MicroRNA‐29b‐3p suppresses oral squamous cell carcinoma cell migration and invasion via IL32/AKT signalling pathway

Abstract Oral squamous cell carcinoma (OSCC) is aggressive accompanied with poor prognosis. We previously isolated the most invasive cells resembling the invasive tumour front by microfluidic technology and explored their differentially expressed microRNAs (miRNAs) in our previous work. Here, we verified the miR‐29b‐3p as a guarder that suppressed migration and invasion of OSCC cells and was down‐regulated in the most invasive cells. Besides that, the invasion suppression role of miR‐29b‐3p was achieved through the IL32/AKT pathway. Thus, miR‐29b‐3p and IL32 might serve as therapeutic targets for blocking the progression and improving the outcome of OSCC.

MiR-29b had antitumour effects in various tumours and is downregulated in pancreatic cancer cells, 10 colorectal cancer 11 and human glioblastoma multiforme. 12 MiR-29b acts as a tumour suppressor in tongue squamous cell carcinoma by inhibiting the expression of genes involved in tumour cell proliferation, migration and invasion by targeting specificity protein 1 and thereby altering phosphatase and tensin homolog/AKT signalling. 13 However, there is little information on the function and mechanism of miR-29b in OSCC invasion.
In the present study, we show that miR-29b-3p is down-regulated in OSCC invasive cells and inhibits OSCC cell migration and invasion by suppressing the interleukin (IL) 32/AKT pathway.

| Cell culture
The human UM-SCC6 cell line was a kind gift from Peking Union Medical University. The OSCC invasion tumour front-like cells termed as UM-SCC6-M were separated from UM-SCC6 cells via the microfluidic chip in vitro. 4 They were cultured in DMEM/HG (HyClone) medium, supplemented with 10% foetal bovine serum (FBS) (Sciencell) and 1% penicillin streptomycin (Hyclone).

| Wound healing assay
Cell migration capacity was validated by wound healing assay. The cells were trypsinized and seeded into six-well plates (5 × 10 5 cells per well). When the cells grew to 90% confluence, the monolayers of cells were scratched by a sterile 200 µL pipette tip and washed with PBS for three times to remove cellular debris and then allowed to culture F I G U R E 1 MiR-29b-3p showed low expression level in high invasive cell line UM-SCC6-M. A, Transwell invasion assay for UM-SCC6 and UM-SCC6-M cells. B, Western blot of migration/invasion biomarkers' expression in UM-SCC6 and UM-SCC6-M cells. C, qPCR analysis of RNA level of miR-29b-3p in UM-SCC6 and UM-SCC6-M cells. Data represent the mean ± standard error of three independent experiments. *, P < .05; **, P < .01; ***, P < .001; ns, not significant in serum-free medium. The wound area at the start and end of each experiment was recorded by inverted microscope (Olympus IX 71) and calculated with Image-Pro Plus 6.0 at 0, 12, 24 and 36 hours after scratch. The cell migration rate was calculated by the decreased wound width in the wound area normalized to the initial wound width. The data represent the mean ± standard error of three independent experiments.
Invasion inserts with 8µm pore were coated with 80 µL Matrigel™ (Corning) and dried overnight at 37°C incubator. About 3 × 10 4 cells in 200 µL serum-free medium were seeded into the top chambers, and 600 µL medium supplemented with 20% serum was used as a chemoattractant in the lower chamber. After incubating for 24 hours at 37°C in 5% CO 2 , the medium in top chambers was removed, and the cells were fixed with 4% paraformaldehyde and stained with 0.1% crystal violet. The non-invading cells on the upper sides of membrane were removed by cotton wool. The cells on the lower sides of membrane were counted. Five random fields (×200) were selected and calculated the average. The data represent the mean ± standard error of three independent experiments.

| Total RNA isolation and Quantitative PCR (qPCR)
Total RNA was isolated from the cells using TRIzol reagent (Invitrogen) according to the manufacturer's instructions. For mRNA analyses, first strand cDNA was synthesized by 5X All-In-One RT Master Mix (ABMgood). qPCR was performed using KAPA SYBR ® FAST Universal qPCR Kits (KAPA Biosystems). Relative quantitation was calculated based on the 2 −ΔΔCt method. Gapdh was used as internal reference. For miRNA analyses, the mature miRNA was reverse transcribed with specific primers by Prime Script™ RT Master Mix (Takara). Then, qPCR was performed and analysed the same as for mRNA. U6 was used as internal reference. All reactions were carried out in triplicate. The primers used in this work were purchased from RiboBio.

| The gene overexpression and RNA interference
Plasmids pcDNA6 and pLKO-tet-on were used to overexpressing and knocking down the IL32, respectively. The sequences of shRNA targeting human IL32 were shIL32-1: TGGGGAGAGCTTTTGTGACAA; F I G U R E 2 MiR-29b-3p suppressed OSCC cell migration and invasion. A, B, Effect of miR-29b-3p mimic on UM-SCC6-M cells' migration (A) and invasion (B), as evaluated with wound healing and transwell invasion assays, respectively. C, D, Effect of miR-29b-3p inhibitor on UM-SCC6-M cells' migration (C) and invasion (D), as evaluated with wound healing and transwell invasion assays, respectively. Scale Bar = 100 μm. Data represent the mean ± standard error of three independent experiments. *, P < .05; **P < .01; ***, P < .001; ns, P > .05. Ctrl, control; In, inhibitor; Mi, mimic shIL32-2: AGAGCTGGAGGACGACTTCAA; shIL32-3: GCTCTGAACCCCAATCCTCAA. The mimic and the inhibitor of miR-29b-3p were purchased from Gene Pharma. The plasmids, mimic or inhibitor was transfected into the OSCC cell lines using Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions. Cells were harvested for analysis 48 hours later. All experiments were performed in triplicates.

| Western blot analysis
Total proteins were extracted from cells with RIPA buffer (Sigma The immunoreactive protein bands were detected by ECL detection system. GAPDH was used as a loading control.

| mRNA sequencing and data analysis
An RNA-seq library was prepared using the protocol of KAPA Stranded mRNA-Seq Kit (Kapa Biosystems) for the Illumina platform, following the manufacturer's instructions. Paired-end 150-bp sequencing was performed on HiSeq 2500 system in Berry Genomics Company. All reads were aligned to the human reference genome hg38 from UCSC using TopHat. Only unique mapped reads were used for further analyses. Cufflinks was used to measure the relative abundance of each transcript using the Ensemble Transcript ID.
Gene functional enrichment analysis was performed using Gene Set

| Statistical analysis
Statistical analysis was performed using SPSS. Student's t test was used to confirm comparisons of binary variables. Statistical significance was defined as P < .05.

| MiR-29b-3p suppressed OSCC cell migration and invasion
In our previous work, using microfluidic chip, we isolated invasion  Figure 1B).
The small RNA sequencing data from our previous work showed that miR-29b-3p was significantly down-regulated in UM-SCC6-M cells, which was supported by qPCR ( Figure 1C). To explore the function of miR-29b-3p in OSCC cells, we employed the inhibitor and mimic of miR-29b-3p ( Figure S2). The results of the wound healing and transwell invasion assays showed that transfection of the miR-29b-3p mimic markedly suppressed both migration ( Figure 2A) and invasion ( Figure 2B) in UM-SCC6-M cells, whereas inhibiting miR-29b-3p in UM-SCC6 cells by inhibitor enhanced these behaviours ( Figure 2C,D).

| AKT signalling mediated the migration and invasion of OSCC cells
The AKT pathway is widely acknowledged as a critical regulator of tumour progression. 14 To determine whether the behaviours of UM-SCC6-M cells were due to the up-regulation of pAKT, cells were treated with the AKT inhibitor, MK-2206. We found that the treatment suppressed cells' migration and invasion ( Figure 3A,B). The same effect was also observed in UM-SCC6 cells, which exhibited only weak migratory and invasive capacities and MK-2206 made them further weaker ( Figure S3A,B). Meanwhile, MK-2206 attenuated the migrating and invading stimulatory effect of miR-29b-3p inhibitor in UM-SCC6 cells ( Figure 3C,D). These results demonstrate that AKT signalling mediates the migration and invasion of OSCC cells and may be a downstream effector of miR-29b-3p.

| IL32 was a downstream target of miR-29b-3p
To clarify the mechanism underlying the effects of miR-29b-3p, we  Figure 4A). We compared these 30 genes with the 842 targets of miR-29b-3p predicted using miRanda. Then, we got IL32 which was the only candidate both in the 30 genes list and in the 842 candidate targets of miR-29b-3p.
So we focused on IL32 to conduct further research. In accordance with the mRNA sequencing data ( Figure 4B), qPCR and Western blotting showed that IL32 was up-regulated in UM-SCC6-M cells at the mRNA and protein levels ( Figure 4C,D). More importantly, it has been reported that miR-29b regulated IL32 via direct binding to the 3′ untranslated region of the IL-32 transcript. 15 Furthermore, by transfecting UM-SCC6 and UM-SCC6-M cells with the miR-29b-3p inhibitor and mimic, respectively, we found that IL32 was under the negative regulation by miR-29b-3p at the post-transcriptional level ( Figures 4E and S5).

| IL32 promoted OSCC cell migration and invasion by activating AKT signalling
IL32 is a cytokine involved in cancer development. 16 It was reported to promote breast cancer cell invasion and metastasis via integrin β3/p38 mitogen-activated protein kinase (MAPK) signalling. 17 We therefore investigated the role of IL32 in OSCC migration and invasion and found that overexpression of IL32 in UM-SCC6 cells stimulated both of these behaviours ( Figure 5A,B and S6A). On the contrary, migration and invasion of UM-SCC6-M cells were suppressed by IL32 knockdown (Figure S6A-C).
IL32 was previously reported to enhance metastatic potential in gastric cancer by activating AKT signalling. 18 So IL32 might be the reason for the activated AKT in UM-SCC6-M cells. As expected, overexpression and knockdown of IL32 altered AKT phosphorylation ( Figure 5C).
Additionally, AKT inhibition completely abolished the stimulatory effect of IL32 on the migration and invasion of UM-SCC6 cells ( Figure 5D,E).

| MiR-29b-3p suppressed migration and invasion via the IL32/AKT pathway
The above results implied that IL32 mediated the effects of miR-29b-3p on AKT signalling. Indeed, miR-29b-3p mimic interfered with the migration and invasion of UM-SCC6-M cells, overexpression of IL32 abrogated its effects and restored the migratory and invasive potentials of the cells (Figure 6A,B). Conversely, knockdown of IL32 reversed the effect of the miR-29b-3p inhibitor on migration and invasion of UM-SCC6 cells ( Figure S7A,B). A similar effect was observed for AKT phosphorylation level, which was found to be regulated by the miR-29b-3p/IL32 axis ( Figure 6C). As expected, the AKT inhibitor MK-2206 suppressed the migration and invasion of UM-SCC6 cells transfected with the miR-29b-3p mimic and IL32 overexpression plasmid ( Figure S7C,D). Taken together, miR-29b-3p suppresses migration and invasion of OSCC via the IL32/AKT axis.
F I G U R E 6 MiR-29b-3p suppressed migration and invasion via the IL32/ AKT pathway. A, B, Effect of IL32 overexpression on the migration (A) and invasion (B) of UM-SCC6-M cells with miR-29b-3p mimic transfected, as evaluated with wound healing and transwell invasion assays, respectively. C, Western blot showed the AKT and pAKT level under the effect of IL32 and miR-29b-3p. Scale Bar = 100 μm. Data represent the mean ± standard error of three independent experiments. **, P < .01; ***, P < .001. Ctrl, control; In, inhibitor; KD, knockdown; Mi, mimic; OE, overexpression