微RNA hsa-miR-125a-5p通过激活p53诱导肺癌SPC-A-1细胞凋亡的研究
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摘要
为了探讨微RNA hsa-miR-125a-5p对肺癌细胞凋亡的影响及其可能的调控机制,通过实时定量RT-PCR (qRT-PCR法)检测hsa-miR-125a-5p在不同细胞系表达的相对含量,流式细胞术检测肺癌细胞的凋亡情况,Western blot和RT-PCR检测p53蛋白和mRNA表达情况,进一步的功能性实验选择SPC-A-1细胞进行。结果显示,与人支气管上皮HBE细胞相比较,hsa-miR-125a-5p在不同的肺癌细胞系中表达下降。在功能获得性实验中,hsa-miR-125a-5p促进凋亡,hsa-miR-125a-5p过表达能够增加野生型p53 mRNA和蛋白表达;阻断野生型p53能够减弱hsa-miR-125a-5p在凋亡中的作用。在功能丧失性实验中,阻断hsa-miR-125a-5p能够降低野生型p53的mRNA和蛋白表达,阻断野生型p53同样能够减弱hsa-miR-125a-5p抑制剂在凋亡中的作用。最后,在p53缺陷细胞系H1299中转染正义或反义miR-125a-5p混合物,结果显示凋亡率均没有明显变化。上述研究表明,微RNA hsa-miR-125a-5p在肺癌细胞SPC-A-1中通过激活p53诱导凋亡。这些结果将为miR-125a家族在肺癌中的作用提供新的视点。
In order to investigate the effect of hsa-mir-125a-5p on apoptosis of lung cancer cells and its possible regulatory mechanism,the relative expression of hsa-mir-125a-5p in different cell lines was detected by real-time quantitative RT-PCR( qRT-PCR),and the apoptosis of lung cancer cells was detected by flow cytometry. The p53 and mRNA expression were tested by Western blot and semi-quantitative RT-5p,respectively. It was found that,compared with human bronchial epithelial( HBE) cells,the expression of hsa-mir-125a-5p decreased in different lung cancer cell lines. In functional acquisition experiments,hsa-miR-125a-5p promoted the apoptosis. Overexpression of hsa-miR-125a-5p could increase wild-type p53 mRNA and protein expression. Further,blocking wild type p53 could attenuate the role of hsa-miR-125a-5p in apoptosis. In the experiment of functional loss,blocking hsa-mir-125a-5p could decrease the expression of wild-type p53 gene and protein. Blocking wild type p53 also attenuated the role of hsa-miR-125a-5p inhibitors in apoptosis. Finally,in p53-deficient cell line H1299,just or antisense miR-125 a-5p mixture was transfected. The results showed that there was no significant change in apoptotic rate. It is concluded that mRNA hsa-miR-125a-5p can induce apoptosis of lung cancer cell SPC-A-1 by activating p53. These results will provide a new perspective for the role of miR-125 a family in lung cancer.
In order to investigate the effect of hsa-mir-125a-5p on apoptosis of lung cancer cells and its possible regulatory mechanism,the relative expression of hsa-mir-125a-5p in different cell lines was detected by real-time quantitative RT-PCR( qRT-PCR),and the apoptosis of lung cancer cells was detected by flow cytometry. The p53 and mRNA expression were tested by Western blot and semi-quantitative RT-5p,respectively. It was found that,compared with human bronchial epithelial( HBE) cells,the expression of hsa-mir-125a-5p decreased in different lung cancer cell lines. In functional acquisition experiments,hsa-miR-125a-5p promoted the apoptosis. Overexpression of hsa-miR-125a-5p could increase wild-type p53 mRNA and protein expression. Further,blocking wild type p53 could attenuate the role of hsa-miR-125a-5p in apoptosis. In the experiment of functional loss,blocking hsa-mir-125a-5p could decrease the expression of wild-type p53 gene and protein. Blocking wild type p53 also attenuated the role of hsa-miR-125a-5p inhibitors in apoptosis. Finally,in p53-deficient cell line H1299,just or antisense miR-125 a-5p mixture was transfected. The results showed that there was no significant change in apoptotic rate. It is concluded that mRNA hsa-miR-125a-5p can induce apoptosis of lung cancer cell SPC-A-1 by activating p53. These results will provide a new perspective for the role of miR-125 a family in lung cancer.
引文
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[5]LEE Y,AHN C,HAN J,et al.The nuclear RNase IIIDrosha initiates microRNA processing[J].Nature,2003,425(6956):415-419.
[6]CHENG C J,BAHAL R,BABAR I A,et al.MicroRNAsilencing for cancer therapy targeted to the tumour microenvironment[J].Nature,2015,518(7537):107-110.
[7]LI S,LIU L,ZHUANG X,et al.MicroRNAs inhibit the translation of target mRNAs on the endoplasmic reticulum in Arabidopsis[J].Cell,2013,153(3):562-574.
[8]DJURANOVIC S,NAHVI A,GREEN R.miRNA-mediated gene silencing by translational repression followed by mRNA deadenylation and decay[J].Science,2012,336(6078):237-240.
[9]KRZESZINSKI J Y,WEI W,HUYNH H,et al.miR-34a blocks osteoporosis and bone metastasis by inhibiting osteoclastogenesis and Tgif2[J].Nature,2014,512(7515):431-435.
[10]ROM U A,NIELSEN F C,LUND A H.MicroRNA-10a binds the 5'UTR of ribosomal protein mRNAs and enhances their translation[J].Mol Cell.2008,30(4):460-471.
[11]BRENNECKE J,STARK A,RUSSELL R B,et al.Principles of microRNA-target recognition[J].PLo S Biol,2005,3(3):e85.
[12]BARTEL D P.MicroRNAs genomics,biogenesis,mechanism,and function[J].Cell,2004,116(2):281-297.
[13]FOSHAY K M,GALLICANO G I.miR-17 family miR-NAs are expressed during early mammalian development and regulate stem cell differentiation[J].Dev Biol,2009,326(2):431-443.
[14]WU H,ZHU S,MO Y Y.Suppression of cell growth and invasion by miR-205 in breast cancer[J].Cell Res,2009,19(4):439-448.
[15]BUENO M J,DE CASTROL I P,MALUMBRES M.Control of cell proliferation pathways by microRNAs[J].Cell Cycle,2008,7(20):3143-3148.
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