Between October 2018 and December 2019, 60 pairs of LUAD and neighboring lung tissues were obtained from LUAD patients in the First Affiliated Hospital of Bengbu Medical College (Anhui Province, China). All subjects underwent surgery after obtaining informed consent. This research was approved by the Ethics Committee of the same institute, and was in compliance with the standards established by the Declaration of Helsinki. After snap-freezing in liquid nitrogen, the samples were kept at –80 ℃ until further analysis.

The tissue specimens were fixed in 4% formalin, embedded in paraffin, and sectioned at 4 $\mu$m thickness using a microtome. The sections were then exposed to 3% H${}_2$O${}_2$ to inhibit endogenous peroxidase activities, followed by heating at 95 ℃ for 20 min in 10 mM citrate buffer (pH 6.0) for antigen retrieval. To prevent non-specific binding, 0.4% triton-X100 (P0096, Beyotime, Shanghai, China) + 10% bovine serum albumin (BSA, ST023, Beyotime, Shanghai, China) was used at room temperature (RT) for 1 h. Next, these sections were cultured with LPGAT1-specific antibody (ab230647, Abcam, Waltham, MA, USA) at 37 ℃ for 60 min, followed by incubation with the secondary antibodies (PV-6000, Zsbio, Beijing, China). DAB chromogenic agent (ZLI-9018, Zsbio, Beijing, China) was then used for nucleus staining at RT for 10 min. Finally, the stained sections were examined using an microscope (CX43, Olympus, Tokyo, Japan).

Human LUAD cell lines (PC9, H1299, A549 and H23) and HEK-293T cell line were supplied by the Institute of Cell Research, Chinese Academy of Sciences (Shanghai, China). All cell lines were tested for mycoplasma and no mycoplasma contamination was observed. All cell lines were detected by short tandem repeat (STR) and all were detected correctly. All cells were cultured in DMEM containing 10% fetal bovine serum (FBS, A3160802, Gibco, Grand Island, NY, USA), and 10 mg/mL penicillin-streptomycin (PS, 15140122, Gibco, Grand Island, NY, USA). Cells were grown at 37 ℃ in an appropriate incubator with 5% CO${}_2$.

LPGAT1 knockdown was performed by siRNA and shRNA viruses. siLPGAT1 and siCtrl were the experimental and control groups, respectively. H1299 and A549 cells were transfected with siLPGAT1 and siCtrl. The target sequences of siLPGAT1 and siCtrl are displayed in Supplementary Table 1. shLPGAT1 and shCtrl were cloned into pLKO.1 plasmid, followed by sequencing. The shRNA-pLKO.1 and helper plasmids (delta8.9 and VSVG) were transfected into HEK293T cells for 3 days. Lentiviruses were generated, collected, and purified via ultracentrifugation. The viral titer was determined by evaluating the infectious capacity of HEK293T cells. Stable, transfected cells were screened with 2 $\mu$g/mL puromycin (ST551, Beyotime, China). Subsequently, quantitative real-time reverse transcription PCR (qRT-PCR) and western blot (WB) analyses were conducted.

The lentivirus-infected H1299 and A549 cells (1000 cells/well) were grown in a 96-well plate, followed by incubation at 37 °C. At 1, 2, 3, 4 and 5 days, 10 $\mu$L of cell counting kit -8 (CCK8, BL001B, Biomiky, Shanghai, China) was added to each well. After 4 h, the absorbance was determined using a microplate reader (F50, Tecan, Männedorf, Switzerland) at 450 nm.

To evaluate the impact of LPGAT1 on apoptosis, the treated H1299 and A549 cells (5 $\times$ 10${}^5$) were collected and stained with Annexin VFITC/PI (22837, AAT Bioquest, Sunnyvale, CA, USA). Cell apoptosis was evaluated using the FACS Calibur (FACSCanto, BD, San Jose, CA, USA).

To assess the effect of LPGAT1 on the cell cycle, A549 and H1299 cells (1 $\times$ 10${}^6$) were harvested and fixed with 70% ethanol at 4 ℃ for 1 h. After rinsing twice with phosphate buffered saline (PBS, C10010500BT, Gibco, Grand Island, NY, USA), the cells were added into 2 mg/mL propidium (PI, C1052, beyotime, Shanghai, China) and 10 mg/mL RNase A (C1052 ,Beyotime, Shanghai, China), followed by incubation at 4 ℃ for 30 min in the dark. PI fluorescence intensity was measured using FACS Calibur.

H1299 (or A549) cells were exposed to sh-LPGAT1 or sh-Ctrl lentivirus and cultured for 2 days. The cells were then dissociated and grown in a 6-well plate for colony formation at six hundred viable cells per well. Three replicate wells were set up for each experimental group. After 14 days, most clones contained more than 50 cells (medium was exchanged every three days). Crystal violet staining (1000 $\mu$L/well) was then performed and visualized using a microscope. Only a colony with more than 50 cells was counted.

After being interfered by siLPGAT1 and siCtrl, the H1299 cell lines were extracted for RNA, and transcriptome sequencing was performed. We selected differentially expressed genes (DEGs) with $|$log2 fold change$|$$\ge$2 and a p-value 0.05. A clustering heat map was drawn with the DEGs, and then these DEGs were detected by GO functional enrichment analysis, and the results were classified into three terms, including biological processes (BPs), cellular components (CCs) and molecular functions (MFs).

Total RNA was extracted from the H1299 cells treated with lentivirus using the TRIzol® Plus RNA Purification Kit (12183-555, Invitrogen, Carlsbad, CA, USA). RNA was reverse-transcribed by SuperScript™ III First-Strand Synthesis SuperMix (11752-050, Invitrogen, Carlsbad, CA, USA), and qPCR was performed by Power SYBR Green PCR Master Mix (4367659, Applied Biosystems, Waltham, MA, USA). The primer sequences are displayed in Supplementary Table 1. The CFX384 multiplex RT-PCR instrument (Bio-Rad, Berkeley, CA, USA) was employed for mRNA expression analysis. The fold-change was measured using the 2${}^{-\mathrm{\Delta }\mathrm{\Delta }\text{Ct}}$ method.

A total protein extraction kit containing protease inhibitor cocktail (BC3710, Solarbio, Beijing, China) was utilized for total protein isolation. A bicinchoninic acid (BCA) quantification kit (B0010, Beyotime, Shanghai, China) was utilized for total protein quantification. After separation through 10% SDS-PAGE, the proteins were subsequently transferred onto PVDF membranes (IPVH00010, Millipore, St. Louis, MO, USA). The membranes were incubated with the primary antibodies at 4 °C overnight, followed by the secondary antibodies (Goat anti-Rabbit IgG (H+L), 31210, Thermo Pierce, Waltham, MA, USA) at RT for 1 h. The following primary antibodies were employed: anti-LPGAT1 (bs-18347r, Bioss, Woburn, MA, USA), anti-GPAM (bs-5063r, Bioss, Woburn, MA, USA), anti-LCLAT1 (bs-18190r, Bioss, Woburn, MA, USA), anti-LPCAT1(16112-1-AP, Proteintech, Wuhan, China) anti-LPCAT4 (17905-1-AP, Proteintech, Wuhan, China), anti-CRLS1 (14845-1-AP, Proteintech, Wuhan, China), and anti-$\alpha$-tubulin (11224-1-AP, Proteintech, Wuhan, China). Protein bands were quantified with ImageJ software (NIH, Bethesda, MD, USA).

All the experimental protocols were conducted in accordance with the institutional ethics and safety guidelines of the same institute. Twenty-two BALB/c nude mice (female, 6 weeks old, 16–18 g) were supplied by Shanghai Lingchang Biotechnology (Shanghai, China), and were maintained at the Experimental Animal Center, and underwent a 10/14-h light-dark cycle (55 $\pm$ 5% humidity and 22 $\pm$ 1 °C). The mice were randomly assigned to four groups. LPGAT1 knockdown and control xenograft models were established by injecting 4 $\times$ 10${}^6$ H1299 (or A549) cells into the right flank of mice. After 2 weeks, the tumor size was examined twice a week. Tumor length and width were determined using a caliper. Tumor volume was calculated as follows: volume (mm${}^3$) = $\pi$/6 $\times$ length $\times$ width $\times$ width. Following six weeks, each mouse was euthanized by cervical dislocation, and tumor tissues were obtained. Tumor volumes were calculated as described above, and tumor weights were measured using an electronic balance.

The mRNA level of LPGAT1 was detected using the TCGA-LUAD cohort. After merging with LPGAT1 expression data, the samples with complete information on patient survival, age, gender, N stage, T stage and tumor stage were used to construct the Kaplan-Meier curve and perform univariate and multivariate Cox analyses.

All values are shown as mean $\pm$ SD. The differences between groups were compared by Student’s t-test or one-way ANOVA. p-values of 0.05 were regarded as statistically significant. All tests were performed with SPSS v18.0 (IBM, Amonk, NY, USA) and GraphPad Prism v6.0 (GraphPad Software, San Diego, CA, USA).

This study assessed the role of LPGAT1 in LUAD. Expression data from the Cancer Genome Atlas (TCGA-LUAD), comprising 497 LUAD tissues and 54 non-tumor tissues, were analyzed. Our results indicated that LPGAT1 was significantly increased in LUAD tissue compared to non-tumor tissue (Fig. 1A). All LUAD patients were classified into LPGAT1 high-expression and low-expression groups based on the median value of LPGAT1 expression. However, Kaplan-Meier analysis revealed that the survival rate of the LPGAT1 low-expression group was not better than that of the LPGAT1 high-expression group (Fig. 1B). In addition, after combining LPGAT1 expression data and survival data from these samples, univariate and multivariate Cox analyses were performed. The results demonstrated that LPGAT1 was an independent poor prognostic factor (Supplementary Tables 2, 3). 60 pairs of LUAD and neighboring normal tissues were acquired, and the following steps were performed: immunohistochemical staining, photography, interpretation, and pathological analysis. The findings were highly consistent with the above data, in which the expression of LPGAT1 was higher in LUAD tissues than in paracancer tissues (Fig. 1C). The immunohistochemical scores of the 60 clinical samples are shown in Fig. 1D.

Fig. 1.

LPGAT1 is highly expressed in lung adenocarcinoma tissues. (A) Statistical results of LPGAT1 expression data from TCGA-LUAD. (B) Kaplan-Meier analysis of LUAD patients’ prognosis with high and low expression of LPGAT1. (C) LPGAT1 expression in tumor tissues and normal tissues via IHC staining. Scale bars: 20 $\mu$m. (D) IHC results of 60 clinical samples were scored: 0 as negative, 1 as 10% of positive cells, 2 as 11–50%, 3 as 51–75%, and 4 as $>$75%.

To explore the role of LPGAT1 in LUAD, a series of functional assays were performed in LUAD cells. H1299 and A549 cells with the highest LPGAT1 expression were selected for the subsequent analyses (Fig. 2A and Supplementary Fig. 1A,B). These LUAD cell lines were transfected with siLPGAT1 and siCtrl, and qPCR assays were then conducted to detect the endogenous LPGAT1 expression. It was observed that the expression of LPGAT1 was downregulated by 95% in H1299 cells (Fig. 2B) and 88% in A549 cells (Supplementary Fig. 1C). Next, CCK-8, FC and CFA were conducted on the stable transfected LUAD cells to assess cell proliferation and apoptosis. The findings of CCK-8 assays demonstrated that the proliferation of H1299 cell line (Fig. 2C) and A549 cell line (Supplementary Fig. 1D,E) was attenuated by LPGAT1 knockdown. FC analysis revealed that, compared to the control group, the LPGAT1-knockdown group inhibited the cell cycle progression, G2/M cells were decreased, and G1 cells were increased in H1299 cell lines (Fig. 2D–F and Supplementary Fig. 1F), while only G1 cells were increased in A549 cell lines (Supplementary Fig. 1G,H). The green-, yellow-, and blue-emitting populations in the latter corresponded to G1, G1/S and S/G2-M cells, respectively. In addition, the data of FC indicated that the apoptotic rate of LUAD cells was dramatically elevated after LPGAT1 knockdown (Fig. 2G–I, Supplementary Fig. 1I,J and Supplementary Fig. 2A). Furthermore, the findings of CFA showed that the number of colonies was decreased in H1299 cells with LPGAT1 knockdown (Fig. 2J,K).

Fig. 2.

Knockdown of LPGAT1 inhibits cell proliferation and promotes cell apoptosis in H1299 cell lines. (A,B) The expression levels of LPGAT1 in four NSCLC cell lines (A549, H1299, PC9, and H23), and the knockdown efficiency of siLPGAT1 in H1299, were detected by qRT-PCR. Unpaired two-tailed t-test; ${}^{****}$p 0.0001. (C) Cell Counting Kit-8 assay was used to detect H1299 cell proliferation, n = 3. Two-way ANOVA. ${}^{*}$p = 0.0262 (day 3), ${}^{**}$p = 0.0030 (day 4), ${}^{***}$p = 0.0006 (day 5). (D,E) The cell cycles in the control (D) and LPGAT1-knockdown groups (E) were detected by flow cytometry, n = 3. (F) Statistical results of the cell cycle assay. Unpaired two-tailed t-test; ${}^{*}$p = 0.0115 (G1 phase), ${}^{ns}$p = 0.0552 (S phase), ${}^{**}$p = 0.0087 (G2/M phase). (G,H) Cell apoptosis in the control (G) and LPGAT1-knockdown groups (H) was detected by flow cytometry, n = 3. (I) Statistical results of cell apoptosis assay. Unpaired two-tailed t-test; ${}^{**}$p = 0.0019. (J) Colony formation assay, n = 3. (K) Statistical results of the colony formation assay. Unpaired two-tailed t-test; ${}^{**}$p = 0.0015.

A BALB/cnu/nu nude mouse xenograft model was constructed by subcutaneously injecting H1299 cells to investigate whether LPGAT1 knockdown can suppress LUAD progression in vivo. H1299 cells were transfected with lentiviruses (shCtrl and shLPGAT1) for 48 h (Fig. 3A). The infection efficiency of the lentivirus was 93.6% and 94.1%, respectively (Supplementary Fig. 2B). Eight mice were subcutaneously injected with shCtrl-infected H1299 cells (4 $\times$ 10${}^6$ each) into the right flanks, while the other eight mice were injected with shLPGAT1-infected H1299 cells (Fig. 3B). Consistent with the in vitro results, the LPGAT1 knockdown group had a more significant inhibitory effect on tumor volume growth in a time-dependent fashion (Fig. 3C). After 4 weeks, tumors were peeled from the nude mouse subcutis. Then, the tumors were weighed (Fig. 3D) and their length and width were measured to calculate the tumor volumes (Fig. 3E–G). The results of the experiments performed with A549 cells were similar to those of the H1299 cells (Supplementary Fig. 2C,D). The tumors in the shLPGAT1 group were smaller in size and lower in weight compared with those in the control group.

Fig. 3.

Knockdown of LPGAT1 inhibits tumor growth in tumor-bearing mice. (A) Infection efficiency of lentiviruses (shCtrl and shLPGAT1) in H1299 cells. Scale bars: 25 $\mu$m. (B) In vivo imaging of tumor-bearing mice in the control and LPGAT1-knockdown groups, n = 8. Scale bars: 1 cm. (C, D) Measurement of the volume and weight of tumors. C, measure the volume of tumors twice a week since 17 days after injection, volume (mm${}^3$) = $\pi$/6 $\times$ length $\times$ width $\times$ width; ${}^{*}$p = 0.0124 (at day 17), ${}^{*}$p = 0.0106 (at day 21), ${}^{*}$p = 0.0110 (at day 24), ${}^{***}$p = 0.0002 (at day 28), ${}^{**}$p = 0.0011 (at day 30); (D) tumors were peeled and then weighed after 4 weeks, Welch t-test, ${}^{**}$p = 0.0015 (tumor weight). (E) Tumor size was measured in the control and LPGAT1-knockdown groups. Scale bars: 1 cm. (F, G) Statistical analysis of the differences in length and width between the two groups of tumors. ${}^{**}$p = 0.0017 (tumor length), ${}^{***}$p = 0.0006 (tumor width).

After the knockdown of LPGAT1, H1299 cells were collected for RNA-Seq. 106 DEGs are shown in the heatmap (Fig. 4A). These genes were detected by GO enrichment analysis; the top 10 results in BPs, CCs, and MFs are shown in Fig. 4B and Supplementary Table 4. We found the most prominent regulation involved the phosphatidylglycerol metabolic pathway, phosphatidylglycerol acyl-chain remodeling, the phosphatidic acid biosynthetic process, the phosphatidic acid metabolic processes, and the phospholipid biosynthetic process in BPs. There were five common genes in the five most prominent pathways, namely LPCAT4, LPCAT1, Lyso-CL acyltransferase 1 (LCLAT1), Glycerol-3-phosphate acyltransferase mitochondrial (GPAM), and cardiolipin synthase 1 (CRLS1). In order to verify the effect of target gene LPGAT1 knockdown on these pathways, we performed qRT-PCR and WB analyses. Based on the qRT-PCR data, these genes were up-regulated in H1299 cells with LPGAT1 knockdown compared to the control group (Fig. 5A–E). However, WB analysis demonstrated that the levels of CRLS1, GPAM and LPLAT1 proteins had an upward trend in H1299 cells with LPGAT1 knockdown, but no statistical significance was observed. Similarly, there was no marked difference in the expression of LPCAT1 and LPCAT4 between the two groups (Fig. 5F,G).

Fig. 4.

Heatmap of DEGs and GO functional enrichment analysis after LPGAT1 knockdown in H1299 cell lines. (A) Heatmap of differentially expressed genes between the control group (on the right) and the shLPGAT1 group (on the left). (B) Top 30 GO terms, including biological processes (BPs), cellular components (CCs) and molecular functions (MFs).

Fig. 5.

Expression of the related proteins after LPGAT1 knockdown in H1299 cell lines. (A–E) The mRNA levels of CRLS1, LCLAT1, GPAM, LPCAT1 and LPCAT4 in the shCtrl and shLPGAT1 groups. Unpaired two-tailed t-test; ${}^{**}$p = 0.0052 (CRLS1), ${}^{**}$p = 0.0021 (LCLAT1), ${}^{****}$p 0.0001 (GPAM), ${}^{***}$p = 0.0001 (LPCAT1),${}^{****}$p 0.0001 (LPCAT4). (F) The protein levels of CRLS1, LCLAT1, GPAM, LPCAT1 and LPCAT4 in the shCtrl and shLPGAT1 groups. (G) Statistical results of protein expression intensity.