ISSN 1007-7626 CN 11-3870 / Q http / / cjbmb bjmu edu cn Chinese Journal of Biochemistry and Molecular Biology 2012 10 28 10 958 ~ 965 Cdk6 ES * 100048 6 cyclin-dependent kinase 6 Cdk6 embryonic stem ES RNA Cdk6 Cdk6 ES RA LIF ES Cdk6 RNA Cdk6 ES Cdk6 ES 6 Cdk6 Q71 The Role of Cdk6 in Maintaining the Self-renewal State of ES Cells WANG Juan-Juan LIU Hui-Xian HOU Jie ZHANG Wei-Wei * College of Life Sciences Capital Normal University Beijing 100048 China Abstract Cyclin-dependent kinase 6 Cdk6 is an important factor in development however whether it is able to maintain the embryonic stem ES cells in undifferentiated state remained obscure In this study we investigated the function of Cdk6 in mouse ES cells by RNA interference and gene expression analyses Our results showed that the expression level of Cdk6 was correlated with the self-renewal of the ES cells with dramatic decrease upon retinoic acid RA treatment or leukaemia inhibitory factor LIF removal for the induction of ES cell differentiation Cdk6 knockdown by RNA interference caused the ES cells into a undifferentiated state where the expression of self-renewal genes were reduced and the expression of differentiation markers such as Cdx2 were induced Our findings suggested that an important function of Cdk6 was to maintain the mouse ES cells into a undifferentiated and self-renewing state Key words embryonic stem cells cyclin-dependent kinase 6 Cdk6 differentiation embryonic stem ES 2012-05-17 2012-08-05 ES ES No 31101055 No KM201110028012 No 2011 1568 ES * TRA-1-60 TRA-1-81 1 2 3 ~ ES 5 Tel 010-68909575-605 ES ES 6 ES SSEA3 SSEA4 SSEA1 ES E-mail zhangww@ mail cnu edu cn Received May 17 2012 Accepted August 5 2012 Supported by National Natural Science Foundation of China No 31101055 Scientific Research Program of Beijing Municipal Commission of Education No KM201110028012 and Scientific Research Foundation for the Returned Overseas Chinese Scholars from the Education Ministry of China No 2011 1568 * Corresponding author Tel 010-68909575-605 ES E-mail zhangww@ mail cnu edu cn
10 Cdk6 ES 959 ES p27 13 ES 14 Cdk6 D CIP / ES KIP Cdk4 Cdk 13 15 7 ~ 9 ES 16 ES G 1 17 - Cdk6 20 Cdk6 ES 10 Oct4 Sox2 Nanog 21 ES Cdk6 ES ES transcriptional regulatory network Cdk6 ES ES RNA Cdk6 ES Cdk6 ES Cdk6 ES RNA ES Cdk6 ES ES ES ES Cdk6 ES ES leukaemia inhibitory factor LIF ES bone morphogenetic protein BMP Wnt wingless int LIF Wnt 1 ES Cartwright 11 LIF Wnt 1 1 ES Myc E14-CRL-1821 ATCC ES LIF DMEM GIBCO 11960- STAT-3 Myc mrna 044 GIBCO 16141-079 β- T58 GSK-3 Myc ES LIF Wnt 081 GIBCO 11140-050 LIF Myc ES ESGRO ESG1107 ES 1 2 ES Oct4 3 10 5 6 Sox2 Nanog 24 h 2 1 Chambers 12 LIF 3 d 6 d 10 d RNA Nanog ES 2 1 μg / ml RA Nanog Sigma 302-79-4 3 d 10 d RNA Nanog 1 3 Nanog ES GIBCO 21985-023 L- GIBCO 25030- lipofeatanine2000 6 3 10 5 6 cyclin-dependent 6 24 h kinase 6 Cdk6 D 4 μg 10 μl lipofeatanine2000
960 28 Invitrogen 11668-019 5 min 250 μl Opti-MEM GIBCO 31985 25 min 24 h 1 0 μg / ml Puromycin 4 d RNA 1 4 RNA real-time PCR Trizol Invitrogen 15596-018 RNA RNeasy minikit fermentas RNA 500 ng RNA RevertAid Fist strand cdna synthesis kit Invitrogen 00075471 cdna 2 μl 5 μl SYBR Green Supermix Bio-RAD #170-8880AP 50 nmol / L mrna β-actin 1 5 3 NP-40 Fluka 74385 30 min 4 12 000 r / min 30 min 1 6 BCA BSA X A Y Reagent A Bio-Rad 500-0113 Reagent B Bio-Rad 500-0114 Reagent C Bio-Rad 500-0115 A Y X 50 μg Fig 1 Inducing ES cell differentiation by LIF removal 6 SDS 100 5 min Supersignal west pico Chemiluminescent Substrate Thermo LJ1504480P PVDF 5 min PVDF ImageQuant TM 2 2 1 LIF ES Cdk6 LIF ES RNA real-time RT-PCR Oct4 Sox2 Nanog Fig 1 LIF 5 d ES Fig 1A Real-time RT-PCR Oct4 mrna 25% Sox2 50 nmol / L 35% Nanog 1% ILF ES 20 μl Bio-rad PCR ES Fig 1B Cultured ES cells by using the medium without LIF and changed the medium every twenty-four hours Analyze results 1 7 SDS-PAGE after five days A The phenotype of ES cells after removing LIF for five days Observe the cytomorphology of ES cells 2 through microscope B Reduction of Oct4 Sox2 and 12% 5% SDS-PAGE Nanog expression levels in ES cells cultured in no LIF 1 SDS-PAGE 100V SDSmedium ES cells were cultured in medium removed LIF for PAGE 1 5 h five days The mrna levels of Oct4 Sox2 and Nanog were 1 8 Western SDS-PAGE 100V 1 5 h determined by real-time PCR Data are presented as the mean ± SEM Cdk6 Santa Cruz SC-177 β-tublin Santa Cruz SC-166729 retinoic acid RA A ES 22 RA ES RNA real-time RT-PCR LAS4000 Oct4 Sox2 Nanog Fig 2 RA ES 5 d
10 Cdk6 ES 961 LIF RNA Western Fig 2A Real- Cdk6 RA LIF time RT-PCR Oct4 mrna 1% Sox2 10% Nanog 1% RA Cdk6 ES ES Fig 2B Fig 3C ES Cdk6 Cdk6 ES 2 3 Cdk6 shrna Cdk6 ES Cdk6 ES Cdk6 2 RNA Cdk6 shrna1 2 Table 1 Table 1 The target sequence of Cdk6 gene Name Cdk6 shrna1 Cdk6 shrna2 Sequence GACCAGCAGTGGACAGATA GAGAAGTTTGTGACAGATA Fig 2 RA-induced ES cell differentiation Treated ES cells with 1μ mol / L RA and changed the medium every twenty-four hours Analyze results after five days A The phenotype of ES cells after RA treatment Observe the cytonorphology of ES cells through microscope B Oct4 Sox2 and Nanog expression levels were reduced in ES cells cultured in RA medium ES cells were cultured in medium with RA for five days The mrna levels of Oct4 Sox2 and Nanog were determined by real-time PCR Data are presented as the mean ± SEM 2 2 ES Cdk6 Cdk6 ES Cdk6 2 4 Cdk6 RA 3 d 10 d Oct4 Sox2 Nanog ES LIF 3 d 6 d 10 d RNA real-time RT-PCR Western Fig 3A mrna RA 3 d 10 d Cdk6 40% 20% Cdk6 RA Real-time RT-PCR RA Cdk6 Cdk6 ES Oct4 Sox2 Nanog Fig 3B mrna LIF 3 d 6 d 10 d Cdk6 2 Cdk6 shrna lipofectamine 2000 Cdk6 shrna ES 1 μg / ml puromycin 4 d RNA real-time RT-PCR Western Fgi 4A Cdk6 shrna ES mrna Cdk6 20% Fgi 4B Cdk6 shrna ES Cdk6 Cdk6 shrna ES Cdk6 ES ES Oct4 Sox2 Nanog Esrrb Tdgf1 Fig 5 Cdk6 ES Oct4 90% 50% 20% 40% Sox2 Nanog Esrrb
962 28 Fig 3 Cdk6 expression level decreased after ES cell differentiation A Reduction of Cdk6 expression level in ES cells cultured in RA-induced cell differentiation ES cells were cultured in the medium with RA for 3 days and 10 days The mrna expression level of Cdk6 was measured by quantitative real-time PCR analysis Data are presented as the mean ± SEM B Reduction of Cdk6 expression level in ES cells cultured in LIF withdrawn induced cell differentiation ES cells were cultured in medium without LIF for 3 6 and 10 days The mrna expression level of Cdk6 was measured by quantitative real-time PCR analysis Data are presented as the mean ± SEM C Reduction of Cdk6 protein expression level in ES cells cultured in RA-treatment or LIF removal-induced cell differentiation The protein expression levels of Cdk6 were measured by Western blotting with RA or no LIF withdrawn treatment for 3 and 6 days The antibody againstβ-tubulin was used as a loading control Fig 4 The expression levels of Cdk6 upon shrna treatment Transfected the construct of Cdk6 shrna to ES cells and selected cells with 1 μg / ml puromycin for 4 days A The mrna levels of Cdk6 were determined by real-time PCR quantification of reverse transcribed RNAs Data are presented as the mean ± SEM B Reduction of Cdk6 protein expression level in ES cells treatmented with shrna The protein expression levels of Cdk6 were measured by western blotting with shrna treatment for 4 days The antibody againstβ-tubulin was used as a loading control Fig 5 The change of self-renewal-related gene expression in ES cells after shrna-mediated Cdk6 depletion The mrna levels of Oct4 Sox2 Nanog Esrrb and Tdgf5 were determined by real-time PCR quantification of reverse transcribed RNAs Transfected the construct of shrna Cdk6 to 60% ~ 70% Tdgf5 ES cells and selected cells with 1 μg / ml puromycin for 4 50% ~ 60% days Data are presented as the mean ± SEM Cdk6 ES Cdk6 Fig 6 trophectoderm Cdx2 Hand1 Cdk6
10 Cdk6 ES 963 ectoderm Fgf5 cyclins mesendoderm Msx1 cyclin-dependent kinase Cdks Cdk6 cyclin-dependent kinase ES inhibitor CKIs Fig 6 The change of differentiation-related gene expression in ES cells after shrna- mediated Cdk6 depletion The mrna levels of differentiation genes were determined by real-time PCR quantification of reverse transcribed RNAs Transfected the construct of shrna Cdk6 to ES cells and selected cells with 1 μg / ml puromycin for four days Data are presented as the mean ± SEM 3 Cdks Cyclins Cdks CKIs 25-26 ES ES G 1 27 ES 11 h G 1 2 h 28-29 E2F G 1 / S 30 ES Cdk2-cyclinA / E cyclin E p21cip1 p27kip1 30 Cdk2 Cdk2 30 ES Cdk6 / CyclinD 31-32 ES Cdk1- Cyclin B Cyclin B Cdk1 Y 15 Cdk1 30 Cdk1 Cdk2 PCNA ES 33 Cdk1 Cdk1 34 Ullah 35 Cdk1 RO3306 Cdk1 Cdk1 36 ES Cdk1 Oct4 Cdk1 Oct4 ES Cdks Cdk1 ES 4 G 1 S G 2 M Cdk6 RA LIF checkpoint ES real-time G 1 / S G 2 / M RT-PCR ES Cdk6 ES Cdk6 23-24 ES Cdk6
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