MiR-504-3p Has Tumor-Suppressing Activity and Decreases IFITM1 Expression in Non-Small Cell Lung Cancer Cells

Abstract

Objective:

To analyze the impact of expression of miR-504-3p on the proliferation, migration, cell cycle transit and rate of apoptosis of NSCLC cells and explore the underlying mechanisms.

Methods:

The Cancer Genome Atlas (TCGA) database was used to compare the expression levels of miR-504 between NSCLC tissues and normal lung tissues. NSCLC cells were transfected with lentiviral vectors that either overexpressed or knocked down miR-504-3p to evaluate its effects on NSCLC biological behavior. Quantitative Real Time Polymerase Chain Reaction was used to measure the levels of miR-504-3p and Interferon-Induced Transmembrane Protein 1 (IFITM1). A luciferase reporter array was used to reveal whether miR-504-3p directly targets IFITM1.

Results:

The expression of miR-504 was significantly down-regulated in lung cancer tissues compared to normal lung tissues. Overexpression of miR-504-3p in NSCLC cell lines inhibited cell proliferation, migration and promoted cell apoptosis. Meanwhile, changes in the expression level of miR-504-3p had no significant effect on NSCLC cell cycle progression. Moreover, over-expressed miR-504-3p following its transfection significantly decreased the expression of IFITM1 in NSCLC cell lines and suppressed the activity of the luciferase reporter containing wild type but not mutant IFITM1 3′ -UTR.

Conclusion:

miR-504-3p inhibits cell proliferation and migration and promotes cell apoptosis in NSCLC cells. MiR-504-3p decreases IFITM1 expression in NSCLC cells, which may be a potential mechanism of its tumor-suppressive functions in NSCLC.

Introduction

Lung cancer is one of the most common human malignant tumors in the world and non-small cell lung cancer (NSCLC) accounts for ∼85% of all cases. Although we have made great progress in the diagnosis and treatment of lung cancer, the prognosis and survival of patients are still unsatisfactory (Bade and Dela Cruz, 2020). Therefore, novel diagnostic and therapeutic strategies for NSCLC are in rapid development.

MicroRNAs (miRNAs) are a type of small molecule noncoding RNA. Half of them are located in the fragile sites of tumor chromosomes (Tutar et al, 2014). Mature miRNA is complementary or noncomplementary to the 3′-untranslated region (3′-UTR) of the target messenger RNA (mRNA) under the guidance of RNA-induced silencing complex after pairing (Krol et al, 2010). It can interfere with or inhibit the expression of mRNA, thereby negatively regulating the target gene at the post-transcriptional level (Correia de Sousa et al, 2019). Emerging evidence has confirmed that miRNAs exert important biological functions in tumorigenesis and development (Mishra et al, 2016; Vishnoi and Rani, 2017), especially in lung cancer (Wu et al, 2018).

Furthermore, the underlying mechanisms were also widely reported. Previous research has found that miR-504 has a biological effect of suppressing cancer in a variety of malignant tumors. For instance, miRNA-504 can suppress hepatocellular carcinoma by inhibiting Frizzled-7-mediated-Wnt/β-catenin signaling (Quan et al, 2018). In addition, miRNA-504 inhibited tumor cell proliferation and invasion by acting on AGE-1 in retinoblastoma (Wang et al, 2019). Meanwhile, research on the role of miRNA-504 in NSCLC has gradually emerged. A recent study incorporating 55 NSCLC cases revealed that the expression of miR-504 was lower in NSCLC tissues and associated with TNM stage and lymph node metastasis in NSCLC patients. Moreover, miR-504 inhibited cell proliferation, invasion, and epithelial-mesenchymal transition (EMT) through targeting LOXL2 in NSCLC (Ye et al, 2018). However, more significant reports on miR-504-3p about its effect or functional mechanism in NSCLC were hardly found.

The interferon-induced transmembrane (IFITM) family were first discovered as interferon-induced genes in human neuroblastoma cells (Friedman et al, 1984). Despite the functions in osteoblast (Hanagata et al, 2011), germ cell specification (Lange et al, 2003; Saitou et al, 2002), adaptive immunity (Bedford et al, 2019; Wakim et al, 2013), and antiviral activity (Bailey et al, 2014; Shi et al, 2020), their crucial roles in tumors have been called for great concern (Liang et al, 2020). Interferon-induced transmembrane protein 1 (IFITM1), which is also known as 9-27 or Leu13, is reported to be highly correlated to prognosis and therapeutic resistance in certain tumor types, such as colorectal cancer (Yu et al, 2015), gallbladder cancer (Li et al, 2019a), and gastric and esophageal adenocarcinoma (Borg et al, 2016). Nevertheless, the role that IFITM1 plays in lung cancer was rarely reported (Sakamoto et al, 2020; Tutar et al, 2014; Yang et al, 2019).

In this study, we investigated the role of miR-504-3p in growth, migration, and cell apoptosis of NSCLC cells. Moreover, we demonstrated that miR-504-3p negatively regulated IFITM1, which may be one of the underlying mechanisms of its tumor-suppressive functions.

Materials and Methods

Cell culture and transfected

H226 and A549 cells (Sangon, Shanghai, China) were used in this study. All cells were cultured with Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum (FBS) in a 5% CO₂ incubator at 37°C. The miR-504-3p overexpression sequence (5′-CGAAACACCGGGAGTGGA-3′) and knockdown sequence (5′-GGGAGTGCAGGGCAGGGT-3′) were amplification by polymerase chain reaction (PCR) and cloned into PCMV-GV309 vector. The vectors were co-transfected with pHelper1.0 and pHelper2.0 (Biovetor, Beijing, China) into lentivirus packaging cells 239T (Sangon). The concentrated and purified lentivirus were transfected to H226 and A549 cells using HitransG (Genechem, Shanghai, China).

Quantitative real-time polymerase chain reaction

Total RNA from transfected cells was extracted using Trizol (Pufei, Shanghai, China) for both miRNA and IFITM1-mRNA analyses. For analysis of miR-504-3p expression, quantitative real-time (qRT)-PCR was performed using Bulge-Loop™ miRNA qPCR Primer Set (Ribobio, Guangzhou, China) according to the manufacturer's instructions. Relative expression was calculated by comparative cycle threshold (Ct) method and normalized to the expression of U6 small RNA. IFITM1-mRNA was quantified by qRT-PCR using Quantitect SYBR Master PCR Kit (Takara Bio, Shiga, Japan) and normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH). PCR primers were as follows: IFITM1, 5′-TCCGTGAAGTCTAGGGACAG-3′ (forward) and 5′-GTCACAGAGCCGAATACCAG-3′ (reverse) and GAPDH, 5′-TGACTTCAACAGCGACACCCA-3′ (forward) and 5′-CACCCTGTTGCTGTAGCCAAA-3′(reverse). All qRT-PCRs were performed in triplicate.

Western blot

Radio immuno precipitation assay buffer (Beyotime Biotechnology, Shanghai, China) blending with phenylmethylsulfonyl fluoride (PMSF) protease inhibitors (Thermo Fisher, Waltham, MA) was used to lyse cells. The protein was extracted using Protein Extraction Reagent A and B (Yeasen, Shanghai, China). Separated protein on 10% sodium dodecyl sulfate-polyacrylamide gelelectrophoresis by equal amounts and transferred it to polyvinylidene difluoride membranes (Pall, NY). Incubated membranes with primary antibodies in 5% nonfat powdered milk configured with Tris-Buffered Saline and Tween 20 and secondary antibody in succession. Enhanced chemiluminescence was used to determine the bands. Primary antibodies anti-IFITM1 and anti-GAPDH were purchased from Abcam (Cambridge, United Kingdom).

Cell proliferation assays

For the cell proliferation assay, cells were plated in 96-well plates (Corning, Glendale, AZ) at 2000 cells/well and incubated for 24 h after transfection of lentiviral vector with different expression levels of miR-504-3p. At the end of incubation, thiazolyl blue tetrazolium bromide (MTT) were added into each well and incubated for 4 h at 37°C. After that, dimethyl sulfoxide was added to dissolve Formazan crystals formed by MTT reduction. Cell viability was tested every 24 h for 120 h in total through evaluating the viable cell numbers by measurement of optical density at 490 nm using the microplate reader (Tecan, Seestrasse, Switzerland).

Cell migration assays

Cell migration was assessed using 3422 Transwell Kit (Corning) according to the manufacturer's introduction. In brief, cells were trypsinized (Thermo Fisher), washed, resuspended in serum-free RPMI1640 (Corning) and placed in the top portion of the chamber. Added 30% FBS to the lower portion of the chamber. The chambers were incubated at 37°C in 5% CO₂ for 24 h, washed with phosphate buffered saline, and fixed in 100% methanol. After fixation, cells were stained with GIEMSA (Sigma-Aldrich, Darmstadt, Germany) and imaged by inverted microscope (Caikon, Shanghai, China). The number of migrating cells was counted in nine random fields at 100-fold magnification. Assays were conducted in duplicates.

Cell apoptosis assay

Cell apoptosis was measured using the Annexin-V-APC Apoptosis Detection Kit (Thermo Fisher). Twenty-four hours after the transfection for overexpression or knockdown of miR-504-3p, cells were trypsinized, washed, and resuspended in RPMI1640. Then stained the cells with Annexin V-APC. Finally, analyzed the percentage of apoptotic cells by flow cytometry (Becton, Dickinson and Company, NJ).

Cell cycle assay

For the cell cycle analysis, cells were cultured at 37°C for 24 h after transfection. Digested cells with trypsin and resuspended them in complete medium. Cells were washed by precooled Dulbecco phosphate-buffered saline (Corning) and then stained with propidium iodide (Sigma-Aldrich) for 30 min protected from light. The distribution of cells in distinct cell cycle phases ModFit LT3.2 (Verity Software House, Topsham, ME) was used to determine the distribution of cells in different cell cycle phases.

Luciferase reporter assay

A 199 nt nucleotide sequence of IFITM1 mRNA 3′-UTR containing the binding site for miR-504-3p seed sequence was synthesized and inserted into the Xbal site of the pGV272 vector (Promega, Madison, WI) to construct IFITM1-WT, whereas the mutation of the aforementioned nucleotide sequence (mutated at the site expected to bind to miR-504-3p) was inserted to construct IFITM1-Mut. After transfection with miR-504-3p overexpression or knockdown lentiviral vector for 24 h, X-tremegene HP Transfection Reagent (Roche, Basel, Schweiz) was used to transfect cells with wild-type or Mut reporter plasmid vector. After 36 h, Luciferase activity was measured by a dual-luciferase reporter assay system (Promega). The fluorescence intensity of Firefly luciferase was normalized to that of Renilla luciferase for each sample.

Statistical analysis

Statistical analysis was performed using SPSS software (version 19.0; SPSS, Inc., Chicago, IL) and GraphPad Prism 5.0 (GraphPad Software, Inc., La Jolla, CA). The relative miR-504-3p or IFITM1 expression was evaluated by comparative CT method and normalized to the expression of U6 or GAPDH. The results of cell experiments were expressed as mean ± standard deviation and the differences between groups were analyzed by Student's t-test. Differences were considered to be statistically significant at p < 0.05.

Results

Abnormal expression levels of miR-504-3p and its target gene IFITM1 between normal and lung cancer tissues

The available data from The Cancer Genome Atlas (TCGA) revealed that the levels of miR-504-3p were significantly lower both in lung adenocarcinoma (LUAD) and lung squamous cell carcinoma (LUSC) tissues compared with normal lung tissues with the fold change of LUAD versus normal to be 0.68 (p < 0.001). Similarly, the fold change of LUSC versus normal was 0.79 (p = 0.003) (Fig. 1A). Likewise, we analyzed the expression of IFITM1, which was predicted to be the target gene of miR-504-3p through miRanda and TargetScan. The results demonstrated that the levels of IFITM1 were significantly higher in LUAD tissues (fold change of cancer vs. normal = 1.296, p = 0.035). However, the levels of IFITM1 showed no significant difference between LUSC and normal lung tissues. (Fold change of cancer vs. normal = 0.944, p = 0.635) (Fig. 1B). We failed to find a correlation analysis between expression of miR504-3p and IFITM1 indicating an inverse relationship in the TCGA database.

FIG. 1.

Comparison of miR-504 and IFITM1 levels between normal and lung cancer tissues. (A) miR-504-3p was significantly downregulated in lung cancer tissues compared with normal lung cancer tissues according to data downloaded from The Cancer Genome Atlas public data set (https://portal.gdc.cancer.gov). (B) IFITM1 was upregulated in lung adenocarcinoma tissues compared with normal lung tissues but showed no significant differences between lung squamous cell carcinoma tissues and normal lung tissues. Error bars indicated S.E.M. miR, microRNA; IFITM1, interferon-induced transmembrane protein 1.

MiR-504-3p suppresses the proliferation of NSCLC cells in vitro

We chose H226 and A549 lung cancer cell lines for transfection with lentiviral vector containing over- or low expression of miR-504-3p to investigate its biological functions. The qRT-PCR results suggested that the level of miR-504-3p in H226 and A549 cells was significantly increased after transfection with overexpression miR-504-3P (p < 0.05). Relatively, transfection with knockdown of miR504-3p markedly decreased the level of miR-504-3p in H226 and A549 cells (p < 0.05) (Fig. 2A). Overexpression of miR-504-3p significantly inhibited the proliferation of H226 and A549 cells 48 h after transfection whereas knockdown of miR-504-3p significantly promoted the proliferation of H226 and A549 cells 48 h after transfection (p < 0.05) (Fig. 2B).

FIG. 2.

MiR-504-3p suppresses the proliferation of NSCLC cells in vitro. (A) Relative levels of miR-504-3p after transfection in H226 and A549 cell lines. The column represented the mean of fold change for three independent experiments, error bars represented S.E.M. (B) The differences of proliferation rates of H226 and A549 cells after transfection with different levels of miR-504-3p suggested miR-504-3p suppressed the proliferation of NSCLC cells. *p < 0.05. KD, knockdown; NC, negative control; NSCLC, non-small cell lung cancer; OE, overexpression.

MiR-504-3p suppresses migration of NSCLC cells

We evaluated the effect of miR-504-3p on migration ability of NSCLC cells. The results suggested that migration ability of H226 and A549 cells was significantly reduced after transfection with overexpression of miR-504-3p (p < 0.05), whereas transfection with knockdown of miR-504-3p significantly promoted the migration ability of H226 and A549 cells (p < 0.05) (Fig. 3A, B).

FIG. 3.

Comparison of the migration ability of H226 and A549 cells between groups with different levels of miR-504-3p. (A) Stained H226 and A549 cells of different groups were counted in nine random areas at 100-fold magnification in three independent samples. (B) Quantities of migratory H226 and A549 cells of different groups suggested that miR-504-3p suppressed the migration of NSCLC cells. Error bars represented S.E.M. Each experiment was performed in triplicates independently, p < 0.05. *p < 0.05.

MiR-504-3p promotes the apoptosis of NSCLC cells and makes no significant impact on cell cycle

We further analyzed the effect of miR-504-3p on NSCLC apoptosis and cell cycle to investigate how miR-504-3p regulating the proliferation of NSCLC cell using the flow cytometry assay. The results revealed that overexpression of miR-504-3p significantly advanced the apoptosis of H226 and A549 cells (p < 0.05). Meanwhile, knockdown of miR-504-3p observably decreased the apoptosis of H226 and A549 cells (p < 0.05) (Fig. 4A, B). Besides, no significant difference of cell cycle was shown no matter how expression level of miR-504-3p changed in NSCLC cells (p > 0.05) (Fig. 4C). These results demonstrated that miR-504-3p regulated cell proliferation principally through increasing the apoptosis of NSCLC cells.

FIG. 4.

Comparison of the H226 and A549 cell apoptosis and cell cycle between groups with different levels of miR-504-3p. (A) The flow cytometry results of different groups. Upper left quadrant represented live cells, upper right quadrant represented late apoptotic cells, right lower quadrant represented dead cells, and left lower quadrant represented early apoptotic cells. (B) The percentage of apoptosis cells of different groups in H226 and A549 cells. (C) The percentage of cells at different stages in different groups showed no significant difference. Error bars meant S.E.M. Each experiment was performed in triplicates, p < 0.05. *p < 0.05. G1, first gap; S, synthesis; G2/M, second gap/mitosis.

IFITM1 is a direct target of MiR-504-3p in NSCLC cells

We predicted the potential target genes of miR-504-3p using bioinformatics algorithms, Tagetscan and miRanda. The results showed that 3′-UTR of the IFITM1 mRNA contains a highly conserved and perfect complementary region from position 26 to 33 for binding the seed sequence of miR-504-3p (GCACUCC paired CGUGAGG) (Fig. 5A), which strongly supported that IFITM1 was one of the target genes of miR-504-3p. To determine whether IFITM1 was truly the target gene of miR-504-3p, we construct the IFITM1-Wt and IFITM1-Mut luciferase reporter plasmids, which contain the wild-type IFITM1 3′-UTR sequence and the 3′-UTR with the 7 nt binding site sequence with miR-504-3p deletion, severally.

FIG. 5.

(A) The sequence information of IFITM1 3′-UTR WT, IFITM1 3′-UTR MUT, and miR-504-3p. (B) Relative IFITM1 expression levels of different groups in H226 and A549 cells. GAPDH mRNA was used as an internal control. The IFITM1 expression levels were significantly decreased by miR-504-3p. Error bars represented S.E.M. Each experiment was performed in triplicates, p < 0.05. (C) IFITM1 protein immunoblotting of different groups in H226 and A549 cells. GAPDH protein was used as an internal control. The numbers above immune stripes represented the corresponding relative IFITM1 protein levels. (D) Relative IFITM1 WT and IFITM1 MUT luciferase activity of different groups in H226 and A549 cells. Error bars represented S.E.M. Each experiment was performed in triplicates independently, p < 0.05. *p < 0.05. 3′-UTR, 3'-untranslated region; MUT, mutant-type; WT, wild-type.

We quantified IFITM1-mRNA after transfection using qRT-PCR and normalized to GAPDH. IFITM1 was markedly reduced after miR-504-3p overexpression, whereas increased after miR-504-3p knockdown in H226 and A549 cells according to the results of qRT-PCR and western blot (p < 0.05) (Fig. 5B, C). The luciferase reporter results suggested that overexpression of miR-504-3p significantly decreased the luciferase activity of H226 cells transfected with IFITM1-Wt compared with the negative control (p < 0.05), whereas the deletion mutation of the binding sites eliminated the effect of miR-504-3p on luciferase activity.

In addition, knockdown of miR-504-3p markedly increased the luciferase activity in A549 cells transfected with IFITM1-Wt (p < 0.05), and the mutation abrogated the effect of the low expression level of miR-504-3p on the luciferase activity (Fig. 5C). These results revealed that miR-504-3p regulates the expression of IFITM1 by directly binding to the IFITM1 3′-UTR sequence in NSCLC cells. In other words, IFITM1 is one of the target genes of miR-504-3p.

Discussion

Lung cancer, especially NSCLC, has always been of great concern for its high morbidity and mortality all over the world (Schabath and Cote, 2019). According to statistics, about 1.6 million people died of lung cancer worldwide in 2012, and this number will reach 3.5 million by 2035. Predictably, with the intensification of aging, environmental pollution, and smoking problems, the incidence of lung cancer will further increase, making the worldwide social medical system face severe challenges (Didkowska et al, 2016). Therefore, the early diagnosis, treatment, and molecular biological mechanisms of lung cancer have always been the popular fields.

Emerging evidence has confirmed that miRNAs exert important biological functions in tumorigenesis and development (Lee and Dutta, 2009; Sandiford et al, 2018), especially in lung cancer (Du et al, 2018; Iqbal et al, 2019). For example, miR-127 is related to the poor prognosis of LUAD (Shi et al, 2017). The expression of miR-105-1 in NSCLC tissue was downregulated compared with normal lung tissue, and it was associated with poor overall survival and disease free survival in NSCLC patients (Lu et al, 2017). miR-221 and miR-222 can suppress tumors in most lung cancer cell lines (Yamashita et al, 2015).

Recent reports demonstrated that miRNA-504-3p acted as a tumor suppressor in various cancers. MiR-504-3p exerted inhibiting effects in the proliferation, migration, and invasion of diverse cancer types by targeting different genes. For instance, miR-504 inhibits cell proliferation and promotes apoptosis by targeting FOXP1 in human glioma (Cui et al, 2016). MiR-504-3p may also serve as a tumor suppressor by targeting MTHFD2 in acute myeloid leukemia (Li et al, 2019b). Moreover, miR-504 has been shown to inhibit cell proliferation through targeting CDK6 in hypopharyngeal squamous cell carcinoma (Kikkawa et al, 2014). However, few reports were found on miR-504-3p in lung cancer.

Therefore, we chose miRNA-504-3p as the research object and conducted functional tests of NSCLC cells in vitro to investigate the effect of miRNA-504-3p on the biological behavior of NSCLC in this study. The function study showed that miRNA-504 inhibited the proliferation and migration of NSCLC cells and promoted cell apoptosis. These results suggested that miR-504-3p acted as an antioncogene in NSCLC and the downregulation of miR-504-3p contributes to the progression and metastasis of NSCLC. Experiments in vivo need to be further performed to determine whether targeting miR-504-3p can become a new therapeutic strategy for NSCLC.

IFITM1 is a member of the IFITM protein family (Yánez et al, 2020). Evidence has shown that IFITM1 is highly expressed in tumor tissues and cancer cell lines (Hatano et al, 2008; He et al, 2015). It has been reported that overexpression of IFITM1 promotes tumor cell proliferation, invasion, metastasis (Lee et al, 2012), and angiogenesis (Baeriswyl and Christofori, 2009), and the high expression level of IFITM1 is often associated with resistance to radiotherapy (Guo et al, 2012), chemotherapy (Bani et al, 2004), and endocrine therapy (Xu et al, 2019).

For example, IFITM1 is closely related to lymph node metastasis, distance metastasis, and higher clinical stages in colorectal cancer (Sari et al, 2016). According to the results of a recent research, silencing of IFITM1 inhibited proliferation, migration, and invasion of lung cancer cells (Yan et al, 2019). However, the mechanism of IFITM1 dysregulation in lung cancer is poorly understood. In this study, we revealed a novel mechanism by which IFITM1 is regulated. The luciferase reporter assay demonstrated that IFITM1 was directly targeted by miR-504-3p in NSCLC cells. Nevertheless, whether the dysregulation of IFITM1 is regulated by other mechanisms in NSCLC is not yet known.

Our study has several limitations. There may be other targets for IFITM1. In addition, further analysis for functions of miR-504-3p in vivo is necessary. Next, we will establish an animal model of NSCLC to evaluate the effect of in vivo.

This study analyzed the expression levels and functions of miR-504-3p in NSCLC. The data showed miR-504-3p is downregulated in NSCLC tissues. Results of functional experiment revealed that miR-504-3p execute a tumor-suppressive effect on NSCLC cells. This effect is probably partially on account of directly targeting 3′-UTR of IFITM1 by miR-504-3p. Thus, our findings may suggest novel therapeutic strategies for NSCLC.

Footnotes

Author Disclosure Statement

No competing financial interests exist.

Funding Information

This study was supported by Science and Technology Development Plan of Soochow (Livelihood Science and Technology) (SS201832) and Medical and Health Scientific and Technological Innovation Program of Soochow (SKJY2021023).

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