Intermittent hypoxia-induced downregulation of microRNA-320b promotes lung cancer tumorigenesis by increasing CDT1 via USP37

Abstract

Obstructive sleep apnea-hypopnea (OSAH) is correlated with an increased incidence of lung cancer. In our study, we explored the functional roles of microRNAs (miRNAs) in lung cancer patients that were complicated with OSAH involving the deubiquitination enzyme. The miR-320b expression pattern in lung cancer tissues and cells was determined. The interactions between ubiquitin-specific peptidase 37 (USP37) and miR-320b were evaluated by a dual-luciferase reporter gene assay, whereas USP37 and Cdc10-dependent transcript 1 (CDT1) was assessed by co-immunoprecipitation and immunofluorescence. After the induction of intermittent hypoxia (IH), a gain-of function approach was performed to investigate roles of miR-320b, USP37, and CDT1 in lung cancer cell proliferation and invasion. In addition, nude mouse xenograft models were used to study their effects on tumor growth in vivo. miR-320b was poorly expressed in lung cancer patients with OSAH. IH treatment downregulated the expression of miR-320b but promoted the proliferation and invasion capabilities of lung cancer cells, both of which were suppressed by the overexpression of miR-320b through decreasing USP37. USP37 interacted with and deubiquitinated CDT1 to protect it from proteasomal degradation. Our study uncovered that IH-induced downregulation of miR-320b promoted the tumorigenesis of lung cancer by the USP37-mediated deubiquitination of CDT1.

Keywords: CDT1; USP37; deubiquitinase; lung cancer; microRNA-320b; obstructive sleep apnea-hypopnea.

Graphical abstract

Figure 1

miR-320b is poorly expressed in lung cancer patients with OSAH- and IH-treated lung cancer cells (A) qRT-PCR detecting the expression of miR-320b in lung cancer patients with OSAH (n = 27) or without OSAH (n = 68) (∗p < 0.05; lung cancer + OSAH versus lung cancer). (B) qRT-PCR detecting miR-320b levels in normal bronchial epithelial cells and lung cancer cells (∗p < 0.05, H1299 and Calu-3 versus NHBE; #p < 0.05, A549 and H1650 versus H1299 and Calu-3). (C) Western blot analysis for determining the expression level of HIF1α normalized to β-actin in lung cancer cells (∗p < 0.05, 6IH versus control). (D) qRT-PCR detecting the expression of miR-320b in lung cancer cells treated with or without 6IH (∗p < 0.05, 6IH versus control). Data are presented as the mean ± standard deviation. Data between two groups were compared using an unpaired t test. Comparisons among multiple groups were performed using one-way ANOVA with Tukey’s post hoc test.

Figure 2

Overexpression of miR-320b inhibits IH-induced lung cancer cell proliferation and invasion (A) qRT-PCR detecting levels of miR-320b in A549 and H1650 cells. (B) Representative images and results of EdU proliferation evaluating the growth of lung cancer cell (A549 and H1650) with altered miR-320b expression levels (magnification 200×). (C) Representative images and results of Transwell invasion assay measuring the effect of miR-320b on lung cancer cell (A549 and H1650) invasion potency (magnification 200×). (D) Representative images and results of immunoblotting analysis detecting MMP-2 and MMP-9 proteins in lung cancer cells with altered levels of miR-320b. ∗p < 0.05, 6IH + mimic NC versus control + mimic NC; #p < 0.05, 6IH + mimic NC versus 6IH + miR-320b mimic. Data are presented as the mean ± standard deviation. Data between two groups were compared using an unpaired t test. Comparisons among multiple groups were performed using one-way ANOVA with Tukey’s post hoc test.

Figure 3

miR-320b targets USP37 in lung cancer cells (A) Different online tools (microT, StarBase, and miRWalk) predicted miR-320b targets visualized by a Venn diagram. (B) Expression of the 12 predicted targets of miR-320b in lung cancer tissues and normal lung tissues analyzed by UALCAN website. (C) Representative images and results of immunoblotting analysis detecting the expression of USP37 in lung tissues obtained from lung cancer patients with OSAH (n = 27) or without OSAH (n = 68) (∗p < 0.05, lung cancer + OSAH versus lung cancer). (D) Correlation analysis between miR-320b and USP37 in patients with lung cancer and OSAH. (E) Representative images and results of western blot analysis detecting the expression of USP37 in lung cancer cells and NHBE cells (∗p < 0.05, A549, H1299, Calu-3, and H1650 versus NHBE). (F) Predicted miR-320b targeting sequences on USP37 3′ UTR. (G) Dual-luciferase reporter gene assay evaluating the interactions between miR-320b and predicted sequences on USP37 3′ UTR (∗p < 0.05, miR-320b mimic versus miR-NC). (H) qRT-PCR evaluating the expression of miR-320b in lung cancer cells transfected with miR-320b mimic, miR-320b inhibitor, and NCs (∗p < 0.05, miR-320b mimic versus miR-NC; #p < 0.05, miR-320b inhibitor versus NC inhibitor). (I) Representative images and results of western blot analysis measuring the expression of USP37 in lung cancer cells transfected with miR-320b mimic, miR-320b inhibitor, and NCs (∗p < 0.05, miR-320b mimic versus miR-NC; #p < 0.05, miR-320b inhibitor versus NC inhibitor). Data are presented as the mean ± standard deviation. Data between two groups were compared using an unpaired t test. Comparisons among multiple groups were performed using one-way ANOVA with Tukey’s post hoc test. The correlation between miR-320b and USP37 was analyzed by Pearson correlation coefficient.

Figure 4

miR-320b inhibits IH-induced lung cancer cell proliferation and invasion by targeting USP37 (A) qRT-PCR evaluating the expression of miR-320b in lung cancer cells under different conditions. (B) Immunoblotting analysis detecting the expression of USP37 in lung cancer cells. (C) Results of EdU proliferation assay measuring lung cancer cell growth. (D) Results of Transwell invasion assay investigating lung cancer cell invasion potency (∗p < 0.05, 6IH + miR-320b mimic + oe-NC versus 6IH + mimic NC + oe-NC; #p < 0.05, 6IH + miR-320b + oe-USP37 versus 6IH + miR-320b + oe-NC). Data are presented as the mean ± standard deviation. Data between two groups were compared using an unpaired t test. Comparisons among multiple groups were performed using one-way ANOVA with Tukey’s post hoc test.

Figure 5

USP37 promotes the expression of CDT1 by deubiquitination (A) Differential expressed genes in the dataset obtained from the GEO database (GEO: GSE106929) represented in a volcano plot (red dots indicate upregulated genes and green dots indicate downregulated genes in lung tumor samples). (B) Differentially expressed genes in the dataset obtained from the GEO database (GEO: GSE33532) represented in a volcano plot (red dots indicate upregulated genes and green dots indicate downregulated genes in lung tumor samples). (C) Overlapped upregulated genes in datasets GEO: GSE106929 and GSE33532 visualized by a Venn diagram. (D) Expression levels of USP37 and CDT1 were co-related by an online tool (Chipbase). (E) Relative expression levels of CDT1 in lung tumor (red box) and normal lung tissue (gray box) analyzed by TCGA database. (F) Representative images and results of immunoblotting analysis evaluating CDT1 expression in lung tissues obtained from lung cancer patients with OSAH (n = 27) or without OSAH (n = 68) (∗p < 0.05, lung cancer + OSAH versus lung cancer). (G) Correlation analysis between miR-320b and CDT1 as well as between USP37 and CDT1 in lung cancer patients with OSAH. (H) Representative images and results of immunoblotting analysis evaluating CDT1 expression in normal bronchial epithelial cells and lung cancer cells (∗p < 0.05, lung cancer cells versus NHBE cells). (I) Representative images and results of immunoblotting analysis validating the overexpression or knockdown of USP37 (∗p < 0.05, oe-USP37 versus oe-NC; #p < 0.05, sh-USP37 versus sh-NC). (J) Representative images and results of immunoblotting analysis evaluating CDT1 expression in lung cancer cells with the overexpression or knockdown of USP37 (∗p < 0.05, oe-USP37 versus oe-NC; #p < 0.05, sh-USP37 versus sh-NC). (K) Representative images of co-immunoprecipitation assay investigating the interactions between USP37 and CDT1. (L) Representative images of immunofluorescent staining showing co-localization of USP37 and CDT1 (magnification 400×). (M) Representative images of immunoblotting analysis evaluating the expressions of USP37 and CDT1. (N) Ubiquitination assay detecting ubiquitination levels of USP37 and CDT1. Data are presented as the mean ± standard deviation. Data between two groups were compared using an unpaired t test. Comparisons among multiple groups were performed using one-way ANOVA with Tukey’s post hoc test. The correlation between two indicators was analyzed by Pearson correlation coefficient.

Figure 6

miR-320b inhibits lung cancer cell proliferation and invasion by inhibiting CDT1 through targeting USP37 (A) qRT-PCR detecting the expression of miR-320b in lung cancer cells (A549 and H1650). (B) Immunoblotting analysis detecting the expressions of USP37 and CDT1 in lung cancer cells. (C) Results of EdU proliferation evaluating lung cancer cell growth. (D) Results of Transwell invasion assay measuring the invasion potency of lung cancer cells (∗p < 0.05, 6IH versus control; #p < 0.05, 6IH + miR-320b mimic + oe-NC versus 6IH + mimic NC + oe-NC; &p < 0.05, 6IH + miR-320b mimic + oe-CDT1 versus 6IH + miR-320b + oe-NC). Data are presented as the mean ± standard deviation. Data between two groups were compared using an unpaired t test. Comparisons among multiple groups were performed using one-way analysis of variance (ANOVA) with Tukey’s post hoc test.

Figure 7

miR-320b inhibits tumorigenesis and development of lung cancer in vivo by regulating USP37/CDT1 (A) qRT-PCR evaluating the expression of miR-320b in lung cancer cells. (B) Representative images and results of immunoblotting analysis measuring the protein levels of USP37 and CDT1 in lung cancer cells. (C) Images of xenograft tumors at endpoint (left panel), tumor growth curve (middle panel), and final tumor weight (right panel) reflecting the development of lung tumor in vivo. (D) Representative images of tumor nodules on the lung surface. (E) Immunohistochemistry detecting the expression of Ki67 in tumor tissues from mice in each group (scale bar = 25 μm) (∗p < 0.05, model versus control; #p < 0.05, model + miR-320b agmoir + oe-NC versus model + agmoir NC + oe-USP37; &p < 0.05, model + miR-320b agmoir + oe-USP37 versus model + miR-320b + oe-NC). Data are presented as the mean ± standard deviation. Data between two groups were compared using an unpaired t test. Comparisons among multiple groups were performed using one-way ANOVA with Tukey’s post hoc test. Comparisons over time were performed using repeated-measurements of ANOVA with Bonferroni post hoc test. n = 8.

Figure 8

The mechanism diagram depicting that IH-induced downregulation of miR-320b promoted the tumorigenesis of lung cancer by the USP37-mediated deubiquitination of CDT1