siRNA抑制HMGB1表达对子宫内膜癌细胞侵袭与迁移的影响
发布时间：2019-03-15 14:51
【摘要】：目的：探讨靶向HMGB1的shRNA对HMGB1表达改变的影响及其对子宫内膜癌HEC-1A细胞侵袭与迁移能力改变的影响。 方法：运用RNA干扰技术,构建针对HMGB1基因的短发夹RNA (pshRNA-1/HMGB1、pshRNA-2/HMGB1、pshRNA-3/HMGB1),同时设置转染空白质粒的阴性对照组(HMGB1/p-NC)和脂质体转染组(Lipo组),用脂质体法转染人子宫内膜癌细胞(HEC-1A),利用反转录-聚合酶链反应(RT-PCR)和免疫印迹法(Western-Blot)检测转染后48h各组细胞HMGB1在mRNA水平和蛋白水平的表达,Transwell小室法观察转染HMGB1shRNA后HEC-1A细胞的侵袭情况,细胞划痕实验观察转染HMGB1shRNA后HEC-1A细胞的迁移情况。 结果：1、RT-PCR结果显示：针对HMGB1构建的三组重组质粒HMGB1-pshRNAs(1、2、3)转染后,HMGB1mRNA相对表达水平分别是0.192±0.006,0.055±0.002和0.123±0.004,均较Lipo组(0.268±0.008)和阴性对照组(0.270±0.004)明显减少(P0.05)；与阴性对照组比较,分别减少了28.9%,79.6%和54.4%。 2、Western-blot结果显示：针对HMGB1构建的三组重组质粒HMGB1-pshRNAs(1、2、3)转染后,HMGB1蛋白相对表达水平分别是0.259±0.013,0.032±0.002和0.104±0.007,均较Lipo组(0.347±0.007)和阴性对照组组(0.349±0.007)明显减少(P0.05)；与阴性对照组比较,分别减少了25.8%,90.8%和70.2%。 3、Transwell小室法检测细胞的侵袭能力显示：转染后48h,pshRNA2转染组穿膜细胞为(20±1)个,阴性对照组和Lipo组穿膜细胞数分别为(36±1)和(36±2)个。与阴性对照组比较,pshRNA2转染组穿膜细胞数明显减少(P0.05),差异有统计学意义；而Lipo组和HMGB1/p-NC组穿膜细胞数差异无统计学意义(P0.05) 4、细胞划痕实验检测细胞迁移能力显示：划痕后培养48h,pshRNA2转染组细胞的迁移率为25.75%±1.70%,脂质体转染组细胞的迁移率为65.25%±1.70%,阴性对照组细胞的迁移率为64.75%±4.57%。与阴性对照组相比,pshRNA2转染组细胞迁移率明显降低(P0.05)；而脂质体转染组和阴性对照组的细胞迁移率比较,差异无统计学意义(P0.05)。 结论：1、靶向干扰HMGB1的三组重组质粒在人子宫内膜癌细胞中均能有效下调HMGB1在mRNA和蛋白水平的表达,其中以pshRNA-2/HMGB1沉默效果最好。 2、靶向干扰HMGB1的shRNA2转染子宫内膜癌细胞HEC-1A后细胞的侵袭与迁移能力明显下降,提示HMGB1可能参与子宫内膜癌侵袭与迁移的发展过程。
[Abstract]:Aim: to investigate the effect of shRNA targeting HMGB1 on the expression of HMGB1 and its effect on invasion and migration of endometrial carcinoma HEC-1A cells. Methods: the short hairpin RNA (pshRNA-1/HMGB1,pshRNA-2/HMGB1,pshRNA-3/HMGB1) targeting HMGB1 gene was constructed by RNA interference technique. At the same time, the negative control group (HMGB1/p-NC) and the liposome transfected group (Lipo group) were transfected into human endometrial carcinoma cell line (HEC-1A) by lipofectamine method. Reverse transcription-polymerase chain reaction (RT-PCR) and immunoblot assay (Western-Blot) were used to detect the expression of HMGB1 at mRNA level and protein level at 48 h after transfection, and the invasion of HEC-1A cells was observed by Transwell chamber assay. Cell scratch assay was used to observe the migration of HEC-1A cells transfected with HMGB1shRNA. Results: 1. The results of RT-PCR showed that the relative expression level of HMGB1mRNA was 0.192 卤0.006,0.055 卤0.002 and 0.123 卤0.004respectively after transfection with recombinant plasmid HMGB1-pshRNAs (1,2, and 3) constructed by HMGB1, and the expression level of HMGB1mRNA was 0.192 卤0.006, 0.055 卤0.002 and 0.123 卤0.004respectively. Compared with Lipo group (0.268 卤0.008) and negative control group (0.270 卤0.004), both of them were significantly decreased (P0.05). Compared with the negative control group, it decreased by 28.9%, 79.6% and 54.4% respectively. 2. Western blot analysis showed that the relative expression level of HMGB1 protein was 0.259 卤0.013,0.032 卤0.002 and 0.104 卤0.007respectively after transfection of three recombinant plasmids HMGB1-pshRNAs (1, 2, 3) constructed by HMGB1, and the expression level of HMGB1 protein was 0.259 卤0.013, 0.032 卤0.002 and 0.104 卤0.007, respectively. Compared with Lipo group (0.347 卤0.007) and negative control group (0.349 卤0.007), it was significantly decreased (P0.05). Compared with the negative control group, it decreased by 25.8%, 90.8% and 70.2% respectively. 3. The invasiveness of pshRNA2 transfected group was (20 卤1), and that of negative control group and Lipo group was (36 卤1) and (36 卤2), respectively. Compared with the negative control group, the number of transmembrane cells in pshRNA2 transfected group decreased significantly (P0.05), the difference was statistically significant. However, there was no significant difference in the number of transmembrane cells between Lipo group and HMGB1/p-NC group (P0.05) 4. The ability of cell migration was detected by cell scratch assay. After 48 hours of culturing, the migration rate of pshRNA 2 transfected group was 25.75% 卤1.70%, and that of pshRNA2 transfected group was 25.75% 卤1.70%. The cell migration rate was 65.25% 卤1.70% in liposome transfected group and 64.75% 卤4.57% in negative control group. Compared with the negative control group, the cell migration rate of pshRNA2 transfection group was significantly lower (P0.05), but there was no significant difference between liposome transfection group and negative control group (P0.05). Conclusion: 1. Three recombinant plasmids targeting HMGB1 can effectively down-regulate the expression of HMGB1 at mRNA and protein levels in human endometrial carcinoma cells, and the silencing effect of pshRNA-2/HMGB1 is the best. 2. The invasion and migration ability of endometrial carcinoma cell line HEC-1A transfected with shRNA2 targeting HMGB1 decreased significantly, suggesting that HMGB1 may be involved in the process of invasion and migration of endometrial carcinoma.
【学位授予单位】：中南大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R737.33
本文编号：2440725
[Abstract]:Aim: to investigate the effect of shRNA targeting HMGB1 on the expression of HMGB1 and its effect on invasion and migration of endometrial carcinoma HEC-1A cells. Methods: the short hairpin RNA (pshRNA-1/HMGB1,pshRNA-2/HMGB1,pshRNA-3/HMGB1) targeting HMGB1 gene was constructed by RNA interference technique. At the same time, the negative control group (HMGB1/p-NC) and the liposome transfected group (Lipo group) were transfected into human endometrial carcinoma cell line (HEC-1A) by lipofectamine method. Reverse transcription-polymerase chain reaction (RT-PCR) and immunoblot assay (Western-Blot) were used to detect the expression of HMGB1 at mRNA level and protein level at 48 h after transfection, and the invasion of HEC-1A cells was observed by Transwell chamber assay. Cell scratch assay was used to observe the migration of HEC-1A cells transfected with HMGB1shRNA. Results: 1. The results of RT-PCR showed that the relative expression level of HMGB1mRNA was 0.192 卤0.006,0.055 卤0.002 and 0.123 卤0.004respectively after transfection with recombinant plasmid HMGB1-pshRNAs (1,2, and 3) constructed by HMGB1, and the expression level of HMGB1mRNA was 0.192 卤0.006, 0.055 卤0.002 and 0.123 卤0.004respectively. Compared with Lipo group (0.268 卤0.008) and negative control group (0.270 卤0.004), both of them were significantly decreased (P0.05). Compared with the negative control group, it decreased by 28.9%, 79.6% and 54.4% respectively. 2. Western blot analysis showed that the relative expression level of HMGB1 protein was 0.259 卤0.013,0.032 卤0.002 and 0.104 卤0.007respectively after transfection of three recombinant plasmids HMGB1-pshRNAs (1, 2, 3) constructed by HMGB1, and the expression level of HMGB1 protein was 0.259 卤0.013, 0.032 卤0.002 and 0.104 卤0.007, respectively. Compared with Lipo group (0.347 卤0.007) and negative control group (0.349 卤0.007), it was significantly decreased (P0.05). Compared with the negative control group, it decreased by 25.8%, 90.8% and 70.2% respectively. 3. The invasiveness of pshRNA2 transfected group was (20 卤1), and that of negative control group and Lipo group was (36 卤1) and (36 卤2), respectively. Compared with the negative control group, the number of transmembrane cells in pshRNA2 transfected group decreased significantly (P0.05), the difference was statistically significant. However, there was no significant difference in the number of transmembrane cells between Lipo group and HMGB1/p-NC group (P0.05) 4. The ability of cell migration was detected by cell scratch assay. After 48 hours of culturing, the migration rate of pshRNA 2 transfected group was 25.75% 卤1.70%, and that of pshRNA2 transfected group was 25.75% 卤1.70%. The cell migration rate was 65.25% 卤1.70% in liposome transfected group and 64.75% 卤4.57% in negative control group. Compared with the negative control group, the cell migration rate of pshRNA2 transfection group was significantly lower (P0.05), but there was no significant difference between liposome transfection group and negative control group (P0.05). Conclusion: 1. Three recombinant plasmids targeting HMGB1 can effectively down-regulate the expression of HMGB1 at mRNA and protein levels in human endometrial carcinoma cells, and the silencing effect of pshRNA-2/HMGB1 is the best. 2. The invasion and migration ability of endometrial carcinoma cell line HEC-1A transfected with shRNA2 targeting HMGB1 decreased significantly, suggesting that HMGB1 may be involved in the process of invasion and migration of endometrial carcinoma.
【学位授予单位】：中南大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R737.33
【参考文献】
相关期刊论文 前4条
1 汤夏冰;沈晓辉;钱晓云;高下;;高迁移率族蛋白B1和基质金属蛋白酶-2、9在喉癌组织中的表达及其与预后的关系[J];临床耳鼻咽喉头颈外科杂志;2013年04期
2 付欣;杜晓琴;郝权;;宫颈鳞癌组织高迁移率族蛋白HMGB1表达及其临床意义的研究[J];中华肿瘤防治杂志;2008年05期
3 马本红;张贵宇;梁静;祖增艳;李越;;高迁移率族蛋白B1在子宫内膜腺癌中的表达及意义[J];山东大学学报(医学版);2011年06期
4 ;Growth suppression and radiosensitivity increase by HMGB1 in breast cancer[J];Acta Pharmacologica Sinica;2007年12期
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