MicroRNA-224 down-regulates Glycine N-methyltransferase gene expression in Hepatocellular Carcinoma

Glycine N-methyltransferase (GNMT) is a tumor suppressor for HCC. It is down-regulated in HCC, but the mechanism is not fully understood. MicroRNA-224 (miR-224) acts as an onco-miR in HCC. This study is the first to investigate miR-224 targeting the coding region of GNMT transcript. The GNMT-MT plasmid containing a miR-224 binding site silent mutation of the GNMT coding sequence can escape the suppression of miR-224 in HEK293T cells. Expression of both exogenous and endogenous GNMT was suppressed by miR-224, while miR-224 inhibitor enhanced GNMT expression. miR-224 counteracts the effects of GNMT on the reduction of cell proliferation and tumor growth. The levels of miR-224 and GNMT mRNA showed a significant inverse relationship in tumor specimens from HCC patients. Utilizing CCl4-treated hepatoma cells and mice as a cell damage of inflammatory or liver injury model, we observed that the decreased expression levels of GNMT were accompanied with the elevated expression levels of miR-224 in hepatoma cells and mouse liver. Finally, hepatic AAV-mediated GNMT also reduced CCl4-induced miR-224 expression and liver fibrosis. These results indicated that AAV-mediated GNMT has potential liver protection activity. miR-224 can target the GNMT mRNA coding sequence and plays an important role in GNMT suppression during liver tumorigenesis.


Supporting Information
Reverse-transcription and real-time polymerase chain reaction (RT-qPCR). Total RNA was extracted using Trizol reagent (Invitrogen, Carlsbad, CA). RNA was reverse-transcribed using Tetro cDNA Synthesis Kit (Bioline, Tauton, MA) according to the manufacturer's instructions. KAPA SYBR FAST cursor Kits (Kapa Biosystems, Woburn, MA) were used for real-time PCR applications. PCR conditions were as follows: 5 min at 95°C followed by 40 cycles of 95°C for 10 sec, 60°C for 30 sec and 72°C for 30 sec.
Primer sequences are shown in Table S4. For the detection of hsa-miR-224, 20 ng of total RNA was reversely transcribed into complementary DNA using TaqMan MicroRNA Assay hsa-miR-224 reverse transcription primer and TaqMan miRNA reverse-transcriptase kit using the instructions provided by the manufacturer (Invitrogen, Carlsbad, CA). The miRNA expression was normalized to the level of RNU48 RNA.

Construction of pGNMT-MT plasmid and 3' UTR reporter assay
To construct plasmid pGNMT-MT containing the cytomegalovirus (CMV) promoter, a FLAG fragment and a miR-224 binding site silent mutation (MT) of full length GNMT coding sequences for FLAG-tagged GNMT, we used pFLAG-CMV-5 (Simga) as a vector and the pGNMT (wild type of GNMT cDNA) plasmid 1 as the PCR template for generating the insert. Mutations of the GNMT cDNA were made using PCR with the QuikChange II (Stratagene) site-directed mutagenesis kit. The presence of correct mutations was confirmed by DNA sequencing. The wild type (WT) and silent mutant (MT) cDNA fragment were subcloned downstream of renilla luciferase gene in a vector psi-CHECK2 (Promega). Detailed procedure of the constructions of the plasmids is illustrated in Figure S6 and primer sequences are shown in Table   S4. HEK293T cells were transiently co-transfected with plasmid DNA from psiCHECK2-GNMT-WT (psi-WT) or a binding site mutant plasmid psiCHECK2-GNMT-MT (psi-MT) along with miRIDIAN mimic-negative control (NC) or hsa-miR-224 mimic (224-mimic) (Dharmacon) using TurboFect (Fermentas) and Trans IT-TKO (Mirus). After 48 hours, luciferase activity was measured using the Dual-Luciferase Reporter Assay System Kit (Promega) and Infinite 200 (TECAN) following the manufacturer's instructions. In the assay, renilla luciferase activities were normalized to firefly luciferase activities.

Cell proliferation
Cell proliferation was determined using the alamarBlue assay (Invitrogen, Carlsbad, CA). Ten thousands cells were seeded in triplicates on 24 well plates for 1, 3, 5, 7 and 9days. 100 μl of alamarBlue solution (final concentration of 10% in medium) and cells were further incubated for 4 h at 37°C for a specified time period. After incubation, 100 μl of the alamarBlue solution from each well of the assay plates was transferred to a new well in 96-well plate, then fluorescence was measured at 530/590 nm. Proliferation was compared to that of the 1 st day group.
Mice Liver tissues were obtained from professor Tsai's group in the National Yang-Ming University. The liver tissues were collected separately from 1.5-, 6-, 12, 16-month-old mice in two different groups: HBx transgenic type and wild-type male (n=3~6). Total RNA was isolated from mouse liver using Trizol Reagent (Invitrogen, Carlsbad, CA).

Masson's Trichrome stain
The collagen deposition in liver tissue was evaluated by Masson's trichrome staining (HT15-1KT, SIGMA-AlDRICH). Brief description: deparaffinize tissue sections and hydrate to deionized water. Stain in working Weigert's Iron Hematoxylin solution for 1 minute. Wash in running tap water for 5 minutes.

Figure S5
A scheme of the mechanism of GNMT down-regulation of chronic hepatitis virus infection and toxic exposure induced-inflammation response and miR-224 over-expression in liver cirrhosis and HCC.