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1 Biomedical Science Letters 2013, 19(4): 348~352 eissn : Brief Communication Down-regulation of mir-34a Expression in Cervical Intraepithelial Neoplasia with Human Papillomavirus Infection and Its Relationship with p53 Expression Kyung Eun Lee Department of Clinical Laboratory Science, College of Health Sciences, Catholic University of Pusan, Busan , Korea micrornas (mirnas) play pivotal roles in controlling cell proliferation and differentiation. mirna expression in human is becoming recognized as a new molecular mechanism of carcinogenesis. microrna-34a (mir-34a), a member of the p53 network, was found to be regulated in multiple types of tumor. The purpose of this study was to define roles of mir-34a expression in cervical intraepithelial neoplasia with human papillomavirus infection, and its relationship with p53 protein expression. This study was performed to analyze expression of mir-34a by using qrt-pcr, and to evaluate p53 protein expression by using immunohistochemistry in 40 cases. Down-regulation of mir-34a expression was detected in 27 (67.5%) out of 40 cases and Immunoreactivity for p53 was found in 17 (42.5%) out of 40 cases. Nineteen (82.6%) of the 23 cases with a negative p53 expression showed a down-regulation mir-34a expression, there was a significant associations between mir-34a and p53 protein expression (P=0.04). These results suggest that mirna-34a expression tend to be reduced depending on the advanced histologic grade, and down-regulation of mir-34a expression might be associated with inactivation of p53 protein expression by human papillomavirus infection. Key Words: mir-34a, p53, Cervical intraepithelial neoplasia, Human papillomavirus 자궁경부암의주요원인으로알려진인유두종바이러스의감염은대부분일시적이지만소실되지않고지속되면자궁경부의세포변화를초래하게되며, 자궁경부상피내종양이진행될수록자연적소실가능성은희박해지고침윤성자궁경부암으로진행된다고한다 (Myers et al., 2000; Pinto and Crum, 2000). 이러한자궁경부종양발생은인유두종바이러스감염에의한 p53의활성화와연관성이있다고알려지면서 p53 단백발현에대한연구가다양하게이루어지고있다. 자궁경부종양에서인유두종바이러스가감염되면 HPV E6 및 HPV E7 단백질발현이증가되고이들단백질이종양억제유전자인 p53 및 prb * Received: August 26, 2013 / Revised: December 27, 2013 Accepted: December 27, 2013 Corresponding author: Kyung-Eun Lee. Department of Clinical Laboratory Science, College of Health Sciences, Catholic University of Pusan, Busan , Korea. Tel: , Fax: kelee@cup.ac.kr C The Korean Society for Biomedical Laboratory Sciences. All rights reserved. 유전자를억제하여세포분열주기의조절기능이제대로작동하지않아서암이발생하는것으로알려져있다 (Benchimol et al., 1985; Finlay et al., 1988; Levin et al., 1991). 최근에는단백질을 coding 하지않는유전자도암발생에관여한다고밝혀지면서, 특히 microrna (mirna) 에대한관심이높아지고있다. mirna는약 20개내외의 nucleotide로이루어진작은단일가닥의 non-coding RNA 이다 (Ma and Weinberg, 2007). mirna는주로표적 mrna 의 3' UTR (untranslated region) 에특이적으로결합하여해당 RNA의번역 (translation) 을억제하거나또는유전자발현을억제하는등전사후조절에관여함으로써세포의증식, 분화, 사멸등을조절하는것으로알려져있다 (Calin and Croc, 2006). p53의발현이종양억제 mirna 의가공을촉진시킨다고하였으며 (Suzuki et al., 2009), 주요종양억제 mirna로알려진 mir-34a가 p53을조절하는또다른단백질 SIRT1 의발현을억제함으로써세포자멸사를조절한다고도보고되었다 (Yamakuchi et al., 2008)

2 또다른연구에서는췌장암세포, 대장암, 원발성신경아세포종에서 mir-34a 발현이감소되었다고하였으며 (Lodygin et al., 2008), HPV E6 종양단백이 p53 유전자기능을억제하게되면 mir-34a 발현에도영향을미칠수있다고도하였다 (Chang et al., 2007). 이와같이 mirna 는다양한발암관련유전자의동시조절기전에관여하는것으로알려져있다. 그러나종양발생에있어서 mir-34a 역할및 p53 단백발현과의연관성은명확히밝혀지지않은상태이며, 특히국내에서는인유두종바이러스에감염된인체종양을대상으로한연구는부족한실정이다. 따라서본연구에서는인유두종바이러스에감염된자궁경부상피내종양파라핀블록을이용하여 mir-34a 발현및 p53 단백발현과의연관성분석을통해자궁경부종양의새로운발생기전을알아보고자한다. 본연구는 2011 년 1월부터 2011 년 12월까지부산모병원병리과에서자궁경부생검을통하여이형성증및상피내암으로진단받은조직을대상으로하였다. 슬라이드표본을검사하여생검전약물요법등의보조적치료를받지않은조직중비교적고정이잘된파라핀블록 40예를사용하였다. 본연구의조직슬라이드는병리의사가재검토하여진단분류의일치성을높였다. 파라핀블록을 10 μm 두께로박절한후자일렌에서탈파라핀을실시하였다. RcoverAll TM Total Nucleic Acid Isolation Kit (Ambion, Austin, TX, USA) 를이용하여 RNA 를추출한후, TaqMan MicroRNA Reverse Transcription kit (Applied Biosystems, Carlsbad, CA, USA) 로 RT-PCR 을실시하였다. mir-34a 정량을위해 TaqMan MicroRNA Assays 1.0 μl, cdna 1.33 μl와 TaqMan Universal PCR master Mix 10 μl를섞은후에 Real-time PCR (BioRad, USA) 에서 95 10분, 95 15초, 60 60초를총 40회반복하였다. 대조군인 RNU6B와 mir-34a의 Ct값을표준화 (normalization) 한후상대정량법으로분석하였다. Applied Biosystems 에서제공하는 mir-34a (Part Number P/N Assay ID TM00042, RT000426) 및 RNU6B (Part Number P/N ) 에대한 RT primer 를사용하였다. 4 μm 두께로절편된조직을자일렌에서탈파라핀하고함수과정을거친후흐르는물에서 10분간씻어냈다. 항원성회복을위하여 ph 9.0 Tris-EDTA 용액으로전자레인지에서 10분간끓이는전처리를하였다. 조직내의내인성과산화효소를비활성화시키기위하여실온에서 Hydrogen peroxide block (Lab vision, Lab Vision, Fremont, Table 1. Expression of mir-34a in cervical intraepithelial neoplasia with human papillomavirus infection Histology Up-regulation mir-34a expression Down-regulation CIN I 6 (60) 4 (40) CIN II 3 (30) 7 (70) CIN III 3 (30) 7 (70) CIS 1 (10) 9 (90) CIN; Cervical Intraepithelial Neoplasia CIS ; Carcinoma in Situ USA) 에서 10분간반응시킨후 Tris buffered saline (ph 7.4) 로세척하였다. 비특이적인반응을줄이기위하여 Blocking solution (Life Science, Mukilteo, USA) 에서 10분간더반응시켰다. 수세과정없이토끼단클론성일차항체 p53 (Lab Vision, Fremont, USA) 을 1:100으로희석하여실온에서한시간동안반응시킨후, Broad antibody enhancer (Life Science, Mukilteo, USA) 에서 10분간, Polymer-HRP (Life Science, Mukilteo, USA) 에서 30분간반응시켰다. 면역염색후 DAB (3,3'-Diaminobenzidine) 로발색시킨후 Gill's hematoxylin으로대조염색하였다. 면역조직화학염색결과 p53 단백은종양세포의핵에갈색으로염색될때를양성으로판정하였다. 100배시야에서염색되지되지않은경우즉, 0% 를 0점, 25% 미만의종양세포가염색되는경우 1점, 26~50% 2점, 50% 이상을 3점으로하였다. 그리고염색강도에따라음성은 0점, 약양성인경우를 1점, 중등도양성인경우를 2점, 강양성을 3점으로분류하였다. 염색강도에따른점수와분포점수를더한값이 0~2점이면음성, 3점이상이면면역염색양성으로판정하였다. 통계처리는 SPSS (Version 17.0, SPSS Inc., Chicago, IL, USA) 을이용하였다. mir-34a 발현과 p53 단백발현과의연관성을평가하기위해서는 χ 2 검정방법을사용하여분석하였고, 통계학적으로 P 0.05일때유의한것으로간주하였다. 인유두종바이러스에감염된자궁경부상피내종양으로진단된파라핀블록총 40예는각각 Cervical intraepithelial neoplasia I (CIN I) 10예 (25%), CIN II 10예 (25%), CIN III 10예 (25%), Carcinoma in situ (CIS) 10예 (25%) 로분류되었다. mir-34a 발현은총 40예중 27예 (67.5%) 에서감소되었고, 13예 (32.5%) 에서증가하였다 (Table 1). 자궁경부상피내종양의등급에따른 mir-34a P

3 A B C Fig. 1. Immunohistochemical studies for p53 in cervical intraepithelial neoplasia with human papillomavirus infection; negative nuclear reaction (A; CIN II 400), but a positive reaction (B; CIN I, C; CIN II, 400) identified by brown staining. Table 2. Relationship between mir-34a and p53 protein expression in cervical intraepithelial neoplasia with human papillomavirus infection Expression mir-34a p53 protein expression Negative Positive Down-regulation 19 (82.6) 8 (47.1) Up-regulation 4 (17.4) 9 (52.9) 발현을살펴보면 CIN I에서는 10예중 4예 (40%) 에서감소되었고, CIN II와 CIN III에서는 10예중 7예 (70%) 에서감소되었고, CIS에서는 10예중 9예 (90%) 에서발현이감소되었다 (Table 1). 따라서자궁경부상피내종양이진행될수록 mir-34a 발현량이감소하는경향을보였으나, 통계적으로는유의하지않았다 (P=0.12). p53 단백은 40예중 17예 (42.5%) 에서양성발현을나타냈고, 23예 (57.5%) 에서음성발현이나타났다 (Table 2, Fig. 1). p53 단백발현과 mir-34a 발현의상관성을분석한결과, p53 단백발현이음성일때 mir-34a 발현이감소되는경우가총 23예중 19예 (82.6%) 로가장높게나타났으며, 통계적으로유의한차이가관찰되었다 (P=0.04, Table 2). p53 단백의불활성화는다양한인체암종의발암기전에관련이있는것으로알려져있다. p53 유전자의돌연변이에의해서 p53 단백의기능이상실되기도하고, HPV 의 E6 단백에의해 p53 단백이분해되어불활성화가일어나기도한다 (Scheffner et al., 1993; Herrington, 1995). 자궁경부종양에서는인유두종바이러스감염에의한 p53 P 0.04 단백의불활성화가대부분을차지하고있으며 p53 유전자의돌연변이에의한단백기능상실은인유두종바이러스감염이없는자궁경부종양에서발생한다고보고되었다 (Scheffner et al., 1993; Herrington, 1995). 많은연구에서자궁경부종양에서의 p53 단백발현은통계적으로차이가없다는보고도있고 (Kim et al., 2003), 자궁경부종양의 80% 이상에서단백발현이관찰된다는결과등다양하게보고되고있다 (Park et al., 2001). 본연구에서 p53 단백발현은자궁경부종양 40예중 17예 (42.5%) 에서양성으로관찰되었으며, 23예 (57.5%) 에서음성으로관찰되었다. 이는인유두종바이러스감염에의한종양억제유전자의기능억제로추측할수있으나, 발암관련 p53 단백발현에관한기전을설명하기위해서는좀더많은추가연구가필요할것으로생각된다. Lu 등 (2005) 의연구에서는 mirna는정상조직에비해암조직에서발현량이낮게나타나므로종양억제유전자로서의가능성을보고하였다. 따라서 mirna 생성에직접적으로관련된유전자를 knock-down 시켜전반적인 mirna를감소시켰을때종양형성이촉진된다고하였다 (Lu et al., 2005; Kumar et al., 2007). 그러나현재까지 200여개의 mirna가보고되고있으나, 대부분의기능이밝혀지지않고있다. 그중에서 mir-34a는췌장암및대장암의발생에관여하는종양억제 mirna라고알려져있으며 (Lodygin et al., 2008), 췌장암세포에서 mir-34a 발현을증가시키면암세포침윤이억제된다고보고되었다 (Ji et al., 2009). 본연구에서도자궁경부종양 40예중 27예 (67.5%) 에서 mir-34a 발현이감소되는것을관찰하였으며, 통계학적으로유의한차이는없었으나자궁경부상피내종양이진행될수록 mir-34a의발현량이감소되는경

4 향을나타내었다. 이는인유두종바이러스에감염되면 HPV E6 단백이 mir-34a 발현을억제시킨다는다른연구결과 (Zheng and Wang, 2011) 와일치하였다. 인유두종바이러스가 HPV E6 종양단백에의해활성화되면종양억제유전자인 p53 유전자의기능이억제되고, p53 유전자를특이적으로인지하는부위가있는 mir-34a 의발현에영향을미친다고알려져있다 (Zheng and Wang, 2011). Wang 등 (2009) 은고위험군인유두종바이러스의 E6 단백은 mir-34a 발현을감소시킨다고하였으며, 다른연구에서는인유두종바이러스에감염된자궁경부암세포내 E6 단백발현을억제시켰더니 p53 유전자와 mir- 34a 발현량이증가되었다고하였다 (Kivi st al., 2008; Zheng and Wang, 2011). 본연구에서도 p53 단백발현이음성으로관찰될때 mir-34a 발현이감소되는경우가 23예중 19예 (82.6%) 로높게관찰되었으며통계적으로유의한차이가나타났다 (P=0.04). 이는인유두종바이러스감염에의한 HPV E6-p53 복합체와 mir-34a의상호작용에의한발암기전을설명해주는결과라고생각된다. 하지만다른연구에서는 p53 유전자와별개로 mir-34a 의프로모터부위의 CpG island에서발생하는메틸화가 mir-34a 발현을불활성화시킬수있다고보고 (Lodygin et al., 2008) 되기도하였으므로향후지속적인연구가필요할것으로생각된다. 본연구에서는인유두종바이러스에감염된자궁경부상피내종양이진행될수록 mir-34a의발현이감소되는경향을확인할수있었으며, mir-34a 발현이감소될때 p53 단백발현이억제되는경향도확인할수있었다. 그러나연구재료의수가적어서상호간의연관성을단정하기는어려우므로향후더많은인체암종을대상으로추가적인연구가필요할것으로생각된다. 또한메틸화실험을추가적으로실시함으로써 mir-34a의불활성화에대한정확한원인규명이이루어져야할것이다. REFERENCES Benchimol S, Lamb P, Crawford LV, Shows TB, Bruns GA. Transformation associated p53 protein is encoded by a gene on human chromosome 17. Somatic Cell Mol Genet : Calin GA, Croce CM. MicroRNA signatures in human cancers. Nat Rev Cancer : Chang TC, Wentzel EA, Kent OA, Ramachandran K, Mullendore M, Lee KH, Feldmann G, Yamakuchi M, Ferlito M, Lowenstein CJ, Arking DE, Beer MA, Maitra A, Mendell JT. Transactivation of mir-34a by p53 broadly influences gene expression and promotes apoptosis. Mol Cell : Finlay CA, Hinds PW, Levine AJ. The p53 proto-oncogene can act as a suppressor of transformation. Cell : Herrington CS. Human papillomaviruses and cervical neoplasia. II. Interaction of HPV with other factors. J Clin Pathol : 1-6. Ji Q, Hao X, Zhang M, Tang W, Yang M, Li L, Xiang D, Desano JT, Bommer GT, Fan D, Fearon ER, Lawrence TS, Xu L. MicroRNA mir-34 inhibits human pancreatic cancer tumorinitiating cells. PLoS One : e6816. Kim MY, Cho SH, Park MH. Analysis of expression of p63 in cervical neoplasia comparing with other immunohistochemical markers. Korean J Pathol : Kivi N, Greco D, Auvinen P, Auvinen E. Genes involved in cell adhesion, cell motility and mitogenic signaling are altered due to HPV 16 E5 protein expression. Oncogene : Kumar MS, Lu J, Mercer KL, Golub TR, Jacks T. Impaired microrna processing enhances cellular transformation and tumorigenesis. Nat Genet : Levin AJ, Momandd J, Finlay CA. The p53 tumor supressor gene. Nature : Lodygin D, Tarasov V, Epanchintsev A, Berking C, Knyazeva T, Körner H, Knyazev P, Diebold J, Hermeking H. Inactivation of mir-34a by aberrant CpG methylation in multiple types of cancer. Cell Cycle : 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 : Ma L, Weinberg RA. MicroRNAs in malignant progression. Cell Cycle : Myers ER, McCrory DC, Nanda K, Bastian L, Matchar DB. Mathematical model for the natural history of human papillomavirus infection and cervical carcinogenesis. Am J Epidemiol : Park IS, Han HS, Kim TS. Immunohistochemical expression of p53, p21, and mdm2 proteins in human papillomavirus positive and negative invasive uterine cervical carcinomas

5 Korean J Pathol : Pinto AP, Crum CP. Natural history of cervical neoplasia: defining progression and its consequence. Clin Obstet Gynecol : Scheffner M, Huibregtse JM, Vierstra RD, Howley PM. The HPV- 16 E6 and E6-AP complex functions as a ubiquitin-protein ligase in the ubiquitination of p53. Cell : Suzuki HI, Yamagata K, Sugimoto K, Iwamoto T, Kato S, Miyazono K. Modulation of microrna processing by p53. Nature : Wang X, Wang HK, McCoy JP, Banerjee NS, Rader JS, Broker TR, Meyers C, Chow LT, Zheng ZM. Oncogenic HPV infection interrupts the expression of tumor-suppressive mir- 34a through viral oncoprotein E6. RNA : Yamakuchi M, Ferlito M, Lowenstein CJ. mir-34a repression of SIRT1 regulates apoptosis. Proc Natl Acad Sci USA : Zheng ZM, Wang X. Regulation of cellular mirna expression by human papillomaviruses. Biochim Biophys Acta :

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