miR-365靶向下调BAX抑制凋亡促进皮肤鳞状细胞癌发生发展的初步研究

发布时间：2018-07-05 04:52

本文选题：miR-365 + 皮肤鳞状细胞癌　；参考：《南方医科大学》2017年硕士论文

【摘要】：研究背景非黑色素瘤皮肤癌(Non-melanoma skin cancer,NMSC)是人类常见的恶性肿瘤之一,包括基底细胞癌(Basal cell carcinoma,BBC)和鳞状细胞癌(squamous cell carcinoma of skin,SCC)等。SCC是源于上皮组织中角质形成细胞的恶性肿瘤,包括宫颈癌、食道癌和皮肤鳞状细胞癌等(squamous cell carcinoma of skin,CSCC)。CSCC是发生在表皮(或粘膜)上皮细胞的一种恶性肿瘤,它的发病率低于基底细胞癌,但是转移率和死亡率较高,男性发病多于女性,好发于脸部、手部和前臂[1,21。已有的报道microRNAs参与CSCC发生发展。microRNA(miRNA)是一类由内源基因编码的长度约为22个核苷酸的非编码单链RNA小分子,microRNA与靶mRNA的3TTR部分序列特异性碱基配对,会抑制靶基因的转录翻译。不同microRNA在不同的癌症中表达不同,同一个microRNA在不同的癌症表达也存在着差异,例如miR-365在CSCC及原发性乳腺癌中表达上调,而miR-365在胃癌、非小细胞肺癌、肝癌及黑色素细胞瘤却表达下调。已知多种microRNA可调控BAX表达。BAX属于BCL-2家族中的促凋亡蛋白,BAX蛋白的过表达、低表达和活化都会导致肿瘤生物学功能的改变。本课题的研究目的是探讨miR-365在CSCC中的生物学作用,进一步明确miR-365与BAX分子之间的调控机制,为CSCC的临床治疗提供理论依据和新的治疗策略。实验方法①采用qPCR的方法检测人永生化表皮细胞与CSCC细胞系、人正常表皮组织与人皮肤鳞癌组织以及裸鼠移植瘤中miR-365和BAX的表达,采用Western Blot检测BAX在蛋白水平上的表达;②免疫组化检测人正常表皮组织与人皮肤鳞癌组织以及裸鼠移植瘤中的BAX表达;③双荧光素酶实验等检测miR-365与BAX之间的靶向调控关系;④使用CCK8法、细胞划痕实验、细胞迁移实验和侵袭实验检测A431细胞转染NC、siBAX后,细胞的增殖能力、修复能力、迁移运动和侵袭能力的变化;⑤用流式细胞仪检测A431细胞转染NC、siBAX后,对细胞凋亡的影响;⑥Tunnel实验检测皮下移植瘤细胞中的凋亡情况。结果①与对照相比,miR-365在CSCC细胞系中高表达(P0.001),BAX在mRNA水平以及蛋白水平低表达(P0.001)。②CSCC组织中,与正常表皮组织相比,BAX在mRNA水平以及蛋白水平低表达(p0.01)。免疫组化中BAX的阳性率低于正常表皮组织(P0.01)。③双荧光素酶实验表明,BAX.3'UTR-WT组的荧光强度明显低于BAX 3'UTR-Mutant组(P0.01)。④分别将miR-365 minic和antagomiR-365转染至A431细胞。miR-365组中miR-365表达上调,BAX mRNA水平及蛋白水平表达下调(P0.001)相反,antagomiR-365组中miR-365表达下调,BAX mRNA水平及蛋白水平表达上调(P0.001)。⑤转染NC、siBAX至A431细胞中,与对照NC相比,siBAX组细胞的增殖能力(P0.001)、修复能力(P0.05)、迁移(P0.001)及侵袭(P0.001)能力均增强,细胞凋亡减少(P0.05)。⑥裸鼠皮下成瘤初步实验揭示,与NC组相比,siBAX处理组的生长速度更快(P0.05),肿瘤体积更大;种植瘤中BAX在mRNA水平和蛋白表达水平均低于对照组;免疫组化中,用siBAX处理组的BAX阳性率低于对照组(P0.05);T nnel实验中,siBAX在肿瘤的凋亡率低于对照组。结论①BAX在人CSCC细胞系以及肿瘤组织中的表达是下调的。②miR-365通过结合BAX的3'UTR区域,靶向下调BAX的表达。③BAX表达的降低使CSCC细胞的增殖、修复以及迁移和侵袭的能力增强,细胞凋亡减少;④体内实验证实BAX表达下调能够抑制皮肤鳞状细胞癌的凋亡并促进CSCC的进展。
[Abstract]:Background non melanoma skin cancer (Non-melanoma skin cancer, NMSC) is one of the most common human malignant tumors, including basal cell carcinoma (Basal cell carcinoma, BBC) and squamous cell carcinoma (squamous cell carcinoma of), which are malignant tumors derived from keratinocytes in epithelial tissue, including cervical cancer, esophagus cancer and carcinoma of the esophagus. Squamous cell carcinoma of skin (CSCC).CSCC is a malignant tumor occurring in epidermis (or mucous) epithelial cells. Its incidence is lower than basal cell carcinoma, but the metastasis rate and mortality rate are higher, male incidence is more than women, good hair is in the face, and [1,21. in hand and forearm [1,21. has been reported to participate in CSCC. Occurrence and development.MicroRNA (miRNA) is a class of non coded single strand RNA molecules encoded by endogenous genes about 22 nucleotides. MicroRNA is paired with the specific base of 3TTR part of the target mRNA, which inhibits the transcriptional translation of the target gene. Different microRNA can be expressed differently in different cancers and the same microRNA in different cancer tables. There are also differences, such as the up-regulated expression of miR-365 in CSCC and primary breast cancer, while miR-365 is down regulated in gastric cancer, non small cell lung cancer, liver cancer and melanocytoma. A variety of microRNA can regulate the BAX expression of.BAX as the apoptotic protein in the BCL-2 family, the overexpression of BAX protein, the low expression and activation of the protein can lead to swelling. The purpose of this study is to explore the biological function of miR-365 in CSCC, to further clarify the regulatory mechanism between miR-365 and BAX, and to provide theoretical basis and new therapeutic strategy for the clinical treatment of CSCC. The experimental method is to detect human immortalized epidermal cells and CSCC cell lines by qPCR. The expression of miR-365 and BAX in normal epidermis and human skin squamous cell carcinoma tissue and nude mice, Western Blot was used to detect the expression of BAX at the protein level; (2) immunohistochemistry was used to detect the expression of BAX in human normal epidermis and human skin squamous cell carcinoma tissue and nude mice. (3) double luciferase test was used to detect miR-365 and BAX The relationship between target regulation and regulation; (4) using CCK8 method, cell scratch test, cell migration experiment and invasion test to detect A431 cells transfected NC, siBAX, cell proliferation, repair ability, migration movement and invasion ability; (5) the effect of A431 cells transferred to NC, siBAX, and apoptosis by flow cytometry; (6) Tunnel experimental detection Compared with the control, miR-365 expressed high expression in CSCC cell line (P0.001), BAX at mRNA level and low protein level (P0.001). In CSCC tissue, BAX was lower at mRNA level and low protein expression (P0.01) than normal epidermal tissue (P0.01). The positive rate of BAX in immunohistochemistry was lower than normal. Epidermal tissue (P0.01). (3) double luciferase test showed that the fluorescence intensity of BAX.3'UTR-WT group was significantly lower than that of BAX 3'UTR-Mutant group (P0.01). Fourth, miR-365 minic and antagomiR-365 were transfected to A431 cell.MiR-365 group, and miR-365 expression was up-regulated. Down regulation, BAX mRNA level and protein level expression up-regulated (P0.001). 5. Transfection of NC, siBAX to A431 cells, compared with the control NC, the proliferation ability (P0.001), repair capacity (P0.05), migration (P0.001) and invasion (P0.001) ability of siBAX group cells were enhanced and apoptosis decreased. The growth rate of the iBAX treatment group was faster (P0.05) and the tumor volume was larger. The level of mRNA and the protein expression level of BAX in the implant were lower than that of the control group; the positive rate of BAX in the siBAX treatment group was lower than that of the control group (P0.05), and the apoptosis rate of siBAX in the T nnel experiment was lower than that of the control group. Conclusion: (1) BAX in the human CSCC cell line and the swelling. The expression in the tumor tissue is down-regulated. (2) miR-365 reduces the expression of BAX by binding to the 3'UTR region of BAX. (3) the decrease of BAX expression makes the proliferation, repair and migration and invasion of CSCC cells enhanced, and the apoptosis decreases. (4) in vivo experiments confirmed that the down regulation of BAX expression can inhibit the apoptosis of squamous cell carcinoma of the skin and promote CSCC Progress.
【学位授予单位】：南方医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R739.5

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