3.1| miR-937-3p is associated with LUAD metastasis.
We obtained the data of miRNA microarray from TCGA. We use edgeR to detect significantly differentially expressed miRNAs (DEmiRNA). First, we explored the differential expression of miRNA in 792 LUAD tumors and 203 normal tissues. Using standard filter analysis (absolute fold change >1.5 and FDR value <0.01), 128 miRNAs between normal and LUAD tissues were explored (Figure 1A, Supplementary Figure S1A).Through online survival prognosis analysis(http://kmplot.com/analysis/), we found that four highly expressed miRNAs (miR-937-3p, miR-195, miR-142, miR-345) have important prognosis for survival (Figure 1B, Supplementary Figure S2A). Compared with primary LUAD, miR-937-3p was most prominent in the metastatic LUAD (10 with metastasis, 10 with non-metastatic) (Supplementary Figure S2B). So we selected miR-937-3p for further research. Next, we found that miR-937-3p expression level was higher in tumor cell lines than in 16HBE cells (Figure 1C). Among them, the expression of miR-937-3p was highest in H1395 cells, while the expression of miR-937-3p in A549 cells was the lowest. Besides, we examined the expression of miR-937-3p in LUAD tissues and adjacent tissues (n = 60) (Figure 1D). The expression level of miR-937-3p was significantly up-regulated in LUAD tissues, indicating that miR-937-3p may be an oncogene involved in the development of LUAD. Beside, ISH showed that miR-937-3p was mainly expressed in malignant epithelial cells compared to adjacent tissues (Figure 1E). Interestingly, we found that the expression of miR-937-3p in LUAD with metastasis was higher than that in LUAD without metastasis, which was further verified by ISH (Figure 1F 1G). These results all indicate that miR-937-3p may be involved in the occurrence and metastasis of LUAD.
3.2|miR-937-3p affects cell angiogenesis, invasion and metastasis.
We selected H1395 cells as high-expressing cells and A549 cells as suppressor cells. Compared with normal A549 cells, the expression of miR-937-3p was up-regulated in cells transfected with lv-miR-937-3p,while the expression of miR-937-3p in H1395 cells transfected with lv-miR-937-3p-inhibitor was down-regulated (Figure 2A). The effects of miR-937-3p on cell invasion, metastasis and angiogenesis were measured by Transwell, scratch experiments and tube formation. It was found that high expression of miR-937-3p promotes the invasion, metastasis and angiogenesis of A549 cells, while low expression of miR-937-3p in H1395 cells exerted an inhibition effect (Figure 2B 2C 2D). At the same time,We use PCR to measure the level of miR-937 in HUVECs after treatment(Supplementary FigureS2C). In order to further explore the effect of miR-937-3p on angiogenesis, we have conducted a recruitment experiment and found that high-expression of miR-937-3p promotes HUVEC migration, while low expression has the opposite effect (Figure 2E). We guessed whether miR-937-3p would generate EMT and cause the invasion and metastasis of LUAD. For this reason, we conducted Western Blotting and Immunofluorescence experiments for further verification. Through Western blot and IF, we found that the expression of epithelial cell markers (E-cadherin) in miR-937-3p overexpression cell lines was down-regulated compared with NC cell lines, and the expression of mesenchymal cell markers (vimentin, Slug and N-catenin) are upregulated. Whereas low expression of miR-937-3p in H1395 cells exerted an inhibition effect (Figure 2F 2G).
3.3|SOX11 is the direct target gene of miR-937-3p in LUAD cell lines
In order to explore the downstream regulatory mechanism of miR-937-3p promoting LUAD metastasis and angiogenesis, we intersected the outputs of three online bioinformatics databases (TargetScan, miRDB, and miRWalk) to predict candidate gene targets.Based on these results, we found that SOX11 was the target gene of miR-937-3p (Figure 3A). We found that SOX11 is low in cancer tissues of LUAD patients and is related to survival prognosis. Through online survival analysis, we found that in lung adenocarcinoma, patients with low SOX11 expression have a better prognosis (Figure 3B-C). At the same time, in order to evaluate the response of SOX11 expression to miR-937-3p, PCR results showed that miR-937-3p has a negative regulatory effect on SOX11. Up-regulating miR-937-3p has resulted in decreased SOX11 expression, while down-regulating miR-937-3p has resulted in increased SOX11 expression (Figure 3D). Therefore, we have performed a luciferase reporter assay to explore whether the 3'-UTR of SOX11 is a direct target of miR-937-3p. The use of vectors containing site mutation sequences is not affected by miR-937-3p, but the relative luciferase intensity of cells with miR-937-3p and SOX11 3'-UTR plasmids is significantly reduced (Figure 3E 3F). Thus, SOX11 was confirmed as a direct downstream target of miR-937-3p. In addition, we speculate that SOX11 can mediate the activation of certain signaling pathways and promote the metastasis and tube formation of NSCLC. We examined several classic signaling pathways, and the results showed that miR-937-3p can up-regulate the phosphorylation level of AKT (p-AKT). In summary, miR-937-3p can regulate SOX11 expression and downstream PI3K/AKT signaling (Figure 3G).
3.4| SOX11 overexpression reversed the effect of miR-937-3p on the malignant progress of LUAD cells.
In order to further confirm the role of SOX11 in miR-937-3p-affected angiogenesis, invasion and metastasis, we performed a rescue test. We stably transfected A549/H1395 cells with the SOX11 plasmid, and detected the expression level of SOX11 in transfected cells by qRT-PCR (Figure 4A). Subsequently, tube formation assays, transwell experiments and scratch experiments were performed on co-transfected cells. The effect of up-regulation of miR-937-3p on the angiogenesis of LUAD cells was reversed by the high expression of SOX11. (Figure 4B). Similarly, it was also found that up-regulation of SOX11 inhibited invasion and metastasis of cells (Figure 4C 4D). At the same time, we found through western blot that SOX11 overexpression can rescue the decline in SOX11 expression induced by lv-miR-937-3p. In addition, the rescue effect also changed the level of p-AKT (Figure 4E 4F). In summary, SOX11 overexpression reversed the effect of miR-937-3p on the malignant progress of LUAD cells. In order to further verify our conjecture, it was found by Western blot that the overexpression of SOX11 can rescue the decrease in SOX11 expression induced by lv-miR-937-3p. In addition, the rescue effect also changed the level of p-AKT (Figure 4E 4F). In conclusion, we found that SOX11 overexpression reversed the effect of miR-937-3p on the malignant progression of LUAD cells.
3.5| miR-937-3p regulates LUAD cell metastasis and angiogenesis in vivo
We injected A549 cells transfected with Lv-miR-937-3p into the tail veins of 8 male mice to further verify the effect of miR-937-3p on the metastasis and angiogenesis of LUAD cells in vivo. After eight weeks, the mice were sacrificed and their lung tissues were taken out and the metastatic nodules on the lung surface were counted. Compared with the control group, Lv-miR-937-3p increased the number and volume of metastatic nodules (Figure 5A-B). The results showed that the increase of miR-937-3p significantly increased the number and size of lung metastases by H&E staining (Figure 5C). We further conduct IHC experiments on the metastatic tumors. MMP2 is known to be involved in the related processes of blood vessel formation and invasion and metastasis. In addition, the expression of SOX11 in Lv-miR-937-3p group was reduced, and the positive rate of MMP2 in Lv-miR-937-3p group was higher than that in control group (Figure 5D). Therefore, our in vivo data complements the results of miR-937-3p in vitro function studies.
3.6| MYC was an upstream regulator of miR-937-3p by directly binding to its promoter region
Since miR-937-3p functions as an onco-miRNA in LUAD, we hypothesized that miR-937-3p is likely to be activated by some pro-tumor factors. We selected 2000 sites upstream of miR-937-3p as regions where transcription factors may exist. Based on the JASPAR and UCSC databases, we found that multiple genes within the miR-937-3p promoter region may affect its transcription. Among them, MYC combined with miR-937-3p had the highest putative score (Figure 6A, Supplementary Figure S2E), and MYC expression was up-regulated in a variety of tumors, promoting tumorigenesis and development. We found that compared with adjacent tissues, MYC expression in LUAD is increased, and MYC is also related to survival prognosis (Figure 6B 6C). Therefore, we continue to verify the potential regulatory relationship between miR-937-3p and MYC, and choose the three highest scoring sites for CHIP experiments (Figure 6D). ChIP experiments showed that MYC binds upstream of the putative binding site of miR-937-3p (Figure 6E). At the same time, we used si-MYC expression to down-regulate the relative expression level of MYC (Supplementary Figure S2F). After 36 hours, the reduction of MYC expression can significantly reduce the level of miR-937-3p and increase the level of SOX11 (Figure6F 6G). These results suggest that miR-937-3p is positively correlated with MYC in LUAD cells. First, after co-transfecting Lv-miR-937-3p or Lv-vector with si-MYC, we used PCR to detect the expression levels of miR-937-3p and SOX11 in LUAD cells (Figure 6H, Supplementary Figure S3A). The results show that high level of miR-937-3p can reduce the influence of si-MYC on the invasion and metastasis of LUAD cells (Figure 6I 6J, Supplementary Figures S3B S3C).