Epigenetic silencing of microRNA-335 contributes to nasopharyngeal carcinoma metastasis.

BACKGROUND: MircoRNA-335 (miR-335), a member of mircoRNAs (miRNAs) family, has been found to be correlated with tumor invasion and metastasis. In this study, we aimed to detect the effect of miR-335 methylation on metastasis of nasopharyngeal carcinoma. METHODS: RT-PCR and methylation-specific PCR were applied to detect the expression levels of miR-335 and miR-335 methylation in nasopharyngeal carcinoma tissues. RESULTS: The levels of miR-335 expression were significantly lower in nasopharyngeal cancer tissues with promoter methylation, compared to those with promoter unmethylation. The levels of miR-335 gene promoter methylation were higher in 14 (46.7%) out of 30 nasopharyngeal cancer tissues. Furthermore, the patients with cervical lymph node metastasis had higher methylation rate in miR-335 promoter (66.7% versus 16.7%) than those without cervical lymph node metastasis. CONCLUSION: Gene methylation contributes the expression of miR-335 in nasopharyngeal carcinoma. The expression of miR-335 methylation is correlated with the metastasis of nasopharyngeal carcinoma.

Effects of DACT1 methylation status on invasion and metastasis of nasopharyngeal carcinoma.

BACKGROUND: The purpose of the present study was to investigate the role of the methylation status of the DACT1 gene on the invasion and metastasis of nasopharyngeal carcinoma cells. METHODS: The levels of methylation and expression of the DACT1 gene in nasopharyngeal carcinoma tissues and CNE2 cells were determined by methylation-specific PCR and RT-PCR, respectively. CNE2 cells were treated with 5-aza-2-deoxycytidine, and the variation in the methylation status of the DACT1 gene was detected, as well as the influence of methylation on invasiveness of nasopharyngeal carcinoma cells. RESULTS: The DACT1 gene was hyper-methylated in 44 of 62 cases of nasopharyngeal carcinoma. The DACT1 gene was hyper-methylated in 32 of 38 cases of nasopharyngeal carcinoma with lymph node metastasis, and the DACT1 gene was hyper-methylated in 7 of 24 cases of nasopharyngeal carcinoma without lymph node metastasis. The DACT1 mRNA level was weakly expressed or not expressed in all nasopharyngeal carcinoma tissues with hyper-methylated DACT1 genes; however, the DACT1 mRNA level was highly expressed in nasopharyngeal carcinoma tissues with low expression of the methylated DACT1 gene. The DACT1 gene was hyper-methylated and not expressed in CNE2 cells that did not have 5-aza-2-deoxycytidine treatment. After 5-aza-2-deoxycytidine treatment, the DACT1 gene was demethylated and the expression of DACT1 was restored. Moreover, the invasion ability was inhibited in CNE2 cells treated with 5-aza-2-deoxycytidine. CONCLUSION: The expression of DACT1 was related to the methylation status. High expression of DACT1 may inhibit the invasion and metastasis of nasopharyngeal carcinoma cells.

Characteristics and Impact Factors of Renal Threshold for Glucose Excretion in Patients with Type 2 Diabetes Mellitus.

Sodium glucose co-transporter 2 (SGLT-2) inhibitors are newly developed but promising medicine for type 2 diabetes. However, patients with a different renal threshold for glucose excretion (RT(G)) may have a different reaction to this medicine. Therefore, the objective of this study was to investigate the characteristics of RT(G) and its impact factors in patients with type 2 diabetes mellitus (T2DM). The clinical and laboratory data of 36 healthy individuals and 168 in-hospital patients with T2DM were collected and analyzed, RT(G) was calculated using blood glucose (BG) measured by dynamic BG monitoring, urinary glucose excretion (UGE) and estimated glomerular filtration rate (eGFR). The characteristics of RT(G) were investigated. The risk factors for high RT(G) were analyzed using non-conditional logistic regression analysis. Our results found that RT(G) of the T2DM group was higher than that of the healthy individuals (P < 0.05); and 22.22% from the healthy individuals group but 58.33% from the T2DM group had high RT(G). Age, duration of diabetes, body mass index (BMI), and homeostasis model assessment insulin resistance index (HOMA-IR) were independently associated with high RT(G) (P < 0.05). Further stratified analysis revealed that RT(G) in T2DM patients increased with age, duration of diabetes, and BMI. In conclusion, RT(G) is increased in patients with T2DM, especially in those with longer diabetic duration, higher BMI, and those who are older. Therefore, these patients may be more sensitive to SGLT-2 inhibitors.

Liraglutide reduces the body weight and waist circumference in Chinese overweight and obese type 2 diabetic patients.

AIM: To investigate the effects of liraglutide, a glucagon-like peptide-1 (GLP-1) receptor activator, on body weight and waist circumference in Chinese overweight and obese type 2 diabetic patients. METHODS: A total of 328 Chinese overweight and obese type 2 diabetic patients were included in this multi-center, open-labeled and self-controlled clinical study. The patients were subcutaneously injected with liraglutide once daily for 24 weeks as add-on therapy to their previous hypoglycemic treatments. Statistical analyses were performed using SPSS software package version 11.5 for Windows. RESULTS: Liraglutide treatment caused significant reduction of the mean body weight (from 86.61±14.09 to 79.10±13.55 kg) and waist circumference (from 101.81±13.96 to 94.29±14.17 cm), resulting in body weight lose of 5%-10% in 43.67% patients, and body weight loss above 10% in 34.06% patients, who had significant lower plasma creatinine levels. Baseline waist circumference, BMI and HOMA-IR were independently correlated with the body weight loss. Furthermore, liraglutide treatment significantly decreased HbA1c levels (from 8.66%±2.17% to 6.92%±0.95%) with HbA1c<7.0% in 35.37% patients, who had a significantly lower baseline level of HbA1c, but higher baseline levels of C peptide and glucagon. Moreover, liraglutide treatment resulted in greater body weight loss in patients with a long duration of diabetes, and better glycemic control in patients with a short duration of diabetes. CONCLUSION: Liraglutide significantly reduces body weight and waist circumference in Chinese overweight and obese type 2 diabetic patients. Patients with apparent visceral obesity, insulin resistance and a long duration of diabetes may have greater body weight loss; whereas patients with high insulin-secreting ability, hyperglucagonemia, and short-duration diabetes may obtain better glycemic control with liraglutide.

Effects of 24-week treatment with acarbose on glucagon-like peptide 1 in newly diagnosed type 2 diabetic patients: a preliminary report.

BACKGROUND: Treatment with the alpha-glucosidase inhibitor (AGI) acarbose is associated with a significant reduction the risk of cardiovascular events. However, the underlying mechanisms of this effect are unclear. AGIs were recently suggested to participate in stimulating glucagon-like peptide 1 (GLP-1) secretion. We therefore examined the effects of a 24-week treatment of acarbose on endogenous GLP-1, nitric oxide (NO) levels, nitric oxide synthase (NOS) activity, and carotid intima-media thickness (CIMT) in newly diagnosed patients with type 2 diabetes (T2D). METHODS: Blood was drawn from 24 subjects (14 male, 10 female, age: 50.7 ± 7.36 years, BMI: 26.64 ± 3.38 kg/m2, GHbA1c: 7.00 ± 0.74%) with drug-naïve T2D at 0 and 120 min following a standard mixed meal for the measurements of active GLP-1, NO and NOS. The CIMT was measured prior to and following 24 weeks of acarbose monotherapy (mean dose: 268 mg daily). RESULTS: Following 24 weeks of acarbose treatment, both fasting and postprandial plasma GLP-1 levels were increased. In patients with increased postprandial GLP-1 levels, serum NO levels and NOS activities were also significantly increased and were positively related to GLP-1 levels. Although the CIMT was not significantly altered following treatment with acarbose, a decreased CIMT was negatively correlated with increased GLP-1 levels. CONCLUSIONS: Twenty-four weeks of acarbose monotherapy in newly diagnosed patients with T2D is associated with significantly increased levels of both fasting and postprandial GLP-1 as well as significantly increased NO levels and NOS activity for those patients in whom postprandial GLP-1 levels were increased. Therefore, the benefits of acarbose on cardiovascular risk may be related to its stimulation of GLP-1 secretion.

[Research on the effect of statins on insulin secretion from pancreatic islet in rats and its mechanisms].

OBJECTIVE: To evaluate the inhibitory effect of statins on glucose-stimulated insulin secretion (GSIS) of pancreatic islet in rat and to explore its mechanisms. METHODS: According to the average volume, freshly isolated or 24-hour cultured pancreatic islets were randomly divided into control group (incubated with Kreb-Ringer bicarbonate buffer), the atorvastatin group (incubated with 100 µmol/L atorvastatin), the fluvastatin group (incubated with 100 µmol/L fluvastatin) and the pravastatin group (incubated with 100 µmol/L pravastatin). Stimulated by 2.8, 5.5, 11.1, 16.7 mmol/L and 25.0 mmol/L glucose respectively, the effect of 100 µmol/L statins on ATP content and GSIS was compared in the four groups. GSIS was performed by the 37°C bath incubation method and ATP content was measured by chemiluminescence method. RESULTS: Incubated with 100 µmol/L atorvastatin for 30 minutes, in the presence of 16.7 mmol/L glucose, the ATP content [(9.54 ± 1.64) pmol/islet vs (12.33 ± 1.89) pmol/islet] and GSIS (1.60 ± 0.21 vs 2.39 ± 0.30) were significantly reduced in comparison with the control group (P < 0.05). Cultured with 100 µmol/L fluvastatin for 24 hours, the ATP content [(10.24 ± 2.01) pmol/islet vs (12.31 ± 2.16) pmol/islet] and GSIS (3.12 ± 0.32 vs 4.17 ± 0.37) were all significantly decreased at the higher glucose concentration of 16.7 mmol/L (P < 0.05). CONCLUSION: Atorvastatin and fluvastatin may inhibit GSIS by decreasing ATP content in pancreatic islet and the inhibitory effect is related to the strength of its lipophilicity.

[Effects of transketolase-like gene TKTL1 on occurrence and metastasis of human nasopharyngeal carcinoma].

OBJECTIVE: To investigate the effects of transketolase-like gene TKTL1 on the occurrence and metastasis of human nasopharyngeal carcinoma (NPC). METHODS: Real-time PCR was used to determine the mRNA expression levels of transketolase gene family (TKT, TKTL1, and TKTL2) in the 65 biopsy specimens of human nasopharyngeal carcinoma, 9 at stage I, 15 at stage II, 16 at stage III, 13 at stage IVA, and 12 at stage IVB, 42 with metastasis and 232 without metastasis, and 9 biopsy specimens of chronic nasopharyngitis. RESULTS: The TKL activity level of the NPC tissues was 21.6 +/- 2.8, significantly higher than that of the chronic nasopharyngitis tissues (6.2 +/- 1.3, P < 0.01). The TKL activity level of the NPC tissues with metastasis was 26.5 +/- 3.2, significantly higher than that of the NPC tissues without metastasis (17.6 +/- 2.1, P < 0.01). There was no significant difference in the expression levels of TKT and TKTL2 genes between the human NPC and chronic nasopharyngitis tissues (both P > 0.05). The expression level of TKTL1 gene in the NPC tissues was 5.25 +/- 0.32, significantly higher than that in the chronic nasopharyngitis tissues (0.98 +/- 0.11, t = 6.23, P < 0.01), and the expression level of TKTL1 gene in the NPC tissues with lymph node metastasis was 5.86 +/- 0.38, significantly higher than that in the NPC tissues without lymph node metastasis (4.18 +/- 0.22, t = 3.28, P < 0.01). CONCLUSION: TKTL1 may play an important role in the occurrence and metastasis of human NPC and become a potential target for novel anti-cancer therapy.

Gene silencing of TKTL1 by RNAi inhibits cell proliferation in human hepatoma cells.

We detected a strong upregulation of the mutated transketolase transcript (TKTL1) in human hepatoma cell line HepG2, whereas transketolase (TKT) and transketolase-like-2 (TKTL2) transcripts were not upregulated. We inhibited the expression of TKTL1 by RNAi in HepG2 cells. It was found that total transketolase activity was dramatically downregulated and the proliferation of cancer cells was significantly inhibited in HepG2 cells. These results indicate that TKTL1 gene influences total transketolase activity and cell proliferation in human hepatoma cells, suggesting that TKTL1 gene plays an important role on glycometabolism in tumors and it might become a novel target for tumor gene therapy.

Early intervention with Didang decoction delays macrovascular lesions in diabetic rats through regulating AMP-activated protein kinase signaling pathway.

The study aimed to investigate the intervening role of Didang decoction (DDD) at different times in macrovascular endothelial defense function, focusing on its effects on the AMP-activated protein kinase (AMPK) signaling pathway. The effects of DDD on mitochondrial energy metabolism were also investigated in rat aortic endothelial cells (RAECs). Type 2 diabetes were induced in rats by streptozotocin (STZ) combined with high fat diet. Rats were randomly divided into non-intervention group, metformin group, simvastatin group, and early-, middle-, late-stage DDD groups. Normal rats were used as control. All the rats received 12 weeks of intervention or control treatment. Western blots were used to detect the expression of AMP-activated protein kinase α1 (AMPKα1) and peroxisome proliferator-activated receptor 1α (PGC-1α). Changes in the intracellular AMP and ATP levels were detected with ELISA. Real-time-PCR was used to detect the mRNA level of caspase-3, endothelial nitric oxide synthase (eNOS), and Bcl-2. Compared to the diabetic non-intervention group, a significant increase in the expression of AMPKα1 and PGC-1α were observed in the early-stage, middle-stage DDD groups and simvastatin group (P < 0.05). The levels of Bcl-2, eNOS, and ATP were significantly increased (P < 0.05), while the level of AMP and caspase-3 were decreased (P < 0.05) in the early-stage DDD group and simvastatin group. Early intervention with DDD enhances mitochondrial energy metabolism by regulating the AMPK signaling pathway and therefore may play a role in strengthening the defense function of large vascular endothelial cells and postpone the development of macrovascular diseases in diabetes.

Pancreatic cancer cell-derived IGFBP-3 contributes to muscle wasting.

BACKGROUND: Progressive loss of skeletal muscle, termed muscle wasting, is a hallmark of cancer cachexia and contributes to weakness, reduced quality of life, as well as poor response to therapy. Previous studies have indicated that systemic host inflammatory response regarding tumor development results in muscle wasting. However, how tumor directly regulates muscle wasting via tumor-derived secreted proteins is still largely unknown. METHODS: In this study, we performed bioinformatics analysis in two datasets of pancreatic ductal adenocarcinoma, which causes cancer cachexia and muscle wasting with the highest prevalence, and uncovered that IGFBP3, which encodes IGF-binding protein-3 (IGFBP-3), is dramatically up-regulated in pancreatic tumor samples. We also verified the wasting effect of IGFBP-3 on C2C12 muscle cells with biochemical and genetic assays. RESULTS: IGFBP-3 potently leads to impaired myogenesis and enhanced muscle protein degradation, the major features of muscle wasting, via IGF signaling inhibition. Moreover, conditioned medium from Capan-1 pancreatic cancer cells, which contains abundant IGFBP-3, significantly induces muscle cell wasting. This wasting effect is potently alleviated by IGFBP3 knockdown in Capan-1 cells or IGFBP-3 antibody neutralization. Strikingly, compared to muscle cells, IGF signaling and proliferation rate of Capan-1 cells were rarely affected by IGFBP-3 treatment. CONCLUSIONS: Our results demonstrated that pancreatic cancer cells induce muscle wasting via IGFBP-3 production.

Relationship between circadian blood pressure variability and function of islet α and ß cell in type 2 diabetes with dyssomnia.

AIMS: To investigate the relationship between circadian blood pressure (BP) variability and function of islet α and ß cell in type 2 diabetes (T2D) with dyssomnia. METHODS: Patients with T2D were divided into dyssomnia group and non-dyssomnia group by PSQI. OGTT, insulin and glucagon-releasing test were tested, and ambulatory BP was monitored for 24 hours to compare two groups with α and ß cell, circadian BP variability and fasting and post-meal BP variability. The correlation and regression analysis were made between PSQI and other indicators. RESULTS: In dyssomnia group, ① Glucagon, glucagon/insulin ratio and AUCG were significantly higher (P < 0.05). ② Fasting insulin (13.32 ± 4.54 mIU/L), AUCI (8.51 ± 0.54) and HOMA-IR (4.62 ± 1.11) were high (P < 0.05). But ISI (-4.27 ± 0.77) was low (P < 0.05). ③ Mean 24-hour and nighttime SBP and DBP, as well as their standard deviations and coefficients of variation, were all higher in the dyssomnia group (P < 0.05). Multiple stepwise regression analysis showed that PSQI score was positively related to AUCG, HOMA-IR, nighttime SBP, and negatively related to ISI and nocturnal BP fall (P < 0.05). CONCLUSION: Dyssomnia may cause abnormal circadian BP variability through various mechanisms. Improving dyssomnia can help to better function the islet α and ß cell and restore normal circadian BP variability.

Renal tubular damage may contribute more to acute hyperglycemia induced kidney injury in non-diabetic conscious rats.

AIMS: Growing evidences suggest that acute hyperglycemia is strongly related to kidney injury. Our study aimed to investigate the effects of acute hyperglycemia on kidney glomerular and tubular impairment in non-diabetic conscious rats. METHODS: Non-diabetic conscious rats were randomly subjected to 6h of saline (control group) or high glucose (acute hyperglycemia group) infusion. Blood glucose was maintained at 16.0-18.0 mmol/L in acute hyperglycemia group. Renal structure and function alterations, systemic/renal inflammation and oxidative stress markers were assessed, and apoptosis markers of renal inherent cells were evaluated. RESULTS: Acute hyperglycemia caused significant injury to structure of glomerular filtration barrier, tubular epithelial cells and peritubular vascular endothelial cells. It increased urinary microalbumin (68.01 ± 27.09 µg/24h vs 33.81 ± 13.81 µg/24h , P=0.014), ß2-microglobulin, Cystatin C, urinary and serous neutrophil gelatinase-associated lipocalin levels (P < 0.05). Acute hyperglycemia decreased megalin and cubilin expression, activated systemic and renal oxidative stress as well as inflammation and promoted renal inherent cell apoptosis. CONCLUSIONS: Acute hyperglycemia causes significant injury to kidney function and structure. Compared with damages of glomerular filtration barrier, renal tubular injury may contribute more to acute hyperglycemia induced proteinuria. Activation of inflammation especially renal inflammation, oxidative stress and enhanced apoptosis may be the underlying mechanisms.

Effects of DACT1 methylation status on invasion and metastasis of nasopharyngeal carcinoma

BACKGROUND: The purpose of the present study was to investigate the role of the methylation status of the DACT1 gene on the invasion and metastasis of nasopharyngeal carcinoma cells. METHODS: The levels of methylation and expression of the DACT1 gene in nasopharyngeal carcinoma tissues and CNE2 cells were determined by methylation-specific PCR and RT-PCR, respectively. CNE2 cells were treated with 5-aza-2-deoxycytidine, and the variation in the methylation status of the DACT1 gene was detected, as well as the influence of methylation on invasiveness of nasopharyngeal carcinoma cells. RESULTS: The DACT1 gene was hyper-methylated in 44 of 62 cases of nasopharyngeal carcinoma. The DACT1 gene was hyper-methylated in 32 of 38 cases of nasopharyngeal carcinoma with lymph node metastasis, and the DACT1 gene was hyper-methylated in 7 of 24 cases of nasopharyngeal carcinoma without lymph node metastasis. The DACT1 mRNA level was weakly expressed or not expressed in all nasopharyngeal carcinoma tissues with hyper-methylated DACT1 genes; however, the DACT1 mRNA level was highly expressed in nasopharyngeal carcinoma tissues with low expression of the methylated DACT1 gene. The DACT1 gene was hyper-methylated and not expressed in CNE2 cells that did not have 5-aza-2-deoxycytidine treatment. After 5-aza-2-deoxycytidine treatment, the DACT1 gene was demethylated and the expression of DACT1 was restored. Moreover, the invasion ability was inhibited in CNE2 cells treated with 5-aza-2-deoxycytidine. CONCLUSION: The expression of DACT1 was related to the methylation status. High expression of DACT1 may inhibit the invasion and metastasis of nasopharyngeal carcinoma cells.

Euonymus alatus in the treatment of diabetic nephropathy in rats.

Diabetic nephropathy is a leading cause of chronic renal failure. Recently, Euonymus alatus showed therapeutic potential in the treatment of diabetes and its chronic complications. In this study, effects of Euonymus alatus and its mechanism in the treatment of diabetic nephropathy were investigated. Diabetic nephropathy was induced in Sprague-Dawley rats by uninephrectomy plus streptozotocin (STZ) administration. Euonymus alatus and irbesartan, as a positive control, were lavaged to these rats for 12 weeks. Our data showed that Euonymus alatus was efficient in lowering HbA1c, improving blood lipids, decreasing 24 h urine protein and protecting kidney function. Pathological studies found kidney damage, including extracellular matrix expansion and glomerulosclerosis, were improved by Euonymus alatus treatment. Further investigation found that the herb had a role in downregulating the expression of transform growth factor ß(1). In conclusion, Euonymus alatus has a protective role in diabetic nephropathy, which may be related to its downregulation of transform growth factor ß(1) expression.

Overexpression of transketolase protein TKTL1 is associated with occurrence and progression in nasopharyngeal carcinoma: a potential therapeutic target in nasopharyngeal carcinoma.

Over 80 years ago, Warburg identified a particular metabolic pathway in carcinomas characterised by the anaerobic degradation of glucose even in the presence of oxygen that leads to the production of large amounts of lactate (known as the Warburg effect). Now, widespread clinical use of positron-emission tomography (PET) has confirmed that there exists enhanced glucose degradation in tumors. Recent research demonstrated that pentose phosphate pathway (PPP) was augmented in some tumors, especially non-oxidative part of PPP. The non-oxidative part of PPP is controlled by transketolase enzyme reactions. The present study designed to evaluate the effect of transketolase activity on nasopharyngeal carcinoma. It was found that the transketolase activity was significantly stronger in human nasopharyngeal carcinoma tissues than those in human chronic nasopharyngitis tissues. There is a strong upregulation of the transketolase-like-1 (TKTL1) in human nasopharyngeal carcinoma tissues and cell line (CNE), whereas transketolase (TKT) and transketolase-like-2 (TKTL2) were not upregulated. After inhibited the expression of (TKTL1) by RNAi, we found that total transketolase activity was dramatically downregulated and the proliferation of cancer cells was significantly inhibited in CNE cells. These results indicate that TKTL1 gene influences total transketolase activity and cell proliferation in human nasopharyngeal carcinoma cells, suggesting that TKTL1 gene plays an important role on glycometabolism in tumors and it might become a novel target for tumor gene therapy.

The TKTL1 gene influences total transketolase activity and cell proliferation in human colon cancer LoVo cells.

A plasmid carrying DNA to be transcribed into a small interfering RNA against transketolase-like-1 mRNA was constructed and transfected into a human colon cancer cell line. The mRNA expression of transketolase gene family in the human colon cell line was determined by real-time polymerase chain reaction. The effect of anti-transketolase-like-1 small interfering RNA on cell proliferation and cell cycle in the human colon cancer cell line cells was detected by flow cytometry and 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl tetrazolium bromide. The transketolase-like-1 gene was significantly downregulated in human colon cancer cell line cells transfected with small interfering RNA transketolase-like-1 constructs compared with the cells transfected with control vector and the cells without transfection. In addition, the anti-transketolase-like-1 small interfering RNA construct significantly decreased the level of transketolase in the transfected human colon cancer cell line cells, arrested them in G0/G1 phase and substantially inhibited cell proliferation. No significant difference was found in the other two genes (transketolase and transketolase-like-2 genes) between the transfected human colon cancer cell line cells and the controls (P>0.05). Our data demonstrated that the transketolase-like-1 gene plays an important role in total transketolase activity and in the cell proliferation of human colon cancer. Transketolase-like-1 may serve as a target for novel anticancer therapies.