Increased Nuclear Transporter Importin 7 Contributes to the Tumor Growth and Correlates With CD8 T Cell Infiltration in Cervical Cancer

Abstract

Background: Importin 7 (IPO7), a karyopherin-β protein, is involved in various tumorigenesis and progression abilities by mediating the nuclear import of oncoproteins. However, the exact biological functions of IPO7 remain to be further elucidated. Materials and Methods: TCGA and GEO datasets were used to identify dysregulated expression of IPO7 in various cancers. Gain-of-function and loss-of-function analyses were used to identify the oncogenic functions of IPO7 in vitro and in vivo. Moreover, LC-MS/MS and parallel reaction monitoring analysis were used to comparatively profiled IPO7-related proteomics and potential molecular machinery. Results: Our works demonstrated that the expression of IPO7 was upregulated and was correlated with a poor prognosis in cervical cancer. In vitro and in vivo experiments demonstrated that knockdown of IPO7 inhibited the proliferation of HeLa and C-4 I cells. LC-MS/MS analysis showed that IPO7-related cargo proteins mainly were enriched in gene transcription regulation. Then independent PRM analysis for the first time demonstrated that 32 novel IPO7 cargo proteins, such as GTF2I, RORC1, PSPC1, and RBM25. Moreover, IPO7 contributed to activating the PI3K/AKT-mTOR pathway by mediating the nuclear import of GTF2I in cervical cancer cells. Intriguingly, we found that the IPO7 expression was negatively correlated with CD8 T cell infiltration via regulating the expression of CD276 in cervical cancer. Conclusion: This study enhances our understanding of IPO7 nuclear-cytoplasmic translocation and might reveal novel potential therapeutic targets. The results of a negative correlation between the IPO7 and CD8 T cell infiltration indicate that the IPO7 might play an important impact on the immune microenvironment of cervical cancer.

Keywords: IPO7; cervical cancer; immune infiltration; mass spectrometry; proteome.

FIGURE 1

IPO7 is up-regulated and associated with a poorer prognosis in CC. (A) The expression of IPO7 in the TCGA dataset compared to corresponding normal tissues of the GTEx dataset. (B) The protein level of IPO7 in primary tumor tissues and normal tissues from CPTAC datasets. (C) Correlation between IPO7 expression and overall survival and disease-free survival of cancers in the TCGA dataset. (D) Analysis of IPO7 expression profiles in cervical cancer (CC), high-grade squamous intraepithelial lesion (HSIL), and normal cervix (NC) specimens from GSE6791 and GSE7803 datasets. (E) Kaplan-Meier analysis of the overall survival of patients with IPO7 high or low expression level. (F) Representative immunohistochemical images and quantification analysis showing IPO7 expression in CC, CIN, and CC specimens. LSIL is ligh-grade squamous intraepithelial lesion. Scale bar: 20 μm. The P-value was calculated by χ2 test or Fisher’s exact test. *p < 0.05.

FIGURE 2

IPO7 promotes the cell proliferation of CC in vitro. (A,B) The expression level in Hela and C-4 I after IPO7 knockdown or overexpression. (C,D) CCK-8 assay analyzes cell viability after IPO7 knockdown or overexpression. (E,F) Representative colony formation and quantification analysis. (G–J) Hela and C-4 I cells were infected with shIPO7 or shCtrl and injected into nude mice. Representative time course of xenograft growth and tumor weight (G,I). Representative typical images of PCNA and Ki67 and quantification analysis from shCtrl and shIPO7 groups (H,J). Error bars represent mean ± standard deviation (SD). Scar bar: 50 μm. Two-tailed t-test, *p < 0.05.

FIGURE 3

Comparative profiling of the proteomes after IPO7 knockdown. (A) Results showing protein expression variations in the nucleus and cytoplasm after IPO7 knockdown, compared with shCtrl groups, n = 3. (B,C) Differentially expressed proteins of the cytoplasm (B) and nucleus (C) after IPO7 knockdown were analyzed with Clusters of Orthologous Groups of proteins (COG/KOG) database. (D) The significantly enriched biological process, cellular component, molecular function, and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway terms were shown.

FIGURE 4

PRM analyses of the differentially expressed proteins. Parallel reaction monitoring (PRM) analysis of the differentially expressed proteins after IPO7 knockdown relative to the shCtrl groups. ACTL6A, BAZ1A, BRIX1, PSPC1, DDB2, DIDO1, DNMT1, MYBBP1A, GTF3C4, GTF2I, TRIP12, H4C1, GTF3C5, NAT10, PABPN1, RRP12, RBM25, POLR1A, PRPF40A, CHD4, POLR1E, SEH1L, RCOR1, HEATR1, NCBP1, POLR2B, PES1, PCID2, CKAP5, DRG1, EDC4, GNAI2, and NMD3 were detected. Two-tailed Student’s t-test, *p < 0.05.

FIGURE 5

IPO7 regulates the activation of PI3K/AKT-mTOR pathway. (A,B) Gene set enrichment analysis (GSEA) shows the enriched gene expression signature of IPO7 and GTF2I in the TCGA datasets. (C,D) Immunoblots analysis shows the GTF2I levels in the nucleoplasm and cytoplasm after transfecting with shIPO7 or Importazole. (E) The correlation of GTF2I with PIK3CA and PIK3C2A. (F) The correlation of IPO7 with PIK3CA and PIK3C2A. (G) Immunoblot analysis of phosphor-mTOR (p-mTOR), mTOR, phosphor-AKT(p-AKT), AKT, phosphor-P70S6K (p-P70S6K), P70S6K expression in shCtrl, shIPO7, and Importazole groups. The relative expressions were quantified by normalizing to GAPDH. (H) Immunoblot analysis of phosphor-mTOR (p-mTOR), mTOR, phosphor-AKT(p-AKT), AKT, phosphor-P70S6K (p-P70S6K), P70S6K expression in siNC and siGTF2I groups. The relative expressions were quantified by normalizing to GAPDH. Error bars represent mean ± standard deviation (SD). *p < 0.05.

FIGURE 6

The IPO7 negatively correlates with CD8 T cell infiltration in cervical cancer. (A) The correlation between IPO7 with immune cell type infiltrates are analyzed by CIBERSORT algorithm. (B) Gene-immune analysis of IPO7 in CC conducted on Sanger box. (C) The correlation between IPO7 with activated CD8 cell infiltrate level is analyzed by TISIDB database. (D) Representative IHC images of IPO7 and CD8⁺ cells in CC tissues. Upper panel is IPO7 with high expression; lower panel is IPO7 with low expression in CC. Scale bar, 50 μm. (E) Correlation analysis of IPO7 and CD8⁺ cells in CC. (F) Gene-immune checkpoints analysis of IPO7 in CC conducted on Sanger box. (G) The mRNA level of CD276 in Hela and C-4 I cells under knockdown of IPO7. Error bars represent mean ± standard deviation (SD). Two-tailed t-test, *p < 0.05.