated region luciferase reporter assays, we show that the direct target of miR-17 is the retinoblastoma gene (RB1), a gene that is pivotal to osteoblastic differentiation. We also confirm that RB1 is essential for the miR-17 effects on osteogenesis. Conclusion Our results indicate that miR-17 expression impairs normal osteogenesis by downregulating RB1 expression and significantly inhibiting the function of BMP9.

scholarly journals STAT1-mediated inhibition of FOXM1 enhances gemcitabine sensitivity in pancreatic cancer

2019 ◽  
Vol 133 (5) ◽  
pp. 645-663 ◽  
Author(s):  
Chao Liu ◽  
Jiaqi Shi ◽  
Qingwei Li ◽  
Zhiwei Li ◽  
Changjie Lou ◽  
...  
Keyword(s):  
Pancreatic Cancer ◽  
Cancer Cells ◽  
Immunohistochemical Analysis ◽  
Human Pancreatic Cancer ◽  
Ductal Adenocarcinoma ◽  
Luciferase Reporter ◽  
Pancreatic Cancer Cells ◽  
Foxm1 Expression

Abstract Forkhead box protein M1 (FOXM1) was identified as an oncogenic transcription factor and master regulator of tumor progression and metastasis. FOXM1 expression often correlates with poor prognosis and chemotherapy resistance. In the present study, we investigated the association of FOXM1 expression and chemoresistance in pancreatic cancer. Elevated FOXM1 protein levels were associated with gemcitabine chemoresistance in patients with pancreatic cancer. In gemcitabine resistance cell line models of pancreatic cancer, FOXM1 expression increased, which induced gemcitabine chemoresistance in vitro. In pancreatic cancer cells treated with gemcitabine, FOXM1 affected nuclear factor κB (NF-κB) signaling activity. Immunohistochemical analysis demonstrated a negative association of FOXM1 expression and the level of phosphorylated signal transducer and activator of transcription 1 (pSTAT1) in human pancreatic cancer tissues. Dual-luciferase reporter assays and chromatin-immunoprecipitation assays demonstrated that pSTAT1 directly binds to the FOXM1 promoter to down-regulate its transcription. Interferon γ (IFNγ) promoted gemcitabine-induced cell apoptosis and inhibited cell proliferation in vitro and in vivo by FOXM1 inhibition. These data suggested that FOXM1 enhances chemoresistance to gemcitabine in pancreatic cancer. IFNγ could be used to down-regulate the expression of FOXM1 through STAT1 phosphorylation, thereby increasing the sensitivity of pancreatic cancer cells to gemcitabine. These studies suggested the sensitization by IFNγ in pancreatic ductal adenocarcinoma (PDAC) chemotherapy, which requires further clinical studies.

scholarly journals LncRNA CTD-3252C9.4 modulates pancreatic cancer cell survival and apoptosis through regulating IFI6 transcription

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Xin Yin ◽  
Jingyan Yang ◽  
Jintian Chen ◽  
Ruiqi Ni ◽  
Yanhao Zhou ◽  
...  
Keyword(s):  
Pancreatic Cancer ◽  
Cancer Cells ◽  
Cancer Cell ◽  
Pancreatic Cancer Cell ◽  
Luciferase Reporter ◽  
Downstream Target ◽  
Pancreatic Cancer Cells ◽  
Cancer Cell Survival

Abstract Background Pancreatic cancer (PC) is one of the most lethal cancer types with high degree of malignancy and poor prognosis. Recent studies have shown that long non-coding RNAs (lncRNAs) were associated with the initiation and progression of pancreatic cancer. In the current study, we have investigated the expression, biological function and mechanism of a lncRNA CTD-3252C9.4 in pancreatic cancer. Methods The expression of CTD-3252C9.4 in pancreatic cancer cells and tissues was measured by qRT-PCR. In vitro and in vivo functional experiments assays were implemented for identifying CTD-3252C9.4 function in pancreatic cancer. Molecular relationships among CTD-3252C9.4, IRF1 and IFI6 were investigated via luciferase reporter assay, pulldown assay and ChIP assays. Results CTD-3252C9.4 was found remarkably decreased in pancreatic cancer cells and tissues. Overexpression of CTD-3252C9.4 suppressed migration, invasion and proliferation, yet facilitated apoptosis of pancreatic cancer cells both in vitro and in vivo. Then, IFI6 was identified as a downstream target that could be down-regulated by CTD-3252C9.4 and IFI6 overexpression could counteract the effects of CTD-3252C9.4 upregulation on the survival and apoptosis of pancreatic cancer cells. Furthermore, mechanism experiments revealed that IRF1 was a transcriptional factor of IFI6 that can be blocked by CTD-3252C9.4 to inhibit IFI6 transcription. Conclusion Our data indicated that CTD-3252C9.4 could promote pancreatic cancer cell apoptosis and restrain cell growth via binding IRF1 and preventing the transcription of IFI6, which may become a potential therapeutic target for pancreatic cancer.

MicroRNA-1252-5p, regulated by Myb, inhibits invasion and epithelial-mesenchymal transition of pancreatic cancer cells by targeting NEDD9 

2020 ◽  
Author(s):  
Yuzheng Xue ◽  
Tielong Wu ◽  
Yingyue Sheng ◽  
Yao zhong ◽  
Benshun Hu ◽  
...  
Keyword(s):  
Pancreatic Cancer ◽  
Cell Lines ◽  
Epithelial Mesenchymal Transition ◽  
Luciferase Reporter ◽  
Mechanistic Study ◽  
Loss Of Function ◽  
Mesenchymal Transition ◽  
Pancreatic Cancer Cells

Abstract Background: MicroRNAs (miRNAs) are known to be involved in the development and progression of pancreatic cancer (PAC). The expression level and role of miR-1252-5p in PAC remain unclear. Methods: qRT-PCR and in situ hybridization were used to detect miR-1252-5p expression in PAC cells and tissues. Associations between miR-1252-5p expression and clinical characteristics or overall survival (OS) were assessed based on 102 patients with PAC who underwent surgical resection. Gain and loss of function of miR-1252-5p was studied in the PAC cell lines, Panc-1 and BxPC 3 in vitro and in vivo. The direct targets of miR-1252-5p were analyzed using public databases and a dual-luciferase reporter assay.Results: The expression levels of miR-1252-5p are downregulated in PAC cell lines and tissue samples compared to control, and its expression is negatively associated with adverse clinical features and poor prognosis. In vitro and in vivo experiments show that miR-1252-5p overexpression inhibits the proliferation, migration, invasion and epithelial-mesenchymal transition of PAC cells, whereas miR-1252-5p knockdown enhances these biological behaviors. In addition, miR-1252-5p negatively regulates neural precursor cell expressed, developmentally downregulated 9 (NEDD9) by directly binding its 3'-UTR. NEDD9 restoration at least partially abolishes this effect of miR-1252-5p in PAC cells. Further mechanistic study revealed that the SRC/STAT3 pathway is involved in miR-1252-5p/NEDD9 mediation of biological behaviors in PAC. We also verified that Myb inhibited miR-1252-5p by directly binding at its promoter.Conclusion: MiR-1252-5p may act as a tumor-suppressing miRNA in PAC and may be a potential therapeutic target for PAC patients.

scholarly journals Involvement of Endoplasmic Reticulum Stress in Capsaicin-Induced Apoptosis of Human Pancreatic Cancer Cells

2013 ◽  
Vol 2013 ◽  
pp. 1-12 ◽  
Author(s):  
Shengzhang Lin ◽  
Jianhong Zhang ◽  
Hui Chen ◽  
Kangjie Chen ◽  
Fuji Lai ◽  
...  
Keyword(s):  
Pancreatic Cancer ◽  
Endoplasmic Reticulum ◽  
Endoplasmic Reticulum Stress ◽  
Cancer Cells ◽  
Human Pancreatic Cancer ◽  
Dependent Manner ◽  
Pancreatic Cancer Cells ◽  
Induced Apoptosis

Capsaicin, main pungent ingredient of hot chilli peppers, has been shown to have anticarcinogenic effect on various cancer cells through multiple mechanisms. In this study, we investigated the apoptotic effect of capsaicin on human pancreatic cancer cells in bothin vitroandin vivosystems, as well as the possible mechanisms involved.In vitro, treatment of both the pancreatic cancer cells (PANC-1 and SW1990) with capsaicin resulted in cells growth inhibition, G0/G1 phase arrest, and apoptosis in a dose-dependent manner. Knockdown of growth arrest- and DNA damage-inducible gene 153 (GADD153), a marker of the endoplasmic-reticulum-stress- (ERS-) mediated apoptosis pathway, by specific siRNA attenuated capsaicin-induced apoptosis both in PANC-1 and SW1990 cells. Moreover,in vivostudies capsaicin effectively inhibited the growth and metabolism of pancreatic cancer and prolonged the survival time of pancreatic cancer xenograft tumor-induced mice. Furthermore, capsaicin increased the expression of some key ERS markers, including glucose-regulated protein 78 (GRP78), phosphoprotein kinase-like endoplasmic reticulum kinase (phosphoPERK), and phosphoeukaryotic initiation factor-2α(phospho-eIF2α), activating transcription factor 4 (ATF4) and GADD153 in tumor tissues. In conclusion, we for the first time provide important evidence to support the involvement of ERS in the induction of apoptosis in pancreatic cancer cells by capsaicin.

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