NIBP在结直肠癌组织中的表达及其对结肠癌细胞增殖的影响

发布时间：2018-07-11 11:27

本文选题：结直肠癌 + NIBP　；参考：《广西医科大学》2015年博士论文

【摘要】：背景和目的结直肠癌是临床最常见的恶性肿瘤之一,在全世界范围内,结直肠癌的发病率和死亡率均处于恶性肿瘤的第3位。在我国,随着生活水平的不断提高,发病率呈上升态势。结直肠癌的发生发展是遗传和环境等多种因素作用导致多基因改变及其相互作用的结果,其中涉及到多个信号转导通路,比NF-κB信号通路。它们通过NF-kB转录因子,调节炎症和肿瘤相关的至关重要的几个途径,影响肿瘤细胞的生存、血管生成、肿瘤细胞的运动性和侵袭力,从而促进肿瘤细胞的增殖,抑制细胞凋亡,并引起肿瘤的侵袭和转移。NIBP是一种支架蛋白,它将NF-κB经典途径中的关键激活子-IKKβ和非经典途径中的关键酶-NIK连接成为三聚体。研究发现,NIBP的表达升高可以促进IKKβ及其下游p65的磷酸化,由此推测,NIBP在NF-κB经典途径中发挥激活剂的作用。另一方面,抑制NIBP的表达则能减少由NIK和IKKβ分别介导的INF-κB信号通路的活化。此外,NIBP可能提高由TNF-a刺激引起NF-κB信号通路的活化从而促进肿瘤细胞的侵袭能力。最近研究还发现,NIBP在人结肠癌组织中的表达明显升高,推测这种升高可能与NF-κB信号通路的活化有关,NIBP可能参与腺瘤向腺癌的演变过程。因此,本研究以NIBP为研究对象,检测结直肠癌组织中NIBP、NF-κB活性因子p-p65和c-myc、cyclinD1的表达,了解其表达与结直肠癌的临床分期及临床病理关系。利用基因工程构建NIBP基因沉默载体,在体外试验中验证NIBP基因表达水平变化对结肠癌细胞增殖的影响,探讨其在结直肠癌的发生、发展及侵袭转移中的作用和意义。方法1.采用免疫组化SP法分别检测16例正常结肠粘膜、21例结直肠腺瘤以及114例结直肠癌患者结直肠组织中NIBP、NF-κB活性因子p-p65、c-myc和cyclinD1的表达,分析NIBP等在结直肠癌临床分期和临床病理的关系,推测其在结直肠癌的发生发展和侵袭转移中的作用。2.利用基因工程,以人结肠癌细胞HCT116株为研究对象,设计并构建人NIBP基因的慢病毒表达载体pLenti6.3-EGFP-NIBP-miR-1/2/3;另外,同时还设计无关的序列作为阴性对照(negative control, NC);人结肠癌细胞HCT1 16作为空白对照。用NIBP基因干扰的慢病毒及慢病毒阴性对照病毒液Lenti-EGFP感染靶细胞、空白细胞作对照,抽提细胞的蛋白,WB检测目的蛋白的表达,筛选出最佳干扰靶点。目的基因干扰靶点慢病毒Lenti-EGFP-NIBP-miR感染HCT 116细胞后加入BSD(Blasticidin, BSD)进行筛选,获得稳定转染NIBP低表达的单克隆HCT116细胞。应用QRT-PCR检测转染效率,选择NIBP干扰效率高的稳转株细胞株用于后续的细胞功能试验。3.以空白对照组(blank control,简称CON组,即未转染的HCT116细胞株)、阴性对照组(NC组,Lenti-EGFP-miR转染HCT116细胞株)、NIBP低表达组(NIBP组,Lenti-EGFP-NIBP-miR转染HCT 116细胞株)和NIBP低表达组细胞加NF-κB经典途径激活剂TNF-a组(NIBPT组)作为研究对象,采用CCK-8检测试验盒检测各组细胞的增殖能力；流式细胞仪检测各组细胞的凋亡和细胞周期分布情况；透射电子显微镜观察细胞凋亡情况；QRT-PCR和Western Blot检测细胞相关周期蛋白c-myc和cyclinD1的表达情况。结果1.免疫组化显示结直肠癌组织中NIBP、NF-κB活性因子p-p65、c-myc和cyclinD1的阳性表达率均高于结直肠腺瘤和正常结肠粘膜组织,P0.05；而且NIBP、NF-κB活性因子p-p65、c-myc和cyclinDl在结肠癌组织中的表达与肿瘤的分化程度、组织类型、浸润深度、TNM和Duke's分期、淋巴结转移及远处转移相关,P0.05。同时,NIBP、c-myc和cyclinD1与NF-κB活化因子p-p65呈正相关关系。2.建立NIBP沉默载体和稳定转染HCT116细胞株。我们利用质粒载体pLenti6.3-MCS/V5 DEST系列慢病毒表达载体成功构建人NIBP基因的慢病毒表达载体Lenti-EGFP-NIBP-miR(NIBP)和Lenti-EGFP-miR(NC);经酶切、重组克隆及鉴定等,证实载体构建成功。设计的NIBP基因1#,2#,3#干扰靶点进行慢病毒包装并感染HCT116细胞后3个靶点的敲减效率均非常明显,NIBP蛋白均没有出现明显的条带。我们选择了其中的第3#靶点进行后续稳转株筛选。将这些载体转染HCT116细胞株,放在荧光显微镜下观察,看到绿色荧光；可以筛选出稳定转染的单克隆细胞株；通过QRT-PCR和Western Blot试验证实NIBP干扰效果满意,HCT116细胞NIBP干扰稳转株与阴性对照稳转株相比,NIBP干扰效率高。3. NIBP对结肠癌细胞生长的影响到试验研究：CCK-8试验显示,NIBP转染细胞组和NIBPT组的细胞生长较空白组和阴性对照组减慢,P0.05；细胞凋亡检测四组细胞的凋亡率差异无统计学意义,P0.05；细胞周期检测结果显示NIBP和NIBPT组的细胞多处于Go/G1期(分别为73.500%±3.061%和70.133%±5.181%),明显高于HCT116细胞组和NC细胞组(55.933%±3.963%和52.000%±2.193%),P0.05；NIBP和NIBPT组细胞的S期分别为6.700%±1.249%和9.467%±4.858%,低于HCT116细胞组和NC细胞组(11.300%±3.751%和13.567%±1.563%),差异有统计学意义,P0.05;NIBP和NIBPT组的细胞处于G2/M期分别为14.967%±1.041%和16.067%±1.380%,明显低于HCT116细胞组和NC细胞组(29.933%±2.309%和31.200%±2.007%),P0.05；说明NIBP转染后结肠癌细胞的凋亡无明显增加,但其增殖能力下降,而且即使使用NF-κB信号通路经典途径激活剂TNF-a也不能使其增殖能力恢复。透射电镜观察发现NIBP转染细胞的凋亡亦无明显增加。而QRT-PCR和WB的检测结果显示四组细胞中c-myc和cyclinD1的表达无显著差异。结论1、NIBP、p-p65、c-myc和cyclinD1在结直肠癌组织中高表达。2. NIBP、p-p65、c-myc和cyclinD1的表达与肿瘤的分化程度、浸润深度、TNM和Duke's分期及有无淋巴结转移或/和远处转移有关。3、NIBP基因促进结肠癌HCT116细胞的增殖能力,可能是通过激活NF-κB经典途径来实现的。4、NIBP对结肠癌HCT116细胞株的凋亡无明显影响。5、NIBP对结肠癌HCT116细胞株的c-myc和cyclinD1的表达也无明显影响。
[Abstract]:Background and objective colorectal cancer is one of the most common malignant tumors in clinical. In the world, the incidence and mortality of colorectal cancer are third in the malignant tumor. In China, with the continuous improvement of the living standard, the incidence of colorectal cancer is on the rise. The development of colorectal cancer is caused by many factors such as heredity and environment. The results of multiple gene changes and their interactions involving multiple signal transduction pathways, which are more important than the NF- kappa B signaling pathway. They regulate several important pathways associated with inflammation and cancer through NF-kB transcription factors, affecting the survival of tumor cells, angiogenesis, motility and invasiveness of tumor cells, thus promoting tumor cells. Proliferation, inhibition of apoptosis, and the invasion and metastasis of tumors,.NIBP is a scaffold protein, which connects the key activator -IKK beta in the classical pathway of NF- kappa B and the key enzyme in the non classical pathway, -NIK, to be a trimer. The study shows that the increase of NIBP expression can promote the phosphorylation of IKK beta and its downstream p65, thus speculating that NIBP is in NF- kappa B. On the other hand, inhibition of the expression of NIBP can reduce the activation of the INF- kappa B signaling pathway mediated by NIK and IKK beta, respectively. In addition, NIBP may improve the activation of the NF- kappa B signaling pathway by TNF-a stimulation to promote the invasion of tumor cells. The recent study also found that NIBP is in human colon cancer tissue. The expression in the NF- kappa B signaling pathway may be related to the activation of the signal pathway. NIBP may be involved in the evolution of adenoma to adenocarcinoma. Therefore, this study uses NIBP as a study object to detect the expression of NIBP, NF- kappa B active factor p-p65, c-myc, cyclinD1, and to understand the expression of the colorectal cancer and the clinical stages of colorectal cancer. The effect and significance of the changes of NIBP gene expression level on the proliferation of colon cancer cells and the role and significance of NIBP gene expression level on the occurrence, development and invasion and metastasis of colorectal cancer were examined in vitro by using gene engineering to construct a NIBP gene silencing carrier. Methods 16 cases of normal colon mucosa were detected by the immunization of SP. 21 cases of colorectal adenoma and 114 cases of colorectal cancer, NIBP, NF- kappa B active factor p-p65, c-myc and cyclinD1 expression, analysis of the relationship between NIBP and the clinical staging and clinicopathological features of colorectal cancer, and speculate that its role in the development of colorectal cancer and invasion and metastasis of.2. using genetic engineering, with human colon cancer cell HC The T116 strain was used to design and construct the Lentivirus Expression Vector pLenti6.3-EGFP-NIBP-miR-1/2/3 of human NIBP gene. In addition, unrelated sequences were designed as negative control (negative control, NC); human colon cancer cell HCT1 16 was used as a blank control. Lentivirus and lentivirus negative control virus liquid Lenti-EG with NIBP gene interference were used as control. FP infected target cells, blank cells as control, extract cell protein, WB to detect the expression of target protein, screening out the best interference target. Target gene interfered with lentivirus Lenti-EGFP-NIBP-miR infection HCT 116 cells after BSD (Blasticidin, BSD) screening, obtained stable transfection of low expression of NIBP of the monoclonal HCT116 cells. QRT application of QRT. The transfection efficiency was detected by -PCR, and the stable cell line with high NIBP interference efficiency was selected for subsequent cell function test,.3. was used as a blank control group (blank control, CON group, or untransfected HCT116 cell line), negative control group (NC group, Lenti-EGFP-miR transfected HCT116 cell line), NIBP low expression group (NIBP group, transfected 116) Cell lines and NIBP low expression group cells plus NF- kappa B classical pathway activator TNF-a group (NIBPT group) were used as the research object. CCK-8 test box was used to detect the proliferation ability of each cell. Flow cytometry was used to detect the cell apoptosis and cell cycle distribution; transmission electron microscopy was used to observe the cell apoptosis; QRT-PCR and Weste. RN Blot was used to detect the expression of cell related cyclin c-myc and cyclinD1. Results 1. immunohistochemical staining showed that NIBP, NF- kappa B active factor p-p65, c-myc and cyclinD1 positive rates were higher than those of colorectal adenoma and normal colon mucosa, P0.05. The expression in cancer tissue is related to the degree of differentiation, type of tissue, depth of invasion, TNM and Duke's staging, lymph node metastasis and distant metastasis, P0.05., NIBP, c-myc and cyclinD1 are positively related to NF- kappa B activator p-p65.2.,.2. to establish NIBP silencing carrier and stable transfection HCT116 cell strain. /V5 DEST series lentivirus vector successfully constructed the Lentivirus Expression Vector Lenti-EGFP-NIBP-miR (NIBP) and Lenti-EGFP-miR (NC) of human NIBP gene. Through enzyme digestion, recombinant cloning and identification, it confirmed the success of the vector construction. The designed NIBP gene 1#, 2#, 3# interfered the target of the lentivirus packaging and infected the HCT116 cells after the knockout effect of 3 targets. The NIBP protein had no obvious bands. We selected the 3# target for subsequent stable strain screening. These vectors were transfected into HCT116 cell lines and observed under the fluorescence microscope to see the green fluorescence; the stable transfected monkon cell lines could be screened; the QRT-PCR and Western Blot tests were used. The effect of NIBP interference was satisfactory. Compared with the negative control stable strain of HCT116 cell NIBP interference, the effect of NIBP interference efficiency.3. NIBP on the growth of colon cancer cells was studied. The CCK-8 test showed that the cell growth of NIBP transfected cell group and NIBPT group was slower than that of the blank group and the negative control group, P0.05; the apoptosis detection was four. There was no significant difference in the apoptosis rate of the group cells, P0.05. The cell cycle detection showed that the cells in the NIBP and NIBPT groups were mostly in the Go/G1 phase (73.500% + 3.061% and 70.133% + 5.181% respectively), obviously higher than the HCT116 cell group and the NC cell group (55.933% + 3.963% and 52% + 2.193%), and the S period of the P0.05; NIBP and NIBPT groups was 6.700%, respectively. 1.249% and 9.467% + 4.858%, lower than the HCT116 cell group and the NC cell group (11.300% + 3.751% and 13.567% + 1.563%), the difference was statistically significant. The cells in the group NIBP and NIBPT were 14.967% + 1.041% and 16.067% +, respectively, lower than the HCT116 cell group and NC cell group (29.933% + 2.309% and 31.200% + 3.751%), P0.05. The apoptosis of colon cancer cells was not significantly increased after NIBP transfection, but its proliferation ability decreased, and the proliferation ability could not be restored even with the use of NF- kappa B signaling pathway activator TNF-a. The transmission electron microscopy showed that the apoptosis of NIBP transfected cells was not significantly increased. The results of QRT-PCR and WB detection showed that c-myc in four groups of cells. Conclusion 1, 1, NIBP, p-p65, c-myc and cyclinD1 express the expression of.2. NIBP, p-p65, c-myc and cyclinD1 in colorectal cancer tissues, which are related to the degree of differentiation, depth of invasion, TNM and Duke's stages, and whether there is lymph node metastasis or / and distant transfer. The ability, possibly through the activation of the classical pathway of NF- kappa B, is.4. NIBP has no significant effect on the apoptosis of HCT116 cell line of colon cancer, and NIBP has no significant effect on the expression of c-myc and cyclinD1 in colon cancer HCT116 cell lines.
【学位授予单位】：广西医科大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R735.34

【相似文献】