CBX7 suppression prevents ischemia-reperfusion injury-induced endoplasmic reticulum stress through the Nrf-2/HO-1 pathway.

Renal ischemia-reperfusion injury (I/R) usually occurs in renal transplantation and partial nephrectomy, which could lead to acute kidney injury. However, the effective treatment for renal I/R still remains limited. In the present study, we investigated whether inhibition of chromobox 7 (CBX7) could attenuate renal I/R injury in vivo and in vitro as well as the potential mechanisms. Adult male mice were subjected to right renal ischemia and reperfusion for different periods, both with and without the CBX7 inhibitor UNC3866. In addition, human kidney cells (HK-2) were subjected to a hypoxia/reoxygenation (H/R) process for different periods, both with or without the CBX7 inhibitor or siRNA for CBX7. The results showed that expression of CBX7, glucose regulator protein-78 (GRP78), phosphorylated eukaryotic translation initiation factor-2α (p-eIF2α), and C/EBP homologous protein (CHOP) were increased after extension of I/R and H/R periods. Moreover, overexpression of CBX7 could elevate the expression of CBX7, GRP78, p-eIF2α, and CHOP. However, CBX7 inhibition with either UNC3866 or genetic knockdown led to reduced expression of GRP78, p-eIF2α, and CHOP through nuclear factor-erythroid 2-related factor 2 (Nrf2)/heme oxygenase-1 activation in I/R and H/R injury. Furthermore, ML385, the Nrf2 inhibitor, could elevate endoplasmic reticulum stress levels, abrogating the protective effects of UNC3866 against renal I/R injury. In conclusion, our results demonstrated that CBX7 inhibition alleviated acute kidney injury by preventing endoplasmic reticulum stress via the Nrf2/HO-1 pathway, indicating that CBX7 inhibitor could be a potential therapeutic target for renal I/R injury.

Diabetes aggravates renal ischemia-reperfusion injury by repressing mitochondrial function and PINK1/Parkin-mediated mitophagy.

Diabetes could aggravate ischemia-reperfusion (I/R) injury, but the underlying mechanism is unclear. In the present study, we aimed to investigate whether diabetes exacerbates renal I/R injury and its possible mechanism. In vitro, HK-2 cells under normal or high glucose conditions were subjected to hypoxia (12 h) followed by reoxygenation (3 h) (H/R). Cell viability, intracellular ATP content, mitochondrial membrane potential, reactive oxygen species production, and apoptosis were measured. In vivo, streptozotocin-induced diabetic and nondiabetic rats were subjected to I/R. Renal pathology, function, and apoptosis were evaluated by hematoxylin and eosin staining, transmission electron microscopy, and Western blot analysis. Compared with the normal glucose + H/R group, mitochondrial function (ATP, mitochondrial membrane potential, and reactive oxygen species) and mitophagy were reduced in the high glucose + H/R group, as was expression of phosphatase and tensin homolog-induced putative kinase 1 (PINK1) and Parkin. Also, cells in the high glucose + H/R group exhibited more apoptosis compared with the normal glucose + H/R group, as assessed by flow cytometry, TUNEL staining, and Western blot analysis. Compared with normal rats that underwent I/R, diabetic rats that underwent I/R exhibited more severe tubular damage and renal dysfunction as well as expression of the apoptotic protein caspase-3. Meanwhile, diabetes alleviated mitophagy-associated protein expression in rats subjected to I/R, including expression of PINK1 and Parkin. Transmission electron microscopy indicated that the mitophagosome could be hardly observed and that mitochondrial morphology and structure were obviously damaged in the diabetes + I/R group. In conclusion, our results, for the first time, indicate that diabetes could aggravate I/R injury by repressing mitochondrial function and PINK1/Parkin-mediated mitophagy in vivo and in vitro.

Diabetes aggravates renal ischemia and reperfusion injury in rats by exacerbating oxidative stress, inflammation, and apoptosis.

Diabetic patients are more susceptible to renal ischemia/reperfusion (I/R) injury (RI/RI) and have a poor prognosis, but the underlying mechanism remains unclear. The present study aimed to examine whether diabetes could worsen acute kidney injury induced by I/R in rats and clarify its mechanism. Control and streptozotocin-induced diabetic rats were subjected to 45 min renal pedicle occlusion followed by 24 h reperfusion. Tert-butylhydroquinone (TBHQ, 16.7 mg/kg) was administrated intraperitoneally 3 times at intervals of 8 h before ischemia. Serum and kidneys were harvested after reperfusion to evaluate renal function and histological injury. Enzyme-linked immunosorbent assays were used to test pro-inflammatory cytokines. Terminal deoxynucleotidyl-transferase-mediated dUTP nick-end labeling assays were used to detect apoptotic cells, and western blotting was performed to determine the expression of B-cell lymphoma-2 (Bcl-2), Bcl-2-associated X protein (Bax), and cleaved caspase-3, as well as oxidative stress and inflammation-related proteins, such as nuclear factor-erythroid 2-related factor 2 (Nrf2), heme oxygenase-1 (HO-1), Toll-like receptor 4 (TLR4), and nuclear factor-κB (NF-κB). Compared with control animals, diabetic rats undergoing I/R exhibited more severe tubular damage and renal dysfunction. Diabetes exacerbated oxidative stress, the inflammatory response, and apoptosis after renal I/R by enhancing TLR4/NF-κB signaling and blocking the Nrf2/HO-1 pathway. RI/RI in diabetic rats was attenuated by pretreatment with TBHQ (a Nrf2 agonist), which exerted anti-inflammatory and anti-apoptotic properties by inhibiting NF-κB signaling. These findings indicate that hyperglycemia exacerbates RI/RI by intensifying oxidative stress, inflammation, and apoptosis. Antioxidant pretreatment may alleviate RI/RI in diabetic patients.

MiR-543 Promotes Proliferation and Epithelial-Mesenchymal Transition in Prostate Cancer via Targeting RKIP.

BACKGROUND/AIMS: MicroRNAs (miRNAs, miRs) have emerged as important post-transcriptional regulators in various cancers. miR-543 has been reported to play critical roles in hepatocellular carcinoma and colorectal cancer, however, the role of miR-543 in the pathogenesis of prostate cancer has not been fully understood. METHODS: Expression of miR-543 and Raf Kinase Inhibitory Protein (RKIP) in clinical prostate cancer specimens, two prostate cancer cell lines, namely LNCAP and C4-2B, were determined. The effects of miR-543 on proliferation and metastasis of tumor cells were also investigated with both in vitro and in vivo studies. RESULTS: miR-543 was found to be negatively correlated with RKIP expression in clinical tumor samples and was significantly upregulated in metastatic prostate cancer cell line C4-2B compared with parental LNCAP cells. Further studies identified RKIP as a direct target of miR-543. Overexpression of miR-543 downregulated RKIP expression and promoted the proliferation and metastasis of cancer cells, whereas knockdown of miR-543 increased expression of RKIP and suppressed the proliferation and metastasis of cancer cells in vitro and in vivo. CONCLUSION: Our study demonstrates that miR-543 promotes the proliferation and metastasis of prostate cancer via targeting RKIP.

Picroside II decreases the development of fibrosis induced by ischemia/reperfusion injury in rats.

In kidney transplantation, renal ischemia and reperfusion injury was one of the leading factors to the development of renal fibrosis, which was the main cause of graft loss. The fibrogenic changes were associated with the long term inflammation elicited by ischemia and reperfusion injury. In the present study, we investigated the role of the Picroside II, the main active constituents of the extract of picrorrhiza scrophulariiflora roots, in attenuating renal fibrosis in a renal ischemia and reperfusion injury model. We induced ischemia and reperfusion injury in kidneys treated with or without Picroside II. We observed that inflammation and tissue fibrosis were increased in ischemia and reperfusion injury group compared to Picroside II group, however, these changes were significantly decreased by the treatment with Picroside II. We concluded that Picroside II can protect the ischemic kidney against renal fibrosis and its mechanism may be through the inhibition of the long term inflammation.

The protective effect of ozone oxidative preconditioning against hypoxia/reoxygenation injury in rat kidney cells.

Abstract Ozone (O3) has been viewed as a novel treatment for different diseases in these years and oxidative stress and apoptosis play a key role in the pathogenesis of kidney diseases including renal ischemia and reperfusion (I/R). In the present study, we investigated the role of ozone oxidative preconditioning (OzoneOP) in attenuating oxidative stress and apoptosis in a hypoxia/reoxygenation (H/R) injury model using rat kidney cells. We induced H/R injury in kidney cells treated with or without OzoneOP. Oxidative stress parameters such as superoxide dismutase (SOD), malondialdehyde (MDA) and lactate dehydrogenase (LDH) were determined, as well as some apoptotic proteins. We observed that oxidative stress and apoptosis were increased in H/R group compared to OzoneOP group; however, these changes were significantly decreased by the treatment with OzoneOP. We concluded that OzoneOP can protect the kidney cells against H/R injury and its mechanism may be through the reduction of oxidative stress and apoptosis.

[RelB-siRNA enhanced the radiosensitivity of murine prostate cancer cell line RM-1].

OBJECTIVE: To explore the transfecting effects of RelB small interfering RNA (RelB-siRNA) on murine prostate cancer cell line RM-1 sensitivity of radiotherapy. METHODS: The RM-1 cells were divided into RelB-siRNA vector, empty vector, non-transfection and normal groups. After transfection, the first three groups were irradiated by X ray. Clone formation array method was used to measure the surviving fraction and the relevant parameter values after 0, 2, 4, 6, 8 Gy irradiation. Apoptotic rate was measured via Annexin V/PI flow cytometry after 6 Gy irradiation. The level of RelBmRNA was estimated by real-time polymerase chain reaction (PCR) after 6 Gy irradiation. And the RelB protein of cytoplasm and nucleus was detected by Western blot after 6 Gy irradiation. RESULTS: Clone formation array showed that RelB group at each dose point corresponding to the survival fraction were lower that the empty vector nd non-transfection groups. The D0, Dq and SF2 values of RelB group were 1.68, 0.60, 0.43, lower than those of empty vector group 1.92, 3.08, 0.89 and non-transfection group 1.93, 2.76, 0.84. Annexin V/PI flow cytometry demonstrated that the apoptotic rate of RelB group was 15.27% ± 1.62% and it was significantly higher than the non-transfection group 7.90% ± 1.50% and the empty vector group 8.40% ± 0.69% respectively (P < 0.01) .Real-time PCR indicated that RelB-mRNA amplification of RelB group was 1.18 ± 0.03 and it was significantly lower than the non-transfection group 2.10 ± 0.61 and the empty vector group 1.97 ± 0.66 respectively (P < 0.01) .Western blot suggested that cytoplasmic gray-scale ratio of RelB group was 0.50 ± 0.08 and it was significantly lower than the non-transfection 1.77 ± 0.19 and the empty group 1.52 ± 0.12 respectively (P < 0.01) . And the nuclear gray-scale radio of the RelB group was 0.18 ± 0.03 and it was significantly lower than the non-transfection group 0.61 ± 0.12 and the empty group 0.54 ± 0.13 respectively (P < 0.01) . The RelB protein inhibition rates of cytoplasmic and nuclear RelB-siRNA were 71.8% and 70.4% respectively. CONCLUSION: RelB-siRNA can effectively inhibit the RelB expression of murine prostate cancer cell line RM-1 and significantly increase its sensitivity to radiotherapy.

Nephroprotective Effects of Cardamonin on Renal Ischemia Reperfusion Injury/UUO-Induced Renal Fibrosis.

Acute kidney injury and chronic renal fibrosis are intractable pathological processes to resolve, yet limited strategies are able to effectively address them. Cardamonin (CAD) is a flavonoid with talented antioxidant, anti-inflammatory capacity, and satisfactory biosafety. In our study, animal and cellular models of renal ischemia/reperfusion (I/R) and unilateral ureteral obstruction (UUO) were successfully constructed to confirm whether CAD confers protective effects and underlying mechanisms. Animal experiments demonstrated that CAD application (100 mg/kg) distinctly ameliorated tissue damage and improved renal function. Meanwhile, the continuous oral administration of CAD after UUO surgery efficiently inhibited renal fibrosis as confirmed by hematoxylin-eosin (H&E), Sirius red, and Masson staining as well as the downregulated mRNA and protein expression of collagen I, α-smooth muscle actin (α-SMA), collagen III, and fibronectin. Interestingly, in transforming growth factor ß1 (TGF-ß1)-stimulated and hypoxia/reoxygenation (H/R)-exposed human kidney-2 (HK-2) cells, protective effects of CAD were again authenticated. Meanwhile, we performed bioinformatics analysis and constructed the "ingredient-target-pathway-disease" network to conclude that the potential mechanisms of CAD protection may be through the regulation of oxidative stress, inflammation, apoptosis, and mitogen-activated protein kinase (MAPK) pathway. Furthermore, experimental data validated that CAD evidently decreased the reactive oxygen species (ROS) production and malondialdehyde (MDA) content while depressing the mRNA and protein expression of inflammatory markers (tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and Il-1ß) and inhibiting apoptosis as evidenced by decreased levels of P53, BAX, cleaved caspase-3, and apoptotic rate in renal I/R and UUO models. In addition, the impact of CAD on restraining oxidative stress and inflammation was attributed to its ability to elevate antioxidant enzyme activities including catalase, superoxide dismutase 1 (SOD1), and superoxide dismutase 2 (SOD2) and to inhibit the inflammation-associated MARK/nuclear factor-κB (MAPK/NF-κB) signaling pathway. In conclusion, cardamonin restored the antioxidative capacity to block oxidative stress and suppressed the MAPK/NF-κB signaling pathway to alleviate inflammatory response, thus mitigating I/R-generated acute kidney injury/UUO-induced renal fibrosis in vivo and in vitro, which indicated the potential therapeutic advantage of cardamonin in attenuating acute and chronic kidney injuries.

[Retracted] Effects of cisplatin on the LSD1-mediated invasion and metastasis of prostate cancer cells.

Following the publication of this paper, it was drawn to the Editor's attention by a concerned reader that the cell invasion assay data shown for the 'LSD1siRNA+DDP' experiment in Fig. 3A on p. 2515 were strikingly similar to data appearing in different form in Fig. 3 in another article written by different authors at different research institutes [Liu Y, Li M, Zhang G and Pang Z: MicroRNA-10b overexpression promotes non-small cell lung cancer cell proliferation and invasion. Eur J Med Res 18: 41, 2013]. Owing to the fact that the contentious data in the above article had already been published prior to its submission to Molecular Medicine Reports, the Editor has decided that this paper should be retracted from the Journal. After having been in contact with the authors, they accepted the decision to retract the paper. The Editor apologizes to the readership for any inconvenience caused. [Molecular Medicine Reports 14: 2511-2517, 2016; DOI: 10.3892/mmr.2016.5571].

Over-expressing transporters associated with antigen processing increases antitumor immunity response in prostate cancer.

As we know, prostate cancer down-regulates expression of HLA-1 Antigen Processing Machinery (APM) and has defects in the antigen presentation pathway. In vitro, the prostate cancer cell (PC-3 cells) infected with Lentivirus TAP1 can efficiently over-express TAP1 and Tapasin, and HLA-1 was also up-regulated on the surface of the infected cells. The lentivirus TAP1 infection increased the apoptosis rate of PC-3 cells. In addition, with the co-cluture PC-3 cells and lymphocytes, TAP1 augmented the expression of CD3⁺CD8⁺CD38⁺ T cell. Importantly, administration of Lentivirus TAP1 to prostate cancer cells in a xenograft mouse model can prolong survival and increase the CD4⁺ T cells, and CD8⁺ T cells as well as decrease Foxp3⁺ T cells in the tumor microenvironment. In summary, a recombinant lentivirus expressing TAP1 can effectively increase prostate cancer tumor-specific immune response.

Tolerogenic dendritic cells generated by RelB silencing using shRNA prevent acute rejection.

It is well known that adoptive transfer of donor-derived tolerogenic dendritic cells (DCs) helps to induce immune tolerance. RelB, one of NF-κB subunits, is a critical element involved in DC maturation. In the present study, our results showed tolerogenic DCs could be acquired via silencing RelB using small interfering RNA. Compared with imDCs, the tolerogenic DCs had more potent ability to inhibit mixed lymphocyte reaction (MLR) and down-regulate Th1 cytokines and prompt the production of Th2 cytokines. They both mediated immune tolerance via the increased of T cell apoptosis and generation of regulatory T cells. Administration of donor-derived tolerogenic DCs significantly prevented the allograft rejection and prolonged the survival time in a murine heart transplantation model. Our results demonstrate donor-derived, RelB-shRNA induced tolerogenic DCs can significantly induce immune tolerance in vitro and in vivo.

STEAP3 Affects Ferroptosis and Progression of Renal Cell Carcinoma Through the p53/xCT Pathway.

Renal cell carcinoma is particularly sensitive to ferroptosis, an iron-dependent non-apoptotic form of cell death. This mechanism does not require activation of caspase or the participation of other apoptotic effector molecules (such as BAX or BAK), nor is it accompanied by the morphological characteristics or biochemical processes of apoptosis. The STEAP3 gene was found because it promotes tumor apoptosis in prostate cancer, but its role in renal cell carcinoma has not been studied in depth. Through real-time quantitative polymerase chain reaction, we found that the expression of the STEAP3 gene was upregulated in renal cell carcinoma tissue samples and cell lines, and it was found to be highly expressed in renal cell carcinoma tissue through immunohistochemistry. This upregulation is related to poor survival and prognosis of patients. We used erastin, a ferroptosis inducer, found that renal cell carcinoma became more susceptible to ferroptosis after knocking down STEAP3. The results indicate that renal cell carcinoma cell lines with knocked down STEAP3 expression are more sensitive to ferroptosis, and this effect occurs through the p53/xCT pathway. In summary, our research helps to identify new biomarkers and provides new targets for the treatment of renal cell carcinoma.

Clinical practice guideline for transurethral plasmakinetic resection of prostate for benign prostatic hyperplasia (2021 Edition).

Benign prostatic hyperplasia (BPH) is highly prevalent among older men, impacting on their quality of life, sexual function, and genitourinary health, and has become an important global burden of disease. Transurethral plasmakinetic resection of prostate (TUPKP) is one of the foremost surgical procedures for the treatment of BPH. It has become well established in clinical practice with good efficacy and safety. In 2018, we issued the guideline "2018 Standard Edition". However much new direct evidence has now emerged and this may change some of previous recommendations. The time is ripe to develop new evidence-based guidelines, so we formed a working group of clinical experts and methodologists. The steering group members posed 31 questions relevant to the management of TUPKP for BPH covering the following areas: questions relevant to the perioperative period (preoperative, intraoperative, and postoperative) of TUPKP in the treatment of BPH, postoperative complications and the level of surgeons' surgical skill. We searched the literature for direct evidence on the management of TUPKP for BPH, and assessed its certainty generated recommendations using the grade criteria by the European Association of Urology. Recommendations were either strong or weak, or in the form of an ungraded consensus-based statement. Finally, we issued 36 statements. Among them, 23 carried strong recommendations, and 13 carried weak recommendations for the stated procedure. They covered questions relevant to the aforementioned three areas. The preoperative period for TUPKP in the treatment of BPH included indications and contraindications for TUPKP, precautions for preoperative preparation in patients with renal impairment and urinary tract infection due to urinary retention, and preoperative prophylactic use of antibiotics. Questions relevant to the intraoperative period incorporated surgical operation techniques and prevention and management of bladder explosion. The application to different populations incorporating the efficacy and safety of TUPKP in the treatment of normal volume (< 80 ml) and large-volume (≥ 80 ml) BPH compared with transurethral urethral resection prostate, transurethral plasmakinetic enucleation of prostate and open prostatectomy; the efficacy and safety of TUPKP in high-risk populations and among people taking anticoagulant (antithrombotic) drugs. Questions relevant to the postoperative period incorporated the time and speed of flushing, the time indwelling catheters are needed, principles of postoperative therapeutic use of antibiotics, follow-up time and follow-up content. Questions related to complications incorporated types of complications and their incidence, postoperative leukocyturia, the treatment measures for the perforation and extravasation of the capsule, transurethral resection syndrome, postoperative bleeding, urinary catheter blockage, bladder spasm, overactive bladder, urinary incontinence, urethral stricture, rectal injury during surgery, postoperative erectile dysfunction and retrograde ejaculation. Final questions were related to surgeons' skills when performing TUPKP for the treatment of BPH. We hope these recommendations can help support healthcare workers caring for patients having TUPKP for the treatment of BPH.

Knockdown of TRIM8 Protects HK-2 Cells Against Hypoxia/Reoxygenation-Induced Injury by Inhibiting Oxidative Stress-Mediated Apoptosis and Pyroptosis via PI3K/Akt Signal Pathway.

BACKGROUND: Acute kidney injury (AKI) emerges as an acute and critical disease. Tripartite motif 8 (TRIM8), one number of the TRIM protein family, is proved to participate in ischemia/reperfusion (I/R) injury. However, whether TRIM8 is involved in renal I/R injury and the associated mechanisms are currently unclear. PURPOSE: This study aimed to investigate the precise role of TRIM8 and relevant mechanisms in renal I/R injury. MATERIALS AND METHODS: In this study, human renal proximal tubular epithelial cells (HK-2 cells) underwent 12 hours of hypoxia and 2 h, 3 h or 4 h of reoxygenation to establish an in vitro hypoxia/reoxygenation (H/R) model. The siRNAs specific to TRIM8 (si-TRIM8) were transfected into HK-2 cells to knockdown TRIM8. The cell H/R model included various groups including Control, H/R, H/R+DMSO, H/R+NAC, si-NC+H/R, si-TRIM8+H/R and si-TRIM8+LY294002+H/R. The cell viability and levels of reactive oxygen species (ROS), hydrogen peroxide (H2O2), mRNA, apoptotic proteins, pyroptosis-related proteins and PI3K/AKT pathway-associated proteins were assessed. RESULTS: In vitro, realtime-quantitative PCR and western-blot analysis showed that the mRNA and protein expression of TRIM8 were obviously upregulated after H/R treatment in HK-2 cells. Compared with the H/R model group, knockdown of TRIM8 significantly increased cell viability and reduced the levels of ROS, H2O2, apoptotic proteins (Cleaved caspasebase-3 and BAX) and pyroptosis-related proteins (NLRP3, ASC, Caspase-1, Caspase-11, IL-1ß and GSDMD-N). Western-blot analysis also authenticated that PI3K/AKT pathway was activated after TRIM8 inhibition. The application of 5 mM N-acetyl-cysteine, one highly efficient ROS inhibitor, significantly suppressed the expression of apoptotic proteins and pyroptosis-related proteins. Moreover, the combined treatment of TRIM8 knockdown and LY294002 reversed the effects of inhibiting oxidative stress. CONCLUSION: Knockdown of TRIM8 can alleviate H/R-induced oxidative stress by triggering the PI3K/AKT pathway, thus attenuating pyropyosis and apoptosis in vitro.

Histone Methyltransferase G9a Promotes the Development of Renal Cancer through Epigenetic Silencing of Tumor Suppressor Gene SPINK5.

BACKGROUND: Renal cell carcinoma (RCC) accounts for approximately 2-3% of malignant tumors in adults, while clear cell renal cell carcinoma accounts for 70-85% of kidney cancer cases, with an increasing incidence worldwide. G9a is the second histone methyltransferase found in mammals, catalyzing lysine and histone methylation. It regulates gene transcription by catalyzing histone methylation and interacting with transcription factors to alter the tightness of histone-DNA binding. The main purpose of this study is to explore the role and mechanism of G9a in renal cell carcinoma. METHODS: Firstly, we investigated the expression of G9a in 80 clinical tissues and four cell lines. Then, we explored the effect of G9a-specific inhibitor UNC0638 on proliferation, apoptosis, migration, and invasion of two renal cancer cell lines (786-O, SN12C). In order to study the specific mechanism, G9a knocking down renal cancer cell line was constructed by lentivirus. Finally, we identified the downstream target genes of G9a using ChIP experiments and rescue experiments. RESULTS: The results showed that the specific G9a inhibitor UNC0638 significantly inhibited the proliferation, migration, and invasion of kidney cancer in vivo and in vitro; similar results were obtained after knocking down G9a. Meanwhile, we demonstrated that SPINK5 was one of the downstream target genes of G9a through ChIP assay and proved that G9a downregulate the expression of SPINK5 by methylation of H3K9me2. Therefore, targeting G9a might be a new approach to the treatment of kidney cancer. CONCLUSION: G9a was upregulated in renal cancer and could promote the development of renal cancer in vitro and in vivo. Furthermore, we identified SPINK5 as one of the downstream target genes of G9a. Therefore, targeting G9a might be a new treatment for kidney cancer.

[Prostate cancer cell vaccine transfected with 4-1BBL induces anti-tumor immunity in vitro].

OBJECTIVE: To explore the anti-tumor immunity in vitro induced by prostate cancer cell vaccine transfected with recombinant adenovirus encoding 4-1BBL in mice. METHODS: The replication-deficient adenovirus AdEasy-1 system was used to construct recombinant adenovirus Ad-m4-1BBL and Ad-eGFP. The prostate cancer cell RM-1 of mice was transfected with Ad-m4-1BBL and Ad-eGFP, and treated with mitomycin (MMC) to produce TCV, TCV-Ad-eGFP and TCV-Ad-m4-1BBL, followed by co-culture with syngeneic murine spleen cells. Then the cytotoxic activity of the lymphocytes against RM-1 cells was analyzed with CCK-8 solution, and IL-2 and INF-gamma were detected by ELISA. RESULTS: The 4-1BBL protein was highly expressed in the TCV-Ad-m4-1BBL of the 4-1BBL-transfected mice. TCV-Ad-m4-1BBL significantly increased the expressions of IL-2 ([180.24 +/- 2.22] pg/ml) and INF-gamma ([1512.46 +/- 23.64] pg/ml) as compared with TCV and TCV-Ad-eGFP (P < 0.05), and induced higher RM-1 cell specific cytotoxicity ([34.24 +/- 2.64]%) than the latter two ([9.82 +/- 1.48]%) and ([14.65 +/- 3. 21]%), (P < 0.05). But none of them exhibited significant cytotoxicity against hepatocellular carcinoma Hepal-6. CONCLUSION: The m4-1BBL-expressing prostate cancer cell vaccine can effectively induce anti-tumor immune responses.

LSD1 Promotes Prostate Cancer Cell Survival by Destabilizing FBXW7 at Post-Translational Level.

Prostate cancer (PCa) is the most common cancer in men and the fifth leading cause of cancer death worldwide. Unfortunately, castration-resistant prostate cancer (CRPCa) is incurable with surgical treat and prone to drug resistance. Therefore, it is of great importance to find a new target for treatment. LSD1 is up-regulated in PCa and related with prognosis. The high-expression LSD1 has been shown to be a potential target for treatment and is widely studied for its demethylase-activity. However, its demethylation-independent function remains to be elusive in PCa. Recent study shows that LSD1 can destabilize cancer suppressor protein FBXW7 without demethylation-function. Hence, we hope to investigate the impact of non-canonical function of LSD1 on PCa cell survival. We over-expressed FBXW7 gene through plasmid vector in LNCaP and PC3 cell lines and the result shows that up-regulated FBXW7 can suppress the viability of PC cell through suppressing oncoproteins, such as c-MYC, NOTCH-1. After FBXW7 function experiment on PC cell, we knock-down LSD1 gene in the same kinds of cell lines. In western blot assay, we detected that down-regulation of LSD1 will cause the increasing of FBXW7 protein level and decreasing of its targeting oncoproteins. And mRNA level of FBXW7 did not change significantly after LSD1 knock-down, which means LSD1 may destabilize FBXW7 by protein-protein interactions. Moreover, exogenous wild type LSD1 and catalytically deficient mutant K661A both can abrogate previous effect of LSD1 knock-down. Consequently, LSD1 may promote PC cell survival by destabilizing FBXW7 without its demethylase-activity. Next, we compared two kinds inhibitors, and found that SP-2509 (Allosteric inhibitor) treatment suppress the cancer cell survival by blocking the LSD1-FBXW7 interaction, which is an effect that GSK-2879552 (catalytic inhibitor) cannot achieve. This work revealed a pivotal function of LSD1 in PCa, and indicated a new direction of LSD1 inhibitor research for PCa treatment.

Inhibition of BRD4 prevents proliferation and epithelial-mesenchymal transition in renal cell carcinoma via NLRP3 inflammasome-induced pyroptosis.

BRD4 has long been implicated in many different pathological processes, in particular, the development of cancer and inflammation. Pyroptosis is a newly recognized type of inflammatory programmed cell death. However, the correlation between BRD4 and pyroptosis in renal cell carcinoma (RCC) remains elusive. The present study demonstrates that BRD4 expression levels are markedly upregulated, while pyroptosis-associated proteins are significantly reduced, in RCC tissues and cells. Inhibition of BRD4, via either genetic knockdown or use of bromodomain inhibitor JQ1, prevented cell proliferation and epithelial-mesenchymal transition (EMT) progression and induced caspase-1-dependent pyroptosis in RCC both in vitro and in vivo. In addition, BRD4 inhibition suppressed proliferation and EMT though pyroptosis in vitro and in vivo. Moreover, NLRP3, which mediates caspase-1-dependent pyroptosis, was increased upon BRD4 inhibition. Furthermore, the transcriptional activity of NLRP3 was enhanced by BRD4 inhibition, and this enhancement was blocked by activation of NF-κB phosphorylation, indicating that NF-κB is an upstream regulator of NLRP3. Collectively, these results show that BRD4 inhibition prevents cell proliferation and EMT, and exerts an antitumor effect in RCC by activating the NF-κB-NLRP3-caspase-1 pyroptosis signaling pathway. Thus, BRD4 is a potential target for RCC treatment, and JQ1 shows promise as a therapeutic agent for this disease.

Identification of hub genes associated with outcome of clear cell renal cell carcinoma.

Clear cell renal cell carcinoma (ccRCC) is one of the most common tumor types of the urinary system. Bioinformatics tools have been used to identify new biomarkers of ccRCC and to explore the mechanisms underlying development and progression of ccRCC. The present study analyzed the differentially expressed genes (DEGs) associated with RCC using data obtained from Gene Expression Omnibus datasets and GEO2R software. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis of these DEGs was performed and analyzed using the Database for Annotation, Visualization and Integrated Discovery. A protein-protein interaction (PPI) network was constructed using the Search Tool for the Retrieval of Interacting Genes to identify the hub genes, defined as the genes with the highest degree of interrelation. Subsequently, differential expression and survival analyses of hub genes was performed using The Cancer Genome Atlas database and Gene Expression Profiling Interactive Analysis (GEPIA) online tool. Using GEO2R, 1,650 DEGs were identified, including 743 upregulated and 907 downregulated genes. GO and KEGG pathway analyses indicated that the upregulated DEGs were primarily involved in blood vessel and vasculature development, whereas downregulated DEGs were primarily involved in organic acid metabolic processes and carboxylic acid metabolic processes. Subsequently, important modules were identified in the PPI network using Cytoscape's Molecular Complex Detection. The 15 most connected hub genes were identified among DEGs, including glycine decarboxylase (GLDC), enolase 2 (ENO2) and topoisomerase II alpha. GEPIA revealed the association between expression levels of hub genes and survival. Specifically, GLDC and ENO2 were associated with the prognosis of patients with RCC and thus, the effects of GLDC and ENO2 involvement in renal cancer were investigated in vitro. GLDC and ENO2 affected the proliferation and apoptosis of renal cancer cells. These hub genes may reveal a new mechanism underlying development or progression of RCC and identify new markers for its diagnosis and prognosis.

MiR-142-3p functions as an oncogene in prostate cancer by targeting FOXO1.

Prostate cancer (PCa) is a heterogeneous malignancy, and is a primary cause of cancer-related death in males. Forkhead box transcription factor O1 (FOXO1) exerts antitumor effects in various cancers, including PCa. However, the regulatory mechanism of miR-142-3p on FOXO1 expression in human PCa has not been characterized. In this study, we showed that FOXO1 protein levels were downregulated in PCa tissues and cells. Moreover, FOXO1 expression was a predictor of disease-free survival in patients with PCa and was a predictor of prognosis. Increased expression of FOXO1 suppressed cellular proliferation and induced cell cycle arrest at G0/G1 in vitro. However, FOXO1 mRNA and protein levels were inconsistent in human PCa tissues and cell lines. We showed that miR-142-3p levels were negatively correlated with FOXO1 protein levels in PCa. We also showed that miR-142-3p suppressed FOXO1 expression by directly targeting its 3'-untranslated region. Furthermore, suppression of miR-142-3p inhibited cell proliferation and induced cell cycle arrest, and these effects were blocked by FOXO1 knockdown. In vivo experiments showed that miR-142-3p knockout impaired tumor growth. Our results validate that FOXO1 acted as a tumor suppressor in PCa and demonstrated that FOXO1 was regulated by miR-142-3p, and miR-142-3p may be a potential target for treatment of PCa.