Long Non-Coding RNA THOR Depletion Inhibits Human Non-Small Cell Lung Cancer Cell Growth

Long non-coding RNA (LncRNA) THOR (Lnc-THOR) is expressed in testis and multiple human malignancies. Lnc-THOR association with IGF2BP1 (IGF2 mRNA-binding protein 1) is essential for stabilization and transcription of IGF2BP1 targeted mRNAs. We tested its expression and potential functions in non-small cell lung cancer (NSCLC). In primary NSCLC cells and established cell lines, Lnc-THOR shRNA or CRISPR/Cas9-mediated knockout (KO) downregulated IGF2BP1 target mRNAs (IGF2, Gli1, Myc and SOX9), inhibiting cell viability, growth, proliferation, migration and invasion. Significant apoptosis activation was detected in Lnc-THOR-silenced/-KO NSCLC cells. Conversely, ectopic overexpression of Lnc-THOR upregulated IGF2BP1 mRNA targets and enhanced NSCLC cell proliferation, migration and invasion. RNA-immunoprecipitation and RNA pull-down assay results confirmed the direct binding between Lnc-THOR and IGF2BP1 protein in NSCLC cells. Lnc-THOR silencing and overexpression were ineffective in IGF2BP1-KO NSCLC cells. Forced IGF2BP1 overexpression failed to rescue Lnc-THOR-KO NSCLC cells. In vivo, intratumoral injection of Lnc-THOR shRNA adeno-associated virus potently inhibited A549 xenograft tumor growth in nude mice. At last we show that Lnc-THOR is overexpressed in multiple NSCLC tissues and established/primary NSCLC cells. Collectively, these results highlighted the ability of Lnc-THOR in promoting NSCLC cell growth by associating with IGF2BP1, suggesting that Lnc-THOR represents a promising therapeutic target of NSCLC.


Ethics
All the methods applied in this study were carried out according to the ethics guidelines of Zhengzhou University.

Cell Culture
Established NSCLC cell lines, A549 and H1299, were from Dr. Li at Wenzhou Medical University (32). Cells were grown in RPMI-1640 medium containing 10% FBS. The primary human NSCLC cells, derived from three different patients, "pCan-1", "pCan-2" and "pCan-3", as well as the primary human lung epithelial cells ("pEpi"), were from Dr. Shi at The Second Affiliated Hospital of Soochow University, and were cultured as described (33). The written-informed consent was obtained from each participant. The protocols of using human cells were approved by the Ethics Committee of Zhengzhou University, in according to the principles of Declaration of Helsinki.

Patients and Tissue Samples
A set of 10 pairs of NSCLC tumor tissues and the corresponding adjacent normal lung tissues (over two cm away from the boundary of tumor tissue) were obtained from primary NSCLC patients with tumor resection. The patients were administrated at the First affiliated Hospital of Zhengzhou University and received no preoperative treatments. Human testis tissues were from a patient with post-traumatic orchiectomy. The written informed consent was obtained each patient. Human tissues were stored in liquid nitrogen immediately after resection. The protocols of using human tissues were approved by the Ethics Committee of Zhengzhou University, in according to the principles of Declaration of Helsinki. NSCLC cells were plated at a density of 1 ×10 5 cells/well into sixwell plates (in polybrene containing complete medium) and were infected with the lentivirus (MOI=20). After 24h, puromycin (2.5 mg/mL) was added to select stable cells for 3-4 passages. Lnc-THOR knockdown in stable cells was verified by qRT-PCR assays. Control cells were infected lentiviral particles with scramble control shRNA ("shC").

Lnc-THOR Knockout (KO)
NSCLC cells were seeded into six-well plates (at 1.0 × 10 5 cells per well) and were transfected with a LentiCas9-puro construct (Genechem). Stable Cas9 NSCLC cells were established after puromycin selection. Cells were then transfected with a pSpCas9 (BB)-2A (PX458) plasmid encoding sgRNA against Lnc-THOR [provided by Dr. Pan at Shanghai Jiao Tong University (34)]. The transfected cells were distributed into 96-well plates and subject to Lnc-THOR KO screening. The single stable Lnc-THOR KO NSCLC cells were then established. Control cells were transduced with the Cas9 control empty vector ("Cas9-C").

Lnc-THOR Overexpression
The GV248 lentiviral construct encoding the full-length Lnc-THOR was provided again by Dr. Pan (34), that was transfected to primary NSCLC cells. Cells were subject to puromycin (2.5 mg/ mL) selection for another 4-5 passages. Two stable lines of NSCLC cells with Lnc-THOR-expressing construct, "OE-L1" and "OE-L2", were established. Lnc-THOR overexpression in stable cells was verified by qRT-PCR assay. Control cells were infected with the empty vector ("Vec").

Cell Viability
NSCLC cells were plated (4 × 10 3 cells per well) in 96-well microplates and cultured for 96h. Afterwards, 10 mL per well of CCK-8 solution was added for 2h. CCK-8 optical density (OD) was measured at 490 nm.

Colony Formation
NSCLC cells were grown in a 10-cm culturing dish at (2 × 10 4 cells per dish). Medium was changed every two days for a total of 12 days. Afterwards, the cell colonies were fixed with methanol, washed with PBS and stained with Giemsa. Finally, the number of colonies (with ≥50 cells per colony) were counted.

Cell Migration and Invasion Assays
The in vitro cell migration was tested by using 24-well "Transwell" chambers (Becton Dickinson, Shanghai, China). In brief, NSCLC cells, at 3 × 10 4 per well, were seeded in the upper surface of the Transwell chamber in basic DMEM. The lower chamber was filled with complete medium (with 10% FBS) to attract cells. After 24h, non-migrated NSCLC cells were removed carefully from the top well with a cotton swab, with NSCLC cells on the bottom fixed and stained. The migrated cells were photographed (Olympus, Tokyo, Japan). For in vitro invasion assays, the "Transwell" chambers were always coated with Matrigel (Sigma). Data quantification was reported early (26).

Caspase-3 Activity Assay
Briefly, NSCLC cells were grown in 12-well plates for 72h. Cells were then harvested and tested for caspase-3 activity by a colorimetric assay kit (BioVision, Mountain View, CA) according to the attached protocol.

ssDNA ELISA
NSCLC cells were grown in 12-well plates at 0.5 × 10 5 cells per well for 72h. Cells were then rinsed with cold PBS, fixed with icecold methanol, and incubated with 100% formamide (Roth, Karlsruhe, Germany). Afterwards, cells were incubated with 3% H 2 O 2 and blocked by non-fat dry milk. A ssDNA ELISA Kit (Millipore, Billerica, MA) was then utilized for detection total ssDNA contents based on the attached protocols. ssDNA absorbance in each well was detected at 405 nm.

TUNEL Assay
Briefly, NSCLC cells were grown into 12-well plates for 96h. Cells were fixed with 4% formaldehyde, followed by permeabilization as described (35). Cells were then incubated with TUNEL reaction mixture containing nucleotide mixture and terminal deoxynucleotidyl transferase (TdT), co-stained with DAPI, washed with PBS, and detected under a fluorescence microscope.

RNA-Immunoprecipitation (RIP)
The pCan-1 primary NSCLC cells and A549 cells were incubated with 0.3% formaldehyde and glycine (0.125 M), and cell pellets re-suspended in RIP buffer described early (36). An anti-IGF2BP1 antibody (#8482, Cell Signaling Tech, Beverly, MA) was added to the cell lysates, and IGF2BP1-bound pellets were washed, re-suspended and the magnetic beads were added. The mixture was incubated on a rotator at 4°C overnight. After collecting the magnetic bead-bound complex, the proteinase K was utilized. qRT-PCRs assay were then performed to examine IGF2BP1-bound RNA.

RNA Pull-Down
Biotin-labeled full-length Lnc-THOR was provided by Dr. Chen's Lab at Jiangsu University (26), and was dissolved in RNA structure buffer (Beyotime, Wuxi, China) to obtain an appropriate secondary structure. For RNA pull-down assay, 600 mg nuclei lysates of the pCan-1 primary NSCLC cells and A549 cells were mixed with folded Biotin-Lnc-THOR and Dynabeads MyOne Streptavidin C1 magnetic beads ["Beads", provided by Dr. Chen (26)]. The mixture was incubated on a rotator at 4°C overnight. Beads were washed three times. The bound proteins were eluted in 60 mL protein lysis buffer, separated by the SDS-PAGE, and examined by Western blotting assays.

IGF2BP1 KO
NSCLC cells were seeded into six-well plates (at 1.0 × 10 5 cells per well) and were transfected with a LentiCas9-puro construct (Genechem). Stable Cas9 NSCLC cells were established after puromycin selection. Cells were then transfected with the CRISPR/Cas9-IGF2BP1-KO construct [from Dr. Cheng's group at Soochow University (37)] and were then distributed to 96-well plates, subject to IGF2BP1 KO screening. The IGF2BP1-KO monoclonal stable cells were then established, with IGF2BP1 expression examined by Western blotting and qRT-PCR assays.

IGF2BP1 Overexpression
The recombinant adenovirus encoding IGF2BP1-expressing pSUPER-puro construct was from Dr. Zhao at Soochow University (38). NSCLC cells were grown in six-well tissue culture plates (at 0.6 × 10 5 cells per well) and were infected with the adenovirus for 48h. Stable cells were established by puromycin selection and IGF2BP1 overexpression verified by Western blotting and qRT-PCR assays.

Xenograft Tumor Formation Assay
The nude mice, half male half female, aged 5-6 weeks, 18.5-19.0g in weights, were randomly divided into two groups, and were inoculated with A549 cells [at 6 ×10 6 cells per mouse subcutaneously (s.c.)]. Nude mice were monitored every day, xenograft tumor weights and volumes were measured with a sliding caliper, and tumor volumes calculated using the formula (L×W 2 )/2. When the tumor volume was close to 100 mm 3 ("Day-0"), mice were subject to intratumoral injection of Lnc-THOR shRNA ("sh-S1") adeno-associated virus (AAV) or the scramble control shRNA ("shC") AAV. All mice were sacrificed at the end of the experiments and the tumors were harvested. All animal studies were performed according to the standards of IACUC of Zhengzhou University, with the protocols approved by the Ethics Committee of Zhengzhou University.

Statistical Analyses
All values were presented as mean ± standard deviation (SD). Statistical comparisons were performed by Student's t-test (Excel 2007) between two groups or one-way ANOVA plus a Scheffe' and Tukey Test (SPSS 23.0) for multiple comparisons. P < 0.05 was considered to indicate a significant difference. In vitro experiments were repeated at least three times, with similar results obtained.
The potential effect of Lnc-THOR in other NSCLC cells was studied next. Primary NSCLC cells derived from two other primary patients, pCan-2 and pCan-3, as well as the established cell lines (A549 and H1299), were tested. The lentiviral particles encoding Lnc-THOR shRNA ("sh-S1") were added to the NSCLC cells. Via selection stable cells were established, showing dramatic Lnc-THOR silencing ( Figure 1I). The long isoform of THOR expression was unchanged ( Figure 1J). The nuclear EdU staining assay results, Figure 1K, showed that Lnc-THOR shRNA potently inhibited proliferation of the primary and established NSCLC cells. "Transwell" ( Figure 1L) and "Matrigel Transwell" ( Figure 1M) assay results further showed that Lnc-THOR silencing largely suppressed migration and invasion of the NSCLC cells.
In pCan-2 and pCan-3 primary cells as well as in established cell lines (A549 and H1299), Lnc-THOR silencing by sh-S1 shRNA (see Figure 1) induced caspase-3 activation ( Figure 2G) and mitochondrial depolarization (JC-1 green monomer intensity increase, Figure 2H). Significant apoptosis was detected as well in the Lnc-THOR-silenced NSCLC cells, as the TUNEL-positive nuclei ratio was significantly increased ( Figure 2I). Together, these results showed that Lnc-THOR depletion induced NSCLC cell apoptosis.

Lnc-THOR-Driven NSCLC Cell Growth Is Through Binding to IGF2BP1
Experiments were carried out to examine the possible association between Lnc-THOR and IGF2BP1 protein in NSCLC cells. Lnc-THOR pull-down assay results confirmed that IGF2BP1 protein in cell nuclei was precipitated with the biotinylated Lnc-THOR in pCan-1 primary NSCLC cells and A549 cells ( Figure 4A). Additionally, by employing a RNA-Immunoprecipitation (RIP) assay, we further demonstrated the direct association between endogenous Lnc-THOR and IGF2BP1 protein in pCan-1 cells and A549 cells ( Figure 4B). These results implied that Lnc-THOR directly associated with IGF2BP1 protein in NSCLC cells. Whether Lnc-THOR-driven NSCLC cell growth was through binding to IGF2BP1 protein was tested next. Using the CRISPR/ Cas9 gene-editing method [see (38)], we established the IGF2BP1-KO stable pCan-1 cells (IGF2BP1-KO). The qRT-PCR ( Figure 4C) and Western blotting ( Figure 4D) results confirmed IGF2BP1 KO in the stable cells, where Lnc-THOR expression was unchanged ( Figure 4E). CRISPR/Cas9-induced IGF2BP1 KO potently inhibited pCan-1 cell proliferation (EdU staining assays, Figure 4F) and migration ( Figure 4G). Importantly, altering Lnc-THOR expression ( Figure 4E), by the Lnc-THOR shRNA ("sh-S1", see Figure 2) or the Lnc-THOR-expressing construct (Lnc-THOR-OE, see Figure 3), failed to further affect cell proliferation ( Figure 4F) and migration ( Figure 4G) in IGF2BP1-KO cells. IGF2BP1 expression was not affected by Lnc-THOR shRNA or OE ( Figures 4C, D). These results implied that Lnc-THOR-driven NSCLC cell growth was indeed through binding to IGF2BP1.

Lnc-THOR shRNA Inhibits A549 Xenograft Tumor Growth in Nude Mice
We tested the potential effect of Lnc-THOR on NSCLC cell growth in vivo. A549 cells were s.c. injected to the flanks of the

A
B D E C FIGURE 5 | Lnc-THOR shRNA inhibits A549 xenograft tumor growth in nude mice. A549 xenograft-bearing nude mice were subject to intratumoral injection of Lnc-THOR shRNA ("sh-S1") AAV or the scramble control shRNA ("shC") AAV. The virus was injected daily for seven consecutive days; Tumor volumes (A) and mice body weights (D) were recorded every seven days for six rounds (total 42 days, "Day-0" to "Day-42"); The estimated daily tumor growth (in mm 3 per day) was calculated (B). At Day-42 mice were sacrificed and A549 xenografts were isolated and weighed (C). At experimental Day-7 and Day-14, one tumor of each group was isolated, expression of Lnc-THOR and listed mRNAs (E) was tested by qRT-PCR assays. Data were presented as mean ± standard deviation (SD). *P < 0.05 vs. "shC" group.
nude mice. When the tumor volume was close to 100 mm 3 ("Day-0"), mice were randomly assigned into two groups (with 10 mice per group). The xenograft-bearing mice were subject to intratumoral injection of Lnc-THOR shRNA ("sh-S1") AAV or the scramble control shRNA ("shC") AAV. The virus was injected daily for seven consecutive days. As shown, A549 xenografts with Lnc-THOR shRNA AAV injection grew significantly slower than control tumors with shC AAV injection ( Figure 5A). By using the formula: (Volume at Day-42 subtracting Volume at Day-0)/42, the estimated daily tumor growth (in mm 3 per day) was calculated. The results further confirmed that Lnc-THOR shRNA virus injection potently inhibited A549 xenograft growth in mice ( Figure 5B). At Day-42 mice were sacrificed by cervical dislocation, and palpable A549 xenografts were isolated and weighed. Results showed that A549 xenografts with Lnc-THOR shRNA injection were significantly lighter than shC A549 xenografts ( Figure 5C).
The body weights were however not significantly different between two groups ( Figure 5D). At experimental Day-7 and Day-14, one tumor of each group was isolated to obtain the tumor tissue lysates. As shown Lnc-THOR was depleted in Lnc-THOR shRNA-injected tumors ( Figure 5E). In line with the in vitro findings, IGB2BP1 target mRNAs, including Gli1, Myc, IGF2 and SOX9, were decreased as well in Lnc-THOR-silenced A549 xenograft tissues ( Figure 5E). Collectively, these results show that Lnc-THOR shRNA inhibited A549 xenograft tumor growth in mice.

Increased Lnc-THOR Expression in NSCLC
At last we tested expression of Lnc-THOR in NSCLC. By employing qRT-PCR assays, we show that Lnc-THOR expression is detected in eight out of 10 human NSCLC tissues ("T", Figure 6A). Lnc-THOR expression in NSCLC tumor Lnc-THOR expression was tested by qRT-PCR assays, with its expression normalized to that of human testis tissues (A, B). The proposed signaling cartoon of this study (C). Data were presented as mean ± standard deviation (SD, n=5). *P < 0.05 vs. "N" tissues or "pEpi" cells. "N. S." stands for non-statistical difference (A). The experiments were repeated three times, with similar results obtained.

DISCUSSION
IGF2BP1 is a primary member of IGF2BP RNA-binding family proteins (21,39), and it regulates stabilization and transcription of several key pro-cancerous/oncogenic genes (21)(22)(23)39). Zhang et al., have shown that IGF2BP1 is important for NSCLC cell progression (40). In NSCLC cells, IGF2BP1 silencing potently suppressed cancer cell proliferation, migration and invasion, as well as induced cell cycle arrest and apoptosis (40). Gong et al., showed that microRNAs-491-5p silenced IGF2BP1 to suppress A549 cell proliferation and migration (41). Studies have demonstrated that the direct binding between Lnc-THOR and IGF2BP1 is critical for IGF2BP1 to maintain its functions (25). Lnc-THOR was firstly identified in 2017 as a conserved cancer and testis specific Lnc-RNA (24). Since then, the cancer-promoting activity of this LncRNA has been confirmed in multiple malignancies (24,25,(27)(28)(29)31). Chen et al., reported that Lnc-THOR expression in osteosarcoma (OS) is required for cancer cell growth in vitro and in vivo (25). In addition, Lnc-THOR is expressed in renal cell carcinoma (RCC). Contrarily, Lnc-THOR silencing or KO suppressed RCC cell proliferation (27). Song et al., have shown that Lnc-THOR increased the stemness of gastric cancer cells by enhancing SOX9 mRNA stability (28). By promoting c-myc mRNA-IGF2BP1 protein association, Lnc-THOR increased c-myc expression and retinoblastoma cell progression (29). Wang et al., have shown that triptonide inhibited nasopharyngeal carcinoma cell growth by downregulating Lnc-THOR (26). Xue et al., reported that Lnc-THOR is expressed in human glioma, and silencing Lnc-THOR largely inhibited glioma cell survival via activating MAGEA6-AMPK signaling (34).
In the present study, we found that Lnc-THOR is overexpressed in NSCLC tissues and cells In established and primary NSCLC cells, Lnc-THOR shRNA or complete KO potently inhibited cell viability, proliferation migration and invasion. Moreover, significant apoptosis was detected in Lnc-THOR-silenced/-KO NSCLC cells. We found that ssDNA contents were significantly increased in Lnc-THOR shRNA or KO cells, which could initiate a DNA damage response to provoke apoptosis. Therefore, ssDNA formation could be an important cause of Lnc-THOR depletion-induced apoptosis induction in NSCLC cells. The underlying mechanisms warrant further characterizations. Conversely, forced Lnc-THOR overexpression, by a lentiviral construct, accelerated NSCLC cell proliferation, migration and invasion. In vivo, Lnc-THOR shRNA potently inhibited A549 xenograft tumor growth the nude mice. These results suggested that Lnc-THOR could be an important therapeutic target and a promising diagnosis marker for NSCLC.
Here we provided evidence to support that Lnc-THORdriven NSCLC cell growth is through binding to IGF2BP1 (see the proposed signaling cartoon in Figure 6C). RNA-IP and RNA pull-down results showed a direct binding between Lnc-THOR and IGF2BP1 protein in NSCLC cells. mRNA expression of IGF2BP1 target mRNAs, including IGF2, Gli1, Myc and SOX9, were significantly downregulated in Lnc-THOR-silenced/-KO NSCLC cells, but increased after Lnc-THOR overexpression. IGB2BP1 target mRNAs were robustly decreased in Lnc-THOR-silenced A549 xenograft tissues. Mimicking Lnc-THOR depletion-induced anti-cancer activity, CRISPR/Cas9-induced IGF2BP1 KO potently inhibited NSCLC cell proliferation and migration. More importantly, Lnc-THOR silencing and overexpression were ineffective in IGF2BP1-KO NSCLC cells. Moreover, forced IGF2BP1 overexpression failed to rescue proliferation, migration and invasion of Lnc-THOR-KO NSCLC cells. Therefore, by direct associating with IGF2BP1 Lnc-THOR promotes NSCLC cell growth.

CONCLUSION
These results highlighted the ability of Lnc-THOR in promoting NSCLC progression by associating with IGF2BP1, suggesting that Lnc-THOR represents a promising and novel therapeutic target of NSCLC.

DATA AVAILABILITY STATEMENT
The original contributions presented in the study are included in the article/Supplementary Material. Further inquiries can be directed to the corresponding authors.

ETHICS STATEMENT
The studies involving human participants were reviewed and approved by the Ethics Committee of Zhengzhou University. The patients/participants provided their written informed consent to participate in this study. All animal studies were performed according to the standards of IACUC of Zhengzhou University, with the protocols approved by the Ethics Committee of Zhengzhou University.

AUTHOR CONTRIBUTIONS
All the listed authors in the study carried out the experiments, participated in the design of the study and performed the statistical analysis, conceived of the study, and helped to draft the manuscript. All authors contributed to the article and approved the submitted version.

FUNDING
This work is supported by the Key R & D and promotion projects in Henan Province (212102310192) and Henan Medical Science and technology research plan (LHGJ20190218). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.