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Abstract

BACKGROUND:

MicroRNAs can regulate tumor metastasis either as oncomiRs or suppressor miRNAs. Here, we investigated the role of microRNA 224 (miR-224) in lymphatic metastasis of non-small-cell lung cancer (NSCLC).

METHODS:

The expression of miR-224 was demonstrated by a validation cohort of 156 lung cancer patients (77 cases with lymphatic metastasis) by quantitative polymerase chain reaction (qPCR). In vitro and in vivo experiments were performed to study the malignant phenotype after upregulation and inhibition of miR-224 expression. Furthermore, the direct target genes of miR-224 were determined by a luciferase reporter assay.

RESULTS:

First, miR-224 was identified as a highly expressed miRNA in tumor tissues with lymphatic metastasis, with an area under the curve (AUC) of 0.57 as determined by qPCR analysis of a validation cohort of 156 lung cancer patients. Then, in vitro and in vivo experiments indicated that forced expression of miR-224 in H1299 cells promoted not only cell viability, plate colony formation, migration and invasion in vitro but also tumor growth and lung metastasis in vivo. Consistently, inhibition of miR-224 suppressed malignant characteristics both in vitro and in vivo. Moreover, molecular mechanistic research suggested that miR-224 enhanced NSCLC by directly targeting the tumor suppressor angiopoietin-like protein (ANGPTL).

CONCLUSIONS:

Overall, the collective findings demonstrate that miR-224 is a potential biomarker for the prediction of lymphatic metastasis of NSCLC.

Keywords

microRNA miR-224 on-small-cell lung cancer lymphatic metastasis ANGPTL

Abbreviations

TCGA	The Cancer Genome Atlas
NSCLC	non-small-cell lung cancer

CAM	chick embryo chorioallantoic
	membrane assay
DEGs	differential expressed genes
qRT-PCR	Real-Time Quantitative Reverse
	Transcription PCR
CCK-8	Cell Counting Kit-8
ANGPTL	Angiopoietin-like Protein 1
YPEL1	yippee-like 1

1. Background

Globally, lung cancer is the leading cause of cancer death [1], and its 5-year survival rate is approximately 4–17% [2]. Among the two pathological subtypes, non-small-cell lung cancer (NSCLC) is the most common type, accounting for approximately 80% of lung cancers [3]. Lymph node metastasis is an independent factor affecting survival and recurrence after surgical resection of the primary malignancy [4]. Patients with mediastinal lymph node dissection are associated with lower local recurrence rates and better survival [5]. Therefore, searching for lymph node metastasis-related microRNAs (miRNAs or miRs) and understanding the molecular mechanisms is deeply meaningful.

MiRNAs are defined as endogenous and small RNAs of $\sim$ 22 nt nucleotides in length by complementary base pairing with the target mRNA, leading to its degradation [6]. Recent discoveries related to miRNA found alterations in cancer, either as an oncomiR or a suppressor miR. Thus, modulating miRNA expression and activity in vivo through miRNA mimics or inhibitors provides an opportunity for the development of innovative therapeutic approaches to cancer [7]. Previous studies demonstrated that numerous miRNAs correlated with lymph node metastasis of NSCLC [8]. In recent years, many miRNAs have been identified to be associated with lymph node metastasis, including miR-137 [9] and miR-144-3p [10] as metastasis inducers and miR-148a [11] and miR-129-5p [12] as metastasis suppressors.

Previously, we identified differentially expressed miRNAs in patients with lymph node metastasis compared with patients without lymph node metastasis by miRNA microarray analysis [13]. In total, we obtained 39 upregulated miRNAs in tissues with lymph node-positive metastasis versus lymph node-negative tissues. miR-224 was upregulated 14.6-fold, ranking ninth among upregulated miRNAs. Moreover, the $P$ value of 0.002 for miR-224 ranked second among 39 upregulated miRNAs [13]. Considering both the high fold change and small $P$ value of miR-224 for the prediction of lymph node metastasis, in this study, we selected miR-224 as a potential promoter of lymph node metastasis to explore its role in metastasis by both in vitro and in vivo experiments.

2. Methods

2.1 Cell lines and culture conditions

Human lung cancer cell lines (H1299 and PC9) were purchased from the Chinese Academy of Medical Sciences & Peking Union Medical College (Beijing, China). Cells were cultivated in RPMI 1640 with 10% FBS, 100 U/mL penicillin, and 100 mg/mL streptomycin in a 5% CO2 humidified incubator at 37 ${}^{\circ}$ C.

2.2 Patients and samples

A cohort of 155 NSCLC patients (77 cases with lymphatic metastasis and 78 patients without lymphatic metastasis) were included from 2003 to 2019 to validate miR-224 expression in cancer tissues. Inclusion criteria: 1) definitive pathologic diagnosis; 2) no chemoradiotherapies before surgery; 3) completed a 5-year follow-up; and 4) provided written informed consent. Exclusion criteria: 1) other types of clinical disorders were observed; and 2) patients lost during follow-up or who died of other clinical disorders. This study was approved by the Ethics Committee of Peking University.

2.3 Reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR)

MiRNAs were extracted from tissue samples from NSCLC patients or cells using an miRNeasy Mini Kit (Qiagen, Valencia, CA, USA) according to the manufacturer’s instructions. The purity and concentration of RNA were estimated using an ND-1000 microspectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). For mature miRNA expression detection, the polyA tailing method was used [14], and cDNA was synthesized from 100 ng RNA using Moloney murine leukemia virus reverse transcriptase (M-MLV RT) (Invitrogen, Carlsbad, CA). For mRNA expression detection, 2 $\mu$ g of total RNA was used to synthesize cDNA. Quantitative real-time PCR (qRT-PCR) was performed using SYBR Green PCR Master Mix (ABI) at 95 ${}^{\circ}$ C for 10 min followed by 40 cycles of 95 ${}^{\circ}$ C for 15 s and 60 ${}^{\circ}$ C for 60 s on an ABI 7500 System (Applied Biosystems, CA). The expression level of the gene was calculated by the 2 ${}^{-\Delta\text{Ct}}$ method, where $\Delta$ Ct $=$ Ct (target gene)-Ct (GAPDH or U6). To calculate the fold change expression, the expression levels of genes were calculated by the 2 ${}^{-\Delta\Delta\text{Ct}}$ method, where the control group was normalized to 1. The sequences of all primers are listed in Supplementary Table S1.

2.4 Cell viability assay

5 $\times$ 10 ${}^{3}$ cells/well were seeded into 96-well plates and cultured for 24, 48, 72, and 96 h. Cell Counting Kit-8 (CCK8; Dojindo, Kumamoto, Japan) reagents were added to cultures and incubated for an additional hour. The absorbance at 450 ${}_{\text{nm}}$ was measured using a microplate reader (iMark, Bio-Rad Laboratories, Hercules, CA).

2.5 Plate colony formation assay

Five hundred cells/well were plated in 6-well plates and cultured for 2 weeks. The number of cell colonies was counted after fixation with 4% formalin and staining with 1% crystal violet.

2.6 Transwell assay

For the cell migration and invasion (coated with extracellular matrix substitute) assay, mitomycin C-treated cells were loaded into the upper chamber of a Transwell insert with 8.0- $\mu$ m polycarbonate membranes (Millipore, Billerica, MA, USA) containing 1% FBS in RPMI-1640 medium. Medium containing 10% FBS was added to the lower chamber as a chemoattractant. Cells on the lower surface were stained with 1% crystal violet and photographed under a microscope in four random fields per well.

2.7 Wound healing assay

Cells were cultured in 24-well plates and scratched with a 20- $\mu$ L pipette tip. Detached cells were removed carefully with PBS wash three times, and the wounded area was photographed at 0, 24 and 48 h (Leica, Wetzlar, Germany). The percentage of wound closure was calculated by the formula [wound area (0 h) $-$ wound area (n h)]/wound area (0 h).

2.8 In vivo tumor growth and metastasis assay

Animal experiments were approved by the Animal Ethics Committee of Peking University Cancer Hospital & Institute. The in vivo tumor growth and metastatic characteristics of the cells were measured by a modified chick embryo chorioallantoic membrane assay (CAM). Briefly, 5 $\times$ 10 ${}^{6}$ cells prestained with the cell tracker dye CM-Dil (red fluorescence) were loaded on the CAM with 10-day-old chick embryos. Seven days later, the tumors on the CAM were dissected and weighed. The lungs of chick embryos were isolated, and metastatic tumor lesions with red fluorescence were evaluated under a fluorescence microscope (Leica, Germany).

2.9 Analysis of predicted microRNA targets by online software

The predicted targets of the microRNAs were analyzed by an online database, miRecords (http://c1.accur ascience.com/miRecords/), which is a resource for animal miRNA-target interactions. The Predicted Targets component of miRecords integrates the predicted targets of the following miRNA target prediction tools: DIANA-microT, MicroInspector, miRanda, MirTarget2, miTarget, NBmiRTar, PicTar, PITA, RNA22, RNAhybrid, and TargetScan/TargetScanS [15].

2.10 Plasmid construction

The ANGPTL1 3’-UTR and YPEL1 3’-UTR or UTR mutant fragments containing the miR-224 complementary binding sites and corresponding mutants were inserted into the pGL3.0-control (Promega, Madison, WI) downstream of the luciferase coding sequence and were confirmed by sequencing.

2.11 Dual-luciferase reporter assays

HEK-293FT cells cultured in 24-well plates were transfected with pGL-3 construct (300 ng), miR-224 mimic or negative control (NC: 50 nM) and Renilla luciferase construct (20 ng) using Lipofectamine 2000 (Invitrogen). Firefly and Renilla luciferase activities were detected by a dual-luciferase assay (Promega) 24 h after transfection, and firefly luciferase activity normalized to Renilla luciferase activity was calculated.

2.12 Western blotting analysis

Cells were lysed with RIPA lysis buffer with protease inhibitors, and the protein was quantified by the BCA method. Equal amounts (30 $\mu$ g) of protein were separated by 10% SDS-PAGE and transferred onto PVDF membranes (Millipore, Bedford, MA). The membranes were incubated with primary antibodies, followed by HRP-linked secondary antibodies. Signals were detected using an enhanced chemiluminescence assay (Thermo Scientific, Rockford, IL).

Figure 1.

MiR-224 was highly expressed in NSCLC tissues with lymphatic metastasis. (A) Heatmap for miR-224 expression in lymphatic metastasis-positive and lymphatic metastasis-negative tissues by microarray analysis. (B) MiR-224 expression in lymphatic metastasis-positive (LN+) and lymphatic metastasis-negative (LN-) tissues by q-PCR analysis. (C) ROC curve analysis for the prediction of lymphatic metastasis by miR-224 expression. (D) Survival analysis according to miR-224 expression using the Kaplan-Meier method.

2.13 Statistical analysis

Statistical analyses were calculated with SPSS 16.0 (IBM, Armonk, NY, USA). Receiver operating characteristic (ROC) curves were established to discriminate metastatic lymph nodes and noncancerous lymph nodes. A two-tailed chi-squared test ( $\chi^{2}$ ) was used to evaluate the relationship between miR-224 expression and clinicopathological factors. Survival was analyzed by the Kaplan-Meier method using the log-rank test. Continuous variables with a normal distribution are expressed as the means $\pm$ SD. Two-sided Student’s $t$ test, unless specified, was used for difference comparisons between the two groups. ANOVA followed by Bonferroni’s post hoc test was used to analyze differences among multiple groups. A $P$ value of $<$ 0.05 was considered significant.

3. Results

3.1 Validation of miR-224 expression in NSCLC with lymphatic metastasis

Previously, 50 miRNAs were identified to be differentially expressed by miRNA microarray analysis in lymph node tissues with metastasis from five NSCLC patients and were compared with the expression levels in the corresponding noncancerous lymph node tissue [13]. Among these miRNAs, miR-224 was significantly upregulated in metastatic lymph nodes with a 14.6-fold change (Fig. 1A). Then, we validated miR-224 in a cohort of 155 patients. Consistently, miR-224 expression in samples with lymphatic metastasis was significantly higher than that in samples without lymphatic metastasis (approximately 6.8-fold change, Fig. 1B, $P=$ 0.0005) and showed an AUC value of 0.57 when evaluating lymphatic metastasis (Fig. 1C). Moreover, a survival prognostic analysis by the Kaplan-Meier method revealed that high expression of miR-224 in NSCLC tissues was associated with poor overall survival (Fig. 1D, $P=$ 0.001).

3.2 Forced expression of miR-224 enhanced cell viability, colony formation, migration and invasion in H1299 cells in vitro

First, the expression of miR-224 was detected by qRT-PCR in six lung cancer cell lines. As shown in Fig. 2A, miR-224 expression levels were relatively high in PC9, A549 and H520 cells but low in GLC82, H1299 and H1975 cells. Therefore, we used H1299 cells for overexpression experiments and PC9 cells for knockdown experiments to reveal the role of miR-224 in the malignant phenotype of lung cancer cells. As shown in Fig. 2B, miR-224 expression was increased by approximately 40-fold after transfection with mimic at 24 hrs. Forced expression of miR-224 markedly promoted cell viability (Fig. 2C, $P<$ 0.0001) and colony formation ability by twofold (Fig. 2D, $P<$ 0.0001). Furthermore, following the increase in miR-224, H1299 cells spread much faster than NC cells, as determined by wound healing assay (Fig. 2E, $P<$ 0.0001). Compared with NC cells, the migration and invasion abilities of H1299 cells transfected with miR-224 mimics were also markedly increased, as determined by Transwell assay (Fig. 2F, $P<$ 0.001).

Figure 2.

Overexpression of miR-224 enhanced cell viability, colony formation, migration and invasion in H1299 cells in vitro. (A, B) miR-224 expression in lung cancer cells was detected by q-PCR. (C) Cell viability assay for miR-224 overexpression and NC control cells was determined by CCK-8 assay. (D) A plate colony formation assay was performed for miR-224-overexpressing and NC control cells. (E) Cell spreading ability calculated as wound closure of monolayers was determined by wound healing assay for different cells. (F) Cell migration and invasion ability quantified by cell numbers were assessed by Transwell assay without or with Matrigel. Scale bars, 200 $\mu$ m. The data in B, D, E, and F are presented as the means $\pm$ SD, and statistical significance is shown as ${}^{**}P<$ 0.01, ${}^{***}P<$ 0.001.

Figure 3.

Inhibition of miR-224 suppresses PC9 cell migration and invasion in vitro. (A) miR-224 expression in different cells was detected by q-PCR. (B) CCK-8 assays were performed to assess cell viability. (C) Plate colony formation assays were performed to monitor cell growth. (D) A wound healing assay was used to study the cell spreading ability, and wound closure of the monolayer was calculated. (E) Transwell assays were performed to determine cell migration and invasion. Cell numbers were counted in four different fields. Scale bars, 100 $\mu$ m. The data in C, D, E are presented as the means $\pm$ SD, and statistical significance is shown as ${}^{**}P<$ 0.01, ${}^{***}P<$ 0.001.

3.3 Inhibition of miR-224 suppresses PC9 cell migration and invasion in vitro

MiR-224 inhibitor was transfected into PC9 cells with relatively high endogenous expression of miR-224. MiR-224 expression was successfully decreased by 71% after cells were transfected with inhibitor (Fig. 3A). Consistent with the results above, inhibition of miR-224 decreased cell viability (Fig. 3B, $P<$ 0.01) and colony formation ability (Fig. 3C, $P<$ 0.01). Moreover, inhibition of miR-224 markedly inhibited wound closure in the wound healing assay (Fig. 3D, $P<$ 0.001). The numbers of migrating and invading cells through Matrigel were also decreased in Transwell assays (Fig. 3E, $P<$ 0.001).

Figure 4.

MiR-224 functions as an oncomiR in vivo. (A, C) Control cells or cells with miR-224 mimics or inhibitors were inoculated onto the CAM. The effect of miR-224 on tumor growth was evaluated based on the tumor weight of each group. (B, D) Representative pictures of lung metastasis lesions by laser scanning confocal microscopy. The effect of miR-224 on lung metastasis was evaluated by the number of metastatic lesions (tumor cells were labeled with the red fluorescent dye CM-Dil). The data in the bar graphs were calculated as the mean $\pm$ S.D. for each group.

3.4 MiR-224 functions as an oncomiR in vivo

To validate the results above, we employed a modified chick embryo chorioallantoic membrane (CAM) assay to assess the role of miR-224 in tumor growth and metastasis in vivo. As shown in Fig. 4A, overexpression of miR-224 in H1299 cells resulted in a 5.2-fold increase in tumor weight compared to control cells (tumor weight, $P=$ 0.03). Moreover, metastatic lung lesions were also increased by 2.8-fold in miR-224-overexpressing cells (Fig. 4B, $P=$ 0.03). Consistently, compared with control cells, the tumor weights formed from the miR-224-inhibited PC9 cells were decreased by 83.8% in the CAM assay (Fig. 4C, tumor weight, $P=$ 0.02). Furthermore, the proportion of metastatic cancer cells to embryonic lungs was also markedly decreased by 83.0% in the miR-224-inhibited group (Fig. 4D, $P=$ 0.02). Collectively, these data indicated that miR-224 functions as an oncomiR both in vitro and in vivo, which can enhance the malignant phenotype of NSCLC.

3.5 ANGPTL1 is a direct target of miR-224 and is involved in its oncogene function

The biological significance of miRNAs relies on the regulation of their targets. First, we analyzed the miRNA targets using the online database miRecords. Given the hypothesis that miR-224 correlated with lymphatic metastasis, we analyzed the differentially expressed genes (DEGs) of lung cancer tissues at the N1-3 stage with lymph node metastasis compared with those at the N0 stage without lymph node metastasis, and we performed correlation analysis between the mRNA and miRNA expression profiles from The Cancer Genome Atlas (TCGA) database. A three-set Venn diagram showed 41 intersections of candidate targets (Fig. 5A). Of the 41 predicted target genes, we selected two tumor suppressor genes, ANGPTL1 and YPEL1, for further validation. We detected the expression levels of ANGPTL1 and YPEL1 in miR-224-overexpressing and miR-224-inhibited cells. As shown in Fig. 5B, the expression levels of the two candidate genes were markedly decreased in miR-224-overexpressing H1299 cells but increased in PC9 cells transfected with miR-224 inhibitor at the protein level. Both ANGPTL1 and YPEL1 had one consensus binding site in their 3’-UTR. Luciferase reporters containing either the wild-type or mutant type 3’-UTR fragment were constructed (Fig. 5C). Dual-luciferase reporter assays showed that the miR-224 mimic significantly reduced the reporter activity of the wild-type ANGPTL1 luciferase construct (Fig. 5D, $P<$ 0.01) but not the corresponding mutant construct (Fig. 5D left graph, $P>$ 0.05). However, there was no significant difference in luciferase activity between the miR-224 mimic and NC groups for either the wild-type or mutant YPEL1 luciferase construct (Fig. 5D right graph, $P>$ 0.05). These data suggested that miR-224 directly regulates ANGPTL1 expression by binding to the 3’-UTR of its mRNA but indirectly inhibits YPEL1 expression. Moreover, the data downloaded from TCGA (Fig. 5E left graph, Spearman $r=-$ 0.1536, $P=$ 0.0009) and from our cohort of 155 lung cancer patients all displayed a negative correlation between miR-224 and ANGPTL1 expression at the RNA level (Fig. 5E right graph, Spearman $r=-$ 0.4947, $P<$ 0.0001).

Figure 5.

ANGPTL1 is a direct target of miR-224. (A) Venn diagram predicting the candidate targets of miR-224. (B) Western blot analysis of ANGPTL1 and YPEL1 expression in miR-224 mimic or inhibitor cells. (C) Schematic model indicating complementary binding sites and corresponding mutations of miR-224 in the ANGPTL1 and YPEL1 3’UTRs. (D) Wild-type or mutant PGL3 control plasmids were transfected into HEK293T cells together with miR-224 mimic or NC. Firefly fluorescence intensity (FL) normalized by Renilla fluorescence (RL) intensity was calculated 24 h after transfection. (E, F) Spearman correlation analysis of miR-224 and ANGPTL1 expression in the TCGA-LUAD database (E) and in our cohort of 155 cases of lung cancer (F). The data in D are presented as the means $\pm$ SD, and statistical significance is shown as ${}^{**}P<$ 0.01, ns: not significant.

Figure 6.

ANGPTL1 rescue expression partially diminishes the miR-224-mediated promotion effect on the malignant phenotype of H1299 cells. (A) Western blot determines ANGPTL1 rescue expression. (B) CCK-8 assays were performed to determine cell viability. (C) Plate colony formation assays were performed to detect cell growth. (D) Transwell assays were used to monitor cell migration and invasion. Cell numbers were counted in four different fields. Scale bars, 200 $\mu$ m. The data in C and D are presented as the means $\pm$ SD, and statistical significance is shown as ${}^{*}P<$ 0.05, ${}^{**}P<$ 0.01, ${}^{***}P<$ 0.001.

3.6 Rescue expression of ANGPTL1 partially reverses the promotion of the malignant phenotype mediated by miR-224

To further determine the role of ANGPTL1 in the promotion effect on the malignant phenotype mediated by miR-224, we reintroduced ANGPTL1 expression in miR-224-transfected H1299 cells (Fig. 6A, $P<$ 0.001). As shown in Fig. 6B–D, rescue expression of ANGPTL1 partially abolished the promoting effect of miR-224 on cell viability (Fig. 6B, $P<$ 0.001), colony formation (Fig. 6C, $P<$ 0.05), and the migration and invasion ability (Fig. 6D, $P<$ 0.05). These results indicated that ANGPTL1 rescue partially reverses the promoting effect of miR-224 on the malignant phenotype of NSCLC cells.

4. Discussion

Lymph node status is of great importance in NSCLC, not only for prognosis but also to guide postoperative therapeutic strategies [16]. Previous reports showed that some miRNAs as molecular markers were correlated with either lymph node metastasis or distant metastasis. MiRNA-130a [17] and miR-196a [18] expression were positively associated with lymph node metastasis, while miRNA-451 [19] expression was negatively associated with lymph node metastasis. MiR-328 [20] was validated to have predictive value for brain metastasis with a sensitivity and specificity of 75% and 81.8%, respectively. These studies revealed that miRNAs might be correlated with and used to predict the lymphatic metastasis of NSCLC. In the present study, we aimed to evaluate the role of miR-224 in the lymph node metastasis of NSCLC. Previously, miRNA microarray analysis identified the differentially expressed miRNAs between lymphatic metastatic nodes and noncancerous lymph nodes from five NSCLC patients [13]. One miRNA, miR-224, exerted higher expression in tissues with lymph node metastasis. Here, a validation cohort consisting of 155 NSCLC patients was recruited to validate the positive correlation of miR-224 and lymph node metastasis.

MiR-224 has been revealed to be involved in the regulation of malignant characteristics in human cancers. It was reported to promote tumor growth and metastasis in gastric [21], colorectal [22], and papillary thyroid cancer [23]. In addition, miR-224 was reported to be associated with aggressive progression and poor prognosis in human cervical [24] and colorectal cancer [25]. In colonic cancer, miR-224 was identified as one of the miRNAs related to lymph node metastasis [26]. As for lung cancer, in 2014, Zhu et al. reported that decreased miR-224 expression was found in cancer tissues and tissues with lymph node metastasis by q-PCR analysis of 115 patients, and overexpression of miR-224 in A549 cells transfected with mimics resulted in suppression of cell migration and invasion by Transwell and wound healing assays [27]. However, contrary to the findings of Zhu et al., in 2015, Cui et al. published their results in the PNAS that miR-224 was significantly upregulated in NSCLC tissues, particularly in resected NSCLC metastasis by both q-PCR and in situ hybridization analysis, and increased miR-224 expression promoted cell migration, invasion, and proliferation [28]. In 2020, miR-224 was predicted to be one of the miRNAs associated with lymph node metastasis in lung adenocarcinoma only by bioinformatics analysis of TCGA and GEO datasets [29]. Here, consistent with the report from Cui et al., our microarray also identified miR-224 as an upregulated miRNA in tissues with lymph node metastasis. More importantly, we also validated its high expression of approximately 7-fold change in tissues with metastasis compared to nonmetastasis in our own cohort consisting of 155 patients. Although miR-224 alone was only weakly predictive for lymphatic metastasis with AUC $=$ 0.57, the combination of miR-224 and other predictors might show an improved ability for predicting lymphatic metastasis of NSCLC. In addition to being a predictor for lymphatic metastasis, high expression of miR-224 in NSCLC tissues was found to be associated with poor overall survival in our cohort, indicating its role as a prognostic marker for patients with NSCLC. Our results from gain- and loss-of-function experiments also validated that miR-224 functioned as an oncogene in lung cancer, which could promote cell migration, invasion, and proliferation both in vitro and in vivo.

Previously, some direct targets of miR-224 were found to be involved in its function, such as by directly targeting the tumor suppressors TNF $\alpha$ -induced protein 1 (TNFAIP1) and SMAD4 in lung cancer [28]. Here, we report another direct target, ANGPTL1, and an indirect target, YPEL1, as downstream regulatory genes of miR-224. Previously, ANGPTL1 was reported to be a tumor suppressor in lung cancer that could inhibit cancer cell motility and metastasis by abrogating the expression of the EMT mediator SLUG [30]. Our rescue ANGPTL1 experiments also validated its suppressive role in the regulation of the malignant phenotype in lung cancer, including cell motility. YPEL1 has been identified as a significantly downregulated gene in pancreatic cancer, and its reduced expression might be related to perineural invasion and prognosis [31]. However, the underlying mechanism of miR-224 in the regulation of YPEL1 and its role in lung cancer require further investigation. Here, we chose ANGPTL1 and YPEL1 as target genes for validation. Whether other genes are also regulated by miR-224 is still worthy of further study.

5. Conclusions

Our findings highlight that miR-224 serves as an oncogene associated with lymph node metastasis in NSCLC patients. The promotion effect of miR-224 on the malignant phenotype might be due to its regulation of two tumor suppressors, ANGPTL1 and YPEL1. Thus, miR-224 may be a predictor for the lymph node metastasis of NSCLC.

Ethics approval and consent to participate

All methods and experiments were approved by the Ethics Committee of Peking University Cancer Hospital & Institute, and the informed consent was obtained from all the participants. All animal experiments were performed in accordance with guidelines and regulation of Animal Ethics Committee of Peking University Cancer Hospital & Institute. Written informed consent was obtained from the owners for the participation of their animals in this study.

Consent to publish

All authors give consent for publication.

Availability of data and materials

All relevant materials and data are available from corresponding author (Dr. Jinfeng Chen) for non-commercial purposes.

Funding

This study was supported by the National Natural Science Foundation of China (Grant No. 81773144, 81772632), Special funds of the Ministry of Finance for Reform and Development and Science Foundation of Peking University Cancer Hospital 2020-9.

Authors’ contributions

Study supervision and conception: JFC; Data acquisition: HBH; Analysis or interpretation of data: BP, FL, LNW, XJL and YY; Statistical analysis: HBH; Preparation of the manuscript: HBH. All authors approved the final manuscript.

Supplementary data

The supplementary files are available to download from http://dx.doi.org/10.3233/CBM-210376.

sj-docx-1-cbm-10.3233_CBM-210376.docx - Supplemental material

Supplemental material, sj-docx-1-cbm-10.3233_CBM-210376.docx

Footnotes

Acknowledgments

Not applicable.

Conflict of interest

All authors declare no competing interest.

References

Siegel

R.L.

Miller

K.D.

and Jemal

, Cancer statistics, 2019, CA Cancer J Clin 69 (2019), 7–34.

Hirsch

F.R.

Scagliotti

G.V.

Mulshine

J.L.

Kwon

Curran

W.J.

, Jr. Wu

Y.L.

and Paz-Ares

, Lung cancer: Current therapies and new targeted treatments, Lancet 389 (2017), 299–311.

Gelatti

A.C.Z.

Drilon

and Santini

F.C.

, Optimizing the sequencing of tyrosine kinase inhibitors (TKIs) in epidermal growth factor receptor (EGFR) mutation-positive non-small cell lung cancer (NSCLC), Lung Cancer 137 (2019), 113–122.

Yang

Pan

Liu

Gao

Liang

and Guan

, A new approach to predict lymph node metastasis in solid lung adenocarcinoma: A radiomics nomogram, J Thorac Dis 10 (2018), S807–S819.

Shen-Tu

Mao

Pan

Wang

Zhang

Cheng

Guo

and Wang

, Lymph node dissection and survival in patients with early stage nonsmall cell lung cancer: A 10-year cohort study, Medicine (Baltimore) 96 (2017), e8356.

Riffo-Campos

A.L.

Riquelme

and Brebi-Mieville

, Tools for Sequence-Based miRNA Target Prediction: What to Choose? Int J Mol Sci 17 (2016).

Rupaimoole

and Slack

F.J.

, MicroRNA therapeutics: Towards a new era for the management of cancer and other diseases, Nat Rev Drug Discov 16 (2017), 203–222.

Fazi

and Fontemaggi

, MicroRNAs and lymph node metastatic disease in lung cancer, Thorac Surg Clin 22 (2012), 167–175.

Min

Wang

Chen

Yang

Liang

and Xu

, Aberrant microRNA-137 promoter methylation is associated with lymph node metastasis and poor clinical outcomes in non-small cell lung cancer, Oncol Lett 15 (2018), 7744–7750.

10.

Chen

Y.J.

Guo

Y.N.

Shi

Huang

H.M.

Huang

S.P.

W.Q.

Z.Y.

Wei

K.L.

Gan

T.Q.

and Chen

, Down-regulation of microRNA-144-3p and its clinical value in non-small cell lung cancer: A comprehensive analysis based on microarray, miRNA-sequencing, and quantitative real-time PCR data, Respir Res 20 (2019), 48.

11.

Chen

Min

Ren

Yang

Sun

Wang

Sun

and Zhang

, miRNA-148a serves as a prognostic factor and suppresses migration and invasion through Wnt1 in non-small cell lung cancer, PLoS One 12 (2017), e0171751.

12.

Cheng

X.K.

Lin

W.R.

Jiang

Z.H.

and Wang

, MicroRNA-129-5p inhibits invasiveness and metastasis of lung cancer cells and tumor angiogenesis via targeting VEGF, Eur Rev Med Pharmacol Sci 23 (2019), 2827–2837.

13.

Zhao

Liu

Yang

Zhang

and Chen

, Circulating microRNA-422a is associated with lymphatic metastasis in lung cancer, Oncotarget 8 (2017), 42173–42188.

14.

Shi

and Chiang

V.L.

, Facile means for quantifying microRNA expression by real-time PCR, Biotechniques 39 (2005), 519–525.

15.

Xiao

Zuo

Cai

Kang

Gao

and Li

, miRecords: An integrated resource for microRNA-target interactions, Nucleic Acids Res 37 (2009), D105–10.

16.

Lardinois

De Leyn

Van Schil

Porta

R.R.

Waller

Passlick

Zielinski

Lerut

and Weder

, ESTS guidelines for intraoperative lymph node staging in non-small cell lung cancer, Eur J Cardiothorac Surg 30 (2006), 787–792.

17.

Wang

X.C.

Tian

L.L.

H.L.

Jiang

X.Y.

L.Q.

Zhang

Wang

Y.Y.

H.Y.

D.G.

She

Liu

Q.F.

Fan

F.Y.

and Meng

A.M.

, Expression of miRNA-130a in nonsmall cell lung cancer, Am J Med Sci 340 (2010), 385–388.

18.

Liu

X.H.

K.H.

Wang

K.M.

Sun

Zhang

E.B.

Yang

J.S.

Yin

D.D.

Liu

Z.L.

Zhou

Liu

Z.J.

and Wang

Z.X.

, MicroRNA-196a promotes non-small cell lung cancer cell proliferation and invasion through targeting HOXA5, BMC Cancer 12 (2012), 348.

19.

Wang

X.C.

Tian

L.L.

Jiang

X.Y.

Wang

Y.Y.

D.G.

She

Chang

J.H.

and Meng

A.M.

, The expression and function of miRNA-451 in non-small cell lung cancer, Cancer Lett 311 (2011), 203–209.

20.

Arora

Ranade

A.R.

Tran

N.L.

Nasser

Sridhar

Korn

R.L.

Ross

J.T.

Dhruv

Foss

K.M.

Sibenaller

Ryken

Gotway

M.B.

Kim

and Weiss

G.J.

, MicroRNA-328 is associated with (non-small) cell lung cancer (NSCLC) brain metastasis and mediates NSCLC migration, Int J Cancer 129 (2011), 2621–2631.

21.

Wang

Zhang

and Xu

, Hypoxia-inducible microRNA-224 promotes the cell growth, migration and invasion by directly targeting RASSF8 in gastric cancer, Mol Cancer 16 (2017), 35.

22.

Yuan

Xie

Fox

Zeng

Gao

Huang

and Wu

, Decreased levels of miR-224 and the passenger strand of miR-221 increase MBD2, suppressing maspin and promoting colorectal tumor growth and metastasis in mice, Gastroenterology 145 (2013), 853–64 e9.

23.

Zang

C.S.

Huang

H.T.

Qiu

Sun

R.F.

and Jiang

L.W.

, MiR-224-5p targets EGR2 to promote the development of papillary thyroid carcinoma, Eur Rev Med Pharmacol Sci 24 (2020), 4890–4900.

24.

Shen

S.N.

Wang

L.F.

Jia

Y.F.

Hao

Y.Q.

Zhang

and Wang

, Upregulation of microRNA-224 is associated with aggressive progression and poor prognosis in human cervical cancer, Diagn Pathol 8 (2013), 69.

25.

Adamopoulos

P.G.

Kontos

C.K.

Rapti

S.M.

Papadopoulos

I.N.

and Scorilas

, miR-224 overexpression is a strong and independent prognosticator of short-term relapse and poor overall survival in colorectal adenocarcinoma, Int J Oncol 46 (2015), 849–859.

26.

Wang

Y.X.

Zhang

X.Y.

Zhang

B.F.

Yang

C.Q.

Chen

X.M.

and Gao

H.J.

, Initial study of microRNA expression profiles of colonic cancer without lymph node metastasis, J Dig Dis 11 (2010), 50–54.

27.

Zhu

Chen

Yang

Chen

Wang

and Yu

, Decreased microRNA-224 and its clinical significance in non-small cell lung cancer patients, Diagn Pathol 9 (2014), 198.

28.

Cui

Meng

Sun

H.L.

Kim

Fassan

Jeon

Y.J.

Vicentini

Peng

Lee

T.J.

Luo

Liu

Tili

Jin

Middleton

Chakravarti

Lautenschlaeger

and Croce

C.M.

, MicroRNA-224 promotes tumor progression in nonsmall cell lung cancer, Proc Natl Acad Sci U S A 112 (2015), E4288–4297.

29.

Wang

Shang

Sun

and Zhao

, miR-224, miR-147b and miR-31 associated with lymph node metastasis and prognosis for lung adenocarcinoma by regulating PRPF4B, WDR82 or NR3C2, PeerJ 8 (2020), e9704.

30.

Kuo

T.C.

Tan

C.T.

Chang

Y.W.

Hong

C.C.

Lee

W.J.

Chen

M.W.

Jeng

Y.M.

Chiou

Chen

P.S.

Wang

M.Y.

Hsiao

J.L.

and Kuo

M.L.

, Angiopoietin-like protein 1 suppresses SLUG to inhibit cancer cell motility, J Clin Invest 123 (2013), 1082–1095.

31.

Abiatari

Kiladze

Kerkadze

Friess

and Kleeff

, Expression of YPEL1 in pancreatic cancer cell lines and tissues, Georgian Med News (2009), 60–62.

MiR-224 promotes lymphatic metastasis by targeting ANGPTL1 in non-small-cell lung carcinoma

Abstract

BACKGROUND:

METHODS:

RESULTS:

CONCLUSIONS:

Keywords

Abbreviations

1. Background

2. Methods

2.1 Cell lines and culture conditions

2.2 Patients and samples

2.3 Reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR)

2.4 Cell viability assay

2.5 Plate colony formation assay

2.6 Transwell assay

2.7 Wound healing assay

2.8 In vivo tumor growth and metastasis assay

2.9 Analysis of predicted microRNA targets by online software

2.10 Plasmid construction

2.11 Dual-luciferase reporter assays

2.12 Western blotting analysis

3. Results

3.1 Validation of miR-224 expression in NSCLC with lymphatic metastasis

3.2 Forced expression of miR-224 enhanced cell viability, colony formation, migration and invasion in H1299 cells in vitro

3.5 ANGPTL1 is a direct target of miR-224 and is involved in its oncogene function

4. Discussion

5. Conclusions

Ethics approval and consent to participate

Consent to publish

Availability of data and materials

Funding

Authors’ contributions

Supplementary data

sj-docx-1-cbm-10.3233_CBM-210376.docx - Supplemental material

Footnotes

Acknowledgments

Conflict of interest

References