[Methylation in Promoter Region of SLC6A2 Gene in Heart Failure Patients and Its Correlation with Qi Deficiency/Blood Stasis Syndrome].

OBJECTIVE: To explore the methylation status in promoter region of norepinephrine transporter gene (NET, SLC6A2) in heart failure ( HF) patients and its correlation with qi deficiency/blood stasis syndrome (QDS/BSS). METHODS: Thirty-six patients with heart failure (NYHA classification III to IV) were recruited in the study (as the heart failure group) and their scores of QDS/BSS were evaluated. Besides, a healthy elderly group (30 cases) and a healthy youth group (30 cases) were also set up. They were recruited from Physical Examination Center of Fujian Provincial Hospital. Pyrosequencing was applied to detect the methylation in promoter region of SLC6A2 gene, and the total methylation index (MTI) of CpG island was calculated. The correlation between the methylation status in promoter region of SLC6A2 and scores of QDS/BSS was assessed using Pearson and Partial analyses. Risk factors were screened and adjusted using Logistic regression. RESULTS: By one-factor analysis of variance, the total MTI in the HF group (219.72% ± 54.03%) was obviously higher than that in the healthy elderly group (194.47% ± 34.92%) and the healthy youth group (161.60% ± 41.11%) (all P < 0.05). Meanwhile, the total MTI was higher in the healthy elderly group than in the healthy youth group (P < 0.01). By covariance analysis , after controlling age and BMI, the total MTI was higher in the HF group than in the healthy elderly group (P = 0.041), while it was higher in the healthy elderly group than in the healthy youth group (P = 0.016). Age was found to play an essential role in affecting MTI of SLC6A2 gene promoter region among the 3 groups (F = 16.447, P = 0.01). The total MTI was quite lower in the healthy youth group. Results of Partial correlation analysis showed MTI was positively correlated with scores of qi deficiency and blood stasis respectively (r = 0.494 and 0.419 respectively, both P < 0.05). Logistic regression analysis showed after adjusting confounding factors, the relative risk (OR value) of total MTI of SLC6A2 gene in promoter region was 1.038 (95% CI, 1.006 to 1.071, P = 0.020). CONCLUSIONS: Abnormally elevated methylation of the promoter region of SLC6A2 gene is one of risk factors for HF. In addition, the degree of methylation of the promoter region of SLC6A2 gene was positively correlated with the severity of QDS/BSS.

Probing the interaction between metastatic breast cancer cells and osteoblasts in a thread-based breast-bone co-culture device.

Breast cancer metastasis to bone is a leading killer in breast cancer patients. A type I collagen-modified thread (Col-I@thread) was prepared for 3-dimensional cell culture and breast cancer bone metastasis co-culture device assembly. First, the coating of Col-I on nylon threads for promoting cell adhesion and growth was studied. Through SEM, XPS, and protein concentration measurements, it was found that the lyophilization method remarkably preserved the Col-I activity and the internal structure of the thread, thereby promoting cell attachment and proliferation. RNA-sequencing (RNA-Seq) and quantitative PCR analysis showed that osteoblast cells (MC3T3-E1) grown on Col-I@thread had elevated RUNX2, ALP, OPN, and Col-I gene expression to promote osteoblast differentiation. Single-cell analysis found that osteoblast MC3T3-E1 cells growing on Col-I@thread had higher Ca2+ secretion activity and mineralized nodules, suggesting robust cell activity and bone matrix formation than cells growing on 2D culture plates. Col-I@threads were knotted in an interdigital cross-finger frame to assemble the breast cancer-bone co-culture model. Confocal microscopy and flow cytometry tests quantified the invasive breast cancer cells. Moreover, the thread-based co-culture devices allowed us to isolate the invasive and non-invasive breast cancer cells to compare their molecular characteristics. qPCR results showed that expression of CX43, CXCR5, and CSPG4 genes was significantly increased in breast cancer cells with bone metastasis. Meanwhile, the expression of RUNX2 and OPG genes in osteoblasts was inhibited. The co-culture model based on the Col-I@thread mimics the bone tissue microenvironment to reveal the cross-talk between cancer cells and bone tissue. Moreover, the thread-based co-culture device is easy to fabricate and operate, providing a platform for exploring the cellular and molecular mechanisms of breast cancer bone metastasis, and holds potential for high-throughput screening of anti-breast cancer bone metastasis drugs.

[The role of peroxisome proliferator-activated receptor gamma in proliferation of cardiac non myocytes].

AIM: To investigate the effect of PPARgamma activators on inhibition of cardiac non myocytes (CNM) proliferation and the PPARgamma-dependent pathway possibly involved. METHODS: Angiotensin II was used to induce proliferation of primarily cultured CNM from neonatal rats, and pioglitazone or 15-deoxy-delta12,14-prostaglandin J2 (15d-PGJ2) was applied to these CNM in various dosages in vitro. MTT assay and 3H-TdR uptake were determined to estimate proliferation of CNM, and transient transfection of reporter gene containing PPRE from ACO promoter (PPRE-pGL3) with or without PPARgamma expression plasmid (PPARgamma-pSG5) to CNM was performed to determine the control of target-gene transcription. RESULTS: Angiotensin II caused a significant increase in MTT value and 3H-TdR uptake in CNM, which could be significantly reversed by pioglitazone and 15d-PGJ2 in a dose-dependent manner. Transient cotransfection of PPRE-pGL3 with PPARgamma-pSG5 to CNM resulted in significant increase in luciferase activity compared with that without PPARgamma-pSG5 cotransfection. Pioglitazone and 15d-PGJ2 induced increase in luciferase activity also in a dose-dependent manner. CONCLUSION: Pioglitazone and 15d-PGJ2, as the activators of PPARgamma, inhibit proliferation of CNM from neonatal rats, the effect may be related to the activation of PPARgamma.