A novel intronic circular RNA, circGNG7, inhibits head and neck squamous cell carcinoma progression by blocking the phosphorylation of heat shock protein 27 at Ser78 and Ser82

Abstract

Background: There is increasing evidence that circular RNAs (circRNAs) play a significant role in pathological processes including tumorigenesis. In contrast to exonic circRNAs, which are the most frequently reported circRNAs in cancer so far, the studies of intronic circRNAs have been greatly lagged behind. Here, we aimed to investigate the regulatory role of intronic circRNAs in head and neck squamous cell carcinoma (HNSCC).

Methods: We conducted whole-transcriptome sequencing with four pairs of primary tumor tissues and adjacent normal tissues from HNSCC patients. Then, we characterized circGNG7 expression in HNSCC tissues and cell lines and explored its association with the prognosis of HNSCC patients. We also identified interactions between circGNG7 and functional proteins, which alter downstream signaling that regulate HNSCC progression.

Results: In this study, we identified a new intronic circRNA, circGNG7, and validated its functional roles in HNSCC progression. CircGNG7 was predominately localized to the cytoplasm, and its expression was downregulated in both HNSCC tissues andCAL27, CAL33, SCC4, SCC9, HN6, and HN30 cells. Low expression of circGNG7 was significantly correlated with poor prognosis in HNSCC patients. Consistent with this finding, overexpression of circGNG7 strongly inhibited tumor cell proliferation, colony formation, in vitro migration, and in vivo tumor growth. Mechanistically, the expression of circGNG7 in HNSCC cells was regulated by the transcription factor SMAD family member 4 (SMAD4). Importantly, we discovered that circGNG7 could bind to serine residues 78 and 82 of the functional heat shock protein 27 (HSP27), occupying its phosphorylation sites and hindering its phosphorylation, which reduced HSP27-JNK/P38 mitogen-activated protein kinase (MAPK) oncogenic signaling. Downregulation of circGNG7 expression in HNSCC increased HSP27-JNK/P38 MAPK signaling and promoted tumor progression.

Conclusions: Our results revealed that a new intronic circRNA, circGNG7, functions as a strong tumor suppressor and that circGNG7/HSP27-JNK/P38 MAPK signaling is a novel mechanism by which HNSCC progression can be controlled.

Keywords: circGNG7; head and neck squamous cell carcinoma; heat shock protein 27 (HSP27); intronic circular RNAs; mitogen-activated protein kinase (MAPK) signaling; phosphorylation; tumor suppressor.

FIGURE 1

Reduced circGNG7 expression was associated with poor prognosis in HNSCC. (A) Clustered heatmap for differentially expressed circRNAs between four pairs of primary tumor tissues and adjacent normal tissues, with rows representing circRNAs and columns representing tissues. The numerical data represent the serial numbers of circRNAs in circBase. (B) The representative images of circGNG7 detected by using an RNAscope® in situ assay in HNSCC and adjacent normal tissues. The red dots pointed by black arrows indicate the circGNG7 blots. (C) The subcellular localization of circGNG7 was detected by FISH in CAL27 and HN6 cells. 18S RNA and U6 indicate cytoplasm and nucleus, respectively. (D) The expression level of circGNG7 in the nuclear and cytoplasm fractions was assessed by qRT‐PCR in CAL27 and HN6 cells. Data are presented as mean ± SD of at least three independent experiments. (E) circGNG7 expression was detected in tumor and adjacent normal tissues from 65 HNSCC patients by RNAscope assay. (F, G) circGNG7 staining score was analyzed according to UICC stage in HNSCC patients. (H) OS and PFS of HNSCC patients were evaluated according to the circGNG7 staining score. *P < 0.05, **P < 0.01; ***P < 0.001. Abbreviations: circRNAs: circular RNAs; HNSCC: head and neck squamous cell carcinoma; qRT‐PCR: quantitative real‐time polymerase chain reaction; UICC: Union for International Cancer Control; PFS: progression‐free survival; OS: overall survival

FIGURE 2

Evidence of circGNG7 as a circular RNA and its subcellular localization. (A) circGNG7 expression was measured in HNSCC cell lines and normal oral keratinocytes. (B) Schematic illustration of the origin and structure of circGNG7. The circular form of circGNG7 in cDNA and gDNA was validated by using divergent primers () and convergent primers () in CAL27 cells. GAPDH was used as a linear RNA control. The head‐to‐tail splicing of circGNG7 was confirmed by Sanger sequencing in CAL27 cells. (C) RNA samples were treated with RNase R to remove linear RNAs, and circGNG7 was evaluated after RNase R digestion in CAL27 and HN6 cells. For qPCR normalization, the abundance of GNG7 was calculated by standardizing over the spike DNA control and setting the PBS control. (D) RT‐PCR for the abundance of circGNG7 and GNG7 in CAL27 and HN6 cells treated with actinomycin D at the indicated time points. Data are presented as mean ± SD of at least three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001. Abbreviations: HNSCC: head and neck squamous cell carcinoma; qPCR: quantitative real‐time polymerase chain reaction; RT‐PCR: reverse transcription PCR; GNG7: G protein subunit gamma 7; cDNA: complementary DNA; gDNA: genomic DNA

FIGURE 3

circGNG7 inhibited the progression of HNSCC cells in vitro and in vivo. HN6 and CAL27 cells were transfected with overexpression plasmid (ov‐circGNG7) or vector plasmid respectively for 48 h. (A) Colony formation assays were performed. Representative images are shown, and relative colony formation rate was evaluated. (B) Cell viability after transfection was accessed by using CCK8 assays. (C) EDU assays were performed after transfection. Representative images are shown, and the proportion of EDU‐positive cells was calculated. Transwell assays for cell migration (D) and invasion (E) were performed. Representative images are shown, and relative migration rate and invasion rate were calculated. (F) Representative images of xenograft tumors derived from stable circGNG7 overexpression or vector control cells are shown. (G) Primary tumor growth curves after cell injection in the stable circGNG7 overexpression or vector control groups were analyzed. (H) Tumor weight after removal from mice in the ectopic overexpression and vector control group was measured. (I) The expression of Ki‐67 in xenograft tissues derived from stable circGNG7 overexpression or vector control cells was detected by immunohistochemistry. Representative images are shown, and the Ki‐67 staining score was evaluated. (J) Representative images of the lungs from mice injected with stable circGNG7 overexpression or vector control cells are shown. Arrows indicate individual metastatic nodules. (K) The metastatic nodules were counted. Data are presented as mean ± SD of at least three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001. Abbreviations: HNSCC: head and neck squamous cell carcinoma; CCK‐8: Cell Counting Kit‐8; EDU: 5‐ethynyl‐2'‐deoxyuridine

FIGURE 4

circGNG7 exerted neither miRNA interaction nor protein‐coding potential in HNSCC cells. (A) The relative enrichments of miRNAs were evaluated by pull‐down assay with biotinylated circGNG7 or negative control primer (NC) in CAL27 and HN6 cells. (B) The fold enrichment of miR‐34c‐5p and negative control was evaluated by AGO2‐RIP in CAL27 and HN6 cells. (C) Dual‐luciferase activities were accessed in CAL27 and HN6 cells after indicated transfection. Data are presented as mean ± SD of at least three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001. Abbreviations: AGO2‐RIP: Argonaute 2‐RNA immunoprecipitation; miRNAs: microRNAs.

FIGURE 5

CircGNG7 inhibited the phosphorylation of HSP27 at Ser78 and Ser82. (A) Schematic diagram of the proteins binding with circGNG7. (B) CAL27 cells were transfected with circGNG7 overexpression plasmid (ov‐circGNG7) or negative control plasmid. The probe‐circRNA complexes were captured and subjected to proteomic analysis by LC‐MS/MS system. (C) Top three enriched peptides pulled down by circGNG7 were validated by Western blotting. (D) The expression of P38, MAPK‐APK‐2, PKD‐1 and phosphorylation level of HSP27 were detected by Western blotting in CAL27 and HN6 cells transfected with si‐circGNG7, scramble, ov‐circGNG7, and control vector. (E) CAL27 cells were transfected with ectopic circGNG7 plasmid. The total protein or phosphorylated protein of HSP27 pulled down by circGNG7 was accessed by Western blotting. (F) circGNG7 and GNG7 expression level pulled down by total protein or phosphorylated protein (S15, S78 and S82) of HSP27 was evaluated by qRT‐PCR. (G) CAL27 cells were transfected with wild‐type, mutant plasmid at phosphoacceptor site 15, 78, or 82 of HSP27 (S15A, S78A, S82A, or co‐transfected with S78A and S82A. The total protein of HSP27 pulled down by circGNG7 was detected by Western blotting. (H) RIP assays were conducted to detect the expression of circGNG7 and GNG7 pulled down by total HSP27 protein, HSP27 mutant protein at phosphoacceptor site 15, 78, or 82 (S15A, S78A, S82A, or S78A and S82A) in CAL27 cells respectively. (I) Schematic diagram of the interaction between HSP27 and circGNG7. Data are presented as mean ± SD of at least three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001. Abbreviations: MAPK‐APK‐2: MAPK‐activated protein kinase‐2; PKD‐1: polycystin 1; RT‐PCR: reverse transcription polymerase chain reaction

FIGURE 6

CircGNG7 inhibited HNSCC progression by regulating the HSP27‐JNK/P38 MAPK pathway. HN6 and CAL27 cells were transfected with HSP27 overexpression plasmid (ov‐HSP27) or vector plasmid respectively for 48 h. (A) Cell viability after transfection was accessed by using CCK8 assays in CAL27 and HN6 cells. (B) Transwell assays for cell invasion were performed. Representative images are shown, and relative invasion rate was calculated. HN6 and CAL27 cells were transfected with vector plasmid, ov‐circGNG7, ov‐HSP27, ov‐circGNG7+ov‐HSP27, and ov‐circGNG7+siHSP27 respectively for 48 h. (C) EDU assays were performed after transfection to test DNA replication activity. EDU‐positive cells were counted. (D) Relative cell migration and invasion rates by transwell assays were calculated. (E) HN6 and CAL27 cells were transfected with HSP27 siRNA (si‐HSP27), scrambled siRNA, ov‐HSP27, vector plasmid, ov‐circGNG7, vector of circRNA [vector(circ)], ov‐circGNG7+ov‐HSP27, ov‐circGNG7+siHSP27, si‐circGNG7, scramble of circRNA [scramble(circ)], si‐circGNG7+ov‐HSP27, and si‐circGNG7+siHSP27 respectively for 48 h. The expression of c‐Jun, p‐c‐Jun, P38, and p‐P38 was evaluated by Western blotting. (F, I) Xenograft tumors were derived from stable circGNG7 overexpression or vector control cells. The expression of HSP27‐S78 (F) and HSP27‐S82 (I) in xenograft tissues was detected by immunohistochemistry, and representative images are shown. The staining scores of HSP27‐S78 (G) and HSP27‐S82 expression (J) were evaluated. The relationships between circGNG7 and HSP27‐S78 (H) and HSP27‐S82 (K) in xenograft tissues were analyzed. Data are presented as mean ± SD of at least three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001. Abbreviations: HNSCC: head and neck squamous cell carcinoma

FIGURE 7

CircGNG7 expression level was regulated by SMAD4 binding on GNG7 promoter. (A) The potential transcription factors binding to GNG7 promoter were predicted by using public database LASAGNA‐Search 2.0 with the threshold P value <0.001. (B) The circGNG7 expression was detected by RT‐PCR in CAL27 and HN6 cells transfected with siRNA of selected genes (NF1, LEF1, SMAD3, SMAD4, SPL1, SP1, JUN, NFATC2, TEAD2, E2F1, CREB1, ATF2, ZEB1, WT1, PLAU, NFKB1, and MYB) for 24 h. (C‐D) CAL27 and HN6 cells were transfected with SMAD4 overexpression plasmid (ov‐SMAD4) or vector for 48 h. (C) The circGNG7 expression was evaluated by RT‐PCR. (D) The binding of SMAD4 at the GNG7 promoter region was detected by a chromatin immunoprecipitation assay. (E) CAL27 and HN6 cells were co‐transfected with circGNG7 mutant/wildtype plasmid and ov‐SMAD4, and the luciferase activity was determined by using a dual luciferase reporter assay after 24 h. (F) The expression levels of SMAD4, total HSP27, HSP27‐S15, HSP27‐S78, and HSP27‐S82 were evaluated by Western blotting in CAL27 and HN6 cells transfected with si‐SMAD4, si‐HSP27, scramble, ov‐SMAD4, and vector for 24 h. (G) Schematic diagram of the regulation of circGNG7 by SMAD4 binding with serine residues 78, and 82, occupying the phosphorylation site of functional protein HSP27, hindering the phosphorylation of HSP27, which reduces the HSP27‐JNK/P38 MAPK oncogenic signaling and inhibits HNSCC progression. Data are presented as mean ± SD of at least three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001. Abbreviations: RT‐PCR: reverse transcription polymerase chain reaction