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Objective To investigate the mechanism of glycoprotein serglycin (SRGN) promoting metastasis of breast cancer cells and the possible mechanism of SRGN expression.Methods Real time quantitative polymerase chain reaction (PCR) and bioinformation retrieval were used to detect the expression of SRGN in lymph node metastasis and non-metastasis breast cancer.MDA-MB-231 shRNA and MCF-7-SRGN of breast cancer stable cell line were established by lentivirus shRNA interferencc and overexpression.Transwell assay was used to test the effect of SRGN on invasion and metastasis of breast cancer cell line in vitro.Western blot assay was used to detect the changes of epithelial-mesenchymal (EMT) related markers.The possible regulatory mechanism of SRGN expression was detected by Western blot assay.Results SRGN expression was significantly increased in lymph node metastasis of breast cancer in clinical specimens.SRGN interference inhibited the invasion and metastasis of tumor cells.SRGN promoted breast cancer cells EMT.Transforming growth factor β1 (TGFβ1) promoted the expression of beta SRGN transcription.Conclusions SRGN can induce the change of EMT in breast cancer cells and promote the invasion and metastasis of breast cancer cells.
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Objective:To construct HIV-1 pseudovirus containing enhanced green fluorescent protein ( EGFP ) gene. To understand the interaction between the virus and the cells. Methods: HIV-1 pseudovirus containing EGFP gene was constructed by lentiviral packaging systems, and its EGFP gene was amplified using RT-PCR. The level of genomic integration and transcription of HIV-1 pseudovirus containing EGFP gene were detected on iDCs infected with HIV-1 pseudovirus. At the same time, research on expression of the EGFP gene in iDCs infected with HIV-1 pseudovirus was performed. Results:The EGFP gene of HIV-1 pseudovirus was detected through RT-PCR. The EGFP gene was identified in iDCs infected with HIV-1 pseudovirus through PCR and RT-PCR. The EGFP was observed in iDCs infected with HIV-1 pseudovirus under fluorescence microscopy. Conclusion: HIV-1 pseudovirus containing EGFP gene has been successfully produced. The HIV-1 pseudovirus that we constructed can infect iDCs,then its RNA can integrate into the genome of iDCs in the way of reverse transcription,and the EGFP gene could express in the iDCs after infected with HIV-1 pseudovirus.
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Objective:To construct HIV-1 pseudovirus containing enhanced green fluorescent protein ( EGFP ) gene. To understand the interaction between the virus and the cells. Methods: HIV-1 pseudovirus containing EGFP gene was constructed by lentiviral packaging systems, and its EGFP gene was amplified using RT-PCR. The level of genomic integration and transcription of HIV-1 pseudovirus containing EGFP gene were detected on iDCs infected with HIV-1 pseudovirus. At the same time, research on expression of the EGFP gene in iDCs infected with HIV-1 pseudovirus was performed. Results:The EGFP gene of HIV-1 pseudovirus was detected through RT-PCR. The EGFP gene was identified in iDCs infected with HIV-1 pseudovirus through PCR and RT-PCR. The EGFP was observed in iDCs infected with HIV-1 pseudovirus under fluorescence microscopy. Conclusion: HIV-1 pseudovirus containing EGFP gene has been successfully produced. The HIV-1 pseudovirus that we constructed can infect iDCs,then its RNA can integrate into the genome of iDCs in the way of reverse transcription,and the EGFP gene could express in the iDCs after infected with HIV-1 pseudovirus.
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Objective To reveal acetylated a-tubulin acts as pressure sensors,sensing the changes in extracellular matrix to impact on the invasion and metastasis of breast cancer.Methods The acetylated alpha microtubule protein expressions were detected,and its relationship with invasion and metastasis in breast cancer clinical specimens and different molecular subtypes of breast cancer cell lines were analyzed.The expression of acetylated a-tubulin was interfered through chemical methods,and cell morphology and the change of the invasion and metastasis ability were detected under the condition of different matrix hardness.Results The expression of acetylated a-tubulin was highest in basal-like breast cancer tissue and cell lines,and was lowest expressed in the luminal B breast cancer tissue and the breast cancer cell lines.The expression of acetylated a-microtubule was positively correlated with the occurrence of breast cancer.Under the condition of soft substrate cultivation,cell polarization was declined,becoming the circular or oval shape.The acetylated a-tubulin caused reduction in cell polarity,accompanied with less invasion and metastasis ability.Conclusions The acetylated a-tubulin acts as pressure sensors,sensing the sclerosis of extracellular matrix in the process of tumorigenesis,promoting invasion and metastasis of breast cancer.
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Objective To explore the Cep70 by adjusting the stability of acetylated alpha tubulin,participate in breast cancer drug resistance mechanisms.Methods (1) In order to induce taxol drug resistance cell line Michigan cancer foundation-7 (MCF-7)/pac,high-dose shock treatments taxol MCF-7 was used for 6 months,until the cells can grow in 3.5 μmol/L of paclitaxel.(2) The 3-(4,5-dimenthylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) method was used to detect inhibition rate by taxol to MCF-7 and MCF-7/pac cell.(3) Immunofluorescence and Western blot were used to test acetylated alpha-tubulin and Cep70 expression levels in MCF-7 and MCF-7/pac cells.(4) Chemical intervention was used to acetylate apha-tubulin expression,Western blot and polymerase chain reaction (PCR) were used to detect the change of acetylated alpha-tubulin and Cep70 in MCF-7 and MCF-7/pac groups.Flow cytometry and Western blot were used to detect the change of cell cycle.Results (1) IC50 of MCF-7 and MCF-7/pac was 22.47 μ mol/L and 31.38 μmol/L,respectively.(2) Immunofluorescence and Western blot results showed that the expression of acetylation of alpha-tubulin in resistant MCF-7 cell/pac was obviously decreased.(3) Real time polymerase chain reaction (RT-PCR) and Western blot showed Cep70 expression was consistent of acetylation of alpha-tubulin.(4) After incubation with paclitaxel for 24 hours,the expressions of acetylation of alpha-tubulin and Cep70 in MCF-7 and MCF-7/pac were increased,but the extent of MCF-7 cell was much higher.Instead,incubation with nocodazole after 24 hours,the acetylation of alpha-tubulin and Cep70 in MCF-7 and MCF-7/pac cells were obviously lowered.(5) After paclitaxel intervention,compared to the same group MCF-7 cells,the G2 phase ratio in MCF-7/pac cells was lower.In addition,given nocodazole after the intervention,compared to the same group MCF-7 cells,the ratio of G2 phase in MCF-7 cell/pac was significantly decreased.Conclusions Cep70 decreased the expression of the acetylated alpha-tubulin,reduced the stability of microtubules,which could be an important mechanism of taxol drug resistance.
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Objective To explore the influence of endoplasmic reticulum stress on fatty liver in mice feeding with high-fat diet.Methods The 8-week-old male C57BL/6J mice were randomly divided into two groups:high-fat diet group (with 60％ calories by high saturated fatty acid) and control group (with chow diet),both groups had been fed for 16 weeks.H&E-staining and Sudan Ⅳ-staining reflected lipid deposition in liver.The levels of 78-kDa glucose-regulated protein (GRP78),protein kinase R-like endoplasmic reticulum kinase (PERK),phosphorylated α subunit of eukaryotic initiation factor 2 (p-eIF1 α),C/EBP homologous protein (CHOP),steroid regulated element binding proteins 1 (SREBP-1),and fatty acid synthetase (FAS) protein were determined by Western blot to reflect the endoplasmic reticulum stress and lipid synthesis.Results In liver of high fat diet (HFD) group,H&E staining showed that the cytoplasm of hepatocytes were filled with vacuoles,Sudan Ⅳ staining also displayed that many different sizes of red lipid drops exist in hepatocytes.Compared to the liver of control group,high-fat diet induced endoplasmic reticulum stress and elevated lipid synthesis,as evidenced by increases in the level of peroxisome proliferator-activated receptor alpha (PPARα) mRNA expression,and the protein levels of GRP78,PERK,phosphorylated eIF2α,CHOP were also significantly increased.In primary normal hepatocytes incubated with exogenous oleic acid intervention for 24-72 hours,the expression of GRP78,PERK,phosphorylated eIF2α,CHOP protein levels,and the expression of SREBP-1 and FAS protein were significantly increased in dose-dependent manner.Conclusions Feeding with high-fat diet led to accumulation of lipid deposition in liver and fatty liver,the underlying mechanisms might be related to induction of endoplasmic reticulum stress.
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[ ABSTRACT] AIM:To observe the effect of endogenous nitric oxide synthase ( NOS) inhibitor asymmetric dimeth-ylarginine ( ADMA ) and its signaling pathways on NO levels and skeletal muscle contractility in 4-week running rats. METHODS:The 4 weeks running rat model was established.The twitch tension, tetanic tension and the fatigue test of sole-us muscle induced by electrical stimulation ex vivo were detected.The ATP content, mitochondrial DNA levels and the mR-NA expression of peroxisome proliferator-activated receptor γcoactivator-1α(PGC-1α), nuclear respiratory factor (NRF) were measured to reflect the mitochondrial function and biosynthesis in the skeletal muscle.Serum ADMA concentration was detected by high performance liquid chromatography.The endogenous ADMA enzymes PRMT1 and 2 subtypes of ADMA me-tabolism enzyme DDAH, 3 subtypes of NOS protein expression in the skeletal muscle were determined by Western blot.NOS activity and nitric oxide ( NO) content were analyzed by colorimetric method.RESULTS: Compared with normal control group, the twitch tension, tetanic tension and the anti-fatigue capability of soleus muscle in running group were significantly enhanced, ATP content, mitochondrial DNA content and the mRNA expression of PGC-1αand NRF were significantly in-creased (P ance of soleus muscle.The mechanism may be that increased cNOS expression feedbacks to increase ADMA concentration, thus maintaining the increase in NO synthesis at a relatively low level, and resulting in promoting skeletal muscle mitochon-dria biosynthesis and mitochondrial function.
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[ ABSTRACT] AIM:To study the influence of insulin resistance on fatty liver in the mice fed with high-fat diet (HFD).METHODS:Male 8-week-old C57BL/6J mice were randomly divided into HFD group (with 60% calories by high saturated fatty acid) and control group (with chow diet).The mice in both groups were fed for 12 weeks.The body weight, liver weight, serum triglyceride (TG) and total cholesterol (TC), and blood glucose and insulin levels were meas-ured.Hyperinsulinemic euglycemic clamp experiment was applied to reflect insulin sensitivity .The lipid deposition in the liver was analyzed by HE staining , Sudan IV staining and measurement of liver fat content .The phosphorylation levels of IRS1 and Akt, and the protein levels of SREBP-1 and FAS were determined by Western blot to reflect the activities of insu-lin signaling and lipid synthesis .RESULTS:Compared with control group , the body weight and liver weight were signifi-cantly increased in HFD group .TG and TC contents in serum and liver tissues were remarkably increased in HFD group . High-fat diet induced insulin resistance , as evidenced by increased serum insulin levels , reduced glucose infusion rate and decreases in IRS1 and Akt phosphorylation levels .In livers of HFD group, HE staining showed that the cytoplasm of hepa-tocytes was filled with vacuoles .Sudan IV staining also displayed that many different sizes of red lipid drops existed in the hepatocytes , and the protein levels of SREBP-1 and FAS were significantly increased .In primary normal hepatocytes with exogenous oleic acid intervention for 48 h, the phosphorylation levels of IRS 1 and Akt were reduced , and the protein ex-pression of SREBP-1 and FAS was significantly increased in a dose-dependent manner .CONCLUSION: Feeding with HFD leads to insulin resistance , resulting in activation of lipid synthesis and accumulation of lipid deposition in the liver , thus inducing fatty liver .
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Aim This study was aimed to explore the influence and mechanism of the long-term exercise on skeletal muscle contraction and mitochondrial biosyn-thesis in different muscle fibers.Methods Soleus (SOL)and extensor digitorum longus (EDL)were i-solated from SD male rats with platform running train-ing for eight weeks.The changes of contractility under different electrical stimulation were observed, mito-chondrial biosynthesis,including ATP content,mito-chondrial DNA,the gene expression of PGC-1αand NRF were also detected.Results Long-term endur-ance exercise can improve twitch tension and titanic tension of SOL and EDL ,but only enhanced the fa-tigue resistance in SOL.ATP contents were significant-ly increased in the two types of muscles,but mtDNA content,PGC-1αexpression and NRF translation were only obviously enhanced in SOL,in accompanied with an increase in p-AMPK/AMPK protein ratio.Conclu-sion Long-term endurance exercise increased skeletal muscle contractility and improved the anti-fatigue abili-ty in SOL,which may be associated with increase in mitochondrial biosynthesis via activated AMPK-PGC-1αaxis.
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AIM:To investigate the influence of calorie restriction ( CR) on contractility and mitochondrial bi-osynthesis in different types of rat skeletal muscles .METHODS:CR rat model was set up by feeding 60%normal food in-take of control rat every day for 8 weeks.Soleus (SOL) and extensor digitorum longus (EDL) were isolated under anesthe-sia.The twitch tension, titanic tension and fatigue resistance of SOL and EDL in response to electrical stimulation were measured to reflect the contractile function of the muscles .The copy number ratio of mitochondrial gene cytochrome C oxi-dase subunit I ( COX I) to nuclear gene β-actin was determined to evaluate the mitochondrial biosynthesis .ATP content was measured to mirror mitochondrial function .RESULTS:Compared with control group , CR for 8 weeks significantly in-creased twitch tension and titanic tension of both SOL and EDL , but only improved fatigue resistance in SOL .Markedly in-crease in ATP content in both skeletal muscles by CR intervention was observed , especially in SOL .Although CR activated AMP-activated protein kinase (AMPK) in both 2 muscles, up-regulation of mitochondrial biosynthesis and transcription of mitochondrial regulatory genes peroxisome proliferator-activated receptor γcoactivator 1α( PGC-1α) and nuclear respirato-ry factor ( NRF) was only observed in SOL .CONCLUSION:CR for 8 weeks enhanced the contractility of both rat SOL and EDL in response to electrical stimulation , especially in SOL composed of slow-twitch fibers.The mechanisms may be related to the activation of AMPK and the promotion of mitochondrial biosynthesis in SOL .
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OBJECTIVE To investigate the effect of tacrolimus on cell proliferation and osteoblastic differentiation of primary human bone marrow-derived mesenchymal stem cells (hBMSCs). METHODS hBMSCs were cultured with tacrolimus 0.001-5 μmol·L-1. BrdU incorporation was used to assess the cell proliferation while cellular alkaline phosphatase (ALP) activity and calcium deposition were measured to evaluate the osteoblastic differentiation of hBMSCs cultures. The calcineurin (CaN) activity was also examined using commercial CaN assay kit, and core binding factor 1 alpha subunit (Cbfα1) protein level was determined by Western blotting. RESULTSTacrolimus 0.001-0.1 μmol·L-1 promoted BrdU incorporation but had no effect on ALP activity and calcium deposition, whereas tacrolimus 0.5-5 μmol·L-1 resulted in significant decrease in both cell proliferation and osteoblastic maturation, by reducing BrdU incorporation, ALP activity, and calcium deposition of hBMSCs cultures in a concentration-dependent manner. In addition, tacrolimus 0.5-5 μmol·L-1 led to concentration-dependent decrement in CaN activity, which was consistent with down-regulated Cbfα1 protein in the tacrolimus treated cells. CONCLUSION High concentration of tacrolimus might inhibit the cell proliferation and osteoblastic differentiation of hBMSCs cultures through a CaN/Cbfα1 pathway.