Knockdown of RNA-binding protein IMP3 suppresses oral squamous cell carcinoma proliferation by destabilizing E2F5 transcript

Human tissue samples

OSCC tissues and their paired adjacent normal tissues (from 40 patients) were obtained from the College of Stomatology, Xi’an Jiaotong University. Informed consent was obtained from each enrolled patient, and the study was approved by the Ethics Committee of Xi’an Jiaotong University (approval number 2020393).

Cell culture and transfection

Human OSCC cell lines SCC-25, SCC-4, and HOK cells (human oral keratinocytes) were purchased from the Cell Bank (Shanghai Institute of Biochemistry and Cell Biology, China). All cells were maintained in DMEM high glucose medium containing 10% FBS (Gibco, USA) and 1% antibiotics (penicillin/streptomycin). Transient transfection of siRNA and their respective negative control RNAs was performed using Polyplus transfection kit (Jetprime, France) following the manufacturer’s instructions.

qRT-PCR

Total RNA was collected from cells and extracted with TRIzol Reagent (Invitrogen, USA). cDNA was made using PrimeScript^™ RT reagent kit (Takara, Japan). qRT-PCR was carried out using the SYBR Premix Ex Taq II Kit (Takara, Japan) on IQ5 Multicolor PCR Detection System (Bio-Rad, USA). β-actin was used as an internal reference and 2^-ΔΔCt method was used to determine relative abundance. Three replicates per reaction were performed for qRT-PCR. The primers used are as follows: β-actin forward 5’-CCAGAGGCGTACAGGGATAG-3’; reverse 5’- CCAACCGCGAGAAGATGA-3’; IMP3 forward 5’-CCTGGTGAAGACTGGCTACG-3’; reverse 5’- ATCCAGCACCTCCCACTGTA-3’; E2F5 forward 5’-TCAGGCACCTTCTGGTACAC-3’; reverse 5’-GGGCTTAGATGAACTCGACTC-3’.

Western blotting assays

Total cell lysates were lysed in RIPA buffer (Roche, Switzerland), and protein concentration was determined using a BCA protein assay kit (Thermo Fisher Scientific, USA). Equal amounts of proteins were separated on 10% SDS-PAGE gels and transferred to PVDF membranes. The membranes were blocked with 5% non-fat milk and then incubated with primary antibodies (anti-IMP3, Abcam, USA; anti-E2F5, Abcam, USA; anti-CDK4 and anti-CyclinD1, 1: 2000, Cell Signaling Technology, USA) at 4° C overnight. Following a wash, membranes were incubated with secondary antibodies for 1 h, washed, and developed using ECL (Pierce, USA).

Immunohistochemistry (IHC)

Tissue sections were deparaffinized in xylene and hydrated with an alcohol gradient. Antigen retrieval and endogenous peroxidase blocking were performed sequentially. Tissues were probed with polyclonal rabbit anti-IMP3 (diluted 1:100) and anti-E2F5 (diluted 1:100) at 4° C overnight, washed, and next incubated with secondary antibody (1:5000) for 1 h at room temperature. Tissues were stained with 3,3′-diaminobenzidine kit (DAB, China) and hematoxylin for histological examination. Tissue sections were fixed and images captured under an inverted microscope.

CCK8 assay

CCK8 assay kit (7Sea Biotech, China) was employed to study the cell proliferation of SCC-25 and SCC-4 cells according to the manufacturer' instructions. Briefly, the transfected cells were seeded into 96-well plated with 3000 cells/well. CCK8 solution (10 μl) was added into each well after 24, 48 and 72 h, then the 96-well plates were placed in a humidified incubator for additional incubation of 4 h. The OD value was obtained at 450 nm with FLUOstar OPTIMA (BMG, Germany).

Cell cycle assay

OSCC cells were seeded in 6-well plates (1 × 10⁶ cells/well) and transfected with siRNA. After 48 hours, the cells were collected and fixed in 70% ethanol and stored at 4° C overnight. The next day the samples were stained with 10 μg/mL propidium iodide according to the manufacturer’s instructions (Sigma, USA) and incubated for 1 hour at room temperature in the dark. Cells were then analyzed by Flow Cytometer (BD Biosciences, USA).

RNA stability assay

The cells were seeded in 6-well plates and treated with 5 μg/mL actinomycin D. The cells were collected at 3 h, 6 h, and 9 h post-treatment. The total RNA was extracted for RT-qPCR as described above. The degradation rate of the mRNAs was estimated following published protocols [24].

RNA immunoprecipitation (RIP) assay

For the RIP assay, the Magna RIP™ Kit (No. 17-701, Millipore, USA) was performed according to the manufacturer’s instructions. OSCC cells were lysed in RIP lysis buffer supplemented with proteinase inhibitors and RNAse inhibitor and then incubated with anti-IMP3 or IgG-coupled protein beads for 6 h to overnight at 4° C. The next day after stringently washing the beads with washing buffer, the immunoprecipitated RNA was purified and analyzed by qRT-PCR.

Mouse xenograft studies

Stable IMP3-deficient SCC-25 cells were injected into six-week-old, female, nude mice (n = 6/group), for tumor engraftment. Tumors were measured every 5 days for 30 days. Four weeks after tumor inoculation, mice were euthanized by cervical dislocation under anesthesia. The tumors were excised and analyzed. All the experimental protocols were approved by the Institutional Animal Care and Use Committee of Xi'an Jiaotong University.

Bioinformatics analysis

The gene expression data were obtained at The Cancer Genome Atlas website for OSCC projects (TCGA, https://tcga-data.nci.nih.gov/tcga/).

Statistical analysis

All the above experiments were repeated three times. Differences between groups were analyzed using Student’s t-test or one-way ANOVA; also to determine if the quantitative data showed normal distribution. If data were not normally distributed, the Wilcoxon-Mann-Whitney test was performed. All statistical analyses were carried out using SPSS 18.0 (IBM, USA). P < 0.05 was considered significant, and data are presented as the mean ± Standard error of the mean (SEM).

IMP3 is overexpressed in OSCC

To identify the roles of IMP3 in OSCC, we examined the expression of IMP3. From TCGA database, we found that IMP3 is overexpressed in OSCC compared to normal tissue (Figure 1A). Western blot and qRT-PCR analysis showed that IMP3 expression was enriched in OSCC cell lines (SCC-25 and SCC-4) compared to HOK cells (Figure 1D, 1E). Meanwhile, methylation sequencing results showed that the high expression of IMP3 in oral squamous cell carcinoma was probably related to CpG island methylation modification (Figure 1C). Patients with high IMP3 expression had markedly lower overall survival rates compared to those with low IMP3 expression (Figure 1B). Next, we measured the expression of IMP3 in 40 paired patient samples of OSCC tissue and adjacent normal tissue with IHC (Figure 1F). Consistent with in vitro results, expression of IMP3 was significantly increased in OSCC, and higher expression of IMP3 was observed in the cytoplasm. The IMP3 protein expression increased in 3 pairs of OSCC tissues compared with normal tissues (Figure 1F). Collectively, these findings demonstrate that IMP3 expression is overregulated in OSCC.

Figure 1. Overexpression of IMP3 in human OSCC tissues and cell lines. (A) TCGA analysis showed that IMP3 mRNA levels were increased in the OSCC tissues (n=352) compared with the normal tissues (n=32). (B) Kaplan-Meier analysis was performed on OSCC patient survival with the GEPIA database according to high or low expression of IMP3. (C) Methylation sequencing results showed that the high expression of IMP3 in oral squamous cell carcinoma was probably related to CpG island methylation modification. Expression level of IMP3 mRNA (D) and protein (E) in OSCC cell lines (SCC-25 and SCC-4) in comparison with HOK cell line. (F) The IHC score of IMP3 was markedly higher in the OSCC tissues than in the adjacent normal tissues (n=40). Magnification ×40. The scale bar indicates 50 μm. Relative expression levels were calculated using the image J software (n = 3). P≤ 0.05 was considered to be statistically significant, **P <0.01, ***P< 0.001.

Knockdown of IMP3 inhibits OSCC cell growth

To further investigate the functional roles of IMP3 in OSCC, we used siRNA depletion of IMP3 in SCC-25 and SCC-4 cells to reduce the mRNA and protein levels of IMP3 to 20~30% tested by qRT-PCR and Western blot (Figure 2A, 2B). Then CCK8 and cell cycle assays were performed on OSCC cells. In the CCK8 assay, knockdown of IMP3 significantly inhibited cell proliferation compared to siRNA control (Figure 2C). In the cell cycle assay, suppression of IMP3 in OSCC cells markedly decreased cell viability and led to a decrease in the number of cells in the G1, owing to the decreased levels of CDK4 and Cyclin D1 (Figure 2D, 2E). Taken together, these findings suggest that IMP3 drives OSCC cell growth in vitro.

Figure 2. Knockdown of IMP3 inhibits OSCC proliferation. The expression of IMP3 in SCC-25 and SCC-4 cells transfected with IMP3 siRNA was determined by qRT-PCR (A) and Western blot (B). (C) Cell growth in OSCC cells transfected with siIMP3 as measured by CCK8 assay(n=5). (D) Effects of suppression of IMP3 on the cell cycle progression of SCC-25 and SCC-4 cells measured by flow cytometric analysis. (E) Expression analysis for cell cycles regulators Cyclin D1 and CDK4 was performed by Western blot. Relative expression levels were calculated using the image J software (n = 3). P ≤ 0.05 was considered to be statistically significant, **P < 0.01, ***P < 0.001, ****P < 0.0001.

IMP3 positively regulates E2F5

Even though IMP3 is overexpressed in OSCC and knockdown of IMP3 suppresses cell growth, the molecular mechanisms of IMP3 regulation remained unknown. Using TCGA database analysis, we identified that E2F5 was upregulated in OSCC tissue and had a positive correlation with IMP3 (Figure 3A, 3B). Examination of the level of E2F5 in OSCC tissue and cell lines, as showed in Figure 3C, 3D, E2F5 was distinctly overexpressed in SCC-25 and SCC-4 cell lines when compared to that in HOK cell. Besides, E2F5 was abundant in OSCC tissue in comparison to normal tissues as demonstrated by IHC staining (Figure 3E). Moreover, knockdown of IMP3 significantly down-regulated E2F5 RNA levels in SCC-25 and SCC-4 cell lines (Figure 3F). Thus, all these data imply that IMP3 positively regulates E2F5 in OSCC.

Figure 3. IMP3 positively regulated E2F5. (A) TCGA analysis showed that E2F5 mRNA levels were increased in the OSCC tissues (n=352) compared with the normal tissues (n=32). (B) Pearson correlation analysis of IMP3 and E2F5 expression in OSCC showed that IMP3 is positively correlated with E2F5. (C, D) qRT-PCR and Western blot assay showed that E2F5 was upregulated in OSCC cell lines (SCC-25 and SCC-4) in comparison with HOK cell line. (E) Representative images of IHC staining of E2F5 protein in OSCC tissues (n=40). Magnification ×40. The scale bar indicates 50 μm. (F) Quantification of E2F5 transcripts in SCC-25 and SCC-4 after transfection with siIMP3. Relative expression levels were calculated using the image J software (n = 3). P ≤ 0.05 was considered to be statistically significant, **P < 0.01, ***P < 0.001.

IMP3 stabilizes E2F5 mRNA

Since IMP3 is an RBP and can promote the stability of bound RNA, its effect may be exerted on E2F5 mRNA. Therefore, we analyzed the function of IMP3 on E2F5 mRNA stability in OSCC cells. Upon analyzing the E2F5 mRNA sequence, we identified IMP3 binding motifs (Figure 4A). We investigated the stability of E2F5 mRNA at different times in the presence of actinomycin D (Figure 4B). Suppression of IMP3 significantly decreased the stability and half-life of E2F5 mRNA. To confirm the predicted binding sites, we performed RIP assays. As shown in (Figure 4C), E2F5 mRNA was enriched in IMP3 IP samples compared to the IgG IP samples, indicating that IMP3 specifically binds to E2F5 mRNA. Taken together, these findings demonstrate that E2F5 mRNA is a direct target of IMP3.

Figure 4. E2F5 is a direct target of IMP3. (A) The schematic image of the binding sites of IMP3 in the TIMM44 mRNA. (B) Analysis of the mRNA half-lives of E2F5 expression in the SCC-25 and SCC-4 cells suppression of IMP3. (C) RIP analysis of IMP3 binding to E2F5 mRNA in OSCC cells. Nonspecific rabbit IgG was used as a negative control. Input was used as a positive control.

Suppression of E2F5 inhibits the OSCC cell proliferation

Given that the expression of E2F5 was strongly correlated with IMP3, we next studied the functions of E2F5 in OSCC. Transient silencing of E2F5 in SCC-25 and SCC-4 with siRNA significantly down-regulated the expression of E2F5 as determined by qRT-PCR and Western blot (Figure 5A, 5B). Suppression of E2F5 inhibited the proliferation of OSCC cells as determined by CCK8 assay (Figure 5C). Additionally, cell cycle assays showed that inhibition of E2F5 in SCC-25 and SCC-4 cells resulted in decreased cell numbers in G1 phase due to reduced levels of CDK4 and Cyclin D1 (Figure 5D, 5E). Collectively, these data demonstrate that downregulation of E2F5 suppresses the growth of OSCC cell which shared similar effects of silencing of IMP3.

Figure 5. Effects of E2F5 inhibition on OSCC cell growth. The expression of E2F5 was analyzed with qRT-PCR (A) and Western blot (B) after transfection with siE2F5 and its control. (C) Cell viability of SCC-25 and SCC-4 cells transfected with siE2F5 was determined by the CCK8 assay (n=5). (D) Effects of suppression of E2F5 on the cell cycle progression of OSCC cells measured by flow cytometric analysis. (E) Expression analysis for cell cycles regulators Cyclin D1 and CDK4 was performed by Western blot. Relative expression levels were calculated using the image J software (n = 3). P ≤ 0.05 was considered to be statistically significant, *P < 0.05, **P < 0.01, ***P < 0.001.

To further explore that IMP3 regulated tumor progression through regulating E2F5, SCC-25 and SCC-4 cells were transfected siIMP3 alone or cotransfected with siIMP3 plus E2F5 vector. As showed in Figure 6A, 6B, knockdown of IMP3 partly reversed the cells from the effects of E2F5 on regulation of E2F5 expression. Moreover, CCK8 assay suggested that silencing of IMP3 strongly suppressed the cell growth of OSCC cell. Nevertheless, E2F5 reversed the inhibitory effects of siIMP3 (Figure 6C). All these findings demonstrate that IMP3 regulates the progression of OSCC by regulating E2F5.

Figure 6. Knockdown of IMP3 reverses E2F5-induced cellular phenotypes in OSCC cells. (A, B) qRT-PCR and Western blot were performed to determine the impact of OSCC cells treated with E2F5 plus siIMP3 expression vectors or related negative control. (C) The growth of OSCC cells were detected using CCK8 assay. Relative expression levels were calculated using the image J software (n = 6). P ≤ 0.05 was considered to be statistically significant, *P < 0.05, **P < 0.01, ***P < 0.001.

Ectopic expression of IMP3 inhibits xenograft tumor growth

Since knockdown of IMP3 expression inhibited the malignant properties of OSCC cells in vitro, we also investigated the effect of IMP3 on the tumorigenicity in a xenograft model. SCC-25 cells stably transfected with LV-si-IMP3 or LV-si-Ctrl were injected subcutaneously into nude mice (Figure 7A). Tumor sizes were measured using a caliper every 5 days. After 30 days, the mice were euthanized, and tumor weights were measured. The results demonstrated that the tumor growth and tumor weight were significantly decreased in IMP3-knockdown cells as compared to controls (Figure 7B, 7C). Furthermore, qRT-PCR, Western blot, and IHC showed that the expression of IMP3 and E2F5 was significantly decreased in LV-si-IMP3 injected subcutaneous xenografts as compared to LV-si-Ctrl (Figure 7D, 7E).

Figure 7. IMP3-depleted inhibits OSCC cell proliferation. (A) Establishment of IMP3-depleted SCC-25 cells. The expression of IMP3 was analyzed with qRT-PCR and Western blot. Relative expression levels were calculated using the image J software (n = 3). (B) Stable IMP3-depleted SCC-25 cells and control cells were subcutaneously injected into nude mice. A representative image showing tumors from mice transplanted with SCC-25 cells with or without IMP3 inhibition. (C) The tumor growth and the tumor weights from each group were statistically analyzed (n=6). (D) qRT-PCR and Western blot analysis showed that IMP3 and E2F5 levels were increased in the tissues compared with the control tissues. (E) IHC staining of IMP3 (up) and E2F5 (down) in tumor tissues from mice implanted with SCC-25-LV-si-IMP3 or SCC-25-LV-si-Ctrl cells. Magnification ×40. Scale bar, 50 μm. Relative expression levels were calculated using the image J software (n = 6). P ≤ 0.05 was considered to be statistically significant, *P < 0.05, **P < 0.01, ***P < 0.001.