SERPINA3 is a key modulator of HNRNP-K transcriptional activity against oxidative stress in HCC

Abstract

Most studies about serpin peptidase inhibitor, clade A member 3 (SERPINA3) has been limited to its inhibitory functions and mechanisms. Herein, we report a novel role of SERPINA3 in transcriptional regulation of HCC progression-related genes. Among 19 selected genes through HCC cell isolation system based on telomere length, microarray analyses, and cell-based studies, SERPINA3 was the strongest determinant of increases in telomere length, HCC cell proliferation, survival, migration, and invasion. We also found that SERPINA3 strongly interacted with heterogeneous nuclear ribonucleoprotein K (HNRNP-K) under H₂O₂ exposure, and the oxidation-elicited SERPINA3-HNRNP-K complex enhanced the promoter activities and transcript levels of a telomere-relating gene (POT1) and HCC-promoting genes (UHRF1 and HIST2H2BE). Intriguingly, the inhibition of SERPINA3 oxidation rendered the transcriptional activity of the SERPINA3-HNRNP-K complex suppressed. Moreover, the co-immunoprecipitated HNRNP-K with SERPINA3 quantitatively correlated with not only the level of SERPINA3 oxidation but also the level of POT1, UHRF1, and HIST2H2BE transcripts and telomere length in HCC tissues. Therefore, the upregulated transcriptional activity of HNRNP-K mediated by SERPINA3 promotes HCC cell survival and proliferation and could be an indicator of poor prognosis for HCC patients.

Keywords: Human liver cancer; Reactive oxygen species; Transcription activity.

Graphical abstract

Fig. 1

SERPINA3 induces telomere elongation, cell migration and invasion, cell proliferation, and resistance to cell toxicity in HCC cells. (A) Strategy for sorting Huh7 HCC cells according to telomeric intensity. The telomere marker TPP1 and nuclei are evident as green and blue fluorescence, respectively. Scale bars, 10 μm. (B–C) Migration (B left) and invasion assay (B right) and tumor growth of mice (C) of Huh7-cells with short telomeres or long telomeres with or without the induction of telomere shortening by treatment with both 300 μM H₂O₂ and 15 μM of the telomerase inhibitor BIBR 1532. (D) Microarray analysis of gene expression of short telomere-exhibiting cell fractions and long telomere-exhibiting cell fractions in H₂O₂-treated Huh7 HCC cells. (E) Fluorescence images of SERPINA3 protein localization in mock- and H₂O₂-treated Huh7 cells (top) and percentage of co-localization of SERPINA3 with telomeres per cell (bottom). Scale bars, 5 μm. (F) Functional profiling of candidate genes by analyses of telomere length (top left), cell migration (top middle), invasion (top right), cell proliferation (bottom left), and cell survival in Huh7 cells treated with the cell toxicant (1 mM H₂O₂) (bottom right). HCC, hepatocellular carcinoma; H₂O_2, hydrogen peroxide; EGFP, enhanced green fluorescent protein; T/S, the ratio of telomere repeat copy number (T) to single copy number (S); E.V, empty vector; POC, percent of control; Mean ± s.e.m. (n = 3), except for Fig. 1C (Mean ± s.d., n = 6). ^∗p < 0.05, ^∗∗p < 0.01, and ^∗∗∗p < 0.001, Student's t-test. Sterile distilled water (pH 7.0) was used as the negative control (mock treatment). . (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 2

SERPINA3 interacts with HNRNP-K and H₂O₂ induces increased association between SERPINA3 and HNRNP-K. (A) Experimental overview of the immunoprecipitation and LC-MS/MS analyses for the identification of interacting proteins with SERPINA3 in Huh7 HCC cells. (B) Diagrams of the FLAG-tagged HNRNP-K structure and its deletion mutants and EGFP-tagged SERPINA3 structure (left) and GFP immunoprecipitation followed by immunoblotting analyses using antibodies against FLAG in Huh7 cells expressing full-length FLAG-tagged HNRNP-K or its deletion mutants and full-length EGFP-tagged SERPINA3 (right). (C) Immunofluorescence of endogenous SERPINA3 and HNRNP-K (left) and immunoprecipitation with anti-SERPINA3 antibody followed by immunoblot analyses with anti-HNRNP-K antibody (right) on Huh7-cells with mock or H₂O₂ treatment. Sterile distilled water (pH 7.0) was used as the negative control (mock treatment). EGFP, enhanced green fluorescent protein; FL, full-length; DL, deletion mutant; KH, HNRNP-K-homology; KI, HNRNP-K-interactive; SERPIN, serine protease inhibitors; RCL, Reactive Center Loop; AAs, amino acids; IP, immunoprecipitation; IB, immunoblot; IgG, immunoglobulin G. . (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 3

Lack of interaction between SERPINA3 and HNRNP-K reduces promoter activities and mRNA expression levels of POT1 PI3Kδ, UHRF1, and HIST2H2BE. (A) A view of the location of the oxidation modified residues M219 and M290 on SERPINA3 protein (left) and GFP immunoprecipitation followed by immunoblotting analyses using antibodies against FLAG in Huh7 cells expressing FLAG-tagged HNRNP-K and EGFP-tagged SERPINA3 WT, M219L, or M290L (right). (B) Transcription levels (top) and promoter activities (bottom) of POT1, PI3Kδ, UHRF1, and HIST2H2BE in HCC cells, following overexpression of EGFP E.V, SERPINA3 WT, SERPINA3 M219L, or SERPINA3 M290L plasmid. (C) Transcription levels of POT1, PI3Kδ, UHRF1, and HIST2H2BE in HCC cells stably expressing the SERPINA3 M290L plasmid, which is not degraded by SERPINA3 siRNA, followed by administration of siControl (siCon), siSERPINA3, and siHNRNP-K. EGFP, enhanced green fluorescent protein; IP, immunoprecipitation; IgG, immunoglobulin G; WT, wild-type; E.V, empty vector; siCon, non-targeting siRNA; siRNA, short-interfering RNA; Mean ± s.e.m. (n = 3). ^∗∗p < 0.01, Student's t-test. NS denotes non-significance. . (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 4

Oxidatively-modified SERPINA3 promotes transcriptional activity of HNRNP-K through the increasing interaction between SERPINA3 and HNRNP-K (A) Enrichment of EGFP-tagged SERPINA3 at the POT1 (left), UHRF1 (middle), andHIST2H2BE(right) gene regulatory DNAs in Huh7 cells expressing stably EGFP-tagged SERPINA3 WT or SERPINA3 M290L. (B) Enrichment of SERPINA3 using anti-SERPINA3 antibody after the first ChIP assay using anti-HNRNP-K antibody at the POT1 (left), UHRF1 (middle), andHIST2H2BE(right) gene regulatory DNAs in Huh7 cells with and without H₂O₂ treatment. Sterile distilled water (pH 7.0) was used as the negative control (mock treatment). (C) Enrichment of HNRNP-K at the POT1 (left), UHRF1 (middle), andHIST2H2BE(right) gene regulatory DNA in Huh7 cells stably expressing the SERPINA3 M290L plasmid not degraded by siSERPINA3 followed by administration of siControl (siCon), siSERPINA3, and siHNRNP-K. Gel images show the amount of HNRNP-K in the indicated gene regulatory DNA. (D) Telomere length, invasion activity, caspase-3/7 activity, and cell viability of Huh7 cells stably expressing the SERPINA3 M290L plasmid not degraded by siSERPINA3 followed by administration of siControl (siCon), siSERPINA3, and siHNRNP-K. siCon, non-targeting siRNA; siRNA, short-interfering RNA; WT, wild-type; ChIP, chromatin-immunoprecipitation; IgG, immunoglobulin G; H₂O_2, hydrogen peroxide; NAC, N-acetylcysteine; Mean ± s.e.m. (n = 3). ^∗p < 0.05, ^∗∗p < 0.01, and ^∗∗∗p < 0.001, Student's t-test. NS denotes non-significance.

Fig. 5

The blockade of SERPINA3 binding to HNRNP-K inhibits murine tumor growth in vivo (A) Subcutaneous tumor volumes from mice that were subcutaneously injected with Huh7 cells stably expressing the SERPINA3 oxidation-defect plasmid not degraded by siSERPINA3 followed by intra-tumoral injection of siSERPINA3 and siHNRNP-K (n = 5). Student's t-test; Mean ± s.e.m. (B) Liver tumor growth inhibition in mice after intrahepatic injection of Huh7 cells stably expressing the SERPINA3 oxidation-defect plasmid not degraded by siSERPINA3 followed by tail injection of siSERPINA3 and siHNRNP-K (n = 5). Photographs of liver tumors (top) and median number of tumor nodules (>4 mm), mRNA levels of UHRF1, HIST2H2BE, and POT1, and telomere fluorescence intensity (bottom). Horizontal bars indicate median values. ^∗∗p < 0.01, Mann–Whitney U test. (C) Molecular model illustrating action of oxidized-SERPINA3 complexed with HNRNP-K protein as a transcriptional activator for HCC cell survival and proliferation. siCon, non-targeting siRNA; siRNA, short-interfering RNA; ROS,reactive oxygen species; KI, HNRNP-K-interactive; HCC, hepatocellular carcinoma.

Fig. 6

High-amount of coimmunoprecipitated HNRNP-K with SERPINA3 is associated with high levels of UHRF1, HIST2H2BE, and POT1 expressions as well as with high HCC recurrence in human HCC tissues. (A) Representative immunohistochemical staining of SERPINA3, HNRNP-K, UHRF1, HIST2H2BE, and POT1 in GIII HCC tumor samples and non-tumor samples. (B, C) Immunoprecipitation (IP) using anti-SERPINA3 antibodies followed by immunoblotting analyses using antibodies against HNRNP-K in GII and GIII HCC tissue lysates (B), and correlation analysis between the oxidized-SERPINA3 and the amount of coimmunoprecipitated HNRNP-K with SERPINA3 determined by linear regression (C). n = 48 (D, E) Median mRNA levels of UHRF1, HIST2H2BE, and POT1, and median telomere length (D) and recurrence-free survival rates (E) in GII and GIII HCC tissues with low- and high-amount of coimmunoprecipitated HNRNP-K with SERPINA3 (n = 47). Horizontal bars indicate median values. IP, immunoprecipitation; IB, immunoblot; GII, Grade-II; GIII, Grade-III; R, Pearson correlation coefficient; HCC, hepatocellular carcinoma.