Compared with hPCL3L, hPCL3S was significantly up-regulated in NSCLC and associated with lower survival in NSCLC patients

There are two isoforms of hPCL3, hPCL3S (848 nt) and hPCL3L (4,149 nt), which share the same sequence as the first 578 nt (Fig 1A). The critical efficiency of hPCL3S or hPCL3L in survival of patients with NSCLC was explored using Kaplan–Meier plotter (www.kmplot.com) (Győrffy, 2021). Specific Affymetrix GeneChip probe was selected as shown in Fig 1A to distinguish the expression level of hPCL3S and hPCL3L. Kaplan–Meier survival analyses were performed in 1925 NSCLC patients. The results indicated that higher hPCL3S or hPCL3L mRNA expression in patients with NSCLC was significantly correlated to lower over survival, in which hPCL3S (HR 1.13 [1.11–1.55], P = 0.0011) (Fig 1B) showed a more significant correlation than that of hPCL3L (HR 1.21 [1.03–1.43], P = 0.022) (Fig 1C).

The expression of hPCL3S and hPCL3L in tumor and normal tissue of 46 patients with NSCLC were quantified by Western blotting (Fig 1D). The results demonstrated that the expression of hPCL3S is significantly increased in the tumor of patients with NSCLC compared with that of normal tissues (Fig 1E), whereas the expression of hPCL3L was not significantly changed (Fig 1F). These results suggested hPCL3S may play a more powerful role in promoting NSCLC progression compared with hPCL3L.

Knockout of hPCL3S shows more powerful effect on suppressing NSCLC cell growth and mobility in vitro compared with that of hPCL3L

Next, we intended to investigate the effect of hPCL3S and hPCL3L on NSCLC progression. Therefore, hPCL3S (Fig S1A and B) or hPCL3L (Fig S1C and D) was knocked out in NSCLC A549 and NCI-H226 cells using the CRISPR/Cas9 system. Knockout efficiencies were confirmed by Western blotting (Fig S2). First, the colony formation assays were performed by growing hPCL3S-KO, hPCL3L-KO, and control cells for 14 d. We found both the colony formation capacity of hPCL3S-KO and hPCL3L-KO cells were severely inhibited compared with control cells (Fig 2A and B). Moreover, the hPCL3S-KO cells reflected lower colony formation rate than hPCL3L-KO cells.

Subsequently, we examined the effects of hPCL3S and hPCL3L on cell cycle progression in A549 cells and NCI-H226 cells by using flow cytometry assay. Cell cycle analysis revealed that hPCL3S-KO increased the proportion of cells in the G0/G1 phase of A549 cells (from 48% to 70%) and NCI-H226 cells (from 40% to 58%) and accompanied with a reduction in the proportion of A549 cells (from 25% to 15%) and NCI-H226 cells (from 22% to 18%) in G2/M phase. Knockout of hPCL3L also led to a moderate increase in the proportion of A549 cells in G0/G1 phase (from 48% to 52%) and accompanied with a reduction in the proportion of A549 cells in the S phase (from 20% to 15%) and G2/M phase (from 25% to 17%) (Fig 2C–E). The Western blotting results showed that both hPCL3S and hPCL3L knockout induced p21 expression and inhibited cyclin D1 (CCND1) and CDK4 in A549 and NCI-H226 cell lines; the change rate in hPCL3S cells was higher than that in hPCL3L cells (Fig 2F). These data demonstrate that knockout of both hPCL3S and hPCL3SL inhibits cell proliferation possibly through arresting cell cycle at G0/G1 phase, whereas hPCL3S knockout has a stronger effect on inhibiting cell proliferation in NSCLC. In addition, results of apoptosis assays indicated that both hPCL3S and hPCL3L knockout significantly increased the percentage of apoptotic cancer cells compared with the control A549 and NCI-H266 cells (Fig 2G and H). Moreover, the hPCL3S-KO cells showed a higher efficiency of inducing apoptosis than that of the hPCL3L cells. The Western blotting results showed that hPCL3S-KO–induced BAX and BAD expression and inhibited BCL2 and MCL1 in A549 and NCI-H226 cells (Fig 2I). Furthermore, hPCL3L-KO showed less effect in regulating the expression changes of these proteins. In the transwell assay, the invasiveness of A549 and NCI-H226 cells both in the hPCL3S-KO and hPCL3L-KO group was significantly reduced compared with control cells. Moreover, the invasiveness of the hPCL3S-KO group was significantly lower than that of the hPCL3L-KO group in both A549 and NCI-H226 cells (Fig 2J–L). Overall, these results indicated that both hPCL3S and hPCL3L knockout inhibited NSCLC cells proliferation, invasion, and induced its apoptosis and migration. Our results also implied the suppression effect of hPCL3S showed a deeper impact than that of hPCL3L.

Knockout of hPCL3S exhibits a higher inhibitory effect on tumor growth and metastasis in NSCLC cells compares with hPCL3L knockout

Furthermore, we confirmed the pro-tumorigenic role of hPCL3S and hPCL3L in vivo. We tested the effect of hPCL3S-KO and hPCL3L-KO cells on tumor growth in Balb/c nude mice. Compared with the control group, both the hPCL3S-KO and hPCL3L-KO groups suppressed tumor growth in mice, resulting in significant reductions in tumor size (Fig 3A), volume (Fig 3B), and weight (Fig 3C). Also, the tumor size, volume, and weight of the hPCL3S-KO group were significantly smaller or less than that of the hPCL3L-KO group. To establish a metastasis model, control, hPCL3S-KO, or hPCL3L-KO cells were injected into the tail vein of Balb/c nude mice. Mice were euthanized, and lung metastases were counted 42 d post-injection. The number of lung metastases was significantly decreased in both hPCL3S-KO and hPCL3L-KO groups compared with the control group, whereas the hPCL3S-KO group had less metastases than the hPCL3L-KO group (Fig 3D and E). The Kaplan–Meier survival assay showed that the cumulative survival rate and median survival time in hPCL3S-KO mice significantly decreased compared with that in hPCL3L-KO and control mice (log-rank tests, P < 0.05) (Fig 3F). These results demonstrate that hPCL3S and hPCL3L played an intrinsic role in promoting tumor formation and progression. Besides, hPCL3S showed a significantly more powerful effect than hPCL3L.

TRIM21 interacts with hPCL3S and hPCL3L

To further explore the subcellular localization and expression of hPCL3S and hPCL3L in NSCLC cells, immunofluorescence assays and Western blotting analysis were performed in A549 cells and NCI-H226 cells. As showed in Fig 4A, hPCL3S was detected in both cytosol and nucleus. But hPCL3L was only detected in the nucleus of both A549 cells and NCI-H226 cells. The Western blotting assay validated this conclusion (Fig 4B). To gain insight into the interacting proteins of hPCL3S and hPCL3L, SDS–PAGE and silver staining were performed after immunoprecipitation (IP) of hPCL3S and hPCL3L (Fig 4C). The top ten ranked proteins of hPCL3S and hPCL3L were identified by mass spectrometry, respectively (Fig 4D). In addition, Western blotting of the IP product confirmed that TRIM21 was one of the proteins that interact with both hPCL3S and hPCL3L (Fig 4E and F). Furthermore, co-immunoprecipitation (Co-IP) experiments in 293T cells revealed that TRIM21 bound to hPCL3L and hPCL3S in 293T cells (Fig 4I). However, FLAG-tag hPCL3L did not interact with GFP-tag hPCL3S (Fig 4G and H). Taken together, these findings suggested that TRIM21 could directly interact with both hPCL3S and hPCL3L.

TRIM21 promotes the degradation of hPCL3S but not hPCL3L in NSCLC cells

Previous studies illustrated that tripartite motif containing-21 (TRIM21) is a cytosolic ubiquitin ligase (Frank et al, 1993; Reymond et al, 2001; Ozato et al, 2008; Strandberg et al, 2008). Protein ubiquitination leads to several posttranslational effects (Bednash & Mallampalli, 2016; Tsuchida et al, 2017). Thus, the effects of TRIM21 on hPCL3S and hPCL3L were explored. FLAG-hPCL3S and GFP-hPCL3L were overexpressed in 293T cells. After overexpressed GST-TRIM21, only FLAG-hPCL3S expression level was reduced (Fig 5A). Moreover, the expression levels of hPCL3S and hPCL3L were detected in TRIM21-KD and TRIM21-overexpressed A549 cells and NCI-H226 cells using Western blotting. The results showed that hPCL3S protein expression level was elevated in TRIM21-KD A549 (Fig 5B) and NCI-H226 cells (Fig 5C) and was reduced in TRIM21-overexpressed A549 (Fig 5D) and NCI-H226 cells (Fig 5E). In contrast, the expression level of TRIM21 did not influence the expression of hPCL3L. In addition, we examined whether the hPCL3S and hPCL3L protein stability could be modulated by TRIM21 using a cycloheximide (CHX) chase assay. As showed in Fig 5F, hPCL3S protein sharply decreased within ∼2 h in TRIM21-overexpressed A549 and NCI-H226 cells compared with control cells (Fig 5G and H). Whereas overexpression of TRIM21 does not affect the half-life of hPCL3L in A549 (Fig 5I) and NCI-H226 cells (Fig 5J).

TRIM21 mediates K48-linked ubiquitination of hPCL3S and K63-linked ubiquitination of hPCL3L

Furthermore, we speculated that TRIM21 might regulate hPCL3S and hPCL3L expression by participating in their ubiquitination. Thus, co-IP and in vitro ubiquitination assay were conducted and revealed that the up-regulation of TRIM21 increased the ubiquitination level of hPCL3S (Fig 6A) and hPCL3L (Fig 6C), whereas TRIM21 overexpression only resulted in the degradation of hPCL3S (Fig 6A), the expression level of hPCL3L was not changed (Fig 6C).

Ubiquitination leads to diverse cellular processes dependent on the linkages in the ubiquitin chain (French et al, 2021). Given that hPCL3S and hPCL3L showed distinguish fate after TRIM21 knockdown or overexpression, we postulated ubiquitin linkages on hPCL3S and hPCL3L are different. There are seven distinct ubiquitin lysine residues associated with ubiquitin linkages (K6, K11, K27, K29, K33, K48, and K63). Thus, we investigated the type of linkage in hPCL3S by transfecting FLAG-hPCL3S and GST-TRIM21 in 293T cells along with ubiquitin mutants containing arginine substitution at the indicated position. Co-IP and Western blotting indicated that the polyubiquitin linkage profiles from K6, K11, K27, K29, K33, and K63 ubiquitin showed consist with WT ubiquitin, but mutations in K48 of ubiquitin severely impaired hPCL3S ubiquitination (Fig 6B). A different result was obtained from hPCL3L. The polyubiquitin linkage profiles from K6, K11, K27, K29, K33, and K48 ubiquitin showed normal performance, nevertheless, mutations in K63 of ubiquitin severely impaired hPCL3L ubiquitination (Fig 6D). These results revealed that hPCL3S is predominantly modified by K48-linked ubiquitin and hPCL3L predominantly modified by K63-linked ubiquitin.

Clinical specimens

Human NSCLC specimens and the adjacent non-cancerous tissues were collected from the First Affiliated Clinical Medical College of Zhejiang Chinese Medical University with informed consent. The study was approved by Ethics Committee of the First Affiliated Clinical Medical College of Zhejiang Chinese Medical University (ethical approval number 2022-KL-099).

Cell lines and culture

Two NSCLC cell lines including A549 (adenocarcinoma) and NCI-H226 (squamous cell carcinoma) were obtained from the National Collection of Authenticated Cell Culture of Chinese Academy of Sciences. All cells were cultured according to their instructions in an incubator with 95% humidified air containing 5% CO₂ at 37°C.

Generation of the knockout cell line with CRISPR/Cas9

gRNA sequences for CRISPR/Cas9 were designed at CRISPick (https://portals.broadinstitute.org/gppx/crispick/public). The sequence of hPCL3S gRNA is 5′-CTCTCCTGCCTCCTCCAGTG-3′. The sequence of hPCL3L gRNA is 5′-GGCCCGGAGAATGGTACCTG-3′. The sequence of control gRNA is 5′-GGGCGAGGAGCTGTTCACCG-3′. The complementary oligonucleotides for gRNA were annealed and cloned into pLentiCRISPRv2 Neo. These plasmids were then co-transfected with psPax2 and pCMV.VSV.G in a ratio of 10:10:1 into HEK293T cells (ATCC) using Lipofectamine 2000 (Thermo Fisher Scientific) or JetPrime (Polyplus). Lentiviral supernatants were harvested in 48 h. A549 cells and NCI-H226 cells were seeded in six-well plates with 80% confluency. Lentiviral supernatants were added into the cells with 8 μg/mL polybrene. Transduced cells were then selected by neomycin for 3–6 d using a concentration based on killing curves. Surviving cells were subjected to single cloning.

Colony formation assay

hPCL3S-KO, hPCL3L-KO, and control A549 cells (cells/well) and hPCL3S-KO, hPCL3L-KO, and control NCI-H226 (100 cells/well) were seeded into six-well dishes. The medium was refreshed every 2 d. Plates were incubated for 9 d until colonies had formed. Then, the cells were fixed with 4% paraformaldehyde for 30 min at room temperature and stained with 0.5% crystal violet aqueous (Beyotime) for 20 min. The cell colonies with 50 cells or more were counted. After being photographed, the dye was extracted using 33% acetic acid and quantified using a spectrophotometer at 595 nm. The assay was repeated three times for each cell line.

Cell cycle analysis

hPCL3S-KO, hPCL3L-KO, and control A549 cells (cells/well) and hPCL3S-KO, hPCL3L-KO, and control NCI-H226 were harvested, washed three times in PBS, and then fixed overnight in 70% alcohol. After being fixed, cells were incubated with RNase digestion (200 μg/mL) at 37°C for 1.30 h, then incubated with PI (10 μg/mL) for 30 min. Cells were analyzed by a BD Accuri C6 flow cytometer (BD Biosiences), and cell cycle phase distribution was analyzed by using FlowJo VX0.7 (FlowJo LLC). The experiments were performed independently in triplicate for each cell line.

Cell apoptosis analysis

Annexin V-FITC Apoptosis Detection Kit (BD Biosciences) was used to quantify the levels of apoptosis according to the manufacturer’s instructions. Briefly, hPCL3S-KO, hPCL3L-KO, and control cells were harvested, washed three times in PBS, and incubated with Annexin V-FITC and propidium iodide. Flow cytometry data were acquired on BD Accuri C6 flow cytometer and the data were analyzed by using FlowJo VX0.7. The experiments were performed independently in triplicate for each cell line.

In vitro transwell invasion assay

Transwell cell invasion assays were performed as previously described (Tao et al, 2016). Briefly, 24-well plate inserts (8 μm) were pre-coated with 30 μl Matrigel matrix (diluted at 1:3 with serum-free culture medium) (BD Biosciences) and incubated at 37°C to form a gel. Then, cells were suspended with 250 μl and added into the upper transwell chamber. The lower chamber was filled with 500 μl culture medium with 10% FBS. After incubation at 37°C for 24 h, non-invaded cells on the upper chamber were scraped with a cotton swab. Invaded cells were fixed with 100% methanol and stained with 0.05% crystal violet. Images were taken, and the invaded cells were counted manually. The experiments were performed independently in triplicate for each cell line.

Western blotting analysis

Total protein of treated cells was lysed by RIPA buffer supplemented with protease and phosphatase inhibitors. Equal amounts of proteins were separated by 10% SDS–PAGE and transferred to polyvinylidene fluoride (PVDF) membranes (Millipore), and 5% non-fat milk was used to block PVDF membranes for 1 h at 37°C and incubated overnight at 4°C with primary antibodies. Then, PVDF membranes were washed three times with PBS. Appropriate HRP-linked secondary antibody were added and incubated for 1 h at room temperature. Finally, ECL (Advansta) was used to visualize the bands of the proteins. Quantification of the immunoblots was performed using ImageJ software (version 1.43u). The antibodies used were anti-hPCL3S (SAB1400484, dilution 1,000; Sigma-Aldrich), anti-hPCL3L (#77271, dilution 1,000; CST), anti-TRIM21 (#92043, dilution 1,000; CST), anti-CCND1 (ab134175, 1,000; Abcam), anti-CDK4 (ab108357, 1,000; Abcam), anti-p21 (ab109520, 1,000; Abcam), anti-BAX (ab32503, 1:10,00; Abcam), anti-BAD (ab32445, 1,000; Abcam), anti-BCL2 (ab182858, 1,000; Abcam), anti-MCL1 (ab28147, 1:1,000; Abcam), anti-β-ACTIN (#ab6276, dilution 1:5,000; Abcam).

Animal studies

All animal procedures were conducted in accordance with the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals and were approved by the Institutional Animal Care and Use Committee of Zhejiang Chinese Medical University (ethical approval number IACUC-2022050706). For the xenograft experiment, 4–6-wk-old male Balb/c nude mice were subcutaneously injected with 5 × 10⁶ of tumor cells (resuspended in PBS with 50% Matrigel) and to the back of the right lower limb. Six mice were performed in each group. Tumor volumes were measured each other day after palpable tumors appeared. After 24 d of post injection, mice were euthanized and tumors were surgically isolated, weighed, and photographed. For experimental lung metastasis, tumor cells were injected into the 4-wk-old Balb/c nude mice through tail veins. 6 wk later, the mice were euthanized, with the liver tissues dissected, fixed in 10% formalin, examined under the microscope for metastatic foci. The use and care of animals were approved by the Laboratory Animal Center of Zhejiang Chinese Medical University (ethical approval number IACUC-2022050706).

Co-IP

Cells were transiently transfected with the indicated plasmids using Lipofectamine 2000 reagents. After 48 h of cultivation, the cells were washed and resuspended in lysis buffer. Equal amounts of cleared cell lysates were subjected to immunoprecipitation with GFP or FLAG antibody and protein G-Sepharose beads. The immune complexes and input lysates were detected by immunoblot analysis.

Immunofluorescence assays

A549 and NCI-H225 cells were seeded onto coverslips and washed with PBS and fixed in 4% paraformaldehyde in PBS for 20 min, permeabilized with 0.1% Triton X-100 for 2 min at room temperature and blocked with 5% normal goat serum. Anti-hPCL3S or anti-hPCL3L antibodies were added and incubated at 4°C overnight. After being washed three times in PBS, secondary antibodies were added and incubated for 1 h at room temperature. Finally, the nucleus was stained with 4′,6-diamidino-2-phenylindole (DAPI) for 2 min. Labeled cells were examined under the fluorescence microscope.

CHX chase assay

Cells were transiently transfected with TRIM21 plasmid or empty control. After 48 h of cultivation, the medium was refreshed before CHX was added. CHX (10 μg/mL) were added to the culture and cells were harvested at the indicated time points. Total protein was extracted as described previously and resolved by Western blotting with following primary antibodies: anti-hPCL3S (SAB1400484, dilution 1:1,000; Sigma-Aldrich), anti-hPCL3L (#77271, dilution 1:1,000; CST), anti-TRIM21 (#92043, dilution 1:1,000; CST), anti-β-ACTIN (#ab6276, dilution 1:5,000; Abcam).

Ubiquitination assay

For in vitro ubiquitination assays, 293T cells were transfected with the indicated plasmids using Lipofectamine 2000. After 48 h, cells were treated with 0.25 μM MG132 for 24 h. After treatment, cell lysates were immunoprecipitated (IP) with the labeled antibodies. The eluted proteins were determined by Western blotting.

Statistical analysis

To compare two groups, the normality of the data is first evaluated with the Shapiro–Wilk test, and variance is assessed using the Levene test. If the data are normally distributed, a t test is applied. For samples that do not follow a normal distribution, a Mann–Whitney test is used for equal variances, and a median test is used for unequal variances. When more than two groups are compared, a residual test is performed to study normality, and homoscedasticity is assessed with the Levene test. Depending on the distribution of the data, an ANOVA, Brown–Forsythe, Kruskal–Wallis, or median test is performed. Paired comparisons are further explored using post hoc tests, either Dunnet or Bonferroni. All analyses are two tailed. Error bars represent either the SD. Survival analyses are conducted using the log-rank test. All statistical analyses are performed with GraphPad software, version 8 (*) P < 0.05, (**) P < 0.01, (***) P < 0.001.