Cellular senescence in hepatocellular carcinoma induced by a long non-coding RNA-encoded peptide PINT87aa by blocking FOXM1-mediated PHB2

Abstract

Rationale: Recently, long non-coding RNAs (lncRNAs), known to be involved in human cancer progression, have been shown to encode peptides with biological functions, but the role of lncRNA-encoded peptides in cellular senescence is largely unexplored. We previously reported the tumor-suppressive role of PINT87aa, a peptide encoded by the long intergenic non-protein coding RNA, p53 induced transcript (LINC-PINT). Here, we investigated PINT87aa's role in hepatocellular carcinoma (HCC) cellular senescence. Methods: We examined PINT87aa and truncated PINT87aa functions in vitro by monitoring cell proliferation and performed flow cytometry, senescence-associated β-galactosidase staining, JC-1 staining indicative of mitochondrial membrane potential, the ratio of the overlapping area of light chain 3 beta (LC3B) and mitochondrial probes and the ratio of lysosomal associated membrane protein 1 (LAMP1) overlapping with cytochrome c oxidase subunit 4I1 (COXIV) denoting mitophagy. PINT87aa and truncated PINT87aa functions in vivo were verified by subcutaneously transplanted tumors in nude mice. The possible binding between PINT87aa and forkhead box M1 (FOXM1) was predicted through structural analysis and verified by co-immunoprecipitation and immunofluorescence co-localization. Rescue experiments were performed in vivo and in vitro following FOXM1 overexpression. Further, chromatin immunoprecipitation, polymerase chain reaction, and dual-luciferase reporter gene assay were conducted to validate FOXM1 binding to the prohibitin 2 (PHB2) promoter. Results: PINT87aa was significantly increased in the hydrogen peroxide-induced HCC cell senescence model. Overexpression of PINT87aa induced growth inhibition, cellular senescence, and decreased mitophagy in vitro and in vivo. In contrast, FOXM1 gain-of-function could partially reduce the proportion of senescent HCC cells and enhance mitophagy. PINT87aa overexpression did not affect the expression of FOXM1 itself but reduced that of its target genes involved in cell cycle and proliferation, especially PHB2, which was involved in mitophagy and transcribed by FOXM1. Structural analysis indicated that PINT87aa could bind to the DNA-binding domain of FOXM1, which was confirmed by co-immunoprecipitation and immunofluorescence co-localization. Furthermore, we demonstrated that the 2 to 39 amino acid truncated form of the peptide exerted effects similarly to the full form. Conclusion: Our study established the role of PINT87aa as a novel biomarker and a key regulator of cellular senescence in HCC and identified PINT87aa as a potential therapeutic target for HCC.

Keywords: FOXM1; PINT87aa; cellular senescence; hepatocellular carcinoma; mitophagy..

Figure 1

PINT87aa was overexpressed in H₂O₂-induced senescent HCC cells. (A) Schematic diagram of NR_109851, transcription of LINC-PINT generating linear PINT (linePNT) and circular PINT (circPINT) that could encode the PINT87aa peptide. (B) qRT-PCR detected circPINT and linePINT expression in senescent and proliferating SMMC-7721 and MHCC-97H cells. (C) FISH detected circPINT expression in senescent and proliferating SMMC-7721 and MHCC-97H cells. Scale bars = 15 μm. (D) Western blotting detected PINT87aa expression in senescent and proliferating SMMC-7721 and MHCC-97H cells. *P < 0.05; **P < 0.01; ***P < 0.001; ns P > 0.05.

Figure 2

Overexpression of PINT87aa induced senescence in HCC cells. (A) Expression of PINT87aa determined in SMMC-7721 and MHCC-97H cells after PINT87aa transfection. (B and C) Cell viabilities of HCC cells with different PINT87aa expression levels detected by colony formation (B) and (C) EdU assay (Magnification is ×100). (D) Cell cycle distribution of HCC cells transfected with PINT87aa overexpression vector and empty vector. (E) SA-β-gal staining in HCC cells with different PINT87aa expression levels (Magnification is ×100). ***P < 0.001; ns P > 0.05.

Figure 3

Overexpression of PINT87aa induced senescence in HCC tissues. (A and B) Expression of circPINT (n=8) (A) and PINT87aa (n=8) (B) in human HCC (T) and adjacent non-tumor (NT) tissues. (C) SA-β-gal staining in human HCC tissues with different PINT87aa expression levels (Magnification is ×200). (D) Effects of overexpressed PINT87aa on tumor growth by tumorigenicity assay. (E) IHC staining of PINT87aa and proliferative biomarker Ki67 in implanted tumors with different PINT87aa expression levels. The scale bar represents 100μm (top) and 50 μm (bottom). (F) SA-β-gal staining in implanted tumor tissues with different PINT87aa expression levels (Magnification is ×200). **P < 0.01; ***P < 0.001.

Figure 4

FOXM1 could reverse the pro-senescence effect of PINT87aa in vitro. (A) Expression of FOXM1 in SMMC-7721 and MHCC-97H cells with stably overexpressed PINT87aa. (B-E) Effects of FOXM1 on cell viabilities (Magnification is ×100) (B and C), cell cycle (D), and cellular senescence (E) (Magnification is ×100) were detected in SMMC-7721 and MHCC-97H cells with stably expressed PINT87aa after FOXM1 transfection. ***P < 0.001; ns P > 0.05.

Figure 5

PINT87aa directly bound to FOXM1. (A) Schematic diagram of PINT87aa binding to the DNA-binding domain of FOXM1. (B) Co-immunoprecipitation with the anti-GFP antibody in FOXM1 and PINT87aa-GFP co-transfected HEK293 cells and anti-FOXM1 antibody in FOXM1 and PINT87aa-GFP co-transfected HEK293 cells. (C) Immunofluorescent localization of FOXM1 and PINT87aa-GFP in SMMC-7721 and MHCC-97H cells. Scale bars = 20 μm. ***P < 0.001.

Figure 6

The effects of PINT87aa and FOXM1 on mitophagy. (A) Expression of PHB2 in HCC cell lines after transfection with PINT87aa, FOXM1, PINT87aa+FOXM1 or empty vector. (B-D) The change of mitophagy in HCC cell lines after transfection with PINT87aa, FOXM1, PINT87aa+FOXM1 or empty vector: (B) the MMP (Magnification is ×100), (C) the proportion of GFP-LC3B spots overlapping with the mitochondria by MitoTracker Red (Scale bars = 20 μm), (D) and the proportion of LAMP1 indicating lysosome spots overlapping with the mitochondria by COXIV (Scale bars = 5 μm). ***P < 0.001.

Figure 7

FOXM1 promoted the transcription of PHB2. (A) Dual-luciferase reporter gene assay and (B) CHIP-PCR verified the transcription of PHB2 by FOXM1. ***P < 0.001; ns P > 0.05.

Figure 8

Restoration effect of FOXM1 on PINT87aa-overexpressing HCC in vivo. (A) Effects of overexpressed FOXM1 on tumor growth by tumorigenicity assay. (B) SA-β-gal staining in implanted tumor tissues with different FOXM1 expression levels (Magnification is ×200). (C) IHC staining of FOXM1, PHB2, and Ki67 in implanted tumors with different FOXM1 expression levels. The scale bar represents 100μm (top) and 50 μm (bottom). ***P < 0.001.

Figure 9

Fragment 1 containing amino acids 2 to 39 inhibited HCC cell proliferation and induced cellular senescence in vitro. (A) PINT87aa was split into two segments: fragment 1 (2-39 aa) and fragment 2 (45-87 aa). (B-D) Effects of fragment 1 and fragment 2 on cell viability via (B) EdU assay (Magnification is ×100), (C) cell cycle, and (D) cellular senescence (Magnification is ×100) **P < 0.01; ***P < 0.001; ns P > 0.05.

Figure 10

Fragment 1 containing amino acids 2 to 39 decreased mitophagy in vitro. (A) The MMP (Magnification×100), (B) proportion of GFP-LC3B spots overlapping with the mitochondria by MitoTracker Red (Scale bars = 20 μm), (C) proportion of LAMP1 indicating lysosome spots overlapping with the mitochondria indicated by COXIV (Scale bars = 5 μm), and (D) expression of mitophagy-related proteins: P62, LC3B-II, PINK1, and Parkin detected in SMMC-7721 and MHCC-97H cells with different fragment 1 and fragment 2 levels. **P < 0.01; ***P < 0.001; ns P > 0.05.

Figure 11

Fragment 1 with amino acids 2 to 39 was the functional executor of PINT87aa in vivo. (A) Effects of overexpressed fragment 1 and fragment 2 on tumor growth by tumorigenicity assay. (B) IHC staining of PHB2 and Ki67 in implanted tumors with different fragment 1 and fragment 2 levels. The scale bar represents 100μm (top) and 50 μm (bottom). (C) SA-β-gal staining in implanted tumor tissues with different fragment 1 and fragment 2 levels (Magnification, ×200). ***P < 0.001.