Construction and expression of circular RNA in vitro / 中国生物制品学杂志

@#Objective To construct encoding RNA that can be cyclized in vitro by using the permuted intron exon(PIE)strategy in the maturation process of eukaryotic mRNA,and transfect it into HEK-293T cells for expression.Methods The sequences of 5'and 3'cyclic arms with groupⅠcatalytic intron,the internal ribosome entry sites(IRES)of Coxsackievirus B3(CVB3)and the target gene were selected to construct the template plasmid. Linearization plasmid template obtained by PCR was used to synthesize linear RNA through in vitro transcription(IVT),which then started in vitro cyclization(IVC)by the addition of cyclization reagents to obtain circular RNA(circRNA). RNA cyclization was confirmed by agarose gel electrophoresis and ribonuclease R(RNase R)digestion. HEK-293T cells were transfected with circRNAs respectively carrying enhanced green fluorescent protein(EGFP),firefly luciferase(Fluc),and influenza virus hemagglutinin(HA)IVR-180 genes,to verify their expression with in vitro.Results With RNA cyclization,the main band of agarose gel electrophoresis became smaller and small fragments appeared. After RNase R digestion,only some circRNA bands remained.HEK-293T cells transfected with EGFP-circRNA showed significant green fluorescence under the fluorescence microscope.The Fluc expression values of HEK-293T cells transfected with Fluc-circRNA were on average 20 times higher than non cyclized RNA,and the relative light unit(RLU)scaled up with the increase of Fluc-circRNA transfection dose. Western blot analysis showed that HA protein was successfully expressed in HEK-293T cells transfected with HA-circRNA.Conclusion In this study,linear RNA was successfully cyclized in vitro and different proteins were expressed,which lays a foundation of the research of new influenza vaccines and mRNA vaccines.

Role of adiponectin in diabetes myocardial ischemia-reperfusion injury and ischemic postconditioning

Abstract Purpose Patients with diabetes are vulnerable to myocardial I/R (ischaemia/reperfusion) injury, but are not responsive to IPO (ischaemic post-conditioning). We hypothesized that decreased cardiac Adiponectin (APN) is responsible for the loss of diabetic heart sensitivity to IPO cardioprotecton. Methods Diabetic rats were subjected to I/R injury (30 min of LAD occlusion followed by 120 min of reperfusion). Myocardial infarct area was determined by TTC staining. Cardiac function was monitored by a microcatheter. ANP, 15-F2t-isoprostane, nitrotyrosine and MDA were measured by assay kits. Levels of p-Akt, total-Akt and GAPDH were determined by Western Blot. Results Diabetic rats subjected to myocardial IR exhibited severe myocardial infarction and oxidative stress injury, lower APN in the plasma and cardiac p-Akt expression ( P <0.05). IPO significantly attenuated myocardial injury and up-regulated plasma APN content and cardiac p-Akt expression in non-diabetic rats but not in diabetic rats. Linear correlation analysis showed that the expression of adiponectin was positively correlated with p-Akt and negatively correlated with myocardial infarction area ( P <0.01). Conclusion Protective effect of IPO was tightly correlated with the expression of adiponectin, exacerbation of I/R injury and ineffectiveness of IPO was partially due to the decline of adiponectin and inactivation of Akt in diabetes mellitus.

Partial nephrectomy for T3aN0M0 renal cell carcinoma: shall we step forward?

ABSTRACT Objectives: To evaluate the prognosis of non-metastatic T3a renal cell carcinoma (RCC) with partial nephrectomy (PN). Patients and Methods: We retrospectively evaluated 125 patients with non-metastatic T3a RCC. Patients undergoing PN and radical nephrectomy (RN) were strictly matched by clinic-pathologic characteristics. Log-rank test and Cox regression model were used for univariate and multivariate analysis. Results: 18 pair patients were matched and the median follow-up was 35.5 (10-86) months. PN patients had a higher postoperative eGFR than RN patients (P=0.034). Cancer-specific survival (CSS) and recurrence-free survival (RFS) did not differ between two groups (P=0.305 and P=0.524). On multivariate analysis, CSS decreased with positive surgical margin and anemia (both P <0.01) and RFS decreased with Furhman grade, positive surgical margin, and anemia (all P<0.01). Conclusions: For patients with non-metastatic pT3a RCC, PN may be a possible option for similar oncology outcomes and better renal function.

The effect of swimming training on the expression of cardiovascular CGRPmRNA in rats / 中国应用生理学杂志

<p><b>AIM</b>Rats cardiovascular expression of CGRPmRNA surveyed after an eight-week swimming training, and molecular mechanism of exercise-induced adaptation were studied.</p><p><b>METHODS</b>24 purebred male SD rats were divided randomly into three groups (n = 8): control (CR), exhaust (ER), train (TR). After swimming training left ventricular muscle and main artery arch were extracted, we inspected the expression of CGRPmRNA by RT-PCR.</p><p><b>RESULTS</b>(1) Comparing with the control group, once exhausting exercise had no significant effect on cardiovascular expression of CGRPmRNA. (2) Comparing with the control group, long-term aerobic swimming training upregulated significantly cardiac expression of CGRPmRNA.</p><p><b>CONCLUSION</b>Long-term aerobic training induced benign cardiac adaptation in molecular level. Long-term aerobic swimming training had no significant effect on vascular expression of CGRPmRNA.</p>