C, Western blot was performed to measure the protein levels of miRNA-21 target proteins, PDCD4 and PTEN in anti-miR-21 transfected cells. (p 0.05) and increased miRNA-21 target protein expression. We conclude that HBV infection enhances cell proliferation, at least in part, via HBx-induced miRNA-21 expression during hepatocellular carcinoma progression. Introduction Hepatocellular carcinoma (HCC) is one of the major malignant neoplasm, affecting more than half a million people worldwide each year, and has a Licochalcone C multifactorial etiology including hepatitis B or hepatitis C infections and alcoholism . Several studies have shown that there is an association between hepatitis B infection and HCC, but precise molecular mechanisms Licochalcone C that regulate the proliferation in these cells remain unknown , . Several studies have shown that the X open reading frame of Hepatitis B oncogenic viral genome, encoding a 17 kD protein, is necessary for in vivo infection and stimulates the HBV replication by increasing mitochondrial calcium uptake as well as a critical contributor in the initiation of neoplastic transformation Rabbit Polyclonal to SPI1 C. Paterlini et al. found that HBV DNA was integrated in the chromosomal DNA of hepatocytes in HBV-related HCC patients negative for HBV surface antigen (HBsAg), but positive for X-transcript, implying a significant role of the integrated-X gene in transformation . HBx knock-in transgenic mice at the p21 locus developed liver tumor at 18 months after birth, suggesting the oncogenic potential of X protein . Several studies reveal that HBx can promote proliferation, motility and invasion in human hepatocytes by up-regulating MEKK2, MIG, MMP-9, IKK and Capn4 C. It is also suggested that HBx may possibly alter the adhesion-de-adhesion balance of the cells in the primary tumor site, favoring integrin-mediated cell migration as well as modulate cell cycle regulatory proteins of G1 phase in a calcium-dependent manner , . Emerging data show that miRNAs are involved in HBx-induced cell proliferation and invasion in HCC C. microRNAs (miRNAs) are endogenous, non-coding 22 nucleotide RNA molecules, shown to modulate gene expression via post-transcriptional manner, thus becoming crucial regulators in complex gene regulatory networks. Several studies are available to show the deregulated expression of miRNAs in cancer and reveal a key role in the initiation and progression of the disease . It was shown that at least 17 miRNAs were down-regulated in HCC, which in turn activated many oncogenic pathways including cell cycle progression, while 6 miRNAs were up-regulated, which were responsible for the anti-tumor immune response . Recently it was shown that miRNA-148a is down regulated by HBx and caused increased tumorigenesis . In another study, HBx up-regulated miRNA-143, thereby promoting metastasis . Very limited data is available on the role of HBx-induced miRNAs in HCC. MicroRNA-21 (miRNA-21) is a multifaceted microRNA, regulating multiple genes involved in several cellular programs. APAF1, the core of the apoptosome, essential for activating caspases to initiate apoptosis, contains a miRNA-21 target Licochalcone C site in its 3UTR, and is found to be down-regulated while miRNA-21 is up regulated in gliomas, augmenting proliferation . MiRNA-21 induces AP-1 activity in response to ras onco-protein by directly repressing tumor-suppressor gene PDCD4, contributing to tumorigenesis by auto-regulatory mechanism . In anaplastic thyroid carcinoma and lung cancer, early activation of ras and its two downstream pathways raf-MAPK and PI3K pathways triggered high miRNA-21 expression in neoplastic transformation in vivo . It has been reported that miRNA-21 plays a vital role in keratinocyte migration and in re-epithelialization during wound healing, directly targeting TIMP3 in vitro and in vivo . By down-regulating PDCD4, miRNA-21 contributes to glioblastoma proliferation while its inhibition induced apoptosis and decreased cell cycle progression, down-regulating EGFR, activated akt, cyclin D and bcl-2 in vitro and in vivo suggesting an important therapeutic potential of miRNA-21 , . Thus, the fact that miRNA-21 is frequently elevated in most malignancies and its in vivo knockdown suppresses tumorous potential, suggests that its high levels are essential for promoting pathological cell growth. In HCC tissues and multiple HCC cell lines, it promoted cell proliferation, invasion and migration by repressing the expression of tumour-suppressor genes Programmed Cell Death Protein-4 (PDCD4) and Phosphatase and Tensin homologue (PTEN) , . HBx and.