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2021 ◽  
Author(s):  
Jing Gao ◽  
Akiko Mizokami ◽  
Hiroshi Takeuchi ◽  
Aonan Li ◽  
Fei Huang ◽  
...  

Insulin signalling is tightly controlled by various factors, but the exact molecular mechanism remains incompletely understood. We previously reported that phospholipase C-related but catalytically inactive protein (PRIP) interacts with Akt, the central molecule in insulin signalling. Here, we investigated whether PRIP is involved in the regulation of insulin signalling in adipocytes. We found that insulin signalling including insulin-stimulated phosphorylation of the insulin receptor (IR), insulin receptor substrate-1 (IRS-1), Akt, and glucose uptake, were impaired in adipocytes from PRIP-knockout (KO) mice compared with those from wild-type (WT) mice. The amount of IR expressed on the cell-surface was decreased in PRIP-KO adipocytes. Immunoprecipitation assay showed that PRIP interacted with IR. The reduced cell-surface IR in PRIP-KO adipocytes was comparable with that in WT cells when Rab5 expression was silenced using specific siRNA. In contrast, the dephosphorylation of IRS-1 at serine residues, some of which were reported to be involved in the internalisation of IR, was impaired in cells from PRIP-KO mice. These results suggest that PRIP facilitates insulin signalling by modulating the internalisation of IR in adipocytes.


2021 ◽  
Vol 7 (1) ◽  
Author(s):  
Yingxue Guo ◽  
Junfeng Li ◽  
Shuang Fan ◽  
Qibo Hu

AbstractThe current study tried to uncover the molecular mechanism of E3 ubiquitin ligase F-box and WD repeat domain-containing 7 (FBW7) in a heritable autoimmune disease, type I diabetes (T1D). After streptozotocin-induced T1D model establishment in non-obese diabetic (NOD) mouse, the protein expression of FBW7, enhancer of zeste homolog 2 (EZH2), and Zinc finger and BTB domain containing 16 (ZBTB16) was quantified. Next, splenocytes and pancreatic beta cells were isolated to measure the production of pro-inflammatory cytokines in splenocytes, as well as islet beta-cell apoptosis. Additionally, the stability of EZH2 induced by FBW7 was analyzed by cycloheximide chase assay. The binding affinity of FBW7 and EZH2 and the consequence of ubiquitination were monitored by co-immunoprecipitation assay. Last, a chromatin immunoprecipitation assay was employed to analyze the accumulation of EZH2 and H3K27me3 at the ZBTB16 promoter region. Our study demonstrated downregulated FBW7 and ZBTB16 and upregulated EZH2 in diabetic NOD mice. Overexpression of FBW7 in the NOD mice inhibited pro-inflammatory cytokine release in the splenocytes and the apoptosis of islets beta cells. FBW7 destabilized EZH2 and accelerated ubiquitin-dependent degradation. EZH2 and H3K27me3 downregulated the ZBTB16 expression by accumulating in the ZBTB16 promoter and methylation. FBW7 upregulates the expression of ZBTB16 by targeting histone methyltransferase EZH2 thus reducing the occurrence of T1D.


2021 ◽  
Vol 22 (22) ◽  
pp. 12302
Author(s):  
Fang-fang You ◽  
Jing Zhang ◽  
Fan Cheng ◽  
Kun Zou ◽  
Xue-qing Zhang ◽  
...  

RCE-4, a steroidal saponin isolated from Reineckia carnea, has been studied previously and has exhibited promising anti-cervical cancer properties by inducing programmed cell death (PCD) of Ca Ski cells. Considering the cancer cells developed various pathways to evade chemotherapy-induced PCD, there is, therefore, an urgent need to further explore the potential mechanisms underlying its actions. The present study focused on targeting the Bcl-2–Beclin 1 complex, which is known as the key regulator of PCD, to deeply elucidate the molecular mechanism of RCE-4 against cervical cancer. The effects of RCE-4 on the Bcl-2–Beclin 1 complex were investigated by using the co-immunoprecipitation assay. In addition, autophagy-related genes (ATG) were also analyzed due to their special roles in PCD. The results demonstrated that RCE-4 inhibited the formation of the Bcl-2–Beclin 1 complex in Ca Ski cells via various pathways, and ATG 4B proteins involved in this process served as a key co-factor. Furthermore, based on the above, the sensitivity of RCE-4 to Ca Ski cells was significantly enhanced by inhibiting the expression of the ATG 4B by applying the ATG 4B siRNA plasmid.


Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 3326-3326
Author(s):  
Jianwei Qu ◽  
Yifan Hou ◽  
Qingxiao Chen ◽  
Jing Chen ◽  
YI LI ◽  
...  

Abstract Background: RNA N 6-methyladenosine (m 6A) plays a critical role in regulating gene expression and determining cell fate. The dysregulation of m 6A modulators, including α-ketoglutarate-dependent dioxygenase AlkB homolog 5 (ALKBH5), has been reported to promote tumor development through their enzymatic function. However, the functions of mRNA m 6A and its modulators in multiple myeloma (MM) are largely unknown. Methods: We queried publicly available MM datasets to study the expression profile of m 6A modulators (METTL3, METTL14, WTAP, FTO, and ALKBH5) in MM and their relationships with clinical outcomes in patients with MM. Both gain- and loss-of-function studies were performed to investigate the role of ALKBH5 in MM. The cell proliferation assay, colony formation assay, Annexin V apoptosis analysis, and 5-ethynyl-2′-deoxyuridine (EdU) assay were performed to evaluate the functions of ALKBH5 in MM in vitro. Human MM cell line xenograft models were constructed to examine the effects of ALKBH5 knockdown or overexpression on MM growth in vivo. The rescue assay using catalytically inactive mutant ALKBH5-H204A was conducted to determine whether demethylation activity was required for the function of ALKBH5 in MM. Then, we performed RNA sequencing and m 6A sequencing to explore the key targets that mediated ALKBH5 function in MM. We investigated the gene regulatory mechanism of ALKBH5 in MM by m 6A immunoprecipitation assay, RNA immunoprecipitation assay, RNA decay assay, dual-luciferase reporter assay, and so forth. Gene set enrichment analysis and Western blotting were employed to determine the downstream signaling pathways regulated by ALKBH5 and the recognized target. Results: ALKBH5 was overexpressed in MM and associated with a poor prognosis in patients with MM. The increased ALKBH5 expression was required for the survival and growth of MM cells in vitro and in vivo. Mechanistically, m 6A demethylation activity was required for ALKBH5 to exert tumorigenic effects in MM. Tumor necrosis factor (TNF) receptor-associated factor 1 (TRAF1) was identified as a functionally important target of ALKBH5. ALKBH5 regulated TRAF1 expression via affecting mRNA stability of TRAF1 in an m 6A- and YTHDF2-dependent manner. ALKBH5 promoted MM cell growth and survival partly through TRAF1-mediated activation of NF-κB and MAPK signaling pathways. Conclusion: Our findings showed that ALKBH5 played an oncogenic role in MM and highlighted that ALKBH5 could potentially be a novel therapeutic target in MM. Disclosures No relevant conflicts of interest to declare.


2021 ◽  
Author(s):  
Lei Li ◽  
Chengfeng Jiang ◽  
Yongchun Gu ◽  
Rongrong Jiang ◽  
Jianpeng Han ◽  
...  

Abstract Pulpitis is an inflammatory condition that can lead to the loss of tooth vitality. The odontogenic differentiation potential of the dental pulp stem cells (DPSCs) is significantly decreased in the pulpitis microenvironment. The aim of this study was to determine the mechanism of the impaired odontogenic differentiation of the DPSCs in an inflammatory microenvironment. Overexpression and inhibition of the JunB were performed to evaluate its effect on the odontogenic differentiation potential of the DPSCs. The results showed that the JunB was positively correlated with the odontogenic differentiation ability of the DPSCs. The co-immunoprecipitation assay coupled with the mass spectrometry showed that peptidyl-prolyl cis-trans Isomerase NIMA-Interacting 1 (PIN1) was a downstream target of the JunB. Moreover, the expression of PIN1 was negatively correlated with the odontogenic differentiation potential of the DPSCs. Taken together, the decreased expression level of the JunB in an inflammatory state can impair the odontogenic differentiation ability of the DPSCs by upregulating the expression of the PIN1, a negative regulator of the odontogenic differentiation of the DPSCs.


Epigenomics ◽  
2021 ◽  
Author(s):  
Yan-Jie Xu ◽  
Jie-Min Zhao ◽  
Xue-Feng Ni ◽  
Wei Wang ◽  
Wen-Wei Hu ◽  
...  

Aim: We aimed to explore the effect of long noncoding RNA HCG18 in colorectal cancer (CRC). Materials & methods: Relative gene and protein expression were screened. Colony formation and flow cytometry assays were performed to determine proliferation and apoptosis. Dual luciferase assay and RNA immunoprecipitation assay were conducted to validate the interaction between indicated molecules. Xenograft in nude mice was applied to verify the conclusion in vivo. Results: HCG18 and PD-L1 were upregulated while miR-20b-5p was downregulated in CRC tissue. Functional analysis revealed that lncRNA HCG18 promoted proliferation, migration and resistance to cetuximab of CRC cells via miR-20b-5p/PD-L1 axis. Conclusion: HCG18 facilitated the progress of tumor, conferred to cetuximab resistance and suppressed CD8+ T cell via miR-20b-5p/PD-L1 axis.


Author(s):  
Huan Liu ◽  
Ping He ◽  
Fei Meng ◽  
Mengwei Jiang ◽  
Jin Xiong ◽  
...  

African swine fever (ASF) is a highly contagious viral disease of domestic pigs and wild boars. For the disease surveillance and control, we developed a rapid and easy luciferase immunoprecipitation assay (MB-LIPS) to detect ASF virus (ASFV) antibody. The MB-LIPS is based on magnetic beads modified with protein A/G and the recombinant fusion protein of ASFV p30 and luciferase, where p30 functioned as the recognition element and the luciferase as the signal component. Incubation and washing could be finished automatically on a machine with magnetic rods. Under the optimal conditions, the MB-LIPS showed 96.3% agreement to a commercial enzyme linked immunosorbent assay (ELISA) kit for detecting ASFV antibody in swine sera. Analyzing serial dilutions of a swine serum sample showed that the MP-LIPS assay was 4 times more sensitive than the ELISA kit. The whole run of the MB-LIPS could be completed within 30 min. With its high sensitivity and simple operation, the MB-LIPS platform has great potentials to be used for the detection of ASFV antibody and ASF control in small labs and farms.


2021 ◽  
Author(s):  
Peipei Zhang ◽  
Changyu Chen ◽  
Jiajia Zhang ◽  
Xin Yu

Abstract Objective: To study the role of long non-coding RNA (lncRNA) CRYM-AS1 in human gastric cancer. Methods: Expression levels of CRYM-AS1 in cell lines and clinical tissues were examined by RT-qPCR. The association between CRYM-AS1 levels and clinicopathological parameters / survival rates of gastric cancer patients was analyzed.Cell functional experiments including MTT assay, glucose consumption / lactate production / ATP production detection were performed to examine the role of CRYM-AS1 in cell aerobic glycolysis and cell proliferation of gastric cancer cells. Subcellular fractionation location detection, western blot, RIP (RNA binding protein immunoprecipitation) assay, CHIP (Chromatin immunoprecipitation) assay and BSP (Bisulfite sequencing PCR) assay were carried out to explore the molecular mechanism of CRYM-AS1 in gastric cancer cells.Results: CRYM-AS1 was low expressed in gastric cancer cells and tissues compared with normal gastric cells and tissues respectively. CRYM-AS1 was negatively correlated with TNM staging, tumor size and overall survival (OS) rate in gastric cancer patients. CRYM-AS1 inhibited gastric cancer cell aerobic glycolysis and cell proliferation. CRYM-AS1 directly bound to EZH2 and mediated the CRYM promoter methylation and consequently negatively regulated the expression of CRYM. Forced expression of CRYM rescued the decreased aerobic glycolysis and cell proliferation induced by CRYM-AS1 in gastric cancer cells.Conclusion: CRYM-AS1 was an important biomarker and could be used for human gastric cancer treatment.


2021 ◽  
pp. 1-58
Author(s):  
Jin-wu Zhou ◽  
Man Zhao ◽  
Wen-liang Rang ◽  
Xiao-yan Zhang ◽  
Zhen-ming Liu ◽  
...  

Background: The toxicity of excessive glutamate release has been implicated in various acute and chronic neurodegenerative conditions. Vesicular glutamate transporters (VGLUTs) are the major mediators for the uptake of glutamate into synaptic vesicles. However, the dynamics and mechanism of this process in glutamatergic neurons are still largely unknown. Objective: This study aimed to investigate the candidate protein partners of VGLUT1 and their regulatory roles in the vesicles in rat brain. Methods: Pull down assay, co-immunoprecipitation assay, or split-ubiquitin membrane yeast two hybrid screening coupled with nanoRPLC-MS/MS were used to identify the candidate protein partners of VGLUT1 in the vesicles in rat brain. The in vitro and in vivo models were used to test effects of AβPP, Atp6ap2, Gja1, and Synataxin on VGLUT1 expression. Results: A total of 255 and 225 proteins and 172 known genes were identified in the pull down assay, co-immunoprecipitation assay, or split-ubiquitin yeast two-hybrid screening respectively. The physiological interactions of SV2A, Syntaxin 12, Gja1, AβPP, and Atp6ap2 to VGLUT1 were further confirmed. Knockdown of Atp6ap2, Gja1, and Synataxin increased VGLUT1 mRNA expression and only knockdown of AβPP increased both mRNA and protein levels of VGLUT1 in PC12 cells. The regulatory function of AβPP on VGLUT1 expression was further confirmed in the in vitro and in vivo models. Conclusion: These results elucidate that the AβPP and VGLUT1 interacts at vesicular level and AβPP plays a role in the regulation of VGLUT1 expression which is essential for maintaining vesicular activities.


Author(s):  
Xiang Li ◽  
Hong Qin ◽  
Xiaotong Han ◽  
Xingwen Zhang ◽  
Luping Wang ◽  
...  

IntroductionThis study aims to explore the effect and mechanism of miR�489-3p on the proliferation and apoptosis of pancreatic acinar cells in acute pancreatitis (AP).Material and methodsX-linked inhibitor of apoptosis protein (XIAP) and miR-489-3p expression in serum of AP patients, pancreatic AR42J cells, and rat AP tissues were measured using quantitative reverse transcription poly�merase chain reaction and western blot. The effect of miR-489-3p on pro�liferation and apoptosis was determined by MTT assay and flow cytometry. The relationship between XIAP and miR-489-3p was verified using luciferase assay RNA immunoprecipitation assay. Histological changes in rat pancreatic tissues were observed via haematoxylin and eosin staining.ResultsMeasurement of miR-489-3p and XIAP expressions in AP patients revealed a negative correlation between miR-489-3p and XIAP. Increased miR-489-3p expression in AP patients indicated a poor prognosis. Cerulein was used to induce AP in AR42J cells and standard deviation rats. Exper�iments in cells and AP rat models showed that miR-489-3p can increase cell apoptosis and inhibit cell proliferation by regulating XIAP, as shown by elevated expressions of pro-apoptotic proteins (p53 and Bax) and decreased expression of proliferation indicator (Ki-67) after transfection of miR-489- 3p mimics. Meanwhile, knockdown of miR-489-3p abrogated the inhibitory effects of miR-489-3p on cell proliferation and the promotion on cell apop�tosis. Luciferase assay and RNA immunoprecipitation assay confirmed that XIAP can directly bind miR-489-3p.ConclusionsWe concluded that miR-489-3p modulates cell proliferation and apoptosis in AP by targeting XIAP. Given that high expression of miR�489-3p in AP indicated poor prognosis, it raises the possibility that miR�489-3p might be a novel and valuable therapeutic target and a prognosis indicator for AP.


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