Hsa-let-7g inhibits proliferation of hepatocellular carcinoma cells by downregulation of c-Myc and upregulation of p16 INK4A Fei-Fei Lan 1 , Hua Wang 2 , Yang-Chao Chen 2 , Chu-Yan Chan 2 , Samuel S. Ng 3 , Kui Li 1 , Dan Xie 4 , Ming-Liang He 2 , Marie C. Lin 5 and Hsiang-Fu Kung 1,2,4 1 Laboratory of Integrated Biosciences, School of Life Sciences, Sun Yat-sen University, Guangzhou, China 2 Stanley Ho Center for Emerging Infectious Diseases, and Li Ka Shing Institute of Health Sciences, The Chinese University of Hong Kong, Hong Kong, China 3 Department of Chemistry, The University of Hong Kong, Hong Kong, China 4 State Key Laboratory of Oncology in Southern China, Cancer Center, Sun Yat-Sen University, Guangzhou, China 5 Brain Tumor Center, Department of Surgery, PWH, The Chinese University of Hong Kong, Hong Kong, China zMicroRNAs (miRNAs) are endogenously expressed small noncoding RNAs that regulate approximately one-third of human genes at post-transcription level. Previous studies have shown that miRNAs were implicated in many cellular processes and participated in the progress of various tumors including hepatocellular carcinoma (HCC). Among all miRNAs, the let-7 family is well recognized to play pivotal roles in tumorigenesis by functioning as potential growth suppressor. In the present study, we aimed to investigate the role of let-7 family, particularly the hsa-let-7g, in the molecular pathogenesis of HCC. By use of MTT, qPCR, Western blotting and 2-dimensional electrophoresis (2-DE), over-expression of hsa-let-7g was found to inhibit the proliferation of HCC cell line via negative and positive regulations of c-Myc and p16 INK4A , respectively. The expression of hsa- let-7g was noted to be markedly lowered in the HepG2, Hep3B and Huh7 cells, yet higher in the Bel-7404 HCC cell line. Proliferation of HCC cell line was significantly inhibited after the transfection of hsa-let-7g mimics, while hsa-let-7g inhibitor transfection exerted an opposite effect. Concurrently, the mRNA and protein levels of c-Myc were found significantly decreased in HepG2 cells after transfection of hsa-let-7g mimics, but obviously increased in Bel-7404 cells after transfection of hsa-let-7g inhibitor. As revealed by 2-DE, a significant upregulation of p16 INK4A was revealed after the gain-of-function study using hsa-let-7g. Therefore, we suggest that hsa-let-7g may act as a tumor suppressor gene that inhibits HCC cell proliferation by downregulating the oncogene, c-Myc, and upregulating the tumor suppressor gene, p16 INK4A . Hepatocellular carcinoma (HCC) is one of the most common malignancies worldwide that accounts for about 90% of all primary liver cancers. 1 It is a highly invasive and destructive tumor with its incidence increased exponentially all over the world in the last decade. 2 MicroRNAs (miRNAs) are a class of single-strand and highly conserved noncoding small RNAs that regulate gene expression. They induce translational repression or mRNA degradation by binding to the 3 0 -untranslated regions (3 0 UTR) of the target mRNAs. 3,4 They exist broadly in eukar- yote and have also been reported in some encoded sequences of viral genomes (http://microrna.sanger.ac.uk/sequences/). To date, there are over 80 species (i.e., 1,000 different mammalian miRNA genes) being identified. 4,5 It is generally believed that miRNAs have multibiological functions includ- ing regulation of cellular activities related to development, differentiation, inflammatory response, metabolism, prolifera- tion, apoptosis, viral infection and tumorigenesis. 5–13 Studies have demonstrated that miRNA expression is frequently aberrant in human malignancies; therefore, they have been suggested as potential oncogenes or tumor suppressors. 11,14– 19 Expression of miRNA is commonly applied in diagnosing, staging, prognosis and evaluation of tumor treatment. 20–22 In recent studies, miRNAs were noted to participate in hepatocarcinogenesis as revealed by microarray analysis. 23,24 Aberrant expressions of some miRNAs such as miR-17-92 cluster and miR-21 have been found to play crucial roles in HCC. 25,26 Among all human cancer-related miRNAs, let-7 Key words: hsa-let-7g, hepatocellular carcinoma, c-Myc, p16 INK4A Abbreviations: c-Myc: v-myc myelocytomatosis viral oncogene homolog; 2-DE: 2-dimensional electrophoresis; DMEM: Dulbecco’s Modified Eagle Media; HCC: hepatocellular carcinoma; miRNAs: microRNAs; p16 INK4A : cyclin-dependent kinase inhibitor 2A; qPCR: quantitative real-time PCR; 3 0 UTR: 3 0 -untranslated regions Additional Supporting Information may be found in the online version of this article. Grant sponsor: National Basic Research Program of China; Grant number: 2010CB912800 DOI: 10.1002/ijc.25336 History: Received 11 Aug 2009; Accepted 9 Mar 2010; Online 22 Mar 2010 Correspondence to: Hsiang-Fu Kung, Room 511A, Stanley Ho Centre for Emerging Infectious Diseases, Basic Medical Sciences Building, The Chinese University of Hong Kong, Shatin, HKSAR, Hong Kong, China, Tel.: þ852-2603-7743, Fax: þ852-2994-4988, E-mail: hkung@cuhk.edu.hk; or Marie C. Lin, Brain Tumor Center, Department of Surgery, PWH, The Chinese University of Hong Kong, Hong Kong, China, Tel.: þ852-2299-0776, E-mail: mcllin@hkusua.hku.hk Cancer Cell Biology Int. J. Cancer: 128, 319–331 (2011) V C 2010 UICC International Journal of Cancer IJC