Long Noncoding RNA HITTERS Protects Oral Squamous Cell Carcinoma Cells from Endoplasmic Reticulum Stress‐Induced Apoptosis via Promoting MRE11‐RAD50‐NBS1 Complex Formation

Abstract Recent studies have proven that long noncoding RNAs (lncRNAs) exhibit regulatory functions of both DNA damage response (DDR) and endoplasmic reticulum (ER) stress. Herein, ER stress‐induced lncRNA transcriptomic changes are reported in human oral squamous cell carcinoma (OSCC) cells and a novel lncRNA HITTERS (H ERPUD1 intronic transcript of ER stress) is identified as the most significantly upregulated lncRNA. It is shown that HITTERS is a nucleus‐located lncRNA including two transcript variants. HITTERS lacks an independent promoter but shares the same promoter with HERPUD1. HITTERS is transcriptionally regulated by Activating Transcription Factor (ATF) 6, ATF4, X‐Box Binding Protein 1 (XBP1), and DNA methylation. In human OSCC tissues, HITTERS is significantly correlated with OSCC clinicopathological features and prognosis. Gain‐ and loss‐of‐function studies reveal that HITTERS promotes OSCC proliferation and invasion via influencing the expression of growth factor receptors and the downstream pathways. Once ER stress is triggered, HITTERS significantly attenuates ER stress‐induced apoptosis both in vivo and in vitro. Mechanically, HITTERS functions as RNA scaffold to promote MRE11‐RAD50‐NBS1 complex formation in the repair of ER stress‐induced DNA damage. To sum up, this study presents a novel lncRNA, namely HITTERS, which links ER stress and DDR together in OSCC.

Apoptosis assay: For apoptosis analysis, cells were stained with Annexin V-FITC and propidium iodide (PI) (Genecopoeia) according to the manufacturer's instructions. Briefly, SCC25 and CAL27 cells were trypsinized, centrifuged and wash twice with cold PBS. Cells were re-suspended in binding buffer containing Annexin V-FITC and PI. After 15 min of incubation in the dark, cells were analyzed by flow cytometry.
Wound healing assay: Confluent monolayers of cells were cultured in 6-well plates. Wounds were scratched in the monolayer with a 200 µl plastic pipette tip. The culture medium was replaced with serum-free medium. The migration of cells into the wounded area was photographed under an inverted phase contrast microscope (Olympus) at 0 h and 48 h time points. Three different wound sites in each group were photographed, and the migratory activity of cells were evaluated/the relative cell motility was calculated.
Transwell assay: Transwell assay was performed using matrigel-coated transwell chambers (Corning) containing 8 μm pores. Cells (5×10 4 cells per well) suspended in serum-free medium were added to the upper chambers. DMEM supplemented with 10% FBS was added to the lower chambers as chemoattractant. Following incubation at 37 °C with 5% CO 2 for 24 h, the non-invading cells on the upper surface of matrigel membrane were scraped off with a cotton swab.
Cells migrating through the membrane to the lower surface were fixed with 4% paraformaldehyde and stained with DAPI (Servicebio). The stained cells were photographed under a fluorescence microscope (Olympus).
TUNEL assay: Apoptotic DNA fragmentation was detected using the terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) kit (Roche) according to the manufacturer's instructions. Briefly, SCC25 and CAL27 cells were seeded in 48-well plates at 3×10 4 cells per well.
Cells were fixed with 4% paraformaldehyde and then permeabilized in 0.1% Triton X-100. After washing twice with PBS, cells were incubated with TUNEL reaction mixture for 1 h at 37 °C in a humidified atmosphere. Cells were then treated with DAPI for nuclear staining. TUNEL-positive cells were counted under a fluorescence microscope (Olympus).

Intracellular Reactive Oxygen Species (ROS) measurement:
The level of intracellular ROS was examined using reactive oxygen species assay kit (Beyotime). Briefly, SCC25 and CAL27 cells were seeded in 48-well plates at 3×10 4 cells per well. Cells were incubated with 10 µM DCFH-DA for 20 min at 37 °C. DCFH-DA is oxidized by ROS to its fluorescent product DCF in viable cells.
Cells were washed three time with PBS and treated with Hochest 33342 (Genecopoia) for nuclear staining. The fluorescence intensity of DCF was examined under a fluorescence microscope (Olympus).

Nucleus-cytoplasm fractionation:
Nuclear and cytoplasmic fractions were isolated from SCC25 and CAL27 cells using the PARIS kit (Ambion) according to the manufacturer's instructions.
Briefly, cells were washed once with PBS and resuspended with ice-cold fractionation buffer. Cells were centrifuged at 500 × g for 5 min, and the supernatant containing the cytoplasmic fraction was aspirated off. The nuclear pellet was lysed with disruption buffer. RNA is harvest by adding ethanol and drawing through a filter cartridge. Nuclear and cytoplasmic RNA was converted to cDNA and analyzed by qPCR.

RNA fluorescent in situ hybridazation (RNA FISH):
The subcellular localization of HITTERS was examined by RNA FISH. The sequence of FITC-labeled FISH probe (Genepharma) is listed in supplementary table 2. Cells were fixed in 4% paraformaldehyde for 15 min, permeabilized with 0.1% Triton X-100, and incubated with 2 × SSC buffer. Cells were incubated with FISH probe in Hybridization buffer (Genepharma) in the dark at 37 °C overnight, washed twice with 2 × SSC buffer, and treated with DAPI for nuclear staining. Images were captured by a fluorescence microscope (Olympus).

Rapid amplification of cDNA ends (5'-and 3'-RACE):
RNA was extracted from SCC25 cells using the Trizol reagent (Invitrogen). Total RNA was polyadenylated by treating with poly(A) polymerase buffer and 0.5 mM ATP. First-strand cDNA was synthesized from poly(A) + RNA with an adaptor primer and a RevertAid First Strand cDNA Synthesis Kit (Thermo Scientific). A poly(G) tail was added to the 3' end of the cDNA using TdT and dGTP. The dG-tailed cDNA underwent two rounds of PCR amplification including nested PCR to increase the specificity. PCR products were separated by 1% agarose gel and individual bands were excised for sequencing. The sequence of primers is listed in supplementary Table 1.

RNA extraction and qPCR:
Total RNA was extracted using RNAiso Plus (Takara) added with Acryl Carrier (Solarbio). First-strand cDNA was synthesized using RevertAid RT kit (Thermo Fisher Scientific). qPCR was carried out using gene-specific primers, cDNA, SYBR Green qPCR Master Mix, and an ABI Q6 Flex platform (Thermo Fisher Scientific). The primers for each gene are listed in supplementary Table 2.

Co-immunoprecipitation (IP), RNA IP (RIP) and
MS2bs-MS2bp RNA pull-down: For co-IP, cells were lysed by IP buffer (Beyotime), incubated with anti-Rad50, anti-MRE11 or anti-NBS1 antibody overnight at 4 °C. Protein A/G magnetic beads (Bimake) were subsequently added and the mixture were incubated at 4 °C for 1 h. The beads were collected by magnetic separation and the non-binding supernatant was discarded. Bound protein was eluted by boiling at 100 °C for 5 min.
Samples were subjected to western blotting to evaluate the interaction of Rad50, Mre11 and NBS1.
For RIP, the Imprint RIP Kit (Sigma) was used following the instruction of the manufacture to determine the interaction between HITTERS and RAD50/MRE11. For MS2bs-MS2bp RNA pull-down, cells were co-transfected with pMS2-GFP (Addgene) and pcDNA3.1(+)-12×MS2bs empty/ HITTERS truncations vector. The pcDNA3.1(+)-12×MS2bs was generated via cloning the 12×MS2bs sequence from pSL-MS2-12X (Addgene) and inserting it into pcDNA3.1(+). After 48h, the co-transfected cells were harvested and lysed. After incubation with anti-GFP antibody overnight at 4 °C, the captured RNA-protein complex was captured by protein A/G magnetic beads microarray. An extensive co-expression pattern between lncRNA and mRNA could be observed (D), and HITTERS was highly correlated (R>0.99) with multiple ER stress-related genes (E). F, The schematic representation of 3xHA tagged HITTERS exploring the coding potential. G, Western blot indicated HITTERS lacked coding capability, no matter whether ER stress was triggered or not.
HEK293 cells were transfected with recombined plasmid for 48h before harvesting. H and I, Online bioinformatic tools CPC (H) and CPAT (I) both predicted that similar to HOTAIR, two HITTERS TVs had no protein coding ability. In contrast, HERPUD1 had strong coding potential.

Supplementary Figure S2
A, qPCR results on the knock-down efficiency of ASO and si-RNA. Target 3 sequence was selected for further experiment. SCC25 cells were transfected with ASO or siRNA for 48h before harvesting.
One-way ANOVA and Dunnett-t test were used for ASO or siRNA, respectively. proteins. Cells were transfected with siRNA or plasmid for 48h and then treated with TM (10 μg/ml) for 24h.D, DCF staining indicated knock-down of HITTERS did not influence ROS production under ER stress. Cells were transfected with siRNA for 48h and treated with TM (10 μg/ml, 24h). E, TUNEL assay on SCC25 cells found knock-down of HITTERS or inhibiting MRN complex promoted DNA damage, whereas overexpression of HITTERS suppressed DNA damage. For C and D, cells were transfected with siRNA or plasmid for 48h and then treated with TM (10 μg/ml) or Mirin (100μM) for 24h. Note: ns, no significance; P< 0.05; **, P < 0.01; ***, P < 0.001.

Supplementary Figure S5
A, Western blot results showed under ER stress, knock-down of HITTERS significantly decreased the protein level of MRE11 and NBS1, but had no effect on RAD50 expression. Cells were transfected with siRNA for 48h and then treated with TM (10 μg/ml) for 12h. B, qPCR results indicated inhibiting MRN complex did not affect HITTERS expression under ER stress. Cells were treated with TM (10 μg/ml) for 6h. One-way ANOVA and Dunnett-t test were used. ns, no significance. C, qPCR results indicated HITTERS had no influence on the RNA level of MRE11 and NBS1. Cells were transfected with siRNA for 48h and then treated with TM (10 μg/ml) for 12h.
The Student t test was used, and all were non-significant. D, Treat SCC25 cells with Salubrinal (50μM, 24h) significantly promoted eif2a phosphorylation, but had no impact on MRE11 and NBS1 protein level. Also, knock-down of HITTERS had no impact on eif2a phosphorylation (cells were transfected with siRNA for 48h and then treated with TM for 3h). E and F, ER stress could significantly decrease the protein level of NBS1 and MRE11, and inhibiting authophagy system via HCQ could not rescue the degradation (E), whereas inhibiting proteasome via MG-132 could rescue this degradation (F). For E, SCC25 cells were treated with HCQ for 24h, and then treated with HCQ and TM for another 24h. For F, cells were treated TM for 12h, and then treated with MG-132 and TM for another 12h. H and I, IP found that knock-down of HITTERS significantly increased the ubiquitination level of NBS1 (H) and MRE11 (I) in both SCC25 and CAL27 cells. Cells were transfected with siRNA for 48h and then treated with TM (10 μg/ml) for 12h. J, Inhibiting proteasome could rescue the degradation of NBS1 and MRE11 caused by HITTERS depletion. Cells were transfected with siRNA for 48h and then treated with TM (10 μg/ml) for 24h.