Huang DH et al. (2019), TPX2 silencing exerts anti‑tumor effects on hep...

XB-ART-56380

Int J Mol Med 2019 Dec 01;446:2113-2122. doi: 10.3892/ijmm.2019.4371.

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TPX2 silencing exerts anti‑tumor effects on hepatocellular carcinoma by regulating the PI3K/AKT signaling pathway.

Huang DH , Jian J , Li S , Zhang Y , Liu LZ .

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Hepatocellular carcinoma (HCC) is one of the primary causes of cancer‑associated deaths worldwide. Current treatment methods include surgical resection, chemotherapy and radiotherapy; however the curative rate remains low, thus novel treatments are required. The aim of the present study was to investigate the role of targeting protein for Xenopus kinesin‑like protein 2 (TPX2) in the growth of HCC and its underlying molecular mechanism. Immunohistochemistry staining, reverse transcription‑quantitative (RT‑q)PCR and western blotting were used to detect the expression of TPX2 mRNA and protein in liver cancer tissue samples, adjacent normal liver tissue samples, and the HCC cell lines Huh7, Hep3B, PLC/PRF/5 and MHCC97‑H. The recombinant plasmid pMagic4.1‑shRNA‑TPX2 was constructed and transfected into Huh7 and Hep3B HCC cells to silence TPX2 expression. The proliferation, apoptosis, migration and invasion of Huh7 cells and Hep3B cells were evaluated before and after TPX2 silencing. The mRNA and protein expression levels of multiple signaling pathway‑associated genes were detected by RT‑qPCR and western blotting. The expression levels of TPX2 mRNA and protein were significantly higher in HCC tissue samples compared with adjacent normal liver tissue sample. TPX2 mRNA and protein expression levels were detected in the different HCC cell lines. The recombinant plasmid pMagic4.1‑shRNA‑TPX2 was successfully transfected into Huh7 and Hep3B cells, resulting in TPX2 silencing. TPX2 knockdown significantly reduced cell proliferation, cell migration and cell invasion of Huh7 and Hep3B cells, whilst also increasing the rate of apoptosis in these cells. Following TPX2 silencing, the expression levels of PI3K, phospho‑AKT, Bcl‑2, c‑Myc and Cyclin D1 were significantly decreased, whereas the expression levels of P21 and P27 were significantly increased. In conclusion, TPX2 may suppress the growth of HCC by regulating the PI3K/AKT signaling pathway and thus, TPX2 may be a potential target for the treatment of liver cancer.

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Species referenced: Xenopus
Genes referenced: bcl2l1 cdkn1a ctrl grap2 kit mapk1 myc pik3cg stat3.2 tpx2 znrd2

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	Figure 1. TPX2 expression levels in HCC tissues and liver cancer cell lines. (A) Representative immunohistochemical stain of TPX2 expression in patients' tissues. (B) mRNA expression levels of TPX2 in HCC tissues and the adjacent normal liver tissue. P<0.01. (C) Representative western blot of TPX2 protein expression levels in HCC tissues and the adjacent normal liver tissue. (D) Densitometry analysis of TPX2 protein expression levels in (C). P<0.01. (E) mRNA and (F) protein expression levels in the liver cancer cell lines, Huh7, Hep3B, MHCC97-H, PLC/PRF/5. (G) Densitometric analysis of TPX2 protein expression levels in (F). Data are presented as the mean ± standard deviation. HCC, hepatocellular carcinoma; TPX2, targeting protein for Xenopus kinesin-like protein 2.
	Figure 2. TPX2 expression is downregulated in cells transfected with pMagic4.1-shRNA-TPX2. (A) Green fluorescence was observed following transfection. Magnification, x100. (B) mRNA and (C) protein expression levels of TPX2 in Huh7 and Hep3B transfected with the shRNA-TPX2 vector. (D) Densitometry analysis of TPX2 protein expression levels in (C). **P<0.01. Data are presented as the mean ± standard deviation. TPX2, targeting protein for Xenopus kinesin-like protein 2; sh, short hairpin; Ctrl, untransfected control; NC, negative control plasmid.
	Figure 3. Effects of TPX2 silencing on the proliferation and apoptosis of Huh7 and Hep3B cells. (A) A Cell Counting Kit-8 assay was used to detect the proliferation of Huh7 and Hep3B cells. P<0.05, P<0.01. (B) Representative dot plots of apoptosis in Huh7 and Hep3B cells. (C) Quantitative analysis of apoptosis in Huh7 and Hep3B cells transfected with an shRNA-TPX2 or shRNA-NC plasmid. *P<0.01. Data are presented as the mean ± standard deviation. TPX2, targeting protein for Xenopus kinesin-like protein 2; sh, short hairpin; Ctrl, untransfected control; NC, negative control plasmid.
	Figure 4. Effect of TPX2 silencing on the migration and invasion of Huh7 and Hep3B cells. (A and B) Effect of TPX2 knockdown on cell migration and (C and D) invasion of Huh7 and Hep3B cells was examined using a Transwell assay. Data are presented as the mean ± standard deviation. TPX2, targeting protein for Xenopus kinesin-like protein 2; sh, short hairpin; Ctrl, untransfected control; NC, negative control plasmid.
	Figure 5. Effect of TPX2 knockdown on multiple signaling pathways in Huh7 and Hep3B cells. mRNA expression levels of PI3K, AKT, P21, Bcl-2, c-Myc, Cyclin D1, P27 and BCL2L1 in (A) Huh7 and (B) Hep3B cells. P<0.05, P<0.01. Western blots of PI3K, p-AKT, AKT, P21 and Bcl-2 expression in (C) Huh7 and (D) Hep3B cells. Quantitative analysis of protein expression levels of PI3K, p-AKT, AKT, P21 and Bcl-2 in (E) Huh7 and (F) Hep3B cells. P<0.05, **P<0.01. (G) Western blots of p-Smad2/3, Smad2/3, p-p38 MAPK, p38 MAPK, p-STAT3 and STAT3 expression in Huh7 cells. β-actin was used as the loading control. (H) Quantitative analysis of p-Smad2/3, Smad2/3, p-p38 MAPK, p38 MAPK, p-STAT3 and STAT3 expression in Huh7 cells. Data are presented as the mean ± standard deviation. TPX2, targeting protein for Xenopus kinesin-like protein 2; sh, short hairpin; Ctrl, untransfected control; NC, negative control plasmid.

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	Figure 1. TPX2 expression levels in HCC tissues and liver cancer cell lines. (A) Representative immunohistochemical stain of TPX2 expression in patients' tissues. (B) mRNA expression levels of TPX2 in HCC tissues and the adjacent normal liver tissue. P<0.01. (C) Representative western blot of TPX2 protein expression levels in HCC tissues and the adjacent normal liver tissue. (D) Densitometry analysis of TPX2 protein expression levels in (C). P<0.01. (E) mRNA and (F) protein expression levels in the liver cancer cell lines, Huh7, Hep3B, MHCC97-H, PLC/PRF/5. (G) Densitometric analysis of TPX2 protein expression levels in (F). Data are presented as the mean ± standard deviation. HCC, hepatocellular carcinoma; TPX2, targeting protein for Xenopus kinesin-like protein 2.
	Figure 2. TPX2 expression is downregulated in cells transfected with pMagic4.1-shRNA-TPX2. (A) Green fluorescence was observed following transfection. Magnification, x100. (B) mRNA and (C) protein expression levels of TPX2 in Huh7 and Hep3B transfected with the shRNA-TPX2 vector. (D) Densitometry analysis of TPX2 protein expression levels in (C). **P<0.01. Data are presented as the mean ± standard deviation. TPX2, targeting protein for Xenopus kinesin-like protein 2; sh, short hairpin; Ctrl, untransfected control; NC, negative control plasmid.
	Figure 3. Effects of TPX2 silencing on the proliferation and apoptosis of Huh7 and Hep3B cells. (A) A Cell Counting Kit-8 assay was used to detect the proliferation of Huh7 and Hep3B cells. P<0.05, P<0.01. (B) Representative dot plots of apoptosis in Huh7 and Hep3B cells. (C) Quantitative analysis of apoptosis in Huh7 and Hep3B cells transfected with an shRNA-TPX2 or shRNA-NC plasmid. *P<0.01. Data are presented as the mean ± standard deviation. TPX2, targeting protein for Xenopus kinesin-like protein 2; sh, short hairpin; Ctrl, untransfected control; NC, negative control plasmid.
	Figure 4. Effect of TPX2 silencing on the migration and invasion of Huh7 and Hep3B cells. (A and B) Effect of TPX2 knockdown on cell migration and (C and D) invasion of Huh7 and Hep3B cells was examined using a Transwell assay. Data are presented as the mean ± standard deviation. TPX2, targeting protein for Xenopus kinesin-like protein 2; sh, short hairpin; Ctrl, untransfected control; NC, negative control plasmid.
	Figure 5. Effect of TPX2 knockdown on multiple signaling pathways in Huh7 and Hep3B cells. mRNA expression levels of PI3K, AKT, P21, Bcl-2, c-Myc, Cyclin D1, P27 and BCL2L1 in (A) Huh7 and (B) Hep3B cells. P<0.05, P<0.01. Western blots of PI3K, p-AKT, AKT, P21 and Bcl-2 expression in (C) Huh7 and (D) Hep3B cells. Quantitative analysis of protein expression levels of PI3K, p-AKT, AKT, P21 and Bcl-2 in (E) Huh7 and (F) Hep3B cells. P<0.05, **P<0.01. (G) Western blots of p-Smad2/3, Smad2/3, p-p38 MAPK, p38 MAPK, p-STAT3 and STAT3 expression in Huh7 cells. β-actin was used as the loading control. (H) Quantitative analysis of p-Smad2/3, Smad2/3, p-p38 MAPK, p38 MAPK, p-STAT3 and STAT3 expression in Huh7 cells. Data are presented as the mean ± standard deviation. TPX2, targeting protein for Xenopus kinesin-like protein 2; sh, short hairpin; Ctrl, untransfected control; NC, negative control plasmid.