Association between CD4+ T cells ATP levels and disease progression in patients with non­small cell lung cancer.

Introducing the exploration of stimulated CD4+ cells adenosine triphosphate (sATPCD4) levels for immune monitoring post non-small cell lung cancer (NSCLC) chemotherapy, the present study aimed to investigate its efficacy in gauging the potential risk of disease progression (PD) in patients with NSCLC. Therefore, a total of 89 patients with advanced NSCLC, who underwent chemotherapy between August 15 2022 and August 30 2023 at the Fifth Affiliated Hospital of Guangzhou Medical University (Guangzhou, China), were retrospectively studied. Patients were divided into the PD (n=21) and disease stability (non-PD; n=68) groups and their clinical data were compared. The thresholds for predicting PD were identified using receiver operating characteristics (ROC) curves. Multivariate logistic regression analysis was carried out to assess the association between peripheral blood markers and the incidence of PD. Therefore, post-chemotherapy, significant differences in white blood cell count, non-stimulated CD4+ cells ATP and sATPCD4 levels were obtained between patients in the PD and non-PD groups (P<0.05). In addition, sATPCD4 levels were notably decreased in the PD group compared with the non-PD group. Furthermore, ROC analysis revealed that the predictive threshold for PD was 224.5 ng/ml [area under the curve=0.887; 95% confidence interval, 0.811-0.963]. Additionally, patients with low immunity (ATP <224.5 ng/ml) exhibited a higher risk of PD compared with the high-immunity group (ATP >224.5 ng/ml; P<0.0001). Finally, multivariate logistic regression analysis suggested that sATPCD4 could serve as an independent factor for predicting NSCLC progression. Overall, the current study predicted that immune function could be possibly associated with the risk of PD in patients with NSCLC.

The predictive value of peripheral blood CD4 cells ATP concentration for immune-related adverse events in advanced non-small cell lung cancer patients.

OBJECTIVE: Lung cancer with the highest incidence and mortality in the world. Immune checkpoint inhibitors (ICIs), can bring long-term survival benefits to patients, but also can bring immune-related adverse events (irAEs) in some patients during therapy. Therefore, the aim of this study was to investigate the predictive effect of peripheral blood WBC, NLR, sATPCD4 and nATPCD4 on irAEs in advanced non-small cell lung cancer (NSCLC). METHODS: Clinical data of 112 patients with advanced NSCLC who were treated with PD -1/PD -L1 inhibitor in the Fifth Affiliated Hospital of Guangzhou Medical University from December 15, 2019 to April 30, 2023 were retrospectively analyzed. These patients were divided into the irAEs group (n = 27) and non-irAEs group (n = 85). The clinical data of the two groups were compared. Receiver operating characteristic (ROC) curves were drawn to determine the threshold value of baseline peripheral blood parameters to predict the occurrence of irAEs. Multivariate logistic regression analysis was used to explore the relationship between peripheral blood markers and the incidence of irAEs. RESULTS: The patient characteristics have no significant difference between irAEs and non-irAEs group. But the baseline peripheral blood WBC, sATPCD4 and nATPCD4 of patients in the irAEs group were higher than those in the non-irAEs group (p < 0.05), and the NLR in irAEs group was similar to in the non-irAEs group (p = 0.639).Univariate analysis showed that high WBC, sATPCD4 and nATPCD4 may the risk factors for the occurrence of irAEs (p < 0.05). Multivariate logistic regression analysis showed that high sATPCD4 and nATPCD4 were independent risk factors for the occurrence of irAEs (p < 0.05). The best critical values of WBC, sATPCD4 and nATPCD4 before treatment for predicting the occurrence of irAEs were 8.165 × 109cells/L (AUC = 0.705) ,484.5 ng/mL (AUC = 0.777), and 156 ng/mL (AUC = 0.840), respectively. CONCLUSIONS: sATPCD4 and nATPCD4 were independent risk factors for the occurrence of irAEs in advanced NSCLC patients. This discovery provides a new method to predict the occurrence of irAEs in patients. Based on the prediction results, corresponding treatment measures can be taken to reduce the incidence of adverse events.

The progresses of relevant factors on the efficacy of immune checkpoint inhibitors in the non-small cell lung cancer patients.

Lung cancer has the highest mortality rate of all cancers worldwide. Although immune checkpoint inhibitor (ICI)-based therapy can improve the survival of patients with lung cancer, its efficacy is affected by many factors. Therefore, it is necessary to identify factors that affect the efficacy of ICI-based treatment and establish a model for predicting drug response and resistance before and during treatment for individualized and accurate treatment of patients. This review summarizes the clinical and biological factors related to ICI-based treatment of non-small cell lung cancer (NSCLC) and the recent research progress of predictive models for assessing ICI efficacy.

Oxidized LDL promotes EMS-induced angiogenesis by increasing VEGF-A expression and secretion by endometrial cells.

BACKGROUND: Endometriosis (EMS) is a "tumour-like" gynaecological disease with distant metastasis, and studies have shown that EMS can induce distant metastasis through vascular vessels, but the driving factors and their mechanism are not clear. METHODS: We used an EMS animal model and gene knockout technique to explore the role of EMS-induced angiogenesis in EMS metastasis in vivo and in vitro and clarify the role and molecular mechanism of oxLDL in promoting EMS-induced angiogenesis. RESULTS: We found that microvascular density (MVD) in metastasized ectopic endometrium and eutopic endometrial tissue was higher than that in normal endometrial tissue, and plasma oxLDL was positively correlated with the distant metastasis of EMS. Furthermore, we clarified that oxLDL enhanced the MVD of endometrial tissue by increasing VEGF-A expression and secretion in endometrial cells. Finally, we illustrated the mechanism by which oxLDL promotes VEGF-A expression through the AKT-HIF-1α signalling pathway. CONCLUSION: OxLDL is a risk factor promoting distant EMS metastasis by increasing VEGF-A expression and secretion through AKT-HIF-1α signalling. This finding may provide theoretical support and therapeutic targets for the clinical prevention and treatment of EMS.

CCT5 interacts with cyclin D1 promoting lung adenocarcinoma cell migration and invasion.

Cyclin D1 (CCND1) has been identified as a metastatic promoter in various tumors including lung adenocarcinoma (LUAD), a subtype of non small cell lung cancer (NSCLC). The previous observation revealed that CCND1 was upregulated in NSCLC and predicted poor prognosis of LUAD patients. In this study, we examined a chaperonin containing TCP1 subunit 5 (CCT5) protein interacts with CCND1 in LUAD. Immunofluorescence demonstrated the co-localization of CCT5 and CCND1 protein in LUAD cells. CCT5 expression was detected with both immunohistochemistry (IHC) and bioinformatics analyses. Similar with the expression pattern of CCND1, CCT5 displayed a high level in LUAD tissues compared to non cancerous lung specimens. Patients with high CCT5 expression showed a significant shorter overall survival relative to those with low expression level. Furthermore, upregulated CCT5 exhibited significant positive correlation with TNM stage of LUAD patients in both IHC analyses and bioinformatics. Knocking down CCT5 remarkably inhibited LUAD cell migration and invasion in vitro by inactivating PI3K/AKT and its downstream EMT signals, which could abrogated the accelerated migration and invasion caused by CCND1 overexpression. In summary, our study discovered a highly expressed protein CCT5 in LUAD which interacted with CCND1 and promoted migration and invasion of LUAD cells by positively moderating PI3K/AKT-induced EMT pathway.

VPS33B modulates c-Myc/p53/miR-192-3p to target CCNB1 suppressing the growth of non-small cell lung cancer.

VPS33B is reported to be a tumor suppressor in hepatocellular carcinoma, nasopharyngeal carcinoma, colon cancer, and lung adenocarcinoma. Here, we observed that reduced VPS33B protein level was an unfavorable factor that promoted the pathogenesis of non-small cell lung cancer (NSCLC) in clinical specimens. We achieved lentivirus-mediated stable overexpression of VPS33B in NSCLC cells. Increased VPS33B reduced cell cycle transition and cell proliferation of NSCLC cells in vivo and in vitro. Knocking down VPS33B restored cell growth. Mechanism analysis indicated that miR-192-3p was induced by VPS33B and acted as a tumor suppressor of cell growth in NSCLC. Further, c-Myc or p53 was identified as a transcription factor that bound to the miR-192-3p promoter and regulated its expression. miR-192-3p directly targeted cell cycle-promoted factor CCNB1 and suppressed NSCLC cell growth. VPS33B modulated c-Myc/p53/miR-192-3p signaling to target CCNB1 by reducing activation of the Ras/ERK pathway. Our study reveals a novel molecular basis for VPS33B as a tumor suppressor to participate in the pathogenesis of NSCLC.

SPEN induces miR-4652-3p to target HIPK2 in nasopharyngeal carcinoma.

SPEN family transcriptional repressor (SPEN), also known as the SMART/HDAC1-associated repressor protein (SHARP), has been reported to modulate the malignant phenotypes of breast cancer, colon cancer, and ovarian cancer. However, its role and the detail molecular basis in nasopharyngeal carcinoma (NPC) remain elusive. In this study, the SPEN mRNA and protein expression was found to be increased in NPC cells and tissues compared with nonmalignant nasopharyngeal epithelial cells and tissues. Elevated SPEN protein expression was found to promote the pathogenesis of NPC and lead to poor prognosis. Knockdown of SPEN expression resulted in inactivation ofPI3K/AKT and c-JUN signaling, thereby suppressing NPC migration and invasion. In addition, miR-4652-3p was found to be a downstream inducer of SPEN by targeting the homeodomain interacting protein kinase 2 (HIPK2) gene, a potential tumor suppressor that reduces the activation of epithelial-mesenchymal transition (EMT) signaling, thereby reducing its expression and leading to increased NPC migration, invasion, and metastasis. In addition, SPEN was found to induce miR-4652-3p expression by activating PI3K/AKT/c-JUN signaling to target HIPK2. Our data provided a new molecular mechanism for SPEN as a metastasis promoter through activation of PI3K/AKT signaling, thereby stimulating the c-JUN/miR-4652-3p axis to target HIPK2 in NPC.

HBX-induced miR-5188 impairs FOXO1 to stimulate ß-catenin nuclear translocation and promotes tumor stemness in hepatocellular carcinoma.

Cancer stem cells (CSCs) are the key factor in determining cancer recurrence, metastasis, chemoresistance and patient prognosis in hepatocellular carcinoma (HCC). The role of miR-5188 in cancer stemness has never been documented. In this study, we investigated the clinical and biological roles of miR-5188 in HCC. Methods: MiRNA expression in HCC was analyzed by bioinformatics analysis and in situ hybridization. The biological effect of miR-5188 was demonstrated in both in vitro and in vivo studies through the ectopic expression of miR-5188. The target gene and molecular pathway of miR-5188 were characterized using bioinformatics tools, dual-luciferase reporter assays, gene knockdown, and rescue experiments. Results: MiR-5188 was shown to be upregulated and confer poor prognosis in HCC patient data from TCGA database. MiR-5188 was subsequently identified as a significant inducer of cancer stemness that promotes HCC pathogenesis. Specifically, the targeting of miR-5188 by its antagomir markedly prolonged the survival time of HCC-bearing mice and improved HCC cell chemosensitivity in vivo. Mechanistic analysis indicated that miR-5188 directly targets FOXO1, which interacts with ß-catenin in the cytoplasm to reduce the nuclear translocation of ß-catenin and promotes the activation of Wnt signaling and downstream tumor stemness, EMT, and c-Jun. Moreover, c-Jun transcriptionally activates miR-5188 expression, forming a positive feedback loop. Interestingly, the miR-5188-FOXO1/ß-catenin-c-Jun feedback loop was induced by hepatitis X protein (HBX) through Wnt signaling and participated in the HBX-induced pathogenesis of HCC. Finally, analyses of transcriptomics data and our clinical data supported the significance of the abnormal expression of the miR-5188 pathway in HCC pathogenesis. Conclusions: These findings present the inhibition of miR-5188 as a novel strategy for the efficient elimination of CSCs to prevent tumor metastasis, recurrence and chemoresistance in patients with hepatocellular carcinoma. Our study highlights the importance of miR-5188 as a tumor stemness inducer that acts as a potential target for HCC treatment.

High-throughput sequencing reveals circular RNA hsa_circ_0000592 as a novel player in the carcinogenesis of gastric carcinoma.

Background/Aim: Gastric cancer is one of the most common malignant tumors, and its complex pathogenesis has not been fully elucidated. Circular RNAs (circRNAs) are involved in various biological processes and human diseases. However, their exact functional roles and mechanisms of action remain largely unclear. We previously discovered the differential expression of non-coding RNAs (ncRNAs) during the malignant transformation of human gastric epithelial cells. In this study, we investigated the functional roles of a significantly up-regulated circRNA (hsa_circ_0000592) in gastric cancer. Methods:N-methyl-N'-nitro-N-nitrosoguanidine (MNNG)-induced malignant-transformed gastric epithelial cells (GES-1-T) and normal gastric epithelial cells (GES-1-N) were analyzed by high-throughput circRNA sequencing. The top 15 up-regulated circRNAs in high-throughput sequencing results were further confirmed by qRT-PCR in different gastric epithelial cell lines. The function of the most significant circRNA (hsa_circ_0000592) was investigated by using RNA interference (RNAi) assays, fluorescence in situ hybridization analysis (FISH), and bioinformatics prediction methods. Results: A total of 1509 genes were up-regulated and 3142 genes were down-regulated in GES-1-T cells when compared with GES-1-N cells. When compared with GES-1-N cells, hsa_circ_0000592 was obviously up-regulated in GES-1-T cells, as well as in other gastric cancer cell lines. The silencing of hsa_circ_0000592 mRNA led to a decrease in cell proliferation, cell cycle arrest at the G0/G1 phase, an increased rate of apoptosis, and a reduction in cell migration. Furthermore, FISH showed that hsa_circ_0000592 was mainly located in the cytoplasm, and a bioinformatics analysis suggested that hsa_circ_0000592 might function by sponging multiple miRNAs, and most notably four conserved miRNAs, including miR-139-3p, miR-200, miR-367-3p, and miR-33a-3p. Conclusion: This study is the first to identify hsa_circ_0000592 as a novel circRNA with a critical role in MNNG-induced gastric cancer. Due to the essential role of hsa_circ_0000592 in gastric carcinoma cells, it may be considered as a potential biomarker for use in diagnosing gastric carcinoma. Our findings provide a new insight into the function of circRNAs in environmental carcinogen-induced gastric cancer.

The naturally occurring xanthone α-mangostin induces ROS-mediated cytotoxicity in non-small scale lung cancer cells.

Small cell lung cancer (NSCLC) accounts for 85% of total deaths globally, and recent studies indicate the increasing risks of NSCLC in China and South Asian countries. Hence, development of new therapeutics against NSCLC has been a major concern. α-Mangostin, a naturally occurring xanthone, found abundantly in pericarps of mangosteen fruit is well known for its medicinal importance. The anticancer properties of α-mangostin against several types of cancer are also well documented. But the mechanism of action of α-mangostin against lung cancer is not well understood and requires further investigation. Therefore in the present study, we explored the therapeutic potential of α-mangostin against A549 cells. Treatment of A549 cells with α-mangostin resulted in a dose-dependent loss of cell viability, while the non-malignant cells such as hPBMC and WI-38 remained unaffected. Further we observed that the ROS plays an important role in α-mangostin -induced apoptosis in A549 cells, and administration of N-acetyl cysteine significantly abrogates α-mangostin -mediated cytotoxicity in lung cancer cells. Overall, α-mangostin induces ROS-mediated cytotoxicity in NSCLC cells.

Nuclear CD133 expression predicts poor prognosis for hepatocellular carcinoma.

Cancer stem cells (CSCs) are responsible for cancer recurrence and metastasis and are related to poor prognosis in patients with hepatocellular carcinoma (HCC). CD133 is one of the most commonly used CSC markers. In this study, expression and the biological significance of CSC marker CD133 was evaluated in HCC, at mRNA and protein levels. We demonstrate that both mRNA and protein levels of CD133 are significantly elevated in HCC relative to that in adjacent non-cancerous tissue based on bioinformatics and immunohistochemical analysis, respectively (P < 0.01). Intriguingly, we detected nuclear distribution of CD133 and found that nuclear CD133 expression was indicative of poor patient prognosis (median survival 12 months versus 34.5 months) (Log-Rank, P = 0.0258). Meanwhile, our findings suggest that nuclear CD133 expression is positively correlated with tumor size and serves as an independent prognostic factor for HCC after surgical resection (HR = 0.564, 95% CI 0.313-1.018, P = 0.057). Nuclear CD133 expression can potentially serve as a biomarker for clinical diagnosis and prognosis of HCC.

Low MYH9 expression predicts a good prognosis for hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is currently one of the most common causes of cancer-related death and one of the most commonly diagnosed cancers. MYH9 is thought to play a critical role in cancer progression. However, the expression and prognostic value of MYH9 in HCC are still unknown. In this study, the expression and biological significance of MYH9 were evaluated in HCC, at mRNA and protein levels. We showed that both mRNA and protein levels of MYH9 were significantly upregulated in HCC relative to the levels in adjacent non-tumor tissues based on the TCGA database and immunohistochemical analysis, respectively (P < 0.001). Additionally, we found that high MYH9 protein levels correlated with poor patient prognosis (median survival 19 months versus 49 months) (Log-Rank, P = 0.0292). Meanwhile, our data suggested that MYH9 expression is an independent prognostic factor for HCC after surgical resection (HR = 0.675, 95% CI 0.452-1.008, P = 0.054) and can potentially serve as a biomarker for the clinical diagnosis and prognosis of HCC.

MiR-4505 aggravates lipopolysaccharide-induced vascular endothelial injury by targeting heat shock protein A12B.

Heat shock protein family A member 12B (HSPA12B) is a heat shock protein primarily expressed in endothelial cells. Our previous study showed that it was protective against endothelial injury induced by lipopolysaccharide (LPS). The present study was performed to investigate whether micro (mi)RNA was involved in HSPA12B expression in endothelial cells challenged by LPS. We first screened the miRNA candidates potentially related to HSPA12B by bioinformatics analysis. Then the mimics of the miRNA candidates were transfected into human umbilical vein endothelial cells (HUVECs) to investigate the miRNAs that negatively regulated HSPA12B expression. The miRNA expression was also determined in LPS­stimulated HUVECs. Dual luciferase activity assay was performed to confirm the relationship between the candidate miRNA and HSPA12B. Role of nuclear factor (NF)­κB in the miRNA expression was investigated by using its inhibitor. Finally, the role of the miRNA on LPS induced injury was investigated. Eleven miRNAs were screened by bioinformatics analysis and 4 of them could inhibit HSPA12B expression at both mRNA and protein levels. Among the 4 miRNA candidates, only miR­4505 was highly expressed in HUVECs stimulated by LPS. Luciferase analysis showed that miR­4505 directly interacted with the 3'untranslated region of HSPA12B. LPS­induced upregulation of miR­4505 was blocked by NF­κB inhibitor. Transfection with miR­4505 mimics reduced the transendothelial electrical resistance and vascular endothelial­cadherin expression. The scratch test demonstrated that miR­4505 inhibited endothelial migration capacity. In conclusion, miR­4505 downregulates the expression of HSPA12B and aggravates the LPS­induced vascular endothelial cell injury.

Long non-coding RNA SPRY4-IT1 promotes development of hepatic cellular carcinoma by interacting with ERRα and predicts poor prognosis.

Hepatocellular carcinoma (HCC) has become one of the most common leading causes of cancer-related deaths worldwide. This study investigates the role of lncRNA, SPRY4-IT1 in the development of HCC. Quantitative real-time PCR (qRT-PCR) was performed and the results showed that SPRY4-IT1 expression was up-regulated in HCC tissues and high expression of SPRY4-IT1 was associated with poor 5-year overall survival in the HCC patient cohort. Clinicopathological analysis showed that the expression of SPRY4-IT1 was significantly correlated with TNM stage in HCC patients. In vitro CCK-8 assay, colony formation assay, cell invasion and migration assays demonstrated that knock-down of SPRY4-IT1 suppressed cell proliferation, colony formation, cell invasion and migration in HCC cells. Flow cytometric analysis showed that knock-down of SPRY4-IT1 induced cell cycle arrest at G0/G1 phase and induced apoptosis. In addition, knock-down of SPRY4-IT1 also suppressed the mRNA and protein expression of estrogen-related receptor α (ERRα). Similarly, knock-down of ERRα inhibited cell proliferation, colony formation, cell invasion and migration in HCC cells. More importantly, ERRα overexpression antagonized the effects of SPRY4-IT1 knock-down on cell proliferation, colony formation, cell invasion and migration in HCC cells. Taken together, our data highlights the pivotal role of SPRY4-IT1 in the tumorigenesis of HCC.

Effect of Hemodilution In Vitro with Hydroxyethyl Starch on Hemostasis.

BACKGROUND Hydroxyethyl starch (HES) solutions are used for volume expansion during surgery. We aimed to investigate how 6%HES 130/0.4 affects hemostasis. MATERIAL AND METHODS Blood samples were collected from 12 healthy adult volunteers, diluting with 6%HES 130/0.4 (HES group) or Ringer lactate solution (RL control group). The hemodilution ratio (HR) of citrated blood volume to plasma substitute volume was 10: 0 (undiluted), 10: 2, 10: 4, and 10: 6. Clotting factors activity was measured. Thrombin generation was monitored. Platelet function was analyzed. RESULTS 1) Activity of coagulation factor was decreased with increasing HR compared to undiluted baseline, and the activity of FVIII was significantly decreased in HES vs. RL. 2) Calibrated automated thrombography (CAT) results showed HES extended lag time, time to peak (ttpeak), start tail, and decreased peak of thrombin generation. Although lag time and ttpeak were significantly prolonged in HES vs. RL, endogenous thrombin potential (ETP) did not change. 3) Flow cytometric (FCM) analysis showed that HES reduced platelet phospholipids serine (PS) vs. baseline and RL. 4) HES significantly decreased antithrombin activity (AT: A) of the anticoagulant system with increasing HR vs. baseline and RL. 5) For fibrinolytic system, HES did not affect fibrinogen degradation products (FDP) and D-dimers (D-D) vs. baseline, or α2-antiplasmin (α2-AP) vs. RL. CONCLUSIONS By reducing FVIII activity and platelet PS expression, HES interfered with PS combining to FXIa, FVIIIa, and FVa, which affected the acceleration and explosion stage of thrombin. The decreased velocity and peak of thrombin generation delays and reduces clot formation. Combined 6%HES 130/0.4 decreased anticoagulant activity and may have clinical utility.

Heat Shock Protein A12B Protects Vascular Endothelial Cells Against Sepsis-Induced Acute Lung Injury in Mice.

BACKGROUND: Pulmonary endothelial injury is a critical process in the pathogenesis of acute lung injury (ALI) during sepsis. Heat shock protein A12B (HSPA12B) is mainly expressed in endothelial cells and protects against several harmful factors. However, the effects of HSPA12B in sepsis-induced ALI and its potential mechanisms of action remain unclear. METHODS: For in vivo experiments, C57BL/6 mice were randomly divided into four groups (n=15): a sham operation group, a cecal ligation and puncture (CLP) group, a HSPA12B siRNA-CLP group and a negative control (NC) siRNA-CLP group. The mice were treated by nasal inhalation of 2-OMe-modified HSPA12B siRNA or NC siRNA. Sepsis was induced by CLP. Samples were harvested 24 and 48 hours post-CLP surgery. Pathological changes and scoring of lung tissue samples were monitored using hematoxylin and eosin staining. Levels of pro-inflammatory cytokines (e.g., interleukin (IL)-1ß, tumor necrosis factor (TNF)-α, and IL-6) and myeloperoxidase activity in bronchoalveolar lavage fluid were analyzed by ELISA. Pulmonary edema was assessed using a wet-to-dry weight ratio. Neutrophils and alveolar macrophages were counted using flow cytometry. Pulmonary endothelial cell apoptosis was detected by TUNEL staining. Expression levels of MAPK family signaling molecules and caspase-3 were measured by Western blot analysis. In addition, 7-day survival was recorded. For in vitro experiments, human umbilical vein endothelial cells were pre-transfected with HSPA12B siRNA or pIRES2-EGFP-HSPA12B-Flag plasmid and treated with lipopolysaccharide; subsequently, the expression levels of MAPK family signaling molecules and caspase-3 were measured by Western blotting. RESULTS: Nasal inhalation of nano-polymer-encapsulated HSPA12B siRNA specifically downregulated mRNA and protein expression levels of HSPA12B in lung tissues. The administration of HSPA12B siRNA aggravated lung pathological injury, upregulated pro-inflammatory cytokine (e.g., IL-1ß, TNF-α, and IL-6) expression, and increased myeloperoxidase activity, neutrophil infiltration, pulmonary edema, and pulmonary endothelial cell apoptosis. Additionally, HSPA12B knockdown worsened survival after CLP surgery. The potential protective mechanisms of HSPA12B may involve the inhibition of ERK phosphorylation and caspase-3 activation in vivo and in vitro. CONCLUSION: HSPA12B protected against sepsis-induced ALI. The potential mechanism may be partly due to the inhibition of ERK phosphorylation and caspase-3 activation. These findings provide a potential therapeutic target for treating sepsis.

MiR-19a, miR-122 and miR-223 are differentially regulated by hepatitis B virus X protein and involve in cell proliferation in hepatoma cells.

BACKGROUND: Hepatitis B virus (HBV) X protein (HBx) is a type of oncogenic protein involved in the progression of hepatocellular carcinoma (HCC) via interacting with host genes. Dysregulation of microRNAs (miRNAs) has been observed in HCC. This study aimed to investigate the role of HBx protein in the regulation of miR-19a, miR-122 and miR-223, and examine if these miRNAs involve in progression of malignant hepatocytes. METHODS: Quantitative real time PCR (qRT-PCR) was used to measure the expression of miR-19a, miR-122 and miR-223 in patient samples and in HepG2 cells transfected with HBx or 1.3 fold HBV genome and also in HepG2.2.15 cells, which stably produces HBV. Their target mRNAs and proteins-PTEN, cyclin G1 and c-myc were measured by qRT-PCR and western blot, respectively. The effect of miR-19a, miR-122 and miR-223, and their respective target genes, on cell proliferation was analyzed using 5-ethynyl-2-deoxyuridine incorporation and MTT assay. RESULTS: MiR-19a showed an up-regulation in HBV-positive HCC patients compared to healthy controls and HBV-negative HCC patients, while miR-122 and miR-223 showed a down-regulation compared to healthy controls, and miR-122 in HBV-positive HCC patients was also down-regulated when compared to HBV-negative HCC patients. MiR-19a was found to be up-regulated in HepG2 cells transfected with HBx or 1.3 fold HBV genome, but down-regulated in HepG2.2.15 cells. MiR-122 and miR-223 were down-regulated in HBx or 1.3 fold HBV transfected HepG2 cells as well as in HepG2.2.15 cell. Their target mRNAs and corresponding proteins-PTEN was down-regulated, while cyclin G1 and c-myc were found to be up-regulated. Modulated expression of miR-19a, miR-122 and miR-223 enhanced cell proliferation of HBx-transfected HepG2 cells, and rescue experiment further showed that their target genes-PTEN, cyclin G1and c-myc involved in cell proliferation of HBx-transfected HepG2 cells. CONCLUSIONS: The expression of miR-19a, miR-122 and miR-223 were differentially regulated by HBx protein, the differential expression of miR-19a, miR-122 and miR-223 plays an important role in cell proliferation of HCC. This study provides new insight into understanding how HBx protein interacts with miRNAs and subsequently regulates host function.

Heat shock protein A12B protects against sepsis-induced impairment in vascular endothelial permeability.

BACKGROUND: As a common and life-threatening infectious syndrome, sepsis contributes significantly to morbidity and mortality in clinical settings. Vascular endothelial injury and hyperpermeability play an important role in the development of sepsis-induced organ dysfunction. Heat shock protein A12B (HSPA12B) is one of the HSP70 superfamily members and is mainly expressed in vascular endothelial cells. The present study was performed to investigate the role of HSPA12B in endothelial barrier dysfunction during sepsis. METHODS: Human umbilical vein endothelial cells (HUVECs) were stimulated with 1 µg/mL of lipopolysaccharide (LPS) and harvested at 0, 3, 6, 9, 12, and 24 h. The messenger RNA and protein levels of HSPA12B were detected by Real Time-polymerase chain reaction and Western blot. Upregulation of HSPA12B was induced by transfection of pIRES2-EGFP plasmid carrying the HSPA12B complementary DNA. The in vitro effect of HSPA12B overexpression on endothelial permeability was manifested by the transendothelial electrical resistance value, expression of the adhesion molecules VE-cadherin, and the level of permeability-related kinase myosin light chain, SRC, and CDC42. Mice received cecal ligation and puncture surgery followed by nasal inhalation of nano-polymer-mediated siRNA. Lung endothelial permeability was assessed via intrajugular vein injection of Evans Blue 30 h after cecal ligation and puncture. RESULTS: After LPS induction, the messenger RNA and protein level of HSPA12B in HUVECs increased and peaked at 12 h, whereas they returned to the baseline level at 24 h. Overexpression of HSPA12B can reduce the permeability of HUVEC stimulated by LPS in vitro, while increasing the expression of VE-Cadherin, myosin light chain, and CDC42. On the other hand, downregulating the expression of HSPA12B can significantly increase lung permeability in mice with sepsis-induced vascular injury. CONCLUSIONS: HSPA12B plays a protective role in vascular endothelial barrier dysfunction by preserving the endothelial permeability during sepsis.

Arsenic trioxide reduces chemo-resistance to 5-fluorouracil and cisplatin in HBx-HepG2 cells via complex mechanisms.

BACKGROUND: Multidrug resistance is one of the major reasons chemotherapy-based treatments failed in hepatitis B virus (HBV) related hepatocellular carcinoma (HCC). Hypoxia is generally associated with tumor chemo-resistance. The aim of the study was to investigate the effect of Arsenic trioxide (As2O3) on the hypoxia-induced chemo-resistance to 5-FU or cisplatin and explored its underlying mechanism in the HBx-HepG2 cells. METHODS: MTT assay was used to examine the cell viability. Mitochondrial membrane potential (MMP) and cell cycle was examined by flow cytometry. qRT-PCR was employed to observe the mRNA expression level; and western blot assay was used to determine the protein expression level. RESULTS: Our results showed that transfection of HBx plasmid established the HBx-HepG2 cells expressing HBx, and the expression of HBx was confirmed by qRT-PCR and western blot. Exposure of HBx-HepG2 cells to hypoxia (5 % O2, 3 % O2, 1 % O2) for 48 h increased the chemo-resistance to 5-fluorouracil (5-FU) (50-1600 µM) and cisplatin (25-800 µM), reduced MMP, and caused the cell cycle arrest at G0/G1 phase in a concentration-dependent manner. Hypoxia also concentration-dependently (5 % O2, 3 % O2, 1 % O2) reduced mRNA expression level of P-glycoprotein (P-gp), multidrug resistance protein (MRP1), lung resistance protein (LRP), and decreased the protein expression level of hypoxia-inducible factor-1α (HIF-1α), P-gp MRP1, and LRP. Following pretreatment with As2O3 at a non-cytotoxic concentration re-sensitized the hypoxia (1 % O2)-induced chemo-resistance to 5-FU and cisplatin in HBx-HepG2 cells. As2O3 pretreatment also prevented MMP reduction and G0/G1 arrest induced by hypoxia. Meanwhile, As2O3 antagonized increase of HIF-1α protein induced by hypoxia, and it also suppresses the increase in expression levels of P-gp, MRP1, and LRP mRNA and proteins. In addition, As2O3 in combination with 5-FU treatment caused up-regulation of DR5, caspase 3, caspase 8, and caspase 9, and down-regulation of BCL-2, but had no effect of DR4. CONCLUSIONS: Our results may suggest that As2O3 re-sensitizes hypoxia-induced chemo-resistance in HBx-HepG2 via complex pathways, and As2O3 may be a potential agent that given in combination with other anti-drugs for the treatment of HBV related HCC, which is resistant to chemotherapy.

Downregulation of miR203 induces overexpression of PIK3CA and predicts poor prognosis of gastric cancer patients.

BACKGROUND: Despite advances in clinical therapies and technologies, the prognosis for patients with gastric cancer is still poor. The aim of this study is to investigate new predictive markers for prognosis of gastric cancer. METHODS: In this study, we evaluated the expression pattern of PIK3CA in 107 gastric cancer specimens and their adjacent nontumorous tissues. PIK3CA siRNA was synthesized and transfected into gastric cancer cell lines. Colony formation and MTT assays were employed to analyze the cell proliferation. PIK3CA expression was examined by using immunohistochemical analysis and Western blot assay. Transwell invasion assay was used to detect the invasion capability of the cells. Luciferase activity was examined by using 3'-untranslated region luciferase reporter assays. RESULTS: We observed that PIK3CA was significantly upregulated in gastric cancer tissues. High expression level of PIK3CA was detectable in 48 (44.86%) of the gastric cancer specimens, and correlated with poor prognosis. In addition, our study indicated that miR203 inhibits cell proliferation and invasion via directly targeting and suppressing the PIK3CA expression. MiR203 expression is downregulated in gastric cancer tissues. Moreover, low expression level of miR203 predicted poor prognosis of gastric patients and induced overexpression of PIK3CA. Our further study also reported that overexpression of miR203 inhibited phosphorylation of AKT, while cotransfection of PIK3CA reversed the effect of miR203. CONCLUSION: Our study suggested a miR203-PIK3CA-AKT signaling pathway in gastric cancer cells. This signaling pathway might play an important role in gastric cancer genesis and development.