TP53-regulated Inhibitor Of Apoptosis 1 Research Articles

Abstract Tu050: Deficiency Of Mitochondrial Disulfide Relay Carrier Leads To Cardiac Hypertrophy

Background: Innate immune activation is critical for cardiac hypertrophy; however, its upstream regulation is less well understood. Coiled-coil-helix-coiled-coil-helix domain 4 (CHCHD4) is an essential carrier for the import of various mitochondrial proteins including TP53 -regulated inhibitor of apoptosis 1 (TRIAP1). CHCHD4 was recently identified as a dysregulated gene in patients with dilated cardiomyopathy and was separately found to mediate innate immune signaling in skeletal muscle. Therefore, we hypothesized that CHCHD4 may play a role in the development of cardiac hypertrophy by regulating innate immunity. Methods: Genetically modified C57BL/6 mice overexpressing CHCHD4 (CHCHD4 Tg) or haploinsufficient in CHCHD4 ( CHCHD4 +/- ) were utilized. Mice were injected either with low dose (2 mg/kg) or high dose (30 mg/kg) isoproterenol (ISO) daily for up to 3 wk to induce hypertrophy. Results: CHCHD4 +/- mice developed cardiac hypertrophy (HW/BW (mg/g): 3.62 ± 0.15 CHCHD4 +/- vs 2.70 ± 0.13 wild-type (WT) or 2.55 ± 0.16 CHCHD4 Tg, p<0.0001) when allowed to age to 28 wk and showed reduced left ventricular ejection fraction (EF) at 1 year when compared with wild-type or CHCHD4 Tg mice (EF (%): 50.94 ± 0.94 CHCHD4 +/- vs 58.33 ± 0.78 wild-type or 57.88 ± 1.32 CHCHD4 Tg, p<0.0001). After low dose ISO treatment, CHCHD4 +/- mice had reduced EF at 2 wk compared to WT or CHCHD4 Tg mice (EF (%): 44.60 ± 1.74 CHCHD4 +/- vs 54.28 ± 0.86 wild-type, p<0.0001; or vs 52.76 ± 1.33 CHCHD4 Tg, p=0.0006). After high dose ISO treatment, WT mice developed cardiac hypertrophy by 1 wk and impaired EF by 3 wk, while CHCHD4 Tg mice remained normal. ISO treatment caused reduction of CHCHD4 levels in both the 24 hr and 1 wk states. This reduction was associated with decreased TRIAP1 and activation of the cGAS-STING-NFkB pathway in wild-type, but not CHCHD4 Tg mice. Further, plasma mitochondrial DNA (mtDNA) was 2.3 times higher in WT mice treated with high dose ISO for 1 wk compared to WT control (n=8-11/group, p=0.011), while that of CHCHD4 Tg mice was not significantly changed by ISO treatment. Conclusions: Our findings suggest that CHCHD4 may be a key upstream regulator of innate immune activation mediating cardiac hypertrophy and that cell-free mtDNA in blood could be a potential biomarker of pathogenesis.

CHCHD4-TRIAP1 regulation of innate immune signaling mediates skeletal muscle adaptation to exercise

Exercise training can stimulate the formation of fatty-acid-oxidizing slow-twitch skeletal muscle fibers, which are inversely correlated with obesity, but the molecular mechanism underlying this transformation requires further elucidation. Here, we report that the downregulation of the mitochondrial disulfide relay carrier CHCHD4 by exercise training decreases the import of TP53-regulated inhibitor of apoptosis 1 (TRIAP1) into mitochondria, which can reduce cardiolipin levels and promote VDAC oligomerization in skeletal muscle. VDAC oligomerization, known to facilitate mtDNA release, can activate cGAS-STING/NFKB innate immune signaling and downregulate MyoD in skeletal muscle, thereby promoting the formation of oxidative slow-twitch fibers. In mice, CHCHD4 haploinsufficiency is sufficient to activate this pathway, leading to increased oxidative muscle fibers and decreased fat accumulation with aging. The identification of a specific mediator regulating muscle fiber transformation provides an opportunity to understand further the molecular underpinnings of complex metabolic conditions such as obesity and could have therapeutic implications.

Transcriptomic Analysis Reveals That Granulocyte Colony-Stimulating Factor Trigger a Novel Signaling Pathway (TAF9-P53-TRIAP1-CASP3) to Protect Retinal Ganglion Cells after Ischemic Optic Neuropathy.

Optic nerve head (ONH) infarct can result in progressive retinal ganglion cell (RGC) death. The granulocyte colony-stimulating factor (GCSF) protects the RGC after ON infarct. However, protective mechanisms of the GCSF after ONH infarct are complex and remain unclear. To investigate the complex mechanisms involved, the transcriptome profiles of the GCSF-treated retinas were examined using microarray technology. The retinal mRNA samples on days 3 and 7 post rat anterior ischemic optic neuropathy (rAION) were analyzed by microarray and bioinformatics analyses. GCSF treatment influenced 3101 genes and 3332 genes on days 3 and 7 post rAION, respectively. ONH infarct led to changes in 702 and 179 genes on days 3 and 7 post rAION, respectively. After cluster analysis, the levels of TATA box-binding protein (TBP)-associated factor were significantly reduced after ONH infarct, but these significantly increased after GCSF treatment. The network analysis revealed that TBP associated factor 9 (TAF9) can bind to P53 to induce TP53-regulated inhibitor of apoptosis 1 (TRIAP1) expression. To evaluate the function of TAF9 in RGC apoptosis, GCSF plus TAF9 siRNA-treated rats were evaluated using retrograde labeling with FluoroGold assay, TUNEL assay, and Western blotting in an rAION model. The RGC densities in the GCSF plus TAF9 siRNA-treated rAION group were 1.95-fold (central retina) and 1.75-fold (midperipheral retina) lower than that in the GCSF-treated rAION group (p < 0.05). The number of apoptotic RGC in the GCSF plus TAF9 siRNA-treated group was threefold higher than that in the GCSF-treated group (p < 0.05). Treatment with TAF9 siRNA significantly reduced GCSF-induced TP53 and TRIAP1 expression by 2.4-fold and 4.7-fold, respectively, in the rAION model. Overexpression of TAF9 significantly reduced apoptotic RGC and CASP3 levels, and induced TP53 and TRIAP1 expression in the rAION model. Therefore, we have demonstrated that GCSF modulated a new pathway, TAF9-P53-TRIAP1-CASP3, to control RGC death and survival after ON infarct.

MiR-103 Derived from Bone Marrow Mesenchymal Stem Cell (BMSC) Retards the Chemo-Resistance Through Targeted-Regulation of TP53 Regulated Inhibitor of Apoptosis 1 (TRIAP1) in Breast Cancer

The chemo-resistance was one of the major reasons for the treatment failure for breast cancer. Our study aimed to discuss the action of miR-103 derived from BMSC on retarding the chemo-resistance through targeted-regulating TRIAP1 in breast cancer. The cisplatin-resistant breast cancer cells were cultivated and transfected with si-RNA targeting miR-103 followed by analysis of cell invasion, migration and apoptosis by Transwell. MiR-103 target gene was analyzed with bioinformatics method and dual-luciferase reporter assay. TRIAP1 expression was measured by Western Blot. The cell apoptosis was reduced when miR-103 expression was restrained along with enhanced cell proliferation. The co-cultivation with BMSC in vitro could upregulate TRIAP1 expression in breast cancer cells. In cells transfected with si-miR-103, the expression of TRIAP1 was reduced, cell apoptosis was increased and invasion was decreased. In conclusion, the chemo-resistance is induced and the malignant invasion of breast cancer cells is retarded by co-cultivation with mikR-103 derived from BMSC which might be through regulating the expression of TRIAP.

Circular RNA circPVT1 Contributes to Doxorubicin (DXR) Resistance of Osteosarcoma Cells by Regulating TRIAP1 via miR-137.

Background Chemoresistance is a major obstacle to the treatment of osteosarcoma patients. Circular RNA (circRNA) circPVT1 has been reported to be related to the doxorubicin (DXR) resistance in osteosarcoma. This study is designed to explore the role and mechanism of circPVT1 in the DXR resistance of osteosarcoma. Methods circPVT1, microRNA-137 (miR-137), and TP53-regulated inhibitor of apoptosis 1 (TRIAP1) levels were detected by real-time quantitative polymerase chain reaction (RT-qPCR). The protein levels of ATP-binding cassette, subfamily C, member 1 (ABCC1), multidrug resistance-associated protein 1 (MRP-1), cleaved- (c-) caspase-3, B-cell lymphoma-2 (Bcl-2), and TRIAP1 were examined by a western blot assay. Cell viability, proliferation, and apoptosis were detected by cell counting kit-8 (CCK-8), colony formation, and flow cytometry assays, severally. The binding relationship between miR-137 and circPVT1 or TRIAP1 was predicted by starbase 3.0 and then verified by dual-luciferase reporter and RNA Immunoprecipitation (RIP) assays. The biological role of circPVT1 in osteosarcoma tumor growth and drug resistance was examined by the xenograft tumor model in vivo. Results. circPVT1 and TRIAP1 were highly expressed, and miR-137 was decreased in DXR-resistant osteosarcoma tissues and cells. Moreover, circPVT1 knockdown could boost DXR sensitivity by inhibiting DXR-caused proliferation and DXR-induced apoptosis in DXR-resistant osteosarcoma cells in vitro. The mechanical analysis discovered that circPVT1 acted as a sponge of miR-137 to regulate TRIAP1 expression. circPVT1 silencing increased the drug sensitivity of osteosarcoma in vivo. Conclusion. circPVT1 boosted DXR resistance of osteosarcoma cells partly by regulating the miR-137/TRIAP1 axis, hinting a promising therapeutic target for the osteosarcoma treatment.

Abnormal expression of TRIAP1 and its role in gestational diabetes mellitus-related pancreatic β cells.

Gestational diabetes mellitus (GDM) is a disease that is typically characterized by insulin resistance and pancreatic β cell dysfunction. Currently, the role of TP53-regulated inhibitor of apoptosis 1 (TRIAP1) in the process of GDM remains to be elucidated. Therefore, the present study investigated the effects of TRIAP1 on GDM-related pancreatic β cells. Reverse transcription-quantitative PCR and western blot assays were conducted to analyze the expression levels of TRIAP1 in the peripheral blood of patients with GDM and subjects with healthy pregnancies. Subsequently, TRIAP1 small interfering RNA (siRNA), control siRNA, TRIAP1 plasmid and control plasmid were transfected into INS-1 cells to assess the effects of TRIAP1 on pancreatic β cells. ELISA was used to assess the total insulin content and insulin secretion of pancreatic β cells. MTT and flow cytometry assays were performed to determine the viability and apoptosis of pancreatic β cells. The results demonstrated that TRIAP1 expression was downregulated in peripheral blood samples from patients with GDM. Transfection with TRIAP1 siRNA significantly decreased the levels of total insulin content and reduced insulin secretion in pancreatic β cells. In addition, downregulation of TRIAP1 in pancreatic β cells significantly induced cell apoptosis and reduced cell viability. Accordingly, transfection of INS1 cells with TRIAP1 siRNA increased the levels of the apoptosis-associated genes apoptotic protease-activating factor 1, caspase-3, caspase-7 and caspase-9. However, transfection of the cells with TRIAP1 plasmid resulted in the opposite effects. TRIAP1 increased the growth of pancreatic β cells and their ability to secrete insulin, thus playing a protective role in GDM. The findings verified the effects and the underlying mechanism of TRIAP1 in pancreatic β cells and may provide additional clinical applications for the therapy of GDM.

Regulation of Apoptosis and Inflammatory Response in Interleukin-1β-Induced Nucleus Pulposus Cells by miR-125b-5p Via Targeting TRIAP1.

The aim of the present study was to determine the function of microRNA (miR)-125b-5p in lumbar disc degeneration (LDD). Nucleus pulposus (NP) cells were stimulated with 10ng/ml IL-1β for 24h to establish an LDD model. Reverse transcription-quantitative PCR was used to assess miR-125b-5p levels in human lumbar degenerative NP samples and IL-1β-treated NP cells. An interaction between miR-125b-5p and TP53-regulated inhibitor of apoptosis 1 (TRIAP1) was revealed by TargetScan 7.1 and dual-luciferase reporter assay. Protein levels of pro-inflammatory factors were determined using ELISA. Cell viability and apoptosis were evaluated by MTT and flow cytometry analysis, respectively. miR-125b-5p was markedly upregulated in both human lumbar degenerative NP specimens and IL-1β-treated NP cells. TRIAP1, which directly targets miR-125b-5p, was markedly downregulated in human lumbar degenerative NP specimens and IL-1β-treated NP cells. The levels of TNF-α and IL-6 were inhibited in IL-1β-treated NP cells transfected with miR-125b-5p inhibitor. Moreover, miR-125b-5p inhibitor increased NP cell viability, prevented apoptosis and repressed the apoptotic peptidase activating factor 1/caspase 9 pathway in IL-1β-treated NP cells. Thus, the present findings suggested that miR-125b-5p could regulate LDD by adjusting NP cell apoptosis and inflammatory responses via TRIAP1.

TRIAP1 knockdown sensitizes non‐small cell lung cancer to ionizing radiation by disrupting redox homeostasis

BackgroundRadioresistance of some non‐small cell lung cancer (NSCLC) types increases the risk of recurrence or metastasis in afflicted patients, following radiotherapy. As such, further improvements to NSCLC radiotherapy are needed. The expression of oncogene TP53‐regulated inhibitor of apoptosis 1 (TRIAP1) in NSCLC is increased following irradiation. Furthermore, gene set enrichment analysis (GSEA) has suggested that TRIAP1 might be involved in maintaining redox homeostasis. This in turn might enhance cell radioresistance.MethodsIn this study we irradiated human NSCLC cell lines (A549 and H460), while knocking down TRIAP1, to determine whether a disrupted redox homeostasis could attenuate radioresistance.ResultsIrradiation notably increased both mRNA and protein levels of TRIAP1. In addition, TRIAP1 knockdown decreased the expression of several antioxidant proteins, including thioredoxin‐related transmembrane protein (TMX) 1, TMX2, thioredoxin (TXN), glutaredoxin (GLRX) 2, GLRX3, peroxiredoxin (PRDX) 3, PRDX4, and PRDX6 in A549 and H460 cells. In addition, silencing TRIAP1 impaired the radiation‐induced increase of the aforementioned proteins. Continuing along this line, we observed a radiation‐induced reduction of cell viability and invasion, as well as increased apoptosis and intracellular reactive oxygen species following TRIAP1 knockdown.ConclusionsIn summary, we identified TRIAP1 as a key contributor to the radioresistance of NSCLC by maintaining redox homeostasis.

MicroRNA-107 may regulate lung cancer cell proliferation and apoptosis by targeting TP53 regulated inhibitor of apoptosis 1.

Lung cancer causes over 1.6 million mortalities worldwide annually. MicroRNAs (miRs) are involved in various types of cancer-associated processes. The present study investigated the possible mechanism of miR-107 in the development of lung cancer in order to identify novel targets for clinical treatment. The expression levels of miR-107 and its putative target gene TP53 regulated inhibitor of apoptosis 1 (TRIAP1) were measured in lung cancer tumor tissues and non-tumor adjacent tissues. Subsequently, the association between TRIAP1 and miR-107 was investigated using a dual-luciferase reporter assay. Following transfection, the effects of miR-107 and TRIAP1 on the proliferation and apoptosis of lung cancer cell lines in vitro were investigated using Cell Counting Kit-8 and flow cytometry assays, respectively. Furthermore, the regulatory effect of miR-107 on the expression levels of TRIAP1 and associated proteins was analyzed using a western blot assay. The results revealed lower expression levels of miR-107 and higher expression levels of TRIAP1 in lung cancer tumor tissues compared with non-tumor adjacent tissues. The dual-luciferase reporter assay demonstrated that TRIAP1 is a target gene of miR-107. Additionally, the results revealed that overexpression of miR-107 resulted in a lower proliferation rate and higher apoptosis rate of A549 cells, compared with the negative control (NC) and control groups (P<0.01). The variation of cell proliferation and apoptosis induced by miR-107 mimics was reversed by co-transfection with pcDNA3.1-TRIAP1. Furthermore, the expression levels of cyclin D1 and proliferating cell nuclear antigen were markedly decreased in the miR-107 mimics group compared with the NC group (P<0.01). The expression levels of BCL2 associated X apoptosis regulator, tumor protein p53 and caspase 3 were upregulated and the expression levels of TRIAP1 and BCL2 apoptosis regulator were significantly reduced in the miR-107 mimics group compared with the NC group (P<0.01). The results of the present study suggested that miR-107 regulates lung cancer cell proliferation and apoptosis by targeting TRIAP1.

Dysregulation of microRNA-770-5p influences pancreatic-β-cell function by targeting TP53 regulated inhibitor of apoptosis 1 in gestational diabetes mellitus.

The purpose of this study was to investigate the role of microRNA-770-5p (miR-770-5p) in gestational diabetes mellitus (GDM). In the present study, the expression levels of miR-770-5p in the peripheral blood from GDM women and healthy women were investigated by quantitative reverse transcription-polymerase chain reaction (qRT-PCR). The relationship between TP53 regulated inhibitor of apoptosis 1 (TRIAP1) and miR-770-5p was determined using dual-luciferase reporter assay. 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) assay and flow cytometry were used to detect pancreatic β-cell proliferation and apoptosis. Enzyme-linked immunosorbent assay (ELISA) was used to measure total insulin content and insulin secretion. Our data indicated that miR-770-5p was up-regulated in GDM patients. TRIAP1 was a direct target of miR-770-5p and it was down-regulated in GDM patients. Besides, miR-770-5p negatively regulated the expression of TRIAP1 in INS-1 cells. Then, we explored the effects of miR-770-5p down-regulation on the insulin secretion of pancreatic β-cells, and the results showed that miR-770-5p inhibitor promoted the generation of insulin secretion or total insulin content in INS-1 cells, while these effects were significantly inhibited by TRIAP1-siRNA. Moreover, we found that miR-770-5p inhibitor enhanced INS-1 cell proliferation and suppressed cell apoptosis, whereas these effects were eliminated by TRIAP1-siRNA. Accordingly, miR-770-5p inhibitor decreased the expression of Bax, apoptotic peptidase activating factor 1 (APAF1) and increased Bcl-2 level in INS1 cells. These results were all reversed by TRIAP1-siRNA. The data demonstrated that miR-770-5p was a vital regulator in pancreatic β-cell proliferation, apoptosis and insulin secretion by targeting TRIAP1, and dysregulation of miR-770-5p resulted in the development of GDM via APAF1 signaling pathway.

High TRIAP1 expression in penile carcinoma is associated with high risk of recurrence and poor survival.

TP53-regulated inhibitor of apoptosis 1 (TRIAP1), also known as p53 cell survival factor or p53CSV, is reported to be associated with resistance of apoptosis in different human malignancies, but the potential role of TRIAP1 in penile carcinoma (PeCa) has not been well studied. This study aimed to analyze the association between TRIAP1 expression and clinical outcome in PeCa patients. Bioinformatics was used to analyze the differential TRIAP1 expression in PeCa compared with normal tissues in Gene Expression Omnibus (GEO) Dataset (GSE57955). The expression of TRIAP1 in tumor specimens from 57 patients undergoing radical penile surgery was detected by immunohistochemistry (IHC). Differential TRIAP1 expression in various human malignancies was also assessed by GEPIA web-tool based on The Cancer Genome Atlas (TCGA) Datasets. Subsequently, the relationship between TRIAP1 expression and clinical prognosis of PeCa patients was analyzed. Both IHC and GEO Dataset (GSE57955) showed that TRIAP1 was significantly overexpressed in PeCa tissues when compared with normal tissues. Based on patient data and IHC on clinical specimens, we found that strong intensity of TRIAP1 expression was significantly related with higher histological grade (P=0.049) and elevated local recurrence rate (P=0.023), suggesting TRIAP1 as a potential predictor in recurrence. Further, high TRIAP1 expression was identified to be a hazardous prognostic factor for local recurrence-free survival (RFS). High TRIAP1 expression in PeCa is associated with high risk of recurrence and poor survival, suggesting TRIAP1 may become a potential prognostic factor for PeCa.

MiRNA-125a-5p inhibits hepatocellular carcinoma cell proliferation and induces apoptosis by targeting TP53 regulated inhibitor of apoptosis 1 and Bcl-2-like-2 protein.

The present study aimed to investigate the role and underlying molecular mechanism of microRNA (miR)-125a-5p in hepatocellular carcinoma. The level of miR-125a-5p was detected using reverse transcription-quantitative polymerase chain reaction. TargetScan was used to investigate the association between miR-125a-5p and TP53-regulated inhibitor of apoptosis 1 (TRIAP1)/B cell lymphoma-2-like 2 protein (BCL2L2). Dual luciferase reporter assay was used to confirm this prediction. To investigate the role of miR-125a-5p in hepatocellular carcinoma (HCC) cells, miR-125a-5p was overexpressed in the human HCC cell line PLC/PRF/5 using miR-125a-5p mimics. Subsequently, cell proliferation, cell apoptosis and cell migration were studied using MTT assay, flow cytometry analysis and Transwell assay, respectively. Protein expression levels in the present study were measured by western blot analysis. Taken together, the present results suggested that miR-125a-5p was markedly downregulated in HCC cells. TRIAP1 and BCL2L2 were direct targets of miR-125a-5p and were upregulated in PLC/PRF/5 cells. miR-125a-5p upregulation inhibited PLC/PRF/5 cell viability and migration and induced cell apoptosis. In addition, miR-125a-5p overexpression increased the expression of caspase9 and apoptotic protease-activating factor 1. Notably, the present study revealed that all the effects on PLC/PRF/5 cells elicited by miR-125a-5p overexpression were eliminated by TRIAP1/BCL2L2 upregulation. In conclusion, miR-125a-5p was shown to be downregulated in hepatocellular carcinoma and its upregulation inhibited hepatocellular carcinoma cell growth and metastasis by targeting TRIAP1 and BCL2L2.

Progression-Related Loss of Stromal Caveolin 1 Levels Mediates Radiation Resistance in Prostate Carcinoma via the Apoptosis Inhibitor TRIAP1.

Tumour resistance to chemo- and radiotherapy, as well as molecularly targeted therapies, limits the effectiveness of current cancer treatments. We previously reported that the radiation response of human prostate tumours is critically regulated by CAV1 expression in stromal fibroblasts and that loss of stromal CAV1 expression in advanced tumour stages may contribute to tumour radiotherapy resistance. Here we investigated whether fibroblast secreted anti-apoptotic proteins could induce radiation resistance of prostate cancer cells in a CAV1-dependent manner and identified TRIAP1 (TP53 Regulated Inhibitor of Apoptosis 1) as a resistance-promoting CAV1-dependent factor. TRIAP1 expression and secretion was significantly higher in CAV1-deficient fibroblasts and secreted TRIAP1 was able to induce radiation resistance of PC3 and LNCaP prostate cancer cells in vitro, as well as of PC3 prostate xenografts derived from co-implantation of PC3 cells with TRIAP1-expressing fibroblasts in vivo. Immunohistochemical analyses of irradiated PC3 xenograft tumours, as well as of human prostate tissue specimen, confirmed that the characteristic alterations in stromal-epithelial CAV1 expression were accompanied by increased TRIAP1 levels after radiation in xenograft tumours and within advanced prostate cancer tissues, potentially mediating resistance to radiation treatment. In conclusion, we have determined the role of CAV1 alterations potentially induced by the CAV1-deficient, and more reactive, stroma in radio sensitivity of prostate carcinoma at a molecular level. We suggest that blocking TRIAP1 activity and thus avoiding drug resistance may offer a promising drug development strategy for inhibiting resistance-promoting CAV1-dependent signals.

MicroRNA-770-5p is involved in the development of diabetic nephropathy through regulating podocyte apoptosis by targeting TP53 regulated inhibitor of apoptosis 1.

Diabetic nephropathy (DN) is a major diabetic micro-vascular complication, and podocyte apoptosis induced by high glucose(HG) is a typical early feature of DN. Studies have shown that microRNAs (miRNAs) play a crucial role in the pathogenesis of DN. The purpose of the current study was to explore the role and molecular mechanism of miR-770-5p in podocyte apoptosis in DN. In vitro podocyte model of DN was conducted by treatment conditionally immortalized mouse podocytes with HG (30 mM D-glucose). The level of miR-770-5p in podocytes was detected using quantitative real-time PCR (qRT-PCR), and protein levels were measured using Western blot assay in our current study. The relationship between miR-770-5p and TP53 regulated inhibitor ofapoptosis1 (TRIAP1) was revealed by TargetScan and dual luciferase reporter assay. Cell proliferation ability and cell apoptosis were determined by using cell counting kit-8 (CCK-8) assay and flow cytometer (FCM), respectively. We found that miR-770-5p was significantly upregulated in podocytes under HG condition. TRIAP1 was a target gene of miR-770-5p and it was down-regulated in podocytes by HG treatment. Further analysis indicated that HG induced cell proliferation ability reduction, cell apoptosis enhancement and apoptotic peptidase activating factor 1(APAF1)/Caspase9 pathway exaltation in podocytes were prevented by miR-770-5p down-regulation. More importantly, the results showed that all the effects of miR-770-5p inhibitor on HG induced podocytes were eliminated by TRIAP1 silencing. S.-Z. Zhang, X.-J. Qiu, S.-S. Dong, L.-N. Zhou, Y. Zhu, M.-D. Wang, L.-W. JinWe showed that miR-770-5p was upregulated in the in vitro model of DN, and it might promote the development of DN through regulating podocyte apoptosis by targeting TRIAP1.

MiR-1301/TRIAP1 Axis Participates in Epirubicin-Mediated Anti-Proliferation and Pro-Apoptosis in Osteosarcoma.

PurposeEpirubicin is one of the most effective drugs against osteosarcoma. miR-1301 is involved in the occurrence and development of osteosarcoma. Whether miR-1301 is responsible for the chemosensitivity of osteosarcoma cells to epirubicin remains largely unknown.Materials and MethodsU2OS and SAOS-2 cells were treated with various concentrations of epirubicin. Flow cytometry was employed to evaluate cell apoptotic rate. Cell proliferation was measured by Cell Counting Kit-8 assay. Western blot and quantitative real-time polymerase chain reaction were utilized to detect the expressions of B-cell lymphoma-2 (Bcl-2), Bcl-2 assaciated X protein (Bax), cleaved-caspase-3, cleaved-poly (ADP-ribose) polymerases (PARP1), TP53-regulated inhibitor of apoptosis 1 (TRIAP1), and microRNA-1301 (miR-1301). The relationship between miR-1301 and TRIAP1 was determined by luciferase reporter assay.ResultsEpirubicin inhibited proliferation in a dose-dependent manner, induced apoptosis, decreased the expression of Bcl-2, and increased the expressions of Bax, cleaved-caspase-3, and cleaved-PARP1 in osteosarcoma cells. miR-1301 was downregulated in U2OS and SAOS-2 cells. Importantly, epirubicin significantly increased the levels of miR-1301. Overexpression of miR-1301 suppressed proliferation and promoted apoptosis. Interestingly, those effects were enhanced by epirubicin. In contrast, miR-1301 depletion attenuated the epirubicin-mediated anti-osteosarcoma effect. miR-1301 negatively regulated the expression of TRIAP1 in U2OS and SAOS-2 cells. Furthermore, epirubicin inhibited the mRNA and protein levels of TRIAP1 by upregulating miR-1301 levels. Epirubicin suppressed cell proliferation by downregulating TRIAP1.ConclusionmiR-1301 was implicated in the chemosensitivity of osteosarcoma to epirubicin by modulating TRIAP1.

MiR-107 inhibits proliferation of lung cancer cells through regulating TP53 regulated inhibitor of apoptosis 1 (TRIAP1)

AbstractAimsAccumulating evidence indicates that aberrant expression of miR-107 plays a crucial role in cancers. This study aims to display the function of miR-107 and its novel target genes in the progression of lung cancer.Methods and MaterialMiR-107 or miR-107 inhibitor was transfected into lung cancer cells A549. The levels of miR-107 and TP53 regulated inhibition of apoptosis 1 (TRIAP1) were examined by quantitative real-time Polymerase Chain Reaction (qRT-PCR) analysis and Western Blot. Functionally, MTT and colony formation assays were carried out to test the effect of miR-107 inhibitor and/or small interference RNA (siRNA) targeting TRIAP1 mRNA on proliferation of lung cancer cells. Levels of miR-107 or TRIAP1 were detected in clinical lung cancer samples by using qRT-PCR analysis.ResultsQRT-PCR analysis revealed that miR-107 inhibitor or miR-107 was successfully transfected into A549 cells. Western Blot indicated that miR-107 decreased the expression of TRIAP1 protein in the cells. In contrast, miR-107 inhibitor augmented the levels of TRIAP1 protein. Functionally, miR-107 inhibitor remarkably suppressed A549 cell proliferation, whereas, TRIAP1 siRNAs could abrogate the miR-107 inhibitor-induced proliferation of cells. Then, we validated that TRIAP1 was increased in clinical lung cancer samples. MiR-107 expression was negatively related to TRIAP1 expression in clinical lung cancer samples.ConclusionsMiR-107 suppresses cell proliferation by targeting TRIAP1 in lung cancer. Our finding allows new insights into the mechanisms of lung cancer that is mediated by miR-107.

TP53 Regulated Inhibitor of Apoptosis 1 (TRIAP1) stable silencing increases late apoptosis by upregulation of caspase 9 and APAF1 in RPMI8226 multiple myeloma cell line

BackgroundTP53 Regulated Inhibitor of Apoptosis 1 (TRIAP1) modulates apoptotic pathways preventing the formation of the apoptosome complex. Our group previous study showed that 90% of patients' multiple myeloma (MM) marrow-derived plasma cells present TRIAP1 overexpression as compared to normal plasma cells. Due to high prevalence and lack of information on TRIAP1's role in MM biology, we decided to explore the impact of TRAIP1 through stable gene silencing in MM cell lines and its effect on cell cycle and apoptosis. MethodsTRIAP1 expression was examined in MM cell lines by quantitative real time PCR. Cell lines were submitted to transduction with lentiviral vector encoding a TRIAP1-specific short hairpin RNA (shRNA) and, as control, encoding a non-targeting shRNA (scramble). Apoptosis was assessed by flow cytometry with annexin V and propidium iodide staining (the later also used for cell cycle), APAF1 and Caspase 9 apoptosome related genes expression and Caspase 9 and Caspase 3/7 activity. ResultsRPMI8226 and U266 cell lines were chosen for transduction experiments since they present higher levels of TRIAP1 expression. Inhibition of TRIAP1 in RPMI8226 cells increased the percentage of apoptotic cells, accompanied by increased expression of APAF1 and Caspase 9, and Caspase 9 and Caspase 3/7 activity. Transduced U266 cell line did not show sustained inhibition of TRIAP1 expression nor apoptosis induction. ConclusionStable silencing of TRIAP1 induces late apoptosis through APAF1/Caspase 9 pathway at least in RPMI8226 cell line, suggesting that it could be exploited as a potential target at least for a subgroup of MM patients. General significanceIn the present study, we demonstrated effects of TRIAP1 silencing on RPMI8226 MM cell line and established its mechanism mediated through APAF1 and Caspase 9. No relevant effect was found after gene silencing in U266 cell line.

Abstract 1017: Silencing of apoptosome regulating genes, HSP70 and TRIAP1, induces apoptosis in MM cell lines

Abstract Introduction: Some evidences suggest that heat shock protein 70 (HSP70) is overexpressed in many types of cancer, providing a selective advantage for tumor cell survival, due in part, to its ability to inhibit cell death via APAF1 (apoptosis protease activating factor 1) and Caspase 9. The TP53 Regulated Inhibitor of Apoptosis 1 (TRIAP1) gene can modulate apoptotic pathways by interaction with HSP70. Although there are several studies on the role of HSP70 gene in apoptosis and drug resistance, there is a lack of information about this gene in multiple myeloma (MM). Objectives: To analyze the importance of HSP70 and TRIAP1 as potential targets for MM therapy through: 1) stable silencing of HSP70 and TRIAP1 in MM cell lines; 2) evaluation of each gene silencing effect on cell cycle and apoptosis. Methods: The expression of HSP70 and TRIAP1 genes in MM cell lines (U266, SKO-007, SK-MM2 and RPMI8226) was examined by quantitative real time PCR (qPCR). Cell lines were submitted to transduction with pLKO lentiviral vector containing short hairpin RNAs (shRNAs) for silencing the target genes (shRNAHSP70 and shRNATRIAP1) - three independent transductions, in triplicate. Lentiviral vectors with control sequences (scramble) were used to transduce the same cell lines. Apoptosis was assessed by flow cytometry with annexin V and propidium iodide (PI) staining. We also evaluated APAF1 and Caspase9 genes expression by qPCR and Caspase 9 and Caspase 3/7 protein activity. Results: The cell lines RPMI8226 (without deletion of TP53 by FISH) and U266 (deletion of one allele of TP53 by FISH) were chosen for the transduction experiments because they showed significant levels of expression of HSP70 and TRIAP1. The efficiency of transduction, with GFP reporter gene, was 70% for both cell lines. Silencing of both genes was confirmed by qPCR and Western blotting. Inhibition of TRIAP1 increased the percentage of cells in late apoptosis (p&amp;lt;0.001 for RPMI8226 and P&amp;lt;0.01 for U266) and was accompanied by increased expression of Caspase9 in both MM cell lines (p&amp;lt;0.001 for RPMI8226 and p&amp;lt;0.05 for U266). Furthermore, the inhibition of TRIAP1 resulted in accumulation of hypodiploid cells after 24 hours of transduction in U266 cell line. Inhibition of HSP70 showed no significant changes in the cell cycle in both MM cell lines. However, we observed an increment in late apoptosis after inhibition of this gene in the two cell lines (p&amp;lt;0.01 for RPMI8226 and p&amp;lt;0.05 for U266) and these results were confirmed by increased activity of Caspase3/7 (p&amp;lt;0.01 for RPMI8226 and p&amp;lt;0.05 for U266). Conclusion: Stable silencing of HSP70 and TRIAP1 in MM cell lines showed a strong impact of this method on the induction of late apoptosis, through APAF1/Caspase9 pathway, suggesting that inhibitors of both genes could be exploited as potential targets for the treatment of MM, helping patients whatever FISH status for TP53 (Support by FAPESP 2010/17668-6). Citation Format: Veruska Lia Fook Alves, Daniela B. Zanatta, mariana bleker oliveira, angela isabel pereira eugenio, rodrigo carlini fernando, Bryan E. Strauss, Gisele Wally Braga Colleoni. Silencing of apoptosome regulating genes, HSP70 and TRIAP1, induces apoptosis in MM cell lines. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1017. doi:10.1158/1538-7445.AM2015-1017

Apoptosis inhibitor TRIAP1 is a novel effector of drug resistance

TP53-regulated inhibitor of apoptosis 1 (TRIAP1) is a novel apoptosis inhibitor that binds HSP70 in the cytoplasm and blocks the formation of the apoptosome and caspase-9 activation. TRIAP1 has been shown to be upregulated in many types of cancers; however, its role remains elusive. We determined the TRIAP1 mRNA levels in a panel of human tissues and found its expression to be ubiquitous. Normal breast, as well as non-tumorigenic breast cells, exhibited lower TRIAP1 mRNA levels than breast cancer cells or their drug-resistant derivatives. TRIAP1 is a small, evolutionarily conserved protein that is 76 amino acids long. We found that yeast cells, in which the TRIAP1 homologue was knocked out, had increased sensitivity to doxorubicin. Equally, RNA interference in breast cancer drug-resistant cells demonstrated that downregulation of TRIAP1 impaired cell growth in the presence of doxorubicin. As expected, caspase-9 activation was diminished after overexpression of TRIAP1 in drug-resistant cells. Importantly, stable transfections of a TRIAP1 expression plasmid in CAL51 cells led to a marked increase in the number of doxorubicin-resistant clones, that was abolished when cells expressed hairpins targeting TRIAP1. In addition, we showed that TRIAP1 expression was also triggered by estrogen deprivation in MCF-7 cells. Although both polyclonal and monoclonal antibodies generated for the present study failed to robustly detect TRIAP1, we demonstrated that TRIAP1 represents a novel marker for drug resistance in breast cancer cells and it may be used in the stratification of breast cancer patients once a suitable antibody has been developed. Equally, these studies open potential drug development strategies for blocking TRIAP1 activity and avoiding drug resistance.

Silencing of HSP70 and TRIAP1 Genes in Multiple Myeloma Cell Lines Induces Apoptosis through Apaf-1/Caspase 9 Pathway: New Potential Targets on the Way?

Introduction: Some evidences suggest that heat shock protein 70 (HSP70) is overexpressed in many types of cancer, and that high expression of this chaperone is linked with increasing tumor grade and/or poor prognosis. The overexpression of HSP70 may provide a selective advantage for tumor cell survival, due in part, to its ability in inhibiting cell death via APAF-1 (apoptosis protease activating factor 1) and Caspase 9. The TP53 Regulated Inhibitor of Apoptosis 1 (TRIAP1) gene can modulate apoptotic pathways by interaction with HSP70. Some studies have shown that HSP70 gene silencing using antisense RNA, abundantly induced cell death in breast cancer lines and it was not toxic to normal breast epithelial cells or human fibroblasts. This cell death was not dependent on P53status or inhibited by Bcl2 pathway. Other studies using HSP70 antisense RNA have also indicated apoptosis of tumor cells in lung cancer, oral cavity, colon, prostate, liver, and brain cell lines. Although there are several studies on the role of HSP70 gene in apoptosis and drug resistance, there is a lack of information about this gene in multiple myeloma (MM).Objectives: To analyze the importance of HSP70 and TRIAP1 as potential targets for MM therapy through: 1) stable silencing of HSP70 and TRIAP1 in MM cell lines; 2) evaluation of each gene silencing effect on cell cycle and apoptosis.Methods: The expression of TRIAP1 and HSP70 genes in MM cell lines (U266, SKO-007, SK-MM2 and RPMI8226) was examined by quantitative real time PCR (qPCR). Cell lines were submitted to transduction with pLKO lentiviral vector containing short hairpin RNAs (shRNAs) for silencing the target genes (shRNAHSP70 and shRNATRIAP1). Lentiviral vectors with control sequences (scramble) were used to transduce the same cell lines. Apoptosis was assessed by flow cytometry after annexin V and propidium iodide (PI) staining. We also evaluated APAF-1 and Caspase 9 gene expression by qPCR and Caspase 9 and Caspase 3/7 protein activity.Results: The cell lines RPMI8226 (without deletion of P53 by FISH) and U266 (deletion of one allele of P53 by FISH) were chosen for the transduction experiments because they showed relevant expression of TRIAP1 and HSP70. The efficiency of transduction, as measured in both cell lines transduced with the pLKO vector containing the GFP reporter gene, was 70%, demonstrating that there would be no technical restriction to perform this experiment. RPMI8226 and U266 were submitted to three independent transductions, in triplicate, with the lentiviral vector containing the constructs pLKO shRNATRIAP1, shRNAHSP70 and shRNAscramble. We obtained the silencing of TRIAP1 and HSP70 genes in both MM cell lines when the transduced cell lines were compared with shRNAscramble. Silencing was confirmed by relative qPCR and Western blotting (for HSP70 only). Inhibition of TRIAP1 expression significantly increased the percentage of cells in late apoptosis (annexin V+/Propidium Iodide+) analysis (p<0.001 for RPMI8226 and P<0.01 for U266) one week after transduction and it was accompanied by increased expression of Caspase 9 in both MM cell lines (p<0.001 for RPMI8226 and p<0.05 for U266 cell lines) (One-Way ANOVA with Tukey´s multiple comparison test) when transduced cells were compared with the respective wild type cell lines. Furthermore, the inhibition of TRIAP1 resulted in accumulation of hypodiploid cells after 24 hours of transduction in U266 cell line. Inhibition of HSP70 showed no significant changes in the cell cycle in both MM cell lines. However, we observed an increment in late apoptosis after inhibition of this gene in the two cell lines when the inhibited cells were compared with cells transduced with shRNAscramble one week after transduction (p<0.01 for RPMI8226 and p<0.05 for U266 cell lines) and the results were confirmed by increased activity of Caspase 3/7 (p<0.01 for RPMI8226 and p<0.05 for U266). We observed significantly increase of Caspase 9 activity when RPMI8226 was transduced with shRNAHSP70 (p<0.001).Conclusion: Stable silencing of HSP70 and TRIAP1 in MM cell lines showed a strong impact on the induction of late apoptosis, through APAF-1/Caspase 9 pathway, suggesting that inhibitors of both genes could be exploited as potential targets for the treatment of MM, helping patients whatever P53 status assessed by FISH, as suggested by previous studies in other types of cancer (Support by FAPESP 2010/17668-6). DisclosuresNo relevant conflicts of interest to declare.