Mutant MEG3 Enhances the Activity of Telomerase by Increasing DNA Damage Repair in Human Liver Cancer Stem Cells

Background: Long noncoding RNAs have recently considered as central regulators in diverse biological processes and emerged as vital players controlling tumorigenesis. Although wild MEG3 acts as a suppressor in several cancers, the function of mutant MEG3 is also unclear during tumorigenesis. Methods: Lentivalus infection,RT-PCR,Western blotting and tumorigenesis test in vitro and in vivo were performed. Results: our results suggest that mutant MEG3 promotes the growth of human liver cancer stem cells in vivo and in vitro.Mechanistically, our results show that mutant MEG3 enhances acetylation modication of HistoneH4 on K16.Then, mutant MEG3 enhances the expression of SETD2 dependent on H4K16Ac.Moreover, mutant MEG3 increases the DNA damage repair through SETD2.Ultimately, mutant MEG3 increases the telomeras activity dependent on DNA damage repair.Strikingly,TERT determines the cancerous function of mutant MEG3 in liver cancer stem cells. Therefore, we shed light on the fact that targeting mutant MEG3 could be a viable approach for cancer treatment. Conclusions: these observations will play an important role in nding effective tumor treatment targets.

In this study,we clearly demonstrate that mutant MEG3 promotes the growth of human liver cancer stem cells in vivo and in vitro. Mutant MEG3 enhances acetylation modi cation of Histone H4K16 and then enhances the expression of SETD2.Therefore, mutant MEG3 increases the DNA damage repair through SETD2 and increases the telomeras activity. These observations will play an important role in nding effective tumor treatment targets.

Materials And Methods
Tetracycline (DOX) inducting lentiviral rLV-tet on-mutant MEG3 The expression plasmid pLVX-tet on-Tight-EF1a-ZsGreen and pLVX-mutant MEG3-ZsGreen-Puro were digested with Spel and NotI, respectively, and the large fragment of plasmid pLVX-tet on-Tight-EF1a -ZsGreen and the small fragment pLVX-mutant MEG3-ZsGreen-Puro were recovered by 1% agarose gel electrophoresis respectively. The two plasmid pLVX-tet on-Tight-EF1a-ZsGreen (Spel + NotI) and pUC57-mutant MEG3(Spel + NotI ) were carried out the ligation reaction at 22 ° C for 3 hours and then the ligation products were transformed into JM109 competent bacterial overnight. Monoclonal colonies were picked for sequencing veri cation. The RT-PCR cDNA was prepared by using oligonucleotide (dT), random primers, and a SuperScript First-Strand Synthesis System (Invitrogen). PCR analysis was performed according to the manufacturer. β-actin was used as an internal control.
Following three washes in Tris-HCl pH 7.5 with 0.1% Tween 20, the blots were incubated with antibody(appropriate dilution) overnight at 4°C. Signals were visualized by enhanced chemiluminescence plus kit(GE Healthcare).
RNA Immunoprecipitation(RIP) Ribonucleoprotein particle-enriched lysates were incubated with protein G/A-plus agarose beads (Santa Cruz) together with antibody or normal rabbit IgG for 4 hours at 4°C. Beads were subsequently washed. RNAs were isolated and then RT-PCR.
Super-RNA-EMSA Cells were washed and scraped in ice-cold PBS to prepare nuclei for electrophoretic gel mobility shift assay with the use of the gel shift assay system (Promega) modi ed according to the manufacturer's instructions.
CHIP assay Cells were cross-linked with 1% (v/v) formaldehyde (Sigma) for 10 min at room temperature and stopped with 125 mm glycine for 5 min. Crossed-linked cells were washed with phosphate-buffered saline, resuspended in lysis buffer, and sonicated for 8-10 min in a SONICS VibraCell to generate DNA fragments. Chromatin extracts were diluted 5-fold with dilution buffer, pre-cleared with Protein-A/G-Sepharose beads, and immunoprecipitated with speci c antibody on Protein-A/G-Sepharose beads. After washing, elution and de-cross-linking, the ChIP DNA was detected by PCR.
DNA damage repair assay DNA damage marker rH2AX (S139) detection, in situ DNA damage analysis and Quantitative analysis of DNA Damgae via 8-OHdG were performed according to the manufacturer's instructions, respectively.
Cell colony-formation e ciency assay cells were plated in six wells and incubated in a humidi ed atmosphere of 5% CO 2 incubator at 37ºC for 14 days. For visualization, colonies were stained with 0. 5% Crystal Violet (sigma) in 50% methanol and 10% glacial acetic acid. Colonies were counted using a dissecting microscope by MacBiophotonics Image J.
Tumorigenesis testin vivo Four-weeks male athymic Balb/c mice were maintained in the Tongji university animal facilities approved by the China Association for accreditation of laboratory animal care. athymic Balb/c mice per group were injected at the armpit area subcutaneously with cells. The mice were observed over 4 weeks for tumor formation. The mice were then sacri ced and the tumors recovered. The wet weight of each tumor was determined for each mouse. A portion of each tumor was xed in 4% paraformaldehyde and embedded in para n for histological examination.

Mutant MEG3 enhances the expression of SETD2 dependent on H4K16Ac
Given that mutante MEG3 enhances H4K16Ac, we consider to com rm whether mutant MEG3 enhances the expression of SETD2 through H4K16Ac. In DOX (0µg/ml) treatment group, DOX (0.5µg/ml) treatment group, DOX (1µg/ml) Treatment group, DOX (1.5µg/ml) treatment group, DOX (2µg/ml) treatment group, the loading of H4K16Ac onto SETD2 promoter were signi cantly increased with the increase of DOX concentration (Fig. 3A). The binding ability of H4K16Ac to SETD2 promoter probe was signi cantly increased with the increase of DOX concentration (Fig. 3B). The ability of RNA polymerase II and H4K16Ac to enter the SETD2 promoter-enhancer loop was signi cantly increased with the increase of DOX concentration (Fig. 3C). The SETD2 promoter transcription activity was signi cantly increased with the increase of DOX concentration (6648. 22 (Fig. 3D). The ability of SETD2 transcription and translation expression was signi cantly increased with the increase of DOX concentration (Fig. 3E).Collectively, these results suggest that mutant MEG3 enhances the expression of SETD2.
Collectively, these results suggest that mutant MEG3 increases the DNA damage repair through SETD2.

Discussion
At the present, we clearly demonstrate that mutant MEG3 promotes the growth of human liver cancer stem cells in vivo and in vitro.Mechanistically,our results shows that mutant MEG3 enhances acetylation modi cation of HistoneH4K16.Then, mutant MEG3 enhances the expression of SETD2 dependent on H4K16Ac.Furthermore, mutant MEG3 increases the DNA damage repair through SETD2.Ultimately, mutant MEG3 increases the telomeras activity dependent on DNA damage repair.In particular,TERT determines the cancerous function of mutant MEG3 in liver cancer stem cells (Fig. 6H). These observations will play an important role in nding effective tumor treatment targets.
First, our results indicate that mutant MEG3 promotes the growth of human liver cancer stem cells in vivo and in vitro. LncRNA-MEG3 inhibits cell proliferation and invasion by modulating Bmi1/RNF2 in cholangiocarcinoma (21). Also, MEG3 inhibits HMEC-1 cells growth, migration and tube formation via sponging miR-147 (22).
Notably, our results suggest that mutant MEG3 increases the DNA damage repair through H3K36me3 dependent on SETD2. There are diverse clues showing H3K36me3 participates in DNA damage response by directly recruiting DNA repair machinery to set the chromatin at a "ready" status (43). Histone H3 trimethylation at lysine 36 guides m 6 A RNA modi cation co-transcriptionally (44,45). Chromosome 3P loss of heterozygosity reduces expression of H3K36me3 in sacral conventional chordoma (46). Furthermore, gene body DNA methylation conspires with H3K36me3 to preclude aberrant transcription (47).DNA damage is related to the balance between survival and death in cancer biology (48).As exempli ed in diverse cancers,disruption or deregulation of DNA repair pathways results in genome instability (49).Moreover, the DNA mismatch repair triggers cell cycle arrest in some cases (50).The DNA damage respons makes it safe to play with knives (51). Cell fate regulation is associated with upon DNA damage (52,53).
Intriguingly, we clearly identity that mutant MEG3 increases the telomeras activity dependent on DNA damage repair. Furthermore, our results indicate that TERT determines the cancerous function of mutant MEG3. Telomerase, an RNA-dependent DNA polymerase with telomerase reverse transcriptase (TERT), regulates cancer formation (54,55).A particular attention is given to the putative connections between TERT transcriptional reactivation and signalling pathways frequently altered in cancer, such as c-MYC, NF-κB and β-Catenin(56). TERT promoter mutations are associated with poor prognosis and cell immortalization in meningioma (57).DNA methylation of the TERT promoter is associated with human cancer(58).TERT and TERC mutations suppress telomerase activity (59).TERT C228T mutation is associated with intravesical recurrence for patients with non-muscle invasive bladder cancer(60).TERC is an RNA component of telomerase and TERC promotes cellular in ammatory response independent of telomerase(61). HuR regulates telomerase activity through TERC methylation(62). Mitochondrionprocessed TERC regulates senescence without affecting telomerase activities(63).C-MYC drives overexpression of telomerase RNA (hTR/TERC) (64). In particular,the TERC haploinsu ciency affcts on the inheritance of telomere length(65) and is involved in the process of genetic instability leading to tumorgenesis (66).
In conclusions, the present study will focus on studying the effective mechanism of mutant MEG3 in carcinogenesis. These studies will play an important role in nding effective tumor treatment targets. All methods were carried out in "accordance" with the approved guidelines. All experimental protocols "were approved by" a Tongji university institutional committee. Informed consent was obtained from all subjects. The study was reviewed and approved by the China national institutional animal care and use committee.     Immunoblot analysis with anti-rH2AX(S139). H2AX serves as an internal reference.G. The assay of DNA damage repair ability. The values of each group were expressed as mean ± SEM (n =3), * *, P < 0.01, and *, P < 0.05, respectively. expressed as mean ± SEM (n =3), * *, P < 0.01, and *, P < 0.05, respectively. E. The assay of telomere length. The values of each group were expressed as mean ± SEM (n =3), * *, P < 0.01, and *, P < 0.05, respectively.