RESUMO
In mammalian cells, long noncoding RNAs (lncRNAs) form complexes with proteins to execute various biological functions such as gene transcription, RNA processing and other signaling activities. However, methods to track endogenous lncRNA dynamics in live cells and screen for lncRNA interacting proteins are limited. Here, we report the development of CERTIS (CRISPR-mediated Endogenous lncRNA Tracking and Immunoprecipitation System) to visualize and isolate endogenous lncRNA, by precisely inserting a 24-repeat MS2 tag into the distal end of lncRNA locus through the CRISPR/Cas9 technology. In this study, we show that CERTIS effectively labeled the paraspeckle lncRNA NEAT1 without disturbing its physiological properties and could monitor the endogenous expression variation of NEAT1. In addition, CERTIS displayed superior performance on both short- and long-term tracking of NEAT1 dynamics in live cells. We found that NEAT1 and paraspeckles were sensitive to topoisomerase I specific inhibitors. Moreover, RNA Immunoprecipitation (RIP) of the MS2-tagged NEAT1 lncRNA successfully revealed several new protein components of paraspeckle. Our results support CERTIS as a tool suitable to track both spatial and temporal lncRNA regulation in live cells as well as study the lncRNA-protein interactomes.
RESUMO
In mammalian cells, long noncoding RNAs (lncRNAs) form complexes with proteins to execute various biological functions such as gene transcription, RNA processing and other signaling activities. However, methods to track endogenous lncRNA dynamics in live cells and screen for lncRNA interacting proteins are limited. Here, we report the development of CERTIS (CRISPR-mediated Endogenous lncRNA Tracking and Immunoprecipitation System) to visualize and isolate endogenous lncRNA, by precisely inserting a 24-repeat MS2 tag into the distal end of lncRNA locus through the CRISPR/Cas9 technology. In this study, we show that CERTIS effectively labeled the paraspeckle lncRNA NEAT1 without disturbing its physiological properties and could monitor the endogenous expression variation of NEAT1. In addition, CERTIS displayed superior performance on both short- and long-term tracking of NEAT1 dynamics in live cells. We found that NEAT1 and paraspeckles were sensitive to topoisomerase I specific inhibitors. Moreover, RNA Immunoprecipitation (RIP) of the MS2-tagged NEAT1 lncRNA successfully revealed several new protein components of paraspeckle. Our results support CERTIS as a tool suitable to track both spatial and temporal lncRNA regulation in live cells as well as study the lncRNA-protein interactomes.
RESUMO
In mammalian cells, long noncoding RNAs (lncRNAs) form complexes with proteins to execute various biological functions such as gene transcription, RNA processing and other signaling activities. However, methods to track endogenous lncRNA dynamics in live cells and screen for lncRNA interacting proteins are limited. Here, we report the development of CERTIS (CRISPR-mediated Endogenous lncRNA Tracking and Immunoprecipitation System) to visualize and isolate endogenous lncRNA, by precisely inserting a 24-repeat MS2 tag into the distal end of lncRNA locus through the CRISPR/Cas9 technology. In this study, we show that CERTIS effectively labeled the paraspeckle lncRNA NEAT1 without disturbing its physiological properties and could monitor the endogenous expression variation of NEAT1. In addition, CERTIS displayed superior performance on both short- and long-term tracking of NEAT1 dynamics in live cells. We found that NEAT1 and paraspeckles were sensitive to topoisomerase I specific inhibitors. Moreover, RNA Immunoprecipitation (RIP) of the MS2-tagged NEAT1 lncRNA successfully revealed several new protein components of paraspeckle. Our results support CERTIS as a tool suitable to track both spatial and temporal lncRNA regulation in live cells as well as study the lncRNA-protein interactomes.
RESUMO
Objective To investigate the effect of postoperatively early stage-function-exercise on the recovery of upper limb function of diseased side after breast cancer modified radical mastectomy.Methods One hundred patients with breast cancer admitted to our hospital from January 2013 to June 2016 were collected.All patients underwent modified radical mastectomy.The patients were divided into the observation group and control group after operation,50 cases in each group.The observation group was given the early stage-function-exercise,while the control group received the routine nursing care.The shoulder joint mobility,affected limb edema degree,self-rating anxiety scale (SAS)score,self-rating depression scale (SDS) score and 36-item short-form health survey (SF-36) score were observed in the both groups.Results When compared with the control group,the patients in the observation group got a significant increase in the shoulder joint activity such as antexion,abduction,internal rotation,external rotation,adduction and rear protraction at postoperative 3,6-months (P0.05).However,when compared with the control group,the patients in the observation group got a significant decrease of upper limb volume at postoperative 3 months[(1 543.38±414.93) mL vs.(1 635.58±401.58) mL,P=0.000)],a significantly decrease of SAS score[(37.87±6.90) points vs.(44.07±12.32) points,P=0.000)] and SDS score[(38.32±6.65) points vs.(45.02±11.07) points,P=0.000)] at postoperative 6 months,and a significant increase of SF-36 score at postoperative 6 months [(77.52±7.28) points vs.(70.28±7.55) points,P=0.000)].Conclusion Postoperatively early stage-function-exercise can help to improve the shoulder joint activity degree and quality of life,and reduce the limb edema degree after modified radical mastectomy in the patients with breast cancer.
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Objective To investigate the clinical values of postoperative aerobic exercise and psychological intervention in patients with breast cancer after operation.Methods One hundred patients with breast cancer admitted to our hospital were prospectively studied.All patients underwent modified radical mastectomy and were randomly assigned into observation group (50 cases)and control group (50 cases).The patients in the observation group were treated with aerobic exercise and psychological intervention,and the health education was given in the control group. The 3 -month postoperative self -rating anxiety scale score,self -rating depression scale score,shoulder mobility, degree of limb edema and 6 -month postoperative health related quality of life (SF -36)of the two groups were observed.Results There were no significant differences between the two groups in preoperative self -rating anxiety scale score,self -rating depression scale score,degree of limb edema and health related quality of life (all P >0.05).However,the self -rating anxiety scale score of the observation group three months after operation significantly decreased compared with the control group[(40.94 ±9.20)points vs.(45.84 ±8.79)points,t =2.722,P =0.008];self -rating anxiety scale score significantly decreased[(40.18 ±9.96)points vs.(45.28 ±8.67)points,t =2.730, P =0.008];the degree of limb edema was significantly reduced[(10.00 ±6.27)mL vs.(18.52 ±7.20)mL,t =6.313,P =0.000].The health related quality of life 6 months after operation was significantly improved[(78.70 ± 8.85)points vs.(72.02 ±9.76)points,t =3.584,P =0.001 ].Compared with the control group,the shoulder flexion,extension,abduction,external rotation and internal rotation of the observational group were significantly increased at 3 months after operation (all P <0.05 ).Conclusion Postoperative rehabilitation and psychological intervention contribute to the rapid recovery in patients with breast cancer after operation.