Effect of CUX1 on the Proliferation of Hu Sheep Dermal Papilla Cells and on the Wnt/ß-Catenin Signaling Pathway.

CUT-like homeobox 1 protein (CUX1), also called CUX, CUTL1, and CDP, is a member of the DNA-binding protein homology family. Studies have shown that CUX1 is a transcription factor that plays an important role in the growth and development of hair follicles. The aim of this study was to investigate the effect of CUX1 on the proliferation of Hu sheep dermal papilla cells (DPCs) to reveal the role of CUX1 in hair follicle growth and development. First, the coding sequence (CDS) of CUX1 was amplified by PCR, and then CUX1 was overexpressed and knocked down in DPCs. A Cell Counting Kit-8 (CCK8), 5-ethynyl-2-deoxyuridine (EdU), and cell cycle assays were used to detect the changes in the proliferation and cell cycle of DPCs. Finally, the effects of overexpression and knockdown of CUX1 in DPCs on the expression of WNT10, MMP7, C-JUN, and other key genes in the Wnt/ß-catenin signaling pathway were detected by RT-qPCR. The results showed that the 2034-bp CDS of CUX1 was successfully amplified. Overexpression of CUX1 enhanced the proliferative state of DPCs, significantly increased the number of S-phase cells, and decreased the number of G0/G1-phase cells (p < 0.05). CUX1 knockdown had the opposite effects. It was found that the expression of MMP7, CCND1 (both p < 0.05), PPARD, and FOSL1 (both p < 0.01) increased significantly after overexpression of CUX1 in DPCs, while the expression of CTNNB1 (p < 0.05), C-JUN, PPARD, CCND1, and FOSL1 (all p < 0.01) decreased significantly. In conclusion, CUX1 promotes proliferation of DPCs and affects the expression of key genes of the Wnt/ß-catenin signaling pathway. The present study provides a theoretical basis to elucidate the mechanism underlying hair follicle development and lambskin curl pattern formation in Hu sheep.

miR-143 Targeting CUX1 to Regulate Proliferation of Dermal Papilla Cells in Hu Sheep.

Wool curvature is the determining factor for lambskin quality of Hu lambs. However, the molecular mechanism of wool curvature formation is not yet known. miRNA has been proved to play an important role in hair follicle development, and we have discovered a differentially expressed miRNA, miR-143, in hair follicles of different curl levels. In this study, we first examined the effects of miR-143 on the proliferation and cell cycle of dermal papilla cells using CCK8, EdU and flow cytometry and showed that miR-143 inhibited the proliferation of dermal papilla cells and slowed down the cell cycle. Bioinformatics analysis was performed to predict the target genes KRT71 and CUX1 of miR-143, and both two genes were expressed at significantly higher levels in small waves than in straight lambskin wool (p < 0.05) as detected by qPCR and Western blot (WB). Then, the target relationships between miR-143 and KRT71 and CUX1 were verified through the dual-luciferase assay in 293T cells. Finally, after overexpression and suppression of miR-143 in dermal papilla cells, the expression trend of CUX1 was contrary to that of miR-143. Meanwhile, KRT71 was not detected because KRT71 was not expressed in dermal papilla cells. Therefore, we speculated that miR-143 can target CUX1 to inhibit the proliferation of dermal papilla cells, while miR-143 can target KRT71 to regulate the growth and development of hair follicles, so as to affect the development of hair follicles and ultimately affect the formation of wool curvature.

Preliminary study on microR-148a and microR-10a in dermal papilla cells of Hu sheep.

BACKGROUND: Hu sheep, a unique Chinese breed with high reproductive performance, are also well known for their rare white lambskin in China. The quality of lambskin is affected by hair follicles, and dermal papilla cells are an important component of hair follicles that plays a key role in hair follicle growth and development. This study helps elucidate the effect of miR-148a and miR-10a on hair follicle growth and development. RESULTS: Based on the results of gene chip and high-throughput sequencing, bone morphogenetic protein 7 (BMP7) was used as a research object. Bioinformatics analysis and the dual-luciferase reporter system indicated that, along with Western blot and quantitative real-time polymerase chain reaction (qRT-PCR) that miR-148a and miR-10a target relationships with BMP7. BMP7 was the target gene both for miR-148a and miR-10a by the dual-luciferase reporter system and Western blot. Hu sheep dermal papilla cells were successfully isolated and purified, and after transfecting miR-148a/miR-10a mimics and inhibitors into dermal papilla cells, a Cell Counting Kit-8 (CCK-8) was used to determine that miR-148a/miR-10a inhibited the proliferation of Hu sheep dermal papilla cells. In addition, after the overexpression of miR-148a, the expression levels of Smad3 (P < 0.05), Smad6 (P < 0.05), Smad4 (P < 0.01), and Smad5 (P < 0.01) were significantly higher than those of the control groups. After the inhibition of miR-148a, the expression levels of Smad3 (P < 0.05), Smad4 (P < 0.05), and TGF-ß (P < 0.01) were significantly lower than those of the control groups. After the overexpression of miR-10a, the expression levels of Smad1 (P < 0.01), Smad2 (P < 0.05), Smad4 (P < 0.01), Smad5 (P < 0.01), and TGF-ß (P < 0.05) were significantly lower than those of the control groups. After the inhibition of miR-10a, the expression levels of Smad1 (P < 0.01) and Smad2 (P < 0.05) were significantly lower than those of the control groups. CONCLUSIONS: These results revealed the target relationship between miR-148a, miR-10a and BMP7, and the effect of miR-148a and miR-10a on the proliferation of dermal papilla cells. They will provide the basis for a follow-up study on how miR-148a, and miR-10a mediate BMP7 regulation of hair follicle growth and development.

Effects of pulsed electromagnetic fields on histomorphometry and osteocalcin in disuse osteoporosis rats.

OBJECTIVE: Bone histomorphometry and the concentration of the serum osteocalcin expression were observed in disuse osteoporosis (DOP) rats in order to explore the mechanism of pulsed electromagnetic fields in treating DOP. METHODS: Female SD rats, weighing 250 ∼ 280 g, were randomly divided into 4 groups: a control group and three experimental groups. The right hindlimbs of all the rats were immobilized by tibia-tail fixation, except for those in the INT group. The ALN group rats were given an alendronate sodium (1 mg kg-1 d-1) treatment, and the rats in the PEMF group received PEMF irradiation. Bone histomorphometry and the concentration of serum osteocalcin expression were detected in 2, 4, 8, and 12 weeks. RESULTS: Four weeks after modeling, as compared with the DOP group, the %Tb-Ar and Tb-N in the ALN group were increased, and the difference was significant (P< 0.05 or P< 0.01). At 8 weeks, as compared with the DOP group, the %Tb-Ar and Tb-N in the ALN group and the PEMF group both increased, and there was a significant difference (P< 0.05 or P< 0.01). At 12 weeks, as compared with the DOP group, the BGP serum concentration of the ALN group was reduced, and there was a statistical difference (P< 0.05). CONCLUSION: Pulse electromagnetic field with drug can prevent disuse osteoporosis by changing the bone microstructure. In the long run, PEMF improves the mechanical performance of biological structures better than alendronate sodium does. PEMF may influence the process of bone remodeling by promoting the level of osteocalcin.

Endpoint visual detection of three genetically modified rice events by loop-mediated isothermal amplification.

Genetically modified (GM) rice KMD1, TT51-1, and KF6 are three of the most well known transgenic Bt rice lines in China. A rapid and sensitive molecular assay for risk assessment of GM rice is needed. Polymerase chain reaction (PCR), currently the most common method for detecting genetically modified organisms, requires temperature cycling and relatively complex procedures. Here we developed a visual and rapid loop-mediated isothermal amplification (LAMP) method to amplify three GM rice event-specific junction sequences. Target DNA was amplified and visualized by two indicators (SYBR green or hydroxy naphthol blue [HNB]) within 60 min at an isothermal temperature of 63 °C. Different kinds of plants were selected to ensure the specificity of detection and the results of the non-target samples were negative, indicating that the primer sets for the three GM rice varieties had good levels of specificity. The sensitivity of LAMP, with detection limits at low concentration levels (0.01%−0.005% GM), was 10- to 100-fold greater than that of conventional PCR. Additionally, the LAMP assay coupled with an indicator (SYBR green or HNB) facilitated analysis. These findings revealed that the rapid detection method was suitable as a simple field-based test to determine the status of GM crops.

[Changes in the expression of T lymphocyte subsets in ageing mice induced by D-galactose].

AIM: To explore the changes and significance of spleen T cell subsets in ageing mice induced by D-galactose. METHODS: Ageing mice model was successfully established by 100 g/L D-galactose. The content of IFN-γ and IL-4 in serum was measured by ELISA. T cell subsets were detected by Immunofluorescence technique and flow cytometry. RESULTS: The content of IFN-γ and IL-4 in the serum was decreased of model group (P<0.01). The naive T cell related molecule, CD45RA was decreased(P<0.05). T cell activation-related molecule, CD25 was decreased(P<0.05). The Foxp3 in CD4(+);CD25(+); T cell was increased(P<0.01). CONCLUSION: In spleen of the ageing mice, the percentage of naive and active T cell are decreased, but The percentage of CD4(+); Tr subset is increased.

[Changes and effects of significant membrane molecules on thymic T cell of aging mice induced by D-galactose].

AIM: To establish subacute aging mice model by D-galactose and to explore the changes and effects of significant membrane molecules on thymic T cell. METHODS: Female Kunming mice of 8 weeks old were injected with D-galactose of 12.5 mL/(kg.d) by subcutaneous in scruff for 42 days. The animals' living conditions and biological behaviors were observed everyday.SOD activities and MDA content of serum were measured to determine whether the aging model was successfully established.On the basis of successfully establishing aging model, detect the significant membrane molecules of thymic T cell by Immunofluorescence technique and Flow Cytometer. RESULTS: During the 42 days, gradually, the model mice showed bending body, loose skin, slow action and so on.The activities of SOD in the serum were significantly decreased(P<0.01), and the content of MDA in the serum was significantly increased(P<0.01). The thymic naive T cell significant molecule, CD45RA was decreased(P<0.05). T cell activation-related molecules, CD28 and CD25 were both decreased(P<0.05), and PD-1 was significantly increased(P<0.01). The memory T cell significant molecule, CD196 was increased, but was not significantly compared to the control mice. CONCLUSION: The D-galactose subacute aging mice model was successfully established.The naive and active T cell were decreased and the memory T cell was increased in the thymic of the aging.

[The changes of spleen B cells of D-galactose-induced aging mice].

AIM: This paper is to analyze the changes of important membrane type molecules of the spleen B cells and their significance on the basis of biology identification by establishing subacute aging mice model with D-galactose. METHODS: Health Kunming mice are injected with 100 ng/L D-galactose, 0.25 mL/20 g, once per day, for 42 days consecutively, into their back necks. The animals' living conditions and biological behaviors are observed everyday and the dynamic changes of their weight are measured regularly. The biology identification of aging model mice is conducted by means of measuring the SOD viability and malondialdehyde (MDA) concentration in serum; the analysis of the activation- and memory-related important membrane type molecules of spleen cells is carried out with immune fluorescence and flow cytometry; the analysis of the concentration of IL-4 in serum is conducted by ELISA. RESULTS: Building a successful subactue aging mice model.The results of immune fluorescence and flow cytometry showed that CD20(+); was 56.8%, CD40(+); was 43.6%, CD20(+);CD45RA(+);B was 14.04%, CD40(+);CD45RA(+);B was 35.4%, CD20(+);CD86(+);B was 2.25%, CD40(+);CD86(+);B was 4.38%, CD20(+);CD196(+);B was 10.68%, and CD40(+);CD196(+);B was 10.98%. The results of ELISA showed that the average level of IL-4 in serum was 7.93 ng/L. The statistical analysis showed that the expressions of CD20, CD40, CD45RA, and CD86 of the spleen cells of the model control group and the average level of IL-4 in serum were lower than the normal control group(P<0.05)while CD196 was higher than the normal control group(P<0.01). CONCLUSION: In the body's aging process, the expressions of the activation- and memory-related important membrane type molecules of spleen cells change, activated cells reduce, memory cells increase, and the expression of IL-4 in serum drop, resulting in the immune system function disorder.

[Construction of human CXCR3B gene transfected cell line, identification and study of its biological function].

AIM: To construct recombinant human CXCR3B gene expression vector and obtain L929-CXCR3B gene transfected cell line for stably expressing human CXCR3B. METHODS: Human CXCR3B gene of full length was amplified by PCR from the plasmid pMD19-T/huCXCR3A. Then, it was inserted into eukaryotic expression vector pIRES2-EGFP to construct recombinant vector pIRES2-EGFP/huCXCR3B. The recombinant vector was transfected into L929 cells with Lipofectamine(TM); 2000, and cell clones stably expressing human CXCR3B molecule were screened by G418.We used FCM and RT-PCR to verify expression of CXCR3B from protein level and gene level. The ability of proliferation of L929-huCXCR3B under the circumstance of CXCR3B ligand called Mig was analyzed via MTT methods. RESULTS: We have constructed recombinant human CXCR3B gene expression vector and obtained L929-huCXCR3B gene transfected cell line which can stably express human CXCR3B molecule. The positive expression rate of CXCR3B on L929-huCXCR3B cells was 93&. The result of MTT assay showed that the proliferation of L929-huCXCR3B cells were inhibited when the cells were cocultured with Mig after 24 h, 48 h and 72 h, and the inhibition ratio were 41.44&, 44.01& and 24.80& respectively. CONCLUSION: Construction of L929-huCXCR3B cell line has laid a good foundation on research of the CXCR3B signal pathway and preparation of the CXCR3B monoclonal antibody.