Glutaminolysis is a Potential Therapeutic Target for Kidney Diseases.

Metabolic reprogramming contributes to the progression and prognosis of various kidney diseases. Glutamine is the most abundant free amino acid in the body and participates in more metabolic processes than other amino acids. Altered glutamine metabolism is a prominent feature in different kidney diseases. Glutaminolysis converts glutamine into the TCA cycle metabolite, alpha-ketoglutarate, via a cascade of enzymatic reactions. This metabolic pathway plays pivotal roles in inflammation, maladaptive repair, cell survival and proliferation, redox homeostasis, and immune regulation. Given the crucial role of glutaminolysis in bioenergetics and anaplerotic fluxes in kidney pathogenesis, studies on this cascade could provide a better understanding of kidney diseases, thus inspiring the development of potential methods for targeted therapy. Emerging evidence has shown that targeting glutaminolysis is a promising therapeutic strategy for ameliorating kidney disease. In this narrative review, equation including keywords related to glutamine, glutaminolysis and kidney are subjected to an exhaustive search on Pubmed database, we identified all relevant articles published before 1 April, 2024. Afterwards, we summarize the regulation of glutaminolysis in major kidney diseases and its underlying molecular mechanisms. Furthermore, we highlight therapeutic strategies targeting glutaminolysis and their potential clinical applications.

Multi-store collaborative delivery optimization based on Top-K order-split.

Regarding the fulfillment optimization of online retail orders, many researchers focus more on warehouse optimization and distribution center optimization. However, under the background of new retailing, traditional retailers carry out online services, forming an order fulfillment model with physical stores as front warehouses. Studies that focus on physical stores and consider both order splitting and store delivery are rare, which cannot meet the order optimization needs of traditional retailers. To this end, this study proposes a new problem called the "Multi-Store Collaborative Delivery Optimization (MCDO)", in which not only make the order-split plans for stores but also design the order-delivery routes for them, such that the order fulfillment cost is minimized. To solve the problem, a Top-K breadth-first search and a local search are integrated to construct a hybrid heuristic algorithm, named "Top-K Recommendation & Improved Local Search (TKILS)". This study optimizes the search efficiency of the breadth-first search by controlling the number of sub-orders and improving the initial solution of the local search using a greedy cost function. Then achieve the joint optimization of order-split and order-delivery by improving the local optimization operators. Finally, extensive experiments on synthetic and real datasets validate the effectiveness and applicability of the algorithm this study proposed.

lncRNA MAGI2-AS3 suppresses castration-resistant prostate cancer proliferation and migration via the miR-106a-5p/RAB31 axis.

Prostate cancer (PCa) is a common malignant cancer in elderly males in Western countries. Whole-genome sequencing confirmed that long non-coding RNAs (lncRNAs) are frequently altered in castration-resistant prostate cancer (CRPC) and promote drug resistance to cancer therapy. Therefore, elucidating the prospective role of lncRNAs in PCa oncogenesis and progression is of remarkable clinical significance. In this study, gene expression in prostate tissues was determined using RNA-sequencing datasets, and the gene diagnostic and prognostic values of CRPC were analyzed using bioinformatics. Further, the expression levels and clinical significance of MAGI2 Antisense RNA 3 (MAGI2-AS3) in PCa clinical specimens were evaluated. The tumor-suppressive activity of MAGI2-AS3 was functionally explored in PCa cell lines and animal xenograft models. MAGI2-AS3 was found to be aberrantly decreased in CRPC and was negatively correlated with Gleason score and lymph node status. Notably, low MAGI2-AS3 expression positively correlated with poorer survival in patients with PCa. The overexpression of MAGI2-AS3 significantly inhibited the proliferation and migration of PCa in vitro and in vivo. Mechanistically, MAGI2-AS3 could play a tumor suppressor function in CRPC through a novel miR-106a-5p/RAB31 regulatory network and could be a target for future cancer therapy.

Disrupted Alpha-Ketoglutarate Homeostasis: Understanding Kidney Diseases from the View of Metabolism and Beyond.

Alpha-ketoglutarate (AKG) is a key intermediate of various metabolic pathways including tricarboxylic acid (TCA) cycle, anabolic and catabolic reactions of amino acids, and collagen biosynthesis. Meanwhile, AKG also participates in multiple signaling pathways related to cellular redox regulation, epigenetic processes, and inflammation response. Emerging evidence has shown that kidney diseases like diabetic nephropathy and renal ischemia/reperfusion injury are associated with metabolic disorders. In consistence with metabolic role of AKG, further metabolomics study demonstrated a dysregulated AKG level in kidney diseases. Intriguingly, earlier studies during the years of 1980s and 1990s indicated that AKG may benefit wound healing and surgery recovery. Recently, interests on AKG are arising again due to its protective roles on healthy ageing, which may shed light on developing novel therapeutic strategies against age-related diseases including renal diseases. This review will summarize the physiological and pathological properties of AKG, as well as the underlying molecular mechanisms, with a special emphasis on kidney diseases.

HepaCAM­PIK3CA axis regulates the reprogramming of glutamine metabolism to inhibit prostate cancer cell proliferation.

Energy metabolism reprogramming is becoming an increasingly important hallmark of cancer. Specifically, cancers tend to undergo metabolic reprogramming to upregulate a cell­dependent glutamine (Gln) metabolism. Notably, hepatocellular cell adhesion molecule (HepaCAM) has been previously reported to serve a key role as a tumour suppressor. However, the possible regulatory role of HepaCAM in Gln metabolism in prostate cancer (PCa) remains poorly understood. In the present study, bioinformatics analysis predicted a significant negative correlation among the expression of HepaCAM, phosphatidylinositol­4,5­bisphosphate 3­kinase catalytic subunit α (PIK3CA), glutaminase (GLS) and solute carrier family 1 member 5 (SLC1A5), components of Gln metabolism, in clinical and genomic datasets. Immunohistochemistry results verified a negative correlation between HepaCAM and PIK3CA expression in PCa tissues. Subsequently, liquid chromatography­tandem mass spectrometry (LC­MS/MS) and gas chromatography­mass spectrometry (GC­MS) assays were performed, and the results revealed markedly reduced levels of Gln and metabolic flux in the blood samples of patients with PCa and in PCa cells. Mechanistically, overexpression of HepaCAM inhibited Gln metabolism and proliferation by regulating PIK3CA in PCa cells. In addition, Gln metabolism was discovered to be stress­resistant in PCa cells, since the expression levels of GLS and SLC1A5 remained high for a period of time after Gln starvation. However, overexpression of HepaCAM reversed this resistance to some extent. Additionally, alpelisib, a specific inhibitor of PIK3CA, effectively potentiated the inhibitory effects of HepaCAM overexpression on Gln metabolism and cell proliferation through mass spectrometry and CCK­8 experiments. In addition, the inhibitory effect of PIK3CA on the growth of tumor tissue in nude mice was also confirmed by immunohistochemistry in vivo. To conclude, the results from the present study revealed an abnormal Gln metabolic profile in the blood samples of patients with PCa, suggesting that it can be applied as a clinical diagnostic tool for PCa. Additionally, a key role of the HepaCAM/PIK3CA axis in regulating Gln metabolism, cell proliferation and tumour growth was identified. The combination of alpelisib treatment with the upregulation of HepaCAM expression may serve as a novel method for treating patients with PCa.

Panobinostat reverses HepaCAM gene expression and suppresses proliferation by increasing histone acetylation in prostate cancer.

Increased expression of histone deacetylases (HDACs) affiliated to the epigenetic regulation is common aberration in prostate cancer (PCa). We have confirmed that hepatocyte cell adhesion molecule (hepaCAM), acting as a tumor suppressor gene, is rarely expressed in PCa previously, However, the mechanisms of which is still unknown. The level of histone acetylation reportedly may involve anti-oncogene transcription and expression. In this study, we investigated the effect of panobinostat, the broad-spectrum histone deacetylases inhibitor, on PCa LNCaP and DU145 cell growth, and observed re-expression of hepaCAM when treated with panobinostat. We demonstrated that intranuclear acetylation of lys9 of histone H3 (Ac-H3K9) were increased, while that of both mRNA and protein of HDAC1, HDAC3, and HDAC4 were decreased when the treating concentration of panobinostat increased. We confirmed the relationship between histone acetylation and the expression of hepaCAM and AR in prostate cancer tissues. We also confirmed that panobinostat could overcome the resistance for androgen deprivation therapy (ADT). Further, we combined panobinostat with Ad-hepaCAM, which resulted in significantly increased antitumor activity and significant attenuation of the proliferation-associated genes CCND1 and PCNA compared to each single treatment. In conclusion, panobinostat may enhance the acetylation of lys9 of histone 3 and reverse the hepaCAM expression through its inhibitory effect on HDACs activity in PCa LNCaP and DU145 cells; Ad-hepaCAM combined with panobinostat may synergistically inhibit the growth of LNCaP and DU145 cells, via a potential mechanism associated with the down-regulation of the expression of CCND1 and PCNA. These findings suggest that this therapeutic strategy should be further developed in clinical trials.

Development and Implementation of Couple-Based Collaborative Management Model of Type 2 Diabetes Mellitus for Community-Dwelling Chinese Older Adults: A Pilot Randomized Trial.

Background: To mobilize family's positive involvement in improving and sustaining self-management activities of older adults with diabetes, we developed a couple-based collaborative management model (CCMM) for community-dwelling older Chinese. Methods: The model was developed stepwise through applying theoretical models, interviewing older couples and community healthcare workers, as well as incorporating expert reviews. A 3-month pilot study was conducted to test the model's feasibility and its treatment effects by linear regression on 18 pairs of older couples aged 60 years+, who were equally divided into a couple-based intervention arm and a patient-only control arm. Results: The developed CCMM covered four theory-driven intervention modules: dyadic assessment, dyadic education, dyadic behavior-change training, and dyadic monitoring. Each module was delivered by community healthcare workers and targeted at older couples as the management units. Based on interviews with older couples and healthcare workers, 4 weekly education and training group sessions and 2-month weekly behavior change booster calls were designed to address older adults' main management barriers. These modules and session contents were evaluated as essential and relevant by the expert panel. Furthermore, the CCMM showed good feasibility and acceptability in the pilot, with non-significant yet more positive changes in physiological outcomes of diabetic participants and couples' well-being and exercise levels of these in the intervention arm than their controlled counterparts. Conclusion: We systematically developed a couple-based collaborative management model of diabetes, which was well-received by healthcare practitioners and highly feasible among older Chinese couples living in the community. The model's treatment effects need to be verified in fully powered randomized controlled trials. Clinical Trial Registration: http://www.chictr.org.cn/showproj.aspx?proj=42964, identifier: ChiCTR1900027137.

CircHIPK3 Facilitates the G2/M Transition in Prostate Cancer Cells by Sponging miR-338-3p.

BACKGROUND: Circular RNAs (circRNAs) play a crucial role in gene expression regulation. CircHIPK3 is a circRNA derived from Exon 2 of HIPK3 gene and its role in prostate cancer (PCa) is still unclear. METHODS: CCK8 assays, flow cytometry and colony formation assays were performed to assess the effects of circHIPK3 in PCa cells. Bioinformatics analysis, RNA pull-down assay, RNA immunoprecipitation assay (RIP), and luciferase activity assay were performed to dissect the mechanism underlying circHIPK3-mediated G2/M transition in PCa cells. RESULTS: CircHIPK3 expression was upregulated in PCa cells and prostate cancer tissues. Overexpression of circHIPK3 or circHIPK3 silencing altered PCa viability, proliferation and apoptosis in vitro. CircHIPK3 could sponge miR-338-3p and inhibit its activity, resulting in increased expression of Cdc25B and Cdc2 in vitro. CONCLUSION: CircHIPK3 promotes G2/M transition and induces PCa cell proliferation by sponging miR-338-3p and increasing the expression of Cdc25B and Cdc2. CircHIPK3 may play an oncogenic role in PCa.

PLCε regulates metabolism and metastasis signaling via HIF-1α/MEK/ERK pathway in prostate cancer.

Phospholipase C-ε (PLCε) is frequently overexpressed in tumors and plays an important role in the regulation of tumorigenesis. Although great progress has been made in understanding biological roles of PLCε, the relevant molecular mechanisms underlying its pro-tumor activity remain largely unclear. Here, we demonstrated that PLCε knockdown reduced cell metastasis, glucose consumption and lactate production in a manner that depended on hypoxia inducible factor 1α (HIF-1α) expression in prostate cancer cells. Interestingly, our findings showed that the expression levels of PLCε were positively associated with those of HIF-1α in clinical prostate carcinoma samples. Knockdown of PLCε impaired HIF-1α levels and transcriptional activity by regulating the extracellular-signal-regulated kinase pathway, and blocking HIF-1α nuclear translocation. Furthermore, PLCε could interact with the von Hippel-Lindau E3 ligase complex to modulate the stability of HIF-1α. Collectively, our findings demonstrate that PLCε could be a crucial positive regulator of HIF-1α, which would promote PLCε-enhanced tumorigenesis.

Reasons for different therapeutic effects of high-intensity focused ultrasound ablation on excised uterine fibroids with different signal intensities on T2-weighted MRI: a study of histopathological characteristics.

OBJECTIVE: The objective of this study was to explore the correlations between the therapeutic effect of high intensity focused ultrasound (HIFU) and histopathological characteristics of excised uterine fibroids with different signal intensities as visualized on T2-weighted magnetic resonance imaging (MRI). METHODS: We collected 47 specimens of uterine fibroids after surgical resection and classified them into four groups according to preoperative T2-weighted MRI hypo-intense, isointense, heterogeneous intense and homogeneous hyper-intense. Then, specimens in each group were irradiated by HIFU with the same parameters and the necrotic tissue volume was calculated. The smooth muscle cell (SMC) count and collagen fiber content were quantitatively measured and compared between different groups. We analyzed the correlation between the necrotic tissue volume and SMC count and the collagen fiber content. RESULTS: Necrotic tissue volume gradually decreased from the hypo-intense group to the homogeneous hyper-intense group (p = .008). The SMC count from the hypo-intense group to the homogeneous hyper-intense group was 215.6 ± 59.3, 237.0(89.5), 232.3 ± 72.5 and 330.5 ± 30.9, respectively; collagen fiber content was 0.65 ± 0.07, 0.64 ± 0.10, 0.53 ± 0.11 and 0.41 ± 0.06, respectively. Comparison among the four groups showed that SMC count progressively increased (p = .001) but collagen fiber content progressively decreased (p = .000) from the hypo-intense group to the homogeneous hyper-intense group. Correlation analysis showed that necrotic tissue volume was negatively correlated with SMC count (R = -0.488, p=.013) but positively correlated with collagen fiber content (R = 0.534, p = .005). CONCLUSIONS: Differences in histopathological characteristics may be one of the reasons for different therapeutic effects of HIFU ablation on uterine fibroids with different signal intensities on T2-weighted MRI.

HepaCAM Regulates Warburg Effect of Renal Cell Carcinoma via HIF-1α/NF-κB Signaling Pathway.

OBJECTIVE: To investigate how hepatocyte cell adhesion molecule (hepaCAM) regulates cancer energy metabolism through hypoxia-inducible factor (HIF-1α) in renal cell carcinoma (RCC). MATERIALS AND METHODS: The expression of hepaCAM and HIF-1α in RCC tissue samples was examined by immunohistochemistry. Glucose consumption and lactate production assays were used to detect metabolic activity in RCC cell lines. P65 and IκB kinase (IKKß) mRNA and protein expression were detected using quantitative real-time polymerase chain reaction and western blotting, respectively. Nuclear translocation of P65 was observed by immunofluorescence staining after re-expressing hepaCAM. The luciferase reporter assay was applied to validate the transcriptional activity of HIF-1α. RESULTS: HIF-1α expression was elevated and hepaCAM suppressed in RCC compared with adjacent normal tissues. Furthermore, hepaCAM re-expression significantly decreased glycolytic metabolism in RCC cell lines, and reduced HIF-1α, IKKß, and P65 expression. The expression of HIF-1α, GLUT1, LDHA, and PKM2 were further reduced with combined hepaCAM overexpression and treatment with the NF-κB inhibitor BAY11-7082, compared to hepaCAM overexpression alone. Additionally, hepaCAM decreased the transcriptional activity of HIF-1α and blocked P65 nuclear translocation by the NF-κB pathway. CONCLUSION: Our data suggest that hepaCAM suppresses the Warburg effect via the HIF-1α/NF-κB pathway in RCC, which is a facilitating factor in hepaCAM-reduced tumorigenesis.

Biological analysis of cancer specific microRNAs on function modeling in osteosarcoma.

Osteosarcoma (OS) is the most common bone tumor characterized with a high risk of amputation and malignant morbidity among teenagers and adolescents. However, relevant pathogenic/biological mechanisms underlying OS-genesis remains to be ambiguous. The aim of this study was to elucidate functional relationship about microRNAs-mRNAs networks and to identify potential molecular markers via a computational method. Gene expression profile (GSE70415) was recruited from Gene Expression Omnibus. 3856 differentially expressed genes and 250 significantly expressed microRNAs were identified by using GCBI. The results of GO and KEGG pathways associated proteomics analysis indicated that extracellular matrix organization, small molecule metabolic process, cell adhesion (GO IDs: 0030198, 0044281, 0007155) and pathways in cancer, PI3K-Akt signaling pathway, metabolic pathways (pathway IDs: 5200, 4151, 1100) were significantly enriched. In addition, CKMT2, miR-93b-5p, miR-29b-3p were found to be positively/negatively correlated with TP53, EGFR, and MMP members mediated OS development, including angiogenesis, migration and invasion. Further visualization of collective effect of 1181 microRNAs-mRNAs pairs and protein-protein interactions was realized by applying with cytosacpe. In summary, our work provided a better understanding of non-coding regulatory mechanisms of transcriptomics and unraveled essential molecular biomarkers in osteosarcoma.

Dickkopf Wnt signaling pathway inhibitor 1 regulates the differentiation of mouse embryonic stem cells in vitro and in vivo.

Embryonic stem cells (ESCs) are pluripotent stem cells derived from early stage embryos. It remains unclear whether inhibiting the Wnt/ß­catenin signaling pathway using dickkopf Wnt signaling pathway inhibitor 1 (DKK1) impacts on the differentiation potential of mouse ESCs in vitro and in vivo. In the present study, immunohistochemical staining was used to measure the expression of markers of the three germ layers in ESCs and teratomas derived from ESCs. The expression of markers for the Wnt/ß­catenin signaling pathway were detected by reverse transcription­polymerase chain reaction (RT­qPCR). Immunohistochemistry and western blotting indicated that the expression levels of octamer­binding transcription factor 4 in the DKK1­treated ESC group were significantly greater compared with the control ESCs. Reduced expression levels of NeuroD and bone morphogenetic protein 4 were observed in the DKK1­treated ESCs and teratomas derived from DKK1­treated ESCs compared with the control group. Increased expression levels of SOX17 were observed in the DKK1­treated ESCs compared with the control group. RT­qPCR indicated that ß­catenin expression was significantly reduced in DKK1­treated ESCs and teratomas derived from DKK1­treated ESCs compared with the control groups. Western blotting indicated no alterations in the expression of GSK­3ß, however, the levels of phosphorylated­GSK­3ß were significantly greater in the DKK1 treatment groups, while cyclin D1 and c­Myc expression levels were significantly reduced in the DKK1 treatment groups compared with the control groups. These results suggest that inhibiting Wnt signaling in ESCs using DKK1 may promote mouse ESCs to differentiate into endoderm in vitro and in vivo, and suppress the tumorigenicity of ESCs.

Expression of hepaCAM inhibits bladder cancer cell proliferation via a Wnt/ß-catenin-dependent pathway in vitro and in vivo.

We previously established that hepatocyte cell adhesion molecule (hepaCAM), a typical structure of immunoglobulin (Ig)-like adhesion molecules, inhibited the proliferation and the progression of cultured human bladder cancer cells. As increasing evidence reveals that aberrant activation of canonical Wnt pathway is involved in the pathogenesis of bladder cancer, and ß-catenin serves as a pivotal molecule of Wnt pathway. Then, we explored whether the anti-proliferation effect of hepaCAM was associated with Wnt/ß-catenin pathway in human bladder cancer cells. The negative correlation between hepaCAM and ß-catenin in transitional cell carcinoma of bladder (TCCB) was found. Follow by, studied the effect of hepaCAM on the key elements of Wnt pathway. Here, Our researches showed that hepaCAM played a central role in modulating the Wnt/ß-catenin signaling pathway by interfering nuclear protein levels of ß-catenin, leading to down-regulate transcriptional activity of LEF/TCF and its target genes c-Myc and cyclinD1. Mechanistically, we demonstrated that hepaCAM-activated GSK3ß led to elevate the phosphorylation of ß-catenin, contributing to the aberrant translocation of ß-catenin. In addition, Anti-proliferation and associated molecular mechanisms of hepaCAM were demonstrated by using vivo experiment. In conclusion, our reports uncover that expression of hepaCAM suppresses the proliferation of bladder cancer cells through a Wnt/ß-catenin-dependent signaling pathway in vitro and in vivo.

Knockdown of PLCε inhibits inflammatory cytokine release via STAT3 phosphorylation in human bladder cancer cells.

Phospholipase Cε (PLCε) is a multifunctional enzyme implicated in inflammatory functions. There are limited data, however, on how PLCε can alter inflammatory cytokine by affecting downstream pathways. Recent studies suggest that inflammation is likely to have an important role in transitional cell carcinoma of bladder (TCCB) and cancer disease progression. Here, we showed that PLCε and p-STAT3 expression were both elevated in TCCB tissues compared to adjacent tissues, and the increase of PLCε level was associated with the increase of p-STAT3 level. Then, knockdown of PLCε using adenovirus-shPLCε significantly decreased inflammatory cytokine (IL-6, TNF-α, IL-1ß) expression and inflammation-associated gene (TLR4, MyD88, p-STAT3) expression. Furthermore, we demonstrated that PLCε knockdown blocked LPS-induced inflammatory cytokine and p-STAT3 expression. Additionally, we found that combined treatment of STAT3 inhibitor S3I-201 with adenovirus-shPLCε exhibited synergistic inhibitory effects on expression of p-STAT3. Our results suggested that STAT3 phosphorylation is involved in PLCε-mediated inflammatory cytokine release. Our research is of potential importance in drug development programs using PLCε as a therapeutic target for TCCB.

PLCε knockdown inhibits prostate cancer cell proliferation via suppression of Notch signalling and nuclear translocation of the androgen receptor.

Phospholipase Cε (PLCε), a key regulator of diverse cellular functions, has been implicated in various malignancies. Indeed, PLCε functions include cell proliferation, apoptosis and malignant transformation. Here, we show that PLCε expression is elevated in prostate cancer (PCa) tissues compared to benign prostate tissues. Furthermore, PLCε depletion using an adenovirally delivered shRNA significantly decreased cell growth and colony formation, arresting the PC3 and LNCaP cell lines in the S phase of the cell cycle. We also observed that PLCε was significantly correlated with Notch1 and androgen receptor (AR). Additionally, we demonstrate that the activation of both the Notch and AR signalling pathways is involved in PLCε-mediated oncogenic effects in PCa. Our findings suggest that PLCε is a putative oncogene and prognostic marker, potentially representing a novel therapeutic target for PCa.

Nuclear factor-κB signaling pathway is involved in phospholipase Cε-regulated proliferation in human renal cell carcinoma cells.

Phospholipase Cε (PLCε), a downstream effector of small GTPase superfamily, has been identified to play a crucial role in tumorigenesis. Previously, our studies have showed that PLCε promotes proliferation of renal cell carcinoma (RCC) cells. However, the molecular mechanisms by which PLCε enhances the survival phenotype of RCC cells are still not fully instructed. In the present study, we first demonstrated that PLCε was highly expressed and had a close correlation with Ki67 protein expression in RCC tissue samples. Further, we found that downregulation of PLCε expression repressed growth and induced apoptosis in RCC cells. In addition, we reported a mechanism by which knockdown of PLCε gene potently suppressed the nuclear factor kappa (NF-κB) signaling pathway through action on inhibitor of κB kinase. Moreover, silencing PLCε gene decreased vascular endothelial growth factor (VEGF) expression, which was a downstream growth factor of NF-κB signaling pathway. Finally, downregulation of VEGF was severely enhanced by treatment cells with NF-κB specific inhibitor BAY11-7028 in PLCε knockdown cells. Taken together, these findings suggest that PLCε promotes RCC cell growth via NF-κB-mediated upregulation of VEGF.

Knockdown of phospholipase C-epsilon by short-hairpin RNA-mediated gene silencing induces apoptosis in human bladder cancer cell lines.

Transitional cell carcinoma of bladder (TCCB) is a common malignancy worldwide, and outcomes for patients with advanced bladder cancer remain poor. To study the pathogenesis of TCCB, we investigated roles of Phospholipase C (PLC)ɛ, an effector of Ras and Rap small GTPases. RNA interference was used to knockdown PLCɛ expression in human bladder cancer cell lines (BIU-87 and T24). The expression levels of PLCɛ mRNA and protein were detected by reverse transcriptase-polymerase chain reaction and Western blot, respectively. Flow cytometry (FCM) was used to detect distribution of cell cycle. Cellular apoptosis was reflected by transmission electron microscopy and the expression of bcl-2 and bax. We found that PLCɛ could be efficiently knocked down by shRNA. FCM assay showed that the pGenesil-PLCɛ-transfected cells were arrested at the G0/G1 phase. Silence of PLCɛ might induce apoptosis via modulation of bcl-2 and bax. In conclusion, our results suggest that PLCɛ plays an important role in the pathogenesis of human bladder cancer cells. PLCɛ may be used as a potential target of gene therapy for bladder cancer in future.

RNA interference suppressing PLCE1 gene expression decreases invasive power of human bladder cancer T24 cell line.

Mutational activation of the ras proto-oncogenes is frequently found in cancers. The phospholipase C epsilon gene (PLCE1) encodes a novel ras-related protein (R-Ras) effector mediating the effects of R-Ras on the actin cytoskeleton and membrane protrusion, because R-Ras is coprecipitated with the PLCE1 protein and can increase its activity. The nature of downstream signaling pathways from Ras involved in bladder cancer remains poorly understood. We aimed to construct a small hairpin RNA (shRNA) expression plasmid against the PLCE1 gene and to observe the inhibition of human bladder carcinoma cell T24 migration by RNA interference suppressing the expression of PLCE1. Two PLCE1 plasmids (P1 and P2) were constructed and inserted into T24 cells. Reverse transcriptase-polymerase chain reaction and Western blot analyses were performed to investigate inhibition of PLCE1 expression after plasmid transfection. Invasive power of the T24 cell line was measured before and after transfection by a membrane invasion culture system (transwell chamber), gelatin enzymography, and immunocytochemistry of cells. The RT-PCR analysis of BCL2 mRNA levels among different groups of T24 cell line indicated that expression of BCL2 mRNA was lower in the two positive plasmid-transfected cell groups than in the blank control or HK-A groups. Silencing of PLCE1 might downregulate the level of MMP and BCL2 gene expression, decreasing the invasive power of bladder cancer T24 cells and thus inhibiting tumor development.