HMGA1 and HMGA2 expression and comparative analyses of HMGA2, Lin28 and let-7 miRNAs in oral squamous cell carcinoma

Abstract

Background: Humans and dogs are affected by squamous cell carcinomas of the oral cavity (OSCC) in a considerably high frequency. The high mobility group A2 (HMGA2) protein was found to be highly expressed in human OSCC and its expression was suggested to act as a useful predictive and prognostic tool in clinical management of oral carcinomas. Herein the expression of HMGA2 and its sister gene HMGA1 were analysed within human and canine OSCC samples. Additionally, the HMGA negatively regulating miRNAs of the let-7 family as well as the let-7 regulating gene Lin28 were also comparatively analysed. Deregulations of either one of these members could affect the progression of human and canine OSCC.

Methods: Expression levels of HMGA1, HMGA2, Lin28, let-7a and mir-98 were analysed via relative qPCR in primary human and canine OSCC, thereof derived cell lines and non-neoplastic samples. Additionally, comparative HMGA2 protein expression was analysed by immunohistochemistry.

Results: In both species, a significant up-regulation of the HMGA2 gene was found within the neoplastic samples while HMGA1 expression did not show significant deregulations. Comparative analyses showed down-regulation of mir-98 in human samples and up-regulation of let-7a and mir-98 in canine neoplastic samples. HMGA2 immunostainings showed higher intensities within the invasive front of the tumours than in the centre of the tumour in both species.

Conclusions: HMGA2 could potentially serve as tumour marker in both species while HMGA1 might play a minor role in OSCC progression. Comparative studies indicate an inverse correlation of HMGA2 and mir-98 expression in human samples whereas in dogs no such characteristic could be found.

Figure 1

Expression analyses of HMGA1 and HMGA2 in human OSCC. The study included 10 non neoplastic control samples (green columns) and 10 tumour samples (red columns). A: relative HMGA1/GUSB real time PCR. B: relative HMGA1/HPRT real time PCR. C: relative HMGA2/GUSB real time PCR. D: relative HMGA2/HPRT real time PCR. Statistical analysis was performed applying the Hypothesis Test using REST 2008 (version 2.0.7.). * indicates a statistical significant expression deregulation of the HMGA genes when compared to non neoplastic control group; p-value is displayed next to *.

Figure 2

Expression analyses of HMGA1 and HMGA2 in canine OSCC. The study included 2 non neoplastic control samples (green columns) and 7 tumour samples (red columns). A: relative HMGA1/GUSB real time PCR. B: relative HMGA1/HPRT real time PCR. C: relative HMGA2/GUSB real time PCR. D: relative HMGA2/HPRT real time PCR. Statistical analysis was performed applying the Hypothesis Test using REST 2008 (version 2.0.7.). * indicates a statistical significant expression deregulation of the HMGA genes when compared to non neoplastic control group; p-value is displayed next to *.

Figure 3

HMGA2 immunohistochemistry in human OSCC. Immunolabelling of a human tumour: overview (A), tumour centre (B) and invasive front (C). In the tumour centre (B) lower numbers of tumour cells with nuclear immunolabelling are present when compared to the respective invasive front (C). The invasive front shows numerous tumour cells exhibiting intense nuclear immunolabelling of HMGA2. Magnification: (A) 50x, (B) and (C) 200x.

Figure 4

HMGA2 immunohistochemistry in canine OSCC. Immunolabelling of a canine tumour grade II: overview (A), tumour centre (B) and invasive front (C). HMGA2 staining in the tumour centre (B) revealed approx. 25% tumour cells with nuclear immunolabelling while cells at the invasive front showed approx. 50% staining (C). Magnification: (A) 100x, (B) and (C) 200x.

Figure 5

Comparative expression analyses of the HMGA2 and Lin28 genes and the let-7a and mir-98 miRNAs in human OSCC. The study included 5 non neoplastic control samples (green columns), 6 tumour samples (red columns) and 2 patient derived cell lines (brown columns). A: relative HMGA2/HPRT real time PCR. B: relative Lin28/HPRT real time PCR. C: relative let-7a/RNU6B real time PCR. D: relative mir-98/RNU6B real time PCR. Statistical analysis of the relative real time PCR results (Hypothesis Test) was performed with REST 2008 software tool. A p-value of <0.05 was considered statistically significant. * indicates a statistical significant expression deregulation of HMGA2 and/or Lin28 and/or let-7a and/or mir-98 when compared to non neoplastic control group; p-value is displayed next to *.

Figure 6

Comparative expression analyses of the HMGA2 and Lin28 genes and the let-7a and mir-98 miRNAs in canine OSCC. The study included 2 non neoplastic control samples (green columns), 7 tumour samples (red columns) and 2 cell line derived samples (brown columns) which derived from patients 1–10. A: relative HMGA2/HPRT real time PCR. B: relative Lin28/HPRT real time PCR. C: relative let-7a/RNU6B real time PCR. D: relative mir-98/RNU6B real time PCR. Statistical analysis of the relative real time PCR results (Hypothesis Test) was performed with REST 2008 software tool. A p-value of <0.05 was considered statistically significant. * indicates a statistical significant expression deregulation of HMGA2 and/or Lin28 and/or let-7a and/or mir-98 when compared to non neoplastic control group; p-value is displayed next to *.