Inhibition of down-regulated miRNA-221 and miRNA-222 in human breast cancer MCF-7 cell line to tumor cell proliferation and migration

doi:10.3969/j.issn.1673-8640.2014.05.006

Abstract

Abstract:

Objective To study the proliferation and migration abilities of human breast cancer MCF-7 cell line through knocking down microRNA (miRNA)-221 and miRNA-222 expressions and up-regulating tissue metalloproteinase inhibitor 3 (TIMP3). Methods Antisense-miRNA-221(AS-miRNA-221), antisense-miRNA-222(AS-miRNA-222), antisense-miRNA-221/222(AS-miRNA-221/222) and scramble were designed based on Homo sapiens (hsa)-miRNA-221 and hsa-miRNA-222 oligonucleotide sequence and nonsense sequence in miRBase. Lipofectamine^TM2000 was used to transfect AS-miRNA-221, AS-miRNA-222, AS-miRNA-221/222 and scramble into MCF-7 cell line . They were classified into control groups with stable expression [untransfected normal cell group(Control group) and scramble group(Scramble group)] and down-regulated miRNA-221 and miRNA-222 groups(AS-miRNA-221 group, AS-miRNA-222 group and AS-miRNA-221/222 group), respectively, according to G418 screening. After transfecting for 24 h, transfection efficiency was determined by fluorescence microscopy. Real-time fluorescence quantitation polymerase chain reaction (PCR) was used to determine the expressions of miRNA-221 and miRNA-222. The neo gene expression on the plasmid vector was detected in each cell line by reverse transcription PCR. The TIMP3 mRNA level was assessed by real-time fluorescence quantitation PCR, and the expression difference of metalloproteinase 17(ADAM17) protein was determined by Western blotting. The growth curves to describe the low-expressed miRNA-221 and miRNA-222 cells′growth were evaluated by CCK-8. The wound healing model was used to represent the migration ability. Results After transfecting for 24 h, there was an increasing expression of green fluorescence, and the transfection efficiency was high. The expressions of neo gene in Control group were lower than those in Scramble, AS-miRNA-221, AS-miRNA-222 and AS-miRNA-221/222 groups. Compared to Scramble group, the expressions of miRNA-221 and miRNA-222 decreased in AS-miRNA-221, AS-miRNA-222 and AS-miRNA-221/222 groups, and the establishment of AS-miRNA-221/222 down-regulated expression-stable cell line had succeeded. Compared with Scramble group, the expressions of TIMP3 mRNA of AS-miRNA-221 group, AS-miRNA-222 and AS-miRNA-221/222 groups increased, and the level of ADAM17 decreased. The growth curves showed that the proliferation of down-regulated miRNA-221 and miRNA-222 groups had been inhibited. The wound healing model showed that the migration of down-regulated miRNA-221 and miRNA-222 groups had been decreased. Conclusions Through up-regulatingp TIMP3, knocking down miRNA-221 and miRNA-222 can inhibit the proliferation and migration of human breast cancer MCF-7 cell line.

Key words: MicroRNA-221, MicroRNA-222, Transfection, Tissue metalloproteinase inhibitor 3, Metalloproteinase 17, Human breast cancer MCF-7 cell

CLC Number:

GAN Rong, YANG Xiao, YANG Yingmei, ZHAO Lingxu, MENG Qinghe.. Inhibition of down-regulated miRNA-221 and miRNA-222 in human breast cancer MCF-7 cell line to tumor cell proliferation and migration[J]. , 2014, 29(5): 452-458.

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