The long non-coding RNA SNHG5 regulates gefitinib resistance in lung adenocarcinoma cells by targetting miR-377/CASP1 axis

Gefitinib resistance is one of the major obstacles for the treatment of lung adenocarcinoma (LAD). The present study aimed to investigate the effects of the long non-coding RNA (lncRNA), small nucleolar RNA host gene 5SNHG5 on gefitinib resistance in LAD and explore the underlying mechanisms. The quantitative real-time PCR (qRT-PCR) results showed that SNHG5 expression was significantly down-regulated in LAD patients with acquired gefitinib resistance and gefitinib resistant LAD cell lines. SNHG5 overexpression sensitized gefitinib resistant LAD cells to gefitinib treatment, while knockdown of SNHG5 rendered gefitinib sensitive LAD cells to gefitinib treatment. Bioinformatics analysis showed that SNHG5 exerted its function through interaction with miR-377, which was further confirmed by luciferase reporter assay in 293T cells. Overexpression of SNHG5 suppressed the expression of miR-377, while the knockdown of SNHG5 increased the miR-377 expression. MiR-377 expression was significantly up-regulated in LAD specimens with acquired gefitinib resistance and was negatively correlated with SNHG5 expression. In addition, CASP1 was predicted as a downstream target of miR-377. Overexpression of miR-377 suppressed the expression of CASP1 in PC9 cells and knockdown of miR-377 increased the CASP1 expression in PC9GR cells. In vitro functional assay showed that knockdown of CASP1 in SNHG5-overexpressed PC9GR cells abolished their gefitinib resistance. Overall, the present study demonstrated, for the first time, that the SNHG5/miR-377/CASP1 axis functions as an important role in LAD cells gefitinib resistance and potentially contributes to the improvement of LAD diagnosis and therapy.


Introduction
Lung cancer has become one of the most common malignancies and the leading cause of cancer-associated mortality worldwide. Lung adenocarcinoma (LAD) is the most common type of primary lung cancer and is usually diagnosed at an advanced stage [1]. Treatment options available for NSCLC include surgery, radiation, chemotherapy, and biological therapy [2]. Gefitinib is clinically used for the treatment of chemoresistant NSCLC patients, which is a selective inhibitor of epidermal growth factor, a growth factor that plays a pivotal role in the control of cell growth, apoptosis, and angiogenesis [3]. However, acquired resistance to targetted therapy with epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs), such as gefitinib, occurs inevitably in almost all the NSCLC patients [4]. It is thus vital to explore novel strategies to restore sensitivity to gefitinib [5].
Long non-coding RNAs (lncRNAs) are a class of mRNA-like transcripts ranging from 200 bp to 100 kbp, which were regarded as transcription noise in the human genome, due to their lack of capability of protein translation [6]. LncRNAs emerged rapidly as a diverse group of essential regulators of genetic information flow that interact with the epigenetic, transcriptional, and post-transcriptional pathways of cell proliferation, differentiation, and survival [7]. Accumulating evidence indicates the association between the expression of certain lncRNAs and gefitinib sensitivity of lung cancer cells.
Previous studies identified a total of 22587 differentially expressed lncRNAs in gefitinib-sensitive and gefitinib-induced acquired-resistant lung cancer cells [8]. Pan et al. [9] confirmed that lncRNA BC087858 could promote cells' invasion and induce non-T790M mutation acquired resistance to EGFR-TKIs by activating PI3K/AKT and MEK/ERK pathways in NSCLC. Dong et al. [10] found that GAS5 enhanced gefitinib induced cell death in innate EGFR-TKI-resistant LAD cells with wide-type EGFR via down-regulation of the IGF-1R expression. Wang et al. [11] demonstrated that overexpression of MIR31HG lncRNAs may contribute to gefitinib resistance in PC9-R cells through the EGFR/PI3K/AKT pathway.
Small nucleolar RNA (snoRNA) host gene 5 (SNHG5 or U50HC) is a member of both the non-protein-coding multiple snoRNA host gene family and the 59-terminal oligopyrimidine class of genes [12]. The SNHG5 gene is 524 bp in size and located on chromosome 6q15 at the breakpoint of chromosomal translocation [13]. SNHG5 has been reported to suppress gastric cancer progression by trapping MTA2 in the cytosol [14]. In addition, LncRNA SNHG5 regulates imatinib resistance in chronic myeloid leukemia via acting as a ceRNA against miR-205-5p [15].
However, the biological role of SNHG5 and its function in gefitinib resistant LAD remain largely unknown. In the present study, SNHG5 down-regulated in LAD patients and SNHG5 expression level was significantly correlated with acquired gefitinib resistance. Our results also showed that SNHG5 overexpression sensitized LAD cells to gefitinib treatment in vitro and in vivo. SNHG5 exerted its function through interaction with miR-377 to modulate its downstream target CASP1. Taken together, our results indicate that SNHG5 plays an important role in gefitinib resistance of LAD and could be a potential therapeutic target for LAD patients.

Patients and tissue samples
Seventy-one advanced LAD tissues were collected from LAD patients who had either an exon 19 deletion (19DEL) or an exon 21-point mutation (L858R) in their EGFRs, treated with or without gefitinib between October 2013 and September 2017, were recruited in the present study. The study protocol was approved by the Ethics Committee of China-Japan Union Hospital of Jilin University, and informed written consent was signed by all the patients participating in the present study. Lung cancer tissue samples were obtained from patients undergoing lung cancer resection, and snap-frozen in liquid nitrogen post surgery.

RNA extraction and quantitative real-time PCR
Total RNA from tissues and cells was extracted using TRIzol reagent (Invitrogen) according to the manufacturer's instructions. First-strand cDNA was generated using the Reverse Transcription System Kit (Takara, Dalian, China). Quantitative real-time PCR (qRT-PCR) analyses utilized SYBR Green I (Takara) and were performed in triplicate. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and U6 snRNA were used as endogenous controls. The relative fold change in expression was calculated by the 2 − C T method. The primers were listed below. SNHG5-Forward:

Cell lines and culture
Human lung cancer PC9 and A549 cells were cultured in RPMI 1640 at 37 • C in a humid atmosphere with 5% CO 2 ; the HEK-293T cell line was cultured in DMEM containing 10% FBS, 100 IU/ml penicillin, and 100 mg/ml streptomycin. The PC9GR and A549GR cells were generated by continually exposing to stepwise increase in the concentration of gefitinib over a period of 24 months. The SNHG5 overexpression or knockdown cell lines were generated by infection of lentivirus vector and were grown in the presence of 5 μg/ml puromycin for selection of stably transfected clones. The empty lentiviral vector was used as control groups.

Cell proliferation assay
Cell proliferation was measured using the Cell Counting Kit-8 (Solarbio). After plating cells in 96-well microtiter plates at 1.0 × 10 3 /well, 10 μl CCK-8 was added to each well at the time of harvest, according to the manufacturer's instructions. Two hours after adding CCK-8, cellular viability was determined by measuring the absorbance of the converted dye at 450 nm.

Cell apoptosis analysis
Cells were stimulated with 0.1 μM gefitinib for 24 h. Cells were washed twice in cold 1× PBS and then fixed with 500 μl of 70% cold ethanol for 2 h. Cells (1 × 10 6 ) per ml were resuspended in 1× binding buffer, and 100 μl cell suspension was mixed with 5 μl FITC and 5 μl PI using the FITC Annexin V Apoptosis Detection Kit (BD Biosciences), according to the manufacturer's instructions. Stained cells were analyzed using a flow cytometer (FACScan; BD Biosciences).

Luciferase reporter assay
Luciferase reporter constructs containing theoretical binding sequence and their mutated sequence were cloned into pMiR-Report firefly luciferase vector (GenePharma). pRL-Tk Renilla luciferase reporter was used for luciferase assay normalization.The assays were performed 48 h after transfection of the indicated constructs. HEK293 cells (2 × 10 4 ) per well (four wells, each samples) were seeded in 96-well plates. The cells were transfected with 50 ng of firefly luciferase vectors and 1 ng of the pRL-Tk Renilla reporter. The reporter activities were measured using the Dual-Glo Luciferase Assay System (Promega) and GloMax-Multi Detection System (Promega).

RNA immunoprecipitation
An RNA immunoprecipitation was used to analyze whether SNHG5 and miR-377 were associated with the RNA-induced silencing complex (RISC).
PC9GR was lysed and incubated with RIPA buffer containing magnetic beads conjugated with human anti-Argonaute2 (Ago2) antibody (Millipore). Normal mouse IgG (Millipore) was used as a negative control. Samples were incubated with Proteinase K, and then immunoprecipitated RNA was extracted. Purified RNA was subjected to qRT-PCR analysis.

Western blotting assay
Cells were seeded and reverse transfected in six-well plates. After 36 h, cells were harvested, washed once with PBS, and the pellets lysed in RIPA buffer (Sigma) containing protease inhibitors (cOmplete Mini Protease Inhibitor Cocktail; Roche Applied Science). Proteins were separated by electrophoresis in polyacrylamide/SDS (8-10% gel) and transferred on to nitrocellulose membranes (Millipore). The primary antibodies used were: anti-GAPDH antibody (Abcam; ab8245), anti-Caspase-1 antibody (Abcam; ab1872).

Statistical analysis
SPSS version 19.0 for Windows (IBM SPSS, U.S.A.) was used for all the analyses. Student's t test was used to compare the differences between groups. P-values were based on the two-sided statistical analysis and P<0.05 was considered to indicate a statistically significant difference.

SNHG5 expression is down-regulated in LAD patients with acquired gefitinib resistance
To assess the role of SNHG5 in LAD, SNHG5 expression was assessed in patients before gefitinib treatment and after harboring acquired resistance to gefitinib. A total of 63 patients were enrolled in the present study ( Table 1). RT-qPCR showed that SNHG5 was significantly down-regulated in acquired resistance group ( Figure 1A). Overexpression of SNHG5 was also observed in lung cancer cells with acquired gefitinib resistance (PC9GR and A549GR) ( Figure 1B). We also measured the mRNA expression of SNHG5 by RT-PCR in eight matched LAD patients' specimens, and found that SNHG5 expression was down-regulated in patients with acquired resistance ( Figure 1C). Whereas it exhibited no difference in patients with primary gefitinib resistance group ( Figure 1D). On the basis of the SNHG5 expression before gefitinib treatment, the patients were divided into a high expression group (n=12) and a low expression group (n=18), depending on whether they were above or below the mean value of SNHG5 expression. When overall survival rate (OS) was assessed, patients in the low SNHG5 expression group had a significantly poorer prognosis than those TNM, Tumor-Node-Metastasis.  in the high expression group ( Figure 1E). The objective response rate (ORR) in the low SNHG5 expression group was significantly lower than in the high expression group (33.3 compared with 75%; Figure 1F). Therefore, SNHG5 might play a critical role in acquired resistance to gefitinib in LAD patients.

SNHG5 treatment enhances gefitinib sensitivity in vitro and in vivo
To clarify the function of SNHG5 in gefitinib resistance, we overexpressed SNHG5 in PC9GR cells (Figure 2A). Overexpressed SNHG5 increased the gefitinib sensitivity in PC9GR cells compared with parental cells (Figure 2B,C).
In addition, when we knocked down SNHG5 in PC9 cells ( Figure 2D), PC9 cells showed resistance to gefitinib treatment ( Figure 2E,F). To evaluate the function of SNHG5 in gefitinib resistance in vivo, PC9GR-LV-NC and PC9GR-LV-SNHG5 cells were subcutaneously administered in both posterior flanks of male BALB/c nude mice. LV-SNHG5 plus gefitinib decreased tumor volumes and weights compared with LV-NC the plus gefitinib group, and no significance of tumor volumes and weights between LV-SNHG5 group and LV-NC group without gefitinib treatment ( Figure 2G,H). These findings suggested that SNHG5 could modulate gefitinib sensitivity in LAD cells.

SNHG5 directly interacts with miR-377
We searched for miRNAs with complementary base pairing with SNHG5 utilizing the online software program star-Base v2.0 (http://starbase.sysu.edu.cn/mirLncRNA.php). From the results, we focussed on miR-377, which has been noted to be down-regulated in other malignancies such as metastatic prostate cancer and epithelial ovarian cancer [16]. The qRT-PCR assay showed that knockdown of SNHG5 increased the expression of miR-377, while overexpression of SNHG5 significantly decreased the expression level of miR-377 ( Figure 3A). To determine whether miR-377 negatively and reciprocally regulates SNHG5, miR-377 mimics and inhibitor were transfected into PC9 and PC9GR cells, respectively. SNHG5 expression was significantly suppressed by miR-377 mimics and markedly enhanced by miR-377 inhibitor ( Figure 3B). To further verify the correlation between SNHG5 and miR-377, we performed a luciferase reporter assay with constructs containing the putative binding sequences between SNHG5 and miR-377 ( Figure 3C). miR-377 mimics inhibited luciferase expression when co-transfected with SNHG5-WT. However, miR-377 mimics did not change luciferase expression when co-transfected with SNHG5-Mut ( Figure 3D). It was well documented that miRNA plays its function by binding to Ago2, a core component of the RISC necessary for miRNA-mediated gene silencing, and potential miRNA targets could be isolated from this complex after Ago2 co-immunoprecipitation [17]. To determine whether SNHG5 and miR-377 were part of the same RISC, we performed RNA precipitation experiments. SNHG5 and miR-377 were enriched in Ago2 immunoprecipitates compared with control IgG immunoprecipitates ( Figure 3E). To further analyze the expression level of miR-377 in tumor specimens, we found that miR-377 was significantly up-regulated in acquired gefitinib resistance group ( Figure 3F) and was negatively correlative with the expression level of SNHG5 ( Figure 3G). These findings demonstrate that miR-377 is a direct target of SNHG5.

miR-377/CASP1 axis modulates the gefitinib sensitivity of SNHG5 on human LAD cells
Based on these results, we used the TargetScan (http://www.targetscan.org) to identify the main target genes of miR-377. CASP1 was predicted to be a target of miR-377 ( Figure 4A). We performed reporter assays with a lu- ciferase plasmid harboring the 3 -UTR sequence of CASP1 containing the predicted miR-377 binding site or mutated binding site. Reporter assays in 293T cells revealed miR-377 was able to markedly reduce the relative luciferase activity of WT-CASP1-3 -UTR in the 293T cells, whereas in the cells transfected with Mut-CASP1-3 UTR was not decreased ( Figure 4B). Consistent with the reporter assay, CASP1 mRNA and protein expression was increased in the presence of miR-377 inhibitor. Whereas, CASP1 mRNA and protein level decreased after treatment with miR-377 mimics ( Figure 4C,D). What is more, addition of miR-377 mimics abolished the SNHG5 overexpression induced CASP1 expression ( Figure 4E,F). These results indicate that CASP1 is a direct target of miR-377. To clarify the role of SNHG5-miR-377-CASP1 axis in gefitinib sensitivity, we transfected PC9GR-LV SNHG5 cells with CASP1-siRNA. We found that CASP1-siRNA also rendered PC9GR-LV-SNHG5 cells resistant to gefitinib ( Figure 4G,H). Taken together, these data suggest that the miR-377/CASP1 axis mediated the gefitinib resistance of SNHG5 on LAD cells.

Discussion
Although therapeutic responses to EGFR-TKIs may persist for as long as 2-3 years; however, drug resistance eventually emerges and this limits the mean duration of response to 68 months [18]. Although EGFR activating mutations have been recognized as the main cause of EGFR-TK resistance, nearly 30% of the acquired resistance cannot be explained by the mechanisms recognized [4]. Recently, more and more studies have focussed on investigating the roles lncRNAs, which have been shown to regulate many key biological processes, including drug resistance. In the present study, we found that SNHG5 expression was significantly down-regulated in LAD patients and LAD cell lines with acquired gefitinib resistance. SNHG5 overexpression sensitized LAD cells to gefitinib treatment in vitro and in vivo.
In the further study, we discovered that SNHG5 and miR-377 negatively regulated each other. MiR-377 has been reported to function as a tumor suppressor via suppressing DNMT1 in pancreatic cancer cells [16], negatively regulated of E2F and MAP3K7/NF-κB signaling pathway in melanoma cells [19]. Furthermore, miR-377 was a target of NEAT1 in NSCLC cells [20]. Amongst all the predicted target genes of miR-377, we discovered CASP1 acted as a crucial effector of miR-377, which belongs to a family of cysteine proteases that cleave proteins following an aspartic acid residue [21]. We showed that overexpression of miR-377 suppressed the expression of CASP1 in PC9 cells and knockdown of miR-377 increased the CASP1 expression in PC9GR cells. In vitro functional assay showed that knockdown of CASP1 in SNHG5-overexpressed PC9GR cells abolished their gefitinib resistance.
In conclusion, the present study demonstrated that SNHG5 might be a predictive biomarker of acquired gefitinib resistance and that SNHG5 may be a therapeutic target in patients with gefitinib resistance. These findings may improve the clinical outcome of LAD patients receiving gefitinib therapy.