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-原著- 微小RNA-21对人乳腺癌细胞株紫杉醇耐药性的 … 材料 人乳腺癌细胞株MCF-7、SKBR-3购自中国科学院细胞库;紫杉醇、四甲基偶氮唑盐(MTT)、

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Text of -原著- 微小RNA-21对人乳腺癌细胞株紫杉醇耐药性的 … 材料...

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    RNA-21

    1, 2

    , 1, 2

    , 1, 2

    , 3,

    3,

    4,

    4,

    4

    1. 233000

    2. 233000

    3. 233000

    4. 233000

    :2014-09-23 2015-03-06

    810718481508085MH159

    KJ2013A184KJ2010A240

    201310367014

    (1989-),E-mail

    [email protected]://orcid.org/0000-0002-4741-2198 1968-

    [email protected]://orcid.org/0000-0001-9646-320X

    [] RNA-21 (miR-21) MCF-7/PR

    SKBR-3/PR

    MDR1BCRPMRP1

    MCF-7 SKBR-3 miR-21Bax

    Bcl-2 miR-21 MCF-7 SKBR-3

    MTT miR-21

    MCF-7/PR

    SKBR-3/PR MDR1BCRPMRP1

    Bcl-2 Bax miR-21 (P

  • 233000, China)

    Corresponding author CHEN [email protected]

    [Abstract] Objective: To explore the effects of miR-21 gene expression changes on

    paclitaxel resistance in human breast cancer cells MCF-7/PR and SKBR-3/PR. Methods:

    Paclitaxel-resistant cell line of human breast cancer (MCF-7/PR and SKBR-3/PR ) was

    established by continuous stepwise selection in increasing concentration of

    paclitaxel. Cellular morphology, mRNA and protein level of MDR1, BCRP and MRP1 were

    investigated to determine the drug resistance features of MCF-7/PR and SKBR-3/PR

    cell lines. RT-PCR and western blotting were used to analyze the expression of Bax,

    Bcl-2 and miR-21 between parental and paclitaxel-resistant cells. After synthetic

    miR-21 inhibitor and miR-21 mimic were respectively transfected into MCF-7/PR,

    SKBR-3/PR and MCF-7, SKBR-3 breast cancer cells with lipofectamine 2000 vector,

    miR-21 level was determined by RT-PCR. Western bloting was employed to detect the

    protein level of P-gp, Bcl-2 and Bax. MTT assay was used to measure the drug

    sensitivity, while flow cytometry was performed to analyze the cell cycle and

    apoptosis. Results: The level of MDR1, BCRP, MRP1, Bcl-2/Bax and miR-21 in MCF-7/PR

    and SKBR-3/PR were significantly higher than that in MCF-7 and SKBR-3. The protein

    levels of P-gp, Bcl-2 were up-regulated, and Bax was down-regulated compared with

    parental cells. All these results showed that paclitaxel-resistant breast cancer

    cell lines MCF-7/PR and SKBR-3/PR were established successfully. miR-21 was

    significantly downregulated after miR-21 inhibitor was transfected. Additionally,

    downregulation of miR-21 in the MCF-7/PR and SKBR-3/PR breast cancer cell lines were

    concurrent with the downregulation of the level of MDR1 , BCRP, MRP1 and Bcl-2/Bax

    (P

  • 1.1

    MCF-7SKBR-3 MTT

    (DMSO)(DEPC) Sigma TrizolLipofectamine 2000

    Opti-DMEM Invitrogen Fermentas

    P-(P-glycoprotein, P-gp)Bcl-2Bcl-2 X Bcl-2 associated X protein

    Bax -actin Santa Cruz (HRP)

    IgG BD (PVDF)

    ECL Millipore has-miR-21 snRNAU6

    miR-21 5UCAACAUCAGUCUGAUAAGCUA3

    5UAGCUUAUCAGACUGAUGUUGA3 5AACAUCAGUCUGAUAAGCUAUU 3

    multidrug resistance-associated

    protein ,MRP1breast cancer resistance protein, BCRP

    1 multidrug resistance 1,MDR1 -3-

    glyceraldehyde-3-phosphate dehydrogenase, GAPDH

    Thermo ScientificSYBRPremix Ex Taq(Perfect

    Real Time) TaKaRa All-in-onemiRNAqRT-PCR Detection Kit

    GeneCopoeia Muse Annexin V & Dead Cell Kit Merck Millipore

    1.2

    MCF-7SKBR-3 10% RPMI1640

    37 5% 3

    1.3 MTT MCF-7SKBR-3

    0.25% MCF-7SKBR-3 1105/mL 100 L

    96 375% 24h 100 L

    MCF-7 0.0010.010.1110100mol/LSKBR-3

    0.010.111050mol/L 5

    72 h 5 mg/mL MTT 20 L 4 h

    150 L DMSO 10 min 490 nm

    %=1-(/

    )100%IC50 3

    1.4 MCF-7/PR SKBR-3/PR

    MCF-7SKBR-3 70%~80% 1 mol/L

    24 h

    24 h 6

    MCF-7/PR SKBR-3/PRMCF-7/PR 10 mol /mL

    SKBR-3/PR 25 mol/mL

    1.5 miR-21

    miR-21 MCF-7/PRSKBR-3/PRmiR-21 MCF-7SKBR-3

    4 miR-21 miR-21

    Lipofectamine 2000

    0.25%

    6 4105/mL 70%~80% 5 L

    20 mol/L miR-21 miR-21 250L

    5L Lipofectamine 2000 250 L 5 min

  • miR-21 miR-21 Lipofectamine 2000

    20 min6 1.5mL 6

    6 h 10% RPMI 1640

    1.6 Annexin V/PI

    4 PBS

    Muse Annexin V & Dead Cell Kit 20 min

    ModFitLTV3.0 3

    1.7 PI

    4 PBS

    4 70%4 70%

    4 PBS 500 L 4 PI 5mg PI+2mg

    RNaseA+0.1 mL 1.0%Trition X-100+3.7 mg EDTA+10 mL PBS 4

    50 g/mL 30 min ModFitLTV3.0

    3

    1.8 PCR mRNA miR-21

    Trizol RNA SYBRPremix Ex

    Taq(Perfect Real Time) All-in-onemiRNA qRT-PCR Detection Kit

    miR-21 PCR GAPDH snRNAU6

    RQ=2-CT

    CT=(CT-CTGAPDH)- CT

    - CTGAPDH) miR-21

    CT=( CTmiRNA- CTU6 RNA)-( CTmiRNA- CT

    U6 RNA)

    1 PCR

    Table 1 The sequences of real time quantitive RT-PCR primers

    (bp)

    MRP1 acttcctctatctctcccgaca aagactgaactcccttcctcct 246

    BCRP ctcagtttatccgtggtgtgtc gattgttcgtccctgcttagac 176

    MDR1 gtacccatcattgcaatagc caaacttctgctcctgagtc 157

    Bcl-2 atgtgtgtggagagcgtcaa acagttccacaaaggcatcc 136

    Bax ggggacgaactggacagtaa cagttgaagttgccgtcaga 122

    GAPDH cagcctcaagatcatcagca tgtggtcatgagtccttcca 106

    1.9

    30min4

    (BCA )10% SDS-PAGE

    20 g Marker 5 L PVDF 5%

    3 h P-gpBcl-2Bax -actin

    4 1:1000TBST 3 10 min

    IgG1:500037 2 h PVDF BIO-RAD

    ,AlphaView SA

    1.10

    SPSS13.0 S

    tP0.05

  • 2.

    2.1 MCF-7 SKBR-3

    MCF-7 SKBR-3 MCF-7

    SKBR-3 MCF-7/PR SKBR-3/PR

    1

    1 MCF-7 SKBR-3 (=50m)

    Fig.1 Optical morphology of MCF-7 and SKBR-3 parental and Paclitaxel resistant

    cells(bar=50m)

    2.2 MCF-7 SKBR-3

    MTT MCF-7 SKBR-3

    mol/L%-

    72 h MCF-7 IC505.640.15 mol/LSKBR-3 IC50

    4.612.17 mol/L 2

    SKBR-3

    MCF-7 MCF-7/PR

    SKBR-3/PR

  • 2 MCF-7 SKBR-3

    Fig. 2 The growth inhibition curve of MCF-7 and SKBR-3 cells treated with Paclitaxel

    2.3 MCF-7SKBR-3 mRNA miR-21

    MCF-7 SKBR MCF-7/PRSKBR-3/PR MDR1BCRPMRP1 Bcl-2

    mRNA Bcl-2/Bax miR-21 ( P

  • 4 MCF-7 SKBR-3 P-gpBcl-2 Bax

    Fig. 4 P-gp, Bcl-2 and Bax protein expression in MCF-7 and SKBR-3 parental and

    Paclitaxel resistant cells

    2.5 miR-21 MCF-7 SKBR-3

    miR-21 MCF-7 SKBR-3 72h MCF-7

    SKBR-3

    5 miR-21 MCF-7 SKBR-3 (=50m)

    Fig. 5 The effects of miR-21 mimic on MCF-7 and SKBR-3 cells morphology (bar=50m)

    2. 6 miR-21 MCF-7/PR SKBR-3/PR

  • MTT miR-21

    miR-21 miR-21

    6 miR-21 MCF-7/PR SKBR-3/PR

    Fig.6 Drug susceptibility of MCF-7/PR and SKBR-3/PR cells with Taxol action after

    transfected miR-21 inhibitor

    2.7 miR-21 MCF-7/PR SKBR-3/PR

    miR-21 MCF-7/PRSKBR-3/PR

    ( P0.05) miR-21 MCF-7/PRSKBR-3/PR 2

    2 miR-21 MCF-7/PR SKBR-3/PR

    Table 2 The cell apoptpsis of MCF-7/PR and SKBR-3/PR cell after transfected miR-21

    inhibitor

    S ,%

    MCF-7/PR SKBR-3/PR

    3.8630.1387 4.5470.2267

    3.6670.1532 4.7600.2050

    miR-21 13.440.3327*# 17.340.2454

    *#

    *P

  • S %

    MCF-7/PR SKBR-3/PR

    G0/G1 S G2/M G0/G1 S G2/M

    45.17

    1.074

    32.27

    1.132

    14.70

    0.6245

    48.27

    1.110

    33.47

    1.260

    12.10

    0.6807

    miR-21

    P

    58.77

    1.244

  • 8 miR-21 MCF-7 SKBR-3 MDR1 mRNA

    Fig.8 Expression of MDR1 mRNA in MCF-7 and SKBR-3 parental and Paclitaxel resistant

    cells by miR-21

    9 miR-21 MCF-7 SKBR-3 Bcl-2 Bax mRNA

    Fig.9 Expression of Bcl-2 and Bax mRNA in MCF-7 and SKBR-3 parental and Paclitaxel

    resistant cells by miR-21

  • 2. 10 miR-21 MCF-7/PRSKBR-3/PR

    miR-21 P-gp

    Bcl-2 Bax P

  • [16]

    Tomimaru [17]

    miR-21 INF-/5-FU

    miR-21

    miR-21

    MCF-7/PR

    SKBR-3/PR

    MDR1P-gpBCRPMRP1

    miR-21

    miR-21MCF-7SKBR-3miR-21

    miR-21miR-21MCF-7/PR

    SKBR3/PRmiR-21,MDR1P-gpmiR-21

    MCF-7SKBR-3MDR1

    P-gpP-gpP-gp

    P-gp

    caspase[18]

    miR-211(TPM1)

    4(PDCD4)p53(TGF-)(PTEN)

    miR-21PTEN 3PTEN[19]

    PTENPI3K/Akt

    Bcl-2Bax[20]

    miR-21

    Bcl-2/BaxMCF-7/PR

    SKBR-3/PRBcl-2/BaxmiR-21Bcl-2/Bax

    miR-21Bcl-2/BaxBcl-2BaxmiR-21

    miR-21

    miR-21G0/G1miR-21

    MCF-7/PRSKBR-3/PR

    miR-21miR-21

    MCF-7/PRSKBR-3/PRmiR-21

    miR-21miR-21Bcl-2Bax

    miR-21

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