Lower Protein Expression Levels Research Articles

Differential Expression and Prognostic Significance of STARD3 Gene in Breast Carcinoma.

StAR related lipid transfer domain containing 3 (STARD3) gene has been reported to be co-amplified with human epidermal growth factor receptor 2 (HER2) in breast carcinoma. STARD3 is necessary for cholesterol transfer and metabolism in tumor cells. The possible role played by STARD3 as a diagnostic and prognostic biomarker was investigated in breast cancer (BC). Data mining was performed using several bioinformatics websites to investigate the correlation of STARD3 with BC and its molecular subtypes, and conventional PCR was used to detect the STARD3 mRNA levels in a panel of BC cell lines. STARD3 was overexpressed in BC more than the other types of cancer. The results also showed that STARD3 expression was significantly associated with HER2+ BC tumors and BC cell lines, and low STARD3 mRNA and protein expression levels were observed in estrogen receptor-positive (ER+) and triple-negative BC (TNBC) patients. Moreover, high STARD3 expression levels predicted worse overall survival (OS), relapse-free survival (RFS) and disease metastasis-free survival (DMFS) in BC, and HER2+ BC. Notably, low expression of STARD3 was associated with poor OS in ER+ BC. Our findings suggest that STARD3 may have strong diagnostic and prognostic value for HER2+ breast carcinoma.

Quantitative analysis of CDX2 protein expression improves its clinical utility as a prognostic biomarker in stage II and III colon cancer.

Better stratification of patients with stage II and stage III colon cancer for risk of recurrence is urgently needed. The present study aimed to validate the prognostic value of CDX2 protein expression in colon cancer tissue by routine immunohistochemistry and to evaluate its performance in a head-to-head comparison with tandem mass spectrometry-based proteomics. CDX2 protein expression was evaluated in 386 stage II and III primary colon cancers by immunohistochemical staining of tissue microarraysand by liquid chromatography with tandem mass spectrometry (LC-MS/MS) analysis using formalin-fixed paraffin-embeddedtissue sections of a matched subset of 23 recurrent and 23 non-recurrent colon cancers. Association between CDX2 expression and disease-specific survival (DSS) was investigated. Low levels of CDX2 protein expression in stage II and III colon cancer as determined by immunohistochemistry was associated with poor DSS (hazard ratio [HR]=1.97 (95% confidence interval [CI]: 1.26-3.06); p=0.002). Based on analysis of a selected sample subset, CDX2 prognostic value was more pronounced when detected by LC-MS/MS (HR=7.56 (95% CI: 2.49-22.95); p<0.001) compared to detection by immunohistochemistry (HR=1.60 (95% CI: 0.61-4.22); p=0.34). This study validated CDX2 protein expression as a prognostic biomarker in stage II and III colon cancer, conform previous publications. CDX2 prognostic value appeared to be underestimated when detected by routine immunohistochemistry, probably due to the semiquantitative and subjective nature of this methodology. Quantitative analysis of CDX2 substantially improved its clinical utility as a prognostic biomarker. Therefore, development of routinely applicable quantitative assays for CDX2 expression is needed to facilitate its clinical implementation.

Little to no expression of angiotensin-converting enzyme-2 on most human peripheral blood immune cells but highly expressed on tissue macrophages.

Angiotensin-converting enzyme-2 (ACE2) has been recognized as the binding receptor for the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Flow cytometry demonstrated that there was little to no expression of ACE2 on most of the human peripheral blood-derived immune cells including CD4+ T, CD8+ T, activated CD4+ /CD8+ T, Tregs, Th17, NKT, B, NK cells, monocytes, dendritic cells, and granulocytes. There was no ACE2 expression on platelets and very low level of ACE2 protein expression on the surface of human primary pulmonary alveolar epithelial cells. The ACE2 expression was markedly upregulated on the activated type 1 macrophages (M1). Immunohistochemistry demonstrated high expressions of ACE2 on human tissue macrophages, such as alveolar macrophages, Kupffer cells within livers, and microglial cells in brain at steady state. The data suggest that alveolar macrophages, as the frontline immune cells, may be directly targeted by the SARS-CoV-2 infection and therefore need to be considered for the prevention and treatment of COVID-19.

Transcriptional readout of neuronal activity via an engineered Ca2+-activated protease

Molecular integrators, in contrast to real-time indicators, convert transient cellular events into stable signals that can be exploited for imaging, selection, molecular characterization, or cellular manipulation. Many integrators, however, are designed as complex multicomponent circuits that have limited robustness, especially at high, low, or nonstoichiometric protein expression levels. Here, we report a simplified design of the calcium and light dual integrator FLARE. Single-chain FLARE (scFLARE) is a single polypeptide chain that incorporates a transcription factor, a LOV domain-caged protease cleavage site, and a calcium-activated TEV protease that we designed through structure-guided mutagenesis and screening. We show that scFLARE has greater dynamic range and robustness than first-generation FLARE and can be used in culture as well as in vivo to record patterns of neuronal activation with 10-min temporal resolution.

Quantitative expression of Ikaros, IRF4, and PSMD10 proteins predicts survival in VRD-treated patients with multiple myeloma

AbstractThe search for biomarkers based on the mechanism of drug action has not been thoroughly addressed in the therapeutic approaches to multiple myeloma (MM), mainly because of the difficulty in analyzing proteins obtained from purified plasma cells. Here, we investigated the prognostic impact of the expression of 12 proteins involved in the mechanism of action of bortezomib, lenalidomide, and dexamethasone (VRD), quantified by capillary nanoimmunoassay, in CD138-purified samples from 174 patients with newly diagnosed MM treated according to the PETHEMA/GEM2012 study. A high level of expression of 3 out of 5 proteasome components tested (PSMD1, PSMD4, and PSMD10) negatively influenced survival. The 5 analyzed proteins involved in lenalidomide's mode of action were associated with time to progression (TTP); low levels of cereblon and IRF4 protein and high levels of Ikaros, AGO2, and Aiolos were significantly associated with shorter TTP. Although the glucocorticoid receptor (GCR) level by itself had no significant impact on MM prognosis, a high XPO1 (exportin 1)/GCR ratio was associated with shorter TTP and progression-free survival (PFS). The multivariate Cox model identified high levels of PSMD10 (hazard ratio [HR] TTP, 3.49; P = .036; HR PFS, 5.33; P = .004) and Ikaros (HR TTP, 3.01, P = .014; HR PFS, 2.57; P = .028), and low levels of IRF4 protein expression (HR TTP, 0.33; P = .004; HR PFS, 0.35; P = .004) along with high-risk cytogenetics (HR TTP, 3.13; P < .001; HR PFS, 2.69; P = .002), as independently associated with shorter TTP and PFS. These results highlight the value of assessing proteins related to the mechanism of action of drugs used in MM for predicting treatment outcome.

The forkhead box M1 (FOXM1) expression and antitumor effect of FOXM1 inhibition in malignant rhabdoid tumor.

Malignant rhabdoid tumor (MRT) is a rare, highly aggressive sarcoma with an uncertain cell of origin. Despite the existing standard of intensive multimodal therapy, the prognosis of patients with MRT is very poor. Novel antitumor agents are needed for MRT patients. Forkhead box transcription factor 1 (FOXM1) is overexpressed and is correlated with the pathogenesis in several human malignancies. In this study, we identified the clinicopathological and prognostic values of the expression of FOXM1 and its roles in the progression of MRT. We investigated the FOXM1 expression levels and their clinical significance in 23 MRT specimens using immunohistochemistry and performed clinicopathologic and prognostic analyses. We also demonstrated correlations between the downregulation of FOXM1 and oncological characteristics using small interfering RNA (siRNA) and FOXM1 inhibitor in MRT cell lines. Histopathological analyses revealed that primary renal MRTs showed significantly low FOXM1 protein expression levels (p = 0.032); however, there were no significant differences in other clinicopathological characteristics or the survival rate. FOXM1 siRNA and FOXM1 inhibitor (thiostrepton) successfully downregulated the mRNA and protein expression of FOXM1 in vitro and the downregulation of FOXM1 inhibited cell proliferation, drug resistance to chemotherapeutic agents, migration, invasion, and caused the cell cycle arrest and apoptosis of MRT cell lines. A cDNA microarray analysis showed that FOXM1 regulated FANCD2 and NBS1, which are key genes for DNA damage repair. This study demonstrates that FOXM1 may serve as a promising therapeutic target for MRT.

Formation of HERV-K and HERV-Fc1 Envelope Family Members is Suppressed on Transcriptional and Translational Level.

The human genome comprises 8% sequences of retroviral origin, so-called human endogenous retroviruses (HERVs). Most of these proviral sequences are defective, but some possess open reading frames. They can lead to the formation of viral transcripts, when activated by intrinsic and extrinsic factors. HERVs are thought to play a pathological role in inflammatory diseases and cancer. Since the consequences of activated proviral sequences in the human body are largely unexplored, selected envelope proteins of human endogenous retroviruses associated with inflammatory diseases, namely HERV-K18, HERV-K113, and HERV-Fc1, were investigated in the present study. A formation of glycosylated envelope proteins was demonstrated in different mammalian cell lines. Nevertheless, protein maturation seemed to be incomplete as no transport to the plasma membrane was observed. Instead, the proteins remained in the ER where they induced the expression of genes involved in unfolded protein response, such as HSPA5 and sXBP1. Furthermore, low expression levels of native envelope proteins were increased by codon optimization. Cell-free expression systems showed that both the transcriptional and translational level is affected. By generating different codon-optimized variants of HERV-K113 envelope, the influence of single rare t-RNA pools in certain cell lines was demonstrated. The mRNA secondary structure also appears to play an important role in the translation of the tested viral envelope proteins. In summary, the formation of certain HERV proteins is basically possible. However, their complete maturation and thus full biologic activity seems to depend on additional factors that might be disease-specific and await elucidation in the future.

IRAK4 Deficiency Presenting with Anti-NMDAR Encephalitis and HHV6 Reactivation

IRAK4 deficiency is an inborn error of immunity predisposing patients to invasive pyogenic infections. Currently, there is no established simple assay that enables precise characterization of IRAK4 mutant alleles in isolation. Anti-N-methyl-D-aspartate receptor (NMDAR) encephalitis is an autoimmune condition that is characterized by psychiatric symptoms, involuntary movement, seizures, autonomic dysfunction, and central hypoventilation. It typically occurs in adult females associated with tumors. Only a few infantile cases with anti-NMDAR encephalitis have been so far reported. We identified a 10-month-old boy with IRAK4 deficiency presenting with anti-NMDAR encephalitis and human herpes virus 6 (HHV6) reactivation. The diagnosis of IRAK4 deficiency was confirmed by the identification of compound heterozygous mutations c.29_30delAT (p.Y10Cfs*9) and c.35G>C (p.R12P) in the IRAK4 gene, low levels of IRAK4 protein expression in peripheral blood, and defective fibroblastic cell responses to TLR and IL-1 (TIR) agonist. We established a novel NF-κB reporter assay using IRAK4-null HEK293T, which enabled the precise evaluation of IRAK4 mutations. Using this system, we confirmed that both novel mutations identified in the patient are deleterious. Our study provides a new simple and reliable method to analyze IRAK4 mutant alleles. It also suggests the possible link between inborn errors of immunity and early onset anti-NMDAR encephalitis.

Low PTEN expression and overexpression of phosphorylated AktSer473 and AktThr308 are associated with poor overall survival in upper tract urothelial carcinoma.

The PI3K/Akt signaling pathway serves an essential role in various cellular processes, including cell growth, survival, cell motility, angiogenesis and cell metabolism. Loss of PTEN expression and hyperactivation of Akt can result in tumorigenesis. Previous studies observed expression of the Akt protein and absence of the PTEN protein in bladder cancer and non-small cell lung carcinoma tissues. The aim of the present study was to evaluate the expression status and prognostic value of PTEN and the PI3K/Akt signaling pathway in Taiwanese patients with upper tract urothelial carcinoma (UTUC). Archival formalin-fixed, paraffin-embedded (FFPE) tissues from 65 UTUC cases were stained via immunohistochemistry for PTEN, phosphorylated (p)Akt serine (Ser)473 and pAkt threonine (Thr)308. The expression levels of each protein were significantly correlated with clinicopathological parameters. PTEN, pAktSer473 and pAktThr308 protein expression levels were higher in adjacent normal tissues compared with those in tumor tissues. Cytoplasmic PTEN protein expression levels were lower in high-stage tumors compared with those in low-stage tumors, and nuclear and cytoplasmic pAktThr308 protein expression levels were higher in high-grade tumors compared with those in low-grade tumors. Univariate analysis showed that high pathological tumor stage (pT2-4) [P=0.01; hazard ratio (HR)=3.40; 95% confidence interval (CI), 1.34–8.60], metastatic status (P=0.003; HR=3.55, 95% CI, 1.55–8.11), low cytoplasmic PTEN protein expression levels (P=0.016; HR=3.14; 95% CI, 1.24–7.95) and high cytoplasmic pAktSer473 protein expression levels (P=0.019, HR=2.71, 95% CI, 1.18–6.21) were predictive of poor overall survival. However, only metastatic status (P=0.031; HR=2.73; 95% CI, 1.10–6.78), low cytoplasmic PTEN protein expression levels (P=0.017; HR=3.29; 95% CI, 1.24–8.73) and high cytoplasmic pAktSer473 protein expression levels (P=0.027; HR=2.64; 95% CI, 1.12–6.23) remained significant in the multivariate analysis. Kaplan-Meier survival analysis showed that high T stage, metastasis, low expression levels of cytoplasmic PTEN protein and high expression levels of cytoplasmic pAktSer473 protein were significantly associated with poor survival (P=0.006, 0.001, 0.011 and 0.014, respectively). Co-expression of PTENlow/pAktSer473/high and pAktThr308/high phenotypes was associated with a less favorable overall survival (P=0.001). Overall, the present findings demonstrated that low expression levels of PTEN and high expression levels of pAktSer473 and pAktThr308 were predictors for poor overall survival in patients with UTUC.

Experimental study of expression profile and specific role of human microRNAs in regulating atrophic bone nonunion

The expression profile and specific roles of microRNAs (miRNAs) in regulation of atrophic bone nonunion are not fully understood. Here, we present evidence that miRNAs are involved in regulation of several osteogenic genes and may contribute to the development of atrophic bone nonunion.The miRNA expression profile of repairing tissues in atrophic bone nonunion patients (group A) and in callus tissues from patients with healed fractures (group B) were quantitatively measured. microRNA microarrays were used to identify differentially expressed miRNAs, and the bioinformatics methods were used to predict the potential target genes. Quantitative real-time polymerase chain reaction (qRT-PCR), western blot, and dual-luciferase reporter assay were performed in human bone marrow stromal cells (hBMSCs) to validate the microarray results.Nine miRNAs in group A were up-regulated 1.5 times compared to group B, while the other 9 miRNAs in group A were down-regulated 1.5 times. Several target regions of these miRNAs were identified in the osteogenic genes, as well as in the other genes in their families or related regulatory factors. Four miRNAs (hsa-miR-149∗, hsa-miR-221, hsa-miR-628-3p, and hsa-miR-654-5p) could play important roles in regulating bone nonunion development. hBMSCs transfected with these miRNAs significantly decreased mRNA levels of alkaline phosphatase, liver/bone/kidney (ALPL), platelet derived growth factor subunit A (PDGFA), and bone morphogenetic protein 2 (BMP2). Lower protein expression levels were observed using western blotting, confirming that ALPL, PDGFA, and BMP2 were directly targeted by hsa-miR-149∗, hsa-miR-221, and hsa-miR-654-5p, respectively.In summary, hsa-miR-149∗, hsa-miR-221, and hsa-miR-654-5p may play important biological roles by repressing osteogenic target genes ALPL, PDGFA, and BMP2, and, therefore, contributing to progression of atrophic bone nonunion.

Mouse liver is more resistant than skeletal muscle to heat-induced apoptosis.

During passive heat stress, shifting of blood flow from the hepato-splanchnic to peripheral regions produces less favorable physiological conditions in the liver than in the skeletal muscle. We were wondering if the two organs differ in susceptibility to heat injury and thus examined the effects of heat shock exposure on apoptotic and heat stress-related markers in the gastrocnemius muscle and liver of mice. During heat exposure, mice had a peak core body temperature of 41.1 ± 0.7°C. Heat-exposed mice showed higher levels of reactive oxygen species (ROS), cleaved caspases, fragmented DNA, and Drp1 protein expression in the gastrocnemius muscles than control mice. These changes were not observed in the livers of heat-exposed mice. Furthermore, the levels of glucocorticoid receptor, HSP70, and HSF1 proteins were significantly elevated in the gastrocnemius muscles of heat-exposed mice compared with that of control mice. The livers of heat-exposed mice also revealed increased expression of HSP70 but no changes in the other proteins. These results demonstrate that heat exposure induces significantly lower levels of the stress response and apoptosis in the liver than in the skeletal muscle of mice. The liver tissue resistance against heat stress is associated with low levels of heat-induced ROS production and mitochondrial fission protein expression.

Engineering G-quadruplex aptamer to modulate its binding specificity.

The use of aptamers in bioanalytical and biomedical applications exploits their ability to recognize cell surface protein receptors. Targeted therapeutics and theranostics come to mind in this regard. However, protein receptors occur on both cancer and normal cells; as such, aptamers are now taxed with identifying high vs. low levels of protein expression. Inspired by the flexible template mechanism and elegant control of natural nucleic acid-based structures, we report an allosteric regulation strategy for constructing a structure-switching aptamer for enhanced target cell recognition by engineering aptamers with DNA intercalated motifs (i-motifs) responsive to the microenvironment, such as pH. Structure-switching sensitivity can be readily tuned by manipulating i-motif sequences. However, structure-switching sensitivity is difficult to estimate, making it equally difficult to effectively screen modified aptamers with the desired sensitivity. To address this problem, we selected a fluorescent probe capable of detecting G-quadruplex in complicated biological media.

Expression and function of an Hac1-regulated multi-copy xylanase gene in Saccharomyces cerevisiae

Saccharomyces cerevisiae-based expression systems, which rely on safe, food-grade strains, are low cost, simple to operate, and can be used for large-scale fermentation. However, low levels of foreign protein expression by S. cerevisiae have limited their widespread application. The ability of the endoplasmic reticulum (ER) to fold and process foreign proteins is an important factor restricting the expression of foreign proteins. In the current study, the effects of transcription factor Hac1p, which is involved in the unfolded protein response pathway, on S. cerevisiae-based expression of xylanase gene xynB from Aspergillus niger were examined. Overlap extension polymerase chain reaction (PCR), rDNA integration and droplet digital PCR technology were used to generate a S. cerevisiae strain (S8) containing eight copies of xynB, allowing high-yield secretory expression of xylanase. The effects of subsequent overexpression of HAC1 in strain S8 on the expression of genes associated with protein folding in the ER were then examined using the GeXP system. Results confirmed the constitutive secretory expression of the multiple copies of xynB following rDNA-based integration of the expression cassette, with a maximum xylanase yield of 325 U/mL. However, overexpression of HAC1 further improved xylanase production by strain S8, resulting in a yield of 381 U/mL.

RNF168-Mediated Ubiquitin Signaling Inhibits the Viability of BRCA1-Null Cancers.

BRCA1 gene mutations impair homologous recombination (HR) DNA repair, resulting in cellular senescence and embryonic lethality in mice. Therefore, BRCA1-deficient cancers require adaptations that prevent excessive genomic alterations from triggering cell death. RNF168-mediated ubiquitination of γH2AX at K13/15 (ub-H2AX) serves as a recruitment module for the localization of 53BP1 to DNA break sites. Here, we found multiple BRCA1-mutant cancer cell lines and primary tumors with low levels of RNF168 protein expression. Overexpression of ectopic RNF168 or a ub-H2AX fusion protein induced cell death and delayed BRCA1-mutant tumor formation. Cell death resulted from the recruitment of 53BP1 to DNA break sites and inhibition of DNA end resection. Strikingly, reintroduction of BRCA1 or 53BP1 depletion restored HR and rescued the ability of cells to maintain RNF168 and ub-H2AX overexpression. Thus, downregulation of RNF168 protein expression is a mechanism for providing BRCA1-null cancer cell lines with a residual level of HR that is essential for viability. Overall, our work identifies loss of RNF168 ubiquitin signaling as a proteomic alteration that supports BRCA1-mutant carcinogenesis. We propose that restoring RNF168-ub-H2AX signaling, potentially through inhibition of deubiquitinases, could represent a new therapeutic approach. SIGNIFICANCE: This study explores the concept that homologous recombination DNA repair is not an all-or-nothing concept, but a spectrum, and that where a tumor stands on this spectrum may have therapeutic relevance.See related commentary by Wang and Wulf, p. 2720.

465-P: Thrombin Induces SGLT1 and SGLT2 Expression to Promote the AT1R/NADPH Oxidase-Mediated Pro-oxidant Response Inducing Senescence in Atrial Endothelial Cells

Selective sodium-glucose cotransporter 2 (SGLT2) inhibitors have shown cardiovascular protection in type 2 diabetes patients with established cardiovascular (CV) disease independently of glycemic control. Although blood-derived coagulation factors are major contributors of CV diseases, their impact on the expression of SGLT1 and 2 in endothelial cells (ECs) has not been studied. This study investigated whether thrombin affects the expression of SGLT1 and 2 and examined their role in the induction of endothelial senescence. ECs were isolated from porcine atrial tissue (AECs) and used at first passage. Endothelial senescence was assessed by determining senescence-associated beta-galactosidase (SA-beta-gal) activity, oxidative stress using dihydroethidium, and protein level by Western blot analysis and immunofluorescence staining. Exposure of AECs to thrombin (1 or 3 U/ml) for 24 h induced an upregulation of the low protein expression level of SGLT1 and SGLT2. Thrombin induced a sustained pro-oxidant response and increased the level of SA-beta-gal activity that were both inhibited by antioxidants, an angiotensin-converting enzyme inhibitor and an AT1 receptor antagonist and also by sotagliflozin (a dual SGLT1 and SGLT2 inhibitor) and by empagliflozin (a selective SGLT2 inhibitor). In addition, thrombin upregulated the expression level of angiotensin-converting enzyme and AT1 receptors, VCAM-1 and down-regulated that of endothelial NO synthase. In conclusion, thrombin up-regulates both SGLT1 and SGLT2 expression in AECs to sustain oxidative stress leading to endothelial senescence and dysfunction. The fact that the AT1R/NADPH oxidase/SGLT1 and 2 pathway acts in a feedforward manner suggests that inhibition of SGLT1 and/or SGLT2 appears as an attractive strategy to perpetuate the protective endothelial function on the cardiovascular system. Disclosure H. Hasan: None. S. Park: None. E. Belcastro: None. C. Auger: None. H. Lee: None. V. Schini-Kerth: Research Support; Self; Boehringer Ingelheim International GmbH.

Enhanced production of recombinant proteins in Corynebacterium glutamicum by constructing a bicistronic gene expression system

BackgroundCorynebacterium glutamicum is a traditional food-grade industrial microorganism, in which an efficient endotoxin-free recombinant protein expression factory is under developing in recent years. However, the intrinsic disadvantage of low recombinant protein expression level is still difficult to be solved. Here, according to the bacteria-specific polycistronic feature that multiple proteins can be translated in one mRNA, efforts have been made to insert a leading peptide gene upstream of target genes as an expression enhancer, and it is found that this can remarkably improve the expression level of proteins under the control of inducible tac promoter in C. glutamicum.ResultsIn this research, the Escherichia coli (E. coli) tac promoter combined with 24 different fore-cistron sequences were constructed in a bicistronic manner in C. glutamicum. Three strong bicistronic expression vectors were isolated and exhibited high efficiency under different culture conditions. The compatibility of these bicistronic vectors was further validated using six model proteins- aldehyde dehydrogenase (ALDH), alcohol dehydrogenase (ADH), RamA (regulator of acetate metabolism), Bovine interferon-α (BoIFN-α), glycoprotein D protein (gD) of infectious bovine rhinotracheitis virus (IBRV) and procollagen type Ι N-terminal peptide (PΙNP). All examined proteins were highly expressed compared with the original vector with tac promoter. Large-scale production of PΙNP was also performed in fed-batch cultivation, and the highest PΙNP production level was 1.2 g/L.ConclusionIn this study, the strength of the inducible tac promoter for C. glutamicum was improved by screening and inserting fore-cistron sequences in front of the target genes. Those vectors with bicistronic expression patterns have strong compatibility for expressing various heterogeneous proteins in high yield. This new strategy could be used to further improve the performance of inducible promoters, achieving double competence of inducible control and high yield.

Effects of miR‑195‑5p on cell proliferation and apoptosis in gestational diabetes mellitus via targeting EZH2.

Gestational diabetes mellitus (GDM) is a type of diabetes mellitus (DM) that occurs during pregnancy. The present study aimed to investigate the roles of microRNA (miR)-195-5p and enhancer of zeste homolog 2 (EZH2) in GDM, and their potential association. Human umbilical vein endothelial cells (HUVECs) were collected from healthy and GDM umbilical cords, and the endothelial properties were detected by flow cytometry. mRNA expression levels of miR-195-5p and EZH2, and EZH2 protein expression levels were detected by reverse transcription-quantitative PCR (RT-qPCR) and western blot analysis, respectively. Cell colony formation and flow cytometry were performed to determine cell proliferation and apoptosis. Furthermore, the target gene of miR-195-5p was predicted and assessed using a dual-luciferase reporter assay. The levels of cell viability, proliferation and apoptosis following the overexpression of miR-195-5p, EZH2 or miR-195-5p + EZH2, were detected using Cell Counting Kit-8, colony formation and flow cytometry assays, respectively. In addition, the mRNA expression levels of miR-195-59 and EZH2, and EZH2 protein expression levels following transfection with overexpression plasmids were detected using RT-qPCR and western blot analysis, respectively. It was identified that high mRNA expression of miR-195-5p, and low EZH2 mRNA and protein expression levels decreased the level of cell proliferation and the high apoptotic rate of GDM-HUVECs. In addition, miR-195-5p was predicted and identified to target EZH2, and miR-195-5p overexpression was identified to inhibit cell proliferation and promote apoptosis. However, it was demonstrated that upregulation of EZH2 could alleviate the inhibition of cell proliferation and the increased apoptotic rate induced by miR-195-5p overexpression. Therefore, the present results suggested that miR-195-5p may inhibit cell viability, proliferation and promote apoptosis by targeting EZH2 in GDM-induced HUVECs.

The Clinical Significance of PIWIL3 and PIWIL4 Expression in Pancreatic Cancer.

P-element-induced wimpy testis (PIWI) proteins have been described in several cancers. PIWIL1 and PIWIL2 have been recently evaluated in pancreatic cancer, and elevated expression of PIWIL2 conferred longer survival to patients. However, PIWIL3’s and PIWIL4’s role in carcinogenesis is rather controversial, and their clinical implication in pancreatic cancer has not yet been investigated. In the present study, we evaluated PIWIL1, PIWIL2, PIWIL3 and PIWIL4 expression in pancreatic cancer-derived cell lines and in one non-tumor cell line as healthy control. Here, we show a differential expression in tumor and non-tumor cell lines of PIWIL3 and PIWIL4. Subsequently, functional experiments with PIWIL3 and/or PIWIL4 knockdown revealed a decrease in the motility ratio of tumor and non-tumor cell lines through downregulation of mesenchymal factors in pro of epithelial factors. We also observed that PIWIL3 and/or PIWIL4 silencing impaired undifferentiated phenotype and enhanced drug toxicity in both tumor- and non-tumor-derived cell lines. Finally, PIWIL3 and PIWIL4 evaluation in human pancreatic cancer samples showed that patients with low levels of PIWIL4 protein expression presented poor prognosis. Therefore, PIWIL3 and PIWIL4 proteins may play crucial roles to keep pancreatic cell homeostasis not only in tumors but also in healthy tissues.

CHK1 Inhibition Is Synthetically Lethal with Loss of B-Family DNA Polymerase Function in Human Lung and Colorectal Cancer Cells.

Checkpoint kinase 1 (CHK1) is a key mediator of the DNA damage response that regulates cell-cycle progression, DNA damage repair, and DNA replication. Small-molecule CHK1 inhibitors sensitize cancer cells to genotoxic agents and have shown single-agent preclinical activity in cancers with high levels of replication stress. However, the underlying genetic determinants of CHK1 inhibitor sensitivity remain unclear. We used the developmental clinical drug SRA737 in an unbiased large-scale siRNA screen to identify novel mediators of CHK1 inhibitor sensitivity and uncover potential combination therapies and biomarkers for patient selection. We identified subunits of the B-family of DNA polymerases (POLA1, POLE, and POLE2) whose silencing sensitized the human A549 non-small cell lung cancer (NSCLC) and SW620 colorectal cancer cell lines to SRA737. B-family polymerases were validated using multiple siRNAs in a panel of NSCLC and colorectal cancer cell lines. Replication stress, DNA damage, and apoptosis were increased in human cancer cells following depletion of the B-family DNA polymerases combined with SRA737 treatment. Moreover, pharmacologic blockade of B-family DNA polymerases using aphidicolin or CD437 combined with CHK1 inhibitors led to synergistic inhibition of cancer cell proliferation. Furthermore, low levels of POLA1, POLE, and POLE2 protein expression in NSCLC and colorectal cancer cells correlated with single-agent CHK1 inhibitor sensitivity and may constitute biomarkers of this phenotype. These findings provide a potential basis for combining CHK1 and B-family polymerase inhibitors in cancer therapy. SIGNIFICANCE: These findings demonstrate how the therapeutic benefit of CHK1 inhibitors may potentially be enhanced and could have implications for patient selection and future development of new combination therapies.

High IGF1R protein expression correlates with disease-free survival of patients with stage III colon cancer

The aim of this study was to investigate the association between expression of insulin-like growth factor-1 receptor (IGF1R) and its ligand, IGF-II, and disease-free survival (DFS) in patients with stage III colon cancer (CC). In this retrospective study we included consecutive patients who underwent curative surgery for stage III CC. IGF1R and IGF-II/IGF2 status were evaluated in tumour samples by immunohistochemistry and quantitative real-time PCR (qRT-PCR). Associations of markers with DFS were analysed using Cox proportional hazards models. Hundred and fifty-one CC patients were included (median age, 66.6years; female, 54.3%). Low levels of IGF1R and IGF-II protein expression were observed in 16.1% and 10.7% of the cases, respectively. No significant differences in clinicopathological characteristics between patients with tumours expressing low IGF1R or IGF-II protein levels and those with high levels were observed. A low IGF1R protein expression was found to be significantly associated with a shorter DFS (HR 3.32; 95% CI, 1.7-6.31; p= 0.0003), while no association was observed between IGF-II protein expression and DFS (HR 0.91; 95% CI, 0.28-2.96; p= 0.87). In a multivariate analysis, IGF1R protein status remained an independent prognostic factor for DFS (HR 2.73; 95% CI, 1.40-5.31; p= 0.003). Furthermore, we found that neither IGF1R nor IGF2 mRNA expression levels as measured by qRT-PCR correlated with the respective protein expression levels as assessed by immunohistochemistry. Neither of the mRNA expression levels was significantly associated with DFS. From our data we conclude that low IGF1R protein expression represents a poor prognostic biomarker in stage III colon cancer.