Deciphering the role of non-coding RNAs involved in sorafenib resistance.

Sorafenib is an important treatment strategy for advanced hepatocellular carcinoma (HCC). Unfortunately, drug resistance has become a major obstacle in sorafenib application. In this study, whole transcriptome sequencing (WTS) was conducted to compare the paired differences between non-coding RNAs (ncRNAs), including long non-coding RNAs (lncRNAs), circular RNAs (circRNAs), microRNAs (miRNAs), and mRNAs, in sorafenib-resistant and parental cells. The overlap of differentially expressed ncRNAs (DENs) between the SMMC7721/S and Huh7/S cells and their parental cells was determined. 2 upregulated and 3 downregulated lncRNAs, 2 upregulated and 1 downregulated circRNAs, as well as 10 upregulated and 2 downregulated miRNAs, in both SMMC7721/S and Huh7/S cells, attracted more attention. The target genes of these DENs were then identified as the overlaps between the differentially expressed mRNAs achieved using the WTS analysis and the predicted genes of DENs obtained using the "co-localization" or "co-expression," miRanda, and RNAhybrid analysis. Consequently, the potential regulatory network between overlapping DENs and their target genes in both SMMC7721/S and Huh7/S cells was explored. The "lncRNA-miRNA-mRNA" and "circRNA-miRNA-mRNA" networks were constructed based on the competitive endogenous RNA (ceRNA) theory using the Cytoscape software. In particular, lncRNA MED17-203-miRNA (miR-193a-5p, miR-197-3p, miR-27a-5p, miR-320b, miR-767-3p, miR-767-5p, miR-92a-3p, let-7c-5p)-mRNA," "circ_0002874-miR-27a-5p-mRNA" and "circ_0078607-miR-320b-mRNA" networks were first introduced in sorafenib-resistant HCC. Furthermore, these networks were most probably connected to the process of metabolic reprogramming, where the activation of the PPAR, HIF-1, Hippo, and TGF-ß signaling pathways is governed. Alternatively, the network "circ_0002874-miR-27a-5p-mRNA" was also involved in the regulation of the activation of TGF-ß signaling pathways, thus advancing Epithelial-mesenchymal transition (EMT). These findings provide a theoretical basis for exploring the mechanisms underlying sorafenib resistance mediated by metabolic reprogramming and EMT in HCC.

Ubenimex suppresses glycolysis mediated by CD13/Hedgehog signaling to enhance the effect of cisplatin in liver cancer.

Background: Liver cancer ranks third in fatalities among all cancer-related deaths. As a traditional chemotherapy drug, the application of cis-Diamminedichloroplatinum (II) (cisplatin, CDDP) for the treatment of liver cancer is greatly limited by its side effects and high drug resistance. Therefore, we are in urgent need of a more effective and less toxic CDDP therapeutic regimen. Our research aimed to clarify the possible mechanism of ubenimex in enhancing the effect of CDDP on liver cancer. Methods: The underlying mechanism was determined using Cell Counting Kit-8 (CCK-8) assay, flow cytometry, immunofluorescence, enzyme-linked immunosorbent assay (ELISA), transwell assay, wound healing assay and western blot assay. Results: The data indicated that ubenimex suppressed the expression levels of glycolysis-related proteins by decreasing the expression levels of cluster of differentiation 13 (CD13), while overexpression of CD13 could restore the activity of glycolysis. The glycolysis inhibitor 2-deoxy-D-glucose enhanced the antiproliferative effect of ubenimex and CDDP. In addition, the inhibition of the activity levels of the Hedgehog (Hh) pathway members was accompanied by a decrease in CD13 expression, which was reversed following CD13 overexpression. Moreover, ubenimex inhibited the production of lactic acid and adenosine triphosphate (ATP), as well as the expression of key proteins involved in glycolysis, which was similar to the effects caused by the Hh inhibitor cyclopamine. However, the effects of ubenimex were mediated by targeting CD13, while cyclopamine exhibited no effects on CD13, suggesting that Hh signaling occurred in the downstream of CD13. The inhibition of glycolysis by cyclopamine was reduced following CD13 overexpression, which further indicated that ubenimex targeted the CD13/Hh pathway to inhibit glycolysis. Finally, wound healing and transwell assays and cell proliferation and apoptosis analysis demonstrated that ubenimex inhibited glycolysis by alleviating the CD13/Hh pathway, which in turn enhanced the effects of CDDP on inhibiting the progression of liver cancer. Conclusions: Ubenimex inhibits glycolysis by targeting the CD13/Hh pathway, thus playing an anti-tumor role together with CDDP. This study demonstrated the adjuvant effect of ubenimex from the perspective of Hh signal-dependent glycolysis regulation.

CD13 downregulation mediated by ubenimex inhibits autophagy to overcome 5-FU resistance by disturbing the EMP3/FAK/NF-κB pathway in gastric cancer cells.

Background: Gastric cancer (GC) is one of the most common malignant tumours in China, but the efficacy of chemotherapy on GC is significantly reduced due to the occurrence of drug resistance. Some studies have shown that the expression level of CD13 is associated with tumour resistance, but whether ubenimex, as a CD13 inhibitor, reverses GC drug resistance and the underlying mechanism remain unclear. Methods: Herein, resistance to 5-fluorouracil (5-FU) was reversed in GC by ubenimex, and the underlying mechanism was determined using Cell Counting Kit-8 (CCK-8) assays, gene chip analysis, high content screening (HCS), transmission electron microscopy, flow cytometry, immunofluorescence and western blot assays. Results: Flow cytometry, transmission electron microscopy and immunofluorescence analyses indicated that ubenimex, an inhibitor of CD13, regulated the autophagy and apoptosis of SGC7901/5-FU cells by downregulating CD13 expression. In addition, Gene chip analysis and HCS demonstrated that epithelial membrane protein 3 (EMP3)/focal adhesion kinase (FAK) was a putative signalling pathway downstream of CD13. Furthermore, western blot analyses showed that ubenimex not only inhibited EMP3, FAK and nuclear factor-κB (NF-κB) expression but also suppressed GC autophagy and activated apoptosis by targeting CD13. These findings indicated a potential mechanism via the CD13/EMP3/FAK/NF-κB pathway and that the activity of which was restrained. Conclusions: Ubenimex affects autophagy and apoptosis to reverse GC cell resistance by targeting the CD13/EMP3/FAK/NF-κB pathway. These results showed that ubenimex is a promising agent that may inhibit GC autophagy to improve chemotherapeutic drug sensitivity and thereby reverse drug resistance.

Comparison of the effects of adenosine, isoproterenol and their combinations on pulmonary transit time in rats using contrast echocardiography.

AIMS: To compare the effects of adenosine (Ade), isoproterenol (Iso) and their combinations on pulmonary transit time (PTT) in rats using contrast echocardiography. MATERIAL AND METHODS: Thirty-two adult Sprague Dawley (SD) rats were divided into four groups (n=8) according the medicines of tail-intravenous injection: Group 1, control; Group 2, Ade; Group 3, Iso; Group 4, Ade+Iso. They all underwent conventional echocardiography and contrast echocardiography with measurements of PTT. RESULTS: With Ade injection, OnsetRV-OnsetLV PTT (PTT1), PeakRV-PeakLV PTT (PTT2) and OnsetRV-PeakLV PPT (PTT3) decreased and PTT3 had the largest decreased percentage, with the highest performance in differentiating the Ade group from the control group [the area under receiver operating characteristic curve (AUC), sensitivity and Youden's index was maximal]. With Iso injection, PTT1, PTT2 and PTT1 all increased and PTT1 had the largest increased percentage, with the highest performance in differentiating the Iso group from the control group (AUC, sensitivity and Youden's index was maximal). With a combination injection of Ade and Iso, the PTT values were similar to the control group and no PTT coulddifferentiate the Ade+Iso group from the control group. CONCLUSIONS: Ade or/and Iso exerted distinct effects on PTT. These findings remind us that it is a necessary to consider the effects of medicine (especially cardiopulmonary vasoactive drugs) on the PTT values. At the same time, it provides the basis for the clinical transformation of consecutive Iso/Ade treatment from the perspective of pulmonary circulation.

Ubenimex Suppresses the Ability of Migration and Invasion in Gastric Cancer Cells by Alleviating the Activity of the CD13/NAB1/MAPK Pathway.

BACKGROUND: Gastric cancer (GC) is one of the most common malignant tumors in China. Most GC patients are diagnosed at an advanced stage, for that the prognosis is dismal and metastasis is common. Although there have been increasing numbers of studies indicating that Ubenimex can suppress metastasis in GC, the underlying mechanism is still unknown. METHODS: Herein, the inhibitory effect of Ubenimex on GC metastasis, in which the underlining mechanism was determined using Gene chip analysis, high content screening (HCS), transwell assays, wound healing assays and Western blot assays. RESULTS: The results obtained from wound healing assays and transwell assays indicated that Ubenimex, an inhibitor of CD13, suppressed the migration and invasion of MKN-28, MGC-803, BGC-823 and SGC-790 cells, by downregulating CD13 expression. In addition, the findings acquired from Gene chip analysis and HCS demonstrated that NGFI-A-binding protein 1 (NAB1) was a putative target downstream of CD13. Furthermore, the results obtained from Western blot assays showed that Ubenimex not only inhibits NAB1 expression by targeting CD13, but also inhibits GC metastasis by mitigating the activity of the MAPK signaling pathway. These findings indicated a possible mechanism via the CD13/NAB1/MAPK pathway of which activity was restrained. CONCLUSION: Ubenimex exert the inhibitory effect on GC metastasis by targeting CD13, in which NAB1 expression and the activation of MAPK signaling pathway were both suppressed. This study identified a promising target for the inhibition of GC metastasis.

Evaluation of regional myocardial perfusion in methamphetamine abusers using real-time myocardial contrast echocardiography.

AIMS: To evaluate the feasibility of assessing regional myocardial perfusion using real-time myocardial contrast echocardiography (MCE) at rest for detecting coronary microcirculation abnormalities in methamphetamine abusers.Material and methods: Twenty-two male methamphetamine abusers (11 without chest pain, 11 with chest pain), free of ascertained coronary artery disease, were enrolled in this study. A control group of 22 age-matched male healthy participants was studied for comparison. Standard 2D, flow and tissue Doppler echo with measurements of cardiac morphologic and functional indicators,MCE with measurements of regional myocardial perfusion were performed, respectively. RESULTS: Compared to healthy participants, methamphetamine abusers had higher blood pressure, greater left ventricular mass index and more impaired diastolic function, with preserved cardiac sizes and systolic function. Methamphetamine abusers with chest pain had a faster heart rate than those without chest pain and healthy participants. MCE in methamphetamine abusers, especially with chest pain, had significant longer contrast agent arrival times, less functional capillary blood volumes, slower microvascular flow velocities and less myocardial perfusion than healthy participants (p<0.05). Moreover, along with the increases of dosage and duration of use (from group A to group C, group A: 1-2 g/day, <2 years; group B: 2-3 g/day, 2-5 years; group C: >3 g/day, >5 years) the reductions in the myocardial perfusion indices were more significant (p<0.01). The cutoff value with 5.1 dB2/s of the myocardial perfusion at the left ventricular apex had a sensitivity of 87.5%, specificity of 75.2% and accuracy of 81.9% for differentiating methamphetamine abusers from normal subjects. CONCLUSIONS: Real-time MCE can effectively detect coronary microcirculation abnormalities in methamphetamine abusers at rest and myocardial perfusion is significantly reduced in methamphetamine abusers. This finding may be involved in the occurrence and development of cardiac damage.

Coronary sinus flow is reduced in methamphetamine abusers: a transthoracic echocardiographic study.

To compare the coronary sinus flow among healthy participants, methamphetamine abusers without chest pain and those with chest pain. One hundred and eight methamphetamine abusers: 53 ones without chest pain, 55 ones with chest pain, free of ascertained coronary artery disease, were enrolled in this study. A control group of 50 age-matched male healthy participants was studied for comparison. Standard 2D, flow and tissue Doppler echo with measurements of cardiac morphologic and functional indicators, coronary sinus flow, and inferior vena cava (IVC) ultrasound with measurements of the IVC dimensions and their collapsibility index were performed, respectively. Compared to healthy participants, methamphetamine abusers had higher blood pressure, greater left ventricular mass index and more impaired diastolic function, with preserved cardiac sizes, systolic function and right atrial pressure. Methamphetamine abusers with chest pain had faster heart rate than those without chest pain and healthy participants. Coronary sinus flow was significantly less in methamphetamine abusers than in healthy participants (P < 0.05), and was extremely lower in those with chest pain than in healthy participants (about one-fourth) (P < 0.01). The area under the curve (AUC) of coronary sinus flow was 0.913 (0.864-0.962), and the cutoff value with 221.65 mL/min had sensitivity of 83.4%, specificity of 87.2% and accuracy of 85.2% for differentiating methamphetamine abusers from healthy participants. While the AUC of coronary sinus flow was 0.996 (0.989-1.003), and the cutoff value with 172.59 mL/min had sensitivity of 100%, specificity of 93.3% and accuracy of 96.5% for predicting methamphetamine abusers with chest pain. Coronary sinus flow is significant reduced in methamphetamine abusers, which is maybe a good indicator for indentifying methamphetamine abusers from normal population, and for predicting methamphetamine abusers with chest pain.

Findings on 3D speckle tracking echocardiography in asymptomatic methamphetamine abusers.

To detect potential cardiac abnormalities in asymptomatic methamphetamine abusers using three-dimensional speckle tracking echocardiography (3D STE). Fifty-three male methamphetamine abusers, free of cardiac symptoms/signs, were enrolled in this study. A control group of 53 age-matched male normal subjects was studied for comparison. Standard 3D, flow and tissue Doppler echo with measurements of left ventricular end-diastolic volume (LVEDV), end-systolic volume (LVESV), ejection fraction (LVEF), the ratio of the early to late diastolic transmitral filling velocity (E/A), the ratio of the early diastolic transmitral filling velocity to the early diastolic septal tissue velocity (E/E') and 3D STE with measurements of global area strain (GAS), global longitudinal strain (GLS), global circumferential strain (GCS) and global radial strain (GRS) were performed, respectively. These echocardiographic parameters were compared between methamphetamine abusers and normal subjects, and receiver operating characteristic curve (ROC) analysis was done to differentiating methamphetamine abusers from normal subjects. LVESV, LVEDV, LVEF, E/A, E/E' ratios and GRS were not significantly different between methamphetamine abusers and normal subjects (p > 0.05). However, GAS, GLS and GCS were significantly less in methamphetamine abusers than in normal subjects (p < 0.05). The areas under ROC (AUC) for GAS were greatest among all the 3D STE derived LV global strains (GAS vs. GLS, GCS and GRS, 0.95 vs. 0.76, 0.69 and 0.61, respectively). The cutoff value with - 30.3% of GAS had sensitivity of 91.8%, specificity of 91.6% and accuracy of 91.3% for differentiating methamphetamine abusers from normal subjects. The potential myocardial function abnormalities can be detected by 3D STE in asymptomatic methamphetamine abusers, and GAS is a good indicator for indentifying methamphetamine abusers from normal population, which can be used to screening and monitor methamphetamine abuse, detect subclinical LV dysfunction, predict potential methamphetamine-related cardiotoxicity, and to initiate early cardioprotective therapy before the onset of overt heart failure in time.

Fetal regional myocardial strain rate in the membranous ventricular septum: changes with gestational age and the left ventricular mass and predictive value for a complete membranous ventricular septum (without defect).

To describe the fetal regional myocardial strain rate in the membranous ventricular septum across gestation and to determine their predictive value for a complete membranous ventricular septum (without defect) after delivery. In 1150 fetuses, the peak systolic strain rate (SRs), peak early diastolic strain rate (SRe) and peak late diastolic strain rate (SRa) in the membranous ventricular septum were measured at four time points across gestation (18-20, 24-26, 30-32 and 36-38 weeks). The integrity of the interventricular septum was examined at 12 weeks' postnatal age. The correlations between myocardial strain rates and gestational age as well as fetal left ventricular mass were analyzed, and the performance of myocardial strain rates in predicting a complete membranous ventricular septum was deducted. Strain rate absolute values in the membranous ventricular septum all increased across gestation. They all significantly correlated with gestational age and left ventricular mass. At 24 weeks during pregnancy, the areas under the receiver operating characteristics curve (AUC) for SRe and SRa were all > 0.72 (p < 0.05) in predicting a complete membranous ventricular septum, while the AUC for SRs was only 0.55. The sensitivity, specificity and accuracy of the cut off value (> 1.53 s-1) for SRe was 62.5, 85.7 and 73.3%, respectively, and the sensitivity, specificity and accuracy of the cut off value (> 1.51 s-1) for SRa was 75.2, 71.9 and 73.8%, respectively. The changes of myocardial strain rates in the membranous ventricular septum across gestation maybe can be used to predict a complete membranous ventricular septum after delivery.

Gastric cancer exosomes trigger differentiation of umbilical cord derived mesenchymal stem cells to carcinoma-associated fibroblasts through TGF-ß/Smad pathway.

BACKGROUND: Mesenchymal stem cells (MSCs) promote tumor growth by differentiating into carcinoma-associated fibroblasts (CAFs) and composing the tumor microenvironment. However, the mechanisms responsible for the transition of MSCs to CAFs are not well understood. Exosomes regulate cellular activities by mediating cell-cell communication. In this study, we aimed to investigate whether cancer cell-derived exosomes were involved in regulating the differentiation of human umbilical cord-derived MSCs (hucMSCs) to CAFs. METHODOLOGY/PRINCIPAL FINDINGS: We first showed that gastric cancer cell-derived exosomes induced the expression of CAF markers in hucMSCs. We then demonstrated that gastric cancer cell-derived exosomes stimulated the phosphorylation of Smad-2 in hucMSCs. We further confirmed that TGF-ß receptor 1 kinase inhibitor attenuated Smad-2 phosphorylation and CAF marker expression in hucMSCs after exposure to gastric cancer cell-derived exosomes. CONCLUSION/SIGNIFICANCE: Our results suggest that gastric cancer cells triggered the differentiation of hucMSCs to CAFs by exosomes-mediated TGF-ß transfer and TGF-ß/Smad pathway activation, which may represent a novel mechanism for MSCs to CAFs transition in cancer.