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1 대한내분비학회지 : 제 24권제 1 호 2009 원 저 /jkes 갑상선암에서 mirna 146a/b, 221 및 222 의발현에대한연구 충남대학교병원내과, 외과 1 조영석 이인석 홍우정 송인상 1 송민호 김제룡 1 Expression of mirna 146a/b, 221 and 222 in Thyroid Cancer Young Suk Jo, Ihn Suk Lee, Woojeong Hong, In Sang Song 1, Minho Shong, Je Ryoung Kim 1 Department of Internal Medicine and Surgery 1, Chungnam National University Hospital ABSTRACT Background: mirnas can be diagnostic markers and therapeutic targets in cancers, but few studies have been conducted in thyroid cancer. We investigated the expression levels of mirna 146a/b, 221, and 222 which are important mirnas in papillary thyroid cancers (PTCa), and verified their impact on clinicopathological factors. Methods: We measured the expression of pre-mirnas 146a/b, 221, and 222 in NPA cells treated with 10% fetal bovine serum (FBS) or in HEK293T cells transfected with RET/PTC3 or BRAF V600E expression vectors. We also investigated the relationship between mirna expression levels in thyroid cancer tissue specimens and clinicopathological parameters. Results: Growth stimulation with 10% FBS induced mirna expressions in NPA cells, and transfection of RET/PTC3 and BRAF V600E also increased the expression of these mirnas in HEK293T cells. Most (25 cases; 50%) of PTCa showed increased expression of mirna-146a/b and 30 cases (60%) had elevated expression of mirna-221 and mirna-222 compared to normal thyroid samples from the contralateral lobe. However, increased mirna expression did not correlate with clinicopathological factors. Conclusion: Expression of mirna 146a/b, 221, and 222 was increased by BRAF V600E and RET/PTC3 rearrangement and might have a role in tumorigenesis in PTCa. However, expression levels of these mirnas did not correlate with clinicopathological parameters of patients with PTCa. (J Korean Endocr Soc 24:17~24, 2009) ꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏ Key Words: microrna, prognosis, thyroid neoplasm 서론 1) microrna는 22개정도의염기쌍 (basepair) 으로구성된 non-coding RNA로 1993년 Ambros와 Ruvkun 등이 C. elegans에서 lin-4를클로닝하여기능을분석함으로써알려지기시작하였다 [1,2]. 최근에는다양한 mirnas가존재하는것이밝혀지고있으며 mirnas의생성과정과생물학적의접수일자 : 2008년 8월 13일통과일자 : 2009년 1월 7일책임저자 : 김제룡, 충남대학교병원외과 의가규명되고있다 [3]. 이들 mirnas의생성과정을살펴보면 DNA로부터 pri-mrnas의형태로전사되어 endonucleolytic cleavage, nuclear export 및 strand seletion 과정을거쳐 mature mirnas로전환된다 [4]. 이렇게생성된 mature mirna는 RNA-induced silencing complexes (RISC) 를이루어표적유전자의발현을억제하게되며, 이를통해세포의증식, 분화또는세포자연사등의생명활동에 negative regulator' 로작용하게된다 [5,6]. 암의생성과정에서 mirna는표적유전자에따라종양억제유전자 (tumor suppressor) 또는종양유전자 (oncogene) 로작용하게되는데 [7,8], 종양의원발부위와아형 (subtype)

2 대한내분비학회지 : 제 24 권제 1 호 2009 에따라이들 mirna의발현양상이달라서이를활용한종양의분류방법이꾸준히연구되고있다 [9]. 이와더불어, 폐암, 유방암및고환암등과같은악성종양에서이들 mirnas의발현양상및기능에대한연구가활발히수행되고있으며이를통해진단, 예후예측및치료등에활용이가능한표지자를발굴하고있다 [10~12]. 갑상선유두암에서도 mirna-146a/b, mirna-221 및 mirna-222 등과같은특정 mirnas의발현이증가되어있다는보고가있었으며 [13], BRAF V600E 돌연변이나 RET/PTC1 재배열에의해 mirna-128a/b, mirna-185, mirna-200a/b 및 mirna-213 등의발현이유도된다는연구결과도있었다 [14,15]. 그러나, 아직은일부연구자들에의해수행된제한적인결과이고, 이들 mirna의임상적의의가충분히규명되지않은상태이다. 이에본연구에서는 mirna-146a/b, mirna-221 및 mirna-222의발현이갑상선암세포주에서유의하게증가되어있는지확인하고, 우태아혈청및종양유전자와같은성장유발신호에의해조절되는지알아보고자하였다. 또한, 수술을통해확보된갑상선유두암조직에서이들 mirnas의발현이증가되어있는지확인하고, 이미알려진예후인자와연관성이있는지규명하고자하였다. 대상및방법 1. 세포배양및항체갑상선암세포주인 TPC1, NPA, FRO 및 ARO를이용하여 mirnas의발현과 10% 우태아혈청 (Invitrogen Life Technologies, Carlsbad, CA) 처리에따른변화를확인하였으며, HEK293T 세포주를이용하여종양유전자의도입에의한 mirnas의발현변화를관찰하였다. 이들의배양은이미기술된방법을사용하였으며성장을위한배지는 TPC1과 HEK293T 세포주는 Dulbecco's Modified Eagle Medium (Invitrogen Life Technologies) 을, NPA, FRO 및 ARO 세포주는 RPMI 1640 (Invitrogen Life Technologies) 을사용하였다. Anti-phospho-p44/42-MAPK (pt202/y204) 항체와 Anti-p44/42-MAPK 항체는 New England Biolabs, Inc. (Danvers, MA) 로부터구입하였으며, Anti-pY 항체및 anti-actin 항체는 Santa Cruz Biotechnology, Inc. (CA) 로부터구입하였다. 2. 플라스미드및형질도입 pcdna3.1-ret/ptc3 및 pcdna3.1-braf는 PCR을통해제작하였으며, pcdna3.1-ret/ptc3 K284M, pcdna3.1 -BRAF V600E 및 pcdna3.1-braf G469A 는 Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA) 를사용하여제작하였다 [16~18]. 형질도입은 LipofectAMINE (Invitrogen Life Technologies) 을사용하여제조사의사용설명서에따라시행하였다. 3. 역전사중합효소연쇄반응및 Western Blot 분석 각각의세포주, 갑상선유두암조직및반대편정상조직에서 Trizol Reagent (Invitrogen Life Technologies) 를이용하여 RNA를추출하였다. Maxime TM RT-PCR Premix Kit 와 Maxime TM PCR Premix Kit (intron Biotechnology, Korea) 를사용하여표준조건에서역전사중합효소연쇄반응을수행하였으며, 사용된 primer는 Table 1과같다. 정량적분석을위해 U6 snrna를 mirvana TM qrt-pcr Primer Sets (Ambion, Austin, TX) 를사용하여증폭한후, end-point RT-PCR product를사용하였다. HEK293T 세포에 IPH 완충액 (50 mm Tris-HCl, ph 8.0, 150 mm NaCl, 5 mm EDTA, 1% NP-40, 100 μm PMSF), 1 μg/ml 단백질분해효소억제제 (Luepetin, Aprotinin), 1 mm DTT를처리하여단백질을추출하였으며, Sodium dodecyl sulfate -polyacrylamide 겔에서전기영동하여단백질을크기별로분리한후 Immobilon-P transfermembrane (Milipore, Billerica, MA) 으로이동시켰다. 차단완충액 (blocking buffer) 과 30분간반응시킨후 Anti-phospho-p44/42-MAPK (pt202/y204) 항체, Anti-p44/42-MAPK 항체, Anti-pY 항체및 anti-actin 항체로 4 에서 16시간반응시키고, horseradish peroxidase 가표지된이차항체로상온에서 1시간반응시켰다. Phototope-HRP Western Blot Detection Kit (New England Biolabs) 를사용하여단백질발현을확인하였다. 4. 연구대상및예후인자분석 2005년 1월부터 12월까지충남대학교병원에서갑상선유두암으로갑상선절제술을시행받은 50명의환자로부터갑상선암조직및종양발생부위의반대편엽에서정상갑상선조직을얻어 RNA를추출하였으며, 추출된 RNA를정량한 Table 1. Primers for RT-PCR Name Forward primer Reverse primer mir-146a CCGATGTGTATCCTCAGCTTT TGACAGAGATATCCCAGCTGAA mir-146b CCTGGCACTGAGAACTGAAT GGCACCAGAACTGAGTCCAC mir-221 TGAATGCAGTAGGCAGTTGTG GGGGTAGCATTGGTGAGACA mir-222 GCTAGAAGATGCCATCAGAGAC AGGTACCCTCAATGGCTCAG

- 조영석외 5 인 : 갑상선암에서 mirna 146a/b, 221 및 222 의발현에대한연구 - 후, pre-mirna-146a/b, pre-mirna-221 및 pre-mirna-222 의발현정도를 RT-PCR을통하여확인하였다. 생성된 RT-PCR 산물을 Gel Pro Analyzer 3.

통계분석 그룹간의빈도를비교하기위해서카이제곱검정 (chi-square test) 을이용하였으며평균의비교를위해서는 independent samples T-test를사용하였다. 모든연속변수는평균 ± 표준편차로기술하였으며, 제시된 P값은양측검정을통하여산출하였다. 통계프로그램은 SPSS Versions 12.0 (for Windows) 을이용하였다. 결 과 1.

3 - 조영석외 5 인 : 갑상선암에서 mirna 146a/b, 221 및 222 의발현에대한연구 - 후, pre-mirna-146a/b, pre-mirna-221 및 pre-mirna-222 의발현정도를 RT-PCR을통하여확인하였다. 생성된 RT-PCR 산물을 Gel Pro Analyzer 3.1 (Media Cybernetics, Silver spring, MD) 을활용하여 U6의발현과정량적으로비교한후, pre-mirna-146a/b, pre-mirna-221 및 pre-mirna -222의발현이정상조직에비해증가된군및감소된군으로분류하고, 양군의임상적특징을후향적방법으로비교, 분석하였다. 5. 통계분석 그룹간의빈도를비교하기위해서카이제곱검정 (chi-square test) 을이용하였으며평균의비교를위해서는 independent samples T-test를사용하였다. 모든연속변수는평균 ± 표준편차로기술하였으며, 제시된 P값은양측검정을통하여산출하였다. 통계프로그램은 SPSS Versions 12.0 (for Windows) 을이용하였다. 결 과 1. 갑상선암세포주에서 mirna 의발현과우태아혈청처리에따른발현유도 갑상선암세포주에서내인성 mirna의발현을확인하기위해, TPC1, NPA, FRO 및 ARO 세포주를배양한후 RNA 를추출하여 RT-PCR을이용하여 precursor mirna의발현을확인하였다. mirna 146a/b의발현은 TPC1 및 FRO 세포주에서증가되어있고 mirna 221 및 mirna 222의발현은 FRO 세포주에서증가되어있었다 (Fig. 1A). 내인성 mirnas의발현이적은 NPA 세포주를이용하여 10% 우태아혈청처리에따른 mirnas의발현변화를관 찰하였다. 우태아혈청처리후 1시간째부터 MAPK의인산화가증가함을알수있었으며, 이와더불어 mirna-146a와 mirna-221의발현이 1시간째부터증가하여 4시간을정점으로 6시간째까지유지됨을알수있었다 (Fig. 1B). mirna -146b와 mirna-222도 10% 우태아혈청을처리하였을때 mirna-146a와 mirna-221과유사한발현증가를확인할수있었다 (data not shown). 2. RET/PTC3 rearrangement 와 V600E BRAF 에의한 mirnas 의발현유도 갑상선유두암의종양유전자인 RET/PTC3 rearrangement 와 V600E BRAF에의한 mirna의발현조절을확인하기위하여 HEK293T 세포주에이들종양유전자를형질도입하였다. RET/PTC3 rearrangement와 BRAF V600E 의도입에의해각각 tyrosine 또는 MAPK의인산화가증가하였고 (Fig. 2A), mirna-146a 및 mirna-221의발현도 RET/PTC3 rearrangement와 BRAF, BRAF V600E 및 BRAF G469A 에의해증가하였다 (Fig. 2B). 특히, mirna-146a의발현은다른종양유전자와비교하여 BRAF V600E 에의해더욱증가함을알수있었다. mirna-146b와 mirna-222도 RET/PTC3 rearrangement와 BRAF, BRAF V600E, BRAF G469A 에의해 mirna-146a와 mirna-221과유사한양상의발현증가가있음을관찰하였다 (data not shown). 키나아제활성이없는 RET/PTC3 KM 발현벡터를사용했을경우 mirna-146a 와 mirna-221의발현에변화가없었다 (Fig. 2B). 이는 RET/PTC3 rearrangement에의한 mirna-146a와 mirna -221의발현유도가키나아제활성에의존적임을시사하는자료로사료된다. A B Fig. 1. mirna expression in human thyroid cancer cell lines and induction of mirna expression by serum stimulation. A. mirna expression in human thyroid cancer cell lines. To find out the endogenous expression of mirna-146a and mirna-221, we performed RT-PCR targeting precursor mirnas of these mirnas. B. Induction of mirna expression by serum stimulation. As we treated NPA cells with 10% fetal bovine serum, mirna-146a/b, -221 and -222 were induced at 1 hour

대한내분비학회지 : 제 24 권제 1 호 2009 A B Fig. 2. RET/PTC3 rearrangement and BRAF induced expression of mirna-146a and mirna-221. A. RET/PTC3 and activating mutant BRAF constructs induced tyrosine phopsphorylation and erk activation, respectively.

Differential expression of mirna in human papillary thyroid cancer. Expression levels of mirna-146a(a), -146b(B), -221(C) and -222(D) were classified into increased or decreased group. 3.

4 대한내분비학회지 : 제 24 권제 1 호 2009 A B Fig. 2. RET/PTC3 rearrangement and BRAF induced expression of mirna-146a and mirna-221. A. RET/PTC3 and activating mutant BRAF constructs induced tyrosine phopsphorylation and erk activation, respectively. B. RET/PTC3 rearrangement and BRAF induced expression of mirna-146a and mirna -221, whereas kinase-dead form of RET/PTC3 rearrangement did not. A B C D Fig. 3. Differential expression of mirna in human papillary thyroid cancer. Expression levels of mirna-146a(a), -146b(B), -221(C) and -222(D) were classified into increased or decreased group. 3. mirnas와예후인자와의관계세포주를이용한실험에서 10% 우태아혈청과같은성장인자나종양유전자들과 mirna 146a/b, mirna-221 및 mirna-222의발현이연관이있음을확인한본연구진은갑상선유두암조직및반대편엽의정상조직에서 RNA를추출한후, 이들 mirnas의발현여부를확인하였다. 그리고, 반정량적분석을통해암조직에서정상조직보다이들 mirnas의발현비율이높은군과낮은군으로분류하였다 (Fig. 3). 양군의나이, 성별, 진단당시의종양의크기, 다발성유무, 현미경적주변조직으로의침윤여부, T 병기, 림프절전이여부및 TNM 병기등을비교, 분석하였으나, mirna-146a/b, mirna-221 및 mirna-222의발현에따른예후인자의의미있는차이를관찰할수없었다 (Table 2,3)

5 - 조영석외 5 인 : 갑상선암에서 mirna 146a/b, 221 및 222 의발현에대한연구 - Table 2. Relationship between expression of mirna-146a/b and clinicopathological factors Decreased Group Increased Group n (%) n (%) P value Age (years) 49.7 ± ± Sex Male 3 (12) 3 (12) 1 Female 22 (88) 22 (88) Size (mm) 10.9 ± ± Multifocality Negative 20 (80) 22 (88) 0.44 Positive 5 (20) 3 (12) Capsule invasion (microinvasion) Negative 7 (28) 9 (36) 0.54 Positive 18 (72) 16 (64) T-stage I 6 (24) 10 (40) 0.23 III 19 (76) 15 (60) Lymph node metastasis N0 18 (72) 21 (84) 0.31 N1a 7 (28) 4 (16) TNM stage I 9 (36) 15 (60) 0.09 III 16 (64) 10 (40) Table 3. Relationship between expression of mirna-221/222 and clinicopathological factors Decreased Group Increased Group n (%) n (%) P value Age (years) 49.5 ± ± Sex Male 1( 5) 5(16.7) 0.21 Female 19(95) 25(83.3) Size (mm) 10.5 ± ± Multifocality Negative 17(85) 25(83.3) 0.87 Positive 3(15) 5(16.7) Capsule invasion (microinvasion) Negative 7(35) 9(30) 0.71 Positive 3(15) 21(60) T-stage I 6(30) 10(33.3) 0.8 III 14(70) 20(66.7) Lymph node metastasis N0 15(75) 24(80) 0.68 N1a 5(25) 6(20) TNM stage I 7(35) 17(56.7) 0.13 III 13(65) 13(43.3) 고찰최근들어, BRAF V600E 돌연변이, RET/PTC 재배열을비롯한갑상선유두암의종양유전자가규명되면서이를이용한진단방법이다양하게제시되고있다 [19~22]. 그러나, 갑상선유두암환자에서임상적예후인자와연관된분자수준의조절물질은명확히규명되지않은상태이다. 본연구에서는 갑상선유두암에서 mirnas의발현양상을조사하여새로운분자표지자로서의활용가능성을살펴보고자하였다. mirna의임상적의의는폐암에서 let-7 mirna의발현이수술후생존기간의감소와연관성이있으며 [10], pre -mirnas에서 mirnas의생성에관여하는 RNAse III 효소인 Dicer의발현이감소되어있는경우나쁜예후를시사하는것으로도보고된바있다 [23]. 이러한연구를바탕으로

6 대한내분비학회지 : 제 24 권제 1 호 2009 갑상선암을비롯한다양한암에서 mirnas의진단및예후인자적가치가연구되고있으나, 폐암을제외한나머지암에서는진단및예후인자와연관된새로운 mirnas가발굴되지못한상태이다 [8]. He 등 [13] 이갑상선유두암에서 mirna 146a/b, mirna 221 및 mirna 222의발현이정상조직에비해 11배내지 19배증가되어있으며, KIT의발현을조절한다고보고한이후이들 mirnas와갑상선암세포의성장과의연관성이연구되고있다 [24]. 본연구에서도 mirna 146a/b, mirna 221 및 mirna 222가갑상선암세포주및갑상선암조직에서증가되어있는지확인하고증가된 mirnas의예후인자적가치를규명하고자하였다. 그결과 mirna 146a/b, mirna 221 및 mirna 222는 TPC1 및 FRO와같은특정갑상선암세포주에서발현되고있으며, NPA에 10% 우태아혈청을처리할경우발현이유도됨을확인할수있었다. 또한, HEK293T세포주에 RET/PTC3 rearrangement와 BRAF V600E 와같은종양유전자를도입할경우이들 mirnas의발현이증가됨도확인하였다. 이러한결과는우태아혈청이나종양유전자와같은성장신호가이들 mirnas의발현을유도하며, 비록증가된이들 mirnas의발현이세포의성장에미치는영향을본연구에서직접규명하지는못하였으나, 기간의연구와종합하여판단할때이들 mirnas가세포의성장과증식에연관성이있음을시사하는자료라고사료된다 [13,25]. 또한, 50예의갑상선암조직에서이들 mirnas의발현을조사한결과, mirna-146a/b의경우는 25예 (50%) 에서그리고 mirna-221과 mirna-222의경우에는 30예 (60%) 에서이들 mirnas의발현이정상조직에비해 2배내지 6 배정도로증가되어있음을확인하였다. 앞서언급한바와같이, 종양에서발현이증가하거나감소하는 mirnas는종양의진단및임상예후를반영하는중요한수단으로주목을받고있다 [26,27]. 예를들어, let-7/ mir-98과같은 mirnas의발현이감소하면, 이들 mirnas 에의한 Ras 종양유전자의발현을억제하는기능이상쇄되어세포의성장신호가증폭되는것으로알려져있다 [28]. 반대로, mirna-17과같은 mirnas는 myc의발현을증가시켜종양유전자와같은역할을하게된다 [29]. 본연구에서관찰한 mirna-221및 mirna-222의경우, 갑상선유두암에서 p27(kip) 의발현을억제하는것으로보고되고있으며, p27의발현이이들 mirnas의발현과역의상관관계를가짐이입증된바있다. 또한, TPC-1과같은갑상선암세포주에 mirna-221 및 mirna-222의발현을유도할경우, 세포주기가 S phase로진행하게된다 [25]. 흥미롭게도, 암주변정상조직에서도 mirna-221 및 mirna -222의발현이증가되어있는경우가확인되고있는데, 이는조직학적으로는정상인조직이지만분자수준에서암세포와 유사한변형이시작되었음을시사하는소견이라할수있겠다 [13]. 이러한연구결과는갑상선암의발생과정에서종양주변부의조직학적정상조직이종양의 epigenetic alteration에유사한변화를가지고있으며, 이러한과정에서 mirna-221과 mirna-222가종양의생성을유도함을반영한다고할수있다. mirna-146a/b는면역반응에서중요한역할을하는것으로알려지고있는데, IL-1 receptor-associated kinase 1과 TNF receptor-associated factor 6의발현을감소시켜 Toll-like receptor와연관된신호체계의 negative regulator 로작용한다 [30]. 갑상선암에서는이들 mirna의발현이증가되어있다는보고와더불어, pre-mirna-146a의다형성 (polymorphism) 이갑상선유두암의유전학적소인으로제시된바있다 [31]. 본연구에서는갑상선암세포주에서성장인자나종양유전자의도입에의해 mirna-146a/b, mirna-221 및 mirna -222의발현이증가되며, 갑상선유두암조직에서도이들 mirna가증가된경우가상당수존재함을입증할수있었다. 이러한결과는 mirnas가갑상선세포의변형에중요한역할을하며갑상선암의진단에유용한방법으로이용될가능성을시사하는것으로, 향후갑상선유두암과연관된새로운 mirnas를발굴하고, 이들의분자생물학적역할및임상적의의를규명하는연구가꾸준히수행되어야하겠다. 요약연구배경 : 최근들어, mirnas을진단표지자또는치료표적으로활용하고자하는연구들이활발히수행중이다. 그러나, 갑상선암에서이들 mirnas와관련된연구는미미한실정이다. 이에저자들은갑상선암에서증가되어있는것으로보고된바있는 mirna 146a/b, 221 및 222과성장인자또는종양유전자와의연관성을조사하고, 이들 mirnas가예후인자에미치는영향을규명하고자하였다. 방법 : 성장신호및종양유전자와의연관성을확인하기위하여 NPA 세포주에 10% 우태아혈청을처리하거나 HEK293T 세포주에 RET/PTC3 또는 BRAF V600E 발현벡터를형질도입한후한후, pre-mirnas 146a/b, 221과 222의발현량을측정하였다. 또한, 예후에미치는영향을파악하기위하여갑상선유두암조직에서이들 mirnas의발현량을측정한후다양한임상예후인자와의연관성을후향적으로분석하였다. 결과 : TPC1, NPA, FRO 및 ARO 세포주에서 mirna -146a/b와 mirna-221/222가발현되고있음을확인하였다. 또한, 10% 우태아혈청을 NPA 세포주에처리할경우 mirna-146a과 mirna-221 의발현이유도됨을알수있었다. HEK293T 세포주에 RET/PTC3 rearrangement와

7 - 조영석외 5 인 : 갑상선암에서 mirna 146a/b, 221 및 222 의발현에대한연구 - BRAF V600E 을발현시킨경우에도이들 mirnas의발현이증가되었다. 임상연구에서도정상조직과비교하여갑상선유두암의 50% (25예) 와 60% (30예) 에서 mirna-146a/b와 mirna -221/222가각각증가되어있었다. 그러나, 이들 mirnas의발현증가와임상예후인자들과의연관성은확인할수없었다. 결론 : 갑상선유두암의종양유전자인 BRAF V600E 와 RET/PTC3 rearrangement에의해 mirna 146a/b, 221 및 222의발현이증가됨을확인하였으며, 이에따라이들 mirna가갑상선유두암의암화과정에관여할것으로추측할수있었다. 그러나, 갑상선유두암환자의임상조직학적예후인자들과의연관성은확인할수없었다. 참고문헌 1. Lee RC, Feinbaum RL, Ambros V: The C. elegans heterochronic gene lin-4 encodes small RNAs with antisense complementarity to lin-14. Cell 75: , Wightman B, Ha I, Ruvkun G: Posttranscriptional regulation of the heterochronic gene lin-14 by lin-4 mediates temporal pattern formation in C. elegans. Cell 75: , Griffiths-Jones S, Grocock RJ, van Dongen S, Bateman A, Enright AJ: mirbase: microrna sequences, targets and gene nomenclature. Nucleic Acids Res 34:D140-D144, Zeng Y: Principles of micro-rna production and maturation. Oncogene 25: , Hammond SM, Boettcher S, Caudy AA, Kobayashi R, Hannon GJ: Argonaute2, a link between genetic and biochemical analyses of RNAi. Science 293: , Cerutti H, Casas-Mollano JA: On the origin and functions of RNA-mediated silencing: from protists to man. Curr Genet 50:81-99, Chen CZ: MicroRNAs as oncogenes and tumor suppressors. N Engl J Med 353: , Esquela-Kerscher A, Slack FJ: Oncomirs - micrornas with a role in cancer. Nat Rev Cancer 6: , Lu J, Getz G, Miska EA, Alvarez-Saavedra E, Lamb J, Peck D, Sweet-Cordero A, Ebert BL, Mak RH, Ferrando AA, Downing JR, Jacks T, Horvitz HR, Golub TR: MicroRNA expression profiles classify human cancers. Nature 435: , Takamizawa J, Konishi H, Yanagisawa K, Tomida S, Osada H, Endoh H, Harano T, Yatabe Y, Nagino M, Nimura Y, Mitsudomi T, Takahashi T: Reduced expression of the let-7 micrornas in human lung cancers in association with shortened postoperative survival. Cancer Res 64: , Iorio MV, Ferracin M, Liu CG, Veronese A, Spizzo R, Sabbioni S, Magri E, Pedriali M, Fabbri M, Campiglio M, Menard S, Palazzo JP, Rosenberg A, Musiani P, Volinia S, Nenci I, Calin GA, Querzoli P, Negrini M, Croce CM: MicroRNA gene expression deregulation in human breast cancer. Cancer Res 65: , Voorhoeve PM, le Sage C, Schrier M, Gillis AJ, Stoop H, Nagel R, Liu YP, van Duijse J, Drost J, Griekspoor A, Zlotorynski E, Yabuta N, De Vita G, Nojima H, Looijenga LH, Agami R: A genetic screen implicates mirna-372 and mirna-373 as oncogenes in testicular germ cell tumors. Cell 124: , He H, Jazdzewski K, Li W, Liyanarachchi S, Nagy R, Volinia S, Calin GA, Liu CG, Franssila K, Suster S, Kloos RT, Croce CM, de la Chapelle A: The role of microrna genes in papillary thyroid carcinoma. Proc Natl Acad Sci U S A 102: , Cahill S, Smyth P, Denning K, Flavin R, Li J, Potratz A, Guenther SM, Henfrey R, O'Leary JJ, Sheils O: Effect of BRAF V600E mutation on transcription and post-transcriptional regulation in a papillary thyroid carcinoma model. Mol Cancer 6:21, Cahill S, Smyth P, Finn SP, Denning K, Flavin R, O'Regan EM, Li J, Potratz A, Guenther SM, Henfrey R, O'Leary JJ, Sheils O: Effect of ret/ptc 1 rearrangement on transcription and post-transcriptional regulation in a papillary thyroid carcinoma model. Mol Cancer 5:70, Jung HS, Kim DW, Jo YS, Chung HK, Song JH, Park JS, Park KC, Park SH, Hwang JH, Jo KW, Shong M: Regulation of protein kinase B tyrosine phosphorylation by thyroid-specific oncogenic RET/ PTC kinases. Mol Endocrinol 19: , Kim DW, Chung HK, Park KC, Hwang JH, Jo YS, Chung J, Kalvakolanu DV, Resta N, Shong M: Tumor suppressor LKB1 inhibits activation of signal transducer and activator of transcription 3 (STAT3) by thyroid oncogenic tyrosine kinase rearranged in

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Jkbcs016(92-97).hwp

Jkbcs016(92-97).hwp Expression of bcl-2 and Apoptosis and Its Relationship to Clinicopathological Prognostic Factors in Breast Cancer - A Study with Long Term Follow-up correlated with the survival rate.(journal of Korean