Overexpression of HMGA2 promotes tongue cancer metastasis through EMT pathway

Abstract

Background: Metastasis to long distance organs is the main reason leading to morality of tongue squamous cell carcinoma (TSCC); however, the molecular mechanisms are still unknown. High mobility group AT-hook 2 (HMGA2) is highly expressed in multiple metastatic carcinomas, in which it contributes to cancer progression, metastasis and poor prognosis by upregulating Snail expression and inducing epithelial mesenchymal transition (EMT). This study focuses on investigating the role and mechanism of regulation of HMGA2 in the metastasis of TSCC.

Methods: HMGA2 mRNA and protein expression were examined in TSCC specimens by quantitative real-time polymerase chain reaction, western blotting and immunohistochemistry (IHC). Western blotting, IHC and immunofluorescence were also used to measure the expression and localization of EMT marker E-Cadherin and Vimentin both in TSCC cells and tissues. Knockdown assay was performed in vitro in TSCC cell lines using small interfering RNAs and the functional assay was carried out to determine the role of HMGA2 in TSCC cell migration and invasion.

Results: TSCC mRNA and protein expression were significantly up-regulated in tumor tissues when compared to adjacent non-tumor tissues, and the overexpression of HMGA2 was closely correlated with lymph nodes metastasis. Clinicopathological analysis indicated that HMGA2 expression was associated with clinical stage (P = 0.001), lymph node metastasis (P = 0.000), histological differentiation (P = 0.002) and survival (P = 0.000). Silencing the HMGA2 expression in Cal27 and UM1 resulted in the inhibition of cell migration and invasion, meanwhile down-regulation of HMGA2 impaired the phenotype of EMT in TSCC cell lines and tissues. The Multivariate survival analysis indicates that HMGA2 can be an independent prognosis biomarker in TSCC.

Conclusion: Our findings demonstrate that HMGA2 promotes TSCC invasion and metastasis; additionally, HMGA2 is an independent prognostic factor which implied that HMGA2 can be a biomarker both for prognosis and therapeutic target of TSCC.

Fig. 1

Overexpression of HMGA2 in human tongue cancer cell lines. a Western blotting analysis of HMGA2 protein in tongue squamous cell carcinoma (TSCC) cell lines. GAPDH was probed as loading control. b Real-time PCR analysis of HMGA2 mRNA expression level in tongue cancer cell lines

Fig. 2

HMGA2 is upregulated in histopathological sections of tongue cancer and high expression of HMGA2 is correlated with tumorigenesis and metastasis. a Western blotting analysis of HMGA2 protein in eight human primary tongue cancer (T) and paired adjacent non-tumor tongue tissues (N), with each pair taken from a same patient. b Quantitative real time RT-PCR analysis of HMGA2 mRNA from the same eight pairs of tongue cancer and adjacent non-tumor tongue tissues. Error bars represent SDs calculated from three parallel experiments. c HMGA2 expression levels were up-regulated in primary tongue tumor tissues (T) compared to the paired adjacent non-tumor tongue tissue (N) from the same patient as examined by immunohistochemistry. d Expression levels of HMGA2 in 20 paired TSCC and adjacent non-tumor tongue tissues. Alteration of expression is shown as box plot presentations and the mean level of HMGA2 expression in TSCCs were significantly higher than that in non-tumor tissues. (P < 0.001, independent t test). e Expression levels of HMGA2 between 60 TSCCs with and without metastasis. The mean level of HMGA2 expression in TSCCs with metastasis were significantly higher than that in TSCCs without metastasis (P < 0.001, independent t test)

Fig. 3

Kaplan–Meier analysis of TSCC patients with positive HMGA2 expression versus those that negative HMGA2 expression. a Overall survival rate for cases with positive HMGA2 expression versus that of cases with negative HMGA2 expression; b HMGA2 expression status for overall survival rate stratified by lymph node metastasis condition; c overall survival rate for cases categorized with clinical stage classification and HMGA2 expression status; d HMGA2 expression level for overall survival rate stratified by tumor stage classification

Fig. 4

Down-regulation of HMGA2 inhibited TSCC cell motility and invasion. a The knockdown efficiency of two specific siRNA against HMGA2 was examined by Western blotting in Cal27 and UM1 cells. b The migration and c invasiveness abilities were analyzed in an epithelial type tongue cancer cell Cal27 by Boyden chamber assay (scale bar: 200 μm, * P < 0.05, ** P < 0.01). d The migration and e invasiveness abilities were analyzed in a mesenchymal like tongue cancer cell UM1 by Boyden chamber assay (scale bar: 200 μm, ** P < 0.01)

Fig. 5

Knockdown of HMGA2 can reversed epithelial–mesenchymal transition (EMT). a The expression of EMT markers of E-Cadherin, Vimentin, N-Cadherin and Snail were analyzed by western blotting both in Cal27 and UM1 tongue cancer cells. GAPDH was probed as the loading control. b Immunofluorescence staining analysis EMT markers of E-Cadherin and Vimentin (red) using confocal and the nuclei were stained with DAPI (blue) (scale bar: 5 μm)

Fig. 6

Overexpression of HMGA2 promote TSCC epithelial–mesenchymal transition (EMT) by upregulating Snail. a Immunohistochemitry analysis detecting the expression of HMGA2 and EMT markers (E-Cadherin, Vimentin and Snail) in abnormal tongue tissues and primary tongue tumor tissues with different histological differentiation. b Immunohistochemical staining showing the high expression and co-localization of HMGA2 and Snail in metastatic lymph node. c The correlation analysis showing the positive correlation between HMGA2 and Snail in TSCC patients