Mechanistic analysis of endothelial lipase G promotion of the occurrence and development of cervical carcinoma by activating the phosphatidylinositol-4,5-bisphosphate 3-kinase/protein kinase B/mechanistic target of rapamycin kinase signalling pathway

Abstract Lipase G, endothelial type (LIPG) is expressed abundantly in tissues with a high metabolic rate and vascularisation. Research on LIPG has focussed on metabolic syndromes. However, the role of LIPG in providing lipid precursors suggests that it might function in the metabolism of carcinoma cells. Analysis in The Cancer Genome Atlas indicated that patients with cervical carcinoma with high LIPG expression had a lower survival prognosis compared with patients with low LIPG expression. The mechanism underlying the effects of LIPG in cervical carcinoma is unclear. The present study aimed to determine the role of LIPG in cervical carcinoma and its mechanism. The results showed that the LIPG expression level was higher in cervical cancer. Downregulation of LIPG expression inhibited cell migration, invasion, proliferation, and the formation of cell colonies, but increased the rate of apoptosis. The Human papillomavirus E6 protein might reduce the expression of miR-148a-3p, relieve the inhibitory effect of miR-148a-3p on LIPG expression, and promote the progression of cervical cancer through the phosphatidylinositol-4,5-bisphosphate 3-kinase/protein kinase B/mechanistic target of rapamycin kinase signalling pathway. IMPACT STATEMENT What is already known on this subject? LIPG provides lipid precursors, suggesting that it might function in the metabolism of carcinoma cells What do the results of this study add? LIPG might be regulated by HPV16 E6/miR-148a-3p and promote cervical carcinoma progression via the PI3K/AKT/mTOR signalling pathway. What are the implications of these finding for clinical practice and/or further research? The results indicated that novel treatment and diagnosis strategies for cervical carcinoma could be developed related to LIPG. However, the detailed relationship between LIPG and cervical carcinoma remains to be fully determined.


Introduction
Every year approximately 500,000 women are diagnosed with invasive cancer of the cervix throughout the world, killing 273,000 of them.In 2020 alone, 604,127 women were diagnosed with cervical cancer worldwide (Kousar et al. 2022).Approximately 59,060 women succumb to cervical carcinoma every year in China, despite preventive Human papillomavirus (HPV) vaccines and conventional cancer treatments (2021).The rapid growth of tumour cells requires a lot of energy.Little is known about the mechanisms that support the metabolism of cervical carcinoma cells to maintain their rapid growth.Whether these mechanisms affect other malignant features (such as metastasis and invasiveness) is unknown.
Lipase G, endothelial type (LIPG) is an important member of the lipase family whose non-enzymatic and enzymatic functions were first reported in 1999 (Jaye et al. 1999).LIPG is essential for the malignant features of LIPG-expressing triple negative breast carcinoma (TNBC) cells, including invasiveness, stemness, and basal and epithelial-mesenchyme transition (EMT) features, tumorigenicity, and metastasis in vivo (Lo et al. 2018).LIPG is associated with poor prognosis, and high LIPG expression is related to shorter metastasisfree survival (MFS) (Cadenas et al. 2019).Studies have shown that LIPG also possesses both enzymatic and non-enzymatic functions in breast cancer cells.The phospholipase function of LIPG is responsible for supporting cell growth and promoting the cell proliferation rate.The phospholipase-independent function of LIPG promotes invasiveness, stemness, and basal/ EMT features of breast cancer cells.Although the mechanism by which LIPG executes its non-enzymatic function is unknown, it is likely to act through protein-protein interactions (Lo et al. 2020).Treatment with statins to inhibit cholesterol synthesis reduced cisplatin-induced apoptosis, whereas silencing of LIPG or withdrawal of lipids from the culture medium increased sensitivity to the drug (Criscuolo et al. 2020).Studies suggest that the synthesis and metabolism of endothelial lipase plays a critical role in tumorigenesis.For example, a recent study stated that elevated LIPG levels are associated with increased risk of breast cancer, especially Luminal A and HER2negative breast cancers (Gago-Dominguez et al. 2021).Considering that a large proportion of the energy comes from fatty acids and that LIPG functions to provide fatty acid precursors, we hypothesised that LIPG plays a role in cervical carcinoma.Consequently, the present study aimed to investigate the mechanism of LIPG in cervical carcinoma.

Cell lines and culture conditions
Cervical carcinoma cell lines (CaSki and SiHa) and HEK293T cells (all from Jennio Biotech, Guangdong, China) were cultured in Dulbecco's modified Eagle's medium (DMEM; Gibco, Grand Island, NY, USA) supplemented with 10% Foetal Bovine Serum (FBS, Gibco), incubated with 5% CO 2 at 37 � C, subcultured, and the medium was changed according to the cell growth and confluence.

Tissue specimens
Cervical carcinoma specimens and normal cervical specimens were obtained from patients undergoing surgical excision who had not received any preoperative chemotherapy or radiotherapy in the Gynaecology Department of The Third Affiliated Hospital of Guangzhou Medical University.Patient consent forms for all samples were signed before tissue acquisition.The Ethics Committee of Third Affiliated Hospital of Guangzhou Medical University approved this study.(Approval No: 2019-037).

Immunohistochemistry
Pre-made tissue paraffin sections were baked in an oven at 60 � C, dewaxed in xylene I and xylene II, placed in gradient alcohol, and washed with distilled water to complete gradual rehydration.Citric acid repair solution was used for antigen retrieval.The endogenous peroxidase inhibitor reagent 1 of the kit was added to the tissue sections, which were placed in an incubator at 37 � C for 40 min to inactivate the endogenous peroxidase activity.Reagent 2 was added to the tissue sections to block the non-specific binding sites.An anti-LIPG antibody was added to the sections, which were incubated in a wet box at 4 � C overnight.The secondary antibody was added next day, incubated for 30 min at 37 � C, followed by the addition of streptavidin-peroxidase (SP), and incubation for a further 30 min.Then, 80 ml of 3,3 0 -Diaminobenzidine colour development solution was added to each section.The colour development was stopped and the sections were observed under a microscope.The sections were then stained again with haematoxylin for 10 min, subjected to gradient alcohol dehydration to make them transparent, fixed to the slide using neutral gum, and then observed under the microscope again.

Quantitative real-time reverse transcription PCR (qRT-PCR) assay
At 48 h after transfection, cells from 6-well plates were digested, and the total RNA was extracted using the TRIZOL reagent.Equal amounts of RNA were then reverse transcribed to cDNA following the manufacturer's instructions (YESEN Technology, Shanghai, China).The expression of LIPG and miR-148a-3p in the samples was detected using quantitative real-time PCR (qPCR) in an ABI Step one Plus instrument (ABI, Foster City, CA, USA).The 18S and U6 genes were used as internal controls.qPCR was performed using the following conditions, 95 � C for 10 min, followed by 40 cycles of 95 � C for 15 s and 60 � C for 60 s.Then, the relative expression level of each mRNA was normalised to the expression level of the housekeeping genes using the 2 À DDCT method (Livak and Schmittgen 2001).

Wound healing assay
CaSki and SiHa cells were counted, inoculated into 6-well plates, and cultured at 37 � C with 5% CO 2 until they reached 70-80% confluence.A wound was then scraped perpendicular to the marked 6-well plate using a 200 ml pipette tip.The wound was observed and photographed under an inverted microscope immediately after wounding (time 0) and every 24 h for 2 days.Image J software was used to measure the migration area of the cells in each group.

Colony-forming assay
The cells were digested and added to each well of a six-well plate.After the cells had grown adherently, they were transfected with the LIPG siRNA and or scrambled siRNA.The cells were then incubated for 1-2 weeks to form colonies.The cells were then fixed with paraformaldehyde and stained with 5% Giemsa solution for 15 min at room temperature.The number and size of the colonies were calculated.

Cell invasion detection
Matrigel was diluted with serum-free medium and added into each Transwell chamber.50,000 cells were resuspended in 200 ml of serum-free culture medium and added to the upper Transwell chamber; the lower chamber was filled with 600 ml of DMEM containing 10% FBS.The transfection reagent was placed in the upper chamber and the lower chamber in a ratio of 1:3.After 48 h of incubation, the cells were fixed with paraformaldehyde for 30 min, stained with crystal violet for 15 min, washed, air-dried, and observed and quantified under a Leica Camera microscope (Leica, Wetzlar, Germany).

Apoptosis assay
Cells in culture medium were digested using EDTA-free trypsin.Then, 2.5 ml of propidium iodide (PI, BD Life Sciences-Biosciences, San Diego, CA, USA) was added to the PI only control tube, 2.5 ml of fluorescein isothiocyanate was added to the FITC control tube, and a mixture of 2.5 ml PI and 2.5 ml FITC was added to experimental groups at the same time.The cells were diluted with 150 ml of buffer before being detecting using a flow cytometer (Attune NxT, Life technologies, Carlsbad, CA, USA) within 1 h.

Raybio V R human phosphorylation pathway profiling array
All cells were stored on dry ice and sent to RayBiotech company for protein chip detection (RayBiotech life, Peachtree Corners, GA, USA).We analysed the data from the protein chips, including basic analysis such as normalisation of original data, screening of differentially abundant proteins (DABs), clustering of DABs, and gene ontology (GO)/Kyoto Encyclopaedia of Genes and genomes (KEGG) pathway enrichment analysis of DABs.

Dual-luciferase reporter assay
Wild-type or mutated LIPG binding site dual luciferase plasmid and miR-148a-3p mimics (Shanghai Genechem Co., Ltd, Shanghai, China) were designed and used to co-transfect HEK293T cells.The cultured HEK293T cells were harvested at 48 h after transfection.Then, 100 ml of Luciferase Assay Buffer II (Promega, Madison, WI, USA) was pre-dispensed into four wells of a light-tight 96-well plate.The cell lysate (20 ml) was added to the well, mixed by pipetting two or three times, and the firefly luciferase activity was measured and recorded.Then, we quickly added 100 ml of Stop&Glo V R reagent, and started recording the Renilla luciferase activity.

Statistical analysis
SPSS software (IBM Corp., Armonk, NY, USA) and GraphPad Prism software (GraphPad Software Inc., San Diego, CA, USA), Image Lab (Bio-Rad) were used for the statistical analyses.All p-values were two-sided, and p < 0.05 was considered statistically significant.

qRT-PCR and immunohistochemistry detection of LIPG expression in cervical tissues
The Cancer Genome Atlas (TCGA) database indicated that patients with cervical carcinoma with high LIPG expression had a lower survival prognosis compared with patients with low LIPG expression (Figure 1(a)).According to the intensity of the immunohistochemical staining, a semi-quantitative score was assigned to the proportion of positive cells: 0 [À ], (1 [þ], 2 [þþ], and 3 [þþþ].The expression of LIPG in cervical carcinoma tissues was higher than that in normal cervical tissues (Table 1, p < 0.05).We also verified the correlation of LIPG expression with different clinicopathological characteristic (Table 2, p > 0.05).The expression of LIPG in cervical carcinoma tissue (n ¼ 81) was higher than that in normal cervical tissue (n ¼ 19) (Figure 1(e)); however, the expression level of LIPG has no significant correlation with the patient's age, FIGO stage, and other clinicopathological features.We compared the expression of LIPG in normal cervical tissues (n ¼ 30) and cervical carcinoma tissues (n ¼ 114) using qRT-PCR.We observed that the expression of LIPG in cervical carcinoma tissues was markedly higher than that in normal cervical tissues (p < 0.01) (Figure 1(b)).

Western blotting and qRT-PCR confirmed the expression of LIPG in SiHa and CaSki cervical carcinoma cells and verified the siRNA transfection efficiency
We confirmed that LIPG is expressed in cervical carcinoma cells using qRT-PCR analysis, and that the LIPG siRNA could reduce the mRNA expression level.Simultaneously, we verified the transfection efficiency of siRNA-LIPG for 48 h and 72 h (Figure 1(c)).The protein level of LIPG in the LIPG siRNA transfection group was lower than that in the control group (Figure 1(f)).

LIPG depletion reduced the wound-healing capacity of cervical carcinoma cells in vitro
The wound was observed and measured under an inverted microscope every 24 h for 2-3 days.The results showed that compared with the untreated cells and the cells transfected with the scrambled siRNA, the wound healing ability in the LIPG siRNA group was reduced markedly, and the cell migration rate slowed down significantly (Figure 2(a)).

LIPG silencing inhibited cervical carcinoma cell proliferation in vitro
The results of the MTT experiment showed that the cervical cells in the non-specific control group maintained a   continuous cell proliferation state up to 24 h after inoculation, while the LIPG-siRNA transfection group showed suppressed cell proliferation (Figure 1(g)), and these effects continued until the cells aggregated into a monolayer.

Silencing of LIPG decreased the clonogenicity of cervical carcinoma cells in vitro
Cervical carcinoma cells were incubated for 2 weeks to form colonies in a six-well plate.After staining and counting the colonies, we observed that knockdown of LIPG expression significantly reduced the colony formation rate, regardless of the number or area of each single colony, and the colonies formed by the LIPG siRNA transfection group were smaller than those of the other groups (Figure 1(d)).

LIPG-depleted cells displayed a reduced invasion capacity
The tropism of the cervical carcinoma cells made the cells gradually migrate from the serum-free supernatant to the serum-containing supernatant in the Transwell apparatus.The cells were fixed and stained after 48 h and observed under a high-power microscope.The results showed less invasion of the cervical carcinoma cells treated with the LIPG siRNA compared with that of the other groups (Figure 2(b)).

LIPG plays a role in inhibiting cell apoptosis
The LIPG siRNA-transfected cells showed a higher level of apoptosis compared with that in the control siRNA-transfected cell group (Figure 2(c)), which indicated that LIPG plays a role in inhibiting apoptosis.

In cervical carcinoma, the change of LIPG expression is regulated by HPV16 E6/miR-148a-3p
Cancer development after human papillomavirus (HPV) infection can occur through the canonical HPV/p53/RB1 pathway mediated by the E2/E6/E7 viral oncoproteins.During the transformation process, HPV inserts its genetic material into host Integration Sites (IS), affecting coding genes and microRNAs (miRNAs) (Da et al. 2022).One study showed that the human papillomavirus 16 oncoprotein (HPVE16) downregulated miR-148a-3p expression in cervical carcinoma (Han et al. 2018).Our results demonstrated that HPV16E6 silencing increased the expression of miR-148a-3p in CaSki and SiHa cells (Figure 3(a)).The wild-type LIPG 3 0 -untranslated region (UTR) and mutant LIPG 3 0 -UTR dual-luciferase vectors were co-transfected into HEK293T cells with miR-148a-3p mimics, separately.The results of the dual luciferase reporter assays showed that after normalisation using the Renilla luciferase activity, miR-148a-3p decreased the relative luciferase activity from the wild-type LIPG 3 0 UTR construct but not from the mutant LIPG 3 0 UTR construct (Figure 3(b)), indicating that miR-148a-3p might bind to LIPG 3 0 UTR.In addition, after knocking down the expression of HPV16E6, western blotting indicated a decreased level of the LIPG protein (Figure 3(e)).

LIPG might affect cell performance by promoting PI3K/AKT/mTOR signalling
The results of the protein chip assay showed that after transient transfection of the LIPG siRNA most of the downregulated proteins were members of the PI3K/AKT/mTOR signalling pathway (Figure 3(d)).We knocked down LIPG expression, and then detected the changes in the levels of key proteins in the PI3K/AKT/mTOR signalling pathway using western blotting.The results showed that the levels of mTOR, PI3K, pPI3K, AKT, and pAKT in the CaSki and SiHa cell lines transfected with the LIPG siRNA were lower than in the corresponding control groups (Figure 3(e)).

Discussion
The International Agency for Research on Carcinoma (IARC) of the World Health Organisation released the latest data in 2020.The total number of new carcinomas in the world was about 19.29 million, and the number of cervical carcinomas was 600,000 cases, which accounted for 3.1% (2021).Cervical carcinoma has its unique biological characteristics, such as invasiveness and metastasis, abnormal cell differentiation, proliferation, and loss of growth control.It can be divided into three processes: Carcinogenesis, carcinoma promotion, and evolution.Cervical carcinoma might occur through a long multi-factor and multi-step process; therefore, earlier diagnosis and effective treatment strategies have become an urgent need to prolong the survival period of patients with cervical carcinoma and to improve their quality of life.
Research has suggested that LIPG plays a crucial role in the development of other malignant carcinomas, such as breast carcinoma, ovarian carcinoma, gastric carcinoma, testicular germ cell tumour, and colorectal carcinoma (Cheng et al. 2022).Through qRT-PCR analysis, western blotting, and immunohistochemical detection, we proved that LIPG is expressed in cervical carcinoma tumour cell lines, as well as in surgically excised cervical carcinoma tissues.We found significantly higher LIPG gene and protein expression levels in cervical carcinoma tissue than in normal cervical tissue.
Histological examination found that LIPG is highly expressed in cervical carcinoma; therefore, we further explored the function of LIPG in the malignant characteristics of cervical carcinoma.The results revealed that after transient transfection of LIPG siRNA, compared with the scrambled control groups, the migration speed of LIPG knockdown cervical carcinoma cells was slower; their invasion, proliferation, and colony formation abilities were decreased, and the rate of cell apoptosis was increased.These data suggested that decreasing the expression of LIPG had a detrimental effect on the malignant characteristics of cervical carcinoma cells in vitro.
The regulation of downstream proteins by upstream proteins is the mainly the result of intracellular and extracellular signal transduction, which was first proposed in 1984 (Hunter et al. 1984).The PI3K/AKT pathway was proposed as early as 1998 in a study of insulin stimulation (Staubs et al. 1998).The PI3K/AKT pathway has been reported to be associated with the occurrence and development of many malignant cancers.In cervical carcinoma, the levels of PI3K, AKT, and mTOR in patients with cervical intra-epithelial neoplasia (CIN) were higher, and the levels of PI3K, AKT, and mTOR proteins in cervical carcinoma were markedly higher than those in non-carcinoma tissues.In addition, the expression levels of mTOR, AKT, and PI3K correlated positively with the degree of malignancy in cervical carcinoma tissues (Zhang et al. 2019).In the present study, the results of protein chip array technology suggested that after knockdown of LIPG, the levels of proteins belonging to the PI3K/AKT/mTOR signalling pathway, including Raf-1, GSK3b, mTOR, RPS6, PRAS40, AKT, and p27, were decreased.To further verify these results, we performed western blotting on cervical carcinoma cells after silencing LIPG.The results showed that the levels of important proteins mTOR, PI3K, pPI3K, AKT, and pAKT in this pathway were decreased in the LIPG-silenced cells.The findings suggested that LIPG might promote the development of cervical carcinoma at least partially through the PI3K/AKT/mTOR signalling pathway.
In addition, a meta-analysis and systemic review showed that the expression level of microRNAs (miRNA) are highly correlated with cancer (Xu et al. 2021).We queried the experimentally verified microRNA-target interaction database.The results suggested a correlation between LIPG and miR-148a-3p, indicating that LIPG might be a target of miR-148a-3p.We constructed 3 0 -UTR wt-LIPG (wild-type) and mut-LIPG (mutant type) dual luciferase reported vector and co-transfection them into HEK293T cells together with an miR-148a-3p mimic.The results showed that, compared with the other groups, the luciferase activity of miR-148a-3p and the wt-LIPG co-transfection group was significantly reduced, indicating that there is a binding site of miR-148a-3p in the LIPG 3 0 -UTR, and the two can bind with each other.
It believed that sexually transmitted HPV infection, especially high-risk human papillomavirus (HR-HPV), is the main Figure 3. (a) When HPV16E6 was silenced, the level of miR-148a-3p in cervical carcinoma cells was higher than that in the control group.(b) After co-transfection of the dual luciferase vector and the miR-148a-3p mimic, the activity of dual luciferase in lysed 293 T cells decreased.(c) Bioinformatic software prediction of the binding sites of miR-148a-3p in the LIPG gene.(d) Protein chip technology using fold-change over 1.2 or less than 0.83 to screen for differentially abundant proteins (DAPs).(e) After the HPV16E6 siRNA was transiently transfected into cervical carcinoma cells, the LIPG protein level in cells in the treatment group showed a downward trend.After knocking down the expression of LIPG, the levels of key proteins in the PI3K/AKT/mTOR signalling pathway decreased.After HPV16E6 siRNA was transiently transfected into cervical carcinoma cells, the LIPG protein level of cells in the treatment group showed a downward trend.�� p < 0.01.� p < 0.05.
trigger of this malignant tumour.One study revealed that 85 miRNAs were abnormally expressed in HPV E6/E7 expressing cells compared with the controls (Laszlo et al. 2021).A study indicated that miR-148a-3p expression was reduced in cervical cancer tissues compared with that in normal cervical tissues.Furthermore, miR-148a-3p overexpression significantly inhibited the proliferation of HeLa and SiHa cells.These findings suggested that miR-148a-3p exerted inhibitory effects in cervical cancer (Chen et al. 2021).A previous study demonstrated that compared with the normal group, the level of miR-148a-3p in the HPV16-positive cervical carcinoma group was significantly reduced, and after silencing of HPV16E6, the level of miR-148a-3p in cervical carcinoma cells increased (Han et al. 2018).Our results confirmed that when HPV16E6 was silenced in cervical carcinoma cells, the expression of miR-148a-3p increased and the LIPG protein level decreased.Therefore, the results of this study indicated that miR-148a-3p might be regulated by HPV16 E6 to affect the development and progression of cervical carcinoma by targeting LIPG.
In summary, the results of the present study suggested that LIPG plays a pivotal role in promoting the malignant characteristics of cervical carcinoma, and might be regulated by the HPV16 E6/miR-148a-3 axis.LIPG exerts these effects on the growth and development process of cervical carcinoma at least partly through the PI3K/AKT/mTOR signalling pathway.Thus, inhibiting the function of LIPG might slowdown the rapid growth of cervical tumour cells, which might be developed as a practical treatment strategy in the future.

Figure 1 .
Figure 1.(a) TCGA evaluation of the clinical results of different levels of LIPG expression in cervical carcinoma.(b) Immunohistochemical detection of LIPG in various cervical tissues.(c) qRT-PCR analysis to verify the expression of LIPG in two cervical carcinoma cell lines and the transfection efficiency of the LIPG siRNA.(d) Silencing LIPG reduced the rate of colony formation of cervical carcinoma cells.(e) qRT-PCR verification of the expression of LIPG in surgically resected tissue specimens.(f) Analysis of LIPG protein levels in cervical carcinoma cells treated with or without the LIPG siRNA.(g) LIPG silencing reduces the cell proliferation of cervical carcinoma cell lines CaSki and SiHa at 24 h after inoculation.

Figure 2 .
Figure 2. (a) Wound-healing ability of LIPG-silenced cells.Downregulation of LIPG expression slows down cell migration.In the figure, the wound healing rate was calculated as: the original wound area À the wound area at each interval/the original wound healing area �; 100%, the scale bar indicates 100 lm.�� p < 0.01.� p < 0.05.(b) After knocking down the expression of LIPG, the number of invasive cells in the non-specific control group was about 2.38 (caski)/3.03(siha) times that of the cells in the LIPG siRNA transfected group.�� p < 0.01.(c) Compared with the control group, in the LIPG siRNA group, the percentage of apoptotic CaSki cells increased from 15.9% to 18.6%, and the percentage of apoptotic SiHa cells increased from 9.57% to 18.23%.�� p < 0.01.

Table 1 .
LIPG expression in normal cervix and cervical cancer tissues.

Table 2 .
Correlation between LIPG expression and different clinicopathological features of cervical carcinoma.