1.Multidisciplinary treatment for a patient with locally advanced esophagogastric junc-tion cancer
Ziyu JIA ; Tao FU ; Zhaode BU ; Xiaotian ZHANG ; Yongheng LI ; Lei TANG ; Zhongwu LI ; Jiafu JI
Chinese Journal of Clinical Oncology 2016;(1):42-46
Since the biological characteristics of esophageal gastric junction (EGJ) cancer are different from those of gastric cancer and esophageal cancer, the choice of initial treatment is particularly important. This article introduces a case of locally advanced EGJ can-cer with single metastasis factor treated with preoperative radiochemotherapy in the Beijing Cancer Hospital. Through the multidisci-plinary team, we aim to achieve a better prognosis for this patient and propose new treatment practices for EGJ cancer.
2.MicroRNA-1 and-16 inhibit cardiomyocyte hypertrophy by targeting cyclins/Rb pathway
Zhixin SHAN ; Jiening ZHU ; Chunmei TANG ; Wensi ZHU ; Qiuxiong LIN ; Zhiqin HU ; Yongheng FU ; Mengzhen ZHANG
Chinese Journal of Pathophysiology 2016;32(8):1496-1496
AIM:MicroRNAs ( miRNAs) were recognized to play significant roles in cardiac hypertrophy .But, it remains unknown whether cyclin/Rb pathway is modulated by miRNAs during cardiac hypertrophy .This study investigates the potential roles of microRNA-1 (miR-1) and microRNA-16 (miR-16) in modulating cyclin/Rb pathway during cardiomyocyte hypertrophy .METHODS:An animal model of hypertrophy was established in a rat with abdominal aortic constriction (AAC).In addition, a cell model of hypertrophy was also achieved based on PE-promoted neonatal rat ventricular cardiomyocyte .RESULTS:miR-1 and-16 expression were markedly de-creased in hypertrophic myocardium and hypertrophic cardiomyocytes in rats .Overexpression of miR-1 and -16 suppressed rat cardiac hypertrophy and hypertrophic phenotype of cultured cardiomyocytes .Expression of cyclins D1, D2 and E1, CDK6 and phosphorylated pRb was increased in hypertrophic myocardium and hypertrophic cardiomyocytes , but could be reversed by enforced expression of miR-1 and -16.CDK6 was validated to be modulated post-transcriptionally by miR-1, and cyclins D1, D2 and E1 were further validated to be modulated post-transcriptionally by miR-16.CONCLUSION: Attenuations of miR-1 and -16 provoke cardiomyocyte hypertrophy via derepressing the cyclins D1, D2, E1 and CDK6, and activating cyclin/Rb pathway.
3.MEF2C mediates the effect of microRNA-214 on inhibiting cardiomyocyte hypertrophy
Chunmei TANG ; Jiening ZHU ; Wensi ZHU ; Qiuxiong LIN ; Zhiqin HU ; Yongheng FU ; Mengzhen ZHANG ; Zhixin SHAN
Chinese Journal of Pathophysiology 2016;32(8):1496-1497
AIM:To investigate the effect of miR-214 on cardiomyocyte hypertrophy and the expression of the potential target genes . METHODS:A cell model of hypertrophy was established based on angiotensin-Ⅱ( Ang-Ⅱ)-induced neonatal mouse ventricular car-diomyocytes (NMVCs).Dual luciferase reporter assay was performed to verify the interaction between miR-214 and the 3’ UTR of MEF2C.The expression of MEF2C and hypertrophy-related genes at mRNA and protein levels was determined by RT-qPCR and Wes-tern blotting, respectively.RESULTS:The expression of ANP, ACTA1,β-MHC and miR-214 was markedly increased in Ang-Ⅱ-in-duced hypertrophic cardiomyocytes .Dual luciferase reporter assay revealed that miR-214 interacted with the 3’ UTR of MEF2C, and miR-214 was verified to inhibit MEF2C expression at the transcriptional level .The protein expression of MEF2C was markedly in-creased in the hypertrophic cardiomyocytes .Moreover, miR-214 mimic, in parallel to MEF2C siRNA, inhibited the expression of hy-pertrophy-related genes in Ang-Ⅱ-induced NMVCs.CONCLUSION:MEF2C is a target gene of miR-214, which mediates the effect of miR-214 on attenuating cardiomyocyte hypertrophy .
4.Construction of recombinant adenovirus vector carrying human miR-133a and its expression in vitro
Bin ZHANG ; Zhixin SHAN ; Qiuxiong LIN ; Zhiling ZHOU ; Chunyu DENG ; Ailin GUO ; Yongheng FU ; Honghong TAN ; Xiyong YU
Chinese Journal of Pathophysiology 2000;0(07):-
AIM:To construct recombinant adenovirus vector carrying human miR-133a and study its expression in human mesenchymal stem cells(hMSCs).METHODS:The PCR product containing miR-133a was amplified from human genomic DNA and inserted into the adenoviral shuttle vector pAdTrack-CMV.Then the recombinant shuttle plasmid linearized by pmeⅠwas cotransformed into competent E.coli.BJ5183 with the adenoviral backbone plasmid pAdEasy-1 to generate the recombinant adenovirus vector rAd-mir-133a.rAd-mir-133a was then packaged and amplified in human embryonic kidney 293(HEK293) cells.The purified rAd-miR-133a was used to infect the hMSCs and the expression of miR-133a was detected by non-quantitative RT-PCR and real-time PCR.RESULTS:The recombinant adenovirus shuttle vector pAdTrack-CMV-miR-133a was constructed and verified by restriction endonuclease analysis and DNA sequence analysis.rAd-miR-133a was successfully packaged and amplified in HEK293 cells.The transcriptions of primary miR-133a and mature miR-133a were over-expressed in the hMSCs infected with rAd-miR-133a.CONCLUSION:The recombinant adenovirus vector carrying human miR-133a is successfully constructed,which lay a foundation for miR-133a function study.
5.Effect of circRNA_000203 on fibrotic phenotypes in mouse cardiac fibro-blasts
Wensi ZHU ; Chunmei TANG ; Jiening ZHU ; Qiuxiong LIN ; Yongheng FU ; Chunyu DENG ; Hui YANG ; Fang RAO ; Shulin WU ; Zhixin SHAN
Chinese Journal of Pathophysiology 2016;32(8):1351-1356
AIM:To determine circular RNA (circRNA) profiles in the diabetic mouse myocardium , and to investigate the effect of circRNA_000203 on fibrotic phenotypes in cardiac fibroblasts .METHODS:Masson trichrome stai-ning was performed on the myocardium of the diabetic db /db mice and the non diabetic db/m control mice .circRNA ex-pression profile in the diabetic myocardium was detected by circRNAs microarray .The expression of circRNA_000203 was determined by real time fluorescence quantitative PCR ( RT-qPCR ) .Recombinant circRNA_000203 adenovirus was pre-pared for enforced the expression of circRNA_000203 in mouse cardiac fibroblasts.The expression of Col1a2, Col3a1andα-SMA was determined in circRNA_000203-modified cardiac fibroblasts , respectively .RESULTS:Masson trichrome stai-ning showed that fibrosis was increased in the diabetic mouse myocardium .The results of circRNA array detection revealed that circRNAs were dysregulated in the diabetic myocardium .circRNA_000203 was up-regulated in the diabetic myocardi-um.Significant over-expression of circRNA_000203 was achieved in the cardiac fibroblasts after infection with the recombi-nant circRNA_000203 adenovirus.The mRNA and protein expression of Col1a2, Col3a1 and α-SMA was significantly in-creased in the cardiac fibroblasts with over-expression of circRNA_000203.CONCLUSION:circRNA_000203 is up-regu-lated in the diabetic mouse myocardium .It has pro-fibrotic effect on the cardiac fibroblasts .
6.Caspase-8 small hairpin RNA attenuates apoptosis of human bone mar-row mesenchymal stem cells under conditions of serum deprivation and hypoxia
Weiwei YUAN ; Qiuxiong LIN ; Jiening ZHU ; Xiaohong LI ; Yongheng FU ; Xiaoying LIU ; Honghong TAN ; Chunyu DENG ; Zhixin SHAN
Chinese Journal of Pathophysiology 2014;(7):1172-1178
AIM:To investigate the effect of caspase-8 small hairpin RNA ( shRNA) on attenuating apoptosis of human mesenchymal stem cells ( hMSCs ) .METHODS: Two recombinant plasmids for over-expression of caspase-8 shRNA, pAd-Cap8 shRNA1 and pAd-Cap8 shRNA2, were constructed.Caspase-8 mRNA was determined in pAd-Cap8 shRNA-transfected human HEK293 cells by Q-PCR.The screened pAd-Cap8 shRNA was used to construct the recombinant adenovirus plasmid , which was linearized and transfected into HEK 293 cells for packaging and amplification of the recombi-nant adenovirus rAd-Cap8 shRNA.The expression of caspase-8 at mRNA and protein levels was determined by Q-PCR and Western blotting .Annexin V/PI staining and determination of caspase-8 activity were performed to assess apoptosis of hM-SCs under the conditions of serum deprivation and hypoxia .The mRNA expression of vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF), insulin-like growth factor 1 (IGF-1), Bcl-2 and Bcl-xL was analyzed by Q-PCR.RESULTS:The pAd-Cap8 shRNA, which efficiently inhibited caspase-8 expression, was screened by Q-PCR.The recombinant adenovirus plasmid for caspase-8 shRNA was constructed and used to package and amplify the recombinant ad-enovirus ( rAd)-Cap8 shRNA successfully .rAd-Cap8 shRNA-mediated caspase-8 shRNA markedly inhibited caspase-8 ex-pression in hMSCs .Over-expression of caspase-8 shRNA by infection of rAd-Cap8 shRNA also efficiently decreased the ap-optotic rate and caspase-8 activity in hMSCs under the conditions of serum deprivation and hypoxia , with up-regulation of the mRNA expression of HGF, IGF-1 and Bcl-2.CONCLUSION:Caspase-8 shRNA attenuates hMSC apoptosis under the conditions of serum deprivation and hypoxia .
7.siRNA suppresses the green fluorescent protein (GFP) gene expression in human umbilical vein endothelial cells
Zhixin SHAN ; Qiuxiong LIN ; Xiyong YU ; Chunyu DENG ; Meng ZHENG ; Honghong TAN ; Yongheng FU ; Min YANG ; Shuguang LIN
Chinese Journal of Pathophysiology 1986;0(02):-
AIM: To establish human umbilical vein endothelial cells (HUVECs) to express green fluorescent protein (GFP), and to study the suppression of GFP by siRNA in HUVECs. METHODS: Using lipofectamine 2000 to transform plasmid pN_3-EGFP encoding GFP into HUVECs. The HUVEC containing pN_3-EGFP, named HUVEC-GFP, was screened and selected by antibiotic G418. Using in vitro transcription T7 kit, GFPsiRNA targeting GFP mRNA and control-siRNA used as control were synthesized. The siRNAs were transfected into HUVEC-GFP with oligofectamine. 48 h later, the expression levels of GFP protein and mRNA in HUVEC-GFP were determined. RESULTS: The HUVEC-GFP was screened to express GFP in the presence of G418. The agarose gel electrophoresis analysis showed that the siRNAs prepared were integrated. 48 h after transfection with siRNAs, compared to control group, the level of GFP fluorescence was obviously decreased in the HUVEC-GFP transfected with GFPsiRNA. The results of RT-PCR detection showed that GFP mRNA expression was obviously suppressed by GFPsiRNA at the rate of 40%, and no obvious suppression of GFP mRNA expression was found in the HUVEC-GFP transfected with control siRNA. CONCLUSION: The siRNA targeting GFP mRNA, synthesized in vitro, efficiently suppresses the GFP expression in HUVECs.
8.MEF2C mediates inhibitory effect of microRNA-214 on cardiomyocyte hypertrophy
Chunmei TANG ; Jiening ZHU ; Wensi ZHU ; Qiuxiong LIN ; Zhiqin HU ; Yongheng FU ; Mengzhen ZHANG ; Chunyu DENG ; Honghong TAN ; Shulin WU ; Zhixin SHAN
Chinese Journal of Pathophysiology 2016;32(8):1345-1350
AIM:To investigate the effect of microRNA-214 ( miR-214) on cardiomyocyte hypertrophy and the expression of the potential target genes .METHODS:A cell model of hypertrophy was established based on angiotensin-Ⅱ( Ang-Ⅱ)-induced neonatal mouse ventricular cardiomyocytes ( NMVCs) .Dual luciferase reporter assay was performed to verify the interaction between miR-214 and the 3’ UTR of MEF2C.The expression of MEF2C and hypertrophy-related genes at mRNA and protein levels was determined by RT-qPCR and Western blot , respectively .RESULTS:The expression of ANP, ACTA1,β-MHC and miR-214 was markedly increased in Ang-Ⅱ-induced hypertrophic cardiomyocytes .Dual lu-ciferase reporter assay revealed that miR-214 interacted with the 3’ UTR of MEF2C, and miR-214 was verified to inhibit MEF2C expression at the transcriptional level .The protein expression of MEF2C was markedly increased in the hypertro-phic cardiomyocytes .Moreover, miR-214 mimic, in parallel to MEF2C siRNA, inhibited the expression of hypertrophy-re-lated genes in Ang-Ⅱ-induced NMVCs.CONCLUSION:MEF2C is a target gene of miR-214, which mediates the effect of miR-214 on attenuating cardiomyocyte hypertrophy .
9.Quantitative comparison of 68Ga-NGR and 18F-FDG uptake in well-differentiated hepatocellular carcinoma bearing mice
Yongheng GAO ; Zhengjie WANG ; Fei KANG ; Xiaowei MA ; Wenhui MA ; Mingru ZHANG ; Mingxuan ZHAO ; Tianming FU ; Guoquan LI ; Shengjun WANG ; Zhe WANG ; Weidong YANG ; Jing WANG
Chinese Journal of Nuclear Medicine and Molecular Imaging 2017;37(3):147-152
Objective To quantitatively compare the diagnostic capability of 68Ga-NGR and 18F-FDG in well-differentiated hepatocellular carcinoma (HCC) bearing mice by microPET/CT imaging.Methods The in vitro cellular uptake, in vivo microPET/CT imaging and biodistribution studies of 68Ga-NGR and 18F-FDG were quantitatively compared in SMMC-7721-based well-differentiated HCC.The human fibrosarcoma (HT-1080) and human colorectal adenocarcinoma (HT-29) cells/xenografts were respectively used as positive and negative reference groups for CD13.The expression of CD13 was qualitatively verified by immunohistostaining.The levels of CD13 and glucose-6-phosphatase (G6Pase) were semi-quantitatively analyzed by Western blot test for all 3 types of tumors.Two-sample t test was used for data analysis.Results The in vitro cellular uptake showed that the 68Ga-NGR uptake in SMMC-7721 and HT-1080 cells was higher than that in HT-29 cells, and the 68Ga-NGR uptake was higher than 18F-FDG uptake in SMMC-7721 cells.The in vivo microPET/CT imaging results revealed that the uptake of 68Ga-NGR in SMMC-7721 tumor was (2.17±0.21) %ID/g, remarkably higher compared to (0.73±0.26) %ID/g of 18F-FDG uptake (t=8.826, P<0.01).The tumor/liver ratio of 68Ga-NGR was 2.05±0.16, which was 2.03-fold higher than that of 18F-FDG.In the HT-1080 tumors, the uptakes of 68Ga-NGR and 18F-FDG were both high, and the values were (2.46±0.23) %ID/g, (3.47±0.31) %ID/g.The uptake of 68Ga-NGR was significantly lower than that of 18F-FDG in HT-29 tumors: (0.67±0.20) %ID/g vs (3.17±0.29) %ID/g;t=4.221, P<0.01.Western blot and immunohistostaining results were as follows: HT-1080(CD13+, G6Pase-), SMMC-7721(CD13+, G6Pase+), HT-29(CD13-, G6Pase-).Conclusions The uptake of 68Ga-NGR is higher than 18F-FDG uptake in SMMC-7721 tumor bearing mice, therefore it is worthwhile to consider the feasibility of clinical translation for PET/CT in diagnosis of HCC.Furthermore, because of the difference in 68Ga-NGR and 18F-FDG avidities in tumors with different molecular phenotypes of CD13 and G6Pase, there is an underlying potential for molecular imaging in the determination of molecular phenotypes.
10.Quantitative comparison of 68Ga-NGR and 18F-FDG uptake in well-differentiated hepatocellular carcinoma bearing mice
Yongheng GAO ; Zhengjie WANG ; Fei KANG ; Xiaowei MA ; Wenhui MA ; Mingru ZHANG ; Mingxuan ZHAO ; Tianming FU ; Guoquan LI ; Shengjun WANG ; Zhe WANG ; Weidong YANG ; Jing WANG
Chinese Journal of Nuclear Medicine and Molecular Imaging 2017;37(3):147-152
Objective To quantitatively compare the diagnostic capability of 68Ga-NGR and 18F-FDG in well-differentiated hepatocellular carcinoma (HCC) bearing mice by microPET/CT imaging.Methods The in vitro cellular uptake,in vivo microPET/CT imaging and biodistribution studies of 68Ga-NGR and 18F-FDG were quantitatively compared in SMMC-7721-based well-differentiated HCC.The human fibrosarcoma (HT-1080) and human colorectal adenocarcinoma (HT-29) cells/xenografts were respectively used as positive and negative reference groups for CD13.The expression of CD13 was qualitatively verified by immunohistostaining.The levels of CD13 and glucose-6-phosphatase (G6Pase) were semi-quantitatively analyzed by Western blot test for all 3 types of tumors.Two-sample t test was used for data analysis.Results The in vitro cellular uptake showed that the 68Ga-NGR uptake in SMMC-7721 and HT-1080 cells was higher than that in HT-29 cells,and the 68Ga-NGR uptake was higher than 18F-FDG uptake in SMMC-7721 cells.The in vivo micro-PET/CT imaging results revealed that the uptake of 68Ga-NGR in SMMC-7721 tumor was (2.17±0.21) %ID/g,remarkably higher compared to (0.73±0.26) %ID/g of 18F-FDG uptake (t =8.826,P<0.01).The tumor/liver ratio of 68Ga-NGR was 2.05±0.16,which was 2.03-fold higher than that of 18F-FDG.In the HT-1080 tumors,the uptakes of 68 Ga-NGR and 18F-FDG were both high,and the values were (2.46±0.23) %ID/g,(3.47±0.31) %ID/g.The uptake of 68Ga-NGR was significantly lower than that of 18F-FDG in HT-29 tumors:(0.67±0.20) %ID/g vs (3.17±0.29) %ID/g;t=4.221,P<0.01.Western blot and immunohistostaining results were as follows:HT-1080(CD13+,G6Pase-),SMMC-7721(CD13+,G6Pase+),HT-29 (CD13-,G6Pase-).Conclusions The uptake of 68Ga-NGR is higher than 18F-FDG uptake in SMMC-7721 tumor bearing mice,therefore it is worthwhile to consider the feasibility of clinical translation for PET/CT in diagnosis of HCC.Furthermore,because of the difference in 68Ga-NGR and 18F-FDG avidities in tumors with different molecular phenotypes of CD13 and G6Pase,there is an underlying potential for molecular imaging in the determination of molecular phenotypes.