DYNLL1 ubiquitinated and degraded by E3 ligase PRKN regulates cell cycle arrest and apoptosis in lung adenocarcinoma cells

Abstract

This study was intended to investigate the role of DYNLL1 in lung adenocarcinoma (LUAD) and reveal the relevant molecular mechanism. To this end, we manipulated the expression of DYNLL1 and PRKN with shRNA and/or overexpression to assess their effects on A549 cell phenotypes including cell proliferation, cycle arrest and apoptosis using CCK-8 assay, EdU staining, colony formation assay, flow cytometry, TUNEL and western blot. DYNLL1 is a carcinogenic factor in LUAD since it is highly expressed and DYNLL1 upregulation is associated with the low overall survival rate of LUAD patients. Silencing DYNLL1 inhibited the proliferation of LUAD cells while accelerating cell cycle arrest and apoptosis. The regulatory effects of silencing DYNLL1 were counteracted by PRKN deficiency, indicating a functional connection between DYNLL1 and PRKN, which could be attributed to the ubiquitination of DYNLL1 by PRKN. This study could help identify the role of DYNLL1 in LUAD pathogenies and develop reliable therapeutic targets for LUAD improvement.

Supplementary Information

The online version contains supplementary material available at 10.1007/s12672-024-01686-7.

Introduction

Lung cancer is considered as the second most prevalent cancer type in 2023 and is a primary cause of cancer-related deaths worldwide [1]. Lung adenocarcinoma (LUAD), a subtype of non-small cell lung cancer (NSCLC) characterized by high proliferation rate, is difficult to diagnose because it typically has no symptoms at an earlier preclinical phase [2, 3]. Consequently, a majority of patients with LUAD are diagnosed in the middle or late stages, making them miss the optimal chance of surgery treatment [4]. Thence, developing novel biomarkers for early detection and specialized therapy is profoundly vital for LUAD patients.

As a previous study mentioned, the stabilization and degradation of proteins are critical for LUAD progression and the ubiquitin–proteasome system (UPS) is considered as the most important protein degradation modality [5, 6]. The protein degraders designed on the basis of UPS show great advantages in developing small molecule drugs, particularly in cancer treatment [7, 8]. Of note, UPS can modulate a variety of biological processes, including cell apoptosis and cell cycle [9]. E3 ubiquitin ligase PRKN has been evidenced to be reduced in LUAD patients and cells and serves as a prospective target for LUAD treatment [10]. However, its specific substrate molecular and mechanisms wait to be further investigated.

DYNLL1 extensively participates in the modulation of various cellular functions, from intracellular trafficking to apoptosis and plays a complicated role in tumor progression [11, 12]. In breast cancer, the elevation of DYNLL1 can facilitate cell proliferation [13]. Besides, Liu et al. have demonstrated that DYNLL1 expression is increased in hepatocellular carcinoma (HCC) cells and correlated with the prognosis of HCC patients [14]. As demonstrated by UALCAN database (https://ualcan.path.uab.edu/), DYNLL1 expression is highly expressed in LUAD patients and is closely related with the poor prognosis of LUAD patients. It is noted that degradation of DYNLL1 mediated by the E3 ubiquitin ligase RNF114 is involved in the progression of esophageal squamous cell carcinoma (ESCC) [15]. Moreover, DYNLL1 can be ubiquitinated as a substrate [15]. According to UbiBrowser database (http://ubibrowser.bio-it.cn/ubibrowser_v3/), E3 ubiquitin ligase PRKN can modulate DYNLL1 ubiquitination.

In short, this study aims to explore the role and the mechanism of DYNLL1 in cell apoptosis and cell cycle arrest in LUAD.

Material and methods

Cell culture and treatment

Human bronchial epithelial cells (BEAS-2B; cat. no. iCell-h023) and lung cancer H661 (cat. no. iCell-h162), H1975 (cat. no. iCell-h156) and A549 cells (cat. no. iCell-0140a) were obtained from iCell Bioscience Inc. BEAS-2B cells were incubated in DMEM. H661 and H1975 cells were maintained in RPMI-1640 medium while A549 cells were incubated in F12K medium. All media were supplemented with 10% FBS and 1% antibiotic and placed in humid atmosphere at 37 °C with 5% CO₂.

Cell transfection

Short hairpin RNAs (shRNA) specific to DYNLL1 (sh-DYNLL1) and PRKN (sh-PRKN), the negative control (sh-NC), the plasmids carrying PRKN (oe-PRKN) and the pcDNA3.1 empty vector (oe-NC) were synthesized by Geneseed Biotech Co., Ltd. Through Lipofectamine 3000 Transfection Reagent, these plasmids were transfected into cells according to the manufacturer’s instructions. After 48 h, cells were harvested for follow-up assays.

RT-qPCR

The total RNA was extracted from A549 cells via TRIzol® reagent and reverse transcribed into cDNA using SynScript™ III cDNA Synthesis Mix according to the manufacturer’s instructions. Real-time PCR was performed with an ABI 7900HT Sequence Detection system (ABI Applied Biosystems). Relative gene expressions of DYNLL1 and PRKN were calculated with 2^−ΔΔCt method.

Western blot

The total proteins were lysed from A549 cells via RIPA lysate and the protein concentration was quantified using BCA assay kits. Separated by 10% SDS-PAGE, the proteins were transferred to PVDF membranes. Then, the membranes inhibited by 5% BSA were successively immunoblotted with primary antibodies against DYNLL1, Bcl-2, Bax, PRKN, or β-actin and HRP-conjugated secondary antibodies. The protein signals were visualized with ECL kit and ImageJ software was used to analyze the protein density.

CCK-8 assay

A549 cell viability was detected using CCK-8 assay. A549 cells were seeded into 96-well plates and cultured for 24 h at 37 °C. Then, the cells were incubated with CCK-8 solution for additional 4 h. The OD value was read at 450 nm using a microplate reader.

EdU staining

A549 cells were seeded into 96-well plates and cultured in 10 µM EdU media for 1 h according to the manufacturer’s instructions. Following the fixation with 4% paraformaldehyde and the permeabilization with 0.5% Trionx-100, A549 cells were incubated with Click reaction solution (Sangon Biotech, Shanghai, China) and stained by Hoechst 33342 (Sangon Biotech, Shanghai, China). The viable cells were observed using a fluorescence microscope.

Colony formation assay

A549 cells were seeded into 6-well plates and cultured for 14 days. The formed colonies were fixed with 100% methanol for 10 min and stained by 0.5% crystal violet solution for 30 min. Finally, the images of colonies were observed using a microscope.

Flow cytometry

A549 cells were trypsinized and immobilized using 70% ethanol, followed by the staining with a Cell Cycle Detection Kit (KGA512; KeyGene, Holland) according to the manufacturer’s instructions. For the analysis of cell cycle, Guava easyCyte HT Flow Cytometer (LUMINEX, Shanghai, China) was used.

TUNEL

A549 cell apoptosis was detected using TUNEL assay kits. In brief, A549 cells were incubated overnight. Then, the cells were fixed with 4% paraformaldehyde and permeabilized with 0.25% Triton-X 100 for 20 min. Subsequently, A549 cells were reacted with 50 μl TUNEL solution and then stained by DAPI for 5 min. The apoptotic cells in five randomly selected fields were detected using a fluorescent microscope.

Measurement of caspase3

Caspase3 activity was detected using corresponding commercial assay kits according to the manufacturer’s instructions. A549 cells were suspended in the lysis buffer and cultured on ice for 12 min. Then, A549 cells were centrifuged at 14,000g for 10 min to obtain cell supernatant. Following, the supernatant was cultured with the regent for 2 h and the OD value was read at 405 nm.

Ubiquitination assay

Before collection, the transfected A549 cells were cultured with 20 µM MG132 for 6 h. The proteins were lysed with RIPA lysis buffer and then incubated with antibodies targeting DYNLL1 and PRKN overnight at 4 °C. Western blot was adopted to analyze the immune-precipitates on the next day for the detection of ubiquitination of DYNLL1.

Cycloheximide (CHX) chase

A459 cells transfected with oe-PRKN or sh-PRKN and their corresponding controls were initially treated with CHX (25 µg/mL), followed by the collection at indicated time points (0, 2, 4, 8 h). RIPA lysis buffer was used to obtain cell lysates and finally western blot analysis was performed to observe the half-life of DYNLL1.

Bioinformatics analysis

UALCAN database (https://ualcan.path.uab.edu/) collects RNA-seq and clinical data of 31 cancer types from The Cancer Genome Atlas (TCGA) database (https://cancergenome.nih.gov/) and it provides a useful platform to analyze gene expression in tumor and normal tissues; it also can estimate the effect of gene expression level and clinicopathologic features on patient survival [16, 17]. According to UALCAN database, DYNLL1 expression is increased in the tissues of LUAD patients. DYNLL1 upregulation is associated with the low overall survival rate of LUAD patients.

Statistical analysis

All experiments were repeated for three times. The collected experimental data were analyzed with GraphPad Prism 8.0 software (GraphPad software, Inc.) and expressed as mean ± SD. The differences were compared using Student’s t test and one-way ANOVA with Tukey’s post-hoc test. P < 0.05 indicated statistical significance.

Results

DYNLL1 expression is increased in LUAD tissues and cells

According to UALCAN database, it was found that DYNLL1 expression is increased in the tissues of patients with LUAD (Fig. 1A). Noticeably, DYNLL1 upregulation is associated with the low overall survival rate of LUAD patients (Fig. 1B). DYNLL1 expression in normal bronchial epithelial cells and LUAD cells was detected with RT-qPCR, and western blot. It was found that the mRNA and protein expressions of DYNLL1 were significantly increased in LUAD cells including H661, H1975 and A549 cells, particularly in A549 cells (Fig. 1C). Thence, A549 cells were selected for follow-up studies. To reduce DYNLL1 expression, sh-DYNLL1 was transfected into A549 cells and it was found that sh-DYNLL-2 exhibited better transfection efficacy. Thus, we chose sh-DYNLL-2 (hereinafter referred as sh-DYNLL) for subsequent assays (Fig. 1D) and A549 cells were divided into Control, sh-NC and sh-DYNLL1 groups.

Fig. 1.

DYNLL1 expression is increased in LUAD tissues and cells. A UALCAN database shows that DYNLL1 expression is upregulated in LUAD tissues. B DYNLL1 upregulation is correlated with the low overall survival rate of LUAD patients. C DYNLL1 expression was detected using RT-qPCR and western blot. **P < 0.01 and ***P < 0.001 vs BEAS-2B. D The transfection efficacy of sh-DYNLL1 was detected using RT-qPCR and western blot. ***P < 0.001 vs sh-NC

Silencing DYNLL1 suppresses A549 cell proliferation

Abnormal cell proliferation is a primary characteristic of cancer biology. Herein, we investigated the role of DYNLL1 in cell proliferation in LUAD through CCK-8 assay, EdU staining and colony formation assay. It was found that the proliferation of A549 cells was significantly suppressed following the transfection of sh-DYNLL1 (Fig. 2A, B). Besides, the number of formed colonies in sh-DYNLL1 was significantly reduced compared with the sh-NC group (Fig. 2C).

Fig. 2.

Silencing DYNLL1 suppresses A549 cell proliferation. A CCK-8 assay detected cell proliferation. B EdU staining detected cell proliferation. C Colony formation assay detected the colony forming ability of LUAD cells. **P < 0.01 and ***P < 0.001 vs sh-NC

Silencing DYNLL1 induces A549 cell cycle arrest and apoptosis

Cell-cycle arrest indicates the accumulating responses to DNA damage, thereby affecting cell growth and division. Flow cytometry was used to assess cell distribution and the results demonstrated that the cell population in S phase was significantly decreased by DYNLL1 silencing (Fig. 3A). In addition, the apoptosis of A549 cells was significantly promoted by DYNLL1 interference (Fig. 3B). Caspase3 is an important executor in apoptosis. Results of caspase3 assay kits showed that DYNLL1 knockdown increased caspase3 activity (Fig. 3C). Moreover, western blot indicated the levels of proteins implicated in apoptosis and it was found that DYNLL1 knockdown increased Bax expression while decreasing Bcl-2 expression in A549 cells (Fig. 3D).

Fig. 3.

Silencing DYNLL1 induced A549 cell cycle arrest and apoptosis. A Flow cytometry analysis detected cell cycle. B TUNEL detected cell apoptosis. C Caspase3 activity was detected using capase3 assay kit. D The expressions of apoptosis-related proteins were detected using western blot. ***P < 0.001 vs sh-NC

PRKN expression is decreased in A549 cells and ubiquitinates DYNLL1

In A549 cells, the mRNA and protein expressions of PRKN were significantly decreased (Fig. 4A). To increase or decrease PRKN expression, oe-PRKN or sh-PRKN was transfected into A549 cells and the transfection efficacy was examined. The results showed that oe-PRKN significantly increased PRKN expression while sh-PRKN significantly decreasing PRKN expression. Of note, sh-PRKN-1 demonstrated better transfection efficacy, and thus it was selected for follow-up studies (Fig. 4B). In vitro ubiquitination experiments demonstrated that the level of DYNLL1 ubiquitination was reduced in PRKN-overexpressed A549 cells and elevated in PRKN-silenced A549 cells (Fig. 4C). Through a CHX test, the effects of PRKN on DYNLL1 stability were investigated and it was found that PRKN elevation facilitated DYNLL1 degradation whereas PRKN downregulation extended the duration of DYNLL1 in A549 cells (Fig. 4D).

Fig. 4.

PRKN expression is decreased in A549 cells and ubiquitinates DYNLL1. A PRKN expression was detected using RT-qPCR and western blot. ***P < 0.001 vs BEAS-2B. B The transfection efficacy of oe-PRKN and sh-PRKN was detected using RT-qPCR and western blot. ***P < 0.001 vs oe-NC, ^###P < 0.001 vs sh-NC. C In vitro ubiquitination experiments were conducted. **P < 0.01 vs sh-NC and ***P < 0.001 vs oe-NC or sh-NC. D A CHX test was conducted to detect ubiquitination level. *P < 0.05, **P < 0.01 and ***P < 0.001 vs 0 h

Silencing PRKN partially counteracted the inhibitory effects of DYNLL1 knockdown on A549 cell proliferation

To explore the mechanism of DYNLL1 related with PRKN in LUAD cell phenotypes, sh-DYNLL1 and sh-PRKN were co-transfected into A549 cells and above functional experiments were performed again. As shown in Fig. 5A, B, DYNLL1 knockdown significantly decreased A549 cell viability, which was then partially revived following the transfection with sh-PRKN. Similarly, the decreased number of colonies in A549 cells transfected with sh-DYNLL1 was increased after silencing PRKN expression (Fig. 5C).

Fig. 5.

Silencing PRKN partially counteracted the inhibitory effects of DYNLL1 knockdown on A549 cell proliferation. A CCK-8 assay detected cell proliferation. B EdU staining detected cell proliferation. C Colony formation assay detected the colony forming ability of LUAD cells. **P < 0.01 and ***P < 0.001 vs Control, ^#P < 0.05, ^##P < 0.01 and ^###P < 0.001 vs sh-DYNLL1 + sh-NC

Silencing PRKN partially counteracted the promotive impacts of DYNLL1 knockdown on A549 cell cycle arrest and apoptosis

It is found that DYNLL1 interference significantly decreased the cell population in S phase, which was subsequently elevated by PRKN knockdown (Fig. 6A). Additionally, PRKN silencing was also found to inhibit A549 cell apoptosis, accompanied with decreased caspase3 activity, reduced Bax content and increased Bcl-2 content (Fig. 6B–D).

Fig. 6.

Silencing PRKN partially counteracted the promotive impacts of DYNLL1 knockdown on A549 cell cycle arrest and apoptosis. A Flow cytometry analysis detected cell cycle. B TUNEL detected cell apoptosis. C Caspase3 activity was detected using capase3 assay kit. D The expressions of apoptosis-related proteins were detected using western blot. ***P < 0.001 vs Control, ^##P < 0.01 and ^###P < 0.001 vs sh-DYNLL1 + sh-NC

Discussion

LUAD still remains to be a primary cause of cancer-related deaths worldwide [18]. DYNLL1 has been implicated to play complicated roles in human cancers [19, 20]. In this study, DYNLL1 was identified as an oncogene in accelerating LUAD development. First of all, DYNLL1 expression was increased in LUAD tissues and cells and DYNLL1 elevation had negative correlation with the clinical prognosis of patients with LUAD. Subsequently, DYNLL1 was evidenced to be a substrate receptor in E3 ligase PRKN and DYNLL1 could be ubiquitinated and degraded by E3 ligase PRKN. Mechanistically, DYNLL1 silencing inhibited A549 cell proliferation while accelerating cell cycle arrest and apoptosis. Moreover, PRKN could ubiquitinate DYNLL1 and PRKN knockdown reversed the protective role of DYNLL1 interference in the malignant biological behaviors of LUAD cells, preliminarily showing the promising of DYNLL1 in anti-pulmonary tumor treatment.

DYNLL1, which is a homodimeric sequence-specific chaperone, can facilitate the ordered oligomerization of more than a hundred protein targets [21, 22]. Existing researches have corroborated the complex roles of DYNLL1 in numerous human cancers. For instance, the upregulation in DYNLL1 can increase the proliferation of breast cancer [13]. DYNLL1 expression is aberrantly expressed in HCC cells and it can accelerate the malignant progression of HCC through the promotion of cell cycle [14]. Additionally, the inhibition of DYNLL1 by DLEU1 can suppress ESCC cell apoptosis to promote tumor growth [15]. Noticeably, according to UALCAN database, DYNLL1 expression is increased in the tissues of LUAD patients and DYNLL1 upregulation is correlated with the low overall survival rate of LUAD patients. However, the specific role of DYNLL1 is yet to be elucidated. Results of this paper found that DYNLL1 expression was highly expressed in A549 cells. When DYNLL1 expression was silenced, the proliferation of A549 cells was inhibited while cell cycle arrest and cell apoptosis were promoted, accompanied with elevated caspase3 activity, reduced Bcl-2 expression and increased Bax expression.

E3 ubiquitin ligases play important roles in cellular processes through ubiquitinating substrate proteins in the development of human diseases, such as cancer [23]. As a 465-aa E3 ubiquitin ligase, PRKN contains an N-terminal ubiquitin-like domain and C-terminal double Ring finger motifs lined with an IBR domain [24]. It is revealed that PRKN is involved in cancer progression. Wu et al. have showed that the deubiquitination of PRKN mediated by USP26 can inhibit mitophagy in colorectal cancer [25]. In addition, PRKN expression is increased in LUAD tissues and cells [10]. Consistent with the above-mentioned findings, we also found a downregulation in PRKN in A549 cells. DYNLL1 can be ubiquitinated as a substrate [15]. According to UbiBrowser database, E3 ubiquitin ligase PRKN can modulate DYNLL1. Our in vitro ubiquitination experiments demonstrated that PRKN overexpression could decrease DYNLL1 ubiquitination while PRKN interference could increase it. The CHX test revealed that PRKN elevation promoted DYNLL1 degradation while PRKN downregulation extended the duration of DYNLL1 in A549 cells.

Importantly, the upregulation of PRKN by miR-181c-5p suppression can inhibit LUAD cell viability, migration and invasion [10]. The upregulation of PRKN can inhibit the proliferation and migration of bladder cancer cells [26]. To explore the mechanism of DYNLL1 related with PRKN in LUAD malignant cell phenotypes, sh-DYNLL1 and sh-PRKN were co-transfected into A549 cells and functional experiments were performed. It was found that the significant effects of DYNLL1 silence in inhibiting LUAD malignant progression was partially abolished by PRKN interference.

To summarize, we firstly revealed the role of DYNLL1 in LUAD cell cycle arrest and apoptosis and the mechanism by which DYNLL1 participates in LUAD through the ubiquitination and degradation by E3 ligase PRKN.

Supplementary Information

Below is the link to the electronic supplementary material.

Author contributions

Hongjie Tang and Changjiang Wei conceived the experiments. Hongjie Tang and Changjiang Wei performed the experiments. Hongjie Tang analyzed the data. Hongjie Tang and Changjiang Wei confirmed the authenticity of all the raw data. Both authors have read and approved the final manuscript.

Funding

None.

Data availability

The analyzed data sets generated during the present study are available from the corresponding author on reasonable request.

Declarations

Competing interests

The authors declare no competing interests.