【摘要】：目的：探討靶向HMGB1的shRNA對HMGB1表達改變的影響及其對子宮內(nèi)膜癌HEC-1A細胞侵襲與遷移能力改變的影響。 方法：運用RNA干擾技術(shù),構(gòu)建針對HMGB1基因的短發(fā)夾RNA (pshRNA-1/HMGB1、pshRNA-2/HMGB1、pshRNA-3/HMGB1),同時設(shè)置轉(zhuǎn)染空白質(zhì)粒的陰性對照組(HMGB1/p-NC)和脂質(zhì)體轉(zhuǎn)染組(Lipo組),用脂質(zhì)體法轉(zhuǎn)染人子宮內(nèi)膜癌細胞(HEC-1A),利用反轉(zhuǎn)錄-聚合酶鏈反應(yīng)(RT-PCR)和免疫印跡法(Western-Blot)檢測轉(zhuǎn)染后48h各組細胞HMGB1在mRNA水平和蛋白水平的表達,Transwell小室法觀察轉(zhuǎn)染HMGB1shRNA后HEC-1A細胞的侵襲情況,細胞劃痕實驗觀察轉(zhuǎn)染HMGB1shRNA后HEC-1A細胞的遷移情況。 結(jié)果：1、RT-PCR結(jié)果顯示：針對HMGB1構(gòu)建的三組重組質(zhì)粒HMGB1-pshRNAs(1、2、3)轉(zhuǎn)染后,HMGB1mRNA相對表達水平分別是0.192±0.006,0.055±0.002和0.123±0.004,均較Lipo組(0.268±0.008)和陰性對照組(0.270±0.004)明顯減少(P0.05)；與陰性對照組比較,分別減少了28.9%,79.6%和54.4%。 2、Western-blot結(jié)果顯示：針對HMGB1構(gòu)建的三組重組質(zhì)粒HMGB1-pshRNAs(1、2、3)轉(zhuǎn)染后,HMGB1蛋白相對表達水平分別是0.259±0.013,0.032±0.002和0.104±0.007,均較Lipo組(0.347±0.007)和陰性對照組組(0.349±0.007)明顯減少(P0.05)；與陰性對照組比較,分別減少了25.8%,90.8%和70.2%。 3、Transwell小室法檢測細胞的侵襲能力顯示：轉(zhuǎn)染后48h,pshRNA2轉(zhuǎn)染組穿膜細胞為(20±1)個,陰性對照組和Lipo組穿膜細胞數(shù)分別為(36±1)和(36±2)個。與陰性對照組比較,pshRNA2轉(zhuǎn)染組穿膜細胞數(shù)明顯減少(P0.05),差異有統(tǒng)計學(xué)意義；而Lipo組和HMGB1/p-NC組穿膜細胞數(shù)差異無統(tǒng)計學(xué)意義(P0.05) 4、細胞劃痕實驗檢測細胞遷移能力顯示：劃痕后培養(yǎng)48h,pshRNA2轉(zhuǎn)染組細胞的遷移率為25.75%±1.70%,脂質(zhì)體轉(zhuǎn)染組細胞的遷移率為65.25%±1.70%,陰性對照組細胞的遷移率為64.75%±4.57%。與陰性對照組相比,pshRNA2轉(zhuǎn)染組細胞遷移率明顯降低(P0.05)；而脂質(zhì)體轉(zhuǎn)染組和陰性對照組的細胞遷移率比較,差異無統(tǒng)計學(xué)意義(P0.05)。 結(jié)論：1、靶向干擾HMGB1的三組重組質(zhì)粒在人子宮內(nèi)膜癌細胞中均能有效下調(diào)HMGB1在mRNA和蛋白水平的表達,其中以pshRNA-2/HMGB1沉默效果最好。 2、靶向干擾HMGB1的shRNA2轉(zhuǎn)染子宮內(nèi)膜癌細胞HEC-1A后細胞的侵襲與遷移能力明顯下降,提示HMGB1可能參與子宮內(nèi)膜癌侵襲與遷移的發(fā)展過程。
[Abstract]:Aim: to investigate the effect of shRNA targeting HMGB1 on the expression of HMGB1 and its effect on invasion and migration of endometrial carcinoma HEC-1A cells. Methods: the short hairpin RNA (pshRNA-1/HMGB1,pshRNA-2/HMGB1,pshRNA-3/HMGB1) targeting HMGB1 gene was constructed by RNA interference technique. At the same time, the negative control group (HMGB1/p-NC) and the liposome transfected group (Lipo group) were transfected into human endometrial carcinoma cell line (HEC-1A) by lipofectamine method. Reverse transcription-polymerase chain reaction (RT-PCR) and immunoblot assay (Western-Blot) were used to detect the expression of HMGB1 at mRNA level and protein level at 48 h after transfection, and the invasion of HEC-1A cells was observed by Transwell chamber assay. Cell scratch assay was used to observe the migration of HEC-1A cells transfected with HMGB1shRNA. Results: 1. The results of RT-PCR showed that the relative expression level of HMGB1mRNA was 0.192 鹵0.006,0.055 鹵0.002 and 0.123 鹵0.004respectively after transfection with recombinant plasmid HMGB1-pshRNAs (1,2, and 3) constructed by HMGB1, and the expression level of HMGB1mRNA was 0.192 鹵0.006, 0.055 鹵0.002 and 0.123 鹵0.004respectively. Compared with Lipo group (0.268 鹵0.008) and negative control group (0.270 鹵0.004), both of them were significantly decreased (P0.05). Compared with the negative control group, it decreased by 28.9%, 79.6% and 54.4% respectively. 2. Western blot analysis showed that the relative expression level of HMGB1 protein was 0.259 鹵0.013,0.032 鹵0.002 and 0.104 鹵0.007respectively after transfection of three recombinant plasmids HMGB1-pshRNAs (1, 2, 3) constructed by HMGB1, and the expression level of HMGB1 protein was 0.259 鹵0.013, 0.032 鹵0.002 and 0.104 鹵0.007, respectively. Compared with Lipo group (0.347 鹵0.007) and negative control group (0.349 鹵0.007), it was significantly decreased (P0.05). Compared with the negative control group, it decreased by 25.8%, 90.8% and 70.2% respectively. 3. The invasiveness of pshRNA2 transfected group was (20 鹵1), and that of negative control group and Lipo group was (36 鹵1) and (36 鹵2), respectively. Compared with the negative control group, the number of transmembrane cells in pshRNA2 transfected group decreased significantly (P0.05), the difference was statistically significant. However, there was no significant difference in the number of transmembrane cells between Lipo group and HMGB1/p-NC group (P0.05) 4. The ability of cell migration was detected by cell scratch assay. After 48 hours of culturing, the migration rate of pshRNA 2 transfected group was 25.75% 鹵1.70%, and that of pshRNA2 transfected group was 25.75% 鹵1.70%. The cell migration rate was 65.25% 鹵1.70% in liposome transfected group and 64.75% 鹵4.57% in negative control group. Compared with the negative control group, the cell migration rate of pshRNA2 transfection group was significantly lower (P0.05), but there was no significant difference between liposome transfection group and negative control group (P0.05). Conclusion: 1. Three recombinant plasmids targeting HMGB1 can effectively down-regulate the expression of HMGB1 at mRNA and protein levels in human endometrial carcinoma cells, and the silencing effect of pshRNA-2/HMGB1 is the best. 2. The invasion and migration ability of endometrial carcinoma cell line HEC-1A transfected with shRNA2 targeting HMGB1 decreased significantly, suggesting that HMGB1 may be involved in the process of invasion and migration of endometrial carcinoma.
【學(xué)位授予單位】：中南大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2014
【分類號】：R737.33

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