Liver-specific Mir-122 Regulates The Biogenesis Of The OnmiR Mir-21 In Nucleus

MicroRNAs(miRNAs),a class of noncoding short RNAs of-22nt in length,play an essential role in gene regulation in animals and plants.MiRNAs are transcribed from genome and undergo several processing steps via a dedicated pathway.In the canonical pathway of miRNA biogenesis,a long primary transcript(pri-miRNA)is initially cleaved by RNase III DROSHA and its cofactor,DGCR8 to release a relative short hairpin intermediate,pre-miRNAs.The pre-miRNAs are then exported by the nuclear transport factor,exportin-5(XP05)to cytoplasm and then cleaved by Dicer,another RNase III type protein to generate a-22 nt miRNA duplexes.One strand of the duplexes becomes a mature miRNA and is preferentially assembled into the effector complex called miRNA-induced silencing complex(RISC).In the RISC,the mature miRNA acts as a guide by base pairing with its cognate mRNAs and induces translational repression or mRNA destabilization in cytoplasm,while the other strand of the duplex is degraded immediately.Although the prevailing view is that miRNAs execute their function in the cytoplasm,accumulating evidence has shown that miRNAs together with functional proteins such as Argonaute 2(Ago2)can localize in nucleus,suggesting that nuclear miRNAs may also regulate protein expression at the level of DNA as well as after transcription.For example,miR-21 was detected in both cytoplasm and nucleus in HeLa cells.Interestingly,miRNA-guided cleavage of sequence-complementary target RNAs in both HeLa cell cytoplasmic and nuclear extracts.Hwang et al.showed that miR-29b was predominantly present in the nuclei of HeLa and 3T3 cells,whereas the relevant miR-29a was mainly localized in the cytoplasm,implying that a unique hexanucleotide sequence(AGUGUU)at the 3' terminus of miR-29b may serve as signal to guide specific miRNA entering the nucleus.Using superquencher molecular beacon probes,Foldes-Papp et al.first showed that the cytoplasm-assembled mature miR-122 could re-enter into the nucleus in human liver cells.Subsequently,the distribution of miRNAs in both nucleus and cytoplasm has been widely shown by many investigators using systematic and microarray profiling approaches,suggesting that the presence of mature miRNAs in the nucleus is a general phenomenon in mammalian cells.It has been also reported that the level of miRNAs in the nucleus was decreased following the cell's conversion to a differentiated state,suggesting that nuclear miRNAs might play a role in maintaining the undifferentiated state and cortical development.Offering further evidence that mature miRNA can influence the maturation of primary miRNA(pri-miRNA),we demonstrated that mouse miR-709 acted as a posttranscriptional regulator of the miR-15a/16-1 transcript expression by directly binding to a recognition element on the pri-miR-15a/16-1 in the nucleus.In C.elegans,Zisoulis et al.showed that mature let-7 miRNA could bind to a specific site at the 3' end of its own primary transcripts and promote the maturation of primary let-7.Although these two studies revealed a novel picture of miRNA transcripts as the targets by other miRNAs,various functions of nuclear miRNAs especially the underlying mechanisms governing the gene regulation mediated by nuclear miRNAs remain largely unknown.Previous studies showed that miR-122,the most abundant miRNA in the liver,could serve as a pro-apoptotic factor in suppressing hepatocellular carcinoma cell migration and invasion.During hepatocyte tumorigenesis,miR-122 was strongly repressed.Although the underlying mechanism remains unclear,Bai et al.have reported that miR-122 sensitizes hepatocellular carcinoma(HCC)cells to sorafenib.In line with this,Xu et al.found reduction of miR-122 in sorafenib-resistant cells,and their study further showed that miR-122 overexpression induced cell apoptosis and re-sensitized drug-resistant tumor cells to sorafenib treatment.Programmed cell death 4(PDCD4),a tumor suppressor protein targeted by miR-21,has been shown to suppresses tumor cell drug-resistance and chemo-resistance.However,it remains unknown whether and how PDCD4 is involved in the suppressive effect of miR-122 on HCC drug-resistance and chemo-resistance.In the present study,we demonstrated that miR-122 promotes liver cancer cell apoptosis through blocking the maturation of cell survival oncomiR miR-21.Using miRNA tracing,in site hybridization and qRT-PCR studies,we found a considerable amount of miR-122 re-entering into liver cell nucleus.Microarray profiling and qRT-PCR assays showed an inverse relationship between miR-122 and miR-21 was validated in HCC tissues and cells,and that increasing or decreasing nuclear miR-122 level in liver cancer cells significantly reduced or increased miR-21 expression,respectively.Mechanistic studies further revealed that nuclear miR-122 bound to a 19-nt UG-containing recognition element in the basal region of pri-miR-21,preventing the processing of pri-miR-21 into pre-miR-21 by Drosha-DGCR8 microprocessor.Finally,functional assays showed that nuclear miR-122 promoted cell apoptosis and decreased liver cancer cell drug-resistance via downregulating miR-21 but increasing cellular events downstream of PDCD4 activity.