Chen et al. BMC Molecular and Cell Biology
https://doi.org/10.1186/s12860-023-00469-2(2023) 24:7

BMC Molecular and Cell
BiologyOpen Access

RESEARCH

RPL11 promotes non-small cell lung cancer
cell proliferation by regulating endoplasmic
reticulum stress and cell autophagy
Jie Chen1,2†, Changda Lei3†, Huahua Zhang1,2†, Xiaoyong Huang1,2, Yang Yang1,2, Junli Liu1,2, Yuna Jia1,2, Haiyan Shi1,2,
Yunqing Zhang4*, Jing Zhang1,2* and Juan Du1,2*
Abstract
Background Abnormal biogenesis and ribosome free function of ribosomal proteins (RPs) is important for
tumorgenesis and development. Ribosomal protein L11 (RPL11) is a component of ribosomal 60 S large subunit withdifferent roles in different cancers. Here, we aimed to unravel the role of RPL11 in non-small cell lung cancer (NSCLC),
especially those affecting cell proliferation.
Methods RPL11 expression in NCI-H1650, NCI-H1299, A549 and HCC827 and normal lung bronchial epithelial cells
HBE was detected using western blotting. The function of RPL11 in NSCLC cells were determined by investigating cell
viablity, colony formation and cell migration. Mechanism expoloration of RPL11 effect on NSCLC cells proliferation
was explored using flow cytometry, and the effect on autophagy was investigated by the additon of autophagy
inhibitor chloroquine (CQ) and endoplasmic reticulum stress (ERS) inhibitor tauroursodeoxycholic acid (TUDCA).
Results RPL11 was highly expressed in NSCLC cells. Extopic expression of RPL11 promoted NCI-H1299 and A549 cells
proliferation, and migration, and promoted the transition from the G1 phase to the S phase of the cell cycle. Small
RNA interference of RPL11 (siRNA) suppressed NCI-H1299 and A549 cells proliferation and migration and arrested thecell cycle in G0/G1 phase. Moreover, RPL11 promoted NSCLC cell proliferation by modulating autophagy and ERS.
Expression levels of autophagy and ERS markers were induced by RPL11 overexpression and inhibited by siRPL11.
CQ partially suppressed RPL11-induced A549 and NCI-H1299 proliferation: CQ addition reduced RPL11-induced
cells viability and clone numbers and reversed the cell cycle process. ERS inhibitor (TUDCA) partially reversed RPL11induced autophagy.

†

Jie Chen, Changda Lei and Huahua Zhang contributed equally tothis work.
*Correspondence:
Yunqing Zhang
zhangyq2881123@163.com
Jing Zhang
yadxzj@163.com
Juan Dudujuan123du@126.com
Full list of author information is available at the end of the article© The Author(s) 2023. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use,sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) andthe source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in thisarticle are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not includedin the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you willneed to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The
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in this article, unless otherwise stated in a credit line to the data.

Chen et al. BMC Molecular and Cell Biology(2023) 24:7

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Conclusion Taken together, RPL11 has a tumor-promoting role in NSCLC. It promotes the cell proliferation of NSCLC
cells by regulating ERS and autophagy.
Keywords Non-small cell lung cancer (NSCLC), RPL11, Autophagy, Endoplasmic reticulum stress

Introduction
Lung cancer is the primary cause of cancer-related mortality worldwide [1]. It can be divided into two types:
small cell lung cancer (SCLC) and non-small cell lungcancer (NSCLC),of which NSCLC is the most common
type, accounts for approximately 85% of primary lungcancer [2]. Unfortunately, most patients with NSCLC(47%) are diagnosed at advanced stage (stage III/IV),
when the tumor has spread to multiple lymph nodes
and/or distant organs; The 5-year relative survival rate
for patients diagnosed with advanced stage disease isapproximately 6% [3].
Ribosomal Protein L11 (RPL11) is an evolutionallyhighly conserved 60s ribosomal subunit [4], which is
involved not only in protein synthesis but also in cell
cycle progression and cell fate determination. Decreasing RPL11 expression has been reported to impede cellproliferation by reducing the ribosome content andtranslation capacity [5, 6]. Further, beyond its function as a ribosome componet, ribosome-free RPL11 wasalso reported a potential tumor suppressor that inhibited tumor cell proliferation mainly by regulating of
MDM/TP53 axis and/or MYC proto-oncogene (MYC).
Ubiquitin-ligase proto-oncogene (MDM2) is a negative
regulator of tumor protein P53 (TP53) that destabilizes
and suppresses TP53 expression by ubiquitin mediated
TP53 degradation. Cytoplasmic ribosome-free RPL11,
although transported from the nucleus could directlyinteract with and inhibit MDM2 function, thus resulting in the stabilization and activation of TP53 [7–11]. In
nucleus, ribosome-free RPL11 inhibits MYC gene transcription directly by binding to its promoter region: by
doing so, it inhibits ransformation/transcription domain
associated protein recruitment and reduces histone H4acetylation at MYC gene promoter region [12]. In cytoplasm, RPL11 also suppresses MYC activitiy by directly
binding to and recruiting RNA-induced silencing complex to MYC mRNA [13, 14], interacting with MYC proteins and competing with transcriptional coactivators
to suppress MYC activities [15, 16]. In contrast to theaforementioned tumor suppression function of RPL11, aprevious report indicated that RPL11 could be a tumor
promoter factor: increasing nuclear RPL11 expression bysmall nucleolar RNA SNORA18L5 promoted hepatocellular carcinoma development [17]. Therefore, RPL11 maypromote tumor proliferation through mechanisms independent of the MDM2/TP53 or MYC route. However,
the role and mechanism of RPL11 in the development of
NSCLC remain unclear.

In this study, we aimed to explore the potential role of
RPL11 and its underlying mechanisms in NSCLC. The
results of RPL11 expression in NSCLC cell lines, RPL11
effects in NSCLC cells proliferation viability, migration,
ERS, and autophagy using NSCLC cell lines, suggested
that RPL11 promotes NSCLC cells proliferation andmigration by regulating endoplasmic reticulum stressand cell autophagy.

Materials and methods
Cell culture and transient transfection

NCI-H1299 and NCI-H1650 cell lines were purchasedfrom Procell Life Science and Technology Co (Wuhan,
China), whereas HBE, A549, and HCC827 cell lines were
gifted by the Key Laboratory of Environment and Genes
Related to Diseases of Xi’an Jiaotong University. NCIH1299, NCI-H1650, HCC827, and HBE cells were cultured in RPMI-1640 medium (Biological Industries, Beit
Haemek, Israel) containing 10% fetal bovine serum (FBS,
Biological Industries, Beit Haemek, Israel), A549 cells
were cultured in F-12 K medium (Hyclone, Thermo Co.,
USA) supplemented with 10% FBS. All cells were cultured in a humidified incubator at 37 ℃ with 5% CO2.
For vector and small interference RNAs (siRNA) transfection, jetPRIME reagent (polyplus-transfection SA) wasused according to the manufacturer’s instructions. RPL11
overexpression vector were constructed by inserting theflag-conjugated RPL11 sequence into the GV141 vector.
siRNA fragments of RPL11 (siRPL11) were synthesized by
GenePharma (Shanghai, China). The siRPL11 sequences
are si-1: Sense, 5’-GUAUGAUGGGAUCAUCCUU-3’,
Antisense, 5’-AAGGAUGAUCCCAUCAUAC-3’; si-2:
Sense, 5’-GCTUAGAUACACUGUCAGAU-3’, Antisense,
5’-AUCUGACAGUGUAUCUAGC-3’;
si-3:
Sense, 5’-GGUGCGGGAGUAUGAGUUA-3’, Antisense,
5’-UAACUCAUACUCCCGCACC-3’.
The
negative control siRNA (si-nc) sequences are Sense,
5’-UUCUCCGAACGUGUCACGU-3’,
Antisense,5’-ACGUGACACGUUCGGAGAA-3’.
Western blotting analysis

Proteins were isolated using a radio Immunoprecipitation assay buffer supplemented with a cocktail of protease and phosphatase inhibitors (TargetMol, MA, USA).
Protein concentration was quantified by a BCA protein
assay kit (Proteintech, Wuhan, China). Next, equivalentamounts of protein samples were separated by sodium
dodecyl sulfate-polyacrylamide gel electrophoresis andtransferred to a polyvinylidene difluoride membrane.

Chen et al. BMC Molecular and Cell Biology(2023) 24:7

For western blotting, the unbound sites of the membrane
were blocked, and the membrane was sequentially incubated with primary and secondary antibodies and finallywith ECL to detect the protein of interest and photographed using Syngene GBox (Syngene, Cambridge, UK).
The primary and secondary antibodies used are list as follows: ribosmal protein L1 (RPL11) (Proteintech, 162771-AP), cyclin D1 (CCND1) (Proteintech, 26939-1-AP),heat shock protein family A (Hsp70) member 5 (HSPA5/BiP) (Proteintech, 66574-1-Ig), activating transcription
factor 4 (ATF4) (Proteintech, 10835-1-AP), DNA damage inducible transcript 3 (DDIT3/CHOP) (Proteintech,
15204-1-AP), microtubule associated protein 1 lightchain 3 beta (MAP1LC3B/LC3) (Proteintech, 14600-1AP), beclin 1 (BECN1) (BOSTER, BM5181), sequestosome 1 (SQSTM1/p62) (BOSTER, M00300-1), CDK4
(Proteintech, 11026-1-AP), FLAG (Proteintech, 660083-1 g), phosphorylated endoplasmic reticulum to nucleus
signaling 1 (phospho-ERN1/phospho-IRE1) (Affinity,
AF7150), endoplasmic reticulum to nucleus signaling
1 (ERN1/IRE1) (Affinity, DF7709), actin beta (ACTB/βactin) (Proteintech, 66009-1-Ig).
Reverse transcriptionquantitative (RT-q) polymerase chain
reaction (PCR)

Total RNA was extracted using TRIzol® reagent (Invitrogen; Thermo Fisher Scientific, Inc.), EasyScript® One-Step
gDNA Removal and cDNA Synthesis SuperMix (TransGen Biotech, Beijing) were used to reverse transcribe
and further examine the gene expression. The qPCR processes were as follows: 95 ˚C for 5 min; 95 ˚C for 10 s,
60 ˚C for 1 min, 72 ˚C for 30 s (40 cycles); and 94 ˚C for
90 s, 60 ˚C for 3 min gradully heating from 60 ˚C to 94 ˚C
at a speed of 1 ˚C per minute. The relative gene expression levels were normalized to that of β-actin. The primer
sequences were as follows:
RPL11
Forward:5’AAAGGTGCGGGAGTATGAGTT-3’,
Reverse:
5’TCCAGGCCGTAGATACCAATG-3’ ;β-actin Forward: 5’CCAACCGCGAGAAGATGA-3’,
Reverse: 5’CCAGAGGCGTACAGGGATAG-3’;
CCND1
Forward:5’ATCTACACCGACAACTCCATC-3’, Reverse: 5’TGTTCTCCTCCGCCTCTG-3’;
CDK4
Forward:
5’ATGGCTACCTCTCGATATGAGC-3’,
Reverse:5’CATTGGGGACTCTCACACTCT-3’.
The above experiments followed the manufacturer’s
protocol. The relative expression levels were calculatedusing the 2-∆∆Ct method.

Page 3 of 12USA) was used according to the manufacturer’s instructions to determine cell viablity in proliferation .
Clone formation assay

Pre-treated cells were seeded into 12-well plates at a density of 800 (A549) or 1000 (NCI-H1299) cells per well.
After 8 days of culture, the cells were fixed using 4% paraformaldehyde, washed with phosphate-buffered saline(PBS), stained with 0.5% crystal violet(Sigma Aldrich,
Merck KGaA), and imaged using a camera.
Scratch wound healing assay

Pre-transfected cells were seeded into 6-well plates,grown to 80-90% confluency, followed by a scratch
made on the monolayer in the center with a 10 µL sterile
pipette tip. The cells were washed with PBS and further
cultured in 1% FBS-containing medium for 72 h. Magnified images of the cells were captured at 0 h, 24 h, 48hand 72 h after wounding (Nikon, Tokyo, Japan).
Transwell migration assay

Cell migration was examined in a 24-well cell culture
transwell insert system. Transfected A549 and NCIH1299 cells suspended in 200 µL serum-free culture
medium were plated in the upper chamber and 600 µL
complete culture medium were added in the lower chamber. After 48 h incubation, cells in the lower chamber
were fixed with 4% paraformaldehyde for 30 min andstained with 0.1% crystal violet for 30 min. The images
were captured using an inverted microscope (Nikon,
Tokyo, Japan). For quantitative data, the imaged cells
were further dissolved in dimethyl sulfoxide and absorbance at 570 nm was read on the SpectraMax 190 microplate reader ( MD, USA).
Cell cycle analysis

Pre-treated cells were trypsin-digested and centrifuged;
The cell pellet was collected, washed with PBS, and fixedin ice-cold 70% ethanol overnight at 4 ℃. Next, the cells
were further washed with PBS and stained with 50 µg/ml
propidium iodide (Biosharp, China) for 30 min. Distribution of the cell-cycle stages was examined by flow cytometry (Syngene, USA).
Acidic vesicular organelle (AVO) staining.
For AVO staining, 48 h after transfection, cells were
further incubated in 2 µg/mL acridine orange (MedChem Express, USA) containing medium for 20 min. The
cells were then imaged using fluorescence microscopy(Nikon, Tokyo, Japan).

Cell counting kit-8 (CCK-8) assay

Autophagy flux measurement

Expression vectors or siRNAs pre-transfected cells wereseeded into 96-well plates and incubated for 0 h, 24 h,
48 h, and 72 h. Then the CCK-8 reagent (TargetMol,

Autophagy flux assessment by the mRFP-GFP-LC3
method was performed to detect autophagy levels. For
this, 24 h after transfection of the RPL11 overexpression

Chen et al. BMC Molecular and Cell Biology(2023) 24:7

vector (marked as RPL11) or its control vector, NSCLC
cells were further infected with the mRFP-GFP-LC3expressing adenovirus (Hanbio, China) for 48 h according to the manufacturer’s instruction. After that, the cells
were imaged using a laser confocal microscope (Zeiss
LSM800, Germany).
Chloroquine (CQ) and tauroursodeoxycholic acid (TUDCA)
treatment

CQ (HY-17,589 A) and TUDCA (HY-19,696) were allpurchased from MedChemExpress (Shanghai, China)
and dissolved in DMSO. For cell treatment, CQ and
TUDCA were all added 6 h after transfection at a concentration of 20 µM and 2 mM respectively.
Statistical analysis

Experiments were all performed in three independent
replicates. Data was analyzed using GraphPad Prism software version 8.0 (GraphPad Software, San Diego, USA)and presented as the mean ± SD. Unpaired student’s t-test
was used to evaluate the differences in the data between
two groups. One-way analysis of variance (ANOVA)
was used to evaluate the differences in that data of multiple-group. P < 0.05 were considered to be statisticallysignificant.

Results
RPL11 is highly expressed in NSCLC cells and promotes the
NSCLC cells proliferation and migration

To clarify the role of RPL11 in NSCLC, RPL11 expression in lung cancer cells was determined by western blotting. As Fig. 1a shows, RPL11 is relatively more highly
expressed in lung cancer cells than in normal lung bronchial epithelial cells (HBE). To further explore the effectof RPL11 on the biological behavior of NSCLC cells, we
overexpressed RPL11 in A549 and NCI-H1299 cells using
a vector and verified its expression efficiency by western blotting and RT-qPCR (Figure S1a, b). Comparedwith the GV141 vector transfected control group, protein and mRNA levels of RPL11 were increased in both
A549 and NCI-H1299 cells in the RPL11-overexpressedgroup (RPL11): these cells exhibited higher cell proliferation and colony formation capabilities than the control
cells (Fig. 1b and c). Wound healing and transwell assays
further confirmed RPL11 overexpression increased themigration abilities of A549 and NCI-H1299 cells (Fig. 1dand e). Meanwhile, we designed three small interfering
RNA fragments of RPL11 (siRPL11), and verified theirefficiency by western blotting and RT-qRCR (Fig. S1c, d):the protein and mRNA levels of RPL11 were decreased
in both A549 and NCI-H1299 cells transfected with
siRPL11 than in those transfected with the negative control siRNA (si-nc). Additionally, siRPL11 reduced theproliferation and colony formation abilities of A549 andPage 4 of 12

NCI-H1299 cells compared with the si-nc transfected
cells (Fig. 1f g). Moreover, the results of the wound healing assay showed that compared si-nc, siRPL11 transfection inhibited wound closure of A549 and NCI-H1299
cells (Fig. 1h). Transwell assay results showed that the
number of A549 and NCI-H1299 cells that migrated
across the chamber was significantly reduced in thesiRPL11 group than in the control group, indicating an
siRPL11-induced decline in cell migration (Fig. 1i). Taken
together, the above results confirmed an oncogenic roleof RPL11 in NSCLC by regulating cell proliferation andmigration.
RPL11 promotes cell division in NSCLC cells

Next, the effects of RPL11 on the distribution of cell cycle
stages in NSCLC cells were examined using flow cytometry. The analysis revealed that overexpression of RPL11
promoted cell division in A549 and NCI-H1299 cells:
RPL11 overexpression reduced the proportion of cells in
the G0/G1 phase and increased the proportion of cells
in the S phase in A549 and NCI-H1299 cells (Fig. 2a).
In contrast, siRPL11 arrested A549 and NCI-H1299
cells in G0/G1 phase, as the proportion of cells in G0/
G1 phase was increased and that of cells in S phase was
reduced (Fig. 2b). Western blotting to determine changes
in G0/G1 phase-related proteins indicated that RPL11
overexpression increased the protein levels of cyclin
D1 (CCND1) and cyclin-dependent kinase 4 (CDK4)(Fig. 2c), whereas siRPL11 decreased the protein levels of
CCND1 and CDK4 (Fig. 2d). Using RT-qPCR, we verified
that RPL11 overexpression increased CCND1 and CDK4mRNA levels, whereas siRPL11 decreased them (Fig. S1).
Collectively, these data indicate that RPL11 promotes cell
division of A549 and NCI-H1299 and regulates cell proliferation in NSCLC cells.
Effects of RPL11 expression levels on NSCLC cellproliferation via autophagy

To further explore the mechanism by which RPL11 regulates NSCLC cell proliferation, an acridine orange staining (AVO staining) that quantifies the acidic componentsof the cells was used. The results indicated that overexpression of RPL11 significantly promoted AVO formation in A549 and NCI-H1299 cells (Fig. S2). Thus, we
speculated that RPL11 overexpression enhanced NSCLC
cell atuotophagy. To confirm this speculation, an mRFPGFP-LC3 adenovirus was used to detect intracellular
autophagy flux. In this assay, a weakened GFP fluorescence indicates the fusion of lysosome with autophagosome, and red and yellow fluorescent puncta represent
autolysosome and autophagosome after merging, respectively. In our study, RPL11 overexpression significantly
decreased the proportion of yellow puncta while increasing the proportion of red puncta which indicated that

Chen et al. BMC Molecular and Cell Biology(2023) 24:7

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Fig. 1 Ribosomal protein 11 (RPL11) is highly expressed in non-small cell lung cancer (NSCLC) cells and promotes cellular proliferation and migration.
(a) RPL11 protein level in human bronchial epithelial HBE cells and NSCLC cell lines were detected by western blotting. (b-i) Effects of RPL11 overexpression (b, c, d and e) and siRNA knockdown (f, g, h and i) on NSCLC cell viablity (b and f ), colony formation ability (c and g), and migration (d,e, h and i)
were evaluated using the CCK8, colony formation, wound healing assays and transwell migration assays. Data are presented as mean ± SD from three
independent trials (n = 3). Statistical data of a and f was analyzed using one-way ANOVA, statistical data of b was analyzed using unpaired student’s t-test.
*P < 0.05, **P < 0.01, ***P < 0.001

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Fig. 2 Effect of ribosomal protein L11 (RPL11) on cell cycle progression in non-small cell lung cancer (NSCLC) cells. Distribution of the cell cycle stages in
NSCLC cells after overexpression (a) or siRNA-mediated knockdown (b) of RPL11 was assessed by flow cytometry. The levels of cell cycle-related proteins
cyclin D1 (CCND1) and cyclin-dependent kinase (CDK4) in RPL11 overexpressed (c) or siRNA-transfected (d) groups were detected by western blotting.
The blots were cut before hybridization with antibodies during blotting (c, d). The data are presented as mean ± SD from three independent trials (n = 3).
Statastic data of a and c was analyzed using unpaired student’s t-test, statistic data of b and d was analyzed using one-way ANOVA. *P < 0.05, **P < 0.01,***P < 0.001

Chen et al. BMC Molecular and Cell Biology(2023) 24:7

RPL11 overexpression enhanced autophagy flux in A549
and NCI-H1299 cells (Fig. 3a). We further examined therelationship between RPL11 and autophagy-related protein expression in NSCLC cells that were transfected with
RPL11 overexpression plasmids or siRPL11. The expression levels of beclin1 (BECN1) and microtubule-associated proteins 1 A/1B light chain 3B (LC3-II) increased
most markedly in RPL11-overexpressed cells, whereasthat of p62 was significantly decreased. Conversely,
siRPL11 significantly reduced BECN1 and LC3-II expression levels, while increasing the p62 protein expression
level (Fig. 3b). Moreover, chloroquine (CQ), an autophagy inhibitor blocked RPL11 overexpression-induced cell
viability (Fig. 3c) and clone numbers (Fig. 3d), cell cycle
progress (Fig. 3e), and reversed the autogphagy-related
gene expression change (Fig. 3f ).
RPL11 promotes ERS-induced autophagy of NSCLC cells

Next, we explored whether ERS was involved in autophagy. Western blotting was performed to examine theexpressions of ERS marker. The results showed that
RPL11 overexpression in A549 and NCI-H1299 significantly increased ERS-related proteins immunoglobulin binding protein (BiP), activating transcription
factor 4 (ATF4), C/EBP homologous protein (CHOP)
and phospho-inositol-requiring enzyme type 1 (P-IRE1)
level (Fig. 4a). Consistent with these results, siRPL11
decreased the ERS-related protein expression levels
in A549 and NCI-H1299 cell compared with the control transfection group (Fig. 4b). The above data suggestthat RPL11 might induce ERS in A549 and NCI-H1299
cells. Next, an ERS inhibitor, tauroursodeoxycholic acid
(TUDCA), was used to block ERS. Interestingly, TUDCA
not only suppressed the expression of RPL11-induced
ERS-related proteins but also markedly mitigated theexpression of LC3-II and BECN1 and increased theexpression of p62 (Fig. 4c). Thus, RPL11 might induce
autophagy in NSCLC cells by activating ERS.

Discussion
In the present study, the role of RPL11 in NSCLC was
explored. We found that RPL11 was highly expressed
in NSCLC cells and it promoted the proliferation andmigration of NSCLC cells. Mechanically, RPL11 could
promoted NSCLC cell proliferation by regulating ERS
and cell autophagy.
Ribosome biosynthesis is an energy consumption process that involves the binding of ribosomal proteins (RPs)to the ribosome RNA in the nucleolus to tightly regulate protein synthesis. In cancer development, RPs are
involved in cell process of proliferation, migration, invasion, DNA damage repair, and drug resistance and thus
perform tumor-promoting or suppressing activities [18,19]. RPL11 is a previously reported cancer regulator RP,

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known to act as a tumor suppressor in several cancers.
In breast cancer, RPL11 suppressed breast cancer cellproliferation and G1/S cell cycle transition and induced
apoptosis in a TP53-MDM2-dependent way [7]. In livercancer, RPL11-mediated MID1 interacting protein 1
depletion reduced cell viability and colony formation by
inhibiting MYC gene expression [16]. In cervical cancer
and gastric cancer, RPL11 contributed to tumorigenesisby translocating to the cytoplasm and further binding
to MDM2 [20, 21]. Additionally, RPL11 cooperatively
interact with ribosomal protein L5 (RPL5) to inhibitedthe cancer cells proliferation in the way activating TP53
or inactivating MYC [22, 23]. As RPL5 performed both
tumor activator and tumor suppressor in types of cancer
cells [7, 24], it’s possible that RPL11 has similar function
in cancer cell growth. In lung cancer research, Xiaoping Ma et al. showed that the loss of ribosomal protein
S27a expression regulated cell cycle, apoptosis, and proliferation via the RPL11-MDM2-TP53 pathway in lungadenocarcinoma cells[11]. However, they showed nodirect evidence of RPL11 in regulating the lung adenocarcinoma cells proliferation. Our results presented here
showed that RPL11 might be a potential tumor activatorin NSCLC. First, RPL11 showed a relatively high expression level in NSCLC cell lines than in normal HBE cells,
which is consistent with the results of Li H et al. whoreported significantly higher levels of RPL11 proteins inlung adenocarcinoma cells tissues than in normal tissues[11]. Additionally, we investigated the functional effect of
RPL11 in A549 and NCI-H1299 NSCLC cells and foundthat overexpression of RPL11 promoted proliferation,
migration, and cell cycle progression in NSCLC cells,
whereas RNA interference of RPL11 inhibited the same.
As A549 is a TP53 wild-type cell line and NCI-H1299 isa TP53-null cell line, the similar effects of RPL11 overexpression or knockdown on A549 and NCI-H1299 growthindicated that, unlike other cancer types, RPL11 might
regulate NSCLC cell growth in a TP53 independent way.
Cancer cells are characterized by their uncontrolled
proliferation ability, unlike the precise and coordinatedcontrol of normal cell proliferation [25]. Cell cycle regulators cyclin and CDKs, which jointly regulate the cell
cycle process, have proved important for cancer development in several cancers [26]. In NSCLC, overexpression of CCND1, a G1/S transition regulator, occurred in
50% of the NSCLC specimens and was considered relevant to NSCLC prognosis [27]. In this study, we foundthat overexpression of RPL11 increased CCND1 and
CDK4 expression in A549 and NCI-H1299 cells, wherease silencing of RPL11 yielded the opposite result. These
results were consistent with the flow cytometry results
wherein RPL11 overexpression promoted, whereas
siRPL11 suppressed the cell cycle transition from G0/
G1 phase to S phase in A549 and NCI-H1299 cells,

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Fig. 3 Effect of ribosomal protein L11 (RPL11) on autophagy in non-small cell lung cancer (NSCLC) cells. (a) Effect of RPL11 on autolysosome and autophagosome formation in NSCLC cells: cells transfected with RPL11 vector or its control vector GV141 (vector) were infected with mRFP-GFP-LC3 containing adenovirus: 48 h later, the cells were photographed using a laser confocal microscopy. (b) Effect of RPL11 overexpression or siRNA knockdown on
autophagy-related proteins (LC3, BECN1 and p62) expression were detected by western blotting. (c-e) Effect of autophagy inhibitor chloroquine (CQ) on
cell growth in RPL11-overexpressed NSCLC cells determined using the CCK-8 (c), clone formation (d), and flow cytometry (e) assays. (f) Expression levelsof autophagy and G0/G1 phase-related proteins in RPL11 or its control vector transfected cells, and with the addition of 20 µM CQ or an equal volume of
CQ solvent, were examined using western blotting. Blots of b and f were cut before hybridization with antibodies during blotting. The data are presented
as mean ± SD from three independent trials (n = 3). Protein expression comparison between RPL11 overexpression group (RPL11) and control vector
group (Vector) in b was analyzed using Student’s t-test. Statistical data of c, e, f, and comparison of proteins expression in si-nc and siRPL11 groups (b) was
analyzed using one-way ANOVA. *P < 0.05, **P < 0.01, ***P < 0.001

Chen et al. BMC Molecular and Cell Biology(2023) 24:7

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Fig. 4 Ribosomal protein L11 (RPL11) promotes non-small cell lung cancer (NSCLC) cell proliferation through endoplasmic reticulum stress (ERS) activation. (a,b) The effect of RPL11 on ERS-related protein expression levels in NSCLC cells were examined by western blotting. (c) The effect of ERS inhibitor(TUDCA) on ERS and atuophagy-related protein expression in RPL11-overexpressing NSCLC cells was detected by western blotting. The blots (a-c) were
cut before hybridization with antibodies during blotting. The data are presented as mean ± SD from three independent trials (n = 3). Statistical data of a
was analyzed using Student’s t-test, statistical data of b and c was analyzed using one-way ANOVA. *P < 0.05, **P < 0.01, ***P < 0.001

Chen et al. BMC Molecular and Cell Biology(2023) 24:7

indicating that RPL11 promoted NSCLC cells proliferation partly by cell cycle control.
Autophagy is a basic cellular metabolic process that
promotes cellular homeostasis, differentiation, development and survival [28]. In recent years, studies haveshown that autophagy acts as a “double-edged sword”
role in the development and progression of tumors[29]. However, the role of autophagy in cancer cells is
relatively complex, and the inhibition or promotion of
autophagy-mediated cancer may depend on the tumor’s
own background or type. In this study, we first evaluated the autophagy level by acridine orange staining,
and used mRFP-GFP-LC3 double fluorescence system tolabel and track the changes of LC3 and autophagy flux:
the results showed that RPL11 overexpression promoted
AVO formation and autophagic flow in A549 and NCIH1299 cells. LC3-II, BECN1, and p62 are autophagy
markers; during autophagy, LC3-I is modified and processed by the ubiquitin-like systems, including Atg7
and Atg3, to produce LC3-II with a molecular weight of
14kD. Thus, the content of LC3-II is proportional to thedegree of autophagy, and the ratio of LC3-II/I estimating
the level of autophagy [30]. BECN1 is an evolutionarily
conserved protein and is recognized as a positive regulator of autophagy [31]. p62 is a multi-domain adaptor
protein and a key player in autophagy-dependent quality control [32]. In our experimental study, western blotting detected the key autophagic molecules LC3, BECN1,
and p62. RPL11 overexpression increased LC3-II and
BECN1 expression and decreased p62 expression in
A549 and NCI-H1299 cells; silencing RPL11 yielded the
opposite result. Interestingly, we found that the autophagy inhibitor CQ reversed RPL11-induced cell proliferation and autophagy, suggesting that autophagy favoured
NSCLC cell proliferation. Moreover, our previous results
had shown that RPL11-induced increase in CCND1 and
CDK4 expression contributed to the cell cycle transition of A549 and NCI-H1299 cells from the G0/G1 phaseto the S phase. Using autophagy inhibitor CQ, we further confirmed that the RPL11-mediated NSCLC cell
cycle process was autophagy-regulated. CQ reversed
RPL11-induced CCND1 and CDK4 expression and thecell numbers in the S phase. Thus, our results indicatedthat RPL11 regulated NSCLC proliferation through an
autophagy-regulated cell cycle process.
ERS is known to be closely related to autophagy. Under
ER homeostasis, BiP protein binds to ERS sensors (three
ER transmembrane proteins, ATF6, IRE1α, PERK) andkeeps them in an inactive state. In the disease state,
protein misfolding and accumulation of unfolded proteins occur. BiP proteins have a higher affinity for misfolded/unfolded proteins, and once the aforementioned
ER sensors are released, they activate ERS, which, inturn, induces an unfolded protein response (UPR). The

Page 10 of 12

activation of UPR regulates tumor cellular processes suchas cellular growth, autophagy, metastases, and angiogenesis [33]. Evidence from existing studies has highlighted
the extra-ribosomal functions of RPs in UPR. RPL19
overexpression was reported to induce RES in breast cancer by pre-activation of the UPR [34]. RPL22 regulated αβT cell development by dysregulating ERS signaling [35].
Ribosomal protein L5, an RPL11 interacting protein of
60 S ribosome unit promoted colon adenocarcinoma cellproliferation and migration, at least in part, by activating the mitogen-activiated protein kinase/extracellularsignal-regulated kinase 1/2 signaling pathway [36] andalso suppressed breast cancer progression by regulating
ERS and autophagy [37]. Thus, targeting the UPR may be
an effective strategy for treating cancer [38]. In this study,
overexpression of RPL11 induced ERS. Moreover, overexpression of RPL11 enhanced LC3-II and reduced p62
protein levels, suggesting that RPL11 promotes ERS andactivates autophagy in NSCLC.

Conclusion
RPL11 was upregulated in NSCLC and promoted cellular proliferation, migration, and cycle progression in
NSCLC cells. Moreover, RPL11 promoted ERS-induced
autophagy of NSCLC cells. This study suggests that
RPL11 might be a potential therapeutic target for NSCLC
treatment.
Supplementary Information

The online version contains supplementary material available at https://doi.
org/10.1186/s12860-023-00469-2.
Supplementary Material 1
Supplementary Material 2
Acknowledgements
Not applicable.
Author Contribution
Juan Du and Jing Zhang conceived the study and designed the experiments.
Jie Chen, Xiaoyong Huang, Yang Yang, Junli Liu, Yuna Jia and Haiyan Shi
contributed to the data collection, Jie Chen, Huahua Zhang and Changda Lei
performed the data analysis and interpreted the results. Jie Chen and Huahua
Zhang wrote the manuscript; Juan Du and Yunqing Zhang contributed to thecritical revision of article. All authors read and approved the final manuscript.
Funding
This work was supported by National Natural Science Foundation of China(Grant Nos. 81760484, 81860444, 82060521, 82260489, 22264023 and82260530), Shaanxi Province Key Science and Technology innovation team(2020TD-039), Shaanxi Province Natural Science Foundation (2018JQ3073,
2020JQ-801 and 2021JQ-637), Scientific Research Project of Shaanxi Provincial
Department of Education (20JK0986 and 20JG041), and Innovation Teamof Yan’an City-Innovation Team for Chronic Disease Prevention and Control(2018CXTD-03).
Data Availability
All of the data generated during this study were included in this article [and its
supplementary information files].

Chen et al. BMC Molecular and Cell Biology(2023) 24:7

Declarations
Ethics approval and consent to participate
Not applicable.
Consent for publication
Not applicable.
Competing interests
The authors declare no confcts of interest.
Author details
1
Medical Research and Experimental Center, Medical College, Yan’an
University, 716000 Yan’an, People’s Republic of China
2
Yan’an Key Laboratory of Chronic Disease Prevention and Research,
716000 Yan’an, Shaanxi Province, China
3
Department of Gastroenterology, Ninth Hospital of Xi ’an, 710054 Xi’an,
Shaanxi Province, China
4
Laboratory of Obstetrics and Gynecology, Affiliated Hospital of Yan’an
University, 716000 Yan’an, Shaanxi Province, China
Received: 24 December 2022 / Accepted: 28 February 2023

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