miR-424 coordinates multilayered regulation of cell cycle progression to promote esophageal squamous cell carcinoma cell proliferation

Abstract

Background: Dysregulation of the cell cycle has been implicated in esophageal squamous cell carcinoma (ESCC) progression. This study aimed to evaluate the role of miR-424 in cell cycle regulation and ESCC proliferation.

Methods: The role of miR-424 in cell proliferation was evaluated in vitro and in vivo. Transcriptional activation of miR-424 was determined using chromatin immunoprecipitation, and binding of miR-424 to targets was verified using miRNA ribonucleoprotein complex immunoprecipitation.

Findings: miR-424 was upregulated and correlated with poor survival in ESCC patients. Repression or overexpression of miR-424 respectively decreased or increased ESCC cell proliferation in vitro and in vivo. miR-424 expression is transcriptionally regulated by E2F1 and increased during G1/S transition. Knockdown or overexpression of miR-424 respectively inhibited or promoted both G1/S and G2/M cell cycle transitions in ESCC cells, and these effects were mediated by two newly identified miR-424 targets, PRKCD and WEE1, respectively. Consequently, elevation of PRKCD by miR-424 knockdown led to enhanced stability of the p21^Cip1 protein via increased activation of PRKCD and downstream p38 MAPK and JNK signaling to block CDK2 activation and G1/S transition, while elevated WEE1 maintained CDC2 in an inactive state to block G2/M transition. However, circLARP4 could sponge the binding of miR-424 to PRKCD, thus compromising the regulation of G1/S progression by miR-424.

Interpretation: miR-424 coordinates a previously unknown, multilayered regulation of ESCC cell cycle progression to promote ESCC proliferation, and may be used as a novel prognostic marker and an effective therapeutic target for ESCCs. FUND: National Natural Science Foundation of China.

Keywords: Cell cycle; Cell proliferation; Esophageal squamous cell carcinoma; miR-424.

Fig. 1

miR-424 is upregulated in ESCC and correlates with poor prognosis. (a) Hierarchical cluster analysis showed differential miRNA expression profiles between 30 ESCC and ten NE samples (fold change >3, p < 0·001 by Student's t-test with the Benjamin-Hochberg correction). Each column represents a miRNA, and each row represents a specimen. Blue and red denote genes that are under- and overexpressed, respectively. (b) qRT-PCR verified the upregulation of miR-424 in 60 ESCC specimens compared with the expression seen in normal counterparts (p < 0·001 by Student's t-test for paired samples). (c) Kaplan-Meier analysis showed that the overall survival (p = 0·025 by log-rank test) of ESCC patients with high miR-424 expression was poorer than that of patients with low miR-424 expression.

Fig. 2

miR-424 promotes ESCC cell proliferation in vitro and in vivo. (a) qRT-PCR analyses showed the relative miR-424 expression levels in KYSE-410 and KYSE-510 ESCC cells and NE1 immortalized esophageal epithelial cells expressing the miR-424 precursor (lenti-miR-424) or control vectors. Data are presented as the mean ± SD of three independent experiments. **, p < 0·01 or ***, p < 0·001 by Student's t-test for unpaired samples. (b) Cell growth curves were generated for miR-424-overexpressing (lenti-miR-424) or miR-424-knockdown (miRZip-424) KYSE-410, KYSE-510, and NE1 cells with a CCK-8 cell proliferation kit. Data are presented as the mean ± SD of three independent experiments. *, p < 0·05; **, p < 0·01; or ***, p < 0·001 by repeated measures ANOVA. (c) Representative images and results of colony formation assays of KYSE-410 and KYSE-510 cells expressing miR-424-knockdown (miRZip-424), miR-424-overexpressing, or the corresponding control vectors. Data are presented as the mean ± SD of three independent experiments. ***, p < 0·001 by Student's t-test for unpaired samples. (d) Tumor xenograft experiments with subcutaneous injection of KYSE-410 or KYSE-510 cells stably expressing miRZip-424 or control vectors into nude mice (n = 5 in each group) demonstrated the in vivo tumorigenic ability of miR-424. Images of tumor-bearing mice and tumors from all mice in each group are shown. Tumor volumes were measured on the indicated days, and each data point represents the mean ± SD of five nude mice. ***, p < 0·001 by repeated measures ANOVA.

Fig. 3

Effects of miR-424 expression on G1/S and G2/M cell cycle progression in KYSE-410 ESCC cells. (a) Stable miR-424-knockdown (miRZip-424), miR-424-overexpressing (lenti-miR-424), and the corresponding control KYSE-410 cells were synchronized in G0/G1-phase by serum starvation and released, and the cell cycle distribution was monitored by flow cytometry at the indicated time points with propidium iodide staining. Representative images of the cell cycle distribution (upper) and the percentage of cells in G1-phase or S-phase (lower) are shown. (b) Stable miR-424-knockdown (miRZip-424), miR-424-overexpressing (lenti-miR-424), and the corresponding control KYSE-410 cells were synchronized at the onset of S-phase by double-thymidine block and released, and the kinetic transition of the cells through S-phase to G2/M-phase was monitored by flow cytometry at the indicated time points with propidium iodide staining. Representative images of the cell cycle distribution (upper) and the percentage of cells in G2/M- or G1-phase (lower) are shown. (c) Stable miR-424-knockdown (miRZip-424), miR-424-overexpressing (lenti-miR-424), and the corresponding control KYSE-410 cells were synchronized at the onset of S-phase by double-thymidine block and released, and the kinetic transition of the cells through G2-phase to M-phase was monitored by flow cytometry at the indicated time points with propidium iodide and anti-pHH3 and Alexa Fluor 647-conjugated secondary antibody double-staining. Representative images of the cell cycle distribution (left) and the percentage of cells in G2-phase (right) are shown. All the data are presented as the mean ± SD of three independent experiments. *, p < 0·05; **, p < 0·01; ***, p < 0·001; or NS, not significant by Student's t-test for unpaired samples.

Fig. 4

Characterization of the miR-424 promoter and transcriptional regulation of miR-424 by E2F1 during G1/S transition. (a) KYSE-410 and KYSE-510 cells were synchronized in G0/G1-phase by serum starvation and released. qRT-PCR analysis shows the expression kinetics of miR-424, pri-miR-424, and pre-miR-424 in KYSE-410 and KYSE-510 cells after release from G0/G1-phase for the indicated time points. Data are presented as the mean ± SD of three independent experiments. (b) A schematic illustration shows two binding sites for E2F1 in the putative promoter region of the miR-424 gene. Specific primers surrounding the promoter were designed. A fragment of the promoter region encompassing the wild-type or mutant E2F1 binding sites was cloned into a reporter vector. (c) Expression of E2F1 was silenced using siRNA (left panel). qRT-PCR showed changes in the expression kinetics of miR-424, pri-miR-424, and pre-miR-424 after knocking down E2F1 in KYSE-410 cells during G1/S transition (right panel). Data are presented as the mean ± SD of three independent experiments. *, p < 0·05; ***, p < 0·001; or NS, not significant by Student's t-test for unpaired samples to compare differences in expression between E2F1-knockdown and control cells at each time point. (d) ChIP was performed with an anti-E2F1 antibody on lysates of KYSE-410 cells after release from G0/G1-phase for 8 h. The quantification of genomic DNA enrichment was performed using specific primers surrounding the promoter, as shown in (b). Putative E2F1-binding sites are indicated by red squares. Quantification of DNA enrichment in the CDC2 gene promoter was used as a positive control, and GAPDH was used as a negative control. Data are presented as the mean ± SD of three independent experiments. **, p < 0·01 by Student's t-test for unpaired samples to compare differences in enrichment between any specific primer pair and the negative control. (e) Luciferase assays showed the different activities of reporters containing either the wild-type or mutated E2F1-binding sites in KYSE-410 cells, which were released from G0/G1-phase for 8 h. Data are presented as the mean ± SD of three independent experiments. ***, p < 0·001 by Student's t-test for unpaired samples to compare differences in relative luciferase activity between reporters with mutant E2F1-binding sites and that with wild-type binding site.

Fig. 5

miR-424 targets PRKCD and WEE1 in ESCC cells during G1/S and G2/M transitions, respectively. (a) Stable miR-424-knockdown (miRZip-424) and control KYSE-410 cells were synchronized in G0/G1-phase by serum starvation and released for the indicated time points. Western blotting analysis showed the protein expression of potential miR-424 targets during G1/S transition. (b) Stable miR-424-knockdown (miRZip-424) and control KYSE-410 cells were synchronized at the onset of S-phase by double-thymidine block and released for the indicated time points. Western blotting analysis showed the protein expression of potential miR-424 targets during G2/M transition. (c) The illustration shows the predicted miR-424 target sequences in the 3′UTRs of the PRKCD and WEE1 mRNAs. (d) Luciferase assays showed different activities of reporters containing the 3′UTRs or mutated 3′UTRs of PRKCD and WEE1 in miR-424-overexpressing, miR-424-knockdown, and the corresponding control HEK293 cells. Data are presented as the mean ± SD of three independent experiments. *, p < 0·05; **, p < 0·01; ***, p < 0·001; or NS, not significant by Student's t-test for unpaired samples. (e) miRNP-IP was performed with an anti-AGO2 antibody on lysates of stable miR-424-overexpressing or control KYSE-410 and KYSE-510 cells, and the cells were released from G0/G1 for 12 h or from S-phase onset for 9 h. The quantification of RISC enrichment of PRKCD, WEE1, and circLARP4 was determined by qRT-PCR. Data are presented as the mean ± SD of three independent experiments. *, p < 0·05; **, p < 0·01; ***, p < 0·001; or NS, not significant by Student's t-test for unpaired samples. (f) Representative immunohistochemical staining images of PRKCD and WEE1 protein in two ESCC cases with low and high miR-424 expression (Scale bar: 50 μM). (g) The correlation between miR-424 expression and PRKCD or WEE1 protein expression in 110 ESCC samples was analyzed. r = −0·282, p = 0·003 for WEE1, and r = −0·179, p = 0·061 for PRKCD by Chi-square test.

Fig. 6

Knockdown of miR-424 modulates the expression or activity of CDKs or CKIs to regulate ESCC cell cycle progression. (a) Western blotting analysis showed the expression of p21^Cip1, p-CDK2 (Thr160), and total CDK2 in stable miR-424-knockdown or control KYSE-410 cells, which were collected at the indicated time points after release from G0/G1-phase. (b) The stability of the endogenous p21^Cip1 protein in stable miR-424-knockdown or control KYSE-410 cells was measured by incubating the cells with 25 μg/ml cycloheximide (CHX). The cells were collected at the indicated time points and analyzed by western blotting (left panel). Quantitation of p21^Cip1 protein levels is shown in the right panel. Data are presented as the mean ± SD of three independent experiments. ***, p < 0·001 by repeated measures ANOVA. (c) Western blotting analysis showed the expression changes in PRKCD, p-PRKCD (Thr505), p-p38 MAPK (Thr180/Tyr182), p38 MAPK, p-JNK (Thr183/Tyr185), and JNK in stable miR-424-knockdown or control KYSE-410 cells, which were collected at the indicated time points after release from G0/G1-phase. (d) Western blotting analysis showed the expression changes in WEE1, p-CDC2 (Tyr15), and CDC2 in stable miR-424-knockdown or control KYSE-410 cells, which were collected at the indicated time points after release from the onset of S-phase.

Fig. 7

Proposed mechanistic model for the role of miR-424 in coordinating ESCC cell cycle progression. During G1/S transition, activation of the transcription factor E2F1 induces the expression of the miR-424 primary transcript. Subsequently, mature miR-424, which is generated from pri-miR-424, targets PRKCD and WEE1 at the G1/S and G2/M borders, respectively, and these proteins further modulate the expression or activity levels of cell cycle regulators such as p21^Cip1, CDK2, and CDC2 to regulate ESCC cell cycle progression. However, circLARP4, a natural sponge of miR-424, can diminish the binding of miR-424 to PRKCD and compromise its regulation of G1/S progression.