Dysregulated genes closely related to melanoma
Differential expression analysis on were conducted on three GEO datasets independently, and the volcano plots of DEGs (P-value<0.05, absolute fold change>2) were displayed in Fig. 1A-C. Subsequently, DEGs common in three SKCM datasets are filtered out and illustrated in Venn diagram. As shown in Fig. 1D-F, there are 580 overlapping DEGs in all three datasets, in which 165 genes were upregulated and 393 genes were downregulated. Moreover, overexpression of TUBB4A was found in melanoma tissues compared with normal tissues in all three datasets (Fig. 1G-I).
To uncover the role of these differentially expressed genes in the pathogenesis of melanoma, GO and KEGG enrichment analysis was carried out. As shown in Fig. 2A-C, they are significantly enriched in skin epidermis-related biological processes, such as skin and epidermis development, epidermal cell and keratinocyte differentiation, and extracellular structure organization. As for cellular component, these DEGs are significantly enriched in the cell-cell junction, collagen-containing extracellular matrix, apical part of cell, cornified envelope, and intermediate filament. For molecular function, they are significantly enriched in cell adhesion molecular binding, actin binding, structure constituent of cytoskeleton, extracellular matrix structural constituent, and serine-type peptidase activity.
The KEGG enrichment analysis shows top 25 significantly enriched pathways are involved in the process of cancerogenesis (Fig. 2D). More specifically, up-regulated genes are mainly enriched in the pathways related to transcription misregulation in cancer, glycosphingolipid biosynthesis-lacto and neolacto series, p53 signaling pathway and melanogenesis. Down-regulated genes are significantly enriched in histidine metabolish, Staphylococcus aureus infection, amoebiasis, drug metabolism and metabolism of xenobiotics by cytochrome P450.
It has been reported that melanin is partially attributed to the secretion of ɑ-melanocyte stimulating hormone (ɑ-MSH) induced by keratinocytes, while ultraviolet (UV) induction of ɑ-MSH in skin is directly regulated by p53 . Key signal molecule p53 activated in response to DNA damage, trafficked in melanoma and thyroid cancer cells and was associated with cell proliferation and progression. Moreover, it also mediates cell-cycle arrest, apoptosis, and senescence .
TUBB4A is over-expressed and correlated to prognosis in melanoma
We conducted differential expression analysis by integration of TCGA and GTEx datasets. The differential expression analysis revealed 1,485 genes that are differentially expressed between tumor and normal samples. Among them, 514 genes are up-regulated and 972 genes are down-regulated. Since dysregulated genes are often main cancerogenic factors, we performed survival analysis on dysregulated genes independently. The Kaplan-Meier analysis results showed that five up-regulated genes are significantly related to the both overall survival and disease-free survival of cancer patients. They are TUBB4A, PSEN2, SLC45A2, QPRT and TRPV2. Table 2 listed the details of the five genes.
We have conducted extensive literature search to verify the genes associated to melanoma survival rate. SLC45A2 is involved in melanosome maturation and pigmentation and already reported to be associated with risk of cutaneous malignant melanoma [28, 29]. The upregulation of PSEN2 is associated with human melanoma aggressiveness and poor prognosis, and it is also the target of MYC . According to previous study , TRPV2 exhibited ectopic distribution both in melanocytes and melanoma cells. Moreover, activation of TRPV2 could lead to the decline of cell viability for melanoma A2058 and A375 cells .
Most importantly, TUBB4A is the gene with the most statistical significant in the prediction of patient survival status. The Kaplan–Meier survival curve showed that patients with high TUBB4A expression had significantly shorter overall survival and disease-free survival than those with low expression (Fig. 3A, B). Then, we preformed co-expression analysis of TUBB4A in SKCM cases through LinkedOmics in the TCGA database. As shown in Fig. 3C, D, fifty significantly co-expressed genes were demonstrated by heatmap, showing that they were positively or negatively correlated with TUBB4A.
TUBB4A knockdown significantly inhibits SKCM cell proliferation
To further explore the role of TUBB4A in SKCM, small interfering RNAs (siRNAs) to silence TUBB4A gene was transfected in A375 and B16-F10 cells. We ensure the transfection resulted in sufficient knockdown of TUBB4A expression. The downregulation at RNA level was evident at 48h past transfection for both cell lines (Fig. 4A, B). Cell viability was determined using the CCK8 assay based on absorbance at 450 nm after 24h, 48h, 72h and 96h. Both A375 and B16-F10 cells with TUBB4A knockdown by siRNA showed reduced proliferation compared to the control group (si-NC, Fig. 4C, D). These results indicate that the higher expression of TUBB4A is closely associated to the proliferation of melanoma cells.
TUBB4A knockdown reduce SKCM cell migration in vitro
SKCM cell migration capacity was detected by wound healing assay. A375 and B16-F10 cells of which TUBB4A is knocked down by siRNA showed a larger open wound area compared with the control group after 24h and 48h (Fig. 5a, c). The difference in open wound area after 24h and 48h was quantified by calculating the percentage of change in the open wound area (Fig. 5B, D, p<0.05).
In addition, transwell assay is also used to evaluate its impact on migration capacity. Results demonstrated that TUBB4A knockdown caused a strong reduction in cell migration in A375 (t test, P<0.05) and B16-F10 (t test, P<0.05) cells, compared to the control group (Fig. 6A-D). These findings indicate that TUBB4A functions as a driver factor in increasing melanoma cell motility.
TUBB4A inhibitors significantly induce apoptosis of melanoma cells
We went further to search for small molecule drugs that target TUBB4A in the DrugBank database. There are five small molecule agents potentially target to TUBB4A. We selected two drugs, Dihydroartemisinin (DHA) and Nocodazole, which have been approved or already in experimental stage. Dihydroartemisinin is an artemisinin derivative and antimalarial agent used in the treatment of uncomplicated Plasmodium falciparum infections, and it has been also reported to bind to TUBB4A protein . Nocodazole is a 16-membered macrolide that mimics the biological effects of taxol and functions as inhibitor of microtubule function. Nocodazole has also been reported to inhibit the activity of TUBB4A .
Next, we went to test whether treatment with these two drugs separately can induce apoptosis of melanoma cell. As A375 is human melanoma cell line, we conducted assays on A375 cell line. The A375 cells were treated with DHA (20μM and 40μM) for 48h and Nocodazole (0.075μM and 0.15μM) for 24h separately. As shown in Fig. 7A, C, microscopy observations revealed that the exposure to DHA and Nocodazole triggered fragmentation in the cancer cells. The A375 cell apoptosis induced by these two agents was confirmed by staining with Annexin V-FITC/PI and subsequent analysis by flow cytometry. As shown in Fig. 7A-D, the proportion of apoptotic cells (quadrant Q2 An+/PI+ -late apoptosis and quadrant Q4 An+/PI- -early apoptosis) increased significantly in a concentration-dependent manner in cell line after the exposure to two drugs. Our results indicate the pro-apoptotic activity of DHA and Nocodazole against A375 cells.
TUBB4A inhibitors modulate melanoma cell cycle progression
We also examined the effect of two TUBB4A inhibitors on cell cycle progression in A375 cells, using propidium iodide staining. The A375 cells were treated by DHA for 48h and Nocodazole for 24h, respectively. With the increase of DHA concentration, the cell cycle of A375 cells (Fig. 8A, B) was significantly blocked in G2/M phase. As shown in Fig. 8C, D, Nocodazole exerted significant tumor cell cycle arrest, and the dosages effect of 0.075μM and 0.15μM differ slightly. The results revealed that DHA and Nocodazole inhibited the proliferation of A375 cells by inducing cell cycle arrest.