【摘要】：目的:探討PLCE1對(duì)食管鱗癌自噬水平的影響;研究下調(diào)食管鱗癌中沉默PLCE1所誘發(fā)的自噬水平對(duì)癌癥細(xì)胞凋亡的影響;初步研究PLCE1調(diào)控食管鱗癌自噬水平的分子機(jī)制。方法:(1)用PLCE1 si RNA轉(zhuǎn)染食管鱗癌細(xì)胞系Eca-109、TE-1,應(yīng)用MDC染色、AO染色及細(xì)胞免疫熒光檢測(cè)方法,檢測(cè)下調(diào)PLCE1表達(dá)對(duì)腫瘤細(xì)胞內(nèi)自噬水平的影響;(2)運(yùn)用自噬特異性抑制劑3-MA或Beclin-1 si RNA,抑制由沉默PLCE1表達(dá)所誘發(fā)的自噬,運(yùn)用MDC染色檢測(cè)食管鱗癌細(xì)胞內(nèi)的自噬水平,通過(guò)MTT方法、流式細(xì)胞儀檢測(cè)凋亡方法檢測(cè)腫瘤細(xì)胞的凋亡和增殖水平受到的影響,應(yīng)用Western Blot方法檢測(cè)自噬相關(guān)分子Beclin-1和LC3的表達(dá)水平,以及凋亡相關(guān)分子cleaved-PARP、Bax、Bcl-2、cleaved-caspase-3的蛋白表達(dá)水平;(3)根據(jù)課題組前期研究結(jié)果,運(yùn)用Western Blot實(shí)驗(yàn)檢測(cè)mi R-106b-5p的宿主基因MCM7的表達(dá),結(jié)合Target Scan,mi Randa,DIANAm T,mi RDB,mi RWalk等靶基因預(yù)測(cè)軟件分析,運(yùn)用熒光素酶報(bào)告試驗(yàn)檢測(cè)mi R-106b-5p與自噬相關(guān)基因Beclin-1之間的靶向關(guān)系;(4)在食管鱗癌細(xì)胞ECA109內(nèi)共轉(zhuǎn)染mi R-106b-5p模擬物與PLCE1 si RNA,通過(guò)MDC染色和AO染色,檢測(cè)腫瘤細(xì)胞內(nèi)的自噬水平,通過(guò)Western Blot方法檢測(cè)食管癌細(xì)胞內(nèi)自噬基因Beclin-1的表達(dá)水平。結(jié)果:(1)使用PLCE1 si RNA轉(zhuǎn)染食管鱗癌細(xì)胞系,MDC染色和AO染色的結(jié)果顯示,沉默PLCE1的表達(dá)后,細(xì)胞內(nèi)的自噬囊泡數(shù)量明顯增多,細(xì)胞免疫熒光檢測(cè)提示LC3蛋白的表達(dá)水平明顯升高;(2)MDC染色結(jié)果顯示沉默PLCE1后所誘發(fā)的自噬上調(diào)可被3-MA抑制;MTT實(shí)驗(yàn)結(jié)果顯示,PLCE1 si RNA和3-MA共同處理的ECA109細(xì)胞系和TE-1細(xì)胞系的增殖水平與PLCE1 si RNA單獨(dú)處理組相比有明顯升高,Beclin-1 si RNA與PLCE1 si RNA共處理組的細(xì)胞增殖水平與對(duì)照組及PLCE1 si RNA處理組相比有明顯升高;流式細(xì)胞術(shù)結(jié)果顯示,PLCE1 si RNA處理組細(xì)胞的凋亡水平,在轉(zhuǎn)染3-MA或Beclin-1 si RNA后明顯降低;Western Blot結(jié)果顯示,下調(diào)PLCE1蛋白表達(dá)后,Beclin-1蛋白及LC3蛋白的表達(dá)水平明顯升高,3-MA或Beclin-1 si RNA與PLCE1 si RNA共處理組中,cleaved-caspase-3、Bax及cleaved-PARP蛋白的表達(dá)水平與PLCE1 si RNA單獨(dú)處理組相比,表達(dá)水平有明顯降低,而B(niǎo)cl-2蛋白的表達(dá)水平升高;(3)Western Blot實(shí)驗(yàn)結(jié)果顯示,mi R-106b-5p的宿主基因MCM7在沉默PLCE1后表達(dá)明顯降低;Target Scan,mi Randa,DIANAm T,mi RDB,mi RWalk等靶基因預(yù)測(cè)軟件分析結(jié)果顯示,mi R-106b-5p可能與自噬基因Beclin-1存在靶向關(guān)系,熒光素酶報(bào)告試驗(yàn)結(jié)果顯示,mi R-106b-5p通過(guò)靶向結(jié)合3’UTR調(diào)控下游基因Beclin-1;Western Blot結(jié)果顯示,與對(duì)照組相比,mi R-106b-5p模擬物組的Beclin-1蛋白表達(dá)量明顯降低;(4)在Eca-109細(xì)胞系和TE-1細(xì)胞系中轉(zhuǎn)染mi R-106b-5p模擬物及PLCE1 si RNA,通過(guò)MDC染色和AO染色后發(fā)現(xiàn),共轉(zhuǎn)染mi R-106b-5p模擬物及PLCE1 si RNA后,腫瘤細(xì)胞內(nèi)的自噬囊泡數(shù)量比PLCE1 si RNA單獨(dú)處理組也明顯降低,Western Blot結(jié)果顯示,共轉(zhuǎn)染mi RNA模擬物及PLCE1 si RNA后,Beclin-1的表達(dá)水平與單獨(dú)轉(zhuǎn)染PLCE1 si RNA的細(xì)胞組相比明顯降低。結(jié)論:(1)PLCE1的過(guò)表達(dá)可以下調(diào)食管鱗癌細(xì)胞內(nèi)的自噬和凋亡水平,從而促進(jìn)食管細(xì)胞的惡性轉(zhuǎn)化;(2)PLCE1對(duì)調(diào)控食管鱗癌細(xì)胞自噬的分子機(jī)制可能是通過(guò)上調(diào)mi R-106b-5p,靶向抑制Beclin-1的表達(dá),進(jìn)而下調(diào)腫瘤細(xì)胞內(nèi)的自噬過(guò)程;(3)本次研究提示通過(guò)靶向干預(yù)PLCE1的表達(dá),誘導(dǎo)食管鱗癌細(xì)胞的自噬,能夠成為食管癌治療的新策略。
[Abstract]:Objective: To investigate the effect of PLCE1 on the autophagy level of esophageal squamous cell carcinoma, to study the effect of autophagy induced by PLCE1 in esophageal squamous cell carcinoma and to investigate the molecular mechanism of autophagy by PLCE1 to regulate the autophagy level of esophageal squamous cell carcinoma. Methods: (1) transfection of Eca-109, TE-1, MDC staining, AO staining with PLCE1 Si RNA The effect of down regulated PLCE1 expression on the autophagy level in tumor cells was detected by color and cell immunofluorescence. (2) autophagy, 3-MA or Beclin-1 Si RNA, was used to inhibit autophagy induced by silent PLCE1 expression. Autophagy was detected by MDC staining in esophageal squamous cell carcinoma cells, using MTT method and flow cytometry to detect the loss of autophagy. The apoptosis and proliferation level of tumor cells were detected by the method of death. Western Blot method was used to detect the expression level of autophagy related molecules Beclin-1 and LC3, as well as the protein expression level of cleaved-PARP, Bax, Bcl-2, cleaved-caspase-3 of apoptosis related molecules. (3) according to the results of earlier study of the group, Western Blot test was used to detect M The expression of the host gene MCM7 of I R-106b-5p, combined with Target Scan, MI Randa, DIANAm T, MI RDB, MI targets and other target gene prediction software analysis, and the use of luciferase report test to detect the target relationship between the autophagy related genes and those of the autophagy related genes. (4) the simulants were co transfected in the squamous carcinoma cells of the esophagus. The autophagy level in tumor cells was detected by MDC staining and AO staining. The expression level of autophagic gene Beclin-1 in esophageal cancer cells was detected by Western Blot method. Results: (1) transfection of esophageal squamous cell carcinoma cell lines using PLCE1 Si RNA. The results of MDC staining and AO staining showed that the number of autophagic vesicles in the cells was clear after the expression of silent PLCE1. The expression of LC3 protein was significantly increased by cell immunofluorescence, and (2) MDC staining showed that the up regulation of autophagy induced by silent PLCE1 could be suppressed by 3-MA. The MTT experiment showed that the proliferation of ECA109 cell lines and TE-1 cell lines, which were treated by PLCE1 Si RNA and 3-MA, was compared with that of PLCE1 alone. The cell proliferation level of Beclin-1 Si RNA and PLCE1 Si RNA co processing group was significantly higher than that of the control group and PLCE1 Si RNA treatment group. The results of flow cytometry showed that the apoptosis level of PLCE1 Si RNA treatment group was significantly lower than that of the PLCE1 Si Group. The expression level of Beclin-1 protein and LC3 protein increased obviously. The expression level of cleaved-caspase-3, Bax and cleaved-PARP protein in 3-MA or Beclin-1 Si RNA and PLCE1 Si RNA co processing group was significantly lower than that of the single treatment group. The expression of the host gene MCM7 of MI R-106b-5p decreased markedly after the silence of PLCE1, and Target Scan, MI Randa, DIANAm T, MI RDB, and other target gene prediction software analysis showed that there might be a targeting relationship with autophagic gene. Control downstream gene Beclin-1; Western Blot results showed that the expression of Beclin-1 protein in the MI R-106b-5p simulation group decreased significantly compared with the control group; (4) the MI R-106b-5p analog and PLCE1 Si were transfected in the Eca-109 cell line and TE-1 cell line. The number of autophagic vesicles in the tumor cells was significantly lower than that in the PLCE1 Si RNA treatment group. The Western Blot results showed that the expression level of the Beclin-1 was significantly lower than that of the MI RNA mimics and PLCE1 Si RNA. Conclusion: (1) the overexpression of the esophageal squamous cell carcinoma cells can be downregulated in the cells of the squamous cell carcinoma of the esophagus. The level of macrophage and apoptosis promotes malignant transformation of esophageal cells; (2) the molecular mechanism of PLCE1 to regulate autophagy in esophageal squamous cell carcinoma may be by up regulation of MI R-106b-5p, targeting the expression of Beclin-1 and down regulation of autophagy in the tumor cells; (3) this study suggested that the expression of PLCE1 was targeted by target intervention to induce the squamous cell carcinoma of the esophagus. Cell autophagy can become a new strategy for the treatment of esophageal cancer.
【學(xué)位授予單位】：石河子大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2016
【分類(lèi)號(hào)】：R735.1

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