MicroRNA-9 (miR-9) dysregulation is implicated in a variety of human malignancies including hepatocellular carcinoma (HCC), but its role remains contradictory. In this study, we explored the expression and methylation status of miR-9 in HCC samples, as well as the tumor-related functions of miR-9 in vitro. Bioinformatics analysis, array-based RNA expression profile, and literature retrieval were used to identify miR-9 targets in HCC. The potential downstream candidates were then validated by luciferase reporter assay, real-time quantitative PCR, and western blot or enzyme linked immunosorbent assay (ELISA). The expression status and clinicopathologic significances of miR-9 target genes in clinical samples were further explored. The results showed that miR-9 was frequently downregulated in primary HCC. Its silencing was largely contributed by a high frequency (42.5%) of mir-9-1 hypermethylation, which was correlated with bigger tumor size (P = 0.0234). In vitro functional studies revealed that miR-9 restoration retarded HCC cell proliferation and migration. IL-6, AP3B1, TC10, ONECUT2, IGF2BP1, MYO1D, and ANXA2 were confirmed to be miR-9 targets in HCC. Among them, ONECUT2, IGF2BP1, and ANXA2 were confirmed to be aberrantly upregulated in HCC. Moreover, upregulation of ONECUT2, IGF2BP1, and IL-6 were significantly associated with poor post-surgery prognosis (P = 0.0458, P = 0.0037 and P = 0.0461, respectively). Mechanically, miR-9 plays a tumor suppressive role partially through a functional miR-9/IGF2BP1/AKT&ERK axis. Our study suggests that miR-9 functions as a tumor suppressor in HCC progression by inhibiting a series of target genes, including the newly validated miR-9/IGF2BP1/AKT&ERK axis, thus providing potential therapeutic targets and novel prognostic biomarkers for HCC patients.

MicroRNA-9 (miR-9) presents to exert distinct and even opposite functions in different kinds of tumors through targeting different cellular genes. However, its role in cervical adenocarcinoma remains uncertain. Here, we report that miR-9 is down-regulated in cervical adenocarcinoma due to its frequent promoter-hypermethylation and exerts its tumor suppressor role through inhibiting several novel target genes, including interleukin-6 (IL-6). The promoters of miR-9 precursors (mir-9-1, -2, and -3) were hypermethylated in cervical adenocarcinoma tissues. Demethylation treatment of HeLa dramatically increased the expression of mature miR-9. Both in vitro and in vivo functional experiments confirmed that miR-9 can inhibit the proliferation, migration, and malignant transformation abilities of HeLa cells. Bioinformatics methods and array-based RNA expression profiles were used to screen the downstream target genes of miR-9. Dualluciferase reporting assay, real-time qPCR, and ELISA or Western blot confirmed four genes (CKAP2,HSPC159, IL-6, and TC10) to be novel direct target genes of miR-9. Pathway annotation analysis of the differently expressed genes (DEGs) induced by ectopic miR-9 expression revealed the enrichment in Jak/STAT3 pathway, which is one of the downstream pathways of IL-6. Ectopic expression of miR-9 in HeLa inhibited Jak/STAT3 signaling activity. Moreover, such effect could be partially reversed by the addition of exogenous IL-6. In conclusion, our results here present a tumor suppressor potential of miR-9 in cervical adenocarcinoma for the first time and suggest that miR-9 could repress tumorigenesis through inhibiting the activity of IL-6/Jak/STAT3 pathway.

The ASCC3 subunit of the activating signal co-integrator complex is a dual-cassette Ski2-like nucleic acid helicase that provides single-stranded DNA for alkylation damage repair by the α-ketoglutarate-dependent dioxygenase AlkBH3. Other ASCC components integrate ASCC3/AlkBH3 into a complex DNA repair pathway. We mapped and structurally analyzed interacting ASCC2 and ASCC3 regions. The ASCC3 fragment comprises a central helical domain and terminal, extended arms that clasp the compact ASCC2 unit. ASCC2–ASCC3 interfaces are evolutionarily highly conserved and comprise a large number of residues affected by somatic cancer mutations. We quantified contributions of protein regions to the ASCC2–ASCC3 interaction, observing that changes found in cancers lead to reduced ASCC2–ASCC3 affinity. Functional dissection of ASCC3 revealed similar organization and regulation as in the spliceosomal RNA helicase Brr2. Our results delineate functional regions in an important DNA repair complex and suggest possible molecular disease principles.

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23The ASCC3 subunit of the activating signal co-integrator complex is a dual-cassette like nucleic acid helicase that provides single-stranded DNA for alkylation damage repair by 25 the α-ketoglutarate-dependent dioxygenase, AlkBH3. Other ASCC components integrate 26 ASCC3/AlkBH3 into a complex DNA repair pathway. We mapped and structurally analyzed 27 interacting ASCC2 and ASCC3 regions. The ASCC3 fragment comprises a central helical 28 domain and terminal, extended arms that clasp the compact ASCC2 unit. ASCC2-ASCC3 29 interfaces are evolutionarily highly conserved and comprise a large number of residues 30 affected by somatic cancer mutations. We quantified contributions of protein regions to the 31 ASCC2-ASCC3 interaction, observing that changes found in cancers lead to reduced ASCC2-32 ASCC3 affinity. Functional dissection of ASCC3 revealed similar organization and regulation 33 as in the spliceosomal RNA helicase, Brr2. Our results delineate functional regions in an 34 important DNA repair complex and suggest possible molecular disease principles. 35 36 Main 37 The human genome is constantly under assault by endogenous or exogenous DNA 38 damaging agents. To ward off these insults, cells have evolved systems to recognize DNA 39 damage, signal its presence and initiate repair processes. 1 Among the diverse repair 40 mechanisms, direct DNA repair processes represent efficient means to revert chemical 41 changes to DNA and involve enzymes such as photolyases, alkyl-transferases or 42 dioxygenases. 2,3 Escherichia coli α-ketoglutarate-dependent dioxygenase, AlkB, homologs 43 (AlkBH's) are one class of important DNA repair factors that reverse N-alkyl lesions. 4 Among 44 3 the nine identified AlkBH enzymes in human, AlkBH2 and AlkBH3 de-alkylate 1-methyl 45 adenosine and 3-methyl cytosine. 5,6 46Several lines of evidence implicate the human activating signal co-integrator complex 47 (ASCC) in AlkBH3-mediated DNA repair. ASCC is composed of four subunits, ASCC1, ASCC2, 48 ASCC3 and ASC1/TRIP4. 7,8 ASCC3 is the largest subunit of ASCC and was characterized as 49 a DNA helicase that unwinds DNA by translocating on one strand in 3'-to-5' direction. 8 The 50 enzyme is thought to provide single-stranded DNA as a substrate for de-alkylation repair by 51AlkBH3. 8 ASCC and alkylated nucleotides co-localize at nuclear foci upon alkylation damage 52 stress, dependent on a coupling of ubiquitin conjugation to ER degradation (CUE) domain in 53 ASCC2, which links DNA alkylation damage repair to upstream ubiquitin signaling via the RING 54 finger protein 113A. 9 ASCC1 is cleared from these foci upon DNA alkylation damage and 55 knockout of ASCC1 leads to loss of ASCC2 from the nuclear foci and increased cellular 56 sensitivity to alkylating insults. 10 57 Both ASCC2 and ASCC3 have been linked to various human diseases. ASCC2 is 58 upregulated in rheumatoid arthritis patients 11 , and ASCC3 is upregulated in peripheral blood 59 mononuclear cells from lung cancer patients 12,13 . A role of ASCC3 in cancer development or 60 pr...