Objective:To investigate the mechanism of long non-coding RNA-LINC00662 on induction of sorafenib resistance in hepatocellular carcinoma(HCC)cells.Methods:HCC cells(HepG2,HCCLM3),sorafenib-resistant hepatocellular car...Objective:To investigate the mechanism of long non-coding RNA-LINC00662 on induction of sorafenib resistance in hepatocellular carcinoma(HCC)cells.Methods:HCC cells(HepG2,HCCLM3),sorafenib-resistant hepatocellular carcinoma cells HCC-SR(HepG2-SR,HCCLM3-SR)were investigated by real-time fluorescence quantitative polymerase chain reaction(RT-qPCR)and Western blot was used to detect LINC00662,miR-106a-5p and cavitin-1(CAV1)expression in each group of cells.106a-5p and cavitin-1(CAV1)expression levels were measured by RT-qPCR and Western blot.The si-LINC00662 and miR-106a-5p mimics were transfected with HCC-SR cells,respectively,and cell sensitivity to sorafenib drug was detected by cell activity kit(CCK-8).And the targeting relationship between LINC00662 and miR-106a-5p,miR-106a-5p and CAV1 was further determined by dual luciferase reporter assay,RT-qPCR,and Western blot.Results:The relative expression of LINC00662 and CAV1 was significantly increased and miR-106a-5p expression was significantly decreased in HCC-SR cells(P<0.01,P<0.001);interference with LINC00662 expression or overexpression of miR-106a-5p significantly increased the sensitivity of HCC-SR cells to sorafenib drug(P<0.05,P<0.01).And LINC00662 targeted to negatively regulate miR-106a-5p expression and miR-106a-5p targeted to negatively regulate CAV1 expression(P<0.05).Conclusion:LINC00662 could act as a competitive endogenous RNA(ceRNA)of miR-106a-5p to promote the expression of CAV1 and mediate the resistance of sorafenib in HCC cells.Interfering with LINC00662 expression can inhibit sorafenib resistance and increase sorafenib drug sensitivity in HCC cells.展开更多
本研究旨在探讨慢病毒介导的CAV1过表达对HL-60细胞增殖与凋亡的影响。构建慢病毒重组表达载体pcDNA-EF1-CAV1,并与pPACK包装质粒混合物共转染至293TN细胞后,收集病毒液感染HL-60细胞,使CAV1基因在细胞中稳定转染并高表达。采用Western ...本研究旨在探讨慢病毒介导的CAV1过表达对HL-60细胞增殖与凋亡的影响。构建慢病毒重组表达载体pcDNA-EF1-CAV1,并与pPACK包装质粒混合物共转染至293TN细胞后,收集病毒液感染HL-60细胞,使CAV1基因在细胞中稳定转染并高表达。采用Western blot方法评价转染后HL-60 CAV1蛋白表达情况。采用CCK-8法、流式细胞术分别检测转染前后HL-60细胞的增殖活性和凋亡情况。结果表明:PCR阳性克隆筛选及核苷酸测序结果证实CAV1基因正确插入表达载体pcDNA-EF1-GFP中。重组慢病毒颗粒Lv-CAV1成功转染HL-60细胞,转染率达90%。Western blot检测结果显示,转染后48 h HL-60细胞中CAV1蛋白表达明显增高。CCK-8检测结果显示转染后48 h HL-60细胞增殖活性降低(P<0.05),流式细胞仪检测结果显示转染后HL-60细胞凋亡率明显增加(P<0.01)。结论:CAV1过表达对HL-60细胞具有抑制细胞的增殖活性、促进细胞凋亡的作用。展开更多
目的研究小窝蛋白1(CAV1)在缺氧相关肺动脉成纤维细胞(PAFs)增殖调控中的作用。方法将体外培养的人PAFs分为:对照组(C:21%O_2);10%氧浓度组(10%O_2);5%氧浓度组(5%O_2)及2%氧浓度组(2%O_2)进行细胞增殖实验,选取细胞增殖最明显组为缺氧...目的研究小窝蛋白1(CAV1)在缺氧相关肺动脉成纤维细胞(PAFs)增殖调控中的作用。方法将体外培养的人PAFs分为:对照组(C:21%O_2);10%氧浓度组(10%O_2);5%氧浓度组(5%O_2)及2%氧浓度组(2%O_2)进行细胞增殖实验,选取细胞增殖最明显组为缺氧组(H)进行后续实验。并构建CAV1高表达质粒(pCAV1),然后再将PAFs分为对照组(C:21%O_2),缺氧组(H),空白对照组(NC:缺氧+空质粒转染组)和CAV1高表达组(pCAV1:缺氧+pCAV1转染组),采用Western-blot法检测各组细胞中CAV1、细胞周期蛋白D1(cyclinD1)和细胞凋亡抑制蛋白2(c-IAP2)含量,四甲基偶氮唑蓝(MTT)及增殖细胞核抗原(PCNA)免疫组化法检测细胞增殖情况。结果缺氧能刺激PAFs增殖,并呈浓度依赖性,于2%氧浓度刺激48小时PAFs增殖达峰值,与对照组相比差异有统计学意义(1.20+0.02 vs 0.54+0.04,P<0.01);缺氧组PAFs中CAV1表达下调(1.23±0.04 vs 0.90±0.02,P<0.01),cyclin D1(0.19±0.03 vs 1.15±0.06,P<0.01)和c-IAP2(0.63±0.04 vs 0.78±0.09,P<0.01)表达上调,细胞增殖增加(MTT:0.78±0.04 vs 1.20±0.02,P<0.01;PCNA:0.29±0.03 vs 0.54±0.03,P<0.01);CAV1在PAFs中高表达后(0.55±0.03 vs 0.90±0.03,P<0.01),cyclin D1(0.88±0.02 vs 0.52±0.02,P<0.01)和c-IAP2(0.87±0.02 vs 0.72±0.02,P<0.01)表达下调,PAFs增殖减少(MTT:1.20±0.02 vs 1.00±0.06,P<0.01;PCNA:0.52±0.03 vs 0.38±0.03,P<0.01)。结论缺氧能通过下调CAV-1在PAFs中的表达,促进PAFs的增殖、抑制其凋亡。cyclinD1和cIAP2可能是CAV1调控PAFs增殖和凋亡的下游靶点。展开更多
基金This study was supported by National Natural Science Foundation of China(No.81360359)2021 Innovation and Entrepreneurship Training Program for University Students,Hainan Medical University(No.202111810003)。
文摘Objective:To investigate the mechanism of long non-coding RNA-LINC00662 on induction of sorafenib resistance in hepatocellular carcinoma(HCC)cells.Methods:HCC cells(HepG2,HCCLM3),sorafenib-resistant hepatocellular carcinoma cells HCC-SR(HepG2-SR,HCCLM3-SR)were investigated by real-time fluorescence quantitative polymerase chain reaction(RT-qPCR)and Western blot was used to detect LINC00662,miR-106a-5p and cavitin-1(CAV1)expression in each group of cells.106a-5p and cavitin-1(CAV1)expression levels were measured by RT-qPCR and Western blot.The si-LINC00662 and miR-106a-5p mimics were transfected with HCC-SR cells,respectively,and cell sensitivity to sorafenib drug was detected by cell activity kit(CCK-8).And the targeting relationship between LINC00662 and miR-106a-5p,miR-106a-5p and CAV1 was further determined by dual luciferase reporter assay,RT-qPCR,and Western blot.Results:The relative expression of LINC00662 and CAV1 was significantly increased and miR-106a-5p expression was significantly decreased in HCC-SR cells(P<0.01,P<0.001);interference with LINC00662 expression or overexpression of miR-106a-5p significantly increased the sensitivity of HCC-SR cells to sorafenib drug(P<0.05,P<0.01).And LINC00662 targeted to negatively regulate miR-106a-5p expression and miR-106a-5p targeted to negatively regulate CAV1 expression(P<0.05).Conclusion:LINC00662 could act as a competitive endogenous RNA(ceRNA)of miR-106a-5p to promote the expression of CAV1 and mediate the resistance of sorafenib in HCC cells.Interfering with LINC00662 expression can inhibit sorafenib resistance and increase sorafenib drug sensitivity in HCC cells.
文摘本研究旨在探讨慢病毒介导的CAV1过表达对HL-60细胞增殖与凋亡的影响。构建慢病毒重组表达载体pcDNA-EF1-CAV1,并与pPACK包装质粒混合物共转染至293TN细胞后,收集病毒液感染HL-60细胞,使CAV1基因在细胞中稳定转染并高表达。采用Western blot方法评价转染后HL-60 CAV1蛋白表达情况。采用CCK-8法、流式细胞术分别检测转染前后HL-60细胞的增殖活性和凋亡情况。结果表明:PCR阳性克隆筛选及核苷酸测序结果证实CAV1基因正确插入表达载体pcDNA-EF1-GFP中。重组慢病毒颗粒Lv-CAV1成功转染HL-60细胞,转染率达90%。Western blot检测结果显示,转染后48 h HL-60细胞中CAV1蛋白表达明显增高。CCK-8检测结果显示转染后48 h HL-60细胞增殖活性降低(P<0.05),流式细胞仪检测结果显示转染后HL-60细胞凋亡率明显增加(P<0.01)。结论:CAV1过表达对HL-60细胞具有抑制细胞的增殖活性、促进细胞凋亡的作用。
文摘目的研究小窝蛋白1(CAV1)在缺氧相关肺动脉成纤维细胞(PAFs)增殖调控中的作用。方法将体外培养的人PAFs分为:对照组(C:21%O_2);10%氧浓度组(10%O_2);5%氧浓度组(5%O_2)及2%氧浓度组(2%O_2)进行细胞增殖实验,选取细胞增殖最明显组为缺氧组(H)进行后续实验。并构建CAV1高表达质粒(pCAV1),然后再将PAFs分为对照组(C:21%O_2),缺氧组(H),空白对照组(NC:缺氧+空质粒转染组)和CAV1高表达组(pCAV1:缺氧+pCAV1转染组),采用Western-blot法检测各组细胞中CAV1、细胞周期蛋白D1(cyclinD1)和细胞凋亡抑制蛋白2(c-IAP2)含量,四甲基偶氮唑蓝(MTT)及增殖细胞核抗原(PCNA)免疫组化法检测细胞增殖情况。结果缺氧能刺激PAFs增殖,并呈浓度依赖性,于2%氧浓度刺激48小时PAFs增殖达峰值,与对照组相比差异有统计学意义(1.20+0.02 vs 0.54+0.04,P<0.01);缺氧组PAFs中CAV1表达下调(1.23±0.04 vs 0.90±0.02,P<0.01),cyclin D1(0.19±0.03 vs 1.15±0.06,P<0.01)和c-IAP2(0.63±0.04 vs 0.78±0.09,P<0.01)表达上调,细胞增殖增加(MTT:0.78±0.04 vs 1.20±0.02,P<0.01;PCNA:0.29±0.03 vs 0.54±0.03,P<0.01);CAV1在PAFs中高表达后(0.55±0.03 vs 0.90±0.03,P<0.01),cyclin D1(0.88±0.02 vs 0.52±0.02,P<0.01)和c-IAP2(0.87±0.02 vs 0.72±0.02,P<0.01)表达下调,PAFs增殖减少(MTT:1.20±0.02 vs 1.00±0.06,P<0.01;PCNA:0.52±0.03 vs 0.38±0.03,P<0.01)。结论缺氧能通过下调CAV-1在PAFs中的表达,促进PAFs的增殖、抑制其凋亡。cyclinD1和cIAP2可能是CAV1调控PAFs增殖和凋亡的下游靶点。
