Colony Formation Test Research Articles

Δ133p53 decreases the chemosensitivity of carcinoma cell line H1299.

The research evaluated the effect of Δ133p53 on the chemosensitivity of lung adenocarcinoma cell line H1299. By this study, the drug-resistant molecular marker and a new target for cancer therapy could be provided. Δ133p53 or negative control plasmid were transferred into H1299 cells by lentivirus vector. The expression of Δ133p53 in transfected cells was examined using immunofluorescence. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazoliumbromide (MTT) method and colony formation test were applied to detect drug sensitivity after cisplatin or 5-fluorouracil (5-FU) treatment. After cisplatin (CDDP)/FU treatment, MTT assay demonstrated that the inhibition rate of H1299/Δ133p53 cell was reduced compared with that of the H1299 and H1299/NEG cells at the same concentration of drug. The 50% inhibitory concentrations (IC 50 ) of CDDP and 5-FU rose by 36.1 and 30.2%, respectively (P < 0.05). The colony formation assay suggested that the cell proliferation ability of H1299/Δ133p53 cell was prominently increased when compared with that of control group H1299 and H1299 /NEG cells (P < 0.05). The present study demonstrated that the transfection of the Δ133p53 gene in H1299 cells led to the reduction of chemosensitivity.

Effect of PI3K/mTOR Signal Pathway Inhibitor XL765 on Human Leukemic KG-1 Cells

To explore the effect and possible mechanism of PI3K/mTOR inhibitor XL765 on KG-1 cells in vitro. The effect of XL765 on cell proliferation was detected by CCK-8 assay. The colony formation test (200 cells were plated in a plate for 9 days) was used to detect the effect of XL765 on the colony forming ability of KG-1 cells. The apoptosis was assessed by flow cytometry with Annexin V-FITC/PI double staining. Quantitative real-time polymerase chain reaction (q-PCR) was used to detect the expression of cell apoptosis-related genes BCL-2, BAX and caspase-3, Western blot was performed to detect the expression levels of BCL-2, BAX, Caspase-3, and the phosphorylation change of p-PI3K, p-AKT and p-S6K. XL765 effectively inhibited the proliferation and the colony formation of KG-1 cells (P=0.0002). XL765 (150 nmol/L) induced KG-1 cell apoptosis (31.87±1.376%), very statistically significant different from (3.533±0.4179% ) in the control group (P＜0.01). Treatment with 150 nmol/L XL765 could in a significantly increase the expression levels of BAX and active caspase-3, and decreases expression level of the BCL-2 (P＜0.01). In accordance with these results, the Western blot further confirmed the expression decrease of BCL-2 protein along with the increase BAX and cleaved caspase-3 activity. XL765 statistically significantly down-regulated the phosphorylation levels of PI3K, AKT and S6K. PI3K/mTOR inhibitor XL765 substantially suppresses KG-1 cell proliferation and induces apoptosis by inhibiting the activation of PI3K-AKT-mTOR signaling pathway, and regulating the apoptosis-related proteins.

Bmi-1-siRNA Regulates the Proliferation of K562 Leukemia Cells in vitro and in vivo by PTEN/pAKT Pathway

To investigate the effect of Bmi-1 gene silence on the proliferation ability of K562 cells in vitro and in vivo, and to explore the relation of molecular mechanism between proliferation ability of K562 cells in vitro and in vivo with PTEN/pAKT signaling pathway. The Bmi-1 small interference RNA (siRNA) sequences were transfected into K562 cells for decreasing Bmi-1 expression. The effect of Bmi-1 siRNA on the proliferation of K562 cells in vitro and in vivo was detected by MTT method and colony-forming test, the effect of Bmi-1 siRNA on the tumorogenicity of K562 cells was observed by subcutaneous inoculation of K562 cells, LY294002 and Bpv treated K562 cells in nude mice, the expression of Bmi-1, PTEN and pAKT proteins were detected by Western blot. The Bmi-1 siRNA could inhibit the proliferation activity, colony-forming and tumor-forming abilities of K562 cells. After the silence of Bmi-1 gene, the PTEN expression in Bmi-1 gene-silenced group was significantly enhanced. While the pAKT expression in Bmi-1 gene-silenced group was significantly reduced; after the K562 cells were treated with LY294002 (an inhibitor of pAKT), the pAKT expression colony-forming and tumor forming abilities were reduced in comparison with untreated K562 cells; after the K562-S1 cells were treated with Bpv (an inhibitor of PTEN), the PTEN expression decreased, while the pAKT expression, colony forming and tumor-forming abilities were restored. The Bmi-1 gene possibly involves in regulation of K562 proliferation in vivo and in vitro, the effect of PTEN/pAKT signaling pathway maybe one of molecular mechanisms mediating this regulation.

Upregulation of NPL4 promotes bladder cancer cell proliferation by inhibiting DXO destabilization of cyclin D1 mRNA

BackgroundNPL4 is an important cofactor of the valosin-containing protein (VCP)–NPL4–UFD1 complex. The VCP–NPL4–UFD1 has been considered as a ubiquitin proteasome system (UPS) regulator and response to protein degradation. While NPL4 plays important roles in various diseases, little is known about its functions in bladder cancer (BC).MethodsMTT assays and colony forming test were performed to evaluate cell proliferation ability and Western blotting was used to detect protein expression. Cyclin D1 mRNA expression was detected using qRT-PCR, and coimmunoprecipitation (CoIP) was used to detect protein–protein interactions.ResultsNPL4 was upregulated in BC tissue and correlated with poor prognosis. Upregulation of NPL4 promoted cell proliferation while suppression of NPL4 reduced BC cell proliferation. Upregulation of NPL4 led to overexpression of cyclin D1 by enhancing its mRNA stability. Moreover, NPL4 was found to bind directly to DXO and induce its degradation. DXO was downregulated in BC tissue and regulated BC cell proliferation by destabilizing cyclin D1 mRNA. DXO-mediated NPL4 regulated BC cell proliferation by stabilizing cyclin D1 expression.ConclusionsThe NPL4/DXO/cyclin D1 axis exert crucial role in BC cell growth and is associated with prognosis and may represent a potential therapeutic target for BC.

ANXA2 Silencing Inhibits Proliferation, Invasion, and Migration in Gastric Cancer Cells.

Annexin A2 (ANXA2) has been well known to associate with the progress of malignant tumor. However, the biological behavior of ANXA2 in gastric cancer (GC) remains unclear. We made a hypothesis in transcriptome level from TCGA datasets. Then, we used immunohistochemical staining to quantify the expression level of ANXA2 protein in GC tissues compared with adjacent tissues. Quantitative real-time PCR and western blot were used for analyzing ANXA2 expression in human GC (SGC-7901, MKN-45, BGC-823, and AGS) cell lines. We investigated the effect of a lentivirus-mediated knock-down of ANXA2 on the proliferation, invasion and migration of gastric cancer AGS cells. Cell proliferation was examined by MTT and colony formation tests. Cell apoptosis and cycle were measured by flow cytometry. Migration and invasion were detected by transwell assay. We found that high expression of ANXA2 can increase the mobility of cancer cells from TCGA datasets. ANXA2 was upregulated in GC tissues compared with adjacent tissues. AGS cell line displayed significantly higher expression of ANXA2 among the four GC cell lines. In addition, ANXA2 silencing led to a weakened ability of proliferation, invasion, and migration in GC cells; targeting of ANXA2 may be a potential therapeutic strategy for GC patients.

Plumbagin inhibits proliferation and induces apoptosis of hepatocellular carcinoma by downregulating the expression of SIVA.

Purpose: Plumbagin is thought to be a bioactive phytochemical drug and exerts an antitumor effect on various cancers. However, few studies focus on the antitumor activity of plumbagin on liver cancer. This study first investigated the antitumor activity of plumbagin on liver cancer and further investigated the molecular mechanism of its antitumor activity against hepatocellular carcinoma, both in vitro and in vivo.Methods: The antiproliferative activity of plumbagin was evaluated through CCK-8, EdU, and colony forming test. The cell cycle and apoptosis were then analyzed by flow cytometer. Western blot was used to detect the expression of apoptosis related protein, SIVA, and mTOR pathway. RNA-seq was performed to determine the gene expression profiles and overexpressed or knocked down SIVA to validate its role in plumbagin’s antitumor activity. Regarding animal experiment, a xenograft model in BALB/c nude mice was built using LM3-Luci cells. Then bioluminescence imaging and further immunohistochemistry were performed to study the antitumor activity and the expression of SIVA and mTOR in the plumbagin-treated group.Results: Plumbagin can inhibit proliferation and induce apoptosis of liver cancer cells in vitro. Further experiment demonstrated that plumbagin could inhibit the expression of SIVA and subsequently downregulate the mTOR signaling pathway, and upregulating the expression of SIVA will alleviate the antitumor activity of plumbagin on liver cancer, which confirmed the important role of the SIVA/mTOR signaling pathway in the antitumor activity of plumbagin. In vivo bioluminescence imaging showed a decreased signal in the plumbagin-treated group, and further immunohistochemistry demonstrated that plumbagin could inhibit the SIVA/mTOR signaling pathway in tumor tissues.Conclusion: Our promising results showed that plumbagin could inhibit proliferation and induce apoptosis of hepatic cancer through inhibiting the SIVA/mTOR signaling pathway for the first time, which indicated that plumbagin might be a good candidate against liver cancer.

Identification of long non-coding RNA HERC2P2 as a tumor suppressor in glioma.

Long non-coding RNAs (lncRNAs) have been reported to play important roles in glioma; however, most of them promote glioma progression. We constructed a competing endogenous (ceRNA) network based on the Chinese Glioma Genome Atlas dataset, and lncRNA hect domain and RLD 2 pseudogene 2 (HERC2P2) is the core of this network. Highly connected genes in the ceRNA network classified the glioma patients into three clusters with significantly different survival rates. The expression of HERC2P2 is positively correlated with survival and negatively correlated with clinical grade. Cell colony formation, Transwell and cell scratch tests were performed to evaluate the role of HERC2P2 in glioblastoma growth. Furthermore, we overexpressed HERC2P2 in U87 cells and established a mouse intracranial glioma model to examine the function of HERC2P2 in vivo. In conclusion, we identified a lncRNA with tumor suppressor functions in glioma that could be a potential biomarker for glioma patients.

SPARC promotes the proliferation and metastasis of oral squamous cell carcinoma by PI3K/AKT/PDGFB/PDGFRβ axis.

Oral squamous cell carcinoma (OSCC) is a highly lethal cancer in the world, and the prognosis of OSCC is poor with a 60% 5-year survival rate in recent decades. Here, we introduced a novel secretory and acid glycoprotein with cysteine rich (secreted protein acidic and rich in cysteine, SPARC), which is correlated with the worst pattern of invasion (WPOI) and prognosis of OSCC. SPARC expression levels were measured in OSCC tissues and normal tissues using quantitative polymerase chain reaction and immunohistochemistry. The influence of SPARC on cell proliferation was examined by cell counting kit-8, colony formation, and Edu tests. Then, the effect of SPARC on the metastasis of OSCC cells was detected by wound healing and transwell migration assays. Next, the biologic characteristics of SPARC shared by STRING were analyzed. Furthermore, the underlying mechanisms were confirmed by western blot analysis. SPARC revealed higher expression in OSCC tissues than nontumor tissues. Higher SPARC expression was correlated with poorer tumor differentiation, poorer WPOI pattern, and significantly and shorter overall survival. Knockdown SPARC significantly restrained OSCC cell growth, migration, and invasion. In addition, bioinformatics analysis found SPARC had a coexpression network with the platelet-derived growth factor-B (PDGFB) and PI3K/AKT signaling pathways with minimal false discovery rate. Furthermore, SPARC promotes OSCC cells metastasis by regulating the expressions of PDGFB, PDGFRβ, p-PDGFRβ , and the PI3K/AKT pathway. Higher SPARC expression was positively correlated with poor WPOI and differentiation in OSCC. SPARC activates the PI3K/AKT/PDGFB/PDGFRβ axis to promote proliferation and metastasis by OSCC cell lines. Therefore, SPARC may be a potential therapeutic target for patients with OSCC.

Genome-based taxonomic classification.

Bacterial populations are routinely characterized based on microscopic examination, colony formation, and biochemical tests. However, in the recent past, bacterial identification, classification, and nomenclature have been strongly influenced by genome sequence information. Advances in bioinformatics and growth in genome databases has placed genome-based metadata analysis in the hands of researchers who will require taxonomic experience to resolve intricacies. To achieve this, different tools are now available to quantitatively measure genome relatedness within members of the same species, and genome-wide average nucleotide identity (gANI) is one such reliable tool to measure genome similarity. A genome assembly with a gANI score of <95% at the intraspecies level is generally considered indicative of a separate species. In this study, we have analysed 300 whole-genome sequences belonging to 26 different bacterial species available in the NCBI Genome database and calculated their similarity at the intraspecies level based on gANI score. At the intraspecies level, nine bacterial species showed less than 90% gANI and more than 10% of unaligned regions. We suggest the appropriate use of available bioinformatics resources after genome assembly to arrive at the proper bacterial identification, classification, and nomenclature to avoid erroneous species assignments and disparity due to diversity at the intraspecies level.

Up-regulation of Wnt5a inhibits proliferation and migration of hepatocellular carcinoma cells.

Increasing evidence suggests that Wnt5a plays an important role in tumorigenesis. In particular, its expression is downregulated in hepatocellular carcinoma (HCC). The aim of this study was to explore the effect of Wnt5a overexpression on HCC cells. We transfected the human HCC cell line SMMC-7721 with pcDNA3.1-Wnt5a overexpression vectors or empty pcDNA3.1 vectors. The expression of Wnt5a in transfected SMMC-7721 cells was confirmed by the western blot. Cell proliferation was examined by the colony formation test and cell cycle assay in vitro. The effect of Wnt5a overexpression on cell migration was studied using a scratch assay. In vivo tumorigenesis was assessed using a mouse xenograft model. Wnt5a overexpression inhibited SMMC-7721 cell proliferation with a significant reduction in S-phase cells and an enrichment of G1-phase cells, a lower colony formation rate, and decreased tumor volumes in the xenograft model compared with those that of control tumors. The in vitro scratch assay revealed that Wnt5a overexpression diminished the capacity of cell migration, which may be mediated by the change in phosphorylated β-catenin and E-cadherin expression. Wnt5a may act as a tumor suppressor in HCC, partly through the β-catenin/E-cadherin signaling pathway.

Structural, Magnetic, Optical and Antibacterial Properties of Magnetite Ferrofluids with PEG-20000 Template

Abstract Ferrofluids based on magnetite nanoparticles by PEG-20000 as template were successfully prepared through a co-precipitation route. In this experiment, TMAH was employed to coat the magnetite nanoparticles from iron sand preventing agglomerations. The structural, magnetic, optical, and antibacterial performances were investigated by means of XRD, SEM/EDX, FTIR, UV-Vis, VSM, and colony formation test. The magnetite particles were formed in a nanometric size of 9.4 nm in a spherical shape with the lattice parameter of 8.4 A. The FTIR spectrum presented the vibration of Fe-O bond resulting from the magnetite particle both in tetrahedral and octahedral sites. The magnetite nanoparticle had band gap energy of 2.26 eV. Furthermore, the ferrofluids exhibited a superparamagnetic character with a saturation magnetization that was lower than the magnetite nanopowders. Interestingly, the ferrofluids had a significant antibacterial activity on E. coli (gram-negative bacteria) up to multiples times compare to control or placebo. Therefore, it provides a big opportunity to be applied for antiseptical agents as well as for water treatment or sanitation. Besides that, the utilization of the iron sand becomes a novel candidate as an alternative main precursor to produce antibacterial materials.

Fluorine-ion-releasing injectable alginate nanocomposite hydrogel for enhanced bioactivity and antibacterial property

The creation of a moist environment and promotion of cell proliferation and migration together with antibacterial property are critical to the wound-healing process. Alginate (Alg) is an excellent candidate for injectable wound dressing materials because it can form a gel in a mild environment. Taking advantage of its gelation property, an injectable nano composite hydrogel containing nano-sized (about 90 nm) calcium fluoride (CaF2) particles was developed using in-situ precipitation process. The amount of released fluorine (F−) ion from the nanocomposite hydrogel increased with increasing CaF2 content inside the composite hydrogel and the ions stimulated both the proliferation and migration of fibroblast cells in vitro. The antibacterial property of the composite hydrogel against E. coli and S. aureus was confirmed through colony formation test where the number of bacterial colonies significantly decreased compared to Alg hydrogel. The in vivo results based on a full-thickness wound model showed that the nanocomposite hydrogel effectively enhanced the deposition of the extracellular matrix compared to that of the Alg hydrogel. This study demonstrates the potential of this nanocomposite hydrogel as a bioactive injectable wound-dressing material with the ability to inhibit bacterial growth and stimulate cell proliferation and migration for accelerated wound healing.

Destruxin B Suppresses Drug-Resistant Colon Tumorigenesis and Stemness Is Associated with the Upregulation of miR-214 and Downregulation of mTOR/β-Catenin Pathway

Background: Drug resistance represents a major challenge for treating patients with colon cancer. Accumulating evidence suggests that Insulin-like growth factor (IGF)-associated signaling promotes colon tumorigenesis and cancer stemness. Therefore, the identification of agents, which can disrupt cancer stemness signaling, may provide improved therapeutic efficacy. Methods: Mimicking the tumor microenvironment, we treated colon cancer cells with exogenous IGF1. The increased stemness of IGF1-cultured cells was determined by ALDH1 activity, side-population, tumor sphere formation assays. Destruxin B (DB) was evaluated for its anti-tumorigenic and stemness properties using cellular viability, colony-formation tests. The mimic and inhibitor of miR-214 were used to treat colon cancer cells to show its functional association to DB treatment. In vivo mouse models were used to evaluate DB’s ability to suppress colon tumor-initiating ability and growth inhibitory function. Results: IGF1-cultured colon cancer cells showed a significant increase in 5-FU resistance and enhanced stemness properties, including an increased percentage of ALDH1+, side-population cells, tumor sphere generation in vitro, and increased tumor initiation in vivo. In support, using public databases showed that increased IGF1 expression was significantly associated with a poorer prognosis in patients with colon cancer. DB, a hexadepsipeptide mycotoxin, was able to suppress colon tumorigenic phenotypes, including colony and sphere formation. The sequential treatment of DB, followed by 5-FU, synergistically inhibited the viability of colon cancer cells. In vivo studies showed that DB suppressed the tumorigenesis by 5-FU resistant colon cells, and in a greater degree when combined with 5-FU. Mechanistically, DB treatment was associated with decreased the mammalian target of rapamycin (mTOR) and β-catenin expression and an increased miR-214 level. Conclusion: We provided evidence of DB as a potential therapeutic agent for overcoming 5-FU resistance induced by IGF1, and suppressing cancer stem-like properties in association with miR-214 regulation. Further investigation is warranted for its translation to clinical application.

Regulating glioma stem cells by hypoxia through the Notch1 and Oct3/4 signaling pathway.

To investigate the effects of hypoxia on the features of cancer stem cells in the glioma cancer U87 cell line and underlying mechanism, stem cell markers and features in U87 were studied under the hypoxic and normoxic culture conditions by reverse transcription-quantitative polymerase chain reaction, western blot analysis, MTT, a colony formation test and flow cytometry. Compared to the normoxic group, the cluster of differentiation 133+ phenotype, clone formation rate and cell vitality were significantly elevated in U87 cells cultured in a hypoxic microenvironment. Also, the mRNA and protein expression of neurogenic locus notch homolog protein 1 (Notch1) and Oct3/4 were significantly elevated in U87 cells cultured in a hypoxic microenvironment, however, transcription factor SOX-2 expression was not significantly changed. These results indicate that hypoxia can promote the proliferation of glioma stem cells and maintain the characteristics of stem cells through the activation of Notch1 and Oct3/4 or Notch1 activation, affecting the biological characteristics of glioma cells.

Radial shockwave treatment promotes human mesenchymal stem cell self-renewal and enhances cartilage healing

BackgroundShockwaves and mesenchymal stem cells (MSCs) have been widely accepted as useful tools for many orthopedic applications. However, the modulatory effects of shockwaves on MSCs remain controversial. In this study, we explored the influence of radial shockwaves on human bone marrow MSCs using a floating model in vitro and evaluated the healing effects of these cells on cartilage defects in vivo using a rabbit model.MethodsMSCs were cultured in vitro, harvested, resuspended, and treated with various doses of radial shockwaves in a floating system. Cell proliferation was evaluated by growth kinetics and Cell Counting Kit-8 (CCK-8) assay. In addition, the cell cycle and apoptotic activity were analyzed by fluorescence activated cell sorting. To explore the “stemness” of MSCs, cell colony-forming tests and multidifferentiation assays were performed. We also examined the MSC subcellular structure using transmission electron microscopy and examined the healing effects of these cells on cartilage defects by pathological analyses.ResultsThe results of growth kinetics and CCK-8 assays showed that radial shockwave treatment significantly promoted MSC proliferation. Enhanced cell growth was also reflected by an increase in the numbers of cells in the S phase and a decrease in the numbers of cells arrested in the G0/G1 phase in shockwave-treated MSCs. Unexpectedly, shockwaves caused a slight increase in MSC apoptosis rates. Furthermore, radial shockwaves promoted self-replicating activity of MSCs. Transmission electron microscopy revealed that MSCs were metabolically activated by shockwave treatment. In addition, radial shockwaves favored MSC osteogenic differentiation but inhibited adipogenic activity. Most importantly, MSCs pretreated by radial shockwaves exhibited an enhanced healing effect on cartilage defects in vivo. Compared with control groups, shockwave-treated MSCs combined with bio-scaffolds significantly improved histological scores of injured rabbit knees.ConclusionsIn the present study, we found that radial shockwaves significantly promoted the proliferation and self-renewal of MSCs in vitro and safely accelerated the cartilage repair process in vivo, indicating favorable clinical outcomes.

The characteristics of CD44+ /CD24+ cervical cancer cells and radioresistant cervical cancer cells

Objective To investigate the characteristics of radiation resistance of cervical cancer cells, and to explore the mechanism of tumor recurrence and migration. Methods Cervical cancer cells (Siha) were fractionally irradiated to get radioresistant subpopulation. CD44+ /CD24+ Siha cells were sorted with a flow cytometry. Colony-formation tests and tumor xenografts tests were used to evaluate thestemnessof resistant cells. Stem cell markers were studied using fluorescence-activated cell sorting analyses. Migration and invasiveness were assessed by a Transwell test. Gene and protein expressions were determined by RT-PCR and immunoblotting assay, respectively. Results Radiation-resistant Siha cells and CD44+ /CD24+ Siha cells expressed more antiapoptotic protein Bcl-2(t=205.26, 198.17, P<0.05), apoptosis-inhibitory protein Survivin(t=896.62, 765.34, P<0.05)and stem cell markers of OCT-4 and ABCG2 (t=92.13, 81.26, 220.45, 216.32, P<0.05). They were more tumorigenic in vitro and in vivo, showed phenotypic and molecular changes of EMT, and had higher abilities of invasion and migration. Conclusions The radioresistant cervical cancer cells and CD44+ /CD24+ cervical cancer cells are similar to CSCs and undergo EMT, suggesting that radiation resistance-induced EMT is linked to the generation of CSCs. Key words: Cervical cancer stem cells; Radioresistance; Metastasis; Epithelial-mesenchymal transition

The Rim Pathway Mediates Antifungal Tolerance in Candida albicans through Newly Identified Rim101 Transcriptional Targets, Including Hsp90 and Ipt1.

Invasive candidiasis (IC) is a major cause of morbidity and mortality despite antifungal treatment. Azoles and echinocandins are used as first-line therapies for IC. However, their efficacy is limited by yeast tolerance and the emergence of acquired resistance. Tolerance is a reversible stage created due to the yeast's capacity to counter antifungal drug exposure, leading to persistent growth. For Candida albicans, multiple stress signaling pathways have been shown to contribute to this adaptation. Among them, the pH-responsive Rim pathway, through its transcription factor Rim101p, was shown to mediate azole and echinocandin tolerance. The Rim pathway is fungus specific, is conserved among the members of the fungal kingdom, and plays a key role in pathogenesis and virulence. The present study aimed at confirming the role of Rim101p and investigating the implication of the other Rim proteins in antifungal tolerance in C. albicans, as well as the mechanisms underlying it. Time-kill curve experiments and colony formation tests showed that genetic inhibition of all the Rim factors enhances echinocandin and azole antifungal activity. Through RNA sequencing analysis of a rim101-/- mutant, a strain constitutively overexpressing RIM101, and control strains, we discovered novel Rim-dependent genes involved in tolerance, including HSP90, encoding a major molecular chaperone, and IPT1, involved in sphingolipid biosynthesis. Rim mutants were also hypersensitive to pharmacological inhibition of Hsp90. Taken together, these data suggest that Rim101 acts upstream of Hsp90 and that targeting the Rim pathway in combination with existing antifungal drugs may represent a promising antifungal strategy to indirectly but specifically target Hsp90 in yeasts.

Effect of PKA Gene on Acute Lymphoblastic Leukemia in Children and Its Mechanism

To explore the effect of PKA gene on acute T lymphocyte leukemia cells in children and its mechanism. Jurkat and Sup-T1 cells were divided into 2 group: control group (Jurkat and Sup-T1 cells treated with non-specific siRNA) and transfected group (Jurkat and Sup-T1 cells transfected with PKA siRNA). The effects of down-regulating the expression of PKA gene on the viability, proliferotion, migration and cell cycle distribution of Jurkat and Sup-T1 cells in 2 groups were analyzed by CCK-8 assay, transwell experiment, cell colony-formation test and flow cytometry; the cyclin-related protein levels after transfection with PKA siRNA were detected by Western blot. It was revealed that the expression of PKA in Jurkat and Sup-T1 cells decreased to different degree after siRNA transfection(P<0.05). CCK-8 assay showed that the proliferation of Jurkat and Sup-T1 cells in the transfected group was slower than that in the control group(P<0.05). Transwell experiment showed that the migration and invasion ability of Jurkat cells in the transfection group was weaker than that in the control group (P<0.05). The cell colong formation number of Jurkat and Sup-T1 cells in the transfection group decreased significantly (P<0.05). The cell level of Jurkat and Sup-T1 cells in G0/G1 phase increased after tansfection (P<0.05). Western blot assay revealed that the expression levels of CDK2, CyclinD1 and p-Rb in the Jurkat and Sup-T1 cells of the transfection group were suppressed (P<0.05). The down-regulating PKA gene expression can decrease the proliferation and migration of tumor cells, and also can restrict the cell proliferation through related cell cycle proteins.

Hsa-miR-212 modulates the radiosensitivity of glioma cells by targeting BRCA1.

Radioresistance remains a major challenge in the treatment of glioma, and the response of patients to radiotherapy varies considerably. MicroRNAs (miRNAs) are involved in various biological processes. The purpose of the present study was to investigate miRNAs involved in the response to radiation in glioma cell lines. Total RNA was isolated from human glioma U251 cells 30 min after γ-ray exposure and hybridized to an miRNA chip array. miRNA expression profiles were analyzed by quantitative real-time PCR. pcDNA3/EGFP-miR-212 mimic transfection was used to verify the function of miR-212 in colony formation tests, and the effect of miR-212 overexpression on U251 cells was examined by western blot analysis of apoptosis-related proteins (Bcl-2, Bax, caspase-3 and cytochrome c). The target genes of miR-212 were predicted using bioinformatic tools including miRNA databases, and breast cancer susceptibility gene 1 (BRCA1) was selected for further confirmation by EGFP fluorescence reporter and loss- and gain-of-function assays. Of the 16 candidate miRNAs showing altered expression, five were assessed by real-time PCR; miR-212 was identified as contributing to the radioresistance of glioma cells and was shown to attenuate radiation-induced apoptosis. miR-212 negatively regulated BRCA1 expression by interacting with its 3′-untranslated region, suggesting a correlation between BRCA1 expression and radiosensitivity in glioma cells. U-118MG and SHG-44 cell lines were used to confirm these observations. The response of glioma cells to radiation involves the miR-212-mediated modulation of BRCA1 gene expression, suggesting that the miR-212/BRCA1 axis may play a potential role in the radiotherapy of gliomas.

MicroRNA-503 suppresses the migration, proliferation and colony formation of prostate cancer cells by targeting tumor protein D52 like 2.

The present study investigated the expression of microRNA-503 (miR-503) and its effect and mechanism of action on prostate cancer. Tumor tissues and tumor-adjacent tissues were collected from 20 patients with prostate cancer. TargetScan was used to predict the miRNA molecule that interacts with tumor protein D52 like 2 (TPD52L2). DU145 cells were transfected with a negative control, miR-503 mimic or miR-503 inhibitor. DU145 cells that had not undergone transfection were used as a control. Levels of miR-503 and TPD52L2 mRNA were determined using reverse transcription-quantitative polymerase chain reaction and the expression of TPD52L2 protein was measured using western blot analysis. The migration ability of DU145 cells was evaluated using a Transwell assay and cell proliferation was examined using an MTT assay. A flat plate colony formation test was conducted to examine the colony formation rate of DU145 cells. The current study demonstrated that TPD52L2 expression is increased while miR-503 expression is decreased in prostate cancer tissues. Overexpression of miR-503 inhibited the transcription and translation of TPD52L2 in DU145 cells and reduced cell migration, proliferation and colony formation. By contrast, inhibition of miR-503 expression increased the expression of TPD52L2 in DU145 cells and increased cell migration, proliferation and colony formation. The present study demonstrated that miR-503 is an oncogene that regulates the migration, proliferation and colony formation of prostate cancer cells by targeting the TPD52L2 gene. Thus, miR-503 has the potential to become a target for the molecular treatment and prognosis of prostate cancer in the future.