HMGB1 is negatively correlated with the development of endometrial carcinoma and prevents cancer cell invasion and metastasis by inhibiting the process of epithelial-to-mesenchymal transition

Figures & data

Figure 1 HMGB1 expression in human endometrial tissues was assessed by IHC.

Notes: (A, B) The proliferative stage of normal human endometrium. (A) Magnification 200×. (B) Magnification 400×. (C, D) The secretory phase of normal human endometrium. (C) Magnification 200×. (D) Magnification 400×. (E, F) The highly differentiated endometrial cancer. (E) Magnification 200×. (F) Magnification 400×. (G, H) The poorly differentiated endometrial cancer. (G) Magnification 200×. (H) Magnification ×400. (I) Patients with low HMGB1 expression (green line, n=195) had a much worse prognosis than those with high HMGB1 expression (blue line, n=45).
Abbreviations: HMGB1, high-mobility group box protein 1; IHC, immunohistochemistry.

Table 1 HMGB1 protein expressions in human endometrial tissues

Human endometrial tissues	n	HMGB1 low (−/+)	HMGB1 high (++/+++)	χ^2	P-value
		n (%)	n (%)
Normal	60	8 (13.3)	52 (86.7)	101.20	P<0.05
Carcinoma	240	195 (81.3)	45 (18.7)
Pathological type				4.59	P>0.05
Endometrioid	188	158 (84)	30 (16)
Serous	33	24 (72.7)	9 (27.3)
Clear cell	19	13 (68.4)	6 (31.6)
Cell differentiation				16.11	P<0.05
High and medium	133	96 (72.2)	37 (27.8)
Low	107	99 (92.5)	8 (7.5)
Tumor stage				16.24	P<0.05
Stage I	88	45 (51.1)	43 (48.9)
Stage II	75	61 (81.3)	14 (18.7)
Stages III and IV	77	71 (92.2)	6 (7.8)
Nodal status				7.53	P<0.05
Positive	85	77 (90.6)	8 (9.4)
Negative	155	118 (76.1)	37 (23.9)

Abbreviation: HMGB1; high-mobility group box protein 1.

Figure 2 Acquisition of endometrial glandular epithelial cells and mesenchymal cells.

Notes: (A) Normal endometrial glandular epithelial cells. (B) Positive cytokeratin staining in the cytoplasm of glandular epithelial cells was measured by ICC. (C) Normal endometrial mesenchymal cells. (D) Positive vimentin staining in the cytoplasm of mesenchymal cells was measured by ICC (magnification 200×).
Abbreviation: ICC, immunocytochemistry.

Figure 3 Different proliferation, migration and invasion abilities of four types of human endometrial cancer cell lines.

Notes: (A) Ishikawa and HEC-1B cells had significantly lower proliferation abilities compared with KLE and HEC-1A cells. (B) Ishikawa and HEC-1B cells had significantly lower colony formation abilities compared with KLE and HEC-1A cells. (C) Ishikawa and HEC-1B cells were detected to have weaker migration abilities. (D) Ishikawa and HEC-1B cells were detected to have weaker invasion abilities. (E) The average migrating and invading cell counts of Ishikawa and HEC-1B cells were much lower than those of KLE and HEC-1A cells (magnification 200×). *P<0.05.
Abbreviation: HMGB1, high-mobility group box protein 1.

Figure 4 HMGB1 expressions in endometrial cancer cells and normal endometrial cells were assessed by ICC, Western blotting and RT-qPCR. HMGB1 expressions in endometrial cancer cell lines KLE, Ishikawa, HEC-1A, HEC-1B, and normal endometrial cell lines were measured by (A) ICC staining. (B) Western blotting. (C) RT-qPCR. The expression of HMGB1 was significantly strong in normal endometrial cells. Endometrial cancer cells such as KLE and HEC-1A, with the higher proliferation and invasion abilities, had lower expression of HMGB1, compared to Ishikawa and HEC-1B, which had the lower proliferation and invasion abilities (magnification 200×). *P<0.05.

Abbreviations: HMGB1; high-mobility group box protein 1; ICC, immunocytochemistry; RT-qPCR, reverse transcription-quantitative polymerase chain reaction.

Figure 5 Effects of HMGB1 knockdown on the proliferation, migration and invasion abilities of the lowly invasive endometrial cancer cells Ishikawa and HEC-1B. HMGB1 protein expression in lentivirus-infected cells was measured by (A) Western blotting. (B) RT-qPCR. (C) HMGB1 knockdown increased the proliferation abilities of the less invasive endometrial cancer cells. (D) HMGB1 knockdown increased the colony-forming capacities of the less invasive endometrial cancer cells. (E) HMGB1 knockdown promoted the migration abilities of the less invasive endometrial cancer cells. (F) HMGB1 knockdown promoted the invasion abilities of the less invasive endometrial cancer cells. (G) The average migrating and invading cell counts of HMGB1 shRNA-infected cells were much higher than those of non-infected and negative control cells (magnification 200×). *P<0.05.

Abbreviations: HMGB1, high-mobility group box protein 1; RT-qPCR, reverse transcription-quantitative polymerase chain reaction; shRNA, small hairpin RNA.

Figure 6 Effects of HMGB1 knockdown on tumor growth in vivo.

Notes: (A) Tumor growth of lentivirus-infected cells observed continuously for 8 weeks. HMGB1 shRNA-infected group had significantly higher tumor growth compared with negative control. (B) The tumor volumes of the negative control were much smaller than those of HMGB1 shRNA-infected cells. (C) The images of lung sections from mice receiving tail vein injection stained by H&E. The negative control group had no lung metastasis, while the lung metastasis rate of HMGB1 shRNA-infected cells was 100% (magnification 200×). *P<0.05.
Abbreviations: H&E, hematoxylin and eosin; HMGB1, high-mobility group box protein 1; shRNA, small hairpin RNA.

Figure 7 Effects of HMGB1 knockdown on EMT markers. After RNA interference, EMT markers, including E-cadherin, N-cadherin, vimentin, Snail and Twist, in the lentivirus-infected cells were measured by (A) Western blotting and (B) RT-qPCR. HMGB1 knockdown significantly increased the expression of N-cadherin, vimentin, Snail and Twist but decreased the expression of E-cadherin. (C) Compared to HMGB1 – high cell lines (Ishikawa, HEC-1B), HMGB1 – low cell lines (HEC-1A and KLE) showed lower expression of N-cadherin, vimentin, Snail and Twist and higher expression of E-cadherin. *P<0.05.

Abbreviations: EMT, epithelial-to-mesenchymal transition; HMGB1, high-mobility group box protein 1; RT-qPCR, reverse transcription-quantitative polymerase chain reaction; shRNA, small hairpin RNA.