LncRNA SNHG5 Suppresses Cell Migration and Invasion of Human Lung Adenocarcinoma via Regulation of Epithelial-Mesenchymal Transition

Long noncoding RNAs (lncRNAs) are gradually being annotated as important regulators of multiple cellular processes. The goal of our study was to investigate the effects of the lncRNA small nucleolar RNA host gene 5 (SNHG5) in lung adenocarcinoma (LAD) and its underlying mechanisms. The findings revealed a substantial drop in SNHG5 expression in LAD tissues, which correlated with clinical-pathological parameters. Transcriptome sequencing analysis demonstrated that the inhibitory effect of SNHG5 was associated with cell adhesion molecules. Moreover, the expression of SNHG5 was shown to be correlated with epithelial–mesenchymal transition (EMT) markers in western blots and immunofluorescence. SNHG5 also had significant effects of antimigration and anti-invasion on LAD cells in vitro. Furthermore, the migration and invasion of A549 cells were suppressed by overexpressed SNHG5 in the EMT progress induced by transforming growth factor β1 (TGF-β1), and this might be due to the inhibition of the expression of EMT-associated transcription factors involving Snail, SLUG, and ZEB1. In LAD tissues, the expression of SNHG5 exhibited a positive association with E-cadherin protein expression but a negative correlation with N-cadherin and vimentin, according to the results of quantitative real-time PCR (qRT-PCR). In summary, the current work demonstrated that the lncRNA SNHG5 might limit cell migration and invasion of LAD cancer via decreasing the EMT process, indicating that SNHG5 might be used as a target for LAD therapeutic methods.


Introduction
Lung cancer has become one of the most common malignant tumors, as well as the leading cause of cancer-related death globally. Lung adenocarcinoma (LAD) is the most familiar type of primary lung cancer and is generally diagnosed at an advanced stage [1]. Te treatment options available for LAD include surgery, chemotherapy, radiation, and biological therapy [2][3][4]. Tumorigenesis is an intricate process that involves various genetic alterations and fnally leads to malignant transformation [4]. Despite the fact that immunebased approaches and chemoprevention initiatives have revolutionized the treatment of metastatic illnesses, medication toxicity and the growth of resistance remain major difculties [3][4][5]. Tus clarifying the process of tumor growth and establishing novel treatment targets is critical for the treatment of cancer. However, the details of the molecular mechanisms promoting LAD progression have yet to be clarifed.
Long noncoding RNA (lncRNA) is defned as a transcript with a length of more than 200 nucleotides, which plays a signifcant role in human cancer through chromatin remodeling, transcription, and post-transcriptional regulation [6]. Te 524-bp small nucleolar RNA host gene 5 (SNHG5 or U50HC) belongs to the non-protein-coding multiple small nucleolar RNA (snoRNA) host gene family and the 5′-terminal oligopyrimidine gene class [7]. It was formerly reported that SNHG5 is functionally related to many kinds of tumors, including chronic myeloid leukemia [8], gastric cancer [7,9], colorectal cancer [10], melanoma [11], and bladder cancer [12]. In addition, a recent document proclaimed that the overexpression of SNHG5 sensitized LAD cells to geftinib treatment by interacting with miR-377 to modulate its downstream target, CASP1 [5]. However, the functions and molecular basis of SNHG5 in LAD cells and their correlation with LAD pathogenesis have not been further explored.
In the present study, SNHG5 was shown to be signifcantly downregulated in LAD tissues. In vitro, SNHG5 overexpression inhibited lung cancer cell migration and invasion, whereas SNHG5 knockdown increased LAD cell migration and invasion. In addition, our results revealed that SNHG5 plays a role in LAD advancement via modulating the epithelial-mesenchymal transition (EMT) of lung cancer tissues, indicating that SNHG5 might be a valuable target in the treatment strategy for LAD.

Tissue Specimens.
Te tumor tissues and normal adjacent tissues from 61 patients with LAD were collected from 2014 to 2017 at the Fourth Hospital of Hebei Medical University. Te noncancerous tissues were obtained at least 5 cm from the tumor site. Patients who underwent surgical resection and received no preoperative radiotherapy or chemotherapy were enrolled. Two independent pathologists confrmed the histopathological diagnosis according to World Health Organization (WHO) standards. Te tissue samples were promptly frozen in liquid nitrogen and preserved at -80°C after surgical excision. Te newest UICC (Union for International Cancer Control) and AJCC (American Joint Committee on Cancer) tumor classifcations and tumor node metastases (TNM) stages were described. Te Fourth Hospital of Hebei Medical University's Ethics Committee accepted this study [Approval # 2019057]. At the beginning of this study, each participant signed a written informed consent form.  [13,14]. All patients meeting a clinical endpoint had >30 days of follow-up. Following this, gene expression levels as FPKM (fragments per kilobase of exon per million fragments mapped) were used for further analysis. We transformed the level three HTSeq-FPKM into transcripts per million reads (TPM) for quantifcation and comparison [15,16]. Te progression-free interval (PFI) refers to the time during which the disease of the participant does not deteriorate during and after treatment [13]. Disease-specifc survival (DSS) refers to the period during which patients have not died from a specifc disease [13]. Cox regression analysis and the Kaplan-Meier technique were used to perform the survival analysis. Statistical analysis and visualization were performed in R version 3.6.3.

Cell Lines and Reagents.
Te cell bank of the Chinese Academy of Sciences' Committee on Type Culture Collection (Shanghai, China) provided the human NSCLC cell lines (A549 and H460). In a humidifed incubator, the cell lines were cultured at 37°C with 5% CO 2 in RPMI 1640 medium (Gibco, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin, and 100 g/mL streptomycin (Biological Industries, Beit Haemek, Israel).

Quantitative Real-Time PCR (qRT-PCR).
TRIzol reagent was used to extract total RNA from cells and LAD tissues (Invitrogen, Waltham, MA, USA). Using a Reverse Transcription Kit, an equivalent quantity of RNA was reverse transcribed into cDNA (Promega, Madison, WI, USA). Te ABI Prism 7900 Sequence Detection System (Applied Biosystems, Foster City, CA, USA) was used to run RT-PCR using the SYBR-Green PCR Master Mix kit (Promega, Madison, WI, USA). Te gene expression was measured using the 2 -ΔΔCt of each response. Te internal control was GAPDH (glyceraldehyde-3-phosphate dehydrogenase). Te primer sequences for qRT-PCR are listed in Table 1. 2.6. MTS Assay. Cell viability was evaluated using the 3-(4, 5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2(4sulfnyl)-2-H-tetrazolium (MTS) assay. For 24, 48, or 72 hours, 2,000 cells per well were planted into 96-well culture plates; 8 µL of MTS solution (Promega, Madison, WI, USA) was added to each well at each time point and incubated at 37°C in the dark. A spectrophotometer was used to measure the absorbance of each well after two hours at 2 Journal of Oncology 492 nm (Anthos, Salzburg, Austria). Each experiment was carried out three times, with six replications in each group.

Cell Invasion and Migration Analysis. A 24-well
Transwell insert (Collaborative Biomedical, Bedford, MA, USA) with an 8 µm pore size, and Matrigel precoating was used to assess cell invasion (BD Biosciences, Bedford, MA, USA). Te upper compartment was flled with A549 cells (1 × 10 5 ) in 200 µL serum-free media, whereas the bottom chamber was flled with 600 µL of medium containing 10% FBS as a chemotactic agent. After 24 hours of incubation, the nonmigratory or noninvasive cells were removed from the top chambers using cotton swabs, and the flters were rinsed with ice-cold PBS. Te cells that reached the lower side of the membrane were fxed with 4% paraformaldehyde, stained with 1% crystal violet, and counted using an inverted fuorescence microscope (magnifcation: ×100). Te migration analysis was carried out in the same way as the invasion assay, with the exception that 1 × 10 5 cells in serumfree RPMI 1640 medium were introduced to the top compartment without the Matrigel, and a chemoattractant of 10% FBS was utilized. All of the experiments were carried out three times.

Wound Healing Assays.
Te capacity of cells to migrate was further investigated using a wound-healing assay. Te transfected cells were cultured for 24 hours in 6-well plates. After that, a monolayer of cells was scraped using a 20 µL sterilized micropipette tip and cultured for another 24 hours. We observed and recorded the progress of the migration at 0, 24, and 48 hours or other indicated time points after wounding and measured and calculated the distance between the two edges of the scratch.

Western Blot Analysis.
Te transfected cells were lysed in a lysis bufer, and the protein concentrations were determined using the BCA protein assay kit (Termo Fisher Scientifc, Waltham, MA, USA). Subsequently, the proteins were separated by 12% SDS-PAGE gels and transferred onto polyvinylidene fuoride (PVDF) membranes. Blocking was carried out in 5% nonfat dry milk at room temperature for one hour before incubation with the following antibodies at

Immunofuorescence
Assays. SNHG5 or empty vectortransfected cells were seeded on chambered slides of 24-well culture plates for immunofuorescence labeling, as previously reported [17]. Antibodies against E-cadherin, Ncadherin, vimentin, and secondary (FITC)-conjugated rabbit antibody (ZSFB-Bio, Beijing, China; ZF-0311) were used to stain the cells. Te images were taken with an Olympus confocal laser scanning microscope and software from Olympus (Nikon, Tokyo, Japan).

2.11
. Transcriptome (RNA-Seq) Analysis. Tree biological replicates for each pair of SNHG5 or empty vectortransfected cells were performed to detect changes in gene expression after SNHG5 overexpression by transcriptome (RNA-seq) analysis. Briefy, the total RNA was obtained using an RNeasy mini kit (Qiagen, Hilden, Germany). A library with paired ends was produced using the TruSeqTM RNA sample preparation kit, according to the TruSeqTM RNA sample preparation protocol (Illumina, San Diego, CA, USA). Briefy, poly-T oligonucleotides were employed to purify poly-A-containing mRNA molecules using magnetic beads. After purifcation, the mRNA was fragmented at 94°C for 8 minutes using divalent cations. Reverse transcriptase and random primers were used to copy the chopped RNA into frst-strand cDNA. Te second-strand cDNA was then synthesized using DNA polymerase I and RNase H. Afterwards, the cDNA fragments were subjected to an end-repair procedure. Te ligation of the adapters and the addition of a single "A" base were completed. To construct the fnal cDNA library, the products were purifed and enriched by PCR. To confrm the insert size and determine Carlsbad, CA, USA) and verifed using the Agilent 2100 bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). Te cBot constructed the cluster with the library diluted to 10 pM. Finally, the fragments were sequenced on the Illu-minaNovaSeq 6000 (Illumina, San Diego, CA, USA). Shanghai Sinomics Corporation was responsible for the development and sequencing of the sequence library. Te GEO database was used to store the sequencing data (GSE143766). All diferentially expressed genes were mapped to the Kyoto Encyclopedia of genes and genomes enrichment analysis (KEGG) database and searched for signifcantly enriched KEGG pathways at p < 0.05 level, as indicated by reference [18].

Gene Expression Profling Interactive Analysis (GEPIA)
Analysis. Correlation of SNHG5 with other clinical biomarkers was performed based on Gene Expression Profling Interactive Analysis (GEPIA) database (http://gepia.cancerpku.cn/index.html), which is an interactive website based on TCGA and Genotype-Tissue Expression (GTEx)-based project [19,20]. Spearman's rank correlation analysis was used to determine the correlation coefcient between two genes.

Statistical Analysis.
Te SPSS (version 21.0) program was used for all statistical analyses (IBM, Armonk, NY, USA). For comparison, one-way analysis of variance, Student's t-test, and the Mann-Whitney U test were used, as described. Spearman's rank correlation analysis was used to look at the relationship between SNHG5 and EMT markers expression. A p value of 0.05 was used for signifcance determination, and all statistical tests were two-sided.

Te Expression of SNHG5 was Decreased in Lung Adenocarcinoma Tissues and Negatively Correlated with LAD
Aggressiveness. Te levels of SNHG5 expression in 61 samples of LAD tissues and surrounding normal tissues were frst determined using qRT-PCR. As shown in Figure 1(a), the expression of SNHG5 in tumor tissues was much lower than in surrounding normal tissues. Furthermore, the results from controlled clinical trials also revealed that higher expression of SNHG5 is signifcantly positively correlated with a smaller size of the tumor (Figure 1(b)), earlier TNM stage (Figure 1(c)), and clinical-pathological negative lymph node metastasis ( Figure 1

Suppressed Efect of SNHG5 on Lung Cancer Cell Lines
Was Associated with EMT. To investigate how SNHG5 affects lung cancer metastasis, we performed transcriptome sequencing to compare the diferential expression of genes in the SNHG5 overexpressed lung cancer cell A549 with the control group. Te sequencing data were uploaded to the GEO database (GSE143766).
Tere were 262 dysregulated genes associated with SNHG5 overexpression, including 27 genes that were upregulated and 235 genes that were downregulated (fold change > 2, p < 0.01), which were found to be overlapping in both groups (Figures 2(a) and 2(b)). Te KEGG pathway enrichment analysis revealed that some downregulated genes were cell adhesion molecules (Figure 2(c)). Six target genes related to migration and invasion were chosen for verifcation by qRT-PCR analysis in order to corroborate the transcriptome sequencing results [21][22][23]. As shown in Figure 2(d), the levels of expression of ADAMTS20, COL5A3, CXCL10, CXCL11, IL22RA1, and MMP13 were consistent with the results of the transcriptome sequencing. We hypothesized that the efect of SNHG5 on lung cancer cell lines was associated with EMT based on these fndings and the fact that EMT has been strongly linked to tumor progression and metastasis [24]. Terefore, western blot and immunofuorescence were further employed to determine the impact of SNHG5 on the EMT process of LAD. Te results showed that overexpressed SNHG5 increased the expression of the epithelial marker Ecadherin while decreasing the expression of the mesenchymal markers N-cadherin and vimentin, whereas SNHG5-shRNA caused a decrease in the expression of Ecadherin while increasing the expression of N-cadherin and vimentin (Figures 2(e) and 2(f )). Tese fndings suggested that SNHG5 might prevent lung cancer cell migration and invasion by suppressing the EMT process.

SNHG5 Inhibited Lung Cancer Cell Migration and
Invasion In Vitro. Next, in order to evaluate the potential biological role of SNHG5 in LAD, several approaches were used to examine variations in cell growth, invasion, and migration following SNHG5 overexpression or silencing. Initially, the CD511B-SNHG5 plasmid was constructed for the cell experiment. Compared with the empty vector, the expression of SNHG5 increased sharply after transfection with the CD511B-SNHG5 plasmid in both A549 and H460 cells (Figure 3(a)). Furthermore, overexpression of SNHG5 showed little efect on A549 cells but drastically slowed H460 cell growth, according to the MTS assay's growth curves (Figure 3(b)). Additionally, the results of the Transwell tests revealed that the upregulation of SNHG5 markedly decreased the invasion and migration of A549 and H460 in comparison with the control group (Figures 3(c) and 3(d)). Results of wound healing assays showed that the SNHG5 upregulation repressed the migration of A549; however, it had no efect in H460 (Figures 3(e) and 3(f )). Terefore, we selected A549 cells for further investigation of the efects of SNHG5 downregulation. 4 Journal of Oncology Two specifc siRNAs (siRNA #1 and #2) were shown to have considerably lowered endogenous SNHG5 expression (Figure 3(g)). Tough the downregulation of SNHG5 had no efect on the proliferation of A549 lung cancer cells (Figure 3(h)), inhibiting SNHG5 increased cell migration and invasion when compared with the control (Figure 3(i)). Consistent with these results, the downregulation of SNHG5 increased the migration ability of A549 cancer cells (Figure 3(j)). Taken together, these fndings suggest that SNHG5 is important in preventing cell metastasis. Factor β1 (TGF-β1). As well known that TGF-β1 is one of the most important extracellular inducers of EMT, which was marked by a reduction in E-cadherin and an increase in Ncadherin [24]. Terefore, to test the infuence of SNHG5 on the EMT process of LAD cells, we established an EMT model of A549 cells induced by TGF-β1 (10 ng/mL) in the present study. After 48 hours of treatment by TGF-β1, A549 cancer cells presented as EMT elongated morphological shapes, while untreated A549 cells still showed oval and epithelial-like morphology (Figure 4(a)). After TGF-β1 treatment, the expression of E-cadherin decreased while the expression of Ncadherin and vimentin was increased signifcantly (Figures 4(b) and 4(c)). Furthermore, the immunofuorescence results further demonstrated that TGF-β1 treatment caused considerable alterations in the elongated shape of A549 cells (Figure 4(c)). Herein, TGF-β1 was able successfully to induce EMT in A549. At this time, the expression of SNHG5 in TGF-β1-induced A549 cells rapidly dropped, reaching its lowest level after 48 hours (Figure 4(d)). Tus, we picked this time period for the ensuing further tests.

Te Overexpression of SNHG5 Inhibited the EMT of A549
Cells Induced by TGF-β1. Next, we overexpressed SNHG5 in A549 cells following TGF-β1-induced EMT to elucidate whether it had an infuence on the EMT. We frst analyzed the SNHG5 expression levels in control and SNHG5overexpressing A549 cells with or without 48 h of TGF-β1 treatment. It was revealed that overexpression of SNHG5 partially blocked the TGF-β1-induced reduction of SNHG5  ( Figure 5(a)). Furthermore, the overexpression of SNHG5 dramatically reduced the downregulation of E-cadherin and the upregulation of N-cadherin and vimentin produced by TGF-β1, as shown in Figures 5(b) and 5(c). Moreover, the overexpression of SNHG5 inhibited TGF-β1-induced morphological alterations in A549 cancer cells ( Figure 5(d)). We further performed real-time PCR and western blotting to identify the EMT-related transcription factors in A549 cells, such as Snail, SLUG, and ZEB1, to further investigate the inhibitory impact of SNHG5 on the EMT of A549 cells caused by TGF-β1. Accordingly, overexpression of SNHG5 decreased the expression of Snail, SLUG, and ZEB1 in TGF-β1 treated A549 cells (Figures 5(e) and 5(f)). Furthermore, overexpressed SNHG5 inhibited the migration and invasion of A549 cells induced by TGF-β1 treatment, which was consistent with the change of EMT-related factors (Figures 5(g)-5(i)). Tese fndings showed that overexpression of SNHG5 in A549 cells partly reversed TGF-β1-induced EMT.

SNHG5 Reversed the Expression of EMT Markers in LAD
Tissues. To further validate the efect of SNHG5 on EMT of LAD tumor tissues, the levels of expression of EMT markers, including CDH1 (E-cadherin encoding gene), N-cadherin, and vimentin, in LAD and the corresponding noncancerous tissues were measured. Our results showed that, in 34 cases of lung cancer, the CDH1 demonstrated lower expression, while Ncadherin and vimentin had higher levels of expression in tumor tissues, which were signifcantly diferent from those in the corresponding noncancerous tissues (n = 34, Figure 6(a)). Furthermore, SNHG5 expression was positively linked with CDH1 but negatively with N-cadherin and vimentin ( Figure 6(b)). All of these fndings indicate that SNHG5 inhibited LAD cancer cell proliferation, migration, and invasion by reversing the EMT process. To further explore the clinical value of SNHG5, we analyzed the correlation of SNGH5 with genes of popular clinical LAD biomarkers, including CEA (CEACAM1, CEACAM3, CEACAM5, CEA-CAM6), NSE (ENO2), and Mannan-binding lectin-associated serine protease 2 (MASP2) using GEPIA database. Te results demonstrated that SNHG5 was negatively correlated with all of these markers (Supplementary Figure 1). Meanwhile, we performed an interaction analysis between popular chemicals and SNHG5 using the Comparative Toxicogenomics Database (CTD) (http://ctdbase.org/) [25]. Te result showed that co-treated with Jinfukang (JFK), an efective herbal medicine formula against lung cancer, cisplatin could increase the expression of SNHG5 mRNA in another study (Supplementary Table 1) [26]. Taken together, these results suggested that SNHG5 might have a therapeutic role in LAD suppression.

Discussion
In the present study, the lncRNA SNHG5 in lung adenocarcinoma cancer cells was identifed, and its expression was found to be downregulated in lung cancer cells with respect to tumor size, lymph node metastasis, and TNM stage. A succession of experiments has shown a relationship between lncRNA SNHG5 and EMT biomarkers, leading to the conclusion that lncRNA SNHG5 reduces LAD cell invasion and metastasis by controlling the EMT process. Te current fndings emphasize the signifcance of the lncRNA SNHG5 in the development of LAD tumors.
It has been demonstrated by several studies that some lncRNAs, including SBF2-AS1, H19, and OR3A4, have a wide range of applications in the diagnosis and treatment of lung cancer [17,27,28]. SNHG5 belongs to a vast family of noncoding genes that host small RNAs such as snoRNAs and microRNAs, which are usually found in the host genes' introns. SNHG5 is known to be highly downregulated in LAD patients with acquired geftinib resistance, despite the fact that it is infrequently explored in lung cancer [5]. In the current study, the tumor tissues were all isolated from LAD patients without preoperative radiotherapy or chemotherapy. Te expression of SNHG5 in adjacent tissues was much greater than that in LAD tissues, as indicated in this study, and downregulation of SNHG5 might increase metastasis and migration in LAD cells. Te transcriptome sequencing analysis demonstrated that SNHG5 functioned as a wide gene suppressor in LAD cells. Some of the suppressed molecules were involved with cell adhesion, including mmp13. Matrix metalloproteinases (MMPs) are a class of enzymes that regulate EMT, which is known to promote metastasis in a variety of cancer cells [29,30]. Moreover, the overexpression of SNHG5 boosted the expression of the epithelial marker E-cadherin while decreasing the expression of mesenchymal indicators N-cadherin and vimentin, according to immunofuorescence and western blot results. Transwell and wound healing assays further revealed that the overexpressed SNHG5 could repress the metastasis and migration of LAD cells in vitro. Tese results indicate that SNHG5 inhibits EMT, thus reducing lung cancer migration and invasion.
TGF-β1 has been shown to promote EMT [31,32]. We also analyzed the role of SNHG5 in TGF-β1-induced EMT in lung cancer cells. In the EMT of cancer tissues, it was found that overexpressed SNHG5 increased the expression of epithelial-related marker E-cadherin while decreasing the expression of mesenchymal-related markers vimentin and N-cadherin, as well as transcription factors. Interestingly, although SNHG5 overexpression had no efect on A549 cell proliferation, as previously identifed, it signifcantly suppressed A549 cell proliferation caused by TGF-β1, which was consistent with the relationship between the level of SNHG5 expression and lung cancer tumor size (Figure 1(b)). Terefore, it was assumed that the inhibitory efect of SNHG5 was more likely to act on the typical EMT process but not on the general state of LAD cells. In addition, upregulation of SNHG5 reduced the invasion and migration of H460 cells in Transwell assays, whereas it had no efect in wound healing assays, which indicates that SNHG5 exerts its efect on H460 cells mainly through the regulation of single cell migration rather than collective cell migration. Te mechanism of action of SNHG5 on H460 cells needs to be explored further. Tese results further confrmed that the overexpression of SNHG5 suppressed the lung cancer cell EMT that occurred at the transcriptional level.
Journal of Oncology 9 SNHG5 has been shown to increase cell migration and invasion in hepatocellular and oral squamous cell carcinomas, whereas we demonstrated the reverse impact of SNHG5 in LAD. Despite the fact that Transwell and wound healing studies in vitro demonstrated that SNHG5 inhibited migration and invasion of LAD cells, MTS testing revealed no diference in A549 cell proliferation. Terefore, additional research is needed to understand the essential  Figure 5: Overexpression of SNHG5 inhibits EMT, preventing LAD cells from migrating and invading. (a) After TGF-β1 or vehicle control treatment, qRT-PCR was used to examine the relative fold change of SNHG5 expression in control and SNHG5-overexpressing A549 cells. * * * p < 0.001. (b) After TGF-β1 exposure, qRT-PCR was used to examine the relative fold change of EMT markers in normal and SNHG5overexpressing A549 cancerous cells. (c) Western blot analysis was used to assess the level of expression of markers in control and SNHG5overexpressing A549 cancer cells after TGF-β1 treatment. (d) Te inverted microscope determined the morphological changes in control and SNHG5-overexpressing A549 cells after TGF-β1 or vehicle control treatment. (e, f ) Western blot and qRT-PCR analysis of EMT-related transcription factors in normal and SNHG5-overexpressing A549 cancer cells following TGF-β1 or vehicle control treatment. (g-i) Transwell and wound-healing experiments were conducted on control and SNHG5-overexpressing A549 cancer tissues after TGF-β1 treatment. Wound-healing distance was measured and expressed as a percentage of the distance at zero hours. a: * * p < 0.01; b: * * p < 0.01; c: * * * p < 0.001; d: * * p < 0.01; e: * * * p < 0.001; f: * * * p < 0.001. functions of SNHG5 in human cancer [33]. However, the negative correlation between SNHG5 and several valuable prognostic biomarkers for LAD, including CEA, NSE, and MASP2, and the increased expression of SNHG5 mRNA induced by cisplatin in the previous strongly suggest that SNHG5 might have a therapeutic role in LAD [26]. Tus, to make the current fndings more accurate and reliable, more therapeutic information related to SNHG5 should have been obtained for the LAD samples. In the future, as technology progresses and research advances, SNHG5 will aid in the diagnosis and treatment of LAD.

Conclusions
In conclusion, the current fndings have provided a better knowledge of the functions of lncRNAs in LAD etiology and development, suggesting that SNHG5 might have a therapeutic role in LAD.

Data Availability
Te datasets used and/or analyzed during the current study are available from the corresponding author upon reasonable request.

Ethical Approval
All procedures performed in studies that involved human participants were in accordance with the ethical standards of the institutional and/or national research committee, the 1964 Helsinki declaration, and its later amendments or comparable ethical standards. Ethical approval of the Institutional Review Board (IRB) was sought and granted from the Fourth Hospital of Hebei Medical University [Approval # 2019057].

Consent
Written informed consent has been obtained from the patient(s) to publish this paper.

Supplementary Materials
Supplementary