EIF3D promoted cervical carcinoma through Warburg effect by interacting with GRP78

Abstract The incidence of cervical cancer ranks third among all female tumours globally and second in developing countries. However, the role of eukaryotic translation initiation factor 3 subunit D (EIF3D) in cervical carcinoma is unknown. This study investigated the effects of EIF3D on cell progression of cervical carcinoma and its underlying mechanism in vivo and vitro models. There were increases of EIF3D expression mRNA and protein expression levels in patients with cervical carcinoma. Disease-free survival (DFS) and overall surviva (OS) of EIF3D lower expression in patients with cervical carcinoma was higher than those of EIF3D higher expression. EIF3D mRNA expression levels in cervical carcinoma cell lines (AV3, Hela229, CaSki and Hela cells) were up-regulated, compared with cervical normal cell line (UVECs). EIF3D promoted cell growth and Warburg effect in vitro model of cervical carcinoma. EIF3D interacting with GRP78 to reduce the activity of GRP78 in vitro model of cervical carcinoma. The inhibition of GRP78 reduced the effects of EIF3D on Warburg effect in vitro model of cervical carcinoma. Our work identifies EIF3D promoted cell growth and Warburg effect in vitro model of cervical carcinoma and the inhibition of EIF3D represents a potential therapeutic strategy for the treatment of cervical carcinoma. IMPACT STATEMENT What is already known on this subject? The incidence of cervical cancer ranks third among all female tumours globally and second in developing countries. What do the results of this study add? This study investigated the effects of EIF3D on cell progression of cervical carcinoma and its underlying mechanism in vivo and vitro models. What are the implications of these findings for clinical practice and/or further research? EIF3D promoted cell growth and Warburg effect in vitro model of cervical carcinoma and the inhibition of EIF3D represents a potential therapeutic strategy for the treatment of cervical carcinoma.


Introduction
The incidence of cervical cancer ranks third among all female tumours globally and second in developing countries (Ghafouri-Fard et al. 2021).According to WHO estimates, approximately 530,000 women develop cervical cancer and 275,000 women die of cervical cancer each year worldwide (Hsu et al. 2021).In most developing countries, cervical cancer is the first leading cause of female tumour death (Huang et al. 2021, Yang et al. 2021).
Warburg effect exists in most tumour cells and is one of the metabolic characteristics of tumour cells.It is also known as aerobic glycolysis.It means that tumour cells are more inclined to glycolytic metabolic pathway than to generate energy by more effective oxidative phosphorylation during carcinogenesis, no matter whether it is hypoxia or not.This article mainly discusses the abnormal expression of alcoholase, phosphoglycerate mutase, hexokinase, pyruvate kinase and lactate dehydrogenase in cervical cancer tissues, which play an important role in the occurrence and development of cervical cancer.
Glucose regulated protein 78 (GRP78), also known as immunog-lobulin heavy chain binding protein (Bip), has a high degree of homology with the heat shock protein (Hsp70) family, which is considered a member of the Hsp family (Kaira et al. 2016c).The GRP78 molecule and its DNA sequence structure, which are highly conserved in many organisms, can promote the correct folding of proteins in the endoplasmic reticulum lumen, thereby playing a role in alleviating the endoplasmic reticulum stress and protecting cells (Kaira et al. 2016a).In recent years, high GRP78 expression has been discovered in patients with tumours, cardiovascular diseases and diabetes by numerous scholars (Soudry et al. 2017;Yadunandam et al. 2012).
To date, the eukaryotic translation initiation factor 3 subunit D (EIF3D) is the non-core subunit of EIF3 that has received the least attention (Huang et al. 2019).It involves many processes such as translation initiation and termination, as well as ribosome recovery (Lamper et al. 2020).Zhang et al. showed that EIF3D promoted disease progression of gallbladder cancer (Zhang et al. 2017).Gao et al. suggested that EIF3D plays an oncogenic role in development and progression of prostate cancer (Gao et al. 2015).EIF3D has been found to be associated with the tumorigenesis and development, and widely expressed in a variety of tumour tissue cells, regulating the tumour cell cycle, and dysregulating the apoptotic and anti-apoptotic signalling pathways by increasing apoptosis (Lee et al. 2016).So, this study investigated the possible effects of EIF3D on cell progression of cervical carcinoma and its underlying mechanism in vivo and vitro models.

Patients
Cancer tissue and paracancerous tissue from patients with cervical cancer (n ¼ 40) were obtained at Guiyang Maternal and Child Health Care Hospital, Guiyang Children's Hospital from January 2019 to May 2020.No patients had received chemotherapy or pre-operative radiotherapy.The written informed consents were obtained from all the subjects and this study was approved by the Ethics Committee of Guiyang Maternal and Child Health Care Hospital, Guiyang Children's Hospital.All the samples were immediately stored at À 80 � C.

Quantitative PCR
The total RNA was extracted from serum and cell samples using a TRIZOL reagent (Life Technologies Inc.).qRT-PCR assays were performed using Light Cycler V R 480 SYBR Mix (Roche, Germany) using LightCycler V R 480 real-time PCR system.The expression levels of mRNA were normalised to the GAPDH expression using the 2 -DDct method.

Microarray analysis
Total RNA was extracted from serum samples, and the amount of RNA was quantified by use of NanoDrop 1000.Total RNA of each sample was used for reverse transcription using an Invitrogen SuperScript double stranded cDNA synthesis kit.Double-stranded cDNA was executed with a NimbleGen one-color DNA labelling kit and then executed for array hybridisation using NimbleGen hybridisation system and washing with the NimbleGen wash buffer kit.Axon GenePix 4000B microarray scanner (Molecular Devices) was used for scanning.

Western blot analysis
Total protein was extracted from cancer tissue and paracancerous tissue samples or cell samples using Radio-Immunoprecipitation Assay (RIPA) and PMSF reagent (Beyotime, Beijing, China).Protein lysates were separated based on their molecular weight on SDS/PAGE gels and transferred onto a Polyvinylidene Fluoride (PVDF, Millipore) membrane.The membrane was blocked with non-fat-milk (5%) for 2 h at room temperature and incubated with ant-EIF3D antibody, anti-GRP78 antibody and anti-b-actin antibody at 4 � C overnight.Then first antibodies were removed and TBST wash membrane using TBST.Membrane were incubated with the secondary antibody for 2 hours at room temperature.The bound antibodies were detected using enhanced chemiluminescence (ECL) with b-actin used as a control.

Immunofluorescence
Cells were fixed with 4% paraformaldehyde, permeabilized with 0.5% Triton X-100 in PBS for 15 min at room temperature, and blocked with 5% BSA for 30 min at 37 � C. Cells were treated with primary antibodies at 4 � C overnight: ant-EIF3D antibody and anti-GRP78.Cells were then incubated with Cy3-conjugated goat anti-rabbit or goat anti-mouse IgG DyLight 488-conjugated secondary antibodies for 2 h at 37 � C. Nuclei were stained with DAPI and cells were observed under a fluorescent illumination microscope (Olympus IX71, Tokyo, Japan).

Glucose consumption, lactate production and ATP level measurement
Glucose was determined by a glucose assay kit (Sigma, St-Louis, MO, USA).Lactate levels were measured by Lactate Colorimetric/Fluorometric Assay Kit (BioVision, Mountain View, CA).ATP level was determined using ATP Determination Kit (Thermo Fisher Scientific, Waltham, MA).
For ethynyl deoxyuridine (EdU) incorporation assay, EdU (10 mM) was added to each well and cells were fixed with 4% formaldehyde for 30 min.After washing, EdU was detected with Click-iTR EdU Kit and images were visualised using fluorescent microscope (Olympus).

Statistical analysis
Graphad Prism 6 was used for the statistical analysis (repeat ¼ 3).All values are expressed as means ± SEM unless specified.p < 0.05 was considered statistically significant.The differences between groups were analysed using Student's t-test.

The expression of EIF3D in cervical carcinoma model
The expression levels of EIF3D in cervical carcinoma model was firstly investigated.
We collected patients with cervical carcinoma (n ¼ 40).Compared with the normal group, there were increases of EIF3D expression mRNA and protein expression levels in patients with cervical carcinoma (Figure 1(A,C)).Meanwhile, compared with cervical carcinoma patients with I-II, had higher the expression of EIF3D mRNA in cervical carcinoma patients with III-IV (Figure 1(B)).Additionally, DFS and OS of EIF3D lower expression in patients with cervical carcinoma was higher than those of EIF3D higher expression (Figure 1(D-E)).The expression of EIF3D mRNA levels in cervical carcinoma cell lines (AV3, Hela229, CaSki and Hela cells) were up-regulated, compared with cervical normal cell line (UVECs) (Figure 1(F)).Taken together, it suggested that EIF3D played a repair factor in disease progression of cervical carcinoma

EIF3D promoted cell growth in vitro model of cervical carcinoma
The experiment was firstly performed to investigate the specific role of EIF3D in cell growth and cell transfer of cervical carcinoma cell in vitro model.The specific research method is to transfect EIF3D plasmid into cervical carcinoma cell (Hela229 cells) and increase EIF3D mRNA level expression (Figure 2(A)).In this si-EIF3D group, EIF3D mRNA expression level was reduced in cervical carcinoma cell (Hela cells) (Figure 2(B)).It was found that over-expression of EIF3D promoted cell growth of cervical carcinoma cell (Figure 2(C)).
Figure 1.The expression of EIF3D in cervical carcinoma.EIF3D mRNA expression (A) in patients with cervical carcinoma; EIF3D protein expression (C) in patients with cervical carcinoma; Disease free survival (D) and overall survival (E) in patients with cervical carcinoma; EIF3D mRNA expression of cervical carcinoma cell lines (AV3, Hela229, CaSki and Hela cells) and cervical normal cell line (UVECs) (F).Normal, Paracancerous tissue; Cervical carcinoma, patients with cervical carcinoma; �� p < 0.01 compared with cervical carcinoma or UVECs.

Conversely, down-regulation of EIF3D reduced cell growth of cervical carcinoma cell (Figure 2(D)).
Over-expression of EIF3D increased the number of EDU cells and promoted migration rate of cervical carcinoma cells (Figure 2(E,F)).Down-regulation of EIF3D inhibited the number of EDU cells and reduced migration rate of cervical carcinoma cell (Figure 2(G,H)).Taken together, our data suggested that EIF3D promoted cell growth of cervical carcinoma.

EIF3D promoted Warburg effect in vitro model of cervical carcinoma
Next, the verification of the protective function of EIF3D on Warburg effect of cervical carcinoma was performed.In the EIF3D over-expression group, the glucose consumption, lactate production and ATP quantity were promoted (Figure 3(A,C)).In the si-EIF3D group, the glucose consumption, lactate production and ATP quantity were reduced (Figure 3(A,C)).Conversely, over-expression of EIF3D promoted extracellular acidification rate (ECAR) and down-regulation of EIF3D reduced ECAR in vitro model of cervical carcinoma (Figure 3(D,E)).Over-expression of EIF3D reduced OCR relative level and down-regulation of EIF3D promoted OCR relative level in vitro model of cervical carcinoma (Figure 3(F,G)).In general, these data suggested that EIF3D promoted cervical carcinoma cells' Warburg effect.

EIF3D interacting with GRP78 to reduce the activity of GRP78 in vitro model of cervical carcinoma
The study further investigated that the mechanism of EIF3D on cell proliferation and progression of cervical carcinoma.Microarray analysis was performed to analyse target spot of EIF3D in cervical carcinoma (Supplemental Figure S1A).It was found that GRP78 may be one important targets of EIF3D in cervical carcinoma cells (Supplemental Figure S1A).
Over-expression of EIF3D increased GRP78 mRNA expression level, and down-regulation of EIF3D reduced GRP78 mRNA expression level in cervical carcinoma cells (Supplemental Figure S1B,C).According to Immunocoprecipitation, there was the association between EIF3D and GRP78 proteins, and EIF3D increased GRP78 expression in cervical carcinoma cells (Supplemental Figure S1D).
In vitro model, the over-expression of EIF3D induced EIF3D and GRP78 protein expression levels (Supplemental Figure S2A,B).In addition, the down-regulation of EIF3D suppressed EIF3D and GRP78 protein expression levels in cervical carcinoma cells (Supplemental Figure S2C,D).According to immunocoprecipitation, there was the association between EIF3D protein and GRP78 protein (Supplemental Figure S2E).In vitro model of cervical carcinoma, the over-expression of EIF3D reduced ubiquitination of GRP78 protein, and the down-regulation of EIF3D increased GRP78 protein ubiquitination in cervical carcinoma cells (Supplemental Figure S2F).

The inhibition of GRP78 reduced the effects of EIF3D on Warburg effect and cell growth in vitro model of cervical carcinoma
Next, the assessment of the mechanism of GRP78 in the effects of EIF3D was performed in vitro model of cervical carcinoma.In addition, compared with over-expression of EIF3D group, GRP78 inhibitor (YUM70, 100 nM) suppressed GRP78 protein expression in cervical carcinoma cells by overexpression of EIF3D (Supplemental Figure S3A).In cervical GRP78 inhibitor reduced cell growth, decreased the number of EDU cells and migration rate of cervical carcinoma cells by over-expression of EIF3D, compared with overexpression of EIF3D group Supplemental Figure S3C,E).Collectively, GRP78 agonist increased cell growth, promoted the number of EDU cells and migration rate of cervical carcinoma cells by down-regulation of EIF3D, compared with down-regulation of EIF3D group (Supplemental Figure S3F,H).Next, compared with over-expression of EIF3D group, GRP78 inhibitor reduced the glucose consumption, lactate production and ATP quantity in cervical carcinoma cells by over-expression of EIF3D (Supplemental Figure S4A,C).In cervical carcinoma cells, compared with down-regulation of EIF3D group, GRP78 agonist promoted the glucose consumption, lactate production and ATP quantity by down-regulation of EIF3D (Supplemental Figure S4A,C).
GRP78 inhibitor reduced ECAR in cervical carcinoma cells by over-expression of EIF3D, compared with over-expression of EIF3D group (Supplemental Figure S4D,E).GRP78 agonist increased ECAR in cervical carcinoma cells by down-regulation of EIF3D, compared with down-regulation of EIF3D group (Supplemental Figure S4D,E).GRP78 inhibitor promoted OCR relative level in cervical carcinoma cells by overexpression of EIF3D, compared with over-expression of EIF3D group (Supplemental Figure S4F,G).GRP78 agonist reduced OCR relative level in cervical carcinoma cells by down-Figure 3. EIF3D promoted Warburg effect in vitro model of cervical carcinoma.Glucose consumption analysis revealed the glucose consumption (A), Lactate production analysis revealed the lactate production (B), ATP quantity analysis revealed the ATP quantity (C), ECAR analysis for lactate-induced acidification of the medium surrounding cells (D and E), OCR analysis for mitochondrial respiratory capacity was conducted using Seahorse XFp assay (F and G).Vector, negative control group; EIF3D, over-expression of EIF3D group; Si-nc, si-negative control group; Si-EIF3D, down-regulation of EIF3D group; �� p < 0.01 compared with negative control group or si-negative control group.
regulation of EIF3D, compared with down-regulation of EIF3D group (Supplemental Figure S4F,G).In general, these data suggested that EIF3D promoted cervical carcinoma cells' Warburg effect by induction of GRP78 expression.
GRP78 inhibitor the effects of EIF3D on cervical carcinoma in mice model.
The experiment further understood the role of GRP78 in the effects of EIF3D on n cervical carcinoma in mice model.EIF3D could increase tumour weight and tumour volume, and reduced caspase-3/9 activity levels in tumer tissue of mice model (Supplemental Figure S5).However, GRP78 inhibitor reversed the effects of EIF3D on cervical carcinoma in mice model (Supplemental Figure S5).So, EIF3D promoted tumour progression of cervical carcinoma cells by induction of GRP78 expression.

Discussion
The incidence of cervical cancer ranks third among all female tumours worldwide (Lee et al. 2021).In China, there are 130,000 new cases of cervical cancer each year, accounting for 28% of the total global new cases (Shapira-Daniels et al. 2020).In developed countries, the annual incidence of cervical cancer is 10/100,000, while in developing countries, this rate is 40/100,000 (Xiao et al. 2022).By 2030, the death toll from cervical cancer is expected to reach 474,000 cases per year, and 95% of them will occur in low-and middle-income countries (Xiao et al. 2022).The current study identified that there was increases of EIF3D expression mRNA and protein expression levels in patients with cervical carcinoma.DFS and OS of EIF3D lower expression in patients with cervical carcinoma was higher than those of EIF3D higher expression.Zhang et al. showed that EIF3D promotes gallbladder cancer cell progression (Zhang et al. 2017).Thus, EIF3D played a repair factor in replantation of severed fingers.
After siRNA silencing of eIF3D expression, mesothelioma cell proliferation decreases and apoptosis increases (Pan et al. 2019).Thus, EIF3D may be a candidate target for inhibiting the mesothelioma growth (Du et al. 2018).Promoting apoptosis is another important mechanism via which the antitumour therapies suppress the tumour cell growth (Jia and Qian 2021).Various subunits of EIF3 are abnormally expressed in tumours, which are also associated with the chemotherapeutic efficacy and the tumour prognosis (Zhang et al. 2017).Our study discovered that over-expression of EIF3DEIF3D promoted cell growth of cervical carcinoma cell.Down-regulation of EIF3DEIF3D reduced cell growth of cervical carcinoma cell.Fan et al. reported that the knockdown of eIF3D inhibits breast cancer cell proliferation and invasion (Fan and Guo 2015).Thus, it is possible that EIF3D might be context-specific and concentration dependent of cervical carcinoma.This experiment only used one cell line, which was one insufficient for this study.
GRP78 is a multifunctional calcium binding protein on the endoplasmic reticulum, which plays a key role in many aspects like synthesis of endoplasmic reticulum proteins, maintenance of endoplasmic reticulum homeostasis, cell signal control, as well as cell survival (Kaira et al. 2016b).GRP78 can participate in cell surface signal transduction, regulate apoptosis, and be involved in endoplasmic reticulum stress response (Lin et al. 2009;Pi et al. 2014).We revealed that EIF3D interacting with GRP78 to reduce the activity of GRP78 in vitro model of cervical carcinoma.Huang et al. indicated that EIF3D promotes renal cell carcinoma by interacting with GRP78 (Fan and Guo 2015).Specifically, EIF3D interacting with GRP78 to promote cervical carcinoma.
Taking together, EIF3D promoted cell growth and Warburg effect in vitro model of cervical carcinoma.On the basis of these findings, our findings demonstrate a potential use of EIF3D levels as an indicator of cervical carcinoma.Moreover, the inhibition of EIF3D represents a potential therapeutic strategy for the treatment of cervical carcinoma.

Figure 2 .
Figure 2. EIF3D promoted cell growth in vitro model of cervical carcinoma.EIF3D mRNA expression (A and B); Cell growth (CCK-8, C and D); EDU assay (E), migration rate (F) in vitro model of cervical carcinoma by over-expression of EIF3D; EDU assay (G), migration rate (H) in vitro model of cervical carcinoma by down-regulation of EIF3D.Vector, negative control group; EIF3D, over-expression of EIF3D group; Si-nc, si-negative control group; Si-EIF3D, down-regulation of EIF3D group; �� p < 0.01 compared with negative control group or si-negative control group.