旨在筛选对黑色素生成起调节作用的小RNA,并探究其对山羊肤色及毛色的调控机制。本研究采集了健康酉州乌羊(Youzhou dark goat, YZDG)、川东白山羊(Chuandong white goat, CDWG)100日龄胎羊皮肤样本(n=3),和健康2~3周岁大足黑山羊(Dazu ...旨在筛选对黑色素生成起调节作用的小RNA,并探究其对山羊肤色及毛色的调控机制。本研究采集了健康酉州乌羊(Youzhou dark goat, YZDG)、川东白山羊(Chuandong white goat, CDWG)100日龄胎羊皮肤样本(n=3),和健康2~3周岁大足黑山羊(Dazu black goat, DBG)、内蒙古绒山羊(Inner Mongolia cashmere goat, IMCG)个体皮肤样本(n=3),利用组织切片染色技术观察皮肤中黑色素沉积情况;通过小RNA测序技术筛选差异miRNAs;培养B16-F10皮肤黑色素瘤细胞,利用细胞转染、qPCR、Western Blot、黑色素含量检测等技术验证miR-129-5p对黑色素生成的影响。结果显示,黑色素颗粒明显在YZDG胎羊皮肤和DBG毛囊的毛球、毛干、外根鞘等部位沉积,而在CDWG胎羊皮肤和IMCG表皮、毛囊中没有被观察到。经测序分析,在肤色差异的YZDG和CDWG中筛选到62个差异表达miRNAs,其中31个在乌皮山羊中上调,31个下调。在毛色差异的DBG和IMCG中,筛选到38个差异表达miRNAs,其中10个在黑色被毛山羊中表达上调,28个表达下调。两组测序结果均显示miR-129-5p在乌皮和黑色被毛山羊皮肤中高表达(P<0.05)。在细胞中过表达miR-129-5p后,相比于对照组,mimics组细胞黑色素沉积量提高了18.9%(P<0.05),TYR、TYRP1基因表达量分别上调57.3%和16.5%(P<0.05),蛋白表达量分别显著上调49.2%和40.2%(P<0.05);但MITF基因及其蛋白表达量无显著变化(P>0.05)。在抑制miR-129-5p后,inhibitor组TYR基因mRNA表达下调38.9%、蛋白表达水平下调21.1%(P<0.05);TYRP1、MITF蛋白表达水平分别下调25.3%及28.4%(P<0.05)。本研究发现,miR-129-5p在不同肤色及毛色的山羊皮肤中差异表达,且可通过调控TYR、TYRP1等关键基因的表达影响黑色素的生成,是山羊肤色和毛色形成过程的重要调节因子。展开更多
Objective:To investigate the role of miR-129-5p in regulating HMGB1 expression in pancreatic cancer cell apoptosis.Methods:The untreated pancreatic cancer SW1990 cells were used as the control group.Mimics-NC(empty ve...Objective:To investigate the role of miR-129-5p in regulating HMGB1 expression in pancreatic cancer cell apoptosis.Methods:The untreated pancreatic cancer SW1990 cells were used as the control group.Mimics-NC(empty vector),miR-129-5p mimics,inhibitor-NC(empty vector)and miR-129-5p inhibitor were transfected into SW1990 cells by liposome transfection method as the mimics-NC group,miR-129-5p overexpression group(miR-129-5p mimics group),inhibitor-NC group and miR-129-5p low expression group(miR-129-5p inhibitor group).The binding site of miR-129-5p and HMGB1 was predicted by online target gene prediction website Target genes,and the targeting relationship between miR-129-5p and HMGB1 was verified by dual luciferase gene report experiment.The expression of miR-129-5p in each group was detected by qRT-PCR,and the expression of HMGB1 protein and apoptosis-related proteins Caspase 3 and Bcl-2 by Western blot.Hoechst staining was used to observe the changes of apoptosis.Results:Compared with the mimics-NC group and control group,miR-129-5p mimics transfection significantly up-regulated miR-129-5p level(P<0.01),inhibited HMGB1(P<0.01)and Bcl-2(P<0.05)protein expression,pro-moted Caspase 3 protein expression(P<0.05),and promoted apoptosis;compared with the inhibitor-NC group and control group,miR-129-5p inhibitor transfection significantly down-regulated miR-129-5p level(P<0.05),promoted HMGB1 and Bcl-2 protein expression(all P<0.05),inhibited Caspase 3protein expression(P<0.01),and inhibited apoptosis.The results of dual luciferase reporter gene assay showed that miR-129-5p could inhibit the fluorescence activity of wildtype HMGB1 cells and target the expression of HMGB1.Conclusion:miR-129-5p promotes the apoptosis of pancreatic cancer SW1990 cells by targeting inhibition of HMGB1 expression.展开更多
目的:探讨miR-129-5p通过调控高迁移率族蛋白B1基因(high mobility group box 1,HMGB1)影响乳腺癌MCF-7细胞对紫杉醇(paclitaxel,PTX)的敏感性。方法:采用脂质体转染技术将miR-129-5p mimics、HMGB1小干扰RNA(si-HMGB1)分别转染入MCF-7...目的:探讨miR-129-5p通过调控高迁移率族蛋白B1基因(high mobility group box 1,HMGB1)影响乳腺癌MCF-7细胞对紫杉醇(paclitaxel,PTX)的敏感性。方法:采用脂质体转染技术将miR-129-5p mimics、HMGB1小干扰RNA(si-HMGB1)分别转染入MCF-7细胞,用PTX刺激培养细胞后,用实时荧光定量PCR检测转染后MCF-7细胞miR-129-5p和HMGB1 m RNA的表达,Western blotting检测转染后MCF-7细胞HMGB1蛋白的表达,CCK-8增殖实验检测转染后PTX对MCF-7细胞增殖的影响,流式细胞术检测转染后对PTX诱导MCF-7细胞凋亡的影响。结果:转染miR-129-5p mimics后,MCF-7细胞中miR-129-5p的表达水平明显高于阴性对照组细胞(P<0.01);过表达miR-129-5p后可明显增强PTX抑制MCF-7细胞的增殖和诱导细胞凋亡的能力(均P<0.05),并显著抑制HMGB1 m RNA和蛋白的表达(均P<0.05)。转染si-HMGB1后,显著降低MCF-7细胞HMGB1 m RNA和蛋白的表达(均P<0.05);干扰HMGB1表达进一步促进PTX抑制MCF-7细胞的增殖并诱导细胞凋亡(均P<0.05)。结论:miR-129-5p通过下调HMGB1的表达增强乳腺癌MCF-7细胞对PTX的敏感性。展开更多
文摘旨在筛选对黑色素生成起调节作用的小RNA,并探究其对山羊肤色及毛色的调控机制。本研究采集了健康酉州乌羊(Youzhou dark goat, YZDG)、川东白山羊(Chuandong white goat, CDWG)100日龄胎羊皮肤样本(n=3),和健康2~3周岁大足黑山羊(Dazu black goat, DBG)、内蒙古绒山羊(Inner Mongolia cashmere goat, IMCG)个体皮肤样本(n=3),利用组织切片染色技术观察皮肤中黑色素沉积情况;通过小RNA测序技术筛选差异miRNAs;培养B16-F10皮肤黑色素瘤细胞,利用细胞转染、qPCR、Western Blot、黑色素含量检测等技术验证miR-129-5p对黑色素生成的影响。结果显示,黑色素颗粒明显在YZDG胎羊皮肤和DBG毛囊的毛球、毛干、外根鞘等部位沉积,而在CDWG胎羊皮肤和IMCG表皮、毛囊中没有被观察到。经测序分析,在肤色差异的YZDG和CDWG中筛选到62个差异表达miRNAs,其中31个在乌皮山羊中上调,31个下调。在毛色差异的DBG和IMCG中,筛选到38个差异表达miRNAs,其中10个在黑色被毛山羊中表达上调,28个表达下调。两组测序结果均显示miR-129-5p在乌皮和黑色被毛山羊皮肤中高表达(P<0.05)。在细胞中过表达miR-129-5p后,相比于对照组,mimics组细胞黑色素沉积量提高了18.9%(P<0.05),TYR、TYRP1基因表达量分别上调57.3%和16.5%(P<0.05),蛋白表达量分别显著上调49.2%和40.2%(P<0.05);但MITF基因及其蛋白表达量无显著变化(P>0.05)。在抑制miR-129-5p后,inhibitor组TYR基因mRNA表达下调38.9%、蛋白表达水平下调21.1%(P<0.05);TYRP1、MITF蛋白表达水平分别下调25.3%及28.4%(P<0.05)。本研究发现,miR-129-5p在不同肤色及毛色的山羊皮肤中差异表达,且可通过调控TYR、TYRP1等关键基因的表达影响黑色素的生成,是山羊肤色和毛色形成过程的重要调节因子。
基金National Natural Science Foundation of China (No.82260134)Natural Science Foundation of Guangxi (No.2023GXNSFAA026118)+1 种基金High-level Talents Research Project of the Affiliated Hospital of Youjiang Medical College for Nationalities (No.R202011702)Guangxi Graduate Education Innovation Project (No.YCSW2023506)。
文摘Objective:To investigate the role of miR-129-5p in regulating HMGB1 expression in pancreatic cancer cell apoptosis.Methods:The untreated pancreatic cancer SW1990 cells were used as the control group.Mimics-NC(empty vector),miR-129-5p mimics,inhibitor-NC(empty vector)and miR-129-5p inhibitor were transfected into SW1990 cells by liposome transfection method as the mimics-NC group,miR-129-5p overexpression group(miR-129-5p mimics group),inhibitor-NC group and miR-129-5p low expression group(miR-129-5p inhibitor group).The binding site of miR-129-5p and HMGB1 was predicted by online target gene prediction website Target genes,and the targeting relationship between miR-129-5p and HMGB1 was verified by dual luciferase gene report experiment.The expression of miR-129-5p in each group was detected by qRT-PCR,and the expression of HMGB1 protein and apoptosis-related proteins Caspase 3 and Bcl-2 by Western blot.Hoechst staining was used to observe the changes of apoptosis.Results:Compared with the mimics-NC group and control group,miR-129-5p mimics transfection significantly up-regulated miR-129-5p level(P<0.01),inhibited HMGB1(P<0.01)and Bcl-2(P<0.05)protein expression,pro-moted Caspase 3 protein expression(P<0.05),and promoted apoptosis;compared with the inhibitor-NC group and control group,miR-129-5p inhibitor transfection significantly down-regulated miR-129-5p level(P<0.05),promoted HMGB1 and Bcl-2 protein expression(all P<0.05),inhibited Caspase 3protein expression(P<0.01),and inhibited apoptosis.The results of dual luciferase reporter gene assay showed that miR-129-5p could inhibit the fluorescence activity of wildtype HMGB1 cells and target the expression of HMGB1.Conclusion:miR-129-5p promotes the apoptosis of pancreatic cancer SW1990 cells by targeting inhibition of HMGB1 expression.
文摘目的:探讨miR-129-5p通过调控高迁移率族蛋白B1基因(high mobility group box 1,HMGB1)影响乳腺癌MCF-7细胞对紫杉醇(paclitaxel,PTX)的敏感性。方法:采用脂质体转染技术将miR-129-5p mimics、HMGB1小干扰RNA(si-HMGB1)分别转染入MCF-7细胞,用PTX刺激培养细胞后,用实时荧光定量PCR检测转染后MCF-7细胞miR-129-5p和HMGB1 m RNA的表达,Western blotting检测转染后MCF-7细胞HMGB1蛋白的表达,CCK-8增殖实验检测转染后PTX对MCF-7细胞增殖的影响,流式细胞术检测转染后对PTX诱导MCF-7细胞凋亡的影响。结果:转染miR-129-5p mimics后,MCF-7细胞中miR-129-5p的表达水平明显高于阴性对照组细胞(P<0.01);过表达miR-129-5p后可明显增强PTX抑制MCF-7细胞的增殖和诱导细胞凋亡的能力(均P<0.05),并显著抑制HMGB1 m RNA和蛋白的表达(均P<0.05)。转染si-HMGB1后,显著降低MCF-7细胞HMGB1 m RNA和蛋白的表达(均P<0.05);干扰HMGB1表达进一步促进PTX抑制MCF-7细胞的增殖并诱导细胞凋亡(均P<0.05)。结论:miR-129-5p通过下调HMGB1的表达增强乳腺癌MCF-7细胞对PTX的敏感性。
