miR-145在多发性骨髓瘤细胞中的功能及其机制研究

发布时间：2018-05-08 03:42

本文选题：多发性骨髓瘤 + miR-145　；参考：《吉林大学》2015年博士论文

【摘要】：目的：探讨miRNA-145在体内外对多发性骨髓瘤（MM）的增殖、凋亡、侵袭、迁移功能的影响，并研究其可能的作用机制，为多发性骨髓瘤的早期诊断和临床治疗提供新的可能靶点和理论依据。方法： RT-qPCR检测MM细胞株H929、MM1S及RPMI-8226中miR-145的表达，得到初步结果后，检测MM患者及正常志愿者血浆miR-145的表达；RT-qPCR法对miR-145模拟物瞬时转染至MM细胞H929的转染效果进行验证；MTT法和BrdU掺入法检测过表达miR-145组细胞增殖情况及细胞克隆形成能力；流式细胞术和Western blot法分别检测过表达miR-145的H929细胞周期分布及细胞周期相关蛋白cyclin D1和p21蛋白的表达；TUNEL法、ELISA法和Western blot法检测miR-145转染48h后H929细胞内Caspase活性，以及重要抗凋亡蛋白Bcl-2和Survivin的表达；Transwell侵袭及迁移实验检测miR-145对H929细胞侵袭及迁移的影响；ELISA法检测miR-145对细胞上清液VEGF的分泌；Western blot法检测miR-145对金属蛋白酶家族成员MMP-2及MMP-9表达的影响，进一步阐明miR-145影响细胞迁移和侵袭的作用机制；Western blot法检测过表达miR-145组细胞内PI3K/AKT信号通路以及MAPK信号通路相关蛋白的表达活性，，进一步探讨miR-145影响MM细胞增殖、凋亡的作用机制；体内实验中，检测小鼠皮下肿瘤的体积和重量，RT-qPCR法检测miR-145的表达，ELISA法检测小鼠肿瘤VEGF浓度，TUNEL法分析小鼠体内MM细胞的凋亡。结果： MM细胞株H929, MM1S及RPMI-8226中miR-145的表达水平均低于正常志愿者对照组，临床收集的25例MM患者血浆中miR-145的表达水平显著低于正常志愿者对照组，即miR-145在MM细胞系及MM患者的血浆中普遍低表达；RT-qPCR法对转染效果进行验证；MTT法检测细胞活力，与阴性对照组比较，miR-145过表达组OD570显著下降，并且在转染后第2天其活力便显著低于阴性对照组，第4天和第5天差异更加显著（p＜0.01）；BrdU掺入法和细胞克隆形成实验的结果与MTT法一致，即miR-145过表达组MM细胞细胞克隆形成能力显著降低；流式细胞术对转染后48h的细胞进行周期分析，结果发现miR-145过表达组细胞分布在G1期的细胞群体显著增多，而S期细胞百分比大大下降，G2/M期细胞比例无明显变化，说明miR-145主要使细胞周期阻滞在G1期进而影响细胞的增殖；进一步研究细胞周期阻滞的作用机制，以Western blot法检测细胞周期相关蛋白cyclin D1和p21的表达，结果发现，过表达miR-145组细胞内cyclin D1水平显著降低，p21蛋白表达水平显著上升；TUNEL法检测发现，miR-145过表达组细胞凋亡显著增加；ELISA法发现，miR-145过表达组Caspase的活性显著升高，与此同时，Western blot法检测到miR-145过表达的H929细胞组Bcl-2蛋白和Survivin蛋白的表达水平显著下降，说明miR-145对细胞凋亡的影响机制可能与Bcl-2蛋白和Survivin蛋白的表达下调相关；Transwell侵袭实验和迁移实验发现，miR-145过表达组中穿过膜的细胞数与迁移出小室的细胞数相比于阴性对照组都显著降低；ELISA法发现miR-145过表达组能显著抑制H929细胞中VEGF的分泌；Western blot法发现miR-145过表达组中MMP-2及MMP-9蛋白的表达显著降低；Western blot法发现过表达miR-145组磷酸化PI3K和AKT蛋白表达下降，PI3K和AKT总蛋白含量不变；小鼠体内实验进一步验证，miR-145过表达组肿瘤体积减小，重量减轻，VEGF浓度显著降低、凋亡细胞数目显著增加。结论： miR-145是多发性骨髓瘤的抑癌基因，miR-145主要通过抑制PI3K/AKT信号通路的活性抑制MM细胞增殖、促进其凋亡，并利用MM小鼠进一步证实了miR-145对MM肿瘤增殖的负性调节作用；上调miR-145表达可抑制MM细胞迁移和侵袭，机制与抑制VEGF的分泌及MMP-2、MMP-9表达相关；因此，miR-145很可能成为MM治疗过程中的一个重要的潜在靶点，深入研究miR-145与MM的关系具有重要的理论和临床价值。
[Abstract]:Purpose :

To investigate the effect of miRNA - 145 on the proliferation , apoptosis , invasion and migration of multiple myeloma ( MM ) in vitro and to study the possible mechanism of miRNA - 145 , which provides a new possible target and theoretical basis for early diagnosis and clinical treatment of multiple myeloma .

Method :

The expression of miR - 145 in MM cell lines H929 , MM1S and RPMI - 8226 was detected by RT - qPCR . After preliminary results , the expression of miR - 145 was detected in MM patients and normal volunteers .
The expression of miR - 145 was transiently transfected into MM cell H929 by RT - qPCR .
The proliferation of miR - 145 cells and the ability of cell clone formation were detected by the MTT method and the incorporation assay .
The expression of cyclin D1 and p21 protein was detected by flow cytometry and Western blot .
TUNEL , ELISA and Western blot were used to detect the Caspase activity in H929 cells and the expression of Bcl - 2 and Survivin in H929 cells after 48h .
Effect of miR - 145 on invasion and migration of H929 cells by Transwell invasion and migration experiments
The secretion of VEGF by miR - 145 was detected by ELISA .
The effects of miR - 145 on the expression of MMP - 2 and MMP - 9 in the family of MMP - 2 and MMP - 9 were detected by Western blot .
Western blot was used to detect the signaling pathway and MAPK signal pathway related protein expression in miR - 145 group , and further study the mechanism of miR - 145 on the proliferation and apoptosis of MM cells .
In vivo experiments , the expression of miR - 145 was detected by RT - qPCR and the expression of miR - 145 was detected by RT - qPCR . The apoptosis of MM cells in mice was analyzed by TUNEL method .

Results :

The expression level of miR - 145 in MM cell line H929 , MM1S and RPMI - 8226 was lower than that of normal volunteers . The expression level of miR - 145 in 25 MM patients with MM was significantly lower than that of normal volunteers , i.e . , miR - 145 was generally low in MM cell line and MM patients .
The transfection efficiency was verified by RT - qPCR .
Compared with the negative control group , the activity of the miR - 145 overexpression group was significantly lower than that of the negative control group , and the difference between the 4th and 5th day was more significant ( p < 0.01 ) .
The results of the experiment were consistent with the MTT assay , that is , the ability to clone the MM cells of the miR - 145 overexpression group was significantly reduced .
The results showed that the cell population of miR - 145 was significantly increased in G1 phase , while the percentage of S phase cells decreased greatly , and the percentage of G2 / M phase cells decreased significantly , suggesting that miR - 145 mainly caused the cell cycle arrest to affect the proliferation of cells in G1 phase .
The mechanism of cell cycle arrest was further studied . The expression of cyclin D1 and p21 was detected by Western blot . The results showed that the level of cyclin D1 was significantly decreased in the expression of miR - 145 , and the level of p21 protein was significantly increased .
TUNEL assay showed that the apoptosis of miR - 145 overexpression group was significantly increased .
The expression level of Bcl - 2 protein and Survivin protein in the H929 cell group detected by Western blot was significantly decreased , suggesting that the mechanism of miR - 145 could be correlated with the down - regulation of Bcl - 2 protein and Survivin protein .
Transwell invasion experiments and migration experiments showed that the number of cells passing through the membrane in the miR - 145 overexpression group was significantly lower than that in the negative control group compared with the number of cells migrating out of the cells ;
ELISA showed that miR - 145 overexpression group could significantly inhibit the secretion of VEGF in H929 cells .
Western blot showed that the expression of MMP - 2 and MMP - 9 in miR - 145 overexpression group was significantly decreased .
Western blot was used to detect the expression of phosphorylated PI3 - 3 in the miR - 145 group , and the total protein content was unchanged in the miR - 145 group .
In vivo experiments , the tumor volume in the miR - 145 overexpression group was decreased , the weight was reduced , the concentration of VEGF was decreased , and the number of apoptotic cells increased significantly .

Conclusion :

miR - 145 is a tumor suppressor gene of multiple myeloma , and miR - 145 mainly inhibits the proliferation of MM cells by inhibiting the activity of a 3 - 3 - 3 - 3 - 3 - 3 - 3 - 3 - 3 - 3 - 3 - 3 - 3 - kinase / 2 - miR - 145 , promotes apoptosis , and further confirms the negative regulation effect of miR - 145 on MM tumor proliferation by using MM mice ;
Up - regulation of miR - 145 expression can inhibit the migration and invasion of MM cells , and the mechanism is related to the inhibition of VEGF secretion and the expression of MMP - 2 and MMP - 9 ;
Therefore , miR - 145 is likely to be an important potential target in MM treatment , and the relationship between miR - 145 and MM has important theoretical and clinical value .

【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R733.3

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