High mobility group box 3 promotes cervical cancer proliferation by regulating Wnt/β-catenin pathway

Abstract

Objective: High mobility group box 3 (HMGB3) plays an important role in the development of various cancer. This study aims to explore whether HMGB3 regulates cervical cancer (CC) progression and elucidate the underlying mechanism.

Methods: HMGB3 expression in clinical patients' tumor samples were determined by real-time quantitative polymerase chain reaction (qRT-PCR) and western blot. HMGB3 overexpression/knockdown were used to investigate its function. Cell apoptosis and cycle were detected by Annexin V/PI staining and flow cytometry. In vivo tumor model was made by subcutaneous injection of HeLa cells transfected with shRNAs targeting HMGB3 (sh-HMGB31) into the flank area of nude mice. Western blot was used to detect the levels of β-catenin, c-Myc, and matrix metalloproteinase-7 (MMP-7) in Hela and CaSki cells transfected with sh-HMGB3 or shRNAs targeting β-catenin.

Results: Both messenger RNA and protein levels of HMGB3 were upregulated in CC tissues from patients. High expression level of HMGB3 had positive correlation with serosal invasion, lymph metastasis, and tumor sizes in CC patient. Functional experiments showed that HMGB3 could promote CC cell proliferation both in vitro and in vivo. The expression levels of c-Myc and MMP-7 were increased, resulting in regulating cell apoptosis, cell cycle, and activating Wnt/β-catenin pathway.

Conclusions: Our data indicated that HMGB3 may serve as an oncoprotein. It could be used as a potential prognostic marker and represent a promising therapeutic strategy for CC treatment.

Keywords: Cell Proliferation; Cervical Cancer; HMGB3 Protein; beta Catenin; shRNA.

Fig. 1. HMGB3 is overexpressed in cervical cancer tissues. (A) HMGB3 mRNA expression was upregulated in cervical cancer tissues. HMGB3 expression was measured by qRT-PCR and normalized to adjacent tissues (n=58 for each group). (B) Representative western blots of HMGB3 protein expressions between cervical cancer tissues and adjacent tissues. Moreover, HMGB3 expression was significantly higher in patients with bigger tumor size (C) (n=26 for <4 cm and n=32 for ≥4 cm) and advanced FIGO stage (D) (n=24 for

FIGO, International Federation of Gynaecology and Obstetrics; HMGB3, high mobility group box 3; mRNA, messenger RNA; qRT-PCR, real-time quantitative polymerase chain reaction. ^*p<0.001 between the indicated groups.

Fig. 2. HMGB3 promotes cervical cancer growth. (A) qRT-PCR was used to analyze the expressions of HMGB3 among different cervical cancer cell lines (H8 was used as negative control). (B) Knockdown efficiency of HMGB3 in Hela and CaSki cells. (C) Overexpression efficiency of HMGB3 in SiHa and C33A cells. (D, E) CCK-8 assay was used to explore the cell viability of Hela and CaSki cells transfected with sh-HMGB3 or sh-NC. (F, G) CCK-8 assay was used to explore the cell viability of SiHa and C33A cells transfected with HMGB3 or vector (n=6–10 for each group). Data are presented as mean±standard deviation. One-way or 2-way analysis of variance analysis followed by post hoc test.

CCK-8, Cell Counting Kit-8; HMGB3, high mobility group box 3; mRNA, messenger RNA; OD, optical density; qRT-PCR, real-time quantitative polymerase chain reaction; sh-HMGB3, shRNAs targeting HMGB3; sh-NC, negative control shRNAs. ^*p<0.05, ^†p<0.01 and ^‡p<0.001 compared to their negative control.

Fig. 3. Effects of HMGB3 knockdown on cell apoptosis and cell cycle progression in Hela and CaSki cells. Flow cytometry was performed to determine the cell cycle (A and B) and apoptosis (C) of HeLa and CaSki cells transfected with sh-HMGB3 or sh-NC. (D) G1 arrest cell cycle markers were analyzed by western blot in HeLa and CaSki cells transfected with sh-HMGB3 or sh-NC and the relative expressions normalized to sh-NC have been quantified (E and F) (n=10 for each group). Data are presented as mean±standard deviation. Two-way analysis of variance analysis followed by post hoc test.

HMGB3, high mobility group box 3; sh-HMGB3, shRNAs targeting HMGB3; sh-NC, negative control shRNAs. ^*p<0.05, ^†p<0.01 and ^‡p<0.001 between the indicated groups.

Fig. 4. Effects of HMGB3 knockdown on HeLa cell growth in vivo. (A) Representative images of the xenograft tumors from HeLa cells transfected with sh-HMGB3 or sh-NC after 24 days and tumor growth curves determined every 3 days after injection. (B) The relative expression levels of HMGB3 in tumor tissues obtained from mice after 24 days of injection were detected by qRT-PCR. (C) The tumor weight was measured after 24 days of injection. (D, E) qRT-PCR was used to determine the expressions of Ki67 and PCNA in tumor obtained from mice after 24 days of injection (n=6–10 for each group). Data are presented as mean±standard deviation. t-tests followed by Mann-Whitney test.

HMGB3, high mobility group box 3; qRT-PCR, real-time quantitative polymerase chain reaction; sh-HMGB3, shRNAs targeting HMGB3; sh-NC, negative control shRNAs. ^*p<0.05, ^†p<0.01 and ^‡p<0.001 compared to their negative control.

Fig. 5. HMGB3 promotes cervical cancer proliferation by regulating Wnt/β-catenin pathway. (A) Western blot was performed to detect β-catenin, c-Myc, and MMP-7 expression after HMGB3 knockdown in HeLa and CaSki cells and relative expressions were normalized to sh-NC (B and C). (D) The expression of c-Myc, MMP-7 in SiHa and C33A cells were detected by western blot after increasing the expression of HMGB3 or increasing the expression of HMGB3 but inhibited β-catenin expression. GAPDH was used as loading control and relative expressions were normalized to vector control (E and F) (n=10 for each group). Data are presented as mean±standard deviation. Two-way analysis of variance analysis followed by post hoc test.

GAPDH, glyceraldehyde 3-phosphate dehydrogenase; HMGB3, high mobility group box 3; MMP-7, matrix metalloproteinase-7; sh-β-catenin, shRNAs targeting β-catenin; sh-HMGB3, shRNAs targeting HMGB3; sh-NC, negative control shRNAs. ^*p<0.05, ^†p<0.01 and ^‡p<0.001 between the indicated groups.