LINC01559 promotes lung adenocarcinoma metastasis by disrupting the ubiquitination of VIM

doi:10.21203/rs.3.rs-3369096/v1

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Research Article

LINC01559 promotes lung adenocarcinoma metastasis by disrupting the ubiquitination of VIM

https://doi.org/10.21203/rs.3.rs-3369096/v1

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published 05 Feb, 2024

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Background: As the predominant proportion of lung cancer, lung adenocarcinoma (LUAD) has emerged as a formidable malignancy that poses a substantial menace to human health. Numerous studies have demonstrated the undeniable involvement of long non-coding RNAs (lncRNAs) in tumorigenesis and tumor progression. Our investigation aims to elucidate the functional role and intrinsic molecular mechanism of LINC01559 in LUAD metastasis.

Methods: The expression and prognosis of LINC01559 in LUAD were analyzed from the database. Quantitative real‐time PCR (qRT-PCR) and In Situ Hybridization (ISH) were performed to detect the expression level of LINC01559 in LUAD cell lines and tissues. With RNA interference (RNAi) technology, the biological function of LINC01559 in LUAD cell lines was clarified through transwell assay. Tail vein injection model was established to observe the effect of LINC01559 on LUAD metastasis in vivo. RNA pull down and RNA immunoprecipitation (RIP) were utilized to explore the binding proteins of LINC01559. The rescue experiment was conducted to investigate the role of LINC01559 in promoting LUAD metastasis through vimentin (VIM). The molecular mechanism underlying the regulation of VIM by LINC01559 was elucidated using CHX-chase and ubiquitination assays.

Results: LINC01559 exhibited conspicuous upregulation in both LUAD tissues and cell lines, and was identified as a prognostic risk factor for patients with LUAD. Notably, knockdown of LINC01559 expression significantly inhibited the migration and invasion capabilities of LUAD cells. In vivo assay revealed that knockdown of LINC01559 curbed lung metastasis of LUAD. Molecular mechanism studies unveiled that LINC01559 interacted with VIM and modulated its protein level. Further investigations suggested that LINC01559 promoted LUAD metastasis by impeding the ubiquitination-mediated degradation of VIM.

Conclusions: Our results demonstrated that LINC01559 played a crucial role in fostering LUAD metastasis by stabilizing the VIM protein, which suggested that LINC01559 might be a potential therapeutic target for inhibiting LUAD metastasis.

LUAD

LINC01559

VIM

ubiquitination

metastasis

biomarker

Lung cancer stands as a significant malignancy of the respiratory system and poses a formidable threat to human health(1). Although early-stage lung cancer can be cured by radical surgery, the absence of evident symptoms in the majority of patients frequently eventuates metastasis at the time of diagnosis and a missed opportunity for curative treatment. LUAD, categorized as a form of non-small cell lung cancer, has emerged the most prevalent subtype(2). Patients diagnosed with advanced LUAD encounter elevated mortality rates and unfavorable prognoses attributed to the cancer's propensity for metastasis(3, 4). Therefore, it is imperative to investigate the pathogenesis of LUAD and innovate novel targeted therapies.

LncRNAs refers to a class of RNA exceeding 200 nucleotides in length and lacks protein-coding function(5, 6), which modulates gene expression across epigenetic, transcriptional, and post-transcriptional levels(7–9). Recent studies have revealed that lncRNAs play pivotal roles in various cellular processes, including cell apoptosis, cell differentiation and metastasis(10, 11). Increasing evidence underscores the critical role of lncRNAs in LUAD metastasis (12, 13). Consequently, targeting lncRNA represents a promising strategy for molecular targeted therapy and biomarker development in LUAD. In our previous research, we discovered that a recently identified lncRNA named LINC01559, also known as C12orf36, played an oncogenic role in gastric cancer. Building on this, we embarked on a preliminary exploration of LINC01559 expression in LUAD using bioinformatics analysis. Intriguingly, the expression of LINC01559 exhibited strong association with tissue type, prognosis, and M stage in LUAD. These findings prompted us to delve deeper into the role of LINC01559 in LUAD.

Epithelial-Mesenchymal Transition (EMT) represents a physiological process wherein epithelial cells undergo molecular and morphological changes, resulting in the attenuation of cell adhesion and polarity, acquisition of mesenchymal characteristics, and diminution of epithelioid traits(14). This process plays a pivotal role in bolstering tumor metastasis by facilitating the invasion and migration of cancer cells. Notably, the downregulation of E-cadherin expression, accompanied by the upregulation of N-cadherin and vimentin, constitutes key molecular hallmarks that delineate the phenomenon of EMT(15, 16). Vimentin is one of the extensively expressed and highly conserved proteins of the type III intermediate filament family (IF)(17). As per the report, increased expression of VIM correlates with the occurrence and progression of bladder cancer, lung cancer, rectal cancer, breast cancer, gastric cancer and other malignancies(18–22). In recent years, VIM has garnered increasing importance as a marker of EMT, which is characterized by the expression of vimentin IF in epithelial cells that normally express only keratin intermediate filaments(23). However, as of our current understanding, the association between lncRNAs and VIM in LUAD metastasis remains unexplored. Therefore, investigating the regulatory relationship between lncRNAs and VIM is imperative for elucidating the molecular mechanism underlying LUAD metastasis and pinpointing novel therapeutic targets.

In this study, we investigated the biological function of LINC01559 in LUAD and elucidated its molecular mechanism in promoting metastasis. Our findings unveiled that LINC01559 was significantly upregulated in LUAD and positively correlated with advanced M stage and poor prognosis. In vitro assays unequivocally demonstrated that LINC01559 could enhance the migration and invasion of LUAD cells, and in vivo assays confirmed the promoting role of LINC01559 in LUAD metastasis. Subsequent experiments demonstrated that LINC01559 could impede the ubiquitination degradation of VIM by forming a binding interaction. Our discoveries may offer a new molecular target and theoretical foundation for targeted therapy of LUAD.

Database analysis

Expression data of LINC01559 in various tumor tissues and adjacent normal tissues were acquired from TCGA(24) and GTEx(25) database. The 5-years overall survival data and corresponding LINC01559 expression data of LUAD patients were obtained from TCGA, and analyzed using Kaplan-Meier survival analysis. Additionally, the relationship between LINC01559 expression and M stage was investigated through GSE11117 as part of the Biomarker Exploration of Solid Tumors database(26). The exploration of the relationship between VIM expression and LUAD stage through GSE13213 was conducted using the same method.

Cell lines and culture

We purchased the A549, H1299, H1975, H838, HEK-293T, and CCC-HSF-1 cell lines from Shanghai Cell Bank Library of the Chinese Academy of Sciences (Shanghai, China). A549, H1299, H1975, and H838 cell lines were cultured in RPMI-1640 medium (Invitrogen, CA, USA) with 10% fetal bovine serum (FBS) (Invitrogen, CA, USA) at 37°C in a 5% CO₂ environment. CCC-HSF-1 and HEK-293T cell lines were cultured in DMEM (Invitrogen, CA, USA) with 10% FBS at 37°C in 5% CO₂.

RNA extraction and quantitative real-time PCR assays

We used TRIZOL reagent (Invitrogen, CA, USA) to extract total RNA from cells according to the product manual. RNA was reverse-transcribed into cDNA utilizing HiScript® III RT SuperMix (Vazyme Biotech, Nanjing, China). cDNA was quantified by qRT-PCR and the result was generate with AceQ® Universal SYBR qPCR Master Mix (Vazyme Biotech, Nanjing, China) utilizing Applied Biosystems 7500 instrument (ThermoFisher Scientific, MA, USA). β-Actin was utilized as an interior control. The detailed primer sequences are listed in Supplemental Table S1.

Cell transfection

Small interfering RNA (siRNA) targeting LINC01559 and VIM were purchased from Ribobio (Guangzhou, China). The pcDNA3.1 plasmid, LINC01559-pcDNA3.1 plasmid and VIM-pcDNA3.1 plasmid were purchased from TSINGKE Biological Technology (Beijing, China). The lipofectamine 3000 Transfection Kit (Invitrogen, CA, USA) was used to aid in the transfection of siRNA or plasmid into cells. A short hairpin RNA (shRNA) targeting LINC01559 embedded into the pGreenPuro vector plasmid was purchased from TSINGKE Biological Technology. The detailed sequences of siRNA and shRNA are presented in Supplemental Table S2.

Transwell assay

For the cell migration assay, A549 or H1299 cells after 48 hours of transfection were uniformly seeded to the transwell upper chambers at density of 5×10⁴ cells per well. Each of the lower chamber contained 700 µL of 1640 medium with 10% FBS. The cell invasion assay was performed similarly using transwell upper chamber coated with Matrigel (BD Biosciences, New Jersey, USA) and cells were seeded to the transwell upper chambers at density of 1×10⁵ cells per well. The cells were then incubated at 37°C with 5% CO₂ for 12–18 hours. 4% paraformaldehyde solution was used to fix cells on the lower surface and 0.1% crystal violet to dye the penetrating cells. Results were evaluated though a digital microscope at 10X scene (Olympus Corporation, Tokyo, Japan).

Tissue Microarray (TMA) and In Situ Hybridization

TMA(#HLugA060PG02) was produced from 60 paraffin-embedded samples by Outdo Biotech (Shanghai, China). ISH assay was performed using the RNAscope 2.5 HD Detection Reagent-BROWN (Advanced Cell Diagnostics, CA, USA) according to the manufacturer’s instructions. Briefly, before dehybridizing in 100% alcohol, TMA was deparaffinized twice for five minutes in xylene at room temperature. After being air-dried, the tissue sections were incubated with hydrogen peroxide for 10 min at room temperature and rinsed in the distilled water for five times. Then the sections were heated in target retrieval reagent to 100°C for 20 min, followed by being treated with proteinase K and incubated in hybridization buffer containing probes at 40°C for 4 hours. After being washed, the sections were incubated with 3,3′-diaminobenzidine (DAB), and counterstaining was carried out using hematoxylin. The percentage of positive cells was ranged from 0 to 100%. The intensity of staining (intensity score) was judged on an arbitrary scale of 0 to 3: no staining (0), weakly positive staining (1), moderately positive staining (2), and strongly positive staining (3). A reactive score (RS) was derived by multiplying the percentage of positive cells with staining intensity.

In vivo assay

Animal experiment was approved by the Medical Ethics Committee of Zhengzhou University. Twelve NOD/SCID (Non-obese diabetic/Server combined immune-deficiency) mice (male, 4 weeks of age) were purchased from Shanghai Model Organisms (Shanghai, China) and housed under SPF condition. The investigators were blinded to the group allocation during the experiment and when assessing the outcome. Twelve mice were randomly divided into two groups. For tail vein injection model, A549 cells with stable LINC01559 knockdown and control A549 cells diluted in 200ul PBS were injected through tail vein (3×10⁶ cells/mice). The mice in each group were weighed every 8 days after the operation. To investigate tumor metastasis, mice were killed after 8 weeks and the number of nodules on the lungs was counted and confirmed by HE staining.

Subcellular fractionation

Cells were harvested by trypsinization and lysed in hypotonic buffer (25 mM Tris-HCl pH 7.4, 5 mM KCl, 1mM MgCl₂, 1 mM DTT, 1mM PMSF) without or with 1% Nonidet P-40 on ice for 20 min successively. After a centrifugation at 5000 × g for 15 min, the supernatants were collected as the cytoplasmic fractions and the pellets were collected to obtain the nuclear fractionation. After two times wash with cold PBS, the pellets were lysed in nucleus resuspension buffer (20 mM Hepes pH 7.9, 400 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, 1mM PMSF) on ice for 30 min with frequent oscillations. After a same centrifugation, the supernatants were collected as the nuclear fractionation.

RNA pull down

A549 and H1299 cell pellets were then lysed in lysis buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 2.5 mM MgCl₂, 1 mM EDTA, 10% Glycerol, 0.5% Nonidet P-40/Igepal CA-630, 1 mM DTT, cOmplete™ EDTA-free Protease Inhibitor Cocktail and RNase inhibitors) and sonicated after washed in PBS for three times. After rotating between 4 µg antisense / sense biotin-labelled probes with streptavidin beads (ThermoFisher Scientific, MA, USA) for 2 hours, cell pellet was added and incubated at 4°C overnight. Beads were followed by RNA isolation and immunoblotting analysis after washed in lysis buffer for six times. Information of probes ang antibodies are shown in Supplemental Table S2 and S3.

RNA immunoprecipitation

To begin, A549 and H1299 cell pellets were lysed using RIP lysis buffer. 1 µg VIM antibody or control IgG were incubated with lysates at 4°C overnight after rotating with magnetic beads (ThermoFisher Scientific, MA, USA) for additional 30min at 37°C. Next, beads were divided into two parts for extracting RNA and protein after washed in lysis buffer for three times. Finally, the purified RNA was subjected to RT-PCR analysis and protein was analyzed by western blot assay. Information of antibodies is shown in Supplemental Table S3.

CHX-chase assay

Cycloheximide (CHX) (MedChemExpress, New Jersey, USA) is an inhibitor of protein synthesis. After treatment with 100 uM CHX, the levels of VIM protein were determined by western blot assay at 0, 3, 6, and 9 hours.

Ubiquitination assay

After transfected with siRNA or overexpression plasmid of LINC01559 for 48h, 10 µM MG132 (Selleckchem, Houston, USA) was added to the cells for a 4-hours duration. Cell lysates were then obtained by lysis buffer, and immunoprecipitation was performed with VIM antibody overnight at 4°C. Magnetic beads (ThermoFisher Scientific, MA, USA) were then added to incubate overnight for 4 hours at 4°C. Finally, the proteins were analyzed using western blot assay using ubiquitin antibody (P4D1). Information of antibodies is shown in Supplementary Table S3.

Statistical analysis

All experiments were repeated at least three times. All data from independent experiments analysed by SPSS 19.0 (SPSS Software, Chicago, USA) or GraphPad Prism 9.0 (GraphPad Software, CA, USA) and indicated by mean ± standard deviation (SD). Differences between groups were analyzed with Student's t test or one-way ANOVA and correlation analysis was based on chi-squared test. P value threshold was set as 0.05 to indicate the statistical significance.

LINC01559 is upregulated in LUAD tissues and cell lines

To gain a comprehensive and in-depth understanding of LINC01559 in pan-cancer, we retrieved the expression data of LINC01559 from TCGA and GTEx database. As depicted in Fig. 1A, the expression of LINC01559 was significantly upregulated in various cancers, including LUAD. Among the 58 pairs of LUAD tissues from TCGA, LINC01559 exhibited higher expression in tumor tissues compared to normal tissues (Fig. 1B), and the ROC curve (AUC = 0.81) underscored that LINC01559 hold significant diagnostic value for LUAD (Fig. 1C). Subsequent analysis revealed that heightened expression of LINC01559 was associated with an adverse prognosis among patients (Fig. 1D). To investigate whether LINC01559 was an independent factor affecting the prognosis of LUAD, we performed COX regression analysis on the Biomarker Exploration of Solid Tumors website and identified it as an independent prognostic factor in datasets including GSE26939, GSE13213, GSE72094, and TCGA (Fig. 1E). To delve deeper into the association between LINC01559 expression and M stage, we conducted an analysis of GSE11117 data on the Biomarker Exploration of Solid Tumors website and the result indicated a significant elevation of LINC01559 expression in M1 stage compared to M0 stage (Fig. 1F). To validate these findings, we employed four LUAD cell lines to egauged the relative expression of LINC01559 via qRT-PCR in comparison with the HSF cell lines. The result indicated that LINC01559 was upregulated in the A549 and H1299 cell lines (Fig. 1G).

LINC01559 is correlated with the histological grade of LUAD patients.

To investigate the expression of LINC01559, we employed TMAs consisting of 60 tissues, including 30 LUAD tissues and paired 30 para-cancer normal tissues. The expression level of LINC01559 was evaluated by ISH assay (Supplemental Fig.SA-B). Results indicated that the expression of LINC01559 in LUAD tissues was higher compared to normal tissues (Fig. 2A-B). Further analysis revealed a significant correlation between high level of LINC01559 expression and histological grade among LUAD patients (Fig. 2C).

LINC01559 enhances invasion and migration of LUAD cells

To elucidate the role of LINC01559 in LUAD cells, we employed siRNA-mediated knockdown and overexpression plasmid system respectively to downregulate and upregulate LINC01559 expression in A549 and H1299 cells (Fig. 3A-B). Furthermore, transwell assays were performed to determine the invasive and migratory abilities of LINC01559 in LUAD cells. Our results demonstrated that knockdown of LINC01559 significantly suppressed the invasion and migration of LUAD cells (Fig. 3C-D), while overexpression of LINC01559 had the opposite effect (Fig. 3E-F).

LINC01559 promoted LUAD metastasis in vivo

Furthermore, we performed in vivo assays to assess the effect of LINC01559 on LUAD metastasis. Initially, we established A549 sh-LINC01559 and A549 sh-NC cell lines to guarantee stable knockdown of LINC01559 expression (Fig. 4A). Subsequently, we proceeded with tail vein injection of LINC01559-knockdown A549 cells and control cells to establish tumor metastasis mouse models. We observed the weight of sh-NC group gradually decreased compared to the sh-LINC01559 group and the gap increased over time (Fig. 4B). Notably, the knockdown of LINC01559 led to a visible decrease in the number of lung metastasis lesions (Fig. 4C-D), and we measured that the lungs in the sh-NC group were heavier than those in the sh-LINC01559 group (Fig. 4E). Tumor metastasis was confirmed by HE staining (Fig. 4F). Therefore, both in vitro and in vivo data corroborated the metastatic effect of LINC01559 in LUAD.

LINC01559 directly interacts with VIM

Coding Potential Calculator version 2 (CPC2) is a widely used tool for predicting the coding potential of RNA transcripts, particularly long non-coding ones(27). By utilizing CPC2, we have enhanced our confidence in the biological functionality of LINC01559 based on its non-coding attributes (Fig. 5A). Furthermore, the results of subcellular fractionation assays indicated that LINC01559 was predominantly localized in the cytoplasm, suggesting its involvement in post-transcriptional regulatory processes (Fig. 5B). To further elucidate the molecular mechanism underlying LINC01559-mediated LUAD metastasis, we employed RNA-protein pull down assays to identify LINC01559-binding proteins. Coomassie brilliant blue staining and mass spectrometry revealed that VIM was the primary protein interacting with LINC01559 (Fig. 5C-D, Supplemental Fig.SC). Subsequently, RIP assays confirmed that VIM specifically binds to LINC01559 in LUAD cells (Fig. 5E). These findings suggest that LINC01559 actively contributes to LUAD metastasis by interacting with VIM.

LINC01559 regulates the protein level of VIM

To further investigate the potential regulatory role of LINC01559 on VIM expression, knockdown experiments targeting LINC01559 were conducted in A549 and H1299 cell lines, followed by assessment of mRNA levels for VIM. The results showed that there was no significant change in the mRNA expression level of VIM (Fig. 6A). Similar results were obtained after overexpression of LINC01559 (Fig. 6B). However, we observed alterations in VIM protein levels after knockdown or overexpression of LINC01559 in A549 and H1299 cell lines (Fig. 6C-D).

LINC01559-mediated metastasis promotion is facilitated by VIM.

To explore the potential role of VIM in promoting metastasis of LUAD, we initially evaluated the mRNA expression levels of VIM in LUAD cells and HSF cell lines. Our findings indicated that VIM was significantly upregulated in LUAD cell lines compared to HSF (Fig. 7A). Bioinformatics analysis suggested that higher expression of VIM was positively correlated with the higher stage of LUAD patients (Fig. 7B). Subsequently, transwell assays were conducted to evaluate the invasive and migratory capabilities of A549 and H1299 cell lines with respect to VIM. After knocking down VIM expression using siRNA(Fig. 7C), we observed that the invasion and migration capabilities of LUAD cells were significantly inhibited (Fig. 7D-E). Importantly, our findings suggested that the upregulation of VIM could rescue the reduced migration and invasion caused by knocking down LINC10559 (Fig. 8A-B), indicating that LINC10559-mediated acceleration of migration and invasion was mediated through VIM.

LINC01559 impedes the degradation of VIM by inhibiting its ubiquitination.

Based on the observation that LINC01559 only affects VIM protein levels, we used CHX to treat A549 and H1299 cells transfected with siLINC01559-1 or siNC and monitored the expression of VIM protein at 0, 3, 6, and 9 hours. As depicted in Fig. 9A, knockdown of LINC01559 expedited the degradation of VIM, indicating that LINC01559 protected VIM from degradation in LUAD cells. Moreover, MG132, a proteasome inhibitor, was able to alleviate the decrease of VIM caused by LINC01559 knockdown (Fig. 9B). Based on the aforementioned results, further examination was conducted on the ubiquitination of VIM protein, revealing that LINC01559 knockdown or overexpression led to a increase or reduction in the ubiquitination levels of the VIM protein (Fig. 9C-D). These findings suggest that LINC01559 plays a protective role in preventing proteasomal degradation of VIM.

The human genome consists of both coding and non-coding genes, with non-coding genes accounting for approximately 98% of the human genome. As humans, who have undergone millions of years of evolution, why do we allow a large number of non-coding genes to still exist in our genome? The phenomenon provokes contemplation regarding the potential substantial roles played by non-coding genes. In the past, non-coding genes were often referred to as the "dark matter" of the genome due to a lack of in-depth understanding. However, as sequencing technologies have advanced, researchers are increasingly focusing on the biological roles of non-coding genes.

LINC01559, a long intergenic non-protein coding RNA, is transcribed from DNA located on Chromosome 12p12-12p13.1 (Supplemental Fig.SD). As a newly discovered lncRNA, LINC01559 has revealed itself to be intimately entwined with tumorigenesis and development(28–30). Lou et al. discover that LINC01559 exhibits heightened expression in PC cell lines relative to normal controls and facilitates PC cell proliferation and migration in vitro(31). Similarly, Yang’s results indicate that LINC01559 is significantly increased in TNBC tissues as compared to matched normal tissues and knockdown of LINC01559 impedes TNBC cell proliferation, migration and invasion in vitro(30). In like wise, we observed LINC01559 upregulation in LUAD and involved the M stage and prognosis of LUAD patients, which indicated that LINC01559 acted as a considerable role for facilitating the metastasis of LUAD. Correspondingly, knockdown of LINC01559 significantly inhibited the LUAD cells migration and invasion in vitro and LUAD metastasis in vivo.

Numerous researches have substantiated that LINC01559 regulates tumor-related biological processes by acting as a microRNA (miRNA) sponge(32–34). Additionally, lncRNA functioning through its interaction with RNA binding proteins is also a prevalent molecular mechanism(35–37). Teng et al. find that lncRNA PLANE promotes cancer pathogenesis via binding to hnRNPM to regulate an alternative splicing program(38). Xu et al. elucidates the SNHG1 interactes with EZH2 to regulate colorectal cancer cell growth(39). After ascertaining the predominant cytoplasmic localization of LINC01559 in LUAD cells, we identified the binding protein VIM of LINC01559 in LUAD cells by RNA-protein pull down assays.

VIM is an essential protein in the process of EMT and directly affects the progression of EMT(40, 41). Huang’ research underscores the oncogenic role of VIM in hepatocellular carcinoma cells(42). Furthermore, research by Sara Monteiro-Reis et al. highlights the expression of VIM associates with invasive and metastatic phenotype of bladder cancer(43). Our research discovered that knocking down VIM expression significantly curbed the migration and invasion ability of LUAD cell lines, which indicating that VIM was a functional protein to promote the metastasis of LUAD. Further rescue experiment indicated that VIM was a critical prerequisite of LINC01559 to exert its promotive function in LUAD metastasis. Based on the observed positive association between LINC01559 and VIM protein, additional investigations were warranted to confirm the moderator effect of LINC01559 on VIM protein. Considering the previous finding that alterations in LINC01559 didn't impact the mRNA expression level of VIM, we speculated that LINC01559 might exert its role in promoting LUAD metastasis through post-transcriptional regulation to modulate the protein levels of VIM. Several researches have demonstrated that lncRNA can exert biological functions through regulating the stability of its binding proteins. Wang’s research results reveal that lncRNA LINRIS contactes with and stabilizes IGF2BP2 to promotes the aerobic glycolysis in colorectal cancer(44). Zhou et al. find that lncRNA SP100-AS1 induces radioresistance of colorectal cancer via inhibiting the ubiquitination mediated degradation of ATG3(45). Likewise, we demonstrated that LINC01559 impeded the ubiquitination-mediated degradation of VIM protein to stabilize VIM, which contributed to the metastasis of LUAD.

Several limitations are present in our study. Our LUAD tissue samples are too few, more LUAD tissues and more integral clinical data are necessary to analyze the relationship between LINC01559 and clinical characteristics as well as prognosis.

In summary, the results from our study indicate that LINC01559 functions as an oncogene in LUAD and positively correlated with an unfavorable prognosis and M stage. Subsequent experiments demonstrated that LINC01559 interacted with VIM and increase the stability of VIM to promote LUAD metastasis. Our results suggest a strategy for targeting LINC01559 as a therapeutic target of LUAD patients and elucidated a novel regulatory mechanism of LINC01559 (Fig. 10).

LUAD

Lung adenocarcinoma IF:Intermediate filament

lncRNAs

Long non-coding RNAs siRNA:Small interfering RNA

qRT-PCR

Quantitative real-time PCR shRNA:Short hairpin RNA

ISH

In Situ Hybridization TMA:Tissue Microarray

RNAi

RNA interference DAB:Diaminobenzidine

RIP

RNA immunoprecipitation RS:Reactive score

VIM

Vimentin CHX:Cycloheximide

EMT

Epithelial-Mesenchymal Transition SD:Standard deviation

NOD/SCID

Non-obese diabetic/Server combined immune-deficiency

CPC2

Coding Potential Calculator version 2 MicroRNA:miRNA

Ethics approval and consent to participate

Tissue microarray was approved by the Ethics Committee of Shanghai Outdo Biotech Company (approval number: SHYJS-CP-1904007). Animal experiment was approved by the Medical Ethics Committee of Zhengzhou University. All participants provided written informed consent prior to participation.

Consent for publication

Not applicable.

Availability of data and materials

The data that support the findings of this study are available from the corresponding author upon reasonable request.

Competing interests

The authors declare that they have no competing interests.

Funding

This work was supported by grants from Science and Technology Project of Henan Province (grant number: LHGJ20220051) and Natural Science Foundation of Henan Province (grant number: 22230042050).

Authors' contributions

All authors contributed to the study conception and design. Hao Feng and Zhilei Cui designed the research, analyzed data and wrote the manuscript. Chenyang Jiang and Yuming Chen collected the online data. Zirui Ren and Xiang Li performed initial draft preparation. Dengfei Xu and Shundong Cang performed funding and resources acquisition.

Acknowledgements

Thanks to all individuals or teams involved in our research.

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No competing interests reported.

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published 05 Feb, 2024

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Editorial decision: Major revision
16 Oct, 2023
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08 Oct, 2023
Reviewers agreed at journal
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Reviewers invited by journal
25 Sep, 2023
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LINC01559 promotes lung adenocarcinoma metastasis by disrupting the ubiquitination of VIM

Status:

Journal Publication

Version 1

Abstract

Figures

Background

Methods

Database analysis

Cell lines and culture

RNA extraction and quantitative real-time PCR assays

Cell transfection

Transwell assay

Tissue Microarray (TMA) and In Situ Hybridization

In vivo assay

Subcellular fractionation

RNA pull down

RNA immunoprecipitation

CHX-chase assay

Ubiquitination assay

Statistical analysis

Result

LINC01559 is upregulated in LUAD tissues and cell lines

LINC01559 enhances invasion and migration of LUAD cells

LINC01559 promoted LUAD metastasis in vivo

LINC01559 directly interacts with VIM

LINC01559 regulates the protein level of VIM

Discussion

Conclusion

Abbreviations

Declarations

References

Additional Declarations

Supplementary Files

Status:

Journal Publication

Version 1