Therapeutic potential and mechanisms of Rifaximin in ameliorating iron overload-induced ferroptosis and liver fibrosis in vivo and in vitro.

Liver fibrosis could progress to liver cirrhosis with several contributing factors, one being iron overload which triggers ferroptosis, a form of regulated cell death. Rifaximin, a non-absorbable antibiotic, has shown promise in mitigating fibrosis, primarily by modulating gut microbiota. This study investigated the effects and mechanisms of rifaximin on iron overload-related hepatic fibrosis and ferroptosis. In an iron overload-induced liver fibrosis model in mice and in ferric ammonium citrate (FAC)-stimulated primary hepatocytes, treatment with rifaximin showed significant therapeutic effects. Specifically, it ameliorated the processes of ferroptosis triggered by iron overload, reduced liver injury, and alleviated fibrosis. This was demonstrated by decreased iron accumulation in the liver, improved liver function, and reduced fibrotic area and collagen deposition. Rifaximin also modulated key proteins related to iron homeostasis and ferroptosis, including reduced expression of TFR1, a protein facilitating cellular iron uptake, and increased expression of Fpn and FTH, proteins involved in iron export and storage. In the context of oxidative stress, rifaximin treatment led to a decrease in lipid peroxidation, evidenced by reduced levels of reactive oxygen species (ROS) and malondialdehyde (MDA), and an increase in the reduced glutathione (GSH) and decrease in oxidized glutathione (GSSG). Notably, rifaximin's potential functions were associated with the TGF-ß pathway, evidenced by suppressed Tgfb1 protein levels and ratios of phosphorylated to total Smad2 and Smad3, whereas increased Smad7 phosphorylation. These findings indicate rifaximin's therapeutic potential in managing liver fibrosis by modulating the TGF-ß pathway and reducing iron overload-induced damage. Further research is required to confirm these results and explore their clinical implications.

ROR1-AS1 might promote in vivo and in vitro proliferation and invasion of cholangiocarcinoma cells.

Long non-coding RNAs (lncRNAs) play important roles in many pathophysiological processes, including cancer progression. Namely, lncRNA Receptor-tyrosine-kinase-like orphan receptor-1 antisense 1 (ROR1-AS1) is crucial for cancer occurrence and progression in organs such as the liver or bladder. However, its expression and role in cholangiocarcinoma (CCA) have not been thoroughly explored.Firstly, we assessed cell viability, proliferation, invasion, and migration using three cell lines (HuCCT-1, QBC399, and RBE) to explore the biological characteristics of ROR1-AS1 in CCA. Secondly, to determine the in vivo effect of ROR1-AS1 on tumor growth, ROR1-AS1 knockdown (KD) HuCCT-1 cells were subcutaneously injected into nude mice to evaluate tumor growth. Finally, we conducted a bioinformatic analysis to confirm the role of ROR1-AS1 in the prognosis and immunity of CCA.In this study, we found that lncRNA ROR1-AS1 was increased in CCA samples and patients with higher ROR1-AS1 expression had a shorter overall survival period. siRNA-mediated KD of ROR1-AS1 significantly reduced cell proliferation and inhibited the migration of CCA cells. In addition, ROR1-AS1 KD HuCCT-1 cells injected into nude mice grew slower than normal CCA cells.In summary, our results show that ROR1-AS1 can promote CCA progression and might serve as a new target for diagnosis and treatment of CCA.

Cardiovascular risk factors and breast cancer incidence in a large middle-aged cohort study.

BACKGROUND: Several studies have demonstrated that cardiovascular risk factors play a role in the etiology of breast cancer. However, the combined effect of cardiovascular risk factors on the risk of breast cancer is still uncertain. METHODS: Data from the Atherosclerosis Risk in Communities (ARIC) study, a prospective cohort of middle-aged women, were used to investigate the association of individual and combined cardiovascular risk factors with breast cancer. Cox proportional hazards models were applied to calculate the hazard ratio (HR) and 95% confidence intervals (CI). RESULTS: A total of 7501 women were included. During a mean follow-up of 19.7 years, 576 women were diagnosed with breast cancer. White women and premenopausal status were independently associated with increased risk of breast cancer. Of the individual cardiovascular risk factors, only obesity was independently associated with an increased risk of breast cancer (HR 1.29, 95% CI 1.04-1.61). Compared with women without cardiovascular risk factors, women having three or greater, but not those with fewer than three cardiovascular risk factors, had a significantly higher risk of developing breast cancer (HR 1.27, 95% CI 1.06-1.53). Subgroup analyses indicated that women with three or greater cardiovascular risk factors had higher risk of breast cancer among postmenopausal Black women, but not among premenopausal Black and White women. CONCLUSIONS: Combinations of cardiovascular risk factors are associated with increased risk of breast cancer in middle-aged women, especially in postmenopausal Black women. Joint interventions to modify cardiovascular risk factors could be used to prevent breast cancer in these higher-risk individuals.

Adenovirus associated with acute diarrhea: a case-control study.

BACKGROUND: Diarrhea is a major source of morbidity and mortality among young children in low-income and middle-income countries. Human adenoviruses (HAdV), particular HAdV species F (40, 41) has been recognized as important causal pathogens, however limited data exist on molecular epidemiology of other HAdV associated with acute gastroenteritis. METHODS: In the present preliminary study, we performed a case-control study involving 273 children who presented diarrheal disease and 361 healthy children matched control in Children's hospital of Hebei Province (China) to investigate the relationship between non-enteric HAdV and diarrhea. HAdV were detected and quantified using quantitative real-time PCR (qPCR) and serotyped by sequencing and phylogenetic analysis. Odds ratio (OR) was used to assess the risk factor of HAdV. RESULTS: HAdV were detected in 79 (28.94%) of 273 children with diarrhea including 7 different serotypes (HAdV 40, 41, 3, 2,1,5 and 57) with serotypes 40, 41 and 3 being the most dominant and in 26 (7.20%) of 361 healthy children containing 9 serotypes (HAdV 40, 41, 3, 2,1,5,57,6 and 31). A majority (91.14%) of HAdV positives occurred in diarrhea children and 65.38% in controls< 3 years of age. No significant difference in the viral load was found between case and control groups or between Ad41-positive patients and healthy controls. In addition to HAdV 40 and 41, HAdV 3 was also associated with diarrhea (OR = 17.301, adjusted OR = 9.205, p < 0.001). CONCLUSIONS: Our results demonstrate a high diversity of HAdV present among diarrhea and healthy children and implicate that non-enteric HAdV3 may lead to diarrhea.

Use of Acellular Dermal Matrices in One-stage Implant-based Breast Reconstruction.

Implant-based breast reconstruction is the most common choice in breast cancer patients. Recently,the acellular dermal matrix (ADM) technique has been widely used in implant-based breast reconstruction in the western countries. This article briefly reviews the biological characteristics,history,types,surgical techniques,and postoperative complications of ADM.

Isolation and Enrichment of Extracellular Vesicles with Double-Positive Membrane Protein for Subsequent Biological Studies.

The isolation and enrichment of specific extracellular vesicle (EV) subpopulations are essential in the context of precision medicine. However, the current methods predominantly rely on a single-positive marker and are susceptible to interference from soluble proteins or impurities. This limitation represents a significant obstacle to the widespread application of EVs in biological research. Herein, a novel approach that utilizes proximity ligation assay (PLA) and DNA-RNA hybridization are proposed to facilitate the binding of two proteins on the EV membrane in advance enabling the isolation and enrichment of intact EVs with double-positive membrane proteins followed by using functionalized magnetic beads for capture and enzymatic cleavage for isolated EVs release. The isolated subpopulations of EVs can be further utilized for cellular uptake studies, high-throughput small RNA sequencing, and breast cancer diagnosis. Hence, developing and implementing a specialized system for isolating and enriching a specific subpopulation of EVs can enhance basic and clinical research in this field.

Simultaneous detection of membrane protein and mRNA at single extracellular vesicle level by droplet microfluidics for cancer diagnosis.

INTRODUCTION: Simultaneous detection of proteins and mRNA within a single extracellular vesicle (EV) enables comprehensive analysis of specific EVs subpopulations, significantly advancing cancer diagnostics. However, developing a sensitive and user-friendly approach for simultaneously detecting multidimensional biomarkers in single EV is still challenging. OBJECTIVES: To facilitate the analysis of multidimensional biomarkers in EVs and boost its clinical application, we present a versatile droplet digital system facilitating the concurrent detection of membrane proteins and mRNA at the single EV level with high sensitivity and specificity. METHODS: The antibody-DNA conjugates were firstly prepared for EVs protein biomarkers recognition and signal transformation. Coupling with the assembled triplex droplet digital PCR system, a versatile droplet digital analysis assay for simultaneous detection of membrane protein and mRNA at a single EV level was developed. RESULTS: Our new droplet digital system displayed high sensitivity and specificity. Additionally, its clinical application was validated in a breast cancer cohort. As expected, this assay has demonstrated superior performance in distinguishing breast cancer from healthy individuals and benign controls through combined detection of EVs protein and mRNA markers compared to any single kind marker detections, especially for patients with breast cancer at early stage (AUC=0.9229). CONCLUSION: Consequently, this study proposes a promising strategy for accurately identifying and analyzing specific EV subgroups through the co-detection of proteins and mRNA at the single EV level, holding significant potential for future clinical applications.

Establishment and validation of a nomogram model for preoperative prediction of the risk of cholangiocarcinoma with perineural invasion.

OBJECTIVE: To establish and validate a nomogram model for predicting the risk of cholangiocarcinoma with perineural invasion. METHODS: We retrospectively collected the clinical data of 356 patients with surgically confirmed cholangiocarcinoma, including 98 cases of extrahepatic cholangiocarcinoma (eCCA), 197 cases of intrahepatic cholangiocarcinoma (iCCA), and 61 cases of perihilar cholangiocarcinoma (pCCA). RESULTS: Based on these data, we determined the influencing factors of preoperative perineural invasion risk in patients with cholangiocarcinoma by forward multivariate regression analysis. Based on these variables, we established two nomogram models. The model variables for predicting perineural invasion of eCCA included prothrombin time, high-density lipoprotein and tumor size (all P<0.05). The consistency index (C-index) of internal and external validation was 0.845 and 0.806, respectively. In addition, the model variables for predicting perineural invasion of iCCA included carcinoembryonic antigen, carbohydrate antigen 19-9 and tumor size (all P<0.05). The internal and external validation of the C-index was 0.735 and 0.886, respectively. Both models have considerable results in terms of calibration accuracy and clinical decision-making. Kaplan-Meier survival analysis showed that the survival time of patients with perineural invasion was significantly reduced (P=0.033). CONCLUSIONS: We established a predictive model for preoperative perineural invasion in patients with iCCA and eCCA, and this model can provide good predictive value for clinicians. However, we have not obtained relevant predictive variables for predicting perineural invasion of pCCA, and the number of modeling cases was relatively small, so this study needs to be further explored.

Engineered Enzyme-Loaded Erythrocyte Vesicles Precisely Deprive Tumoral Nutrients to Induce Synergistic Near-Infrared-II Photothermal Therapy and Immune Activation.

Starvation therapy has been considered a promising strategy in cancer treatment for altering the tumor microenvironment (TME) and causing a cascade of therapeutic effects. However, it is still highly challenging to establish a therapeutic strategy for precisely and potently depriving tumoral nutrition. In this study, a glucose oxidase (GOx) and thrombin-incorporated erythrocyte vesicle (EV) with cyclic (Arg-Gly-Asp) (cRGD) peptide modification, denoted as EV@RGT, were synthesized for precisely depriving tumoral nutrition and sequentially inducing second near-infrared region (NIR-II) photothermal therapy (PTT) and immune activation. The EV@RGT could specifically accumulate at the tumor site and release the enzymes at the acidic TME. The combination of GOx and thrombin exhausts tumoral glucose and blocks the nutrition supply at the same time, resulting in severe energy deficiency and reactive oxygen species (ROS) enrichment within tumor cells. Subsequently, the abundant clotted erythrocytes in tumor vessels present outstanding localized NIR-II PTT for cancer eradication owing to the hemoglobin. Furthermore, the abundant ROS generated by enhanced starvation therapy repolarizes resident macrophages into the antitumor M1 phenotype via a DNA damage-induced STING/NF-κB pathway, ultimately contributing to tumor elimination. Consequently, the engineered EV@RGT demonstrates powerful antitumor efficiency based on precise nutrition deprivation, sequential NIR-II PTT, and immune activation effect. This work provides an effective strategy for the antitumor application of enzyme-based reinforced starvation therapy.

Biomarkers for predicting the prognosis of intrahepatic cholangiocarcinoma: A retrospective single-center study.

The aim of this retrospective study was to investigate the association between preoperative serological and clinical indicators and postoperative recovery in patients who had undergone resection of intrahepatic cholangiocarcinoma (ICC). We collected data form the medical records of patients who underwent operations for the treatment of ICC at Qingdao University Affiliated Hospital from 2015 to 2021. We analyzed the data to explore the independent predictors of disease prognosis after surgery for ICC. By univariate analysis, we found that the following factors were significantly associated with overall survival and tumor-free survival in patients with ICC: TNM stage; degree of vascular invasion; levels of hemoglobin, carcinoembryonic antigen, carbohydrate antigen 125, direct bilirubin, alkaline phosphatase, and albumin; prothrombin time; neutrophil to lymphocyte ratio; prothrombin time to albumin ratio; albumin to alkaline phosphatase ratio; albumin to gamma-glutamyl transferase ratio; prognostic nutrition Index, and incisional margin. However, only carbohydrate antigen 24-2 and glutamyl transpeptidase were correlated with overall survival in patients with ICC. However, only a positive history of biliary surgery was significantly associated with tumor-free survival in patients with ICC. Preoperative prothrombin time, vascular invasion, N-stage, incisal edge, and carcinoembryonic antigen levels may be simple predictors of disease progression in ICC after hepatectomy.

Polyphyllin VII protects from breast cancer-induced osteolysis by suppressing osteoclastogenesis via c-Fos/NFATc1 signaling.

Bone is a preferred metastatic site of advanced breast cancer and the 5-year overall survival rate of breast cancer patients with bone metastasis is only 22.8%. Targeted inhibition of osteoclasts can treat skeletal-related events (SREs) in breast cancer patients. Polyphyllin VII (PP7), a pennogenyl saponin isolated from traditional Chinese herb Paris polyphylla, exhibits strong anti-inflammatory and anti-cancer activities. In this study, we evaluated the effect of PP7 on metastatic breast cancer-induced bone destruction in vivo and the underlying mechanisms. We found that intraperitoneal injection of 1 mg/kg PP7 significantly ameliorated the breast cancer MDA-MB-231 cell-induced osteolysis in mice. Mechanistically, PP7 (0.125-0.5 µM) inhibited the conditioned medium of MDA-MB-231 cells (MDA-MB-231 CM)-induced osteoclast formation in bone marrow-derived macrophages (BMMs). Furthermore, PP7 markedly reduced MDA-MB-231 CM-induced osteoclastic bone resorption and F-actin rings formation in vitro. During MDA-MB-231 CM-induced osteoclastogenesis, the activation of c-Fos and NFATc1 signaling was significantly downregulated by PP7, and finally osteoclast-related genes such as Oscar, Atp6v0d2, Mmp9 and ß3 integrin were decreased. In addition, the formation of osteoblast was promoted by PP7 treatment. Our current findings revealed PP7 as a potential safe agent for preventing and treating bone destruction in breast cancer patients with bone metastases.

Long non-coding RNA NEAT1 promotes angiogenesis in hepatoma carcinoma via the miR-125a-5p/VEGF pathway.

The study's purpose was to investigate the biological function of long non-coding RNA nuclear paraspeckle assembly transcript 1 (NEAT1) in hepatoma carcinoma (HCC). HCC tissues and cells exhibited increased levels of NEAT1 and decreased levels of miR-125a-5p. Reduction in the expression of NEAT suppressed HepG2 cell proliferation and increased apoptosis. This was accompanied by suppression of the AKT/mTOR and ERK pathways, while the opposite was observed for miR-125a-5p. Angiogenesis assay results indicated that NEAT was proangiogenic. A dual-luciferase reporter assay indicated that NEAT1 was bound to miR-125a-5p and miR-125a-5p was bound to vascular endothelial growth factor (VEGF). The proangiogenic effects of NEAT and its stimulation of AKT/mTOR and ERK were reversed by miR-125a-5p. The anti-angiogenic effects of miR-125a-5p and its inhibitory effect on AKT/mTOR and ERK pathways were reversed by co-incubation with VEGF. The conclusion was that NEAT1 enhances angiogenesis in HCC by VEGF via a competing endogenous RNA (ceRNA) of miR-125a-5p that regulates AKT/mTOR and ERK pathways.

Forkhead box M1 recruits FoxP3+ Treg cells to induce immune escape in hilar cholangiocarcinoma.

OBJECTIVE: Hilar cholangiocarcinoma (HCCA) is a malignancy related to chronic biliary tract inflammation. Tumor immune escape is a necessary process of tumorigenesis. Forkhead box M1 (FoxM1) could affect the progression of various carcinomas. This study attempted to elaborate on the mechanism of FoxM1 in HCCA immune escape. METHODS: HCCA cell lines were collected to measure the expression of FoxM1 and FoxP3. CD8+ T cells were extracted to establish the co-culture system with HCCA cells and Treg cells. pcDNA3.1-FoxM1 or si-FoxP3 was transfected into HCCA cells in the co-culture system. HCCA cell viability, mobility, and invasiveness as well as levels of transforming growth factor (TGF)-ß and interleukin (IL)-6 were evaluated. The binding relation between FoxM1 and FoxP3 promoter was verified. HCCA cells with pcDNA3.1-FoxM1 were subcutaneously injected into mice to establish the xenograft mouse models. RESULTS: FoxM1 and FoxP3 were overexpressed in HCCA cells. The co-culture of CD8+ T and HCCA cells inhibited HCCA cell activity and Treg cells limited CD8+ T killing. FoxM1 overexpression strengthened the inhibiting role of Treg cells in CD8+ T killing, upregulated TGF-ß and IL-6 levels, and encouraged HCCA immune escape. FoxM1 bound to the FoxP3 promoter region to promote FoxP3 transcription. Silencing of FoxP3 neutralized the promoting role of FoxM1 overexpression in Treg cell immunosuppression and HCCA cell immune escape. FoxM1 aggravated tumor development, upregulated FoxP3 expression, increased Treg cells, and reduced CD8+ T cells. CONCLUSION: FoxM1 bound to the FoxP3 promoter region to promote FoxP3 transcription and recruited FoxP3+ Treg cells, thereby inducing HCCA immune escape.

Prognostic influence for hilar cholangiocarcinoma and comparisons of prognostic values of Mayo staging and TNM staging systems.

The study was designed to discuss the effect of stratification factors in the Mayo staging on the prognosis of hilar cholangiocarcinoma (HCCA) patients, and to evaluate the predictive value of the Mayo staging on the prognosis. The Kaplan-Meier survival curve and Log-rank test were used to perform univariate analysis on each index and obtain statistically significant influencing factors. The Kaplan-Meier survival curve and Log-rank test were used to analyze the correlation between the two staging systems and the survival period. The receiver operating characteristic (ROC) curves were used for each single staging system trend analysis, and comparison of their curve area to determine prognosis prediction ability for patients with HCCA. According to Kaplan-Meier survival curve changes and Log-rank test results, it was found that both staging systems were correlated with the survival time of the patients (P < .001). Through a pairwise comparison within the stages, it was found that the heterogeneity between the stages within the Mayo staging is very good, which was better than the TNM staging. A single trend analysis of the prognostic assessment capabilities of the two systems found that the area under the ROC curve of Mayo staging system (AUC = 0.587) was the largest and better than the TNM staging system (AUC = 0.501). Mayo staging can be used for preoperative patient prognosis assessment which can provide better stratification ability based on a single-center small sample study, and the predictive value is better than TNM staging.

Rapid and efficient isolation platform for plasma extracellular vesicles: EV-FISHER.

Extracellular vesicles (EVs) have found diverse applications in clinical theranostics. However, the current techniques to isolate plasma EVs suffer from burdensome procedures and limited yield. Herein, we report a rapid and efficient EV isolation platform, namely, EV-FISHER, constructed from the metal-organic framework featuring cleavable lipid probes (PO4 3- -spacer-DNA-cholesterol, PSDC). The EV-FISHER baits EVs from plasma by cholesterol and separates them with an ordinary centrifuge. The captured EVs could be released and collected upon subsequent cleavage of PSDC by deoxyribonuclease I. We conclude that EV-FISHER dramatically outperforms the ultracentrifugation (UC) in terms of time (∼40 min vs. 240 min), isolation efficiency (74.2% vs. 18.1%), and isolation requirement (12,800 g vs. 135,000 g). In addition to the stable performance in plasma, EV-FISHER also exhibited excellent compatibility with downstream single-EV flow cytometry, enabling the identification of glypican-1 (GPC-1) EVs for early diagnosis, clinical stages differentiation, and therapeutic efficacy evaluation in breast cancer cohorts. This work portrays an efficient strategy to isolate EVs from complicated biological fluids with promising potential to facilitate EVs-based theranostics.

Preparing PAMAM-NK4 nano complexes and examining their in vitro growth suppression effects in breast cancer.

BACKGROUND: This study sought to examine the suppression of the NK4 (which is a fragment that originates from the trypsin digestion of the hepatocyte growth factor) gene as mediated by new nano material polyamidoamine (PAMAM) dendrimers in the growth of breast cancer cells MDA-MB-231 and MCF-7, and the therapeutic effects in a nude mice model of transplanted tumor cell MDA-MB-231. METHODS: We built PAMAM-NK4 nano particles and detected the in vitro transfection rate. Nano complexes and blank plasmid PAMAM dendrimers were transfected to MDA-MB-231 and MCF-7 cells, respectively. The western-blotting method, MTT experiment method, and bead method were used to detect the effects of the nano complexes on NK4 protein expression, cell proliferation, and cell apoptosis. The nude mice model of transplanted tumor cell MDA-MB-231 comprised 40 nude female mice who were subject to injections. The mice were randomly divided into four groups, comprising 10 mice per group. The control, blank plasmid and treatment groups were subcutaneously injected with 0.2 mL of 0.9% NaCl (Sodium chloride) solution, 0.2 mL of plasmid solution (including 100 µg PAMAM pcDNA3.1(-) blank plasmid nano complexes) and 0.2 mL of plasmid solution (including PAMAM-NK4 100 µg) beside the tumor inoculation spot, respectively. The positive control group was intraperitoneally injected with 0.2 mL of doxorubicin solution, including 100 µg doxorubicin. Western blotting was used to detect the NK4 protein expression of the transplanted tumor tissues of the various groups. RESULTS: NK4 protein was successfully expressed in MDA-MB-231 and MCF-7 cells transfected with PAMAM-NK4 nano particles, and cell proliferation was suppressed and cell apoptosis was induced. The tumor volumes and masses of the treatment and positive control groups were obviously smaller than those of the control group. The differences were statistically significant (P<0.05). The treatment group had an obviously higher mean value of NK4 protein expression than the control group. The differences were statistically significant (P<0.05). CONCLUSIONS: PAMAM-NK4 nano complexes suppress the growth of the breast cancer cells MDA-MB-231 and MCF-7, and had a treatment effect on this tumor nude mice model of breast cancer cells.

Methylated biomarkers for breast cancer identified through public database analysis and plasma target capture sequencing.

BACKGROUND: Aberrant methylation is common during the early stage of cancer development. This study was designed to investigate DNA methylation as biomarker for breast cancer. METHODS: Public database analysis and methylation-specific whole-gene sequencing were conducted to identify methylated biomarkers that would enable early non-invasive diagnosis of breast cancer. Firstly, the data was obtained from the TCGA Database and the Blueprint Epigenome Database. Secondly, methylation-specific whole-gene sequencing was conducted in 10 female patients with early-stage breast cancer and 10 healthy female volunteers from Nanfang Hospital of Southern Medical University between March 2018 and July 2018. Thirdly, the R language was used for data analysis, and KEGG and DAVID online tool was used for annotations. RESULTS: We found that methylation levels at 13 cytosine-phosphate-guanine (CpG) sites (cg04066177, cg04281344, cg05995576, cg06221609, cg08642731, cg11388802, cg12665414, cg14557216, cg19404723, cg19457909, cg24570211, cg25818763, and cg26215982) in the malignant tissue DNA were highly comparable to those of circulating cell-free DNA (cfDNA) of breast cancer patients, but were significantly different from those of normal tissue DNA, cfDNA of healthy women, and leukocyte DNA. In addition, three CpG sites (cg04281344, cg24570211, and cg26215982) were confirmed in clinical research, which showed that the sensitivity and specificity of these CpGs as biomarkers for breast cancer were 69.4-83.7% and 85.7-88.6%, respectively. CONCLUSIONS: New biomarkers were identified and confirmed for breast cancer by comparing the methylation of tumour tissues, leukocytes, and non-plasma DNA.

Integrated analyses of long noncoding RNAs and mRNAs in the progression of breast cancer.

OBJECTIVE: The objective was to explore the expression and potential functions of long noncoding RNA (lncRNA) and mRNAs in human breast cancer (BC). METHODS: Differentially expressed lncRNAs and mRNAs were identified and annotated in BC tissues by using the Agilent human lncRNA assay (Agilent Technologies, Santa Clara, CA, USA) and RNA sequencing. After identification of lncRNAs and mRNAs through quantitative reverse transcription polymerase chain reaction, we conducted a series of functional experiments to confirm the effects of knockdown of one lncRNA, TCONS_00029809, on the progression of BC. RESULTS: We discovered 238 lncRNAs and 200 mRNAs that were differentially expressed in BC tissues and para-carcinoma tissue. We showed that differentially expressed mRNAs were related to biological adhesion and biological regulation and mainly enriched in cytokine-cytokine receptor interaction, metabolic pathways, and PI3K-Akt signaling pathway. We created a protein-protein interaction network to analyze the proteins enriched in these pathways. We demonstrated that silencing of TCONS_00029809 remarkably inhibited proliferation, invasion, and migration of BC cells, and accelerated their apoptosis. CONCLUSIONS: We identified a large number of differentially expressed lncRNAs and mRNAs, which provide data useful in understanding BC carcinogenesis. The lncRNA TCONS_00029809 may be involved in the development of BC.

MAPK4 silencing together with a PARP1 inhibitor as a combination therapy in triple­negative breast cancer cells.

Triple­negative breast cancer (TNBC) is the most common type of cancer among females worldwide and is associated with poor prognosis. Poly ADP­ribose polymerase­1 (PARP1) inhibitors are effective against TNBC with mutations in the breast cancer type 1 susceptibility protein (BRCA1) and/or BRCA2 genes; however, the development of resistance to PARP1 inhibitors limits their use. Thus, identifying strategies to overcome this resistance is urgently required. The aim of the present study was to investigate the potential function and mechanism of small interfering (si)RNA­MAPK4 (siMAPK4) in enhancing the efficacy of a PARP1 inhibitor and reducing the resistance. In the present study, data on the mRNA expression level of MAPK4 in normal breast tissues and TNBC tissues were obtained from The Cancer Genome Atlas database. The mRNA and protein expression levels of MAPK4 in normal breast cells and TNBC cells were analyzed using reverse transcription­quantitative PCR and western blotting, respectively. The phosphorylated (p) histone H2AX (γH2AX) protein expression was assessed via immunofluorescence. Cell Counting Kit­8, wound healing and TUNEL assays were used to determine the proliferative, migratory and apoptotic abilities of HCC1937 cells. MAPK4 was highly expressed in TNBC patient tissues and cell lines. Moreover, overexpression of MAPK4 could promote HCC1937 cell proliferation. Treatment of HCC1937 cells with the combination of siMAPK4 and a PARP1 inhibitor olaparib decreased their proliferation and migration and increased their apoptosis. The protein expression levels of the DNA repair­related proteins p­DNA­dependent protein kinase catalytic subunit (DNA­PK) and RAD51 recombinase (RAD51) were inhibited in the siMAPK4 and siMAPK4 + olaparib groups. However, the marker of a double­stranded break γH2AX showed increased protein expression in the siMAPK4 + olaparib group. As MAPK4 could phosphorylate AKT at threonine 308 (AKTT308), the current study restored p­AKTT308 using a constitutively active AKT plasmid (AKT­CA). p­DNA­PK and RAD51 showed high expression and γH2AX exhibited lower protein expression in the AKT­CA group. The present findings suggested that siMAPK4 can enhance the sensitivity of TNBC cells to PARP1 inhibitors.