X-linked Inhibitor Of Apoptosis Protein mRNA Research Articles

Effect of Butylphthalide Capsules on Smac and XIAP Expression in Rats after Ischemia Reperfusion

IntroductionLittle is known about the protective effects of butylphthalide on cerebral ischemia-reperfusion injury. This study aims to investigate the impact on the second mitochondrial-derived activator of Caspases (Smac) and X-linked inhibitor of apoptosis protein (XIAP) expression in the ischemic semidark area using a rat model of carotid artery stenosis. MethodsThirty Sprague–Dawley rats were randomly divided into the sham-operated group, carotid stenosis model controls, low-dose (20 mg/kg), medium-dose (40 mg/kg), and high-dose (80 mg/kg) butylphthalide groups. The neurological function was scored by the balance beam test (BBT). The morphological changes of brain tissue were detected by Hematoxylin-eosin (HE) staining, with apoptosis detected by Terminal Deoxynucleotidyl Transferase mediated dUTP Nick-End Labeling (TUNEL) staining. Smac and XIAP protein expression were detected by immunohistochemistry (IHC). The expressions of Smac and XIAP mRNA were detected by real-time quantitative polymerase chain reaction (RT-qPCR). ResultsHE showed that neuronal loss, nuclear consolidation, and vacuolar degeneration were significantly reduced in the medium and high-dose butylphthalide groups compared with the model controls. The BBT scores and apoptotic index were significantly lower in the medium and high doses of butylphthalide compared with the model controls. RT-qPCR and IHC showed that Smac, XIAP mRNA and protein expressions in the ischemic hemispheric region were significantly reduced in low, medium, and high doses of butylphthalide compared with the model controls (P < 0.05), showing some concentration effect. ConclusionsButylphthalide can significantly reduce Smac and XIAP mRNA and protein expression, inhibit neuronal apoptosis induced by ischemia-reperfusion injury in rats with carotid stenosis, and exert neuroprotective effects.

Abstract P1-04-07: Xiap expression is associated with infiltration of cd163+ tumor-associated macrophages in the tumor micro-environment of inflammatory breast cancer

Abstract BACKGROUND: Inflammatory breast cancer (IBC) is a rare, but aggressive type of locally advanced breast cancer for which a specific immune response seems to be an important driver (Van Laere et al., CCR 2013). The tumor immune micro-environment (TIME) of IBC is characterized by an influx of tumor-associated macrophages (TAMs) and patients with spatial colocalization of TAMs with tumor cells seem to have a worse prognosis (Van Berckelaer et al., SABCS 2019). In contrast with TAMs, the presence of CD8+ T cells was associated with a better prognosis in IBC. Cytotoxic T cells mediate killing of the tumor by inducing apoptosis after activation of effector caspases-3/-7, regulated by the X-Linked Inhibitor of Apoptosis protein (XIAP). Recently, we reported that XIAP in breast cancer is associated with shorter survival and resistance to chemotherapy (Devi et al, Cancers 2021). In IBC, XIAP is translationally upregulated during cellular stress and induces, via NFkB, an immunosuppressive signaling (Evan et al, Cancer Res 2018). Therefore, we investigated the interplay between tumor cell driven XIAP expression and the TIME. METHODOLOGY: IBC patient clinicopathological variables (n=75) along with Affymetrix data (n=30/75) were collected. Immunostainings for XIAP, CD8 and CD163 (Hematoxylin-DAB) were done according to validated protocols. All slides were digitized and evaluated in Visiopharm to quantify the number of DAB+ immune cells and the relative marker area (RMA: the DAB+ area/total area of interest). Spatial interactions were examined using an effector index (EI) that counts the number of immune cells in a circle with a radius of 30 mcm around a CD8+ T cell or a tumor cell. We evaluated correlations between XIAP protein and mRNA expression, immune cell signatures (using CIBERSORT) &amp; gene-expression pathways as a validation of our IHC findings. RESULTS: High XIAP expression was detected in 45.3% of the IBC samples (n= 34/75). Although, XIAP expression was not associated with any clinicopathological parameters like grade, molecular subtype, or disease stage. High XIAP expressing tumors had more sTIL infiltration (P= 0.008), PDL1 expression (P= 0.012) and infiltration with TAMs (RMA: 1.03% (0.03% – 5.97%) vs. 2.73% (0.01% – 11.3%), P= 0.003). Furthermore, in these XIAP high samples TAMs were also located closer to the tumor cells (EI: 0.95 cells (0.06 – 3.48) vs. 2.04 cells (0.28 – 5.20), P&amp;lt; 0.001) and the CD8+ T cells (EI: 2.85 cells (0.26 – 8.85) vs. 5.13 cells (0.87 – 10.16), P&amp;lt; 0.001), while there was no correlation between XIAP and the total number of CD8+ cells. Although gene expression data analysis did not reveal any correlation between protein and mRNA expression of XIAP, there was a significant correlation between XIAP activation signature and protein expression (P&amp;lt;0.001). Furthermore, we discovered a strong association between XIAP protein and cellular stress response- &amp; inflammation-related gene sets. Finally, in the CIBERSORT data, we observed the strongest correlation between XIAP expression and M2 macrophages &amp; activated dendritic cells. CONCLUSION: This is the first study showing high XIAP expressing IBC tumors have increased infiltration of immunosuppressive TAMs, which co-localized near tumor and cytotoxic T cells. We also demonstrated an association between XIAP protein expression and inflammation related &amp; cellular stress response gene signatures. This is relevant as XIAP function depends on translational upregulation during cellular stress which leads to immunosuppressive signaling and an aggressive breast cancer phenotype. XIAP antagonists are in clinical trials and these data suggest potential benefit in targeting XIAP to enhance immunotherapeutic efficacy in IBC therapy. FUNDING: DoDs-W81XWH-17-1-0297,W81XWH-20-1-0153, ACS Mission Boost Grants(GRD) Citation Format: Christophe Van Berckelaer, Steven Van Laere, Joseph Gerardts, Luc Dirix, Michael Morse, Mark Kockx, François Bertucci, Peter Van Dam, Gayathri R Devi. Xiap expression is associated with infiltration of cd163+ tumor-associated macrophages in the tumor micro-environment of inflammatory breast cancer [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P1-04-07.

Downregulation of miR-181a-5p alleviates oxidative stress and inflammation in coronary microembolization-induced myocardial damage by directly targeting XIAP.

BACKGROUNDCoronary microembolization (CME) is a complicated problem that commonly arises in the context of coronary angioplasty. MicroRNAs play crucial roles in cardiovascular diseases. However, the role and mechanism of miR-181a-5p in CME-induced myocardial injury remains unclear.METHODSWe established CME rat models. Cardiac function was detected by echocardiography. Haematoxylin-basic fuchsin-picric acid staining was used to measure micro-infarction size. Serum samples and cell culture supernatants were evaluated via enzyme-linked immunosorbent assay. Cellular reactive oxygen species were determined by dichloro-dihydro-fluorescein diacetate assay, and the other oxidative stress related parameters were assayed by spectrophotometry. The dual-luciferase reporter (DLR) assay and RNA pulldown were conducted to validate the association between miR-181a-5p and X-linked inhibitor of apoptosis protein (XIAP). The expression of miR-181a-5p and XIAP mRNA were determined by quantitative reverse transcription polymerase chain reaction. Proteins were evaluated via immunoblotting. The viability of the cell was evaluated via cell counting kit-8 assay.RESULTSThe miR-181a-5p level was significantly increased in CME myocardial tissues. Downregulation of miR-181a-5p improved CME-induced cardiac dysfunction and alleviated myocardial oxidative stress and inflammatory injury, whereas miR-181a-5p exhibited the opposite effects. Then, the DLR assay and RNA pulldown results revealed that miR-181a-5p directly targeting on XIAP. The XIAP level was found to be remarkably decreased after CME. XIAP overexpression attenuated CME-induced myocardial oxidative stress and inflammatory injury. Finally, in vitro rescue experiments revealed that knockdown of XIAP could abolish the protective effects of miR-181a-5p knockdown on hypoxia-induced cardiomyocyte oxidative stress and inflammatory injury. CONCLUSIONSDownregulation of miR-181a-5p alleviates CME-induced myocardial damage by suppressing myocardial oxidative stress and inflammation through directly targeting XIAP.

Expression of X-Linked Inhibitor of Apoptosis Protein (XIAP) in Breast Cancer Is Associated with Shorter Survival and Resistance to Chemotherapy.

Simple SummaryThe X-linked inhibitor of apoptosis protein (XIAP) is considered the most potent inhibitor of cell death, and it is well established that XIAP promotes resistance to chemotherapy, radiation, and anti-cancer immune responses. This study evaluates the correlations between XIAP expression and clinicopathological features, including disease-free survival (DFS) and pathological complete response (pCR) to chemotherapy, in more than 2300 invasive primary breast cancer samples. We found a significant association of XIAP expression with younger patients’ age (≤50 years), pathological ductal type, lower tumor grade, node-positive status, and PAM50 luminal B subtype. Analysis of molecular subtypes revealed a stronger prognostic value in HR+/HER2− tumors. Higher XIAP expression was associated with shorter DFS and lower pCR rate to chemotherapy in both uni- and multivariate analyses. All these correlations were observed at both the RNA and protein level, indicating the potential of XIAP as a promising therapeutic target in primary invasive breast cancer.XIAP, the most potent inhibitor of cell death pathways, is linked to chemotherapy resistance and tumor aggressiveness. Currently, multiple XIAP-targeting agents are in clinical trials. However, the characterization of XIAP expression in relation to clinicopathological variables in large clinical series of breast cancer is lacking. We retrospectively analyzed non-metastatic, non-inflammatory, primary, invasive breast cancer samples for XIAP mRNA (n = 2341) and protein (n = 367) expression. XIAP expression was analyzed as a continuous value and correlated with clinicopathological variables. XIAP mRNA expression was heterogeneous across samples and significantly associated with younger patients’ age (≤50 years), pathological ductal type, lower tumor grade, node-positive status, HR+/HER2− status, and PAM50 luminal B subtype. Higher XIAP expression was associated with shorter DFS in uni- and multivariate analyses in 909 informative patients. Very similar correlations were observed at the protein level. This prognostic impact was significant in the HR+/HER2− but not in the TN subtype. Finally, XIAP mRNA expression was associated with lower pCR rate to anthracycline-based neoadjuvant chemotherapy in both uni- and multivariate analyses in 1203 informative patients. Higher XIAP expression in invasive breast cancer is independently associated with poorer prognosis and resistance to chemotherapy, suggesting the potential therapeutic benefit of targeting XIAP.

Apoptosis in HUVECs induced by microRNA-616-3p via X-linked inhibitor of apoptosis protein targeting.

Atherosclerosis causes stroke and coronary heart disease and is associated with a high mortality rate worldwide. However, the pathogenesis of atherosclerosis remains unclear. Endothelial cell apoptosis is one of the early changes observed in atherosclerosis. Previous studies have found that microRNA (miR)-616-3p may be involved in the development of atherosclerosis, but the specific mechanism is not clear. The present study aimed to investigate whether miR-616-3p is involved in endothelial cell apoptosis and its underlying mechanism. The present study demonstrated that compared with normal HUVECs, HUVECs treated with oxidized low-density lipoprotein expressed higher miR-616-3p and lower X-linked inhibitor of apoptosis protein (XIAP) levels. In the present study, HUVECs were transfected with miR-616-3p mimic and Cell Counting Kit-8 (CCK-8), flow cytometry and TUNEL staining assays demonstrated that compared with miR-616-3p mimic control, the miR-616-3p mimic promoted HUVEC apoptosis. In addition, using StarBase 3.0 for bioinformatics analysis it was predicted that miR-616-3p may bind to the 3'untranslated region (UTR) of XIAP mRNA. The present study performed the CCK-8, flow cytometry, TUNEL staining and dual-luciferase reporter assays and demonstrated that miR-616-3p binds to the 3'UTR of the XIAP mRNA and inhibits its expression and that this further promotes apoptosis in HUVECs. In addition, western blotting demonstrated that compared with miR-616-3p mimic control, the miR-616-3p mimic increases the level of cleaved caspase-3 in HUVECs. In summary, the present study demonstrated that miR-616-3p can directly inhibit the expression of XIAP mRNA by targeting its 3'UTR which promoted apoptosis in HUVECs. miR-616-3p and XIAP may be used as therapeutic targets of atherosclerosis in the future.

Discovery of N-(3,4-Dimethylphenyl)-4-(4-isobutyrylphenyl)-2,3,3a,4,5,9b-hexahydrofuro[3,2-c]quinoline-8-sulfonamide as a Potent Dual MDM2/XIAP Inhibitor.

Murine double minute 2 (MDM2) and X-linked inhibitor of apoptosis protein (XIAP) are important cell survival proteins in tumor cells. As a dual MDM2/XIAP inhibitor reported previously, compound MX69 has low potency with an IC50 value of 7.5 μM against an acute lymphoblastic leukemia cell line EU-1. Herein, we report the structural optimization based on the MX69 scaffold, leading to the discovery of a 25-fold more potent analogue 14 (IC50 = 0.3 μM against EU-1). We demonstrate that 14 maintains its mode of action by dual targeting of MDM2 and XIAP through inducing MDM2 protein degradation and inhibiting XIAP mRNA translation, respectively, which resulted in cancer cell growth inhibition and cell death. The results strongly suggest that the scaffold based on 14 is promising for further optimization to develop a new therapeutic agent for leukemia and possibly other cancers where MDM2 and XIAP are dysregulated.

In vivo Study of siRNA Silencing XIAP Gene to Reverse Taxol-resistance in Human Ovarian Cancer Cells

To study the relationship between down-regulated expression of X linked inhibitor of apoptosis protein (XIAP) gene and the reversal effect of taxol-resistance by using siRNA interference technology in the taxol-resistant ovarian cancer. Randomly assigned the nude mice into six groups (6 in each group) . Group A: normal saline; Group B: taxol; Group C: siRNA-NC+normal saline; Group D: siRNA-NC+taxol; Group E: siRNA XIAP+normal saline; Group F: siRNA XIAP+taxol. Each group was dealt with the corresponding processing depending on the agreed protocol and the transplanted tumors had a multi-point injection with reagents related siRNA, one time every 3 days, 9 times (27 d) in total. Taxol (2 mg/kg) was used in the intraperitoneal injection, 0.2 mL every time, once a week, for four weeks. After 27 d of siRNA treatment, xenograft volumes and qualities were measured and the inhibitory rate was calculated; RNA expression levels and protein levels of XIAP gene in xenografts were detected respectively by real-time fluorescent quantitative PCR and Western blot. Apoptosis of the transplanted tumor cells was examined by TUNEL method. Among the six groups, the proliferation of transplanted tumor in Group F was the slowest, and the tumor inhibition rate was the highest compared with control Group A, followed by Group E, and the tumor inhibition rate was the lowest in Group C. Group F and E expressed the lowest XIAP mRNA and protein expressions ( P<0.05, vs. the other 4 groups) .The apoptosis rate was highest in Group F, followed by Group E, and lowest in Group A and C ( P<0.05). XIAP siRNA has synergy with taxol in taxol-resistant ovarian cancer cells.

275P - Expression of x-linked inhibitor of apoptosis protein (XIAP) and its association with clinicopathological parameters in invasive breast cancers

BackgroundXIAP is the most potent inhibitor of both the extrinsic and intrinsic cell death pathways, which is linked to chemotherapy resistance and tumor aggressiveness. We analyzed the correlations between XIAP expression in invasive breast cancers and clinicopathological parameters including metastasis-free survival (MFS) and pathological complete response (pCR) to chemotherapy. MethodsBreast cancer databases comprising gene expression profiles with clinicopathological annotations from 8,636 non-redundant non-metastatic, non-inflammatory, primary, invasive patients were analyzed for XIAPexpression (binary variable, ”high” vs.“low” by using median as cut-off). XIAP immunohistochemistry was conducted in a cohort of post-chemotherapy mastectomy samples. ResultsHigh XIAP mRNA expression was associated with pathological ductal type, pT1 tumor size, ER-positive, PR-positive, HR+/HER2-, luminal A and B PAM50 subtypes. Analysis of MFS (3,454 non-stage 4) revealed shorter MFS (p=8.1E-03, log-rank test) associated with “XIAP-high” group. The hazard ratio for metastatic relapse was 1.20 (95%CI 1.05-1.38, p=8.17E-03, Wald test) in “XIAP-high” vs.“XIAP-low” group.In multivariate analysis, two variables (GGI and pT) remained significant, whereas the TN subtype and XIAPexpression tended to be (p=0.059). The prognostic value of XIAPwas significant in the multivariate analysis including two major signatures (70-gene signature, Recurrence Score), suggesting independent prognostic value. The same analysis, but in each molecular subtype separately, showed significant difference in the TN subtype (p=1.0E-03).Univariate analysis of pCR to anthracycline-based neoadjuvant chemotherapy (1,203 patients) identified 20% pCR in the “XIAP-high” group vs.26% in the “XIAP-low” group (p=0.015, logit function). Immunohistochemistry revealed high XIAP cytoplasmic staining only in invasive tumors and identified correlates with variables like grade, T, N, M and subtype status. ConclusionsXIAP-high” tumors are more prone to metastatic relapse and resistance to chemotherapy, suggesting the therapeutic benefit of targeting XIAP in the neoadjuvant setting. Legal entity responsible for the studyThe authors. FundingDepartment of Defense W81XWH-17-1-0297 (to G.R. Devi) and Duke School of Medicine Bridge Fund (to G.R.Devi). DisclosureAll authors have declared no conflicts of interest.

CYP1B1 prevents proteasome-mediated XIAP degradation by inducing PKCε activation and phosphorylation of XIAP

Cytochrome P450 1B1 (CYP1B1) is a key enzyme that catalyzes the metabolism of 17β-estradiol (E2) into catechol estrogens, such as 2-hydroxyestradiol (2-OHE2) and 4-hydroxyestradiol (4-OHE2). CYP1B1 is related to tumor formation and is over-expressed in a variety of cancer cells. In particular, CYP1B1 is highly expressed in hormone-related cancers such as breast cancer, ovarian cancer, or prostate cancer compared to other cancers. However, the detailed mechanisms involving this protein remain unclear. In this study, we demonstrate that CYP1B1 affects X-linked inhibitor of apoptosis protein (XIAP) expression. When CYP1B1 was over-expressed in cells, there was significant increase in the XIAP protein level, whereas the XIAP mRNA level was not affected by CYP1B1 expression. Treatment with 4-OHE2, mainly formed by CYP1B1 activity, also increased XIAP protein levels, whereas treatment with 2-OHE2 did not have a significant effect. Treatment with 4-OHE2 significantly prevented proteasome-mediated XIAP degradation. In addition, phosphorylation of XIAP on serine 87, which is known to stabilize XIAP, was up-regulated by 4-OHE2, indicating that 4-OHE2 affects XIAP stability through XIAP phosphorylation. We also found that phosphorylation of protein kinase C (PKC)ε, which is required for XIAP phosphorylation, increased when cells were treated with 4-OHE2. In summary, our data show that CYP1B1 may play an important role in preventing ubiquitin-proteasome-mediated XIAP degradation through the activation of PKCε signaling in cancer cells.

The reduction of XIAP is associated with inflammasome activation in RPE: implications for AMD pathogenesis

BackgroundAge-related macular degeneration (AMD) is a multifactorial chronic disease of the eye. Several candidate pathways have been hypothesized to play a role in AMD pathogenesis. Our work and those of others suggests inflammasome activity as a mechanism associated with retinal pigment epithelial (RPE) cell demise. X-linked inhibitor of apoptosis protein (XIAP), an anti-apoptosis factor, has recently been shown to regulate inflammasome activity in non-ocular cells. The purpose of this study is to characterize XIAP’s regulatory role in RPE.MethodsProtein lysates of eye tissues from rats (vinpocetine- or aurin tricarboxylic acid complex-treated, ATAC, vs naïve) and mice (wild type vs Caspase-4−/−) were utilized to analyze XIAP protein levels. Immunohistochemistry was used to detect NLRP3 levels in the RPE layer. In vitro inflammasome activation on RPE cells was achieved with L-leucyl-L-leucine methyl ester (Leu-Leu-OMe) stimulation. Levels of XIAP mRNA and 18S RNA were quantified by RT-PCR. Cell culture supernatants were tested directly for secreted IL-1β by ELISA or concentrated for the detection of secreted IL-18 by western blot. Protein lysates from RPE in cell culture were collected for the measurement of cleaved caspase-1 p20, XIAP, and GAPDH. Data are presented as Mean ± SD. p < 0.05 is considered statistically significant.ResultsThe XIAP protein level was significantly increased when the inflammasome was inhibited at the “activation” step by ATAC, but not the “priming” step, in vivo. Concomitantly, NLRP3 immunoreactivity was lower in the RPE layer of animals fed with ATAC. In mice where caspase-1 cleavage was impaired by the genetic deficiency in caspase-4, the XIAP protein level increased in eye tissues. In RPE cell culture, Leu-Leu-OMe stimulation led to caspase-1 cleavage, cytokine secretion, and XIAP reduction, which can be abolished by Z-YVAD-FMK. When XIAP siRNA was given as a pre-treatment to RPE in vitro, Leu-Leu-OMe induced IL-1β/IL-18 secretion was enhanced, whereas overexpressing XIAP reduced IL-1β secretion under inflammasome activation, both compared to controls cells.ConclusionsTogether, these data suggest XIAP-mediated inhibition of inflammasome activity in RPE may provide insights into the biological consequences of inflammasome activation in RPE and reveals the caspase-1/XIAP/IL-1β/IL-18 axis as a target for broader applications in AMD biology and treatment design.

Abstract 1062: Downregulation of miR-200c stabilizes XIAP mRNA and contributes to invasion and lung metastasis of bladder cancer

Abstract Our previous studies have demonstrated that X-linked inhibitor of apoptosis protein (XIAP) plays a regulatory role in cancer invasion via mediation of RhoGDIα protein SUMOylation at Ser138 in colorectal cancer HCT116 cells. However, the alteration/association of XIAP in invasion, progression, and lung metastasis of bladder cancer (BC) as well as the molecular mechanisms leading to this XIAP alteration during BC development have not been explored yet. Herein, we found that XIAP was overexpressed in human high grade BCs, high metastatic human BC T24T cells, and in mouse invasive BCs. Mechanistic studies indicated that XIAP overexpression in the highly metastatic T24T cells was due to increased mRNA stability of XIAP in comparison to the parental non-metastatic T24 cells. Results from XIAP mRNA stability regulation revealed that miR-200c was profoundly downregulated in T24T cells and in highly invasive BCs, and that such miR-200c downregulation was mediated by CREB inactivation. Furthermore, miR-200c downregulation reduced its binding to the 3'-UTR region of XIAP mRNA, and resulted in its mRNA stabilization, protein overexpression, and invasive capabilities in T24T cells. Moreover, our results from XIAP-specific shRNA and miR-200c ectopic expression showed that suppression of XIAP profoundly attenuated lung metastasis of T24T cells in nude mice. In conclusion, our findings demonstrate the molecular basis leading to XIAP overexpression and the crucial role of XIAP in BC invasion in vitro and lung metastasis in nude mice. Citation Format: Zhongxian Tian, Honglei Jin, Jingxia Li, Chao Huang, Chuanshu Huang. Downregulation of miR-200c stabilizes XIAP mRNA and contributes to invasion and lung metastasis of bladder cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1062.

MicroRNA-381 enhances radiosensitivity in esophageal squamous cell carcinoma by targeting X-linked inhibitor of apoptosis protein.

BackgroundIncreasing evidence indicates that radioresistance remains a major problem in the treatment of patients with esophageal squamous cell carcinoma (ESCC). This study was designed to investigate the expression of microRNA-381 (miR-381) and its function in the radioresistance of ESCC.MethodsIn this study, miR-381 expression was first detected in ESCC cell lines and tissue samples by quantitative real-time polymerase chain reaction (qRT-PCR). Then, the effects of miR-381 expression on growth, apoptosis, and radiosensitivity of ESCC cells were analyzed by MTT, colony formation, and flow cytometry, respectively. Dual-luciferase reporter assays were performed to validate the regulation of a putative target of miR-381, in corroboration with qRT-PCR and Western blotting assays.ResultsESCC cell lines or tissues were found to express significantly lower miR-381 than normal esophageal epithelial cells or adjacent normal tissues, respectively. Ectopic expression of miR-381 in ESCC cell lines blocked proliferation, reduced colony formation, enhanced apoptosis, and increased radiosensitivity by enhancing irradiation-induced apoptosis. In addition, dual-luciferase reporter assays showed that miR-381 binds to the 3′-untranslated region of X-linked inhibitor of apoptosis protein (XIAP), suggesting that XIAP should be a direct target of miR-381. Re-expression of miR-381 suppressed XIAP protein expression in ESCC cells, and the effects of miR-381 upregulation on ESCC cells were found to be similar with silencing of XIAP. In addition, XIAP mRNA expression significantly increased in ESCC tissues and was inversely correlated with miR-381 expression.ConclusionThe results of this study suggest that miR-381/XIAP pathway contributed to the growth inhibition, increase in apoptosis, and enhancement of radiosensitivity in ESCC cells Therefore, miR-381 may be a potential therapeutic target in human ESCC.

Effect of matrine combined with cisplatin on the expression of XIAP in human rhabdomyosarcoma RD cells.

The combined effects of matrine (Mat) and cisplatin on the survival and apoptosis of rhabdomyosarcoma (RMS) RD cells, as well as the possible mechanism of the synergistic effect of Mat and cisplatin were investigated in the present study. RMS RD cells were divided and treated as follows: control group, 5 mg/l cisplatin group, Mat groups (0.5, 1.0 and 1.5 g/l), and Mat (0.5, 1.0 and 1.5 g/l) combined with 5 mg/l cisplatin groups. An MTT assay and flow cytometry were applied to detect the survival and apoptotic rates, respectively, while RT-PCR was applied to detect the expression levels of X-linked inhibitor of apoptosis protein (XIAP) mRNA in the RD cells of each group. The survival rates of RD cells in each experimental group were lower than in the control group, and the apoptotic rates were higher than those in the control group (P<0.05). An increase in drug concentrations led to the cell proliferation inhibitory and apoptotic rates of the single Mat groups increasing as a function of dose (pairwise comparison among the groups, P<0.05), while the proliferation inhibitory and apoptotic rates of Mat combined with the cisplatin groups under different concentration were significantly higher than those of the single Mat and single cisplatin groups under the same concentration (P<0.01). The expression levels of XIAP mRNA in the RD cells of each experimental group were lower than those in the control group (P<0.05). Additionally, the expression levels of XIAP mRNA in the group treated with Mat and cisplatin were significantly lower than those of the single cisplatin and single Mat groups (P<0.01). In conclusion, Mat and cisplatin are capable of inhibiting the proliferation of RD cells and inducing apoptosis by suppressing the XIAP mRNA expression levels.

A functional microRNA library screen reveals miR-410 as a novel anti-apoptotic regulator of cholangiocarcinoma.

BackgroundCholangiocarcinoma is characterized by late diagnosis and a poor survival rate. MicroRNAs have been involved in the pathogenesis of different cancer types, including cholangiocarcinoma. Our aim was to identify novel microRNAs regulating cholangiocarcinoma cell growth in vitro and in vivo.MethodsA functional microRNA library screen was performed in human cholangiocarcinoma cells to identify microRNAs that regulate cholangiocarcinoma cell growth. Real-time PCR analysis evaluated miR-9 and XIAP mRNA levels in cholangiocarcinoma cells and tumors.ResultsThe screen identified 21 microRNAs that regulated >50 % cholangiocarcinoma cell growth. MiR-410 was identified as the top suppressor of growth, while its overexpression significantly inhibited the invasion and colony formation ability of cholangiocarcinoma cells. Bioinformatics analysis revealed that microRNA-410 exerts its effects through the direct regulation of the X-linked inhibitor of apoptosis protein (XIAP). Furthermore, overexpression of miR-410 significantly reduced cholangiocarcinoma tumor growth in a xenograft mouse model through induction of apoptosis. In addition, we identified an inverse relationship between miR-410 and XIAP mRNA levels in human cholangiocarcinomas.ConclusionsTaken together, our study revealed a novel microRNA signaling pathway involved in cholangiocarcinoma and suggests that manipulation of the miR-410/XIAP pathway could have a therapeutic potential for cholangiocarcinoma.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-016-2384-0) contains supplementary material, which is available to authorized users.

Impact of X-linked inhibitor of apoptosis protein on survival of nasopharyngeal carcinoma patients following radiotherapy.

This study aims to investigate CNE1 and CNE2 cell proliferation and apoptosis of nasopharyngeal cancer (NPC) and X-linked inhibitor of apoptosis protein (XIAP) expression in NPC patients after radiotherapy. Quantitative real-time quantitative polymerase chain reaction (qRT-PCR) and Western Blot detected XIAP and XIAP-associated factor1 (XAF1) messenger RNA (mRNA) and protein expression of CNE1 and CNE2 in NPC cells irradiated by γ-ray; MTT and flow cytometry assays detected CNE2 cells proliferation and apoptotic rate, respectively. With a retrospective analysis of 109 NPC patients in Xinxiang Central Hospital, immunohistochemistry (IHC) method detected XIAP expression, followed by a 5-year clinical analysis of the prognosis relevance after radiotherapy. In vitro, the inhibition and apoptotic rates of cells increased with the growth of radiation dose. qRT-PCR and Western blot detection declared that XIAP mRNA and protein expression increased, whereas XAF1 mRNA and protein expression decreased with the growth of radiation dose and exposure time. And XIAP mRNA and protein expression were negatively correlated with proliferation and apoptotic rates of the cells. In vivo, positive XIAP expression rate was negatively correlated with pathological tumor-node-metastasis (p-TNM) staging and tumor differentiation. Further, high XIAP expression, high p-TNM staging, and lower degree of differentiation were significantly correlated with the decrease of NPC patients' survival rate. Additionally, XIAP expression, p-TNM staging, and degrees of differentiation were independent risk factors for the survival of the NPC patients after radiotherapy. Increased XIAP expression and decreased XAF1 expression may be one reason for the apoptosis delays of CNE1 and CNE2 cells after irradiation, and the XIAP expression or the p-TNM staging and degree of differentiation are independent risk factors for NPC patients' survival after radiotherapy, providing a molecular rationale for radiotherapy and prognosis of NPC.

Effect of Matrine on the expression of X-linked inhibitor of apoptosis protein in human rhabdomyosarcoma RD cells

Objective To investigate the effect of Matrine on the expression of X-linked inhibitor of apoptosis protein (XIAP) in human rhabdomyosarcoma RD cells in vitro. Methods Cultured human rhabdomyosarcoma RD cells were divided into Matrine intervention groups (0.5 g/L, 1.0 g/L and 1.5 g/L) and a control group.The proliferation-inhibition rates in RD cells treated with different concentrations of matrine were detected by methylthiazolyl blue colorimetric assay.Flow cytometry analysis was performed for the apoptosis rates of RD cells.Reverse transcription-polymerase chain reaction analysis was used to measure the XIAP mRNA expression. Results There was a significant difference in the proliferation-inhibition rates [0.5 g/L Matrine group: (15.84±2.58)%, 1.0 g/L Matrine group: (23.13±4.19)%, 1.5 g/L Matrine group: (30.32±3.02)%, and the control group: (8.92±1.23)%]; apoptotic rates [0.5 g/L Matrine group: (12.33±1.15)%, 1.0 g/L Matrine group: (16.67±0.99)%, 1.5 g/L Matrine group: (25.33±1.91)%, and the control group: (9.47±0.96)%]; XIAP mRNA expression(0.5 g/L Matrine group: 0.633±0.046, 1.0 g/L Matrine group: 0.441±0.055, 1.5 g/L Matrine group: 0.326±0.065, control group: 0.794±0.029)in RD cells among 0.5 g/L, 1.0 g/L, 1.5 g/L Matrine groups and the control group (F=14.15, 83.37, 50.57, all P<0.05). The proliferation-inhibition and apoptotic rates in RD cells were gradually increased with the increasing Matrine concentration.The expression of XIAP mRNA was significantly decreased in different Matrine groups compared with the control group, exhibiting a dose-dependent manner. Conclusions Matrine can inhibit the proliferation of RD cells and induce the apoptosis in a dose-dependent manner, which may be related to the down-regulated XIAP mRNA. Key words: Human rhabdomyosarcoma RD cell; Matrine; X-linked inhibitor of apoptosis protein

MicroRNA-24 inhibits growth, induces apoptosis, and reverses radioresistance in laryngeal squamous cell carcinoma by targeting X-linked inhibitor of apoptosis protein.

BackgroundIncreasing evidence indicates that dysregulation of microRNAs is involved in tumor progression and development. The aim of this study was to investigate the expression of microRNA-24 (miR-24) and its function in laryngeal squamous cell carcinoma (LSCC).MethodsQuantitative RT-PCR (qRT-PCR) was used to detect miR-24 expression in LSCC cell lines and tissue samples. MTT, colony formation, and flow cytometry was performed to analyze the effects of miR-24 expression on growth, apoptosis, and radiosensitivity of LSCC cells. Dual-luciferase reporter assays were performed to examine regulation of putative miR-24 targets. Expression of X-linked inhibitor of apoptosis protein (XIAP) mRNA and protein, cleaved or total caspase-3, and cleaved or total PARP protein were detected by qRT-PCR and western blotting assays, respectively.ResultsmiR-24 expression levels in LSCC cell lines or tissue were significantly lower than in a normal human keratinocyte cell line or adjacent normal tissues. Functional analyses indicated that re-expression of miR-24 inhibits growth, reduces colony formation, and enhances apoptosis in LSCC cells. In addition, miR-24 upregulation increases LSCC sensitivity to irradiation by enhancing irradiation-induced apoptosis, and luciferase activity indicated that miR-24 binds to the 3′-untranslated region (3′-UTR) of XIAP mRNA. Upregulation of miR-24 inhibits XIAP protein expression in LSCC cells, and silencing of XIAP mimics the effects of miR-24 upregulation on LSCC cells. In addition, XIAP mRNA expression significantly increases in LSCC tissues and is inversely correlated with miR-24 expression.ConclusionsOur data suggest that miR-24 inhibits growth, increases apoptosis, and enhances radiosensitivity in LSCC cells by targeting XIAP. Therefore, miR-24 may be a potential molecular target for the treatment of human LSCC.

Turner syndrome patients with bicuspid aortic valves and renal malformations exhibit abnormal expression of X-linked inhibitor of apoptosis protein (XIAP).

We analyzed mRNA expression of X-linked inhibitor of apoptosis protein (XIAP) in patients with Turner syndrome (TS) and examined its association with phenotypic features. XIAP mRNA expression levels were investigated in 98 patients with TS in total RNA extracted from blood leucocytes by real time quantitative polymerase chain reaction. Levels of XIAP mRNA were significantly lower in patients with bicuspid aortic valves (BAV; n=13) than those without (log XIAP -1.17±0.3 vs. -0.94±0.2, p=0.002). Significantly higher expression of XIAP mRNA was seen in patients with a mosaic karyotype and renal malformations (log XIAP -0.79±0.3 vs. -1.0±0.3, p=0.03). No correlations were seen between XIAP and other manifestations. Abnormal expression of XIAP may be an important underlying mechanism in the development of BAV and renal malformations in TS. However, abnormal XIAP mRNA expression, as determined from peripheral mononuclear cells, does not appear to explain all the somatic and visceral stigmata of TS.

Gene transfection mediated by polyethyleneimine-polyethylene glycol nanocarrier prevents cisplatin-induced spiral ganglion cell damage.

Polyethyleneimine-polyethylene glycol (PEI-PEG), a novel nanocarrier, has been used for transfection and gene therapy in a variety of cells. In our previous study, we successfully carried out PEI-PEG-mediated gene transfer in spiral ganglion cells. It remains unclear whether PEI-PEG could be used for gene therapy with X-linked inhibitor of apoptosis protein (XIAP) in the inner ear. In the present study, we performed PEI-PEG-mediated XIAP gene transfection in the cochlea of Sprague-Dawley rats, via scala tympani fenestration, before daily cisplatin injections. Auditory brainstem reflex tests demonstrated the protective effects of XIAP gene therapy on auditory function. Immunohistochemical staining revealed XIAP protein expression in the cytoplasm of cells in the spiral ganglion, the organ of Corti and the stria vascularis. Reverse transcription-PCR detected high levels of XIAP mRNA expression in the cochlea. The present findings suggest that PEI-PEG nanocarrier-mediated XIAP gene transfection results in XIAP expression in the cochlea, prevents damage to cochlear spiral ganglion cells, and protects hearing.

MiR-618 inhibits anaplastic thyroid cancer by repressing XIAP in one ATC cell line

X-linked inhibitor of apoptosis protein (XIAP) is a major factor in cancer growth and progression. Reduction of XIAP induces apoptosis of anaplastic thyroid cancer (ATC), which accounts for more than 50% of thyroid cancer mortality. MicroRNAs (miRNAs) are short non-coding RNA molecules, which modulate gene expression via interaction with mRNA by binding to the 3′-untranslated region (3′-UTR), playing a critical role in cell proliferation, migration, and invasion. In this study, we recruited the ATC cell line 8305C and normal human thyroid cell Nthy-ori 3-1, aiming to find the miRNA which could regulate XIAP and therefore inhibit the growth and invasion of ATC. We first used quantitative real-time PCR (qPCR) to reveal that XIAP mRNA expression was 4.6±0.56 folds (P=0.029) up-regulated in 8305C cells, compared with Nthy-ori 3-1 cells. Then miR-618, predicted to target XIAP directly, was detected 0.24±0.06 folds (P=0.019) down-regulated in 8305C cells. Next we used Luciferase assay showing that XIAP was a target gene of miR-618, which could repress the XIAP expression at both mRNA and protein levels. After that, CCK-8 assay was performed to show that over-expression of miR-618 could inhibit the growth of 8305C cells. Finally, we employed transwell method to prove that miR-618 could prevent the invasion and migration of 8305C cells. In conclusion, our collective data showed that over-expression of miR-618 could inhibit ATC cells by targeting XIAP gene.