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miR-9-5p Promotes Lung Adenocarcinoma Cell Proliferation, Migration and Invasion by Targeting ID4

Objectives Evidence reveals that microRNAs (miRNAs) are abnormally expressed in lung adenocarcinoma (LUAD) tissue and are crucial in LUAD occurrence. Therefore, this study aims to find the miRNA which could regulate LUAD and to further explore its regulatory mechanism, thus providing a potential molecular target for LUAD. Methods miR-9-5p and ID4 expression in LUAD cells were measured by real-time quantitative PCR and western blot. Cell functional assays were conducted to detect the biological functions of LUAD cells. A dual-luciferase reporter assay was utilized to validate the binding relationship between miR-9-5p and ID4. Results miR-9-5p was highly expressed whereas ID4 was lowly expressed in LUAD. miR-9-5p facilitated LUAD cell progression by targeting ID4. Conclusion miR-9-5p promotes LUAD cell progression by modulating ID4 and may become a potential target for LUAD.


Introduction
Lung cancer has become a major cause resulting in high mortality in world. 1,2 Lung adenocarcinoma (LUAD) approximately makes up 50% of total lung cancer cases, which has overtaken lung squamous cell carcinoma (LACC). 3,4 Despite the latest advancements in diagnosis, the prognosis of lung cancer patients is still poor. 5,6 Hence, finding new therapeutic approaches for lung cancer patients is in urgent need.
MicroRNAs (miRNAs) can hinder the target gene level through binding to the 3 ′ -untranslated region (UTR) and inducing translational suppression. 7 Up to now, a lot of miRNAs have been confirmed to be implicated with cell proliferation, differentiation, apoptosis, migration, cell cycle, and other biological processes. 8 Besides, by mediating target gene expression, loads of miRNAs are uncovered to play an inhibitory or promoting role in the malignant progression of human carcinoma. 9 For instance, miR-26a modulates ovarian cancer cell behaviors by targeting TCF12. 10 miR-125 suppresses colorectal cancer progression through TAZ. 11 Besides, research suggests that miRNA is important in the development of LUAD. For example, miR-218 modulates epithelial-mesenchymal transition and PI3K/Akt pathway by targeting BMI-1 to suppress LUAD progression. 12 miR-142-3p suppresses LUAD cell progression through targeting NR2F6. 13 Collectively, these findings fully clarify the significance of miRNAs in LUAD and that miRNA-dependent treatment can be one of the therapeutic approaches for LUAD. miR-9-5p is proven to be able to mediate the development of cancers, by regulating different mRNAs [14][15][16] Currently, there have been few studies on miR-9-5p and LUAD except that one study displayed an increase of miR-9-5p in LUAD. 17 Given this, mechanism by which miR-9-5p regulates LUAD is yet to be confirmed and validated. We attempted to validate the regulatory function of miR-9-5p in the disease as well as explored related downstream regulatory mechanisms.

Bioinformatics Approach
LUAD miRNA and corresponding clinical data (normal: n = 58, tumor: n = 522) were acquired from the TCGA-LUAD dataset. 1 Fujian Medical University Cancer Hospital, Fujian Cancer Hospital, Fuzhou, China miRDB, TargetScan, and miRTarBase were used in the prediction of downstream targets of the target miRNA. Meanwhile, DE_mRNAs were screened with the "DESeq2R" package, and normal samples were taken as control (|logFC|>1.5, Padj<.05). The screened results were then intersected with predicted mRNAs for identification of the mRNA of interest.

Real-Time Quantitative PCR
Total RNA was isolated using Trizol solution (TaKaRa) and then came with reverse transcription of mRNA and miRNA into cDNA by m-MLV reverse transcriptase kits (TaKaRa) and SuperScript II kits (Invitrogen), respectively. qRT-PCR was completed on the Applied Biosystems 7500 with TaKaRa SYBR Premix E × Taq kit. U6 and GAPDH were internal references for miR-9-5p and ID4, respectively. Primers were exhibited in Table 1.

Colony Formation Assay
Transfected cells (2 × 10 2 cells/mL) were placed on 6-well plates. The medium was replaced every 4 d and discarded after 12 d. Cells were then treated with 4% paraformaldehyde for 15 min and 0.1% crystal violet for 10 min. Finally, crystal violet was rinsed off with PBS, and cells were photographed.

Wound Healing Assay
Calu-3 cells were digested after transfection and 2 ml of cells (4 × 10 5 cells/mL) were used. With 90% cell confluence, we made a scratch with the tip of a sterile 200 μL pipette. Floating cells were rinsed with PBS. Remained cells were cultured in an FBS-free medium under standard conditions. An inverted microscope was used to capture photos of the scratch area at 0 and 48 h. Cell migration rate was assessed using the Image J software.

Transwell Assay
Calu-3 cells were digested after transfection and 200 μL of cells in FBS-free medium were then inoculated into the upper Table 1. Primer Sequences.
Gene Sequences chamber of Transwell laid with a matrix. In the lower chamber, 650 μL medium plus 10% FBS was added. 24 h after continuous cell culture, cells in the upper chamber were swabbed, after which cells invaded were treated with paraformaldehyde (4%) for 15 min and stained with crystal violet (0.1%) for 10 min. Finally, 5 random visual fields were observed under a microscope to count invading cells.

Statistical Analysis
Statistical analysis and visualization of experimental data from 3 independent replicates were completed using GraphPad Prism 7 software (CA). All data met normal distribution and were represented by mean ± SD. The t-test was conducted to compare data differences between the 2 groups. Analysis of variance was conducted to compare data differences among multiple groups. Kruskal-Wallis test was used to analyze the correlation between miR-9-5p/ID4 and clinicopathological characteristics of LUAD patients. P < .05 indicated statistically significant.

miR-9-5p is at a High Level in LUAD
After data acquirements from the bioinformatics database, differential gene expression analysis performed by the "edgeR" package suggested that 164 DE_miRNAs were acquired ( Figure 1A). There are few reports about miR-9-5p in LUAD.
In addition, compared with normal samples, miR-9-5p in tumor samples was markedly increased (Figure 1B), and the miR-9-5p level was significantly different in different stages ( Figure 1C). Hence, miR-9-5p was taken as a research target. Additionally, we assayed miR-9-5p levels in control cells BEAS-2B, human nonmetastatic LUAD cell lines, and human metastatic LUAD cell line NCI-H1299 through qRT-PCR. The result demonstrated miR-9-5p expression was increased in LUAD cells ( Figure 1D). Due to the fact that tumor cell behaviors are more often observed in early tumor stages, the Calu-3 cell line which had a relatively high miR-9-5p expression of the 2 nonmetastatic cell lines was chosen for our further study.

ID4 is a Direct Target of miR-9-5p
We predicted downstream targets of miR-9-5p by employing TargetScan, miRDB, and miRTarBase databases. Meanwhile, differential analysis of mRNAs in the TCGA-LUAD dataset was conducted ( Figure 3A), and then the down-regulated DE_mRNAs in LUAD were intersected with the predicted mRNAs. As a result, 10 mRNAs (TGFBR2, AMOTL1, PTX3, MYOCD, SLC19A3, CD34, STARD13, ID4, CBX7, and COLEC12) could serve as the regulatory targets of miR-9-5p ( Figure 3A). Among these 10 mRNAs, ID4 was found to be poorly expressed in LUAD ( Figure 3B). ID4 expression was significantly different in different stages ( Figure 3C). In addition, Pearson correlation analysis noted that ID4 was inversely associated with miR-9-5p in LUAD tissue ( Figure 3D). Research showed that ID4 could inhibit pancreatic cancer cell proliferation, migration, and invasion. 18 Therefore, it was hypothesized that miR-9-5p regulates ID4 in LUAD cells. To confirm the hypothesis, we assessed the ID4 level in control cells and cancer cell lines through qRT-PCR and western blot. ID4 level was poor in all LUAD cells ( Figure 3E and F). After that, ID4 level was measured upon miR-9-5p silencing/overexpression in LUAD cells, which uncover that silencing/over-expressing miR-9-5p up-regulated/down-regulated ID4 ( Figure 3G and H). These results validated the inverse correlation of miR-9-5p and ID4. Subsequently, dual-luciferase reporter assay unveiled that luciferase activity of ID4-wt was suppressed by over-expression of miR-9-5p whereas that of ID4-mut was not affected ( Figure 3I), which suggested that miR-9-5p could target ID4. Taken together, ID4 was directly targeted by miR-9-5p.

miR-9-5p Promotes LUAD Cell Progression by Targeting ID4
We proved in the section "ID4 is a Direct Target of miR-9-5p" that ID4 was a direct target of miR-9-5p, and then we observed whether miR-9-5p could promote LUAD cell behaviors by targeting ID4. After transfection, the ID4 level in these groups was assayed through qRT-PCR and western blot. ID4 expression in the NC-mimic + oe-ID4 group was markedly higher than that in the control group. ID4 expression was significantly lower after miR-9-5p overexpression than that in the control group. ID4 expression in the miR-9-5p-mimic + oe-ID4 group was significantly higher than that in the miR-9-5p-mimic + oe-NC group ( Figure 4A and B). From the MTT assay and colony formation assay, we noted that ID4 hindered proliferation and colony formation of LUAD cells. Simultaneously increased miR-9-5p and ID4 expression could reverse promoting impacts of miR-9-5p on proliferation and colony formation of LUAD cells ( Figure 4C and D). In wound healing and Transwell assays, we also observed that ID4 reduced cell migratory and invasive abilities, which could be rescued by miR-9-5p overexpression ( Figure 4E and F). Hence, miR-9-5p could promote LUAD cell progression by suppressing ID4.

Discussion
Studies that have been done over the recent few years confirm the close correlation between the part of miRNAs and malignant tumors, including the regulation of miRNAs on tumor growth and metastasis. 19,20 As a member of numerous miRNAs, miR-9-5p was confirmed as a modulatory factor of neural progenitor cells. 21 Deeper exploration into miR-9-5p uncovered that miR-9-5p modulates other cancers, such as colorectal cancer, cervical cancer, NSCLC, carcinoma of paranasal sinuses, and so on, in promotion of tumor proliferation, invasion, and metastasis. 14,[22][23][24] Here, we validated the up-regulation of miR-9-5p in LUAD. Additionally, the influence of miR-9-5p on the biological functions of LUAD cells was assayed by cell functional assays. It was suggested that miR-9-5p could enhance the proliferative activity, colony formation ability, migratory and invasive properties of LUAD cells. Collectively, miR-9-5p is able to facilitate LUAD cell progression.  ID4 can act as a dominant inverse modulatory factor of a basic HLH transcription factor. 25 Up to now, research has uncovered that ID4 can play an important regulatory role in cancers. For example, ID4 can facilitate HCC cell proliferation, 26 suppress prostate cancer cell proliferation, and raise cell apoptosis. 27 However, no research has reported the correlation between ID4 and LUAD. Herein, after confirming the oncogenic role of miR-9-5p, we explored targets of miR-9-5p and identified ID4. Meanwhile, rescue experiments unveiled the reverse effect of ID4 on miR-9-5p in exacerbating LUAD progression. Given the literature which displayed that miR-9-5p in NSCLC suppresses TGFBR2 to induce cell proliferation, invasion, and metastasis, 22 we reasoned that promoting effect of miR-9-5p on LUAD cell behaviors was partially realized by suppressing ID4 expression. In conclusion, miR-9-5p expression was at a high level in LUAD and could promote LUAD cell progression by targeting ID4, which provides a theoretical basis for the possibility that miR-9-5p/ID4 axis is a possible target for LUAD.

Availability of Data and Materials
The data used to support the findings of this study are included within the article. The data and materials in the current study are available from the corresponding author on reasonable request.