Objective To observe the effects of leucine on adipogenesis in 3T3-L1 preadipocyte during and after differentiation, and to investigate possible mechanisms. Methods Respectively, 0.0 (control), 0.5, 1.0 and 2.0 mmol/L leucine was added in 3T3-L1 cells and cell proliferation was measured by MTT. Then, 3T3-L1 preadipocyte was induced to differentiate. Leucine was added during whole differentiation period, or after differentiation for 4 days. The cells were stained with Oil Red O dye to observe lipid droplet. The culture media were collected and used to determine glycerol contents. Meanwhile, protein expressions related to lypolytic enzymes, leptin signaling pathway were determined by Western blot. Results MTT result showed that cell viabilities were (100.00 ± 12.10)%, (102.73 ± 12.38)%, (103.94 ± 14.65)%, (108.70 ± 5.05)% in 0.0, 0.5, 1.0 and 2.0 mmol/L leucine groups, respectively, there were no significant differences in cell proliferation among 4 groups (F=1.07, P=0.383). When 0.0, 0.5, 1.0 and 2.0 mmol/L leucine was added during differentiation, the relative number of lipid droplet was 1.00 ± 0.06, 0.94±0.09, 0.82±0.08 and 0.79±0.04, respectively (F=11.74, P<0.001), and it was significantly lower in 1.0 and 2.0 mmol/L leucine groups than in control group(P=0.002 and P<0.001, respectively). There was no significant difference in lipid droplet when leucine was added after differentiation (F=0.16, P=0.924). When leucine was added during differentiation, the increment of glyceride contents in medium was (65.04 ± 11.75) , (71.45 ± 23.71), (79.37 ± 17.63) and (110.32 ± 25.36) μmol/L, respectively (F=2.92, P=0.100) . And it was significantly higher in 2.0 mmol/L leucine group (110.32 ± 25.36)μmol/L than in control group (65.04 ± 11.75)μmol/L(t=2.73, P=0.026). No significant difference of the increment of glyceride contents among 4 groups was observed when leucine was added after differentiation (F=0.80, P=0.528). Western blot results showed that leucine treatment during differentiation upregulated expression level of hormone-sensitive lipase phosphorylation (after 0.0 and 2.0 mmol/L leucine treatment,the protein levels were 1.00 ± 0.08 vs. 2.54 ± 0.27, P<0.001), and downregulated the protein expression levels of perilipin A, leptin and leptin-related pathway, such as leptin receptor, Janus kinase 2 and suppressor of cytokine signaling-3 (after 0.0 and 2.0 mmol/L leucine was added, the protein levels were(1.00 ± 0.03)vs.(0.31 ± 0.07),(1.00 ± 0.08)vs.(0.22±0.07),(1.00±0.07)vs.(0.21 ± 0.04),(1.00 ± 0.03)vs.(0.35 ± 0.05),(1.00 ± 0.06)vs.(0.34 ± 0.05), P<0.001). Leucine treatment after differentiation had no effects on these protein expressions (all P>0.05). Conclusion Leucine inhibits adipogenesis during 3T3-L1 preadipocyte differentiation by the regulation of lypolytic enzymes and leptin signaling pathway;however, leucine has no effect on adipogenesis when differentiation completed.

Objective To observe the effects of leucine on adipogenesis in 3T3-L1 preadipocyte during and after differentiation, and to investigate possible mechanisms. Methods Respectively, 0.0 (control), 0.5, 1.0 and 2.0 mmol/L leucine was added in 3T3-L1 cells and cell proliferation was measured by MTT. Then, 3T3-L1 preadipocyte was induced to differentiate. Leucine was added during whole differentiation period, or after differentiation for 4 days. The cells were stained with Oil Red O dye to observe lipid droplet. The culture media were collected and used to determine glycerol contents. Meanwhile, protein expressions related to lypolytic enzymes, leptin signaling pathway were determined by Western blot. Results MTT result showed that cell viabilities were (100.00 ± 12.10)%, (102.73 ± 12.38)%, (103.94 ± 14.65)%, (108.70 ± 5.05)% in 0.0, 0.5, 1.0 and 2.0 mmol/L leucine groups, respectively, there were no significant differences in cell proliferation among 4 groups (F=1.07, P=0.383). When 0.0, 0.5, 1.0 and 2.0 mmol/L leucine was added during differentiation, the relative number of lipid droplet was 1.00 ± 0.06, 0.94±0.09, 0.82±0.08 and 0.79±0.04, respectively (F=11.74, P<0.001), and it was significantly lower in 1.0 and 2.0 mmol/L leucine groups than in control group(P=0.002 and P<0.001, respectively). There was no significant difference in lipid droplet when leucine was added after differentiation (F=0.16, P=0.924). When leucine was added during differentiation, the increment of glyceride contents in medium was (65.04 ± 11.75) , (71.45 ± 23.71), (79.37 ± 17.63) and (110.32 ± 25.36) μmol/L, respectively (F=2.92, P=0.100) . And it was significantly higher in 2.0 mmol/L leucine group (110.32 ± 25.36)μmol/L than in control group (65.04 ± 11.75)μmol/L(t=2.73, P=0.026). No significant difference of the increment of glyceride contents among 4 groups was observed when leucine was added after differentiation (F=0.80, P=0.528). Western blot results showed that leucine treatment during differentiation upregulated expression level of hormone-sensitive lipase phosphorylation (after 0.0 and 2.0 mmol/L leucine treatment,the protein levels were 1.00 ± 0.08 vs. 2.54 ± 0.27, P<0.001), and downregulated the protein expression levels of perilipin A, leptin and leptin-related pathway, such as leptin receptor, Janus kinase 2 and suppressor of cytokine signaling-3 (after 0.0 and 2.0 mmol/L leucine was added, the protein levels were(1.00 ± 0.03)vs.(0.31 ± 0.07),(1.00 ± 0.08)vs.(0.22±0.07),(1.00±0.07)vs.(0.21 ± 0.04),(1.00 ± 0.03)vs.(0.35 ± 0.05),(1.00 ± 0.06)vs.(0.34 ± 0.05), P<0.001). Leucine treatment after differentiation had no effects on these protein expressions (all P>0.05). Conclusion Leucine inhibits adipogenesis during 3T3-L1 preadipocyte differentiation by the regulation of lypolytic enzymes and leptin signaling pathway;however, leucine has no effect on adipogenesis when differentiation completed.

This study was to develop a new method for detecting circulating tumor cells (CTCs) with high sensitivity and specificity, therefore to detect the colorectal cancer as early as possible for improving the detection rate of the disease. To this end, we prepared some micro-column structure microchips modified with graphite oxide-streptavidin (GO-SA) on the surface of microchips, further coupled with a broad-spectrum primary antibody (antibody1, Ab₁), anti-epithelial cell adhesion molecule (anti-EpCAM) monoclonal antibody to capture CTCs. Besides, carboxylated multi-walled carbon nanotubes (MWCNTs-COOH) were coupled with colorectal cancer related antibody as specific antibody 2 (Ab₂) to prepare complex. The sandwich structure consisting of Ab₁-CTCs-Ab₂ was constructed by the microchip for capturing CTCs. And the electrochemical workstation was used to detect and verify its high sensitivity and specificity. Results showed that the combination of immunosensor and micro-nano technology has greatly improved the detection sensitivity and specificity of the immunosensor. And we also verified the feasibility of the immunosensor for clinical blood sample detection, and successfully recognitized detection and quantization of CTCs in peripheral blood of colorectal cancer patients by this immunosensor. In conclusion, the super sandwich immunosensor based on micro-nano technology provides a new way for the detection of CTCs, which has potential application value in clinical diagnosis and real-time monitoring of disease.
Humans ; Nanotubes, Carbon/chemistry* ; Neoplastic Cells, Circulating/pathology* ; Biosensing Techniques ; Immunoassay/methods* ; Antibodies ; Colorectal Neoplasms/diagnosis* ; Electrochemical Techniques/methods* ; Gold/chemistry*

Alternative splicing is a tightly regulated process that contributes to cancer development. CRNDE is a long noncoding RNA with alternative splicing and is implicated in the pathogenesis of several cancers. However, whether deregulated expression of CRNDE is common and which isoforms are mainly involved in cancers remain unclear. In this study, we report that CRNDE is aberrantly expressed in the majority of solid and hematopoietic malignancies. The investigation of CRNDE expression in normal samples revealed that CRNDE was expressed in a tissue- and cell-specific manner. Further comparison of CRNDE expression in 2938 patient samples from 15 solid and hematopoietic tumors showed that CRNDE was significantly overexpressed in 11 malignancies, including 3 reported and 8 unreported, and also implicated that the overexpressed isoforms differed in various cancer types. Furthermore, anti-cancer drugs could efficiently repress CRNDE overexpression in cancer cell lines and primary samples, and even had different impacts on the expression of CRNDE isoforms. Finally, experimental profiles of 12 alternatively spliced isoforms demonstrated that the spliced variant CRNDE-g was the most highly expressed isoform in multiple cancer types. Collectively, our results emphasize the cancer-associated feature of CRNDE and its spliced isoforms, and may provide promising targets for cancer diagnosis and therapy.