Effect of naringenin regulating RIP1-RIP3-MLKL signaling pathway on apoptosis of ovarian granulosa cells in rats with polycystic ovary syndrome / 中国药理学通报

Aim Based on the apoptotic pathway mediated by receptor interacting protein kinase(RIP)1-RIP3-mixed spectrum kinase domain like protein(MLKL), to explore the effects of naringenin on ovarian granulosa cell apoptosis in rats with polycystic ovary syndrome(PCOS). Methods SD rats were randomly assigned into normal control group, model group, naringenin group, RIP1 inhibitor(Nec-1)group, RIP1-RIP3-MLKL necrosis signal activator(Z-VAD-fmk)group, naringenin+Z-VAD-fmk group, 15 rats per group. ELISA method was performed to measure the levels of IL-1β and TNF-α in ovarian tissue. HE method was performed to observe the shape of the ovary. Granular cells were isolated from ovarian tissue, and flow cytometry was performed to measure apoptosis rate and necrosis rate. Immunohistochemistry was performed to measure the positive expression of p-RIP1 in ovarian tissue. Western blot was employed to detect the expression of RIP1-RIP3-MLKL pathway. Results RIP1 specific inhibitor Nec-1 and naringenin could block the phosphorylation and activation of RIP1, inhibit the RIP1-RIP3-MLKL signaling pathway, reduce the inflammation level in PCOS rats, and alleviate the necrosis and apoptosis of ovarian granulosa cells(P<0.05). Z-VAD-fmk could promote the activation of RIP1-RIP3-MLKL pathway, aggravate the apoptosis of ovarian granulosa cells, and partially weaken the anti-apoptosis effect of naringenin(P<0.05). Conclusions Naringenin may inhibit the apoptosis of ovarian granulosa cells in PCOS rats by blocking the activation of the necrotic apoptotic pathway mediated by RIP1-RIP3-MLKL.

Receptor-interacting Protein Kinase 3 Promotes Osteoarthritis-related Changes by Upregulating Integrin Subunit Beta 3 / 中国生物化学与分子生物学报

Osteoarthritis (OA) is the most common chronic disabling joint disease, and currently there is no effective treatment for the cause. Necroptosis plays a key role in many diseases, and receptor-interacting protein kinase 3 (RIP3) is a key regulator during necroptosis process. Studies have shown that the expression level of RIP3 was significantly upregulated in human and mouse OA degenerative cartilage tissues, suggesting the occurrence of necroptosis. However, the specific pathophysiological role of RIP3 in cartilage is still unclear. This study intends to sequence and analyze the transcriptome of chondrocytes before and after RIP3 overexpression, and explore the specific functional mechanism of RIP3 in OA pathogenesis. RNA sequencing results showed that overexpression of RIP3 induced upregulation of 244 genes and downregulation of 277 genes in chondrocytes. Sixteen candidate target genes were screened out by constructing gene co-expression network for further verification at mRNA level, and the results suggested that RIP3 had the most significant inductive effect on the expression of phosphoinositide-3kinase, regulatory subunit 5 (Pik3r5), integrin subunit beta 3 (Itgb3) and MYB proto-oncogene like 2 (Mybl2). Results from CCK-8 and lactate dehydrogenase activity analysis showed that silencing the expression of Itgb3 by siRNA significantly rescued chondrocyte viability decline and necroptosis induced by RIP3, and it also inhibited the upregulating effect of RIP3 on the expression of catabolism-related genes Mmp1, Mmp13 and Il6, as well as the downregulating effect of RIP3 on the expression of anabolism-related genes Acan, Col2a1 and Sox9. This study has demonstrated that RIP3 promotes chondrocyte necrosis and cartilage matrix metabolism disorders by upregulating the expression of Itgb3 in chondrocytes, and ultimately leads to cartilage degeneration. These findings provided potential novel targets for the clinical treatment of OA, and further clarified the pathophysiological significance of necroptosis.

Ligand-based substituent-anchoring design of selective receptor-interacting protein kinase 1 necroptosis inhibitors for ulcerative colitis therapy

Receptor-interacting protein (RIP) kinase 1 is involved in immune-mediated inflammatory diseases including ulcerative colitis (UC) by regulating necroptosis and inflammation. Our group previously identified TAK-632 (

RIP1-dependent linear and nonlinear recruitments of caspase-8 and RIP3 respectively to necrosome specify distinct cell death outcomes

There remains a significant gap in our quantitative understanding of crosstalk between apoptosis and necroptosis pathways. By employing the SWATH-MS technique, we quantified absolute amounts of up to thousands of proteins in dynamic assembling/de-assembling of TNF signaling complexes. Combining SWATH-MS-based network modeling and experimental validation, we found that when RIP1 level is below ~1000 molecules/cell (mpc), the cell solely undergoes TRADD-dependent apoptosis. When RIP1 is above ~1000 mpc, pro-caspase-8 and RIP3 are recruited to necrosome respectively with linear and nonlinear dependence on RIP1 amount, which well explains the co-occurrence of apoptosis and necroptosis and the paradoxical observations that RIP1 is required for necroptosis but its increase down-regulates necroptosis. Higher amount of RIP1 (>~46,000 mpc) suppresses apoptosis, leading to necroptosis alone. The relation between RIP1 level and occurrence of necroptosis or total cell death is biphasic. Our study provides a resource for encoding the complexity of TNF signaling and a quantitative picture how distinct dynamic interplay among proteins function as basis sets in signaling complexes, enabling RIP1 to play diverse roles in governing cell fate decisions.

Controlled decompression alleviates early brain injury in rabbit intracranial hypertension model by regulating apoptosis/necroptosis

ABSTRACT Purpose To evaluate the effects of controlled decompression and rapid decompression, explore the potential mechanism, provide the theoretical basis for the clinical application, and explore the new cell death method in intracranial hypertension. Methods Acute intracranial hypertension was triggered in rabbits by epidural balloon compression. New Zealand white rabbits were randomly put into the sham group, the controlled decompression group, and the rapid decompression group. Brain water content, etc., was used to evaluate early brain injury. Western blotting and double immunofluorescence staining were used to detect necroptosis and apoptosis. Results Brain edema, neurological dysfunction, and brain injury appeared after traumatic brain injury (TBI). Compared with rapid decompression, brain water content was significantly decreased, neurological scores were improved by controlled decompression treatment. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining and Nissl staining showed neuron death decreased in the controlled decompression group. Compared with rapid decompression, it was also found that apoptosis-related protein caspase-3/ tumor necrosis factor (TNF)-a was reduced markedly in the brain cortex and serum, and the expression levels of necroptosis-related protein, receptor-interacting protein 1 (RIP1)/receptor-interacting protein 1 (RIP3) reduced significantly in the controlled decompression group. Conclusions Controlled decompression can effectively reduce neuronal damage and cerebral edema after craniocerebral injury and, thus, protect the brain tissue by alleviating necroptosis and apoptosis.

The mechanism of anti-hepatocellular carcinoma cell line HepG2 by Chinese medicine Astragalus protein / 药学学报

To detect the inhibitory effect of Astragalus protein on the proliferation of hepatocellular carcinoma cell line HepG2, transcriptomics was used to explore the anti-tumor mechanism of Astragalus protein. The dried roots of Astragalus was precipitated by ammonium sulfate to obtain Huang Qi protein (HQP) with different molecular weights. The effect of HQP on HepG2 and its toxic effect were detected by hemocytometry. Cell necrosis was detected by flow cytometry and Hoechst/propidium iodide (PI) double staining. The necrotic marker protein receptor interacting serine/threonine kinase 1 (RIP1) was determined by Western blot. Transcriptome sequencing was performed on the control group and dosing group RNA, and differential expression genes were analyzed for RNA-seq results. qRT-PCR was used to verified the relative mRNA expression levels of candidate genes. The results showed that the inhibition of HepG2 proliferation was more obvious with the increase of HQP concentration. When the concentration of HQP was 100 μg·mL-1, the necrosis rate increased to 18.78%, and the number of red necrotic cells stained with PI was observed under the microscope. The Western blot results showed an increase in RIP1 protein levels. The results of RNA-seq analysis showed that 26 000 related genes were regulated by HQP, and 979 genes were more regulated. KEGG analysis found that some differentially expressed genes were associated with p53 signaling pathway, and qRT-PCR further verified that the sequencing results were reliable. HQP may cause programmed necrosis of HepG2 cells and may be involved in the p53 signaling pathway.

Effect of Baicalin on the Necroptosis of Mouse Colon Cancer in Vitro / 医药导报

Objective To investigate the effect of baicalin on CT26.WT cells of colon cancer in mice, and to discuss the cell death form. Methods CT26.WT cells were divided into four groups including of control group , routine cultured in fresh medium, the baicalin group, added with concentration of 100 μmol·L-1 baicalin, the z-VAD-fmk group, was added with final concentration of 20 μmol·L-1 z-VAD-fmk, and the combination group, added final concentration of 20 μmol·L-1 z-VADfmk,1 h before adding 100 μmol·L-1 baicalin. Then the inhibitory effect of baicalin on cell proliferation and cell viability were detected by CCK-8 method. The changes of nucleus were detected by DAPI staining, the ultrastructure of cells was observed by TEM, and the effect of baicalin on the expression of RIP3 gene and protein in cells was detected by QPCR method and Western blotting. Results Compared with control group, the differences of baicalin group and combination group had statistically significance (P<0.05) . cell death rate for control group was (10.54±0.19) ％ ,for baicalin group was (34.93±0.16) ％ ,for z- VAD group was (11.23±0.59) ％, and combination group was (23.27±1.20) ％ (P<0.01) . Compared with the normal control group, baicalin group showed nuclear concentration and fragmentation. there was obvious nuclear fragmentation in the combination group against baicalin group. The results of electron microscopy showed that the cells of baicalin were necrotic, cell swelling, mitochondria swelling and contents leaking. Baicalin group significantly up - regulated RIP3 mRNA expression (P < 0. 01) and enhanced RIP3 protein expression (P < 0. 05) . Conclusion Baicalin induces the necrosis of ct26. WT cells, and can significantly increase the gene and protein expression of RIP3.

Complex roles of necroptosis in cancer / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

Necroptosis is a tightly regulated form of necrosis that requires the activation of receptor-interacting protein (RIP) kinases RIPK1 and RIPK3, as well as the RIPK3 substrate mixed lineage kinase domain-like protein (MLKL). Because of membrane rupture, necroptotic cells release damage-associated molecular patterns (DAMPs) that evoke immune responses. Necroptosis is emerging as an important cellular response in the modulation of cancer initiation, progression, and metastasis. Necroptosis of cancer cells is considered to be an immunogenic cell death capable of activating anti-tumor immunity. Necroptosis also participates in the promotion of myeloid cell-induced adaptive immune suppression and thus contributes to oncogenesis. In addition, necroptosis of endothelial cells and tumor cells is conducive to tumor metastasis. In this review, we summarize the current knowledge of the complex role of necroptosis in cancer and discuss the potential of targeting necroptosis components for cancer therapies.

RIP140 and TNF-α regulate energy metabolism in cardiomyocytes / 中国药理学通报

Aim: To explore whether RIP140 and TNF-a regulate energy metabolism in cardiomyocytes. Methods: H9c2 cardiomyocytes were infected with Ad-RIP140, simultaneously with or without TNF-α treatment. The mRNA levels of PPAR-α, PPAR-β/δ, and PDK4 were measured. H9c2 was exposed to adenovirus expressing RIP140-specific or nonspecific control. Expression of p65 in the nucleus and IκB-α: in cytoplasm were measured by Western blotting, and mRNA levels of IL-1β, IL-2 and TNF-α were measured by real-time PCR. H9c2 was treated with or without TNF-α. The mRNA and protein levels of RIP140 were measured. Results: Overexpression of RIP140 led to a decrease in mRNA levels of PPAR-α, PPAR-β/δ, PDK4, while TNF-α aggravated down-regulation of key metabolic genes by superabundant RIP140. A marked increase of p65-NF-κB in nuclear, a significant decrease of IκB-α in cytoplasm and a notable increase in mRNA levels of TNF-α, IL-β and IL-2 in H9c2 cell line were observed following overexpression of RIP140. The mRNA and protein levels of RIP140 were up-regulated by TNF-α treatment. Conclusions: RIP140 and TNF-a may collaborate in mediating proinflammatory processes and metabolic dysregulation in cardiomyocytes.

Complex roles of necroptosis in cancer / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

Necroptosis is a tightly regulated form of necrosis that requires the activation of receptor-interacting protein (RIP) kinases RIPK1 and RIPK3, as well as the RIPK3 substrate mixed lineage kinase domain-like protein (MLKL). Because of membrane rupture, necroptotic cells release damage-associated molecular patterns (DAMPs) that evoke immune responses. Necroptosis is emerging as an important cellular response in the modulation of cancer initiation, progression, and metastasis. Necroptosis of cancer cells is considered to be an immunogenic cell death capable of activating anti-tumor immunity. Necroptosis also participates in the promotion of myeloid cell-induced adaptive immune suppression and thus contributes to oncogenesis. In addition, necroptosis of endothelial cells and tumor cells is conducive to tumor metastasis. In this review, we summarize the current knowledge of the complex role of necroptosis in cancer and discuss the potential of targeting necroptosis components for cancer therapies.

Protective effects of naringenin against hypoxic-ischemic brain damage in neonatal rats through suppression of NOD2/RIP2/NF-κB pathway / 中国药理学通报

Aim To explore the protective effects of naringin on hypoxic ischemic brain injury in neonatal rats and its mechanism. Methods Ninety-six healthy 7-day neonatal SD rats were randomly divided into sham operation group, hypoxic-ischemic brain damage group (HIBD group),HIBD with low-dose naringenin group(50 mg·kg-1, NG-L) and HIBD with high-dose naringenin group(100 mg·kg-1,NG-H). Neu-rological deficit, HE staining and brain water content were measured 48h after operation. Immunoblotting was used to detect the expressions of NOD2,RIP2 and NF-κB. Enzyme linked immunosorbent assay(ELISA) method was adopted to detect TNF-α and IL-1β ex-pression. Results Compared with HIBD group, the neurological deficit score decreased, the pathological damage was reduced, and the water content of brain tissues markedly decreased by naringenin(50,100 mg ·kg-1) treatment(P<0.05). Western blot revealed the down-regulation of NOD2,RIP2 and NF-κB by na-ringenin (50,100mg·kg-1) treatment (P<0.05). The content of TNF-α and IL-1β in brain tissues was lower than that of HIBD group (P <0.05). Conclu-sion Naringenin is likely to exert a protective role in neonatal rats of hypoxic ischemic brain injury perhaps through decreasing the expression of NOD2, RIP2 and NF-κB,and reducing the secretion of TNF-α and IL-1β.

Ricin and its antidote agents: Research advances / 国际药学研究杂志

Ricin is a highly toxic plant protein produced by the seeds of the castor plant. It belongs to the ribosome inactivating proteins (RIP) family and causes cell death by inhibiting the protein synthesis activity of ribosome. Ricin takes a unique pathway called "retrograde trafficking pathway" to enter the cytosol, where it interacts with ribosome and then exerts its inhibitory activity. No effective antidote agents have been developed for the treatment of ricin poisoning. In this paper, the structure, cell trafficking process, toxicological mechanism and the research progress in ricin antitoxin agents are reviewed.

TNF-α induced RIP1-dependent apoptosis in L929-A cells by interrupting mitochondrial respiratory chain complex Ⅲ / 军事医学

Objective To explore the mechanism by which tumor necrosis factor alpha(TNF-α) induces RIP1 kinase-dependented apoptosis in L929-A fibroblastoma cells.Methods The sub-mitochondrial localization of receptor-interacting protein 1(RIP1),caspase-8 and Bid proteins was detected by dose-gradient trypsin digestion and Western blotting.The levels of reactive oxygen species (ROS),intracellular calcium concentration,mitochondrial membrane potential (MMP),and cellular adenosine triphosphate(ATP) content were determined by fluorescent probe labeling and flow cytometry assay.The mitochondrial respiratory chain complex Ⅰ and Ⅲ activities were detected by commercial kits.Nec-1,A RIP1 kinase specific inhibitor,and RIP1-/-or Bid-/-L929-A cells were used to detect the roles of RIP1 kinase and Bid protein in cell death.Results RIP1,caspase-8 and Bid proteins were co-located in the outer membrane of mitochondrial.TNF-α exposure for 3 h could induce Bid cleavage,inhibit mitochondrial respiratory chain complex Ⅲ activity and reduce MMP.Following these changes and after TNF-α exposure for 6-12 h,the intracellular calcium concentration and ROS were increased,whereas the ATP concentration was decreased,and the cells were killed.Inhibiting RIP1 kinase or knockdown RIP1 or Bid protein could suppress all the cytotoxic effects of TNF-α.Conclusion TNF-α treatment can result in RIP1 kinase-mediated Bid cleavage and inhibit mitochondrial respiratory chains and cell energy metabolism,which ultimately leads to the death of L929-A cells.

Repression of SIRT5 is Involved in RIP140-mediated Metabolic Dysregulation in Cardiomyocytes / 中山大学学报(医学科学版)

[Objective]To investigate the effect of desuccinylase Sirtuin5 (SIRT5) on receptor-interacting protein 140 (RIP140)- mediated metabolic dysfunction in cardiomyocytes.[Methods]RIP140 was overexpressed by Adenovirus infection and SIRT5 was overexpressed by plasmid transfection. RIP140 and SIRT5 were knocked down by siRNA interference. The expression of RIP140 and SIRT5 were measured by qRT-PCR and western blot. The transcription levels of mitochondrial DNA-encoded genes were detected by qRT-PCR. Mitochondrial membrane potential was detected by tetramethylrhodamine ethyl ester(TMRE)fluorescence anl?ysis. Cellular oxygen consumption and ATP production were investigated by assay kits. All data are from at least three independent ex?periments.[Results]RIP140 overexpression significantly attenuated SIRT5 expression(P<0.05),whereas knockdown of endogenous RIP140 elevated SIRT5 expression(P<0.05)in cardiomyocytes. Superabundant RIP140 also induced hypersuccinylation of mitochon?drial proteins,suggesting RIP140 could repress the desuccinylase activity of SIRT5. Moreover,SIRT5 overexpression reversed RIP140-mediated mitochondrial dysfunction and energy metabolic impairment ,such as repression of mitochondrial DNA-encoded genes(P<0.05),decrease of mitochondrial membrane potential(P<0.05),as well as reduction of cellular oxygen consumption(P<0.05)and ATP production(P<0.05). Furthermore,the regulation of RIP140 on SIRT5 was dependent on the peroxisome proliferator-activated receptorα(PPARα)in cardiomyocytes.[Conclusion]RIP140 induces mitochondrial dysfunction and metabolic impairment through repression of SIRT5 in cardiomyocytes.

RIP3 expression and function in the liver of rats following ischemic postconditioning / 中国现代普通外科进展

Objective:To investigate the expression and function of RIP3 in the liver of rats following ischemic postconditioning.Methods:The model of 70％ hepatic ischemia and reperfusion was established,then a total of forty healthy adult male Sprague-Dawley(SD) rats were divided randomly into four groups,ten rats in each group:a sham-operation group (Sham group);an ischemia reperfusion injury group(IR group);an ischemic postconditioning group(IPO group);an ischemic postconditioning and necrostatin-1 group (Nec-1 group).The blood samples and liver tissues were collected.The serum levels of ALT and AST were detected,and the liver histological examination was performed.Western-bolt was used to detect the TNF-α and RIP3 levels.Results:Compared with the IR group,ALT and AST in serum were significantly declined in the IPO group (P＜0.05);The liver damage after ischemia and reperfusion was improved obviously in the IPO group compared to which in the IR group;The Suzuki's scores was increased in the IR group compared to which in the IPO group (P＜0.05);There was a low grade of TNF-α and RIP3 in the Sham group,whereas the level of TNF-α and RIP3 significantly increased in the IR and IPO and Nec-1 group(P＜0.05);Compared with the IR group,the level of RIP3 was further decreased in the IPO group (P＜0.05);Compared with the IPO group,the level of RIP3 was further decreased in the Nec-1 group (P＜0.05).Conclusion:RIP3-mediated necroptosis was involved during hepatic ischemia postconditioning,and the protective effect of ischemia postconditioning may act as reducing necroptosis by cutting down the levels of RIP3.

LRRK2 enhances Nod1/2-mediated inflammatory cytokine production by promoting Rip2 phosphorylation

The innate immune system is critical for clearing infection, and is tightly regulated to avert excessive tissue damage. Nod1/2-Rip2 signaling, which is essential for initiating the innate immune response to bacterial infection and ER stress, is subject to many regulatory mechanisms. In this study, we found that LRRK2, encoded by a gene implicated in Crohn's disease, leprosy and familial Parkinson's disease, modulates the strength of Nod1/2-Rip2 signaling by enhancing Rip2 phosphorylation. LRRK2 deficiency markedly reduces cytokine production in macrophages upon Nod2 activation by muramyl dipeptide (MDP), Nod1 activation by D-gamma-Glu-meso-diaminopimelic acid (iE-DAP) or ER stress. Our biochemical study shows that the presence of LRRK2 is necessary for optimal phosphorylation of Rip2 upon Nod2 activation. Therefore, this study reveals that LRRK2 is a new positive regulator of Rip2 and promotes inflammatory cytokine induction through the Nod1/2-Rip2 pathway.

Ricin and its antidote agents:research advances / 国际药学研究杂志

Ricin is a highly toxic plant protein produced by the seeds of the castor plant. It belongs to the ribosome inactivat-ing proteins(RIP)family and causes cell death by inhibiting the protein synthesis activity of ribosome. Ricin takes a unique pathway calledretrograde trafficking pathwayto enter the cytosol,where it interacts with ribosome and then exerts its inhibitory activity. No effective antidote agents have been developed for the treatment of ricin poisoning. In this paper,the structure,cell trafficking process, toxicological mechanism and the research progress in ricin antitoxin agents are reviewed.

RNA-Seq, and ye shall find: Sexual-stage-specific A-to-I RNA editing in fungi.

Fusarium graminearum, a pathogen of wheat and barley, is a haploid homothallic ascomycete filamentous fungus (Goswami and Kistler 2004). It overwinters as saprophytic hyphae in plant debris and undergoes the sexual cycle in spring to produce fruiting bodies (perithecia) bearing the progeny ascospores. The genome sequence of the F. graminearum PH-1 strain was reported last year (King et al. 2015). This year, Jin-Rong Xu and colleagues in Northwest A&F University, China, and Purdue University, USA, re-sequenced the PH-1 genome and also performed RNA-Seq analysis on two independent biological replicates each of RNA from conidia, hyphae, and 8-day post-fertilization perithecia (Liu et al. 2016). Alignment of the two replicate perithecial RNA-Seq reads with the reference genome sequence revealed 23,041 and 19,764 single-nucleotide variants (SNVs), of which, respectively, 22,578 and 19,261 corresponded to A (adenosine)- to-G (guanosine) transitions, and 17,613 A-to-G transitions were common to both the replicates. Non- A-to-G variants were far fewer (463 and 503) and only 35.9% were common between the two perithecial replicates, suggesting that the non-A-to-G variants were false-positives. In sum, 26,056 A-to-G variants were identified as putative A-to-I RNA editing sites at which hydrolytic deamination at the C6 position of the purine ring of A produces I (inosine). Since I preferentially base-pairs with C (cytidine), an I within a transcript is read as G by the translation machinery. Also, during reverse transcription, I directs the incorporation of C; thus, it appears as a G in double-stranded cDNA. The conidial and hyphal RNA-Seq data showed only 68 and 112 A-to-G transitions and 335 and 452 non-A-to-G conversions, indicating that the A-to-I RNA editing is specific to the sexual stage. Xu and colleagues had initially set out to do a yeast two-hybrid experiment to identify proteins that interact with a protein kinase named Puk1 (perithecium unique kinase 1). Ascospores from the puk1 mutant have an abnormal morphology. Additionally, qRT-PCR showed that PUK1 transcription is markedly upregulated in perithecia, suggesting that PUK1 expression and function might be restricted to the sexual stage. Therefore, to generate the PUK1 ORF bait, they synthesized cDNA using RNA isolated from perithecial cultures of the PH-1 strain. Sequencing of the construct revealed that two tandem stop codons – UAG UAG – in the PUK1 ORF were changed to UGG UGG in the cDNA, presumably via A-to-I RNA editing. More than 90% of PUK1 reads in perithecial RNA-Seq showed the A-to-I editing, and experiments with site-specific mutant alleles showed that the editing was essential for PUK1 function. This was an unexpected discovery because fungi lack orthologs of the Adenosine Deaminase Acting on RNA (ADAR) family of enzymes that in metazoans converts A to I in double-stranded RNA. Presumably, A-to-I RNA editing in fungi uses different enzymes than animals. This discovery motivated Xu and colleagues to expand the search for RNA editing genome-wide in transcriptomes from vegetative and sexual-stage tissues (conidia, hyphae, and perithecia). The percentage of reads with the A-to-G variant was taken as the RNA editing level at the site. Editing levels varied from 3% to 100%. Strikingly, 47% of genes bearing sites with editing levels >60% tended to be up-regulated or specifically expressed in perithecia compared to conidia and hyphae. A majority of the editing events resulted in amino acid substitutions, which suggested that Ato- I editing might be important for adaptation of protein functions during sexual reproduction. Editing events similar to those in PUK1 were found 69 other genes, including the rid (RIP defective) ortholog and genes important for meiosis (see below). All these genes displayed UAG-to-UGG change in exons that automated annotation had incorrectly predicted as introns.

Activation of Rip1 promotes necroptosis in LNCaP-AI cells via inhibiting SHARPIN / 中国病理生理杂志

[ ABSTRACT] AIM:To explore the role of SHARPIN in regulation of Rip1 in castration-resistant prostate cancer LNCaP-AI cells.METHODS:The LNCaP-AI cells were treated with TNF-α+Z-VAD ( an inhibitor of pan-caspase) to activate necroptosis, which were compared to the cells treated with TNF-α+Z-VAD+Nec-1 ( an inhibitor of Rip1 ) .A blank group and a TNF-α-treated group were set up as controls.The cell viability in each group was measured by MTS as-say.In addition, SHARPIN was knocked down by siRNA, and the inhibitory efficiency was evaluated by RT-qPCR.The expression of Rip1 at mRNA and protein levels after knocking down SHARPIN was determined by RT-qPCR and Western blot to explore the underlying mechanism of regulatory network of necroptosis in prostate cancer.RESULTS: Compared with blank control group and TNF-α-treated group, the viability of LNCaP-AI cells treated with TNF-α+Z-VAD decreased by 28%(P LNCaP-AI cells.CONCLUSION:Necroptosis is an important way of cell death .Inhibition of oncogenic factor SHARPIN enhances necroptosis via activating Rip1 in LNCaP-AI cells.