IMP4 Silencing Inhibits the Malignancy of Lung Adenocarcinoma via ERK Pathway

Our study aimed to elucidate the function of IMP U3 small nucleolar ribonucleoprotein 4 (IMP4) in lung adenocarcinoma (LUAD) and its potential molecular mechanisms. Cell counting kit-8, 5-ethynyl-20-deoxyuridine, flow cytometry, wound healing, and transwell assays were performed to examine the biological behaviour of LUAD cells. mRNA and protein expression levels were determined using quantitative real-time PCR, Western blotting, and immunohistochemistry. In addition, a mouse tumour xenograft model was used to evaluate the role of IMP4 in tumour progression. Furthermore, glycolysis-related indicators were measured. The levels of IMP4 were up-regulated in both human LUAD tissues and cells. IMP4 silencing significantly suppressed proliferation, migration, invasion, and glycolysis; promoted apoptosis; and induced cell cycle arrest in LUAD cells. IMP4 silencing also inactivated the extracellular signal-regulated kinase (ERK) pathway. Moreover, rescue experiments demonstrated that the function of LUAD cells induced by IMP4 overexpression could be reversed by treatment with an ERK pathway inhibitor (SCH772984). In vivo experiments further verified that IMP4 silencing repressed the growth of subcutaneous tumours and glycolysis. IMP4 silencing suppressed the malignancy of LUAD by inactivating ERK signalling.


Introduction
Lung adenocarcinoma (LUAD) is a histological phenotype of lung cancer and one of the leading causes of cancerrelated mortality and morbidity worldwide [1,2]. Despite the progress in prevention, detection, and treatment of LUAD, the prognosis of LUAD patients remains poor. erefore, there is an urgent need to explore the potential molecular mechanisms of LUAD and identify new therapeutic targets for LUAD. IMP U3 small nucleolar ribonucleoprotein 4 (IMP4) is a component of U3 small nucleolar ribonucleoproteins and is involved in the maturation of 18S rRNA [3]. Moreover, it has been reported that IMP4 is a new telomeric DNAbinding protein, which can play important roles in telomeres [3]. Recently, a study by Chang et al. showed that IMP4, as a potential passenger gene, may be involved in endometrial cancer [4]. Moreover, IMP4 was shown to be up-regulated and was verified to be a novel target for lung cancer therapy [5]. However, the specific effects and molecular mechanisms of IMP4 in LUAD have not been fully explored.
Aerobic glycolysis is a hallmark of cancer [6,7]. Accumulating studies show that aerobic glycolysis plays a critical role in uncontrolled proliferation, metabolism, and metastasis of tumour cells [8]. Interestingly, extracellular signal-regulated kinase (ERK) signalling is required for the responses to extracellular stimuli and has recently emerged as an important modulator of glycolysis during tumorigenesis [9]. Du et al. [10] reported that ATPR can induce acute myeloid leukaemia cell differentiation and cycle arrest by regulating the ERK-glycolysis signalling axis. However, it is unclear whether IMP4 plays a role in LUAD by regulating glycolysis and ERK signalling.
In this study, we showed that IMP4 could promote the progression of LUAD through activating the ERK pathway. ese findings demonstrate the functions of IMP4 in LUAD and highlight its potential as a biomarker for LUAD.

Flow Cytometry.
After transfection for 48 h, the A549 and H1299 cells were harvested, washed twice with ice-cold PBS, and resuspended in 1× binding buffer. For cell apoptosis analysis, cells were incubated in Annexin-V/PI double staining solution (KGA101, KeyGen Biotech, Nanjing, China) for 20 min, according to the manufacturer's instructions, and then analysed using a FACSCalibur flow cytometer. e cell cycle was evaluated using a cell cycle and apoptosis analysis kit (C1052, Beyotime) according to the manufacturer's instructions using a FACS Calibur flow cytometer.

In Vivo Xenograft
Model. BALB/c nude mice (female, 6 weeks old) were supplied by GemPharmatech (Jiangsu, China). A549 cells (3 × 10 6 ) transfected with sh-NC or sh-IMP4 (Genechem) were injected subcutaneously into the left dorsal flanks. Tumour volumes were recorded every three days from the 10th day. e mice were then killed on day 30 after inoculation and the subcutaneous tumour weight of each mouse was determined.

H&E Staining and Immunohistochemistry.
Xenograft tumours and human lung tissues were fixed in formalin, embedded in paraffin, and sectioned into 5 μm thickness after embedding. For H&E staining, the sections were double stained with haematoxylin and eosin, and histopathological changes were analysed under a light microscope.

Terminal Dexynucleotidyl Transferase-Mediated dUTP
Nick End Labelling (TUNEL) Assay. A colorimetric TUNEL apoptosis assay kit (C1086, Beyotime) was used to assess apoptotic cells in mouse lung tissues. e lung sections were immersed in TUNEL reaction mixture in the dark at 37°C in a humidified atmosphere for 1 h. After 3 times of PBS washing, apoptotic cells in lung tissues were observed under a fluorescence microscope.

Dual-Luciferase Reporter Assay.
e sequence of wild type or mutant IMP4 promoter including E2F4 binding sites was subcloned into a pGL3-luciferase reporter vector. e promoter sequence was synthesised by Genscript Biotech Corporation. e luciferase reporter vector and E2F4 were cotransfected into HEK-293Tcells using Lipofectamine 3000 reagent (Invitrogen, USA). After culturing at 37°C for 48 hours, the cells were evaluated by a dual-luciferase reporter assay system (D0010, Solarbio, Beijing, China) following the manufacturer's instructions.

Bioinformatics Analysis.
e Gene Expression Profiling Interactive Analysis (GEPIA) database was used to analyse patient survival data. e median was the criterion for patients to be divided into two groups on the prognosis curve. e differential expression of IMP4 across the cancer genome Atlas (TCGA) and in LUAD was performed using UALCAN according to the database obtained from TCGA. Following transfection with sh-IMP4, the transcriptome of A549 cells was sequenced by Novogene (Beijing, China).
Gene set enrichment analysis (GSEA) was used to investigate the potential mechanisms by which IMP4 regulates LUAD.

Statistical Analysis.
e data are presented as the mean ± standard deviation (SD). Statistical analyses were performed using SPSS version 22.0. Results were compared using Student's t-test or one-way ANOVA. Statistical significance was set at p < 0.05.

High IMP4 Expression Was Found in LUAD and Predicted Poor Prognosis for LUAD Patients.
e data from TCGA showed that IMP4 levels were up-regulated in LUAD tissues (Figures 1(a) and 1(b)) and confirmed that high IMP4 expression predicted poor prognosis in LUAD patients ( Figure 1(c)).
e immunohistochemistry results further demonstrated that IMP4 levels were up-regulated in LUAD patient tissues (Figure 1(d)). Additionally, we showed that the levels of IMP4 were significantly up-regulated in A549 and H1299 cells (Figure 1(e)).

IMP4 Silencing Inhibits Proliferation, Promotes Apoptosis, and Induces Cell Cycle Arrest in LUAD Cells.
e transfection efficiency of si-IMP4-1 and si-IMP4-2 in A549 and H1299 cells was determined using qRT-PCR (Figure 2(a)). e results showed in Figures 2(b) and 2(c) revealed that IMP4 silencing notably repressed A549 and H1299 cell proliferation. Moreover, IMP4 silencing dramatically increased A549 and H1299 cell apoptosis (Figure 2(d)). To confirm its accelerating effect on apoptosis, we measured the levels of apoptosis-related proteins using Western blotting and demonstrated that the protein levels of cleaved PARP and Bax were increased, whereas Bcl-2 was decreased (Figure 2(e)). As shown in Figure 2(f), IMP4 silencing caused accumulation of A549 and H1299 cells in the G1 phase of the cell cycle. Accordingly, typical cell cycle markers such as cyclin D1 were down-regulated, whereas p21 and p53 were up-regulated upon IMP4 downregulation (Figure 2(g)). Together, these results show that IMP4 silencing represses proliferation, accelerates apoptosis, and induces cell cycle arrest in LUAD cells. Figures 3(a) and 3(b) show that IMP4 silencing represses the migration and invasion of both A549 and H1299 cells. To further verify this effect, we performed Western blotting and found that IMP4 silencing significantly increased E-cadherin expression and decreased N-cadherin and vimentin expression in A549 and H1299 cells (Figure 3(c)).

Silencing of IMP4 Suppresses the Malignant Phenotypes
Via Inhibiting the ERK Pathway. GSEA analysis revealed that several signalling pathways were down-regulated by sh-IMP4, including the MAPK pathway (Figures 6(a) and 6(b)). We selected the ERK pathway, one of the MAPK pathways, for further investigation. As shown in Figure 6(c), IMP4 silencing decreased the expression of p-MEK and p-ERK in A549 and H1299 cells (Figure 6(c)). We analysed the transfection efficiency of pcDNA3.1-IMP4 in A549 cells ( Figure 6(f )). Moreover, IMP4 overexpression dramatically elevated proliferation and invasion, and induced accumulation of A549 cells in the S phase of the cell cycle (Figures 6(g)-6(j)). Meanwhile, knockdown of IMP4 significantly reduced p-MEK and p-ERK expression in xenograft tumours (Figures 6(d) and 6(e)). All these functions induced by IMP4 overexpression were reversed following the treatment of A549 cells with SCH772984 (an inhibitor of the ERK pathway) (Figures 6(e)-6(h)).

E2F4 Up-Regulates IMP4 and Promotes the Progression of LUAD.
e Venn chart identified E2F4 in the Animal TFDB, hTFtarget, Chipbase, and the genes positively associated with IMP4 expression in TCGA-LUAD (Figure 7(a)). e TCGA-LUAD database showed that IMP4 positively correlated with E2F4 levels (Figure 7(b)). Moreover, the data from TCGA demonstrated that E2F4 levels were up-regulated in LUAD tissues (Figure 7(c)) and further confirmed that high E2F4 expression predicted poor prognosis in LUAD patients (Figure 7(d)). To further verify that E2F4 mediates upregulation of IMP4, a dual luciferase reporter assay was performed. As seen in Figure 7(e), luciferase activity was increased in the IMP4 promoter-WT + E2F4 group compared to the NC + E2F4 group. Meanwhile, luciferase activity was decreased in the IMP4 promoter-MUT + E2F4 group compared with the IMP4 promoter-WT + E2F4 group. Moreover, after A549 cells were transfected with the pcDNA3.1-E2F4 vector, IMP4 expression was significantly elevated (Figure 7(f )), further indicating that IMP4 is positively correlated with E2F4 expression in LUAD cells. Subsequently, the transfection efficiency of the pcDNA3.1-E2F4 vector in A549 cells was analysed using qRT-PCR (Figure 7

Discussion
Owing to the adverse results reported in cancer statistics, there is an urgent need to identify new markers for LUAD [2]. A previous study showed that IMP4 is highly expressed in human lung cancer and verified it as a new therapeutic target for lung cancer [5]. In this study, we hypothesised that IMP4 can promote the progress of LUAD by activating the ERK pathway (Figure 8). e experimental results confirm our hypothesis that IMP4 was overexpressed in LUAD, meanwhile the silencing of IMP4 significantly inhibited proliferation, migration, invasion, and glycolysis; promoted apoptosis and induced LUAD cell cycle arrest; and suppressed tumour growth and glycolysis in a nude mouse xenograft model. Subsequently, we investigated the IMP4related molecular mechanisms in LUAD cells.
Tumour cells can undergo unrestricted divisions and proliferation. Rapid proliferation of cancer cells requires rapid energy expenditure. Glycolysis in tumour cells accelerates glucose uptake and lactic acid generation, resulting in an acidic environment that induces rapid proliferation and metastasis [12][13][14][15]. Moreover, glycolysis is reported to be involved in ATP synthesis and cell pathway activation [16]. Some genes have been reported to be related to glycolysis in cancers [17,18]. For instance, PPFIA4 has been shown to promote colon cancer cell proliferation and migration by enhancing tumour glycolysis [19]. PFK1 has been shown to accelerate proliferation and migration, and reduce radiosensitivity by promoting glycolysis in colorectal cancer [20]. In this study, we demonstrated that IMP4 dramatically increased glucose uptake, lactate generation, and ATP synthesis in LUAD cells and tumour tissues, suggesting that IMP4 could enhance glycolysis in LUAD. To further verify this hypothesis, the expression levels of glucose transporters and key glycolytic enzymes, including GLUT1, HK2, PFKP,       1-IMP4, qRT-PCR was applied to assess IMP4 levels. After transfection with pcDNA3.1-IMP4 or treatment with SCH772984, in A549 cell proliferation was evaluated using the CKK-8 (g) and EdU (h); (i) After transfection with pcDNA3.1-IMP4 or treatment with SCH772984, A549 cell cycle was assessed using flow cytometry. (j) After transfection with pcDNA3.1-IMP4 or treatment with SCH772984, A549 cell invasion was assessed using transwell. * P < 0.05, * * P < 0.01; # P < 0.05, ## P < 0.01.   PKM2, and LDHA, were detected by Western blotting. e results revealed that IMP4 silencing reduced the levels of GLUT1, HK2, PFKP, PKM2, and LDHA in LUAD cells and tumour tissues.

Journal of Oncology
In recent years, increasing number of studies have shown that the activation of ERK signalling can contribute to the growth, invasion, and epithelial-mesenchymal transition in tumour cells [21,22]. In addition, a variety of genes can play important roles in LUAD by regulating ERK signalling. For example, DLC1 inhibits LUAD cell proliferation and invasion by repressing MAPK/ERK signalling [23]. Another study confirmed that FANCI can act as an oncogene in LUAD by cooperating with IMPDH2 to accelerate cell proliferation through activation of the MEK/ERK pathway [24]. ERK signalling has recently emerged as a critical modulator of glycolysis during tumorigenesis [9]. He et al. (e) Luciferase activities were evaluated via performing dual luciferase reporter assay. After pcDNA3.1-E2F4 vector was transfected into A549 cells, the mRNA levels of IMP4 (f ) and E2F4 (g) were determined using qRT-PCR. After pcDNA3.1-E2F4 vector and si-IMP4-2 co-transfection, A549 cell proliferation was analysed using the EdU assay (h); apoptosis was assessed using FITC-Annexin V/PI apoptosis detection kit (i); the cell cycle was evaluated using cell cycle and apoptosis analysis kit (j); invasion was examined using the transwell assay (k). * P < 0.05, * * P < 0.01, ## P < 0.01. [25] reported that PGK1 contributes to the growth of renal clear cell carcinoma by activating CXCR4/ERK signalling and promoting glycolysis. In this study, we demonstrated that IMP4 silencing inhibited the activation of ERK signalling in LUAD cells. In addition, to further verify this hypothesis, we performed rescue experiments using IMP4 overexpression and the ERK pathway inhibitor SCH772984. We found that the effects of IMP4 overexpression were reversed following treatment with SCH772984.

Conclusion
In our study, we demonstrated that IMP4 silencing significantly inhibited proliferation, migration, invasion, and glycolysis; promoted apoptosis; induced cell cycle arrest in LUAD cells; and suppressed tumour growth and glycolysis in a nude mouse xenograft model. We also showed that IMP4 silencing suppressed the malignancy of LUAD by inactivating ERK signalling. Our study provides strong evidence that IMP4 is an outstanding diagnostic marker as well as a potential therapeutic target for LUAD.
Data Availability e datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.

Ethical Approval
e experimental protocol of our study was performed in accordance with the Guide for the Care and Use of Laboratory Animals and approved by Shandong Provincial Hospital Affiliated to Shandong First Medical University. e protocol of this research has been approved by the Ethics Committee of Shandong Provincial Hospital Affiliated to Shandong First Medical University. All patients have signed written informed consent.