Long noncoding RNA LGALS8-AS1 promotes angiogenesis and brain metastases in non-small cell lung cancer

Brain metastases (BM) are associated with poor prognosis in patients with non-small cell lung cancer (NSCLC). Considering that, LGAS8-AS1-mediated progression of BM was probed in NSCLC. The clinical characteristics of 60 NSCLC patients (30 without BM and 30 with BM) were analyzed. NSCLC patients with BM had higher levels of LGALS8-AS1 than NSCLC patients without BM. Depleting LGALS8-AS1 prevented NSCLC cell proliferation, migration, invasion, and angiogenesis in vitro , and NSCLC tumorigenesis and BM in vivo . LGALS8-AS1 targeted miR-885-3p to mediate Fascin actin-bundling protein 1 (FSCN1) expression. Restoring miR-885-3p inhibited NSCLC growth, angiogenesis, and BM, and FSCN1 induction rescued the performance of LGALS8-AS1 depletion on NSCLC cells. Our results provide new insights into LGALS8-AS1-mediated NSCLC metastasis and suggest that LGALS8-AS1 may be a useful biomarker for identifying NSCLC with metastatic potential.


INTRODUCTION
Non-small cell lung cancer (NSCLC) is one of the deadliest malignancies, accounting for more than 80% of lung cancer cases worldwide, with a current 5-year survival rate of only 15% (Salehi et al., 2020).Patients with advanced NSCLC generally die within 18 months of diagnosis mainly due to metastatic spread (Wood et al., 2014).Brain metastases (BM) are a common complication of advanced NSCLC (Chen et al., 2012), for which the management always involves systemic therapy (Fang et al., 2021).Since angiogenesis is associated with aggressiveness in NSCLC (Schettino et al., 2012), targeting inhibition of angiogenesis is promising in the treatment of NSCLC and many clinical trials have evaluated the addition of anti-angiogenic therapy to standard therapy in patients with NSCLC (Alshangiti et al., 2018).Therefore, exploring the regulatory mechanism of angiogenesis is helpful for understanding the pathogenesis of NSCLC and developing new therapeutic drugs.
Long non-coding RNAs (lncRNAs) are of significance in the physiological and pathological processes of diseases (Xie et al., 2018;Zhang et al., 2019).The occurrence of human malignant tumors is often accompanied by the deregulation of lncRNA (Kondo et al., 2017).More and more studies have shown that lncRNAs play a key role in tumorigenesis and metastasis (Hanniford et al., 2020;Yang et al., 2018;Gupta et al., 2010).LncRNAs usually regulate their downstream target genes by competing endogenous RNAs with microRNAs (miRNAs), which can affect the proliferation and metastasis of various cancer types (Zheng et al., 2019;Wang et al., 2018;Hao et al., 2019).For example, lncRNA GAN1 inhibits tumor progression in NSCLC via decoying miR-26a-5p [9]; lncRNA LINC00473 promotes proliferation, migration, invasion, and inhibition of apoptosis of NSCLC cells by acting as a sponge of miR-497-5p (Xu et al., 2021).
LGALS8-AS1 has been confirmed to be highly expressed in breast cancer and promotes breast cancer metastasis by targeting miR-125b-5p (Zhai et al., 2021).However, the expression and role of LGALS8-AS1 in NSCLC remain unclear.
MiRNAs are a group of short endogenous non-coding RNAs with a length of about 18-22 nucleotides, which can regulate post-transcriptional gene expression by binding to the 3'-UTR of target gene mRNA to inhibit mRNA translation and reduce mRNA stability (Ahn et al., 2020).MiRNAs are involved in almost all biological processes, including tumor angiogenesis (Mao et al., 2015).For example, miR-543 promotes tumorigenesis and angiogenesis in NSCLC (Wang et al., 2020) whereas miR-20a-5p inhibits tumor angiogenesis (Han et al., 2021).It is studied that miR-885-3p can architect cell autophagy and apoptosis in squamous cell carcinoma cells (Huang et al., 2011).However, the biological function of miR-885-3p in NSCLC has not been fully elucidated.
Therefore, this study aims to explore the role of LGAS8-AS1 in NSCLC metastasis and angiogenesis through regulating miR-885-3p expression and its potential mechanism.The study identified a lncRNA, LGALS8-AS1, which is associated with NSCLC metastasis and angiogenesis and elucidated the molecular regulatory mechanism of LGALS8-AS1 in NSCLC and provides a new reference for the treatment of NSCLC patients.

Cell counting kit (CCK)-8
Cells growing on the 96-well plates were tested by a CCK-8 kit (Liji, Shanghai, China) to determine proliferative activity.Quantitative results were obtained on a microplate reader (SAFAS Xenius XL, Ruixuan, Shanghai, China) at 450 nm.

Flow cytometry
Cells were stained with annexin V-fluorescein isothiocyanate and propidium iodide according to the manufacturer's instructions (Bioscience, Shanghai, China).Then the apoptosis rate was detected by flow cytometry (Beckman, USA).The percentage of cells in the Q3 quadrant represents early apoptosis, and the percentage of cells in the Q2 quadrant represents late apoptosis (Wang et al., 2020).

Transwell assay
Transwell chambers (8-μm pore size; Corning Costar, Cambridge, MA, USA) measure cell migration and invasion capacity.For cell invasion, cells suspended in serum-free RPMI-1640 medium were seeded into the upper chamber pre-coated with Matrigel.The lower chamber was PMI-1640 medium with 20% serum as a chemotactic agent.After 24 h culture, the cells were fixed with 90% formaldehyde and stained with 0.1% crystal violet.The cells were photographed under a microscope and counted.In the cell migration experiment, there was no matrigel coating, and the other operations were the same as the invasion experiment (Yang et al., 2018).

Tube formation
Matrigel (0.5mmol/L) was coated with pre-cooled 96well plates.HUVECs were starved without serum for 1 h and then re-suspended in Dulbecco's modified Eagle medium to make a cell suspension.Next, cell suspensions (1×10 5 cells/mL) were inoculated into a matrigel coating containing cell conditioned culture-medium with 3 repeat wells per treatment.The plates were then in-cubated at 37°C for 6 to 8 h.The tube formation was observed under a microscope (Olympus).The number of tubes in a branch (a branch point is a skeleton part where three or more tubes meet) and the number of rings (a ring is a background area surrounded by [or almost] tubular structures) were counted.

Real-time reverse transcriptase-polymerase chain reaction (RT-qPCR)
Total RNA was extracted using a Trizol reagent according to the manufacturer's instructions (Invitrogen, Carlsbad, CA, USA).RNA purity was determined using a NanoDrop spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA).cDNA reverse transcription kit (Promega, Madison, WT, USA) was used to reverse transcribed 1 μg total RNA into the first strand cDNA.Power SYBR Green PCR Master Mix (Promega) quantitative PCR was used to detect RNA levels.LncRNA and protein-coding gene were normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA, and miRNA was normalized to U6.The primer sequences used are presented in Table 1.
A lung cancer BM model was established according to previous studies (Li et al., 2017;Nguyen et al., 2009).A549 cells after transfection were resuspended in 100 μL of PBS and injected into the right ventricle of mice (1×10 6 , n=6/group).Finally, brain tissues were collected and prepared for HE staining and observations of metastatic nodules.

Statistical analysis
The selected way to perform statistical analysis was SPSS 19.0, and that to construct graphs was GraphPad Prism 6. Two-tailed paired Student's t-test and one-way analysis of variance were of utility for data comparison of two groups and more than two groups, respectively.Tukey's post hoc test validated pairwise comparisons.
Pearson correlation analysis assessed gene correlation.P<0.05 was considered to indicate a statistically significant difference.

RESULTS
LGALS8-AS1 is more expressed in NSCLC patients with BM A total of 60 patients with NSCLC were included in the study, of which 30 had BM and the remaining 30 did not.The clinical characteristics are shown in Table 2. Examinations of LGALS8-AS1 found that LGALS8-AS1  was increased in tumor tissues, and it was higher in tumor tissues of NSCLC patients with BM (Fig. 1A, B).

Targeting suppression of LGALS8-AS1 blocks NSCLC development
Firstly, the effect of LGALS8-AS1 on the progression of NSCLC cells was investigated.A549 cells were transfected with sh-LGALS8-AS1 to silence the expression of LGALS8-AS1, and the transfection was verified by RT-qPCR (Fig. 2A).Cell proliferation was then detected by CCK-8, and it was found that A549 cell proliferation decreased after down-regulating LGALS8-AS1 (Fig. 2B).Apoptosis was detected by flow cytometry, and the results showed that apoptosis of A549 cells increased after downregulation of LGALS8-AS1 (Fig. 2C).Transwell was used to detect cell migration and invasion, and it was found that A549 cell migration and invasion decreased after downregulating LGALS8-AS1 (Fig. 2D,  E).Angiogenesis was also examined by tube formation assays, which showed that angiogenesis was attenuated after down-regulating LGALS8-AS1 (Fig. 2F).These results indicate that down-regulation of LGALS8-AS1 inhibits proliferation, migration, invasion, and angiogenesis of NSCLC cells, and promotes cell apoptosis.

LGALS8-AS1 deficiency suppresses tumor growth and BM in vivo
Based on our findings that LGALS8-AS1 is involved in NSCLC cell progression, the study further explored its role in tumor growth and brain metastasis in vivo.Tumor growth was studied by subcutaneous injection of A549 cells stably transfected with sh-LGALS8-AS1 into   3A, B).BM were explored by injecting A549 cells stably transfected with sh-LGALS8-AS1 into the right ventricle of nude mice.As presented in HE staining results, the number of brain metastatic nodules was reduced in sh-LGALS8-AS1-treated mice (Fig. 3C, D).

miR-885-3p represses NSCLC development in vitro
In order to investigate the effect of miR-885-3p on NSCLC cells, miR-885-3p mimic or mimic NC was transfected into A549 cells, and successful transfection was verified by RT-qPCR (Fig. 5A).In A549 cells overexpressing miR-885-3p, it could be recognized that proliferative, invasive, migratory, and an-apoptotic activities were all in a weakened status, and the same was true for angiogenic capacity (Fig. 5B-F).These results suggest that up-regulation of miR-885-3p inhibits proliferation, migration, invasion, and angiogenesis of NSCLC cells, and promotes cell apoptosis.
Then, dual luciferase assay was performed to verify the targeting relationship between them.The results showed that the luciferase activity of A549 cells could be significantly reduced by co-transfection of FSCN1-WT with miR-885-3p mimic (Fig. 6B).RT-qPCR detected FSCN1 mRNA expression in lung cancer tissues, and the results showed that FSCN1 mRNA expression was up-regulated (Fig. 6C) and was negatively correlated with the expression of miR-885-3p (Fig. 6D).Moreover, mRNA and protein expression of FSCN1 in A549 cells decreased after up-regulation of miR-885-3p (Fig. 6E).These results suggest that targeting miR-885-3p regulates FSCN1 expression.

DISCUSSION
As studies indicate, 90% of lung cancer deaths are caused by distant metastasis, and BM is a common site of distant metastasis in NSCLC (Rybarczyk-Kasiuchnicz et al., 2021).LncRNA dysregulation is fundamental for tumorigenesis and distant metastasis of NSCLC and serves as a biomarker for NSCLC (Pan et al., 2020).In the present study, LGALS8-AS1 was up-regulated in NSCLC patients' tumor tissues and was more expressed in NSCLC patients with BM.Silencing LGALS8-AS1 blocked the malignant phenotype and angiogenesis in NSCLC cells, as well as inhibiting tumor growth and BM in vivo.Overall, LGALS8-AS1 plays a critical role in NSCLC metastasis and serves as a potential therapeutic target for NSCLC metastasis.
Abnormally expressed lncRNAs are involved in regulating NSCLC development by acting as miRNA sponges.For example, lncRNA plasmacytoma variant translocation 1 promotes angiogenesis in NSCLC by competitive absorption of miR-29c (Wang et al., 2018).LncRNA DNAH17 antisense RNA 1 induces the occurrence and metastasis of NSCLC by binding to miR-877-5p (Du et al., 2020).LGALS8-AS1 has only been reported in breast cancer considered an oncogenic gene regarding its potential to induce malignant phenotype and metastasis.Here, the study observed high LGALS8-AS1 expression in NSCLC, and LGALS8-AS1 expression was able to discriminate whether NSCLC patients developed BM.Cell and animal experiments consistently confirmed that LGALS8-AS1 knockdown played a negative role in  NSCLC for cell growth, angiogenesis, and tumor growth and metastasis.In addition, the RNA22 database predicted that LGALS8-AS1 had a binding site with miR-885-3p.miR-885-3p was mediated by LGALS8-AS1 in this study.miR-885-3p has received academic attention in the field of cancer.miR-885-3p expression decline has previously been found in lung adenocarcinoma to be associated with pathological stage and poor survival in patients (Yang et al., 2021).Functionally discussed, miR-885-3p impairs gastric cancer cell activities and oxidative stress (He et al., 2021), and tumor angiogenesis in colon cancer (Xiao et al., 2015).This study confirmed the reduction in miR-885-3p expression in NSCLC, and the suppressive effects of miR-885-3p on NSCLC cell growth and angiogenesis.
miRNAs usually function by binding to target gene mRNAs (Datta et al., 2019).Here, FSCN1 was selected to be a target of miR-885-3p.FSCN1 is a structurally unique and highly conserved actin cross-linking protein that mediates cellular interaction (Liu et al., 2019).It has been described that dysregulation of FSCN1 aggrandizes tumor motility and invasiveness by altering the structure of cell protrusions and focal extracellular matrix adhesions (Gao et al., 2019).Therefore, FSCN1 is considered an oncogene in cancers, including adrenocortical carcinoma (Liang et al., 2019), ovarian cancer (Li et al., 2018), laryngeal squamous cell carcinoma (Gao et al., 2018), and NSCLC (Xiao et al., 2016).Here, FSCN1 was overexpressed in NSCLC and can mitigate the effects of LGALS8-AS1 knockdown, promoting NSCLC cell growth and angiogenesis.This study did not address a possible direct relationship between LGALS8-AS1 expression and clinical factors, including tumor node metastasis staging and survival analysis, due to insufficient clinical sample size and lack of a long-term follow-up of all included patients.Also, this study did not further explore the effects of overexpression of LGALS8-AS1 and downregulation of miR-885-3p alone on NSCLC cells.In addition, FSCN1 has been confirmed to mediate NSCLC cell migration and invasion by altering the Mitogen-activated protein kinase pathway (Zhao et al., 2018).Whether the MAPK pathway or other signaling pathways are involved in the regulation of LGALS8-AS1 in NSCLC still needs to be further explored to determine detailed and comprehensive mechanics.

CONCLUSION
Our study provides new insights into the mechanism by which lncRNAs regulate NSCLC progression.Our findings suggest that the LGALS8-AS1/miR-885-3p/ FSCN1 axis is an essential signaling pathway for NSCLC cell growth, angiogenesis, and BM.

Declarations
Acknowledgments.Not applicable.Funding Statements.Not applicable.Declaration of Conflicting Interests.Authors declared no conflict of interest.
Ethical statement.All procedures performed in this study involving human participants were in accordance with the ethical standards of the Affiliated Brain Hospital of Nanjing Medical University research committee and with the 1964 Helsinki Declaration and its later amendments or comparable ethical standards.All subjects were approved by the Affiliated Brain Hospital of Nanjing Medical University.

Figure 1 .
Figure 1.LGALS8-AS1 is more expressed in NSCLC patients with BM LGALS8-AS1 expression in clinical samples (A-B); the values were expressed as mean ± standard deviation (n=60).

Figure 3 .
Figure 3. LGALS8-AS1 deficiency suppresses tumor growth and BM in vivo In nude mice injected with sh-LGALS8-AS1, tumor volume and weight were measured (A-B) and BM was evaluated (C-D); values are presented as mean ± standard deviation (n=6) *P<0.05 vs. sh-NC.