MKRN1/2 serve as tumor suppressors in renal clear cell carcinoma by regulating the expression of p53.

BACKGROUND: Kidney renal clear cell carcinoma (KIRC) belongs to renal cell carcinoma which is a very aggressive malignant tumor with poor prognosis and high mortality. The MKRN family includes three members MKRN1, MKRN2 and MKRN3, which are closely related to cancers, and have been involved in many studies. OBJECTIVE: This study aimed to explore the roles of MKRN family in KIRC. METHODS: The expression of MKRNs was analyzed using the UALCAN database, prognostic analysis was performed with the GEPIA2 and Kaplan-Meier Plotter database, and correlation analysis was assessed by GEPIA2. The CCK-8 and colony formation assay were performed to detect cell proliferation, wound healing assays were performed to detect cell migration, cell cycles were detected by flow cytometry analysis, GST pull-down and co-immunoprecipitation assays were performed to detect the interaction of proteins, and the expression of MKRNs, p53 and other proteins were detect by immunoblotting analysis or quantitative PCR (qPCR). RESULTS: MKRN1 and MKRN2 were lowly expressed in KIRC samples compared to the corresponding normal tissues, and KIRC patients with high levels of MKRN1 and MKRN2 showed higher overall survival (OS) and disease free survival (DFS) rates. The overexpression of MKRN1 and MKRN2 inhibited the proliferation of human KIRC cells by arresting the cell cycles, but shows little effect on cells migration. The expression of MKRN1 and MKRN2 are correlated, and MKRN1 directly interacts with MKRN2. Moreover, both MKRN1 and MKRN2 were closely correlated with the expression of TP53 in KIRC tumor, and promoted the expression of p53 both at protein and mRNA levels. CONCLUSIONS: Our study suggests that MKRN1 and MKRN2 serve as tumor suppressors in KIRC, and act as promising therapeutic targets for KIRC treatment.

A CRISPR-based approach for targeted DNA demethylation.

In mammalian cells, DNA methylation critically regulates gene expression and thus has pivotal roles in myriad of physiological and pathological processes. Here we report a novel method for targeted DNA demethylation using the widely used clustered regularly interspaced short palindromic repeat (CRISPR)-Cas system. Initially, modified single guide RNAs (sgRNAs) (sgRNA2.0) were constructed by inserting two copies of bacteriophage MS2 RNA elements into the conventional sgRNAs, which would facilitate the tethering of the Tet1 catalytic domain (Tet-CD), in fusion with dCas9 or MS2 coat proteins, to the targeted gene loci. Subsequently, such system was shown to significantly upregulate transcription of the target genes, including RANKL, MAGEB2 or MMP2, which was in close correlation to DNA demethylation of their neighboring CpGs in the promoters. In addition, the dCas9/sgRNA2.0-directed demethylation system appeared to afford efficient demethylation of the target genes with tenuous off-target effects. Applications of this system would not only help us understand mechanistically how DNA methylation might regulate gene expression in specific contexts, but also enable control of gene expression and functionality with potential clinical benefits.

Profiling human protein degradome delineates cellular responses to proteasomal inhibition and reveals a feedback mechanism in regulating proteasome homeostasis.

Global change in protein turnover (protein degradome) constitutes a central part of cellular responses to intrinsic or extrinsic stimuli. However, profiling protein degradome remains technically challenging. Recently, inhibition of the proteasome, e.g., by using bortezomib (BTZ), has emerged as a major chemotherapeutic strategy for treating multiple myeloma and other human malignancies, but systematic understanding of the mechanisms for BTZ drug action and tumor drug resistance is yet to be achieved. Here we developed and applied a dual-fluorescence-based Protein Turnover Assay (ProTA) to quantitatively profile global changes in human protein degradome upon BTZ-induced proteasomal inhibition. ProTA and subsequent network analyses delineate potential molecular basis for BTZ action and tumor drug resistance in BTZ chemotherapy. Finally, combined use of BTZ with drugs targeting the ProTA-identified key genes or pathways in BTZ action reduced BTZ resistance in multiple myeloma cells. Remarkably, BTZ stabilizes proteasome subunit PSMC1 and proteasome assembly factor PSMD10, suggesting a previously under-appreciated mechanism for regulating proteasome homeostasis. Therefore, ProTA is a novel tool for profiling human protein degradome to elucidate potential mechanisms of drug action and resistance, which might facilitate therapeutic development targeting proteostasis to treat human disorders.

Zoledronic acid inhibits growth of hepatocellular carcinoma cells in vitro and in vivo.

BACKGROUND: Growing preclinical evidence shows that zoledronic acid (ZOL) exhibits direct antitumor activity in various cancer cell lines. However, the cytotoxic effects of ZOL on human hepatocellular carcinoma (HCC) cells have not been established. In the present study, we investigated the effect of ZOL on HCC both in vitro and in vivo. METHODS: Cytotoxicity and cell cycles were assessed with Sulforhodamine B colorimetric assay and flow cytometry. Expression levels of cell cycle phase-linked proteins were examined. The effect of ZOL on HCC in vivo was explored based on H22-subcutaneous injection (s.c.) and H22-intraperitoneal injection (i.p.) mice model. RESULTS: ZOL inhibited the growth of SK-HEP-1 and H22 cells and induced S-phase arrest through downregulating cdc2 protein and upregulating cyclin A. It inhibited the growth of s.c tumors, and increased the survival of both H22-s.c. and H22-i.p. mice in vivo. CONCLUSION: ZOL inhibits growth of HCC cells in vitro and in vivo.

[Proteomic study of the hippocampus tissue from rats with chronic Toxoplasma gondii infection].

OBJECTIVE: To observe the proteome changes in the hippocampus tissue of rats with chronic Toxoplasma gondii infection. METHODS: Six male SD rats were randomly divided into control group and infection group. Each rat in infection group was intraperitoneally injected with 4 x 10(7) purified T. gondii tachyzoites. Rats in the control group received equivalent volumes of sterile normal saline. At the fifth day post-infection, blood samples were taken from the lateral tail vein and Ciemsa staining of blood cells was performed to find Toxoplasma gondii. Rats were dissected at the 10th week post-infection, total protein in the hippocampus was separated by using two-dimensional gel electrophoresis (2-DE). After Coomassie blue staining, the Image Analysis software was used to select and separate proteins on the gel. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) was used for peptide mass fingerprint PMF). Proteins were identified by using Mascot software to search the MSDB and SwissProt databases. RESULTS: Microscopy examination of blood smears confirmed that the rats in infection group were all infected by 11 gondii. The number of protein spots of rats from infection group and control group was 311 +/- 19 and 327 +/- 13 respectively. Compared with the control group, 5 protein spots disappeared, 4 protein spots were up-regulated and 7 were down-regulated in the infection group. The 9 differentially expressed protein spots were identified by MALDL-TOF-MS: phosphoglycerate kinase 1, similar to alpha-enolase, glutamine synthetase, creatine kinase, creatine kinase B-type, ATP synthase, aconitase 2, mitochondrial precursor, actin and an unnamed protein. The first three proteins were up-regulated and the other five proteins were down-regulated in infection group. CONCLUSION: Nine differential expression proteins are found from the hippocampus tissue in rats chronically infected with T. gondii and normal SD rats.

Conformal hyperthermia of superficial tumor with left-handed metamaterial lens applicator.

With proper source spacing, low loss left-handed metamaterial (LHM) lens should be useful for hyperthermia treatment of large area tumors. With a flat LHM lens applicator, conformal hyperthermia can be performed by joint heating of multiple microwave sources (antennas). In the hyperthermia, we restrict distance of two neighboring sources within a critical source interval, arrange the sources in a specific array of general shape in accord with the tumor, and adjust the source-to-lens distance to acquire desired inclination of the heating zone for better fit to tumor region. It is shown that inclination can also be adjusted by the phases of microwave sources. A maneuverable LHM-based hyperthermia scheme is thus proposed to generate a relatively large and even tilted heating pattern in tissue.

[Preliminary study on serum proteomics in mice with acute toxoplasma gondii infection by using functionalized magnetic beads and MALDI-TOF-MS technique].

OBJECTIVE: To detect and analyze the serum protein biomarkers in mice with acute Toxoplasma gondii infection. METHODS: The serum samples from 8 C57BL/6J mice with acute Toxoplasma gondii infection and 8 normal healthy paired mice were prepared with WCX magnetic beads, and then analyzed on PBS II -C mass spectrometer reader. The protein spectra of the serum samples were normalized by the Ciphergen Protein Chip software. The peak labeling was performed by the Biomarker Wizard software. The specific protein biomarkers were screened by the Biomarker Pattern software to construct a diagnostic model for acute Toxoplasma gondii infection. RESULTS: A total of 13 distinguished proteomic peaks were detected. Nine peaks were of up-regulated expressions including m/z values of 1 932.76, 1 976.85, 2 090.53, 5 004.5, 5 776.01, 5 803.05, 5 847.99, 5 877.51 and 7 501.58, respectively; and four peaks were of down-regulated expressions including m/z values of 1 866.40,4 063.71, 8 120.31 and 8 203.83, respectively. CONCLUSION: The potential protein biomarkers for acute Toxoplasma gondii infection are discovered in mouse serum by MALDI-TOF-MS combined with WCX magnetic beads.

Gene silencing of Toll-like receptor 2 inhibits proliferation of human liver cancer cells and secretion of inflammatory cytokines.

BACKGROUND: Toll-like receptors (TLRs) are key factors in the innate immune system and initiate the inflammatory response to foreign pathogens such as bacteria, fungi and viruses. In the microenvironment of tumorigenesis, TLRs can promote inflammation and cell survival. Toll-like receptor 2/6 (TLR2/6) signaling in tumor cells is regarded as one of the mechanisms of chronic inflammation but it can also mediate tumor cell immune escape and tumor progression. However, the expression of TLR2 and its biological function in the development and progression of hepatocarcinoma have not been investigated. This study aimed to determine the expression of TLRs 1-10 in the established human hepatocellular carcinoma cell line BLE-7402, to investigate the biological effect of TLR2 on cell growth and survival. METHODS: TLR expression in BLE-7402 cells was assayed by RT-PCR, real-time PCR and flow cytometry (FCM). To further investigate the function of TLR2 in hepatocarcinoma growth, BLE-7402 cells were transfected with recombinant plasmids expressing one of three forms of TLR2 siRNA (sh-TLR2 RNAi(A, B and C)). TLR2 knockdown was confirmed using RT-PCR, real-time PCR and fluorescence microscopy. Tumor cell proliferation was monitored by MTT assay and secreted cytokines in the supernatant of transfected cells were measured by bead-based FCM, the function of TLR2 siRNA was also investigated in vivo. RESULTS: The BLE-7402 cell line expressed TLRs 2 to 10 at both mRNA and protein levels. TLR2 was the most highly expressed TLR. While all the three siRNAs inhibited TLR2 mRNA and protein expression, sh-TLR2 RNAi(B) had the strongest knockdown effect. TLR2 knockdown with sh-TLR2 RNAi(B) reduced cell proliferation. Furthermore, secretion of IL-6 and IL-8 was also reduced. The result showed a drastic reduction in tumor volume in mice treated with sh-TLR2 RNAi(B). DISCUSSION: These results suggest that TLR2 knockdown inhibit proliferation of cultured hepatocarcinoma cells and decrease the secretion of cytokines. It is suggested that TLR2 silencing may worth further investigations for siRNA based gene therapy in treatment of hepatocarcinoma.

Effect of the in vivo activity of dihydroartemisinin against Schistosoma mansoni infection in mice.

Dihydroartemisinin, formerly known as an antimalarial drug, is the main metabolite of the mother compound artemisinins, as well as of artemether and artesunate. It has been shown that the drug exhibits antischistosomal efficacy against Schistosoma japonicum. The purpose of the current study was to assess the in vivo effect of dihydroartemisinin against Schistosoma mansoni infection in mice. Drugs at a single oral dose of 300 mg/kg were given to mice to assess the efficacy against different developmental stages of the parasite; juvenile and adult S. mansoni were treated with single doses of dihydroarteminisin with different regimens (at 200, 300, 400 or 600 mg/kg) in the stage of drug sensitivity, and the dose-response relationship was assessed; and the effect of multiple doses (at 200, 300 or 400 mg/kg) on juvenile and adult S. mansoni was also observed. The results showed that a single oral dose (300 mg/kg) of dihydroartemisinin reduced total worm burdens by 13.8-82.1% and female worm burdens by 13-82.8%, and the greatest reductions were seen when treatment was given on day 21 post-infection, with total and female worm burden reductions of 82.1% and 82.8%. Administration of a single oral dose of dihydroartemisinin on day 21 post-infection with different drug dosage (at 200, 300, 400 or 600 mg/kg) reduced total worm burdens by 70.3-87.3% and female worm burdens by 73.5-92.4%, depending on dosage. Similar treatments given on day 49 post-infection reduced total worm burdens by 48.7-68.73% and female worm burdens by 63.25-94.6%. There was obvious dose-response relationship of dihydroartemisinin against the schistosomula and adult worms of S. mansoni observed. Administration with dihydroartemisinin at oral doses of 200, 300 and 400 mg/kg, given once on each of days 20-22 post-infection of three successive days, reduced total worm burdens by 88.5-90.1% and female worm burdens by 89.2-92.1%, depending on dosage. Similar treatments given once on each of days 48-50 post-infection reduced total worm burdens by 60-70.3% and female worm burdens by 77.5-94.9%. It is concluded that dihydroartemisinin exhibits in vivo activity against various developmental stages of S. mansoni, particularly the 21-day schistosomula, and there is obvious dose-response relationship of dihydroartemisinin against the schistosomula and adult worms of S. mansoni observed.

[Studies on spray of niclosamide ethanolamine salt II observation on prevention of Schistosoma japonicum infection in bovine].

OBJECTIVE: To investigate the spray of niclosamide ethanolamine salt on prevention of bovine schistosomiasis in the field so as to provide a technical support for the improvement of schistosomiasis control strategy. METHODS: A total of 160 buffalo were selected as experimental objects marked by ear-mark numbers. All the buffalo were administered with praziquantel and then randomly divided into 3 groups, which were sprayed with niclosamide ethanolamine salt (500 ml per head) every 15 d (Group A), every 30 d (Group B) and an agent without niclosamide ethanolamine salt every 15 d (Group C as the control), respectively. The buffalo's droppings were collected to examine the eggs of schistosome every 30 days during the trial. RESULTS: Ninety days after the spraying, the prevalence rates of schistosomiasis were 4.00%, 4.08%, and 24.49% in the Group A, Group B, and Group C, respectively. Compared with the control group (Group C), the decline prevalence rates of schistosomiasis were 83.67% and 83.34% in the Group A and Group B, respectively. CONCLUSIONS: The buffalo spraying with 1% niclosamide ethanolamine salt can reduce schistosomiasis prevalence in bovine, that is this intervention has an obvious protective effect.

[Effect of latent asymptomatic toxoplasmosis on glucose metabolism in brain of mice].

OBJECTIVE: To explore the effect of latent asymptomatic Toxoplasma gondii infection on glucose metabolism in brain of mice. METHODS: Twenty mice were randomly divided into two groups: a Toxoplasma infected group and normal control group. The mice in the Toxoplasma infected group were inoculated with 0.3 ml of brain suspension in saline containing ten Toxoplasma gondii tissue cysts, avirulent Toxoplasma gondii Prugniaud (PRU, a Type II strain). The mice in the control group received 0.3 ml of saline orally. Six monthes after the infection, the glucose metabolism changes in the mouse brain were evaluated by MicroPET, then all the mice were sacrificed and the brain tissues were observed histopathologically. RESULTS: Compared with the normal controls, the infected mice demonstrated profound and widespread brain pathology, and MicroPET indicated a significant glucose metabolism reduction in the brain of asymptomatic Toxoplasma gondii infected mice. CONCLUSION: Chronic Toxoplasma gondii infection maybe results in the glucose metabolism reduction in the brain of mice.

Effects of artemether, artesunate and dihydroartemisinin administered orally at multiple doses or combination in treatment of mice infected with Schistosoma japonicum.

Artemether and artesunate, derivatives of the antimalarial artemisinin, as well as their main metabolite, dihydroartemisinin, all exhibit antischistosomal activities. The purpose of the current study was to compare the effects of artemether, artesunate and dihydroartemisinin administered orally at multiple doses or combination in treatment of mice infected with Schistosoma japonicum. We carried out experiments with mice, infected with 40 cercariae of S. japonicum, and treated with artemether, artesunate and dihydroartemisinin (all at a single dose of 300 mg/kg, and the dose of the mixed three drugs is also 300 mg/kg) at multiple doses or combination therapy on days 6-8 or 34-36 post-infection. Administration with artemether, artesunate or dihydroartemisinin for 3 successive days reduced total worm burdens by 79.5-86% (30.86 ± 4.98 of mean total worm burden in control), female worm burdens by 79.4-86.7% (11.29 ± 2.63 of mean female worm burden in control) (all P values <0.01 vs. control), depending on different treatment protocols given on days 6-8 post-infection. However, no differences were seen between each treatment group (all P > 0.05). While the same treatment was given on days 34-36 post-infection, total worm burden reductions of 73.8-75.8% were achieved (29.44 ± 3.36 of mean total worm burden in control), which were significant when compared with the untreated control group (all P values <0.01). In all different treatment groups, female worm reductions (ranging from 88.7% to 93.1%, while the mean female worm burden in control is 10.33 ± 1.80) were consistently higher than the total worm reductions, resulting always in significantly lower female worm burdens when compared to the corresponding control (all P values < 0.01). However, there were no significant differences found between each treatment group (all P values >0.05). It is concluded that artemether, artesunate and dihydroartemisinin can be used to control schistosomiasis japonica, as a strategy to prevent S. japonicum infection. Administration with artemether, artesunate and dihydroartemisinin at multiple doses or in combined treatment damages both juvenile and adult S. japonicum, without statistically significant differences among the three drugs at the same dose.

[Studies on resistance of Schistosoma to praziquantel XIV experimental comparison of susceptibility to praziquantel between PZQ-resistant isolates and PZQ-susceptible isolates of Schistosoma japonicum in stages of adult worms, miracidia and cercariae].

OBJECTIVE: To investigate the changes of sensitivity to praziquantel (PZQ) about PZQ-resistant isolates of Schistosoma japonicum established in laboratory by means of the resistance-inducement method during the stages of adult worms, cercariae and miracidia, so as to provide the basis for establishing the sensitivity-detecting technique to praziquantel. METHODS: A Jiangsu laboratory-maintaining isolate and a Hunan field-collecting isolate of S. japonicum that were never treated with PZQ were as PZQ-susceptible isolates, and two PZQ-induced isolates that were established via drug-treated passage in laboratory were as PZQ-resistant isolates. Mice were infected with S. japonicum cercariae collected from above four isolates each. Thirty-five days after the infection, the mice were divided into 6 groups and administered orally with PZQ at dosages of 0, 37.5, 75, 150, 300 mg/kg and 600 mg/kg, respectively. All the mice were sacrificed two weeks after the treatment, and all the adult worms in the hepatic and portomesenteric veins were recovered and counted. The mean worm burden and reductions were calculated and input into Graphpad Prism 5.0 software, and the PZQ ED50 values of four isolates were calculated by the software. The cercariae of above four isolates were exposed to 10(-5), 5 x 10(-6), 10(-6), 5 x 10(-7), 10(-7) mol/L PZQ solutions for 20, 40, 60, 80, 100 min and the changes of tail shedding were observed under a dissecting microscope, then the tail shedding rates of cercariae were calculated. The miracidia of above four isolates were exposed to 5 x 10(-6), 10(-6), 5 x 10(-7), 10(-7) mol/L PZQ solutions for 1, 3 and 5 min and the morphological changes were observed under a dissecting microscope, then the morphological change rates of miracidia were calculated. RESULTS: The PZQ ED50 values of PZQ-susceptible and PZQ-resistant isolates of Jiangsu were 147.7 mg/kg and 565.5 mg/kg, respectively, and the PZQ ED50 values of PZQ-susceptible and PZQ-resistant isolates of Hunan were 151.8 mg/kg and 467.2 mg/kg, respectively. When the cercariae were exposed to 10(-5) mol/L PZQ solution over 20 min, the tail shedding rate of cercariae from PZQ-susceptible isolate of Jiangsu was 68.8%, and the tail shedding rate of cercariae from PZQ-resistant isolate of Jiangsu was 38.2% (P < 0.01). When the cercariae were exposed to 10(-7) mol/L PZQ solution over 100 min, the tail shedding rate of cercariae from PZQ-susceptible isolate of Jiangsu was 15.9%, and the tail shedding rate of cercariae from PZQ-resistant isolate of Jiangsu was 6.7% (P < 0.01). When the cercariae were exposed to 10(-5) mol/L PZQ solution over 20 min, the tail shedding rates of cercariae from PZQ-susceptible isolate of Hunan was 59.4%, and the tail shedding rates of cercariae from PZQ-resistant isolate of Hunan was 54.6% (P < 0.05). When the cercariae were exposed to 5 x 10(-7) mol/L PZQ solution over 40 min, the tail shedding rates of cercariae from PZQ-susceptible isolate of Jiangsu was 34.3%, and the tail shedding rates of cercariae from PZQ-resistant isolate of Jiangsu was 18.4% (P < 0.01). When the miracidia were exposed to 5 x 10(-7) mol/L and 10(-7) mol/L PZQ solutions for 1, 3 and 5 min respectively, the morphological change rates of miracidia from PZQ-susceptible isolates of Jiangsu and Hunan were significantly higher than those of PZQ-resistant isolates (P < 0.01). CONCLUSIONS: PZQ-resistant isolates of S. japonicum has been established in mice with sub-curative doses of PZQ by artificial selection in laboratory, and their sensitivities to PZQ are significantly lower than those of the isolates never treated with PZQ. The drug-resistance could exhibit in the stages of adult worms, cercariae and miracidia. The PZQ ED50 value of adult worms, the tail shedding rates of cercariae and the morphological change rates of miracidia as quantitative indicators can be used for monitoring the S. japonicum sensitivity to PZQ.