XPA serves as an autophagy and apoptosis inducer by suppressing hepatocellular carcinoma in a PI3K/Akt/mTOR dependent manner

Abstract

Background: To explore the potential biological function of XPA (Xeroderma pigmentosum group A) in hepatic neoplasms and the underlying molecular mechanisms.

Methods: Liver cells were used as experimental models to establish HCC (hepatocellular carcinoma) in vitro. Protein extractions were subjected to Western blotting to detect the proteins expression. The lentivirus transfection efficiency was confirmed by Western blot and RT-qPCR, Tunnel staining was used to detect apoptosis, and Transwell assays were used to observe cell migration and invasion. Cell proliferation was detected with colony formation and CCK-8 (cell counting kit-8) assays.

Results: XPA expression was obviously lower in HCC tissue and liver cancer cell lines. XPA overexpression induced autophagy and apoptosis by increasing LC3B II/I, Beclin1, cleaved-caspase-3, and Bax expression and decreasing p62 and Bcl2 protein levels. XPA also suppressed HCC EMT (Epithelial-Mesenchymal Transition) by increasing E-cadherin and decreasing N-cadherin and vimentin protein expression. Cell proliferation, migration and invasion in vivo were significantly inhibited by the overexpression of XPA, and p-PI3K, p-Akt, and p-mTOR expression were decreased in LV-XPA cells. In general, XPA inhibited HCC by inducing autophagy and apoptosis and by modulating the expression of PI3K/Akt/mTOR proteins.

Conclusions: XPA overexpression was found to suppress HCC by inducing autophagy and apoptosis and repressing EMT and proliferation. Each of these effects may be involved in modulating the PI3K/Akt/mTOR signaling pathway.

Keywords: EMT; PI3K/Akt/mTOR; XPA; apoptosis; autophagy.

Figure 1

XPA is poorly expressed in HCC tissues and cells in vivo and in vitro. (A) Western blot analysis of XPA in human HCC tissues and ANLTs; (B,C) Western blot analysis of XPA in human normal and HCC cells and semiquantitative analysis of the data for each cell; (D) IHC detects XPA protein expression in human HCC tissues and ANLTs. The red arrow indicates XPA expression in hepatocytes. ***, P<0.001 vs. HL7702 cells, n=3/group. HCC, hepatocellular carcinoma; XPA, xeroderma pigmentosum group A; ANLTs, adjacent non tumor liver tissues.

Figure 2

XPA inhibits HCC cell proliferation and colony formation. (A) The mRNA expression of XPA was detected by RT-qPCR analysis; (B,C) the protein expression of XPA was detected by Western blot analysis and semiquantitative analysis of the data for each group; (D) confocal laser microscopy reveals the lentivirus transfection efficiency in Hep3B and Huh7 cells; (E,F) colony formation assay showing Hep3B and Huh7 cell growth were stained by crystal violet staining solution (magnification: 100×); (G,H) cell proliferation was measured by performing a CCK-8 assay. *, P<0.05; **, P<0.01; ***, P<0.001 vs. LV-NC group, n=3/group. HCC, hepatocellular carcinoma; XPA, xeroderma pigmentosum group A; RT-qPCR, real time quantitative PCR.

Figure 3

Autophagy activation was induced by upregulating XPA in Hep3B and Huh7 cells. (A,B) LC3B, Beclin1, and p62 protein expression was detected by Western blot analysis and semiquantitative analysis of data for each group in Hep3B and Huh7 cells. *, P<0.05; **, P<0.01; ***, P<0.001 vs. LV-NC group, n=3/group. (C,D) Confocal laser scanning microscopy of Hep3B and Huh7 cells demonstrated the puncta number of GFP/RFP-LC3; colocalization of GFP and mRFP was visualized as yellow puncta (autophagosomes) in the LV-NC and LV-XPA groups. The red puncta represent autolysosomes, and the GFP puncta represent degradation depending on the acidity (scale bars: 10 µm), **, P<0.01; ***, P<0.001 vs. LV-NC group, n=3/group. (E,F) LC3B and Beclin1 protein expression was detected by Western blot analysis and semiquantitative analysis of data for each group after treatment with rapamycin (200 nm) and 3-MA (2 µM). *, P<0.05 vs. LV-NC group; ^#, P<0.05 vs. LV-NC group; ^$, P<0.05 vs. LV-NC group; *^$, P<0.05 vs. LV-XPA group; and ^#$, P<0.05 vs. LV-XPA group, n=3/group.

Figure 4

XPA overexpression triggers Hep3B and Huh7 cell apoptosis. (A,B) Cleaved-Caspase3, Bax, and Bcl2 protein expression was detected by Western blot analysis and semiquantitative analysis of the data for each group; (C,D) TUNEL staining revealed apoptotic puncta in Hep3B and Huh7 HCC cells and quantitative puncta number analysis (400×). **, P<0.01; ***, P<0.001 vs. LV-NC group, n=3/group. HCC, hepatocellular carcinoma.

Figure 5

XPA overexpression inhibits migration, invasion, and EMT. (A,B) E-cadherin, N-cadherin, and vimentin protein expression was detected by Western blot and semiquantitative analysis of data for each group. (C,D) Representative images of the Transwell assay demonstrating Hep3B and Huh7 cell migration and invasion and the quantitative cell number of each group were stained by crystal violet solution. (E,F) Cell scratch test to evaluate the migration ability of Hep3B and Huh7 (magnification: 200×). *, P<0.05; **, P<0.01; and ***, P<0.001 vs. LV-NC group, n=3/group. XPA, xeroderma pigmentosum group A; EMT, epithelial-mesenchymal transition.

Figure 6

XPA induces autophagy and suppresses HCC by modulating the PI3K/AKT/mTOR signaling complex. (A-D) p-Akt and Akt protein expression was detected by Western blot and semiquantitative analysis of data for each group. *, P<0.05; **, P<0.01; and ***, P<0.001 vs. LV-NC group, n=3/group. (E-H) LC3B, Beclin1, p-PI3K, PI3K, p-Akt, Akt, p-mTOR, and mTOR protein expression was detected by Western blot and semiquantitative analysis of data for each group in Hep3B and Huh7. HCC, hepatocellular carcinoma; XPA, xeroderma pigmentosum group A. NS: no significance.