MicroRNA-155對喉鱗癌細(xì)胞株Hep-2惡性行為的影響
發(fā)布時間：2018-10-15 13:07
【摘要】：目的：探討：microRNA-155(miR-155)過表達(dá)對喉鱗狀細(xì)胞癌細(xì)胞株Hep-2增殖、遷移及凋亡方面的影響及其可能的作用機(jī)制。 方法： 1.將miR-155模擬物(miR-155mimics)瞬時轉(zhuǎn)染至Hep-2細(xì)胞,以無義序列轉(zhuǎn)染組(NC組)為陰性對照,以未經(jīng)任何轉(zhuǎn)染處理空白轉(zhuǎn)染組(Blank組)為空白對照。 2.熒光顯微鏡觀察、測量轉(zhuǎn)染效率。Real-time PCR檢測不同轉(zhuǎn)染組Hep-2細(xì)胞轉(zhuǎn)染后miR-155表達(dá)水平。 3.CCK-8法和平板克隆形成實(shí)驗(yàn)評價不同轉(zhuǎn)染組Hep-2細(xì)胞的增殖能力。 4.流式細(xì)胞術(shù)及Hoechst33342-PI雙染法檢測不同轉(zhuǎn)染組LHep-2細(xì)胞的凋亡情況。 5.劃痕愈合試驗(yàn)檢測不同轉(zhuǎn)染組LHep-2細(xì)胞的遷移能力。 6.統(tǒng)計(jì)學(xué)分析應(yīng)用SPSS13.0統(tǒng)計(jì)軟件,采用x±s描述定量資料,兩組間比較采用t檢驗(yàn),多組間比較采用單因素方差分析(one-way ANOVA), P0.05認(rèn)為差異有統(tǒng)計(jì)學(xué)意義。 結(jié)果： 1.熒光顯微鏡下觀察計(jì)數(shù)顯示,轉(zhuǎn)染24h后,miR-155mimics和無義序列轉(zhuǎn)染入Hep-2細(xì)胞,其轉(zhuǎn)染率均可達(dá)90%。 2. Real-time PCR檢測不同轉(zhuǎn)染組轉(zhuǎn)染24h后miR-155相對表達(dá)水平,miR-155組分別與Blank(?)及NC組相比miR-155表達(dá)明顯增加(P0.05),約16倍。 3.CCK-8法顯示1niR-155組細(xì)胞增殖能力更強(qiáng),并且隨著轉(zhuǎn)染后培養(yǎng)時間的延長,細(xì)胞增殖速度更加明顯(P0.05)。 4.平板克隆形成實(shí)驗(yàn)結(jié)果miR-155(?)且細(xì)胞集落數(shù)(832+49)明顯高于Blank組(496+35)和NC組(488+39),差異具有統(tǒng)計(jì)學(xué)意義(P0.05)。 5.流式細(xì)胞術(shù)結(jié)果miR-155組細(xì)胞總凋亡率(0.139±0.022)明顯低于Blank組(0.139±0.022)和NC組(49.23±11.21),差異具有統(tǒng)計(jì)學(xué)意義(P0.05)。 6. Hoechst33342-PI雙染法結(jié)果示miR-155組細(xì)胞凋亡和壞死比例明顯降低。 7.劃痕愈合試驗(yàn)顯示劃痕48h后,miR-155組細(xì)胞愈合率(83.14±12.73)明顯高于Blank組(43.31+9.69)和NC組(488+39),差異具有統(tǒng)計(jì)學(xué)意義(P0.05)。 結(jié)論：過表達(dá)miR-155可以促進(jìn)喉鱗癌Hep-2細(xì)胞的增殖與遷移愈合能力,這可能與其抗凋亡作用有關(guān)。miR-155可能在喉鱗癌的發(fā)生發(fā)展中通過多種途徑作為癌基因發(fā)揮功能。
[Abstract]:Aim: to investigate the effect of microRNA-155 (miR-155) overexpression on the proliferation, migration and apoptosis of laryngeal squamous cell carcinoma cell line Hep-2 and its possible mechanism. Methods: 1. MiR-155 mimics (miR-155mimics) were transiently transfected into Hep-2 cells. The Hep-2 cells were transfected with nonsense sequence transfection group (NC group) as negative control, and blank transfection group without any transfection treatment (Blank group) as blank control. 2. Fluorescence microscope was used to observe the transfection efficiency. Real-time PCR was used to detect the level of miR-155 expression after transfection of Hep-2 cells in different transfection groups. The proliferation ability of Hep-2 cells in different transfection groups was evaluated by 3.CCK-8 assay and plate clone formation assay. 4. Flow cytometry and Hoechst33342-PI double staining were used to detect the apoptosis of LHep-2 cells in different transfection groups. The migration ability of LHep-2 cells in different transfection groups was detected by scratch healing test. 6. 6. Statistical analysis using SPSS13.0 statistical software, using x 鹵s to describe the quantitative data, two groups of comparison using t test, multi-group comparison using single factor analysis of variance (one-way ANOVA), P0.05 that the difference is statistically significant. Results: 1. After 24 hours of transfection, miR-155mimics and nonsense sequence were transfected into Hep-2 cells, and the transfection efficiency was 90. 2%. Real-time PCR was used to detect the relative expression of miR-155 in different transfection groups 24 hours after transfection, miR-155 group and Blank (?) Compared with NC group, the expression of miR-155 was significantly increased (P0.05), about 16 times. 3.CCK-8 assay showed that the proliferation ability of 1niR-155 group was stronger, and with the extension of culture time after transfection, the cell proliferation rate was more obvious (P0.05). MiR-155 (?) The number of cell colonies (832.49) was significantly higher than that of Blank (496.35) and NC (488.39), and the difference was statistically significant (P0.05). Flow cytometry showed that the total apoptosis rate in miR-155 group (0.139 鹵0.022) was significantly lower than that in Blank group (0.139 鹵0.022) and NC group (49.23 鹵11.21), the difference was statistically significant (P0.05). The results of Hoechst33342-PI double staining showed that the percentage of apoptosis and necrosis was significantly decreased in miR-155 group. Scratch healing test showed that the cell healing rate of miR-155 group (83.14 鹵12.73) was significantly higher than that of Blank group (43.31.9.69) and NC group (48839) after 48 h scratch (P0.05). Conclusion: overexpression of miR-155 can promote the proliferation and migration of Hep-2 cells, which may be related to its anti-apoptosis effect. MiR-155 may play a role as a oncogene in the pathogenesis and development of laryngeal squamous cell carcinoma.
【學(xué)位授予單位】：山西醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2013
【分類號】：R739.65
本文編號：2272645
[Abstract]:Aim: to investigate the effect of microRNA-155 (miR-155) overexpression on the proliferation, migration and apoptosis of laryngeal squamous cell carcinoma cell line Hep-2 and its possible mechanism. Methods: 1. MiR-155 mimics (miR-155mimics) were transiently transfected into Hep-2 cells. The Hep-2 cells were transfected with nonsense sequence transfection group (NC group) as negative control, and blank transfection group without any transfection treatment (Blank group) as blank control. 2. Fluorescence microscope was used to observe the transfection efficiency. Real-time PCR was used to detect the level of miR-155 expression after transfection of Hep-2 cells in different transfection groups. The proliferation ability of Hep-2 cells in different transfection groups was evaluated by 3.CCK-8 assay and plate clone formation assay. 4. Flow cytometry and Hoechst33342-PI double staining were used to detect the apoptosis of LHep-2 cells in different transfection groups. The migration ability of LHep-2 cells in different transfection groups was detected by scratch healing test. 6. 6. Statistical analysis using SPSS13.0 statistical software, using x 鹵s to describe the quantitative data, two groups of comparison using t test, multi-group comparison using single factor analysis of variance (one-way ANOVA), P0.05 that the difference is statistically significant. Results: 1. After 24 hours of transfection, miR-155mimics and nonsense sequence were transfected into Hep-2 cells, and the transfection efficiency was 90. 2%. Real-time PCR was used to detect the relative expression of miR-155 in different transfection groups 24 hours after transfection, miR-155 group and Blank (?) Compared with NC group, the expression of miR-155 was significantly increased (P0.05), about 16 times. 3.CCK-8 assay showed that the proliferation ability of 1niR-155 group was stronger, and with the extension of culture time after transfection, the cell proliferation rate was more obvious (P0.05). MiR-155 (?) The number of cell colonies (832.49) was significantly higher than that of Blank (496.35) and NC (488.39), and the difference was statistically significant (P0.05). Flow cytometry showed that the total apoptosis rate in miR-155 group (0.139 鹵0.022) was significantly lower than that in Blank group (0.139 鹵0.022) and NC group (49.23 鹵11.21), the difference was statistically significant (P0.05). The results of Hoechst33342-PI double staining showed that the percentage of apoptosis and necrosis was significantly decreased in miR-155 group. Scratch healing test showed that the cell healing rate of miR-155 group (83.14 鹵12.73) was significantly higher than that of Blank group (43.31.9.69) and NC group (48839) after 48 h scratch (P0.05). Conclusion: overexpression of miR-155 can promote the proliferation and migration of Hep-2 cells, which may be related to its anti-apoptosis effect. MiR-155 may play a role as a oncogene in the pathogenesis and development of laryngeal squamous cell carcinoma.
【學(xué)位授予單位】：山西醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2013
【分類號】：R739.65
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