Enzymatic Galvanic Redox Potentiometry for In Vivo Biosensing.

Redox potentiometry has emerged as a new platform for in vivo sensing, with improved neuronal compatibility and strong tolerance against sensitivity variation caused by protein fouling. Although enzymes show great possibilities in the fabrication of selective redox potentiometry, the fabrication of an enzyme electrode to output open-circuit voltage (EOC) with fast response remains challenging. Herein, we report a concept of novel enzymatic galvanic redox potentiometry (GRP) with improved time response coupling the merits of the high selectivity of enzyme electrodes with the excellent biocompatibility and reliability of GRP sensors. With a glucose biosensor as an illustration, we use flavin adenine dinucleotide-dependent glucose dehydrogenase as the recognition element and carbon black as the potential relay station to improve the response time. We find that the enzymatic GRP biosensor rapidly responds to glucose with a good linear relationship between EOC and the logarithm of glucose concentration within a range from 100 µM to 2.65 mM. The GRP biosensor shows high selectivity over O2 and coexisting neurochemicals, good reversibility, and sensitivity and can in vivo monitor glucose dynamics in rat brain. We believe that this study will pave a new platform for the in vivo potentiometric biosensing of chemical events with high reliability.

Repetitive temporal interference stimulation improves jump performance but not the postural stability in young healthy males: a randomized controlled trial.

BACKGROUND: Temporal interference (TI) stimulation, an innovative non-invasive brain stimulation technique, has the potential to activate neurons in deep brain regions. The objective of this study was to evaluate the effects of repetitive TI stimulation targeting the lower limb motor control area (i.e., the M1 leg area) on lower limb motor function in healthy individuals, which could provide evidence for further translational application of non-invasive deep brain stimulation. METHODS: In this randomized, double-blinded, parallel-controlled trial, 46 healthy male adults were randomly divided into the TI or sham group. The TI group received 2 mA (peak-to-peak) TI stimulation targeting the M1 leg area with a 20 Hz frequency difference (2 kHz and 2.02 kHz). Stimulation parameters of the sham group were consistent with those of the TI group but the current input lasted only 1 min (30 s ramp-up and ramp-down). Both groups received stimulation twice daily for five consecutive days. The vertical jump test (countermovement jump [CMJ], squat jump [SJ], and continuous jump [CJ]) and Y-balance test were performed before and after the total intervention session. Two-way repeated measures ANOVA (group × time) was performed to evaluate the effects of TI stimulation on lower limb motor function. RESULTS: Forty participants completed all scheduled study visits. Two-way repeated measures ANOVA showed significant group × time interaction effects for CMJ height (F = 8.858, p = 0.005) and SJ height (F = 6.523, p = 0.015). The interaction effect of the average CJ height of the first 15 s was marginally significant (F = 3.550, p = 0.067). However, there was no significant interaction effect on the Y balance (p > 0.05). Further within-group comparisons showed a significant post-intervention increase in the height of the CMJ (p = 0.004), SJ (p = 0.010) and the average CJ height of the first 15 s (p = 0.004) in the TI group. CONCLUSION: Repetitive TI stimulation targeting the lower limb motor control area effectively increased vertical jump height in healthy adult males but had no significant effect on dynamic postural stability.

The Effects of Antofine on the Morphological and Physiological Characteristics of Phytophthora capsici.

Phytophthora capsici is an important plant pathogenic oomycete that causes great losses to vegetable production around the world. Antofine is an important alkaloid isolated from Cynanchum komarovii Al. Iljinski and exhibits significant antifungal activity. In this study, the effect of antofine on the mycelial growth, morphology, and physiological characteristics of P. capsici was investigated using colorimetry. Meanwhile, the activity of mitochondrial respiratory chain complexes of P. capsici was evaluated following treatment with a 30% effective concentration (EC30), as well as EC50 and EC70, of antofine for 0, 12, 24, and 48 h. The results showed that antofine had a significant inhibitory effect against P. capsici, with an EC50 of 5.0795 µg/mL. After treatment with antofine at EC50 and EC70, the mycelia were rough, less full, and had obvious depression; they had an irregular protrusion structure; and they had serious wrinkles. In P. capsici, oxalic acid and exopolysaccharide contents decreased significantly, while cell membrane permeability and glycerol content increased when treated with antofine. Reactive oxygen species (ROS) entered a burst state in P. capsici after incubation with antofine for 3 h, and fluorescence intensity was 2.43 times higher than that of the control. The activities of the mitochondrial respiratory chain complex II, III, I + III, II + III, V, and citrate synthase in P. capsici were significantly inhibited following treatment with antofine (EC50 and EC70) for 48 h compared to the control. This study revealed that antofine is likely to affect the pathways related to the energy metabolism of P. capsici and thus affect the activity of respiratory chain complexes. These results increase our understanding of the action mechanism of antofine against P. capsici.

Deciphering the network of cholesterol biosynthesis in Paris polyphylla laid a base for efficient diosgenin production in plant chassis.

Cholesterol serves as a key precursor for many high-value chemicals such as plant-derived steroidal saponins and steroidal alkaloids, but a plant chassis for effective biosynthesis of high levels of cholesterol has not been established. Plant chassis have significant advantages over microbial chassis in terms of membrane protein expression, precursor supply, product tolerance, and regionalization synthesis. Here, using Agrobacterium tumefaciens-mediated transient expression technology, Nicotiana benthamiana, and a step-by-step screening approach, we identified nine enzymes (SSR1-3, SMO1-3, CPI-5, CYP51G, SMO2-2, C14-R-2, 8,7SI-4, C5-SD1, and 7-DR1-1) from the medicinal plant Paris polyphylla and established detailed biosynthetic routes from cycloartenol to cholesterol. Specfically, we optimized HMGR, a key gene of the mevalonate pathway, and co-expressed it with the PpOSC1 gene to achieve a high level of cycloartenol (28.79 mg/g dry weight, which is a sufficient amount of precursor for cholesterol biosynthesis) synthesis in the leaves of N. benthamiana. Subsequently, using a one-by-one elimination method we found that six of these enzymes (SSR1-3, SMO1-3, CPI-5, CYP51G, SMO2-2, and C5-SD1) were crucial for cholesterol production in N. benthamiana, and we establihed a high-efficiency cholesterol synthesis system with a yield of 5.63 mg/g dry weight. Using this strategy, we also discovered the biosynthetic metabolic network responsible for the synthesis of a common aglycon of steroidal saponin, diosgenin, using cholesterol as a substrate, obtaining a yield of 2.12 mg/g dry weight in N. benthamiana. Our study provides an effective strategy to characterize the metabolic pathways of medicinal plants that lack a system for in vivo functional verification, and also lays a foundation for the synthesis of active steroid saponins in plant chassis.

CRISPR/Cas9-HPV-liposome enhances antitumor immunity and treatment of HPV infection-associated cervical cancer.

Increasing evidence shows that human papillomavirus (HPV) E6/E7 deletion in cervical cancer cells may be related to the immunosuppressive tumor microenvironment and adverse reactions or resistance to immune checkpoint blockade. Here, we demonstrate that liposome delivery of CRISPR/cas9 can effectively knock out HPV, which, in turn, induces autophagy and triggers cell death-related immune activation by releasing damage-related molecular patterns. The results of in vivo experiments showed that HPV-targeting guide RNA-liposomes could promote CD8+ T cell infiltration in tumor tissues; enhance the expression of proinflammatory cytokines, such as interleukin-12, tumor necrosis factor-α, and interferon-γ, and reduce regulatory T cells and myeloid suppressor cells. The combination of HPV-targeting guide RNA-liposomes with immune checkpoint inhibitors and antiprogrammed death-1 antibodies produced highly effective antitumor effects. In addition, combination therapy induced immune memory in the cervical cancer model.

Intravaginal delivery for CRISPR-Cas9 technology: For example, the treatment of HPV infection.

The increasing incidence of sexually transmitted diseases in women, including human papillomavirus (HPV) infection, has led to the need to develop user-friendly potential prevention methods. At present, although there are several therapeutic parts, none of them has a preventive effect, but they are only limited to providing patients with symptom relief. Researchers have now recognized the need to find effective local preventive agents. One of the potential undiscovered local fungicides is the vaginal delivery of CRISPR/Cas9. CRISPR/Cas9 delivery involves silencing gene expression in a sequence-specific manner in the pathogenic agent, thus showing microbicidal activity. However, vaginal mucosal barrier and physiological changes (such as pH value and variable epithelial thickness in the menstrual cycle) are the main obstacles to effective delivery and cell uptake of CRISPR/Cas9. To enhance the vaginal delivery of CRISPR/Cas9, so far, nano-carrier systems such as lipid delivery systems, macromolecular systems, polymer nanoparticles, aptamers, and cell-penetrating peptides have been extensively studied. In this paper, various nano-carriers and their prospects in the preclinical stage are described, as well as the future significance of CRISPR/Cas9 vaginal delivery based on nano-carriers.

Association of major dietary patterns and different obesity phenotypes in Southwest China: the China Multi-Ethnic Cohort (CMEC) Study.

PURPOSE: Dietary behavior is an important part of lifestyle interventions for obesity and its cardiovascular comorbidities. However, little is known about associations between dietary patterns and obesity phenotypes in Southwest China, a region with unique dietary patterns and significant heterogeneity in obesity. METHODS: Data from the baseline survey of the China Multi-Ethnic Cohort in Southwest China were analyzed (n = 64,448). Dietary intakes during the past year were measured with the semi-quantitative Food Frequency Questionnaire (s-FFQ). Principal component factor analysis (PCFA) was used to identify dietary patterns. Multinomial logistic regressions were used to examine the associations between dietary patterns and obesity phenotypes and stratified analyses were performed to assess whether the associations differed across demographic variables. RESULTS: Three dietary patterns were identified and then named according to their apparent regional gathering characteristics: the Sichuan Basin dietary pattern (characterized by high intakes of various foods), the Yunnan-Guizhou Plateau dietary pattern (characterized by agricultural lifestyles), and the Qinghai-Tibet Plateau dietary pattern (characterized by animal husbandry lifestyles), respectively. Higher adherence to the Sichuan Basin dietary pattern was positively associated with metabolically healthy overweight/obesity (MHO, OR 1.13, 95% CI 1.05-1.21) but negatively associated with metabolically unhealthy normal weight (MUNW, OR 0.78, 95% CI 0.65-0.95). Higher adherence to the other two dietary patterns was positively associated with MHO and metabolically unhealthy overweight/obesity (MUO). Besides, differences in socioeconomic status also affected the relationship between dietary patterns and obesity phenotypes. CONCLUSIONS: Adherence to the more diverse Sichuan basin dietary pattern performed a mixed picture, while the other two may increase the risk of obesity phenotypes, which indicates nutritional interventions are urgently needed.

Associations between major dietary patterns and blood pressure among Southwest Chinese: A cross-sectional analysis based on the China Multi-Ethnic Cohort (CMEC) study.

BACKGROUND AND AIMS: In the less developed multi-ethnic regions (LEMRs) of Southwest China, the associations between dietary patterns and blood pressure (BP) values remain unclear. We aimed to investigate such associations and related effect modifiers. METHODS AND RESULTS: This study included 81,433 participants from the China Multi-Ethnic Cohort Study. Dietary intakes during the year before the interview were measured with the Quantitative Food Frequency Questionnaire. Three major dietary patterns that were highly in line with geographical and ethnic distributions of the study population, i.e., "Sichuan Basin," "Yunnan-Guizhou Plateau," and "Qinghai-Tibet Plateau," were derived using principal component factor analysis. The multilinear regression model combined with inverse probability of exposure weighting was used to estimate the associations between dietary patterns and BP values. Comparing the highest with the lowest quintiles, the Sichuan Basin dietary pattern (characterized by urban lifestyles) was associated with 2.67 mmHg lower systolic blood pressure (SBP) (95% CI: -3.07 to -2.27) and 0.89 mmHg lower diastolic blood pressure (95% CI: -1.12 to -0.65). In contrast, both the Yunnan-Guizhou Plateau (characterized by agricultural lifestyles) and the Qinghai-Tibet Plateau dietary patterns (characterized by nomadic lifestyles) showed positive associations with BP. In the stratified analysis, the associations between dietary patterns and SBP were significantly stronger in women than in men for all three kinds of dietary patterns. CONCLUSION: Both major dietary patterns and their associations with BP showed a substantial disparity in LEMRs of Southwest China. Dietary patterns in regions of higher socioeconomic status are more conducive to reducing the rising of BP, especially for women and urban residents, which might provide insights into the BP control in LEMRs of Southwest China.

Association of dietary patterns with obesity and metabolically healthy obesity phenotype in Chinese population: a cross-sectional analysis of China Multi-Ethnic Cohort Study.

Metabolically healthy obesity (MHO) might be an alternative valuable target in obesity treatment. We aimed to assess whether alternative Mediterranean (aMED) diet and Dietary Approaches to Stop Hypertension (DASH) diet were favourably associated with obesity and MHO phenotype in a Chinese multi-ethnic population. We conducted this cross-sectional analysis using the baseline data of the China Multi-Ethnic Cohort study that enrolled 99 556 participants from seven diverse ethnic groups. Participants with self-reported cardiometabolic diseases were excluded to eliminate possible reverse causality. Marginal structural logistic models were used to estimate the associations, with confounders determined by directed acyclic graph (DAG). Among 65 699 included participants, 11·2 % were with obesity. MHO phenotype was present in 5·7 % of total population and 52·7 % of population with obesity. Compared with the lowest quintile, the highest quintile of DASH diet score had 23 % decreased odds of obesity (OR = 0·77, 95 % CI 0·71, 0·83, Ptrend < 0·001) and 27 % increased odds of MHO (OR = 1·27, 95 % CI 1·10, 1·48, Ptrend = 0·001) in population with obesity. However, aMED diet showed no obvious favourable associations. Further adjusting for BMI did not change the associations between diet scores and MHO. Results were robust to various sensitivity analyses. In conclusion, DASH diet rather than aMED diet is associated with reduced risk of obesity and presents BMI-independent metabolic benefits in this large population-based study. Recommendation for adhering to DASH diet may benefit the prevention of obesity and related metabolic disorders in Chinese population.

An ultrasensitive electrochemiluminescence biosensor for the detection of total bacterial count in environmental and biological samples based on a novel sulfur quantum dot luminophore.

In this paper, the electrochemiluminescence (ECL) of sulfur quantum dots (SQDs) in a potassium persulfate cathodic co-reactant was studied. Based on the selective quenching of the ECL emission from the SQDs by ß-nicotinamide adenine dinucleotide (NADH), an ultrasensitive ECL biosensor with NADH as an important parameter was established for the highly sensitive and selective detection of total bacterial count (TBC). A linear response between the ECL intensity and the NADH concentration in the range of 1 pM to 10 µM was obtained, thus achieving a detection limit of 1 pM. As the content of NADH in cells is positively correlated with the TBC, a sensor has been successfully applied to detect the TBC in actual water samples with a good recovery rate of 103-107 CFU mL-1. This study provides a green and feasible method for TBC detection in the environment.

Integrated metabolome and transcriptome analysis of the anthocyanin biosynthetic pathway in relation to color mutation in miniature roses.

BACKGROUND: Roses are famous ornamental plants worldwide. Floral coloration is one of the most prominent traits in roses and is mainly regulated through the anthocyanin biosynthetic pathway. In this study, we investigated the key genes and metabolites of the anthocyanin biosynthetic pathway involved in color mutation in miniature roses. A comparative metabolome and transcriptome analysis was carried out on the Neptune King rose and its color mutant, Queen rose, at the blooming stage. Neptune King rose has light pink colored petals while Queen rose has deep pink colored petals. RESULT: A total of 190 flavonoid-related metabolites and 38,551 unique genes were identified. The contents of 45 flavonoid-related metabolites, and the expression of 15 genes participating in the flavonoid pathway, varied significantly between the two cultivars. Seven anthocyanins (cyanidin 3-O-glucosyl-malonylglucoside, cyanidin O-syringic acid, cyanidin 3-O-rutinoside, cyanidin 3-O-galactoside, cyanidin 3-O-glucoside, peonidin 3-O-glucoside chloride, and pelargonidin 3-O-glucoside) were found to be the major metabolites, with higher abundance in the Queen rose. Thirteen anthocyanin biosynthetic related genes showed an upregulation trend in the mutant flower, which may favor the higher levels of anthocyanins in the mutant. Besides, eight TRANSPARENT TESTA 12 genes were found upregulated in Queen rose, probably contributing to a high vacuolar sequestration of anthocyanins. Thirty transcription factors, including two MYB and one bHLH, were differentially expressed between the two cultivars. CONCLUSIONS: This study provides important insights into major genes and metabolites of the anthocyanin biosynthetic pathway modulating flower coloration in miniature rose. The results will be conducive for manipulating the anthocyanin pathways in order to engineer novel miniature rose cultivars with specific colors.

20(S)-Rg3 upregulates FDFT1 via reducing miR-4425 to inhibit ovarian cancer progression.

We previously found that ginsenoside 20(S)-Rg3 diminishes the proliferative and invasive capacities of ovarian cancer cells by decreasing miR-4425 level. Yet the mechanism of action of miR-4425 in ovarian cancer remains unclear. Here we report that miR-4425 is upregulated in ovarian cancer tissues relative to normal ovarian tissues, and transfection of miR-4425 inhibitor impairs the proliferation, migration and invasion of SKOV3 and 3AO ovarian cancer cells. Further, miR-4425 antagomiR reduces cell proliferation in a subcutaneous SKOV3 xenograft model using BALB/c nude mice. We identifies farnesyl-diphosphate farnesyltransferase 1 (FDFT1) as a direct target of miR-4425 by Western blotting and a luciferase reporter assay. Forced expression of FDFT1 via transfection of an FDFT1-expressing plasmid into ovarian cancer cells not only retards cell proliferation, motility and invasiveness, but also negates the tumorigenic properties of a miR-4425 mimic. By contrast, silencing of FDFT1 by siRNAs abrogates suppression of the proliferation, migration and invasion of ovarian cancer cells treated with a miR-4425 inhibitor. Finally, transfection of either a miR-4425 mimic or FDFT1 siRNAs into 20(S)-Rg3-treated ovarian cancer cells counteracts the tumor-inhibitory activity of the ginsenoside. In conclusion, 20(S)-Rg3 exerts anti-ovarian cancer activity by downregulating oncogenic miR-4425 that inhibits the expression of the tumor suppressor gene FDFT1. These results expand our current understanding of the molecular pathways leading to ovarian cancer progression, and unveil the mechanism of action of 20(S)-Rg3 in ovarian cancer inhibition.

Ginsenoside 20(S)-Rg3 upregulates HIF-1α-targeting miR-519a-5p to inhibit the Warburg effect in ovarian cancer cells.

The Warburg effect, one of the metabolic hallmarks of cancer, is responsible for rapid energy production through a high rate of aerobic glycolysis. Ginsenoside 20(S)-Rg3 antagonizes the Warburg effect in ovarian cancer cells by upregulating some microRNAs, including miR-519a-5p, that target key enzymes involved in aerobic glycolysis. How 20(S)-Rg3-upregulated miR-519a-5p influences the Warburg effect in ovarian cancer cells remains poorly defined, however. Here we report that while overexpression of miR-519a-5p in ovarian cancer cells inhibited the Warburg effect, inhibition of miR-519a-5p negated the suppressive action of 20(S)-Rg3 against the Warburg effect as evidenced by a decrease in glucose consumption, lactate production and HK2 expression. We identified HIF-1α as a direct target of miR-519a-5p via luciferase reporter assays and demonstrated the counteraction by overexpressed HIF-1α of 20(S)-Rg3-suppressed Warburg effect. Further, 20(S)-Rg3 decreased DNMT3A-mediated DNA methylation in the promoter region of its precursor gene, leading to an increase in the level of miR-519a-5p. In conclusion, 20(S)-Rg3 upregulates miR-519a-5p via reducing DNMT3A-mediated DNA methylation to inhibit HIF-1α-stimulated Warburg effect in ovarian cancer.

Effects of the Inertia Barbell Training on Lumbar Muscle T2 Relaxation Time.

Sun, M-Y, Lu, J-Q, Ma, Z-C, Lü, J-J, Huang, Q, Sun, Y-N, and Liü, Y. Effects of the inertia barbell training on lumbar muscle T2 relaxation time. J Strength Cond Res 34(12): 3454-3462, 2020-The purpose of this study was to investigate variations in T2 relaxation time in normal human lumbar muscles caused by inertia barbell training. Thirty undergraduate healthy men (mean age = 19 ± 1.2 years, body mass = 72 ± 10.0 kg, and height = 1.78 ± 0.1 m) were recruited to participate in this study. Subjects were randomly assigned into 2 groups: an inertia barbell training group (IBTG) (n = 15) and a normal barbell-training group (NBTG) (n = 15). All subjects participated in lumbar flexion and extension muscle strength training for 1 hour per time, 3 times per week for a total of 8 weeks. The lumbar area of each subject was scanned before and after the experiment using a 3.0T superconductive magnetic resonance imaging system. The T2 values measured after intervention were significantly different compared with the T2 values measured before the experiment in both the IBTG and NBTG groups (p < 0.001). After intervention, there was no significant difference in T2 values between the IBTG and NBTG groups (p = 0.17). The ([INCREMENT]T2)/T2 percentage was significantly different in the IBTG group (p < 0.01). This study demonstrated that 8 weeks of strength training led to significant improvements in the values for T2 relaxation time of the lumbar muscles. Furthermore, the ([INCREMENT]T2)/T2 percentage for IBTG was higher than that for NBTG, which suggested that lumbar muscle activity increased more with inertial barbell training.

miR-603 targeted hexokinase-2 to inhibit the malignancy of ovarian cancer cells.

The Warburg effect, characterized by energy production through a high rate of aerobic glycolysis, is a metabolic hallmark of cancer cells. We previously found that ginsenoside 20(S)-Rg3 upregulated miR-603 and impaired the malignancy of ovarian cancer cells by inhibiting the Warburg effect. However, the precise functional role of miR-603 in ovarian cancer progression remains poorly defined. Here, we report that the level of miR-603 in ovarian cancer tissues is significantly lower than that in para-tumor tissues. Overexpression of miR-603 in ovarian cancer cells inhibits the Warburg effect as evidenced by a decrease in glucose consumption, lactate production and hexokinase-2 (HK2) expression, reduces cell proliferation in vitro, and weakens their migration and invasion. Further, miR-603 directly targets HK2 as indicated in a luciferase reporter assay. In contrast to agomiR-NC, agomiR-603 treatment significantly inhibits tumor growth in vivo and the Warburg effect, which is illustrated by a decreased uptake of 18F-FDG in subcutaneous xenografts and HK2 downregulation. Finally, miR-603 is negatively regulated by DNMT3A-mediated DNA methylation in the promoter region of its precursor gene, suggesting that 20(S)-Rg3 antagonizes DNMT3A-mediated DNA methylation to impair growth, migration and invasion of ovarian cancer cells. In conclusion, miR-603 is a tumor suppressor targeting HK2 in ovarian cancer and its low level may result from DNMT3A-mediated methylation.

Ginsenoside 20(S)-Rg3 Inhibits the Warburg Effect Via Modulating DNMT3A/ MiR-532-3p/HK2 Pathway in Ovarian Cancer Cells.

BACKGROUND/AIMS: The Warburg effect is one of the main energy metabolism features supporting cancer cell growth. 20(S)-Rg3 exerts anti-tumor effect on ovarian cancer partly by inhibiting the Warburg effect. microRNAs are important regulators of the Warburg effect. However, the microRNA regulatory network mediating the anti-Warburg effect of 20(S)-Rg3 was largely unknown. METHODS: microRNA deep sequencing was performed to identify the 20(S)-Rg3-influenced microRNAs in SKOV3 ovarian cancer cells. miR-532-3p was overexpressed by mimic532-3p transfection in SKOV3 and A2780 cells or inhibited by inhibitor532-3p transfection in 20(S)-Rg3-treated cells to examine the changes in HK2 and PKM2 expression, glucose consumption, lactate production and cell growth. Dual-luciferase reporter assay was conducted to verify the direct binding of miR-532-3p to HK2. The methylation status in the promoter region of pre-miR-532-3p gene was examined by methylation-specific PCR. Expression changes of key molecules controlling DNA methylation including DNMT1, DNMT3A, DNMT3B, and TET1-3 were examined in 20(S)-Rg3-treated cells. DNMT3A was overexpressed in 20(S)-Rg3-treated cells to examine its influence on miR-532-3p level, HK2 and PKM2 expression, glucose consumption and lactate production. RESULTS: Deep sequencing results showed that 11 microRNAs were increased and 9 microRNAs were decreased by 20(S)-Rg3 in SKOV3 cells, which were verified by qPCR. More than 2-fold increase of miR-532-3p was found in 20(S)-Rg3-treated SKOV3 cells. Forced expression of miR-532-3p reduced HK2 and PKM2 expression, glucose consumption and lactate production in SKOV3 and A2780 ovarian cancer cells. Inhibition of miR-532-3p antagonized the suppressive effect of 20(S)-Rg3 on HK2 and PKM2 expression, glucose consumption and lactate production in ovarian cancer cells. Dual-luciferase reporter assay showed that miR-532-3p directly suppressed HK2 rather than PKM2. miR-532-3p level was controlled by the methylation in the promoter region of its host gene. 20(S)-Rg3 inhibited DNMT3A expression while exerted insignificant effect on DNMT1, DNMT3B and TET1-3. 20(S)-Rg3 reversed DNMT3A-mediated methylation in the promoter of the host gene of miR-532-3p, and thus elevated miR-532-3p level followed by suppression of HK2 and PKM2 expression, glucose consumption and lactate production. CONCLUSIONS: 20(S)-Rg3 modulated microRNAs to exert the anti-tumor effect in ovarian cancer. 20(S)-Rg3 lessened the DNMT3A-mediated methylation and promoted the suppression of miR-532-3p on HK2 to antagonize the Warburg effect of ovarian cancer cells.

Ginsenoside 20(S)-Rg3 Prevents PKM2-Targeting miR-324-5p from H19 Sponging to Antagonize the Warburg Effect in Ovarian Cancer Cells.

BACKGROUND/AIMS: The Warburg effect is one of the main metabolic features for cancers, with long non-coding RNA (lncRNA) being involved as a class of crucial regulators. Our previous studies have shown that ginsenoside 20(S)-Rg3, an active saponin monomer extracted from red ginseng, inhibits the Warburg effect in ovarian cancer cells. However, the detailed lncRNA regulatory network modulated by 20(S)-Rg3 to prevent the Warburg effect in ovarian cancer cells has not been explored. METHODS: High-throughput sequencing was used to screen out the differentially expressed lncRNAs between 20(S)-Rg3-treated and non-treated SKOV3 cells. The levels of lncRNA H19 and miR-324-5p were manipulated in SKOV3 and A2780, and the glucose consumption, lactate production and PKM2 protein level were detected. Dual-luciferase reporter assay and RIP were utilized to verify the direct binding of H19 to miR-324-5p and miR-324-5p to PKM2. Cell proliferation was examined by CCK8 and colony formation assay. Nude mice subcutaneous xenograft tumor models were established to evaluate the impact of miR-324-5p on tumor growth in vivo. RESULTS: 20(S)-Rg3 downregulated 67 lncRNAs, and H19 was one of the most decreased lncRNAs. Suppression of H19 by siRNA transfection reduced glucose consumption, lactate production and PKM2 expression in ovarian cancer cells, while H19 overexpression in 20(S)-Rg3-treated ovarian cancer cells enhanced glucose consumption, lactate production and PKM2 expression. Dual-luciferase reporter assay and RIP results showed that H19 directly bound to miR-324-5p. Dual-luciferase reporter assay showed that miR-324-5p directly targeted PKM2, and miR-324-5p negatively regulated glucose consumption and lactate production in ovarian cancer cells. miR-324-5p overexpression inhibited cell proliferation in vitro and in vivo. CONCLUSION: Our study revealed that 20(S)-Rg3 blocked the competitive inhibition of H19 on miR-324-5p, which enhanced the suppression of miR-324-5p on PKM2 and therefore inhibited the Warburg effect and repressed tumorigenesis. In a word, 20(S)-Rg3 inhibited the Warburg effect in ovarian cancer cells via H19/miR-324-5p/PKM2 pathway.

FSCN1 Promotes Epithelial-Mesenchymal Transition Through Increasing Snail1 in Ovarian Cancer Cells.

BACKGROUND/AIMS: Epithelial-mesenchymal transition (EMT) is one of the key mechanisms mediating cancer progression. Snail1 has a pivotal role in the regulation of EMT, involving the loss of E-cadherin and concomitant upregulation of vimentin, among other biomarkers. We have found FSCN1 promoted EMT in ovarian cancer cells, but the precise mechanism of FSCN1 in EMT process has not been clearly elucidated. METHODS: The levels of FSCN1 and snail1 were determined in epithelial ovarian cancer(EOC) specimen and in ovarian cancer cells by RT-qPCR. The changes of EMT makers and effects on snail1 by FSCN1 were examined by overexpression or depletion of FSCN1 in EOC cells by RT-qPCR and western blotting. The invasiveness of the FSCN1-modified EOC cells was examined in transwell assay. Co-immunoprecipitation (IP) was performed to detect the interaction between snail1 and FSCN1 in EOC cells. RESULTS: We found FSCN1 and snail1 significantly increased in EOC, and especially in EOC with metastasis. FSCN1 was positively correlated with snail1 expression at the cellular/histological levels. Moreover, we further showed that FSCN1 physiologically interacted with and increased the levels of snail1 to promote ovarian cancer cell EMT. CONCLUSION: FSCN1 promote EMT through snail1 in ovarian cancer cells. FSCN1 is an attractive novel target for inhibiting invasion and metastasis of EOC cells.

WITHDRAWN: CRISPR/Cas9 mediated HPV and PD1 inhibition produces a synergistic anti-tumor effect on cervical cancer.

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Glycosylation status of bone sialoprotein and its role in mineralization.

The highly glycosylated bone sialoprotein (BSP) is an abundant non-collagenous phosphoprotein in bone which enhances osteoblast differentiation and new bone deposition in vitro and in vivo. However, the structural details of its different glycosylation linkages have not been well studied and their functions in bone homeostasis are not clear. Previous studies suggested that the O-glycans, but not the N-glycans on BSP, are highly sialylated. Herein, we employed tandem mass spectrometry (MS/MS) to demonstrate that the N-glycanson the recombinant human integrin binding sialoprotein (rhiBSP) are also enriched in sialic acids (SAs) at their termini. We also identified multiple novel sites of N-glycan modification. Treatment of rhiBSP enhances osteoblast differentiation and mineralization of MC3T3-E1 cells and this effect could be partially reversed by efficient enzymatic removal of its N-glycans. Removal of all terminal SAs has a greater effect in reversing the effect of rhiBSP on osteogenesis, especially on mineralization, suggesting that sialylation at the termini of both N-glycans and O-glycans plays an important role in this regulation. Moreover, BSP-conjugated SAs may affect mineralization via ERK activation of VDR expression. Collectively, our results identified novel N-glycans enriched in SAs on the rhiBSP and demonstrated that SAs at both N- and O-glycans are important for BSP regulation of osteoblast differentiation and mineralization in vitro.