LncRNA LINC00337 Sponges miR-1285-3p to Promote Proliferation and Metastasis of Lung Adenocarcinoma Cells Through Up-Regulating YTHDF1

Background: Emerging studies have attested that long noncoding RNAs (lncRNAs) predominantly functioned in carcinogenesis of multiple developing human tumors. The current research aimed at probing the underlying participation and mechanisms of LINC00337 in lung adenocarcinoma. Methods: Here we analyzed TCGA and GTEx datasets and chose LINC00337 as research object. Cell proliferation, cell apoptosis, cell cycle, and invasion were detected in gain and loss experiment of LINC00337 both in vitro and in vivo. Moreover, RNA pull-down, luciferase reporter assays, western blotting analysis, rescue experiment were performed to investigate underlying molecular mechanisms of LINC00337 function. Results: LINC00337 was remarkably increased in lung adenocarcinoma. Also, LINC00337 knock-down was unraveled to repress cell invasion and proliferation as well as cell cycle, and gear up apoptosis in lung adenocarcinoma in vitro and in vivo. With respect to mechanism, LINC00337 knock-down boosted miR-1285-3p to be expressed and then restrained YTHDF1 to be expressed post-transcriptionally. Crucially, both miR-1285-3p decrement and YTHDF1 overexpression successfully countered the inuence on cell proliferation, invasion and apoptosis caused by LINC00337 shRNA. Conclusions: These results suggest that LINC00337 acted as an oncogenic lncRNA, targeting miR-1285-3p and regulating YTHDF1 expression, to promote the progression of lung adenocarcinoma.


Background
As a frequently seen malignant tumor, lung cancer turns out to be the chief cause of global cancerinvolved death [1,2]. Lung cancer is histologically assorted into large cell carcinoma, squamous cell carcinoma, adenocarcinoma and bronchoalveolar carcinoma [3]. Lung cancer patients have low overall ve-year survival rate (about 18.1%). Lung adenocarcinoma cases take up near 40% of lung cancer ones [4,5]. Therefore, it is of great signi cance to look for novel biomarkers and targets for early diagnosing and treating lung cancer.
Human genomic sequencing uncovers the active transcription of exceeding ninety percent of genomes, among which two percent is the RNA that encodes proteins and the remaining is the RNA unable to encode proteins [6,7]. Long noncoding RNA (LncRNA) are ncRNAs longer than 200 nt [8,9], and quite a few lncRNAs are expressed with speci city to cell types [10][11][12] and possess exact positions of subcellular compartments [13][14][15]. Additionally, expression of numerous lncRNAs has been demonstrated to interrelate to the progression of diverse cancers, which are able to modulate cancer cell proliferation and apoptosis. [16][17][18][19] According to reports, LINC00337 is a pro-tumor factor in gastric cancer [20] and esophageal cancer [21], but its function in lung adenocarcinoma remains elusive.
In current research, we analyzed the TCGA and GTEx datasets, and LINC00337 was dramatically higher in lung adenocarcinoma tissues relative to para-tumor ones. In TCGA dataset, high LINC00337 level Page 3/20 indicated shorter overall survival. We also examined samples surgically resected from 46 lung adenocarcinoma cases at our institution to gure out differences in expression level of LINC00337 between lung adenocarcinoma tissues and normal ones, and the result was in consistency with the analyses of TCGA and GTEx datasets. Then, we conducted a series of experiments to explore whether LINC00337 participated in the onset and development process of lung adenocarcinoma and the mechanism of its function.

Collection of clinical samples
From 2017 to 2019, 46 paired fresh lung adenocarcinoma and para-tumor tissues were harvested in our hospital. We snap-freezed these tissues at -80°C. Patients received no preoperative chemotherapy or radiotherapy. All included subjects offered an informed consent and the research got approval from the Institutional Review Board of Xinxiang Central Hospital. Thorough clinical and pathological features of patients with lung adenocarcinoma are summed up in Table 1. shRNAs and anti-miRNA inhibitors Genechem (Shanghai, China) synthetized shRNAs targeting LINC00337 (shRNA#1, 2) and YTHDF1 (sh-YTHDF1) and negative control shRNA (sh-NC) without exact target. RiboBio (Guangzhou, China) provided inhibitors against miR-1285-3p (anti-miR-1285-3p) and miR-NC (anti-miR-NC). Subsequent to seeding of PC-9 or A549 cells in six-well plates for twenty-four hours, they received transfection with Lipofectamine 2000 offered by Invitrogen (Shanghai, China) in the case of 40-60% con uence as per manufacturer's guidance. Forty-eight hours later, cells were collected, and stable cells were chosen for four-week therapy with neomycin (500 µg/mL).
Western blot evaluation RIPA extraction liquid from Beyotime (Jiangsu, China) in the presence of Protease Inhibitor Cocktail and PMSF (Roche, Shanghai, China) was utilized for lysis of assembled cells. Subsequent to examination of protein sample concentration via BCA Protein Assay Kit from Beyotime, the harvested proteins in commensurable amounts were set apart by SDS-PAGE (10% gel) and transferred to PVDF membranes, which underwent 1-h sealing by Tris-buffered saline (5% defatted milk) and 12-h primary antibody incubation at 4°C. Next, optical density method was adopted to quantitate autoradiographs using Quantity One software (Bio-Rad) with GAPDH (#2118; CST, Shanghai, China) as a reference. Anti-YTHDF1 (#86463), anti-E-cadherin (#3195), anti-Vimentin (#5741) antibodies were provided by CST.

Immunohistochemistry
Nude mouse tumor tissues implanted by para n received immunostaining, and expression level and position of target proteins were ascertained using avidin-biotin-peroxidase method. Next, primary antibodies against E-cadherin and Vimentin were diluted at 1:200 for later application. Tumor apoptosis and proliferation were appraised by independently probing Ki-67 and Bax. Lastly, slice visualization was achieved using a microscope from Olympus (Japan).

5-ethynyl-20-deoxyuridine assay (EdU) Assay
Cell proliferation was also proved by Ethynyl-2-deoxyuridine incorporation assay using an EdU Apollo DNA in vitro kit (RIBOBIO, Guangzhou, China) as per the manufacturer's guidance. Brie y, subsequent to transfection with the corresponding vector, cells received respective 2-h incubation at 37 °C, with 100 μl of 50 μM EdU/well. Via a uorescence microscopy, the cells were identi ed. We carried out each experiment for three times.
Cell Counting Kit-8 assay Cell Counting Kit-8 (Beyotime Inst Biotech, China) was used to determine cell proliferation. In a word, 5×10 3 cells/well underwent 1-day raising in a 96-well broad-bottomed plate at 37°C, followed by transfecting them with corresponding vectors. Finally, with a microplate reader from Bio-Rad (Shanghai, China), the absorbance was nally evaluated at 450 nm, and we carried out each experiment for three times.

Apoptosis and cell cycle experiments
As per the manufacturer's guidance, apoptosis determination was implemented via FACS using PE-Annexin V apoptosis detection kit from BD Pharmingen (Shanghai, China) subsequent to 48-h transfection, and cell cycle was assessed utilizing PI cell cycle assessment kits (BD Pharmingen). Each assay was implemented thrice.

Transwell assay
Transwell chambers were used to observe the invasion of lung adenocarcinoma cells. We seeded cancer cells in the upper chamber coated by Matrigel (Corning, USA, dilution ratio: 1:6) at a density of 10 5 cells per well and supplemented DMEM with 1% FBS. Later we lled 600μL DMEM with 10% FBS into the lower chamber, xed cells by 4% methanol and stained them with crystal violet. Then we counted them in 5 random 200 microscopic elds, after cells being invaded to the lower surface of membrane and incubated at 37°C for 24 hours. We carried out each experiment for three times.

Dual-Luciferase Reporter Assay
Genechem designed and synthesized YTHDF1 full-length promoter reporter vector. Human YTHDF1 3'untranslated region (UTR) fragment with supposed binding sites for miR-1285-3p reporter vector was offered by RiboBio. 48-h transfection later, Dual-Luciferase Reporter Assay System from Promega was adopted for luciferase activity determination as per the manufacturer's guidance, and luciferase activity ratio (Fire y/Renilla) was ascertained. Each assay was implemented thrice.
RNA pull-down assay The DNA fragment with the full length LINC00337 or NC sequence received PCR ampli cation using a primer with T7 and was cloned into GV394 from Genechem, Shanghai, China). Restriction enzyme XhoI was utilized for linearization of DNAs. Next, T7 RNA polymerase (Takara) and Biotin RNA Labeling Mix from Roche (China) were employed for reverse transcription of biotin-labeled RNAs underwent reverse transcription. Thereafter, the products received DNase I (RNase-free, Roche) treatment and puri cation using the RNeasy Mini Kit (Qiagen, USA), and the extracted RNAs were employed for qRT-PCR assessment.

Tumor xenograft implantation in nude mice
We divided six-week-old nude mice into two groups (4 mice/group) randomly, raised them with unceasing food and water in sterile conditions without pathogens.

LINC00337 is elevated in lung adenocarcinoma tissues and cells and dramatically present in the cell cytoplasm
Through the analysis of TCGA and GTEx database, we unraveled that LINC00337 was dramatically raised in lung adenocarcinoma tissues (from TCGA database) relative to normal tissues (from GTEx database) (Fig. 1A, B). We then veri ed in 46 lung adenocarcinoma tissues and adjacent nontumorous tissues by qRT-PCR assays, and the result was consistent with previous analyses of TCGA and GTEx database (Fig. 1C). Similarly, higher LINC00337 levels were showed in lung adenocarcinoma cells (PC-9, H1373, HCC827 and A549) rather than normal human lung epithelial cell line BEAS2B (Fig. 1D). Additionally, PC-9 and A549 cells were picked up for later assays. In addition, LINC00337 expression levels in lung adenocarcinoma evidently interrelated to high-grade cancer, lymph node metastasis and tumor size instead of other parameters such as age or gander (Table 1). TCGA database showed that the overall survival rate of patients with low LINC00337 level was higher relative to that of patients with high LINC00337 level (Fig. 1E). Then, we examined the subcellular localization of LINC00337 and found that most of LINC00337 was present in the cell cytoplasm in lung adenocarcinoma cells (Fig. 1F).
Knockdown of LINC00337 curbs cell cycle, proliferation and invasion but boosts apoptosis of lung adenocarcinoma cells To determine whether LINC00337 functions in lung adenocarcinoma cells, we implemented a variety of in vitro assays to assess the impact of shRNA knockdown of LINC00337 and overexpression of LINC00337 on cell functions including proliferation, apoptosis, and invasion. PC-9 transfected with LINC00337 overexpression vector and A549 cells transfected with sh-LINC00337 ( Fig. 2A). CCK-8 and EdU assays showed that overexpression of LINC00337 promoted proliferation of PC-9 cells, sh-LINC00337 attenuated proliferation of A549 cells (Fig. 2B, C). LINC00337 knockdown reduced cell cycle arrest at S phase in A549 cells compared with negative control, and overexpression of LINC00337 brought on cell cycle arrest at S phase in PC-9 cells (Fig. 2D). In Fig. 2E, evidently elevated apoptotic cell ratio was observed in sh-LINC00337 group relative to sh-NC cells, and brought down apoptotic cell proportion in LINC00337transfected cells relative to vector-transfected cells. Meanwhile, the invasion of cells was considerably elevated by overexpression of LINC00337, and decreased by sh-LINC00337 (Fig. 2F). All above-mentioned data ascertained that knockdown of LINC00337 curbs cell cycle, proliferation and invasion, and boosts apoptosis of lung adenocarcinoma cells.
Later, we utilized the pull-down system labeled by biotin to continuously probe miRNAs interplaying with LINC00337 in a direct way. We unraveled an enormous body of miR-1285-3p in the LINC00337 pull-down pellet relative to control group as examined by qRT-PCR, but the proportions of miR-492, miR-1273a, miR-5095, miR-1273g-3p, and miR-1304-5p in the LINC00337 pull-down pellet displayed unobvious elevation relative to control group (Fig. 3B, C). Moreover, miR-1285-3p was expressed in lung adenocarcinoma samples at a declined level relative to normal samples (Fig. 3D). And overexpression of LINC00337 decreased miR-1285-3p expression level tested by qRT-PCR (Fig. 3E). All above-mentioned data ascertained that LINC00337 could sponge miR-1285-3p directly and speci cally.
LINC00337/miR-1285-3p /YTHDF1 axis regulates behaviors of lung adenocarcinoma cells Subsequently, we explored the effect of LINC00337/ miR-1285-3p / YTHDF1 axis on lung adenocarcinoma. We transfected sh-YTHDF1 in PC-9 cells and transfected YTHDF1 overexpression vector in A549 cells (Additional le 1: Fig. S1B). From Fig. 5A, we found that both knockdown of miR-1285-3p and up-regulated of YTHDF1 reversed the cell proliferation reduced by LINC00337 shRNA. And both knockdown of YTHDF1 and up-regulated of miR-1285-3p reversed the cell proliferation induced by LINC00337 overexpression. Cell apoptosis and invasion assays showed the same trend, that both knockdown of YTHDF1 and up-regulation of miR-1285-3p reversed the in uence caused by overexpression of LINC00337 on cell apoptosis and invasion (Fig. 5B, C). These data suggested that LINC00337 modulated lung adenocarcinoma in vitro through miR-1285-3p / YTHDF1 axis.

Inhibition of LINC00337 suppresses lung adenocarcinoma tumor to grow and metastasize in vivo
For continuously ascertaining the capability of inhibiting anti-tumorigenesis potential of LINC00337 inhibition in vivo, stable A549 cells transfected with sh-NC or sh-LINC00337 were inoculated into nude mice. Mice in sh-LINC00337 group had decreased tumor volume and weight after assay relative to sh-NC group (Fig. 6A-C). Further, LINC00337 knock-down curbed tumor to proliferate and boosted cell apoptosis (Fig. 6D). Western blot assay, qRT-PCR, and histological research of excised tumor tissues implied positive interrelation of LINC00337 expression with YTHDF1 and Vimentin as well as inverse interrelation with miR-1285-3p and E-cadherin in LINC00337 repression and control groups (Fig. 6E-H). Furthermore, HE staining of mouse lung slices unveiled that suppressing LINC00337 cut down metastatic nodule number in the lung relative to NC group (Fig. 6I). Above-mentioned ndings uncovered potential of LINC00337 in tumor metastasis and proliferation and offered more support for treatment regimen targeting LINC00337 in lung adenocarcinoma.

Discussion
In this research, we analyzed TCGA and GTEx dataset and chose LINC00337 as research object, which was expressed at a notably higher level in lung adenocarcinoma tissues and paired para-tumor tissues. Besides, LINC00337 knock-down signi cantly curbed lung adenocarcinoma cells to proliferate and invade and arrested cell cycle, but boosted apoptosis in vitro and in vivo.
Like proteins, the function of lncRNAs depends on their subcellular localization [29]. Cytoplasmic lncRNAs that mostly localize and function in the cytoplasm act as decoys for miRNAs and proteins to affect gene modulation [30,31]. Several studies reveal that lncRNAs are sponges of many miRNAs, exerting the same function as ceRNAs in tumorigenesis [32,33].
Through subcellular localization experiment, we found that most of LINC00337 was present in the cytoplasm of lung adenocarcinoma cells, suggesting that LINC00337 might function at posttranscriptional level. In this case, LINC00337 may act as a ceRNA to disturb miRNA pathways and control the contra-suppression of miRNA targets. So, we predicted miRNA and its downstream targets which may be associated with LINC00337via searching in RegRNA 2.0, miRDB. RNA pull-down assay, dual-luciferase reporter assay, qRT-PCR, and western blot were implemented to prove the association.
Results showed that LINC00337 functions as miR-1285-3p sponge to control YTHDF1 in a positive manner. Subsequent cell function tests con rmed that both knockdown of YTHDF1 and up-regulated of miR-1285-3p reversed the in uence caused by overexpression of LINC00337 on cell invasion, proliferation and apoptosis.

Conclusion
In conclusion, we identi ed that LINC00337 was upregulated in lung adenocarcinoma and correlated with poor survival outcome in lung adenocarcinoma patients. And LINC00337 acted as an oncogenic lncRNA, targeting miR-1285-3p and regulating YTHDF1 expression, to promote the progression of lung adenocarcinoma.

Declarations Ethics statement
The present research gained approval from the Ethics Committee of Xinxiang Central Hospital and Written informed consent from patients was obtained. Animal experiments took place in SPF Animal Laboratory at Xinxiang Medical University. All animal assays were implemented as per the Guide for the Care and Use of Laboratory Animals by NIH.

Consent for publication
All authors involved in the study had given their consent for submitting this article for publication Availability of data and materials Datasets used and/or analyzed during this study are available from the corresponding author on reasonable request.

Competing interests
The authors declare that they have no con ict of interest.

Funding
This research did not receive any speci c grant from funding agencies in the public, commercial, or notfor-pro t sectors.
Authors' contributions RZ designed and conducted the majority of the experiments and manuscript writing. DW assisted with the results collection and processing. LW and GG instructed data analysis and gure production.    expression level in stable A549 and PC-9 cells after transfection. Data represent the mean ± SD; ** P < 0.01, ***P < 0.001. was assessed. Data were presented as represent the mean ± SD of 3 respective experiments; *P < 0.05, ** P < 0.01, ***P < 0.001.