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. 2022 May 5:2022:8016102.
doi: 10.1155/2022/8016102. eCollection 2022.

HOXC6 Regulates the Epithelial-Mesenchymal Transition through the TGF- β/Smad Signaling Pathway and Predicts a Poor Prognosis in Glioblastoma

Sun Eryi  1 , Li Zheng  1 , Cai Honghua  2 , Zhao Su  3 , Xie Han  1 , Pan Donggang  1 , Zhou Zhou  1 , Zhan Liping  1 , Chen Bo  1
Affiliations

HOXC6 Regulates the Epithelial-Mesenchymal Transition through the TGF- β/Smad Signaling Pathway and Predicts a Poor Prognosis in Glioblastoma

Sun Eryi et al. J Oncol. .

Abstract

Background: The HOX gene family of transcription factors, characterized by conserved homeodomains, is positively correlated with the resistance to chemotherapy drugs and poor prognosis, as well as the initiating potential of gliomas. However, there are few studies regarding the HOXC6 gene in glioma cells. Therefore, in the present study, we explored the regulatory roles and detailed mechanisms underlying the relationship between HOXC6 and the progression of GBM.

Methods: The expression levels and prognostic value of HOXC6 in GBM were evaluated using the data obtained from the GCCA, GEPIA, and ONCOMINE databases. The relationship between GBM prognosis and levels of HOXC6 was identified using Kaplan-Meier curves. The protein levels of HOXC6 in GBM and adjacent normal tissues were identified via Western blot and immunohistochemistry (IHC) staining methods. Lentiviruses containing full-length HOXC6 and HOXC6 specific siRNA sequences were used to overexpress and knock down, respectively, the expression of HOXC6 in U87 and U251 cells. The role of HOXC6 in the regulation of migration and proliferation of GBM cells was accessed using Transwell, wound healing, CCK-8, and colony formation assays. The activation of the TGF-β/Smad signaling pathway was detected via Western blotting.

Results: Compared to normal tissues and control cells, GBM tissues and cell lines showed higher expressions of HOXC6. The expression of HOXC6 was associated with disease-free and the overall survival of GBM patients. Additionally, positive correlations between the expression of HOXC6 and the migration and proliferation of GBM cells were observed in vitro. The mechanistic analyses indicated that HOXC6 exerts its promotive effect on the progression and invasion of glioma cells by promoting the activation of the EMT and TGF-β/Smad signaling pathways.

Conclusions: HOXC6 enhances the migration and proliferation of GBM by activating the EMT signaling pathway.

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Conflict of interest statement

We have no conflicts of interest to disclose.

Figures

Figure 1
Figure 1
High expression and significant association of HOXC6 in GBM. The mRNA levels of HOXC6 were analyzed using the ONCOMINE gene profile database. In the images, the best gene rank percentile was used to define the color of the cells. The thresholds were gene rank = top 10%, fold change = 1.5, and p value = 1E − 4. (b) In the heat map of HOXC6 from the TCGA datasets, the topmost bar represents the sample type, in which blue represents normal control samples, and red represents tumor samples; the color gradation at the upper right represents gene expression, in which the color changes from red to green from the top to the bottom, indicating gene expression changes from high to low; the abscissa represents sample number, and the ordinate represents gene names; each rectangle corresponds to a sample expression value. The dendrogram on the left represents cluster analysis based on differences in gene expression. (c) HOXC6 was highly expressed in high- and low-grade gliomas in the GEPIA database. (d) HOXC6 expression data in the CGGA databases with high and low expressions of HOXC6. (e) The prognostic survival rates of GBM patients with low and high HOXC6 expressions were analyzed using the GEPIA database. (f) The prognostic survival rates of LGG GBM patients with low and high HOXC6 expression were analyzed using the GEPIA database. The prognostic survival rate of glioma patients with high HOXC6 expression was low, while the risk ratio was significantly higher (HR = 3.5; p = 4.3E − 8). (g) Using the CGGA database, we constructed Kaplan-Meier curves for patients with high and low HOXC6 expressions. ∗p < 0.05,  ∗∗p < 0.01, and∗∗∗p < 0.001. Data are shown as the mean ± SD.
Figure 2
Figure 2
HOXC6 is highly expressed in human GBM samples. (a, b) Western blot results represent the protein levels of HOXC6 in indicated human tissues. (c) Representative images showing the expression of HOXC6 in noncancerous and glioma tissues measured by IHC. The scale bar represents 50 μm. (d) The levels of HOXC6 in U251, U87, A172, T98G, H4, and SHG44 cell lines by Western blot.
Figure 3
Figure 3
Effects of HOXC6 on the clonogenicity and proliferation of GBM cells. (a) The CCK-8 assay showed that HOXC6 inhibited the proliferation of glioma in U87 and U251 cell lines. (b, c) In the plate cloning experiment, the proliferation of U87 and U251 glioma cells in the siNC group was significantly higher compared to the siRNA1 and siRNA2 groups. ∗p < 0.05,  ∗∗p < 0.01, and∗∗∗p < 0.001. Data are shown as means ± SD.
Figure 4
Figure 4
Effects of HOXC6 on the invasion and migration of GBM cells. (a, b) Images and bar graphs showing the migrative activity measured by the wound healing assay in control or HOXC6-deficient U87 and U251 cells. (c, d) The migrative activity of control or HOXC6-deficient U87 and U251 cells was also examined using the Transwell assay. (e, f) The invasive activity of control or HOXC6-deficient U87 and U251 cells was determined by the Transwell invasion experiment. ∗∗∗p < 0.01. Data are shown as means ± SD.
Figure 5
Figure 5
Association of HOXC6 with the EMT pathway. (a) The GSEA showed that the high expression of HOXC6 was correlated with enhanced expression of EMT signaling pathway components in the TCGA database. (b, c) Representative Western blot results showing the protein levels of vimentin, N-cadherin, E-cadherin, and HOXC6 in U87 and U251 cell lines and HOXC6-deficient GBM cells. ∗p < 0.05,  ∗∗p < 0.01, and∗∗∗p < 0.001. Data are shown as means ± SD.
Figure 6
Figure 6
Activation of the EMT by HOXC6 in GBM. Expressions of TGF-β1, TGF-β2, p-Smad2, Smad4, and Smad2 in U87 and U251 cell lines and HOXC6 knockdown GBM cell models. ∗p < 0.05,  ∗∗p < 0.01, and∗∗∗p < 0.001. Data are shown as means ± SD.
Figure 7
Figure 7
Function of HOXC6 in vivo. (a, b) Glioblastoma cells infected with HOXC6-deficient U87 siRNA1 or U87 control siNC. Representative pictures of subcutaneously implanted tumors and tumor volume quantification (b). ∗p < 0.05,  ∗∗p < 0.01, and∗∗∗p < 0.001. Data are shown as means ± SD.
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