siRNA抑制CNN2表达对肝癌细胞生物学行为及裸鼠成瘤的影响
发布时间：2018-08-02 16:24
【摘要】：目的：建立稳定转染CNN2 siRNA的肝癌细胞株,用以观察CNN2蛋白在表达沉默时对细胞生物学行为和裸鼠成瘤的影响。方法：(1)将插入有CNN2 siRNA的慢病毒以及空载慢病毒LV3分别转染SK-hep-1肝癌细胞,通过嘌呤霉素筛选建立稳定转染的“基因表达下调”细胞模型。Quantitative Real Time-PCR (qRT-PCR)和Western-blot法分别观察分析转染前后目的基因CNN2在转录和翻译水平的表达变化。(2)通过Transwell实验检测细胞的迁移能力和侵袭能力的改变；MTT实验检测细胞增殖能力,观察转染细胞株与对照组细胞增殖能力的差异；流式细胞仪检测细胞周期,观察抑制CNN2表达对细胞周期的影响。(3)构建SK-hep-1-siRNA, SK-hep-1-LV3, SK-hep-1三种细胞株的裸鼠皮下成瘤模型,每组12只裸鼠,雌雄各半。每日观察裸鼠生活情况,待肿瘤长出后,每周测一次瘤体积,称裸鼠体重。第4周处死裸鼠,取瘤体测体积,称重,解剖裸鼠观察是否有肿瘤转移。瘤组织石蜡包埋切片,HE染色进行病理观察,TUNEL法检测细胞凋亡,免疫组织化学检测CNN2表达,磷酸化MEK,磷酸化ERK,磷酸化AKT以及MEK, ERK, AKT蛋白表达。结果：(1) SK-hep-1-siRNA细胞株及空载SK-hep-1-LV3细胞株在慢病毒转染24 h后,带有绿色荧光,证实转染成功,用2μg/mL嘌呤霉素筛选72 h后,在荧光显微镜下观察,细胞100%带有绿色荧光,说明已将未转染成功的肝癌细胞清除,以及成功构建了稳定转染的SK-hep-1-siRNA和SK-hep-1-LV3细胞株。qRT-PCR、Western-blot显示SK-hep-1-siRNA细胞株中目的基因CNN2在mRNA和蛋白质水平上表达均较SK-hep-1-LV3细胞株和未转染细胞株降低(P0.001),转录水平CNN2表达抑制率约为68.3%,翻译水平CNN2表达抑制率约为63.9%。(2)对三种细胞株的观察表明,CNN2表达沉默能抑制SK-hep-1细胞的增殖,使细胞周期阻滞在S期(P0.01),并使迁移运动(P0.01)和侵袭能力降低(P0.001)。(3)成功构建裸鼠皮下移植瘤模型。相比于SK-hep-1-LV3裸鼠成瘤组和SK-hep-1裸鼠成瘤组,SK-hep-1-siRNA裸鼠成瘤组肿瘤生长速度较慢,瘤体较小,成瘤率降低,4周后剥离瘤组织测得瘤体重量较轻,三组裸鼠瘤体积差异与重量差异均有统计学意义(P0.001)。所有组别的裸鼠均未发生肿瘤转移,裸鼠重量三组相当,无显著性差异。HE染色结果显示,三组裸鼠瘤组织均呈现典型的肿瘤组织特征,即细胞核大,染色深,细胞排列紧密。TUNEL法测凋亡结果显示三组瘤组织均未出现细胞凋亡的情况。SK-hep-1-siRNA组瘤组织CNN2、磷酸化MEK、磷酸化ERK表达下调,与阴性对照组和空白对照组相比差异有统计学意义(P0.01,P0.05,P0.01),磷酸化AKT, AKT, MEK, ERK表达在三组之间无显著性差异。结论：(1)成功构建了SK-hep-1-siRNA细胞株和SK-hep-1-LV3细胞株。(2)CNN2 siRNA慢病毒稳定转染可以抑制肝癌SK-hep-1细胞增殖,迁移,侵袭能力,并使细胞周期阻滞在S期。(3) CNN2 siRNA慢病毒稳定转染可以抑制SK-hep-1细胞成瘤能力。CNN2可能通过提高MAPK通路蛋白MEK, ERK的磷酸化水平而发挥促进肿瘤发生发展的作用。
[Abstract]:Objective: to establish a hepatocellular carcinoma cell line stably transfected with CNN2 siRNA to observe the effect of CNN2 protein on cell biological behavior and tumor formation in nude mice. Methods: (1) transfection of CNN2 siRNA with lentivirus and empty lentivirus LV3 to SK-hep-1 hepatoma cells respectively, and to establish a stable transfected "base" by purinomycin screening. The expression of CNN2 at the transcriptional and translation levels of the target gene CNN2 before and after transfection was observed by.Quantitative Real Time-PCR (qRT-PCR) and Western-blot method. (2) the changes in the cell migration ability and invasion ability were detected by Transwell test. The proliferation ability of the cells was detected by MTT test, and the transfection rate was observed. The cell cycle was detected by flow cytometry and the effect of inhibiting the expression of CNN2 on the cell cycle was observed by flow cytometry. (3) the subcutaneous tumor formation model of SK-hep-1-siRNA, SK-hep-1-LV3, and SK-hep-1 cells in nude mice was constructed, and 12 nude mice in each group were half. The daily life of nude mice was observed, after the tumor grew out. The tumor volume of nude mice was measured once a week, and the nude mice were weighed. Fourth weeks were killed in nude mice. The tumor was measured and weighed and weighed. The tumor metastases were observed in nude mice. The paraffin embedded section of the tumor tissue, HE staining for pathological observation, TUNEL assay of apoptosis, immunohistochemical detection of CNN2 expression, phosphorylated MEK, phosphorylated ERK, phosphorylated AKT, MEK, ERK, Results: (1) (1) the SK-hep-1-siRNA cell line and the unloaded SK-hep-1-LV3 cell line had green fluorescence after 24 h transfection of lentivirus, confirmed the successful transfection. After screening 72 h with 2 mu g/mL puramycin, the cells were observed under the fluorescence microscope, and the cells were 100% with green fluorescence, indicating that the untransfected hepatoma cells were cleared and formed. The stable transfected SK-hep-1-siRNA and SK-hep-1-LV3 cell strain.QRT-PCR were constructed, and Western-blot showed that the expression of target gene CNN2 in SK-hep-1-siRNA cell line was lower than that of SK-hep-1-LV3 cell line and untransfected cell line (P0.001). The inhibition rate of transcription level CNN2 expression was about 68.3%, and the expression level of translation level was inhibited. The observation of the rate of 63.9%. (2) on three cell lines showed that the expression of CNN2 could inhibit the proliferation of SK-hep-1 cells, block the cell cycle at S (P0.01), and reduce the migration (P0.01) and invasion ability (P0.001). (3) the subcutaneous tumor model of nude mice was successfully constructed. Compared to the tumor formation and SK-hep-1 nude mice of SK-hep-1-LV3 nude mice, the tumor cells were successfully constructed. In the group of SK-hep-1-siRNA nude mice, the tumor growth rate was slower, the tumor body was smaller, the tumorigenesis rate decreased. The weight of the tumor body was light in the dissection tissue after 4 weeks. The difference of tumor volume between the three groups of nude mice and the weight difference were statistically significant (P0.001). There was no tumor metastasis in all groups of nude mice, the weight of nude mice was equivalent to three groups, no significant difference.HE The staining results showed that the tumor tissues of the three groups of nude mice showed typical tumor tissue characteristics, that is, the nucleus was large, the staining was deep, and the cells arranged closely.TUNEL method to determine the apoptotic results in the three groups of tumor tissues. The tumor tissue was CNN2, phosphorylated MEK, and phosphorylated ERK expression was down regulated, and the negative control group and the blank were in the blank. There were statistically significant differences (P0.01, P0.05, P0.01), phosphorylated AKT, AKT, MEK, and ERK expression between the three groups. Conclusion: (1) SK-hep-1-siRNA cell line and SK-hep-1-LV3 cell line were successfully constructed. (2) CNN2 siRNA lentivirus stable transfer can inhibit the proliferation, migration, invasion ability of hepatocellular carcinoma SK-hep-1 cells, and make it possible Cell cycle arrest in S phase. (3) CNN2 siRNA lentivirus stable transfection can inhibit the tumorigenesis of SK-hep-1 cells,.CNN2 may play a role in promoting the development of tumor by increasing the phosphorylation level of MAPK pathway protein MEK and ERK.
【学位授予单位】：广西医科大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R735.7
本文编号：2160005
[Abstract]:Objective: to establish a hepatocellular carcinoma cell line stably transfected with CNN2 siRNA to observe the effect of CNN2 protein on cell biological behavior and tumor formation in nude mice. Methods: (1) transfection of CNN2 siRNA with lentivirus and empty lentivirus LV3 to SK-hep-1 hepatoma cells respectively, and to establish a stable transfected "base" by purinomycin screening. The expression of CNN2 at the transcriptional and translation levels of the target gene CNN2 before and after transfection was observed by.Quantitative Real Time-PCR (qRT-PCR) and Western-blot method. (2) the changes in the cell migration ability and invasion ability were detected by Transwell test. The proliferation ability of the cells was detected by MTT test, and the transfection rate was observed. The cell cycle was detected by flow cytometry and the effect of inhibiting the expression of CNN2 on the cell cycle was observed by flow cytometry. (3) the subcutaneous tumor formation model of SK-hep-1-siRNA, SK-hep-1-LV3, and SK-hep-1 cells in nude mice was constructed, and 12 nude mice in each group were half. The daily life of nude mice was observed, after the tumor grew out. The tumor volume of nude mice was measured once a week, and the nude mice were weighed. Fourth weeks were killed in nude mice. The tumor was measured and weighed and weighed. The tumor metastases were observed in nude mice. The paraffin embedded section of the tumor tissue, HE staining for pathological observation, TUNEL assay of apoptosis, immunohistochemical detection of CNN2 expression, phosphorylated MEK, phosphorylated ERK, phosphorylated AKT, MEK, ERK, Results: (1) (1) the SK-hep-1-siRNA cell line and the unloaded SK-hep-1-LV3 cell line had green fluorescence after 24 h transfection of lentivirus, confirmed the successful transfection. After screening 72 h with 2 mu g/mL puramycin, the cells were observed under the fluorescence microscope, and the cells were 100% with green fluorescence, indicating that the untransfected hepatoma cells were cleared and formed. The stable transfected SK-hep-1-siRNA and SK-hep-1-LV3 cell strain.QRT-PCR were constructed, and Western-blot showed that the expression of target gene CNN2 in SK-hep-1-siRNA cell line was lower than that of SK-hep-1-LV3 cell line and untransfected cell line (P0.001). The inhibition rate of transcription level CNN2 expression was about 68.3%, and the expression level of translation level was inhibited. The observation of the rate of 63.9%. (2) on three cell lines showed that the expression of CNN2 could inhibit the proliferation of SK-hep-1 cells, block the cell cycle at S (P0.01), and reduce the migration (P0.01) and invasion ability (P0.001). (3) the subcutaneous tumor model of nude mice was successfully constructed. Compared to the tumor formation and SK-hep-1 nude mice of SK-hep-1-LV3 nude mice, the tumor cells were successfully constructed. In the group of SK-hep-1-siRNA nude mice, the tumor growth rate was slower, the tumor body was smaller, the tumorigenesis rate decreased. The weight of the tumor body was light in the dissection tissue after 4 weeks. The difference of tumor volume between the three groups of nude mice and the weight difference were statistically significant (P0.001). There was no tumor metastasis in all groups of nude mice, the weight of nude mice was equivalent to three groups, no significant difference.HE The staining results showed that the tumor tissues of the three groups of nude mice showed typical tumor tissue characteristics, that is, the nucleus was large, the staining was deep, and the cells arranged closely.TUNEL method to determine the apoptotic results in the three groups of tumor tissues. The tumor tissue was CNN2, phosphorylated MEK, and phosphorylated ERK expression was down regulated, and the negative control group and the blank were in the blank. There were statistically significant differences (P0.01, P0.05, P0.01), phosphorylated AKT, AKT, MEK, and ERK expression between the three groups. Conclusion: (1) SK-hep-1-siRNA cell line and SK-hep-1-LV3 cell line were successfully constructed. (2) CNN2 siRNA lentivirus stable transfer can inhibit the proliferation, migration, invasion ability of hepatocellular carcinoma SK-hep-1 cells, and make it possible Cell cycle arrest in S phase. (3) CNN2 siRNA lentivirus stable transfection can inhibit the tumorigenesis of SK-hep-1 cells,.CNN2 may play a role in promoting the development of tumor by increasing the phosphorylation level of MAPK pathway protein MEK and ERK.
【学位授予单位】：广西医科大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R735.7
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