發(fā)布時間：2018-07-05 04:52

  本文選題：miR-365 + 皮膚鱗狀細胞癌　； 參考：《南方醫(yī)科大學》2017年碩士論文

【摘要】：研究背景非黑色素瘤皮膚癌(Non-melanoma skin cancer,NMSC)是人類常見的惡性腫瘤之一,包括基底細胞癌(Basal cell carcinoma,BBC)和鱗狀細胞癌(squamous cell carcinoma of skin,SCC)等。SCC是源于上皮組織中角質(zhì)形成細胞的惡性腫瘤,包括宮頸癌、食道癌和皮膚鱗狀細胞癌等(squamous cell carcinoma of skin,CSCC)。CSCC是發(fā)生在表皮(或粘膜)上皮細胞的一種惡性腫瘤,它的發(fā)病率低于基底細胞癌,但是轉(zhuǎn)移率和死亡率較高,男性發(fā)病多于女性,好發(fā)于臉部、手部和前臂[1,21。已有的報道m(xù)icroRNAs參與CSCC發(fā)生發(fā)展。microRNA(miRNA)是一類由內(nèi)源基因編碼的長度約為22個核苷酸的非編碼單鏈RNA小分子,microRNA與靶mRNA的3TTR部分序列特異性堿基配對,會抑制靶基因的轉(zhuǎn)錄翻譯。不同microRNA在不同的癌癥中表達不同,同一個microRNA在不同的癌癥表達也存在著差異,例如miR-365在CSCC及原發(fā)性乳腺癌中表達上調(diào),而miR-365在胃癌、非小細胞肺癌、肝癌及黑色素細胞瘤卻表達下調(diào)。已知多種microRNA可調(diào)控BAX表達。BAX屬于BCL-2家族中的促凋亡蛋白,BAX蛋白的過表達、低表達和活化都會導致腫瘤生物學功能的改變。本課題的研究目的是探討miR-365在CSCC中的生物學作用,進一步明確miR-365與BAX分子之間的調(diào)控機制,為CSCC的臨床治療提供理論依據(jù)和新的治療策略。實驗方法①采用qPCR的方法檢測人永生化表皮細胞與CSCC細胞系、人正常表皮組織與人皮膚鱗癌組織以及裸鼠移植瘤中miR-365和BAX的表達,采用Western Blot檢測BAX在蛋白水平上的表達;②免疫組化檢測人正常表皮組織與人皮膚鱗癌組織以及裸鼠移植瘤中的BAX表達;③雙熒光素酶實驗等檢測miR-365與BAX之間的靶向調(diào)控關系;④使用CCK8法、細胞劃痕實驗、細胞遷移實驗和侵襲實驗檢測A431細胞轉(zhuǎn)染NC、siBAX后,細胞的增殖能力、修復能力、遷移運動和侵襲能力的變化;⑤用流式細胞儀檢測A431細胞轉(zhuǎn)染NC、siBAX后,對細胞凋亡的影響;⑥Tunnel實驗檢測皮下移植瘤細胞中的凋亡情況。結(jié)果①與對照相比,miR-365在CSCC細胞系中高表達(P0.001),BAX在mRNA水平以及蛋白水平低表達(P0.001)。②CSCC組織中,與正常表皮組織相比,BAX在mRNA水平以及蛋白水平低表達(p0.01)。免疫組化中BAX的陽性率低于正常表皮組織(P0.01)。③雙熒光素酶實驗表明,BAX.3'UTR-WT組的熒光強度明顯低于BAX 3'UTR-Mutant組(P0.01)。④分別將miR-365 minic和antagomiR-365轉(zhuǎn)染至A431細胞。miR-365組中miR-365表達上調(diào),BAX mRNA水平及蛋白水平表達下調(diào)(P0.001)相反,antagomiR-365組中miR-365表達下調(diào),BAX mRNA水平及蛋白水平表達上調(diào)(P0.001)。⑤轉(zhuǎn)染NC、siBAX至A431細胞中,與對照NC相比,siBAX組細胞的增殖能力(P0.001)、修復能力(P0.05)、遷移(P0.001)及侵襲(P0.001)能力均增強,細胞凋亡減少(P0.05)。⑥裸鼠皮下成瘤初步實驗揭示,與NC組相比,siBAX處理組的生長速度更快(P0.05),腫瘤體積更大;種植瘤中BAX在mRNA水平和蛋白表達水平均低于對照組;免疫組化中,用siBAX處理組的BAX陽性率低于對照組(P0.05);T nnel實驗中,siBAX在腫瘤的凋亡率低于對照組。結(jié)論①BAX在人CSCC細胞系以及腫瘤組織中的表達是下調(diào)的。②miR-365通過結(jié)合BAX的3'UTR區(qū)域,靶向下調(diào)BAX的表達。③BAX表達的降低使CSCC細胞的增殖、修復以及遷移和侵襲的能力增強,細胞凋亡減少;④體內(nèi)實驗證實BAX表達下調(diào)能夠抑制皮膚鱗狀細胞癌的凋亡并促進CSCC的進展。
[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.
【學位授予單位】：南方醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R739.5

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相關期刊論文 前1條

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本文編號：2098997