Shikonin Suppresses Cell Tumorigenesis in Gastric Cancer Associated with the Inhibition of c-Myc and Yap-1.

AIM: The study aimed to study the potential roles and mechanisms of shikonin in gastric cancer by network pharmacology and biological experiments. METHODS: The key genes and targets of shikonin in gastric cancer were predicted by network pharmacology and molecular docking study. The effect of shikonin on the proliferation, migration, and invasion of gastric cancer cells was detected by the CCK8 method, and wound healing and transwell assays. The expression levels of c-Myc and Yap-1 were detected via western blotting in gastric cancer cells after shikonin intervention. RESULTS: The results of network pharmacology revealed the key target genes of shikonin on gastric cancer cells to be c-Myc, Yap-1, AKT1, etc. GO and KEGG analysis showed regulation of cell migration, proliferation, adhesion, and other biological processes, including the PI3K-Akt signaling pathway, HIF-1 signaling pathway, necroptosis, and other cancer pathways. Molecular docking showed shikonin to be most closely combined with protooncogenes c-Myc and Yap-1. In vitro experiments showed that the proliferation rate, migration, and invasion ability of the gastric cancer cell group decreased significantly after shikonin intervention for 24h. The expression levels of c-Myc and Yap-1 in gastric cancer cells were found to be significantly decreased after shikonin intervention. CONCLUSION: This study showed protooncogenes c-Myc and Yap-1 to be the core target genes of shikonin on gastric cancer cells. Shikonin may suppress gastric cancer cells by inhibiting the protooncogenes c-Myc and Yap-1. This suggests that shikonin may be a good candidate for the treatment of gastric cancer.

Detection of Klebsiella pneumoniae cfDNA in pleural fluid and its clinical value.

BACKGROUND: Klebsiella pneumoniae (KP) is an important opportunistic pathogen that can easily cause pneumonia and pleural effusion when body resistance is reduced. However, the positive rate of KP detected from clinical pleural effusion by traditional methods, including bacterial culture, is meager. Therefore, new detection methods are urgently needed to improve the positive detection rate of KP and other bacteria in pleural effusion. METHODS: Simulated pleural fluid of KP infection was first set up. Then circulating cell-free DNA (cfDNA) was extracted from cultured hydrothorax and detected by fluorescence polymerase chain reaction (PCR) to verify KP cfDNA in the pleural fluid. The specificity, sensitivity, and repeatability of this method are verified by detecting the cfDNAs in pleural effusion, samples of malignant pleural effusion, tuberculous pleural effusion, and other common microbial infections. Finally, this method was compared with three traditional methods, pleural effusion, precipitation DNA, sputum culture, and pleural effusion culture to explore the clinical diagnostic value of this method. RESULTS: KP cfDNA was positive by fluorescence PCR from the simulated KP infected pleural effusion, which confirmed KP cfDNA in pleural effusion. KP cfDNA was positive by fluorescence PCR from the pleural effusion of KP infected patients, while with the same detection method, KP cfDNA in clinical carcinomatous hydrothorax, tuberculosis hydrothorax, and other standard microbial infection samples was negative, which confirmed the method had high specificity, high sensitivity, and reproducibility. Compared with the three traditional methods, this method has a higher positive rate. Compared with the gold standard, sputum bacterial culture, the sensitivity, specificity, positive predictive value, and negative predictive value of this method were 91.67%, 95.45%, 91.7%, and 95.5%, respectively. CONCLUSIONS: The detection of cfDNA by fluorescence PCR is possible. Moreover, the positive rate of this method in clinical pleural effusions is high.

Detection of ALK Gene Rearrangement in Cell-free RNA from Lung Cancer Malignant Pleural Effusion.

The aim of this study was to evaluate the feasibility of measuring ALK gene rearrangement in cell-free RNA (cf-RNA) of the supernatant from malignant pleural effusion (MPE). Supernatant, cell blocks, and matched sera samples were collected. Cf-RNA was isolated from the supernatant and sera, and cellular RNA was isolated from cell blocks. The ALK gene rearrangement in the cf-RNA was tested by the real-time polymerase chain reaction. Results showed that the concentration of cf-RNA was higher in the supernatant than in matched sera. ALK status concordance rates were 100% between the supernatant and cell blocks, while they were 0% between sera and cell blocks in ALK gene rearrangement cases. This suggests that using cf-RNA in MPE supernatant, but not in sera, could offer a reliable and robust surrogate strategy for the detection of ALK gene rearrangement.

Prostatic epithelial cells and their high expressions of CKIP-1 affect the TGF-ß1 expression levels which might reduce the scar formation in remodeling stage at prostatic urethral wounds after wound repair.

OBJECTIVE: There are less scar formations in some wounds after wound repair. Our earlier study had shown that the amount of collagen fibers in canine prostatic urethra wound were less than in bladder neck wound after 2-µm laser resection of the prostate (TmLRP) and partial bladder neck mucosa at 4 weeks. The purpose of this study was to observe the amount of scar tissue and characterize the probable causes of "less scar healing" in prostatic urethra wound. METHODS: A total of 12 healthy adult male crossbred canines underwent resection of prostate and partial bladder neck mucosa using 2-µm laser. The prostatic urethra and bladder neck wound specimens were harvested at 3, 4, 8 and 12 weeks after operation, respectively. The histopathologic characteristics were observed by hematoxylin and eosin(HE)staining, and the expression of transforming growth factor-ß1 (TGF-ß1) and casein kinase-2 interacting protein-1 (CKIP-1) were examined by immunohistochemistry in prostatic urethra and bladder neck wound, respectively. Overexpressed CKIP-1 human prostate epithelial cells (BPH-1 cells) were established and the expression of TGF-ß1 was detected by Western blotting. Furthermore, a non-contact co-culture system of BPH-1 cells and human fibroblast (HFF-1) cells was used to observe the effects of BPH-1 cell and their high CKIP-1 levels on the expression of TGF-ß1 in HFF-1 in vitro. RESULTS: The histology showed that there were a large number of prostatic epithelium and a small amount of scar tissue in prostatic urethra wound, while no epithelial cells and more scar tissue in bladder neck wound at 4, 8 and 12 weeks after repair. There were a higher expression level of TGF-ß1 in prostate epithelial cells and fibroblasts and a lower expression level of CKIP-1 in prostate epithelial cells at 3 weeks after surgery in prostatic urethral wound. Compared to week 3, the TGF-ß1 expression decreased both in prostate epithelial cells and fibroblasts at 4, 8 and 12 weeks in prostatic urethral wound (p < 0.05 or p < 0.01). The CKIP-1 expression increased in prostate epithelial cells at 4, 8 and 12 weeks compared to 3 weeks in prostatic urethra wound (p < 0.01). A higher TGF-ß1 expression level of fibroblasts was observed in bladder neck wound at 3 weeks. And there was no significant change in the expression of TGF-ß1 of fibroblasts in 3, 4, 8 and 12 weeks after operation in bladder neck wound. Both the prostate urethra and bladder neck wound fibroblasts showed weak expression of CKIP-1 and there was no significant change in 3, 4, 8 and 12 weeks. The vitro experiments showed that the TGF-ß1 expression in BPH-1 cells with CKIP-1 overexpression decreased 25% compared with control group (p < 0.05). Furthermore, the expression of TGF-ß1 in HFF-1 cells of co-cultured group decreased by 20% compared with Control group (p < 0.05); the expression of TGF-ß1 in HFF-1 cells of overexpression co-culture group were reduced by 15% compared with co-cultured group (p < 0.01). CONCLUSIONS: A large number of prostate epithelial cells in prostatic urethra wound may be one of the causes of less formation of scar tissue after repair. The prostate epithelial cells might reduce expression level of TGF-ß1 by raising CKIP-1 expression and inhibit expression of TGF-ß1 in peripheral fibroblasts at remodeling stage to reduce the excessive proliferation of fibrous cells and the excessive scar formation.

Casein kinase 2 interacting protein 1 positively regulates caudal-related homeobox 1 in intestinal-type gastric cancer.

BACKGROUND: Gastric cancer (GC) is one of the most common malignancies, and intestinal-type GC is the main histopathologic type of GC in China. We previously reported that casein kinase 2 interacting protein 1 (CKIP-1) acts as a candidate tumor suppressor in intestinal-type GC. CKIP-1 participates in the regulation of multiple signaling pathways, including the Wnt/ß-catenin pathway, of which caudal-related homeobox 1 (CDX1) may be a downstream target gene. The purpose of this study was to investigate the relationship between CKIP-1 and CDX1 in intestinal-type GC. METHODS: Sixty-seven gastroscopy biopsy specimens and surgically resected gastric specimens were divided into four groups: gastric mucosa group, intestinal metaplasia (IM) group, dysplasia group, and intestinal-type GC group. The expression levels of CKIP-1 and CDX1 were detected in these groups and GC cell lines, and the correlations between these expression levels were analyzed. SGC7901 and BGC823 cells were divided into CKIP-1 shRNA groups and CKIP-1 over-expression groups, and CDX1 expression was detected. ß-Catenin expression was detected in intestinal-type GC tissue samples and CKIP-1 shRNA and CKIP-1 over-expression SGC7901 cells, and its correlation with CKIP-1 expression in intestinal-type GC tissue was analyzed. The Wnt/ß-catenin pathway inhibitor DKK-1 and activator LiCl were incubated with SGC7901 cells, BGC823 cells, and CKIP-1 shRNA and CKIP-1 over-expression SGC7901 and BGC823 cells, following which CDX1 and Ki-67 expression were detected. RESULTS: The expression levels of CKIP-1 and CDX1 were lower in patients with intestinal-type GC than in patients with IM and dysplasia (both P < 0.05). CKIP-1 and CDX1 expression levels were positively correlated in IM, dysplasia, and intestinal-type GC tissue and cell lines (r = 0.771, P < 0.01; r = 0.597, P < 0.01; r = 0.654, P < 0.01; r = 0.811, P < 0.01, respectively). CDX1 expression was decreased in the CKIP-1 shRNA groups and increased in the CKIP-1 over-expression groups of SGC7901 and BGC823 cells compared to that in the corresponding control groups (both P < 0.05). CKIP-1 expression was negatively correlated with ß-catenin expression in intestinal-type GC patients (r = -0.458, P < 0.01). Compared to the control group, ß-catenin expression was increased in the CKIP-1 shRNA SGC7901 cell group and decreased in the CKIP-1 over-expression SGC7901 cell group (P < 0.05). CDX1 expression was increased in SGC7901 and BGC823 cells treated with DKK-1, DKK-1 increased CDX1 expression and decreased Ki-67 expression in the CKIP-1 shRNA group; the opposite result was observed in SGC7901 and BGC823 cells treated with LiCl, and LiCl decreased CDX1 expression and increased Ki-67 expression in the CKIP-1 over-expression group (both P < 0.05). CONCLUSIONS: Through the Wnt/ß-catenin signaling pathway, CKIP-1 may positively regulate CDX1 in intestinal-type GC.

PARP6 acts as an oncogene and positively regulates Survivin in gastric cancer.

Poly (ADP-ribose) polymerases 6 (PARP6) is a novel member of the PARP family. Previous studies focused mostly on the role of PARP6 in colorectal cancer; however, the role of PARP6 in gastric cancer is currently unclear. In the present study, we found a high-level expression of PARP6 in gastric cancer cells and PARP6 promoted cell proliferation, migration and invasion. Moreover, we found a positive correlation exists between PARP6 and Survivin, which contributes to tumor ongoing survival. In sum, our data suggest that PARP6 may contribute to gastric cancer progression by activating the Survivin pathway.

Impact of age on EGFR mutations in never-smoking female lung adenocarcinoma patients with malignant pleural effusion.

Our aim was to evaluate EGFR mutations in never-smoking female lung adenocarcinoma patients with malignant pleural effusion and to reveal the relationship between age and EGFR mutations. Never-smoking female lung adenocarcinoma patients were retrospectively studied, including 301 biopsy samples and 80 cytological specimens. Our results showed a significant increase of EGFR mutation prevalence by increase of age in cytological specimens, but not in biopsy samples. Our data suggests that age at the time of diagnosis may be associated with presence of EGFR mutations in patients with malignant pleural effusion.

P1-HNF4α expression negatively correlates with HER2 levels and is associated with good prognosis in gastric adenocarcinoma.

HNF4α, a member of the steroid/thyroid nuclear receptor super family, is a transcriptional factor expressed in various tissues and cells. In this study, we aimed to investigate the clinical significance of P1-HNF4α protein expression in gastric adenocarcinoma. We examined P1-HNF4α and HER2 protein levels in the tissue of 245 gastric adenocarcinoma samples by immunohistochemistry, and analyzed the association between P1-HNF4α levels, and clinicopathologic factors or prognosis. In gastric adenocarcinoma, positive staining of P1-HNF4α was shown in 150 (61.2%) of 245 cases, while positive expression of HER2 was shown in 36 (14.7%) of 245 cases as detected by immunohistochemistry. The expression of P1-HNF4α was negatively correlated with that of HER2. Furthermore, P1-HNF4α and HER2 highly expressed in intestinal-type adenocarcinoma according to the Lauren classification, tubular adenocarcinoma according to WHO classification, and well-to-moderately differentiated tumors. In gastric adenocarcinoma, P1-HNF4α was significantly associated with histological type, Lauren grade, degree of tumor differentiation, vascular invasion, lymph node metastases, and pTNM stage. Moreover, survival analysis showed that P1-HNF4α expression was an independent prognostic factor of good survival in gastric adenocarcinoma (P<0.05). Our results indicate that a negative correlation exists between P1-HNF4α and HER2 expression levels and P1-HNF4α is significantly correlated with tumor progression and a good prognosis in gastric adenocarcinoma.

Numb-/low Enriches a Castration-Resistant Prostate Cancer Cell Subpopulation Associated with Enhanced Notch and Hedgehog Signaling.

Purpose: To elucidate the role and molecular mechanism of Numb in prostate cancer and the functional contribution of Numb-/low prostate cancer cells in castration resistance.Experimental Design: The expression of Numb was assessed using multiple Oncomine datasets and prostate cancer tissues from both humans and mice. The biological effects of the overexpression and knockdown of Numb in human prostate cancer cell lines were investigated in vitro and in vivo In addition, we developed a reliable approach to distinguish between prostate cancer cell populations with a high or low endogenous expression of Numb protein using a Numb promoter-based lentiviral reporter system. The difference between Numb-/low and Numbhigh prostate cancer cells in the response to androgen-deprivation therapy (ADT) was then tested. The likely downstream factors of Numb were analyzed using luciferase reporter assays, immunoblotting, and quantitative real-time PCR.Results: We show here that Numb was downregulated and negatively correlated with prostate cancer advancement. Functionally, Numb played an inhibitory role in xenograft prostate tumor growth and castration-resistant prostate cancer development by suppressing Notch and Hedgehog signaling. Using a Numb promoter-based lentiviral reporter system, we were able to distinguish Numb-/low prostate cancer cells from Numbhigh cells. Numb-/low prostate cancer cells were smaller and quiescent, preferentially expressed Notch and Hedgehog downstream and stem-cell-associated genes, and associated with a greater resistance to ADT. The inhibition of the Notch and Hedgehog signaling pathways significantly increased apoptosis in Numb-/low cells in response to ADT.Conclusions: Numb-/low enriches a castration-resistant prostate cancer cell subpopulation that is associated with unregulated Notch and Hedgehog signaling. Clin Cancer Res; 23(21); 6744-56. ©2017 AACR.

CKIP-1 serves as a negative regulator and correlates with the degree of differentiation in gastric cancer.

Gastric cancer (GC) is one of the most commonly diagnosed malignancies worldwide. CKIP-1 is a casein kinase-2 α-subunit (CK2α) interacting protein. Though previous reports have shown that CKIP-1 plays a critical role in several types of cancers, hardly there are any studies that examined the role of CKIP-1 in the progression of GC. Our present study aimed to investigate the role of CKIP-1 in GC. Results demonstrated low-level expression of CKIP-1 in GC tissues and cell lines. Moreover, knockdown of CKIP-1 promoted cell proliferation, migration, and invasion in GC cell lines, whereas CKIP-1 overexpression inhibited proliferation, migration, and invasion in the cells. Altogether, our data suggests that CKIP-1 may act as a novel tumor suppressor gene in GC and is related to GC differentiation.

Regulation of Prostate Development and Benign Prostatic Hyperplasia by Autocrine Cholinergic Signaling via Maintaining the Epithelial Progenitor Cells in Proliferating Status.

Regulation of prostate epithelial progenitor cells is important in prostate development and prostate diseases. Our previous study demonstrated a function of autocrine cholinergic signaling (ACS) in promoting prostate cancer growth and castration resistance. However, whether or not such ACS also plays a role in prostate development is unknown. Here, we report that ACS promoted the proliferation and inhibited the differentiation of prostate epithelial progenitor cells in organotypic cultures. These results were confirmed by ex vivo lineage tracing assays and in vivo renal capsule recombination assays. Moreover, we found that M3 cholinergic receptor (CHRM3) was upregulated in a large subset of benign prostatic hyperplasia (BPH) tissues compared with normal tissues. Activation of CHRM3 also promoted the proliferation of BPH cells. Together, our findings identify a role of ACS in maintaining prostate epithelial progenitor cells in the proliferating state, and blockade of ACS may have clinical implications for the management of BPH.

Regulation and methylation of tumor suppressor miR-124 by androgen receptor in prostate cancer cells.

Prostate cancer (PCa) is the most frequently diagnosed cancer for men in the developed world. Androgen receptor signaling pathway plays an important role in prostate cancer progression. Recent studies show that microRNA miR-124 exerts a tumor suppressive function in prostate cancer. However, the relationship between AR and miR-124 is unclear. In the present study, we found a negative feedback loop between AR and miR-124 expression. On one hand, miR-124 was a positively regulated target gene of the AR, on the other hand, overexpression of miR-124 inhibited the expression of AR. In addition, we found that miR-124-2 and miR-124-3 promoters were hypermethylated in AR-negative PCa cells. Furthermore, overexpression of miR-124 inhibited proliferation rates and invasiveness capacity of PCa cells in vitro, and suppressed xenograft tumor growth in vivo. Taken together, our results support a negative feedback loop between AR and miR-124 expression. Methylation of miR-124-2 and miR-124-3 may serve as a biomarker for AR-negative PCa cells, and overexpression of miR-124 might be of potential therapeutic value for the treatment of PCa.

The Sigma-2 Receptor and Progesterone Receptor Membrane Component 1 are Different Binding Sites Derived From Independent Genes.

The sigma-2 receptor (S2R) is a potential therapeutic target for cancer and neuronal diseases. However, the identity of the S2R has remained a matter of debate. Historically, the S2R has been defined as (1) a binding site with high affinity to 1,3-di-o-tolylguanidine (DTG) and haloperidol but not to the selective sigma-1 receptor ligand (+)-pentazocine, and (2) a protein of 18-21 kDa, as shown by specific photolabeling with [(3)H]-Azido-DTG and [(125)I]-iodoazido-fenpropimorph ([(125)I]-IAF). Recently, the progesterone receptor membrane component 1 (PGRMC1), a 25 kDa protein, was reported to be the S2R (Nature Communications, 2011, 2:380). To confirm this identification, we created PGRMC1 knockout NSC34 cell lines using the CRISPR/Cas9 technology. We found that in NSC34 cells devoid of or overexpressing PGRMC1, the maximum [(3)H]-DTG binding to the S2R (Bmax) as well as the DTG-protectable [(125)I]-IAF photolabeling of the S2R were similar to those of wild-type control cells. Furthermore, the affinities of DTG and haloperidol for PGRMC1 (KI = 472 µM and 350 µM, respectively), as determined in competition with [(3)H]-progesterone, were more than 3 orders of magnitude lower than those reported for the S2R (20-80 nM). These results clarify that PGRMC1 and the S2R are distinct binding sites expressed by different genes.

TOP2Ahigh is the phenotype of recurrence and metastasis whereas TOP2Aneg cells represent cancer stem cells in prostate cancer.

Recurrence and metastasis are the main causes of death for prostate cancer patients and cancer stem cells (CSCs) are proposed to play important roles in cancer recurrence and metastasis. It is generally thought that genes upregulated in recurrent/metastatic disease are likely biomarkers of CSCs. Hence we analyzed multiple microarray datasets on prostate tumor tissues to identify upregulated genes associated with cancer recurrence/metastasis, and tried to explore whether those genes were true biomarkers of prostate CSCs. Our results indicated that TOP2A was the most highly upregulated gene in recurrent/metastatic prostate cancer, and its high expression was positively correlated with poor prognosis in patients. Using a promoter reporter system, we unexpectedly discovered enrichment of CSCs in TOP2Aneg cells. Compared to TOP2Ahigh cells, TOP2Aneg cells formed spheres and tumors more efficiently, and became enriched in the presence of stresses. Analysis of cell divisions by time lapse imaging indicated that more slow-cycling cells were observed in TOP2Aneg cells while the proportion of abnormal divisions was higher in TOP2Ahigh cells. Our studies demonstrate that TOP2Ahigh is the phenotype of recurrence/metastasis but TOP2Aneg cells show slow cycling and have CSCs properties in prostate cancer, which has significant implications for prostate cancer therapy.

Symmetrical and asymmetrical division analysis provides evidence for a hierarchy of prostate epithelial cell lineages.

Although symmetrical and asymmetrical divisions of stem cells have been extensively studied in invertebrate and mammalian neural epithelia, their role remains largely unknown in mammalian non-neural epithelial development, regeneration and tumorigenesis. Here, using basal and luminal cell-specific markers and cell lineage tracing transgenic mice, we report that in developing prostatic epithelia, basal and luminal cells exhibit distinct division modes. While basal cells display both symmetric and asymmetric divisions leading to different cell fates, luminal cells only exhibit symmetrical divisions. Examination of cell division modes in prostate-specific Pten-null mice indicates that both luminal and basal cells can be cellular origins for prostate cancer. Furthermore, analysis of Sox2-expressing cells in p63 and Pten-null mice suggests that basal cells contribute to the luminal population and tumorigenesis. These findings provide direct evidence for the existence of a hierarchy of epithelial cell lineages during prostate development, regeneration and tumorigenesis.

Zeb1 promotes androgen independence of prostate cancer via induction of stem cell-like properties.

Current androgen deprivation therapy often leads to androgen independence. However, mechanism of the therapeutic failure is still not well understood. Here, we demonstrate elevated expression of Zeb1 in androgen-independent prostate cancer cells and prostate tumors of castrated PTEN conditional knockout mice. While Zeb1 shRNA resulted in a sensitization of androgen-independent prostate cancer cells, forced Zeb1 expression caused androgen-dependent prostate cancer cells to be more resistant to androgen deprivation. Moreover, such effects appeared to be mediated by induction of pluripotent genes or stem cell-like properties. Collectively, these findings suggest that inhibition of Zeb1 might be a potential therapeutic strategy for treatment of androgen-independent prostate cancer.

Hypermethylation-mediated transcriptional repression of TMPRSS2 in androgen receptor-negative prostate cancer cells.

Prostate cancer is the most common type of cancer for men in the developed world. Androgen receptor (AR) is very important in prostate cancer progression. TMPRSS2 is an AR signaling downstream gene and closely related to prostate carcinogenesis. DNA methylation is a key mechanism to influence gene expression. Though previous reports have shown that AR signaling plays a critical role in the regulation of TMPRSS2 in prostate cancer, hardly any studies have examined whether the DNA methylation has been involved in the regulation of TMPRSS2. In the present study, we demonstrated that AR-negative prostate cancer (PCa) cells showed low expression levels and hypermethylation of TMPRSS2. In contrast, AR-positive PCa cells displayed high levels and hypomethylation of TMPRSS2. Treatment with the DNA methylation inhibitor 5-Aza-2'-deoxycytidine reversed the low expression levels of TMPRSS2 in the AR-negative PCa cells. Additionally, we found that the level of DNA methyltransferases 1 (DNMT1) was high in AR-negative PCa cells, in which hypermethylation of TMPRSS2 and low expression level of TMPRSS2 were observed. Collectively, these data suggest that the high level of DNMT1 might be the mechanism for the hypermethylation-mediated transcriptional repression of TMPRSS2 in AR-negative PCa cells.

Androgen receptor is negatively correlated with the methylation-mediated transcriptional repression of miR-375 in human prostate cancer cells.

Androgen receptor (AR) plays a critical role during the development and progression of prostate cancer in which microRNA miR-375 is overexpressed and correlated with tumor progression. Although DNA methylation is a key mechanism for the repression of gene expression, the relationship between AR and the expression or the hypermethylation of miR-375 is unknown. In this study, we found that AR-positive prostate cancer (PCa) cells showed high expression levels and hypomethylation of the miR-375. In contrast, AR-negative PCa cells displayed low levels and hypermethylation of the miR-375. Addition of 5-Aza-2'-deoxycytidine, a specific inhibitor of DNA methylation, into the culture medium reversed the low expression levels of miR-375 in the AR negative PCa cells. In addition, the total activity levels of DNA methyltransferases (DNMTs) were high in AR-negative PCa cells, in which hypermethylation of miR-375 promoter and low expression levels of miR-375 were observed. Taken together, these findings indicate that the negative correlation between AR and total DNMT activity is one of mechanisms to influence the methylation status of miR-375 promoter, which in turn regulates the expression of miR-375.

Elevated serum osteopontin levels and genetic polymorphisms of osteopontin are associated with Vogt-Koyanagi-Harada disease.

PURPOSE: Osteopontin (OPN) is a proinflammatory cytokine involved in chronic inflammatory diseases. This study aimed to analyze the role of OPN in the pathogenesis of Vogt-Koyanagi-Harada (VKH) disease. METHODS: Serum levels of OPN in VKH patients and healthy controls were assayed by enzyme-linked immunosorbent assay (ELISA). Peripheral blood mononuclear cells (PBMCs) or CD4+ T cells were cultured with anti-CD3 and anti-CD28 antibodies in the absence or presence of recombinant OPN for the determination of cell proliferation and cytokines. Cell proliferation was detected using a cell counting kit. Levels of interferon (IFN)-γ and interleukin (IL)-17 were detected by ELISA. Four single nucleotide polymorphisms (SNPs) of OPN and four SNPs of OPN receptors were genotyped in 601 VKH patients and 605 healthy controls using a polymerase chain reaction-restriction fragment length polymorphism assay. RESULTS: OPN serum levels were significantly higher in patients with active VKH than in patients with inactive VKH and in healthy controls. PBMCs or CD4+ T cells cultured with recombinant OPN induced a marked cell proliferation and profound secretion of IFN-γ and IL-17 from patients with active VKH. A significantly increased frequency of the OPN rs4754 TT genotype (P = 0.004, pc = 0.048) was observed in VKH patients compared with healthy controls. No association could be detected among the four selected SNPs of OPN receptors and VKH. CONCLUSIONS: OPN may be relevant to the pathogenesis of VKH disease. The TT genotype of rs4754 may be a susceptible factor for VKH disease in a Chinese Han population.