LncRNA TMEM99 Complexes with IGF2BP2 to Inhibit Autophagy in Lung Adenocarcinoma

Abstract

Lung adenocarcinoma (LUAD), the most common type of lung cancer, has a poor prognosis. Long noncoding RNAs (lncRNAs) play a key role in LUAD progression, yet the biological role of lncRNA TMEM99 remains unexplored. In this study, its function in inhibiting autophagy in LUAD is explored. Using RNA sequencing and quantitative reverse transcription PCR (qRT-PCR), TMEM99 is found upregulated in LUAD tissues and cell lines, correlating with poor patient outcomes. In vivo and in vitro assays confirmed that TMEM99 promotes cell proliferation, migration, and invasion, and inhibits autophagy. Mechanistically, the 3' end of TMEM99 binds to the K‑homology domain 1 (KH1) and KH4 domains of far upstream element‑binding protein 3 (FUBP3), stabilizing its protein. The TMEM99-FUBP3 complex binds to p21 mRNA and recruits IGF2BP2 in an N⁶‑methyladenosine (m⁶A)-dependent manner, which enhances mRNA stability and translation efficiency. This study reveals that TMEM99 plays a crucial regulatory role in LUAD autophagy and presents a novel cytoplasmic regulatory mechanism contributing to LUAD progression.

Keywords: FUBP3; Insulin‐like growth factor 2 mRNA‐binding protein 2 (IGF2BP2); TMEM99; autophagy; lung adenocarcinoma.

Figure 1

TMEM99 is a key oncogene in lung adenocarcinoma development. A) Significantly up‐regulated lncRNAs (FC > 1.5, p < 0.01) in AIS versus N, MIA versus N, and LUAD versus N are shown via a Venn diagram. B‐D) Overlapping lncRNAs from (A) in AIS (B), MIA (C), and LUAD (D). E) Top 5 enriched lncRNAs in LUAD from TCGA. F) TMEM99 expression in LUAD and adjacent normal tissues from FUSCC (p < 0.0001). G) TMEM99 expression in AIS/MIA, invasive adenocarcinoma, and normal lung tissues in FUSCC (p < 0.0001). H) TMEM99 expression in LUAD and normal tissues in TCGA‐LUAD cohort (p < 0.0001). I) TMEM99 expression in AAH, invasive adenocarcinoma, and normal lung tissues in Kadara's cohort (p < 0.0001). J) TMEM99 impact on Recurrence‐Free Survival (FRS) in LUAD from FUSCC (p = 0.014). K) TMEM99 expression in Beas‐2B, 293T, and LUAD cell lines by qRT‐PCR (± SEM; **p < 0.01; ***p < 0.001; n.s. not significant). L‐O) H1299 (L), PC9 (M), A549 (N), and H1975 (O) cells transfected with control or TMEM99‐specific siRNAs (si1‐TMEM99 and si2‐TMEM99) assessed by proliferation assays (± SEM; **p < 0.01; ***p < 0.001). P, Q) TMEM99 subcellular localization via nuclear‐cytoplasmic assays in H1299 (P) and PC9 (Q) cells, using MALAT1 as nuclear and circHIPK3 as cytoplasmic markers. The data are shown as the mean ± SD (n = 3). **p < 0.01, ***p < 0.001, by two‐tailed unpaired Student's t test (F‐I), one‐way ANOVA (K‐O) with Dunnett's post hoc test, and by the log‐rank test (J). AIS, Adenocarcinoma in situ; MIA, Minimally invasive adenocarcinoma; N, Normal tissue; AAH, Atypical adenomatous hyperplasia; FUSCC, Fudan University Shanghai Cancer Center.

Figure 2

LncRNA TMEM99 promotes proliferation, migration, and invasion of H1299 and PC9 cells. A, B) H1299 (A) and PC9 (B) cells infected with control shRNA (sh‐CTL) or TMEM99‐specific shRNAs (sh1‐TMEM99, sh2‐TMEM99) were analyzed for proliferation (± SEM; ***p < 0.001). C) H1299 and PC9 cells, as described in (A‐B), were analyzed by colony formation assay. D) Quantification of colonies from (C) (± SEM; ***p < 0.001). E, G) H1299 and PC9 cells, as described in (A‐B), subjected to migration (E) and invasion (G) assays. F, H) Quantification of migrated (F) and invaded (H) cells from (E, G) (± SEM; **p < 0.01; ***p < 0.001). I, J) H1299 (I) and PC9 (J) cells transfected with control vector (PCDH) or TMEM99‐expressing vector, followed by proliferation assay (± SEM; *p < 0.05; ***p < 0.001). K) H1299 and PC9 cells, as described in (I‐J), were analyzed by colony formation assay. L) Quantification of colonies from (K) (± SEM; *p < 0.05; **p < 0.01). M, O) H1299 and PC9 cells, as described in (I‐J), subjected to migration (M) and invasion (O) assays. N, P) Quantification of migrated (N) and invaded (P) cells from (M, O) (± SEM; **p < 0.01; ***p < 0.001). Q) RNA‐FISH analysis in H1299 cells from (A) using TMEM99‐specific probe (Cy5‐TMEM99). Red: TMEM99; Blue: DAPI. R; Scale bar = 5 µm.) Quantification of fluorescence intensity (Cy5) (± SEM; ***p < 0.001). The data are shown as the mean ± SD (n = 3). **p < 0.01, ***p < 0.001, by two‐tailed unpaired Student's t test (I, J, L, N, and P) and one‐way ANOVA (A, B, D, F, H, and R) with Dunnett's post hoc test. shRNA, short hairpin RNA; sh‐CTL, control shRNA; sh1‐TMEM99/sh2‐TMEM99, TMEM99‐specific shRNA sequences 1 and 2; PCDH, control vector; RNA‐FISH, RNA fluorescence in situ hybridization; Cy5, cyanine 5, a fluorescent dye; DAPI, 4′,6‐diamidino‐2‐phenylindole, a nuclear stain.

Figure 3

LncRNA TMEM99 interacts with FUBP3 protein. A) In vitro biotinylated TMEM99 RNA pull‐down followed by silver staining in H1299; antisense (Anti) used as a negative control. B) TMEM99‐associated proteins were purified and analyzed by mass spectrometry; the top 10 identified proteins are shown. C) TMEM99 RNA‐pull down assay with FUBP3, PTBP1, PTBP3, NOVA1, IGF2BP2 levels analyzed by IB in H1299; GAPDH as loading control; Anti and 10% input (10% IN) included. D) H1299 cells infected with control or TMEM99‐specific shRNAs to examine PTBP1, PTBP3, NOVA1, IGF2BP2 expression by IB; GAPDH as loading control. E) H1299 and PC9 cells infected with sh‐CTL, sh1/2‐TMEM99, PCDH, TMEM99‐expressing vector to examine FUBP3 expression by IB; GAPDH as control. F, G) FUBP3 RIP assay in H1299 (F) and PC9 (G) with TMEM99 expression measured by qRT‐PCR (± SEM; ***p < 0.001); 10% input as positive and IgG as negative controls. H, I) H1299 cells infected with sh‐CTL, sh1/2‐TMEM99 followed by qRT‐PCR to examine TMEM99 (H) and FUBP3 mRNA (I) expression (± SEM; ***p < 0.001; n.s. not significant). J, K) H1299 cells infected with sh‐CTL, sh1/2‐FUBP3 followed by qRT‐PCR to examine FUBP3 (J) and TMEM99 (K) expression (± SEM; **p < 0.01; n.s. not significant). L) TMEM99 isoforms shown: Full Length (FL), Fragments (F1‐F5), and Anti. M) In vitro biotinylated TMEM99 isoforms RNA‐pull down assay in H1299 or PC9 cells to examine FUBP3 by IB; 10% input included. N) Schematic of FUBP3 domain architecture and truncations (KH1‐KH4) with Flag tag, Full Length (FL), Fragments (F1‐F9). O, P) RIP assay with FUBP3 truncations and qRT‐PCR to examine TMEM99 enrichment in H1299 (O) or PC9 (P) cells (± SEM; ***p < 0.001; n.s. not significant). Q, R) H1299 or PC9 cells infected with sh‐CTL or sh1‐TMEM99 treated with CHX (Q) or MG132 (R), followed by IB using FUBP3 antibody; GAPDH as control. The data are shown as the mean ± SD (n = 3). *p < 0.05, **p < 0.01, ***p < 0.001, by two‐tailed unpaired Student's t test (F, G) and one‐way ANOVA followed by Dunnett's post hoc test (H‐K, O, and P). Anti, antisense RNA; IB, immunoblotting; RIP, RNA immunoprecipitation; sh1/2‐FUBP3, FUBP3‐specific shRNA sequences 1 and 2; CHX, cycloheximide, a protein synthesis inhibitor; KH1‐KH4, K homology domains 1–4 in FUBP3; FL, full‐length protein/RNA; F1‐F5/F1‐F9, fragments 1–5 or 1–9 of TMEM99 RNA or FUBP3 protein.

Figure 4

FUBP3 promotes proliferation, migration, and invasion of H1299 and PC9 cells. A‐C) FUBP3 expression in lung adenocarcinoma and adjacent normal tissues from FUSCC (p = 0.51) (A), TCGA (p < 0.0001) (B), and CPTAC (p < 0.0001) (C) databases. D) H1299 cells infected with sh‐CTL, sh1/2‐FUBP3 were analyzed for proliferation (± SEM; ***p < 0.001). E, F) H1299 (E) and PC9 (F) cells infected with sh‐CTL, sh1/2‐TMEM99, and transfected with or without FUBP3‐expressing vector, followed by proliferation assay (± SEM; *p < 0.05; **p < 0.01; ***p < 0.001). PCDH: control vector. G) H1299 and PC9 cells infected with sh‐CTL, sh1/2‐FUBP3 subjected to colony formation assay. H, I) Quantification of colony numbers from (G) (± SEM; *p < 0.05; **p < 0.01). J, K) H1299 cells from (E) subjected to colony formation assay (J), with colony quantification in (K) (± SEM; *p < 0.05; **p < 0.01; ***p < 0.001). L) H1299 and PC9 cells infected with sh‐CTL, sh1/2‐FUBP3 subjected to migration assay. Scale bar = 100 µm. M,N) Quantification of migrated cells from (L) (± SEM; *p < 0.05; **p < 0.01; ***p < 0.001). O, Q) H1299 cells from (E) subjected to migration (O) and invasion (Q) assays. Scale bar = 100 µm. P, R) Quantification of migrated and invaded cells from (O, Q) (± SEM; *p < 0.05; **p < 0.01; ***p < 0.001). S) H1299 cells transfected with TMEM99 or FUBP3‐expressing vectors analyzed by FISH‐immunofluorescence using probes for TMEM99 (Cy5‐TMEM99) and FUBP3 (FITC‐FUBP3). Blue: DAPI; Red: TMEM99; Green: FUBP3. Scale bar = 5 µm. The data are shown as the mean ± SD (n = 3). *p < 0.05, **p < 0.01, ***p < 0.001, by two‐tailed unpaired Student's t test (A–C) and one‐way ANOVA followed by Dunnett's post hoc test (D‐F, H, I, K, M, N, P, and R). CPTAC, Clinical Proteomic Tumor Analysis Consortium; FITC, fluorescein isothiocyanate, fluorescent dye.

Figure 5

TMEM99 regulates p21 via the FUBP3 signaling axis. A, B) qRT‐PCR analysis of FUBP3 and p21 expression in H1299 (A) and PC9 (B) cells infected with sh‐CTL, sh1/2‐FUBP3 (± SEM; *p < 0.05; **p < 0.01; ***p < 0.001). C, D) qRT‐PCR analysis of TMEM99 and p21 expression in H1299 (C) and PC9 (D) cells infected with sh‐CTL, sh1/2‐TMEM99 (± SEM; *p < 0.05; ***p < 0.001). E, F) IB analysis of FUBP3, N‐MYC, C‐MYC, and p21 protein levels in H1299 cells infected with sh‐CTL, sh1/2‐FUBP3 (E) or sh1/2‐TMEM99 (F); GAPDH as control. G, I) FUBP3 RIP assay in H1299 (G) and PC9 (I) cells with p21 expression measured by qRT‐PCR (± SEM; ***p < 0.001); 10% input as positive, IgG as negative controls. H, J) In vitro FUBP3 truncation RIP assay and qRT‐PCR analysis to examine p21 enrichment in H1299 (H) and PC9 (J) cells (± SEM; *p < 0.05; **p < 0.01; ***p < 0.001; n.s. not significant). K) p21 isoforms: p21, p21 5′UTR, p21 CDS, p21 3′UTR, and Anti. L, M) RNA‐pull down assay with biotinylated p21 isoforms in H1299 (L) and PC9 (M) cells to examine FUBP3 protein level by IB; 10% input included. N‐Q) H1299 (N, O) and PC9 (P, Q) cells infected with sh‐CTL or sh1‐TMEM99 treated with MG132 (N, P) or CHX (O, Q), followed by IB analysis using p21 antibody; GAPDH as control. R) FISH‐immunofluorescence analysis of H1299 cells transfected with p21 or FUBP3‐expressing vectors using p21 (FITC‐p21) and FUBP3 (RSred‐FUBP3) probes. Blue: DAPI; Red: p21; Green: FUBP3. Scale bar = 5 µm. S, T) qRT‐PCR analysis of p21 expression in H1299 (S) and PC9 (T) cells infected with sh‐CTL or sh1‐TMEM99, transfected with or without FUBP3‐expressing vector (± SEM; *p < 0.05; **p < 0.01; ***p < 0.001); PCDH as control vector. The data are shown as the mean ± SD (n = 3). *p < 0.05, **p < 0.01, ***p < 0.001, by two‐tailed unpaired Student's t test (I, G) and one‐way ANOVA followed by Dunnett's post hoc test (A‐D, H, J, S, and T). UTR, untranslated region; CDS, coding sequence; RSred, red fluorescent dye.

Figure 6

IGF2BP2 is involved in the TMEM99‐FUBP3‐p21 regulatory axis to affect cell autophagy. A) TMEM99 RNA‐pull down assay in H1299 and PC9 cells with IGF2BP2 protein levels analyzed by IB; GAPDH as loading control; 10% input included. B, C) IGF2BP2 RIP assay in H1299 cells with TMEM99 (B) and p21 (C) levels measured by qRT‐PCR (± SEM; **p < 0.01; ***p < 0.001); 10% input as positive, IgG as negative controls. D) H1299 and PC9 cells infected with sh‐CTL, sh1/2‐TMEM99, PCDH, or TMEM99‐expressing vector to examine IGF2BP2 expression by IB; GAPDH as control. E‐J) H1299 (E‐G) and PC9 (H‐J) cells infected with control or IGF2BP2‐specific siRNAs (si1/2‐IGF2BP2) were analyzed for IGF2BP2 (E, H), TMEM99 (F, I), and p21 (G, J) expression by qRT‐PCR (± SEM; **p < 0.01; ***p < 0.001; n.s. not significant). K) RNA‐pull down assay with TMEM99 isoforms in H1299 and PC9 cells to examine IGF2BP2 by IB. L) Schematic of IGF2BP2 domain architecture and truncations (RRM1, RRM2, KH1‐KH4) with Flag tag. M, N) RIP assay with IGF2BP2 truncations and qRT‐PCR to examine TMEM99 (M) and p21 (N) enrichment in H1299 cells (± SEM; *p < 0.05; ***p < 0.001; n.s. not significant). O, P) H1299 (O) and PC9 (P) cells infected with sh‐CTL, sh1/2‐TMEM99, transfected with or without p21‐expressing vector, were analyzed by electron microscopy; PCDH as a control vector. Scale bar = 1 µm. Q, R) Western blot analysis of H1299 (Q) and PC9 (R) cells transfected with sh‐CTL or sh1‐TMEM99, with or without p21 vector, examining Actin, beclin1, LC3, Atg5, ATG7, p62, and p21 expression; PCDH as control. S, T) Flow cytometry analysis of H1299 (Q) and PC9 (R) cells transfected with sh‐CTL or sh1‐TMEM99, with or without p21 vector, examining cell cycle. U, V) Quantification of cell cycle (G1, S, G2/M) from (S, T). W, X) FISH‐immunofluorescence analysis of H1299 (S) and PC9 (T) cells using probes for TMEM99 (Cy5‐TMEM99), IGF2BP2 (IGF2BP2‐RSred), and p21 (FITC‐p21). Blue: DAPI; Red: TMEM99; Green: IGF2BP2. Scale bar = 5 µm. The data are shown as the mean ± SD (n = 3). *p < 0.05, **p < 0.01, ***p < 0.001, by two‐tailed unpaired Student's t test (B, C) and one‐way ANOVA followed by Dunnett's post hoc test (E‐G, M, N). siRNA, small interfering RNA; si1/2‐IGF2BP2, IGF2BP2‐specific siRNA sequences 1 and 2; RRM1/RRM2, RNA recognition motif domains 1 and 2 in IGF2BP2; KH1‐KH4, K homology domains 1–4 in IGF2BP2; Flag tag, a peptide tag for protein detection; LC3, microtubule‐associated protein 1A/1B‐light chain 3, autophagy marker; Atg5, autophagy‐related 5; ATG7, autophagy‐related 7; p62, sequestosome‐1, autophagy substrate.

Figure 7

TMEM99 enhances p21 mRNA stability by interacting with FUBP3 and IGF2BP2. A, C) H1299 (A) and PC9 (C) cells infected with sh‐CTL or sh1‐IGF2BP2, transfected with control vector (PCDH) or FUBP3 vector, treated with Actinomycin D (Act D), followed by qRT‐PCR for p21 expression (± SEM; *p < 0.05; **p < 0.01). B, D) H1299 (B) and PC9 (D) cells infected with sh‐CTL or sh1‐FUBP3, transfected with PCDH or FUBP3 vector, treated with Act D, followed by qRT‐PCR for p21 expression (± SEM; *p < 0.05; **p < 0.01; n.s. not significant). E) Immunoprecipitation and IB of H1299 and PC9 cells transfected with IGF2BP2 or FUBP3 vectors; 10% input included. F, G) Mapping of m6A‐modified regions in TMEM99 and p21 mRNA via m6A RIP. H) qRT‐PCR of TMEM99 expression in H1299 cells transfected with PCDH, TMEM99‐wt, or TMEM99‐mut vectors (± SEM; **p < 0.01; ***p < 0.001). I) qRT‐PCR of p21 expression in H1299 cells transfected with PCDH, p21‐wt, or p21‐mut vectors (± SEM; **p < 0.01; ***p < 0.001). J, K) IGF2BP2 RIP in H1299 cells from (H, I) with TMEM99 (J) or p21 (K) enrichment measured by qRT‐PCR (± SEM; **p < 0.01; n.s. not significant). L, M) IGF2BP2 RIP in H1299 cells infected with si‐CTL or si1/2‐METTL3, transfected with p21‐wt (L) or p21‐mut (M), followed by qRT‐PCR for p21 enrichment (± SEM; *p < 0.05; **p < 0.01; ***p < 0.001). N, O) IGF2BP2 RIP in H1299 cells infected with sh‐CTL, sh1/2‐FUBP3, transfected with p21‐wt (N) or p21‐mut (O), followed by qRT‐PCR for p21 expression (± SEM; **p < 0.01; ***p < 0.001). P, Q) FUBP3 RIP in H1299 cells infected with sh‐CTL, sh1/2‐IGF2BP2, transfected with p21‐wt (P) or p21‐mut (Q), followed by qRT‐PCR for p21 expression (± SEM; n.s. not significant). R, S) IGF2BP2 RIP in H1299 cells transfected with PCDH, FUBP3, or IGF2BP2 vectors, followed by qRT‐PCR for p21‐wt (R) or p21‐mut (S) expression (± SEM; *p < 0.05; **p < 0.01). T, U) m6A RIP in H1299 cells from (R, S), followed by qRT‐PCR for p21 m6A site expression (± SEM; *p < 0.05; **p < 0.01). The data are shown as the mean ± SD (n = 3). *p < 0.05, **p < 0.01, ***p < 0.001, by one‐way ANOVA followed by Dunnett's post hoc test. Act D, actinomycin D, a transcriptional inhibitor; m6A, N6‐methyladenosine, an RNA methylation modification; wt, wild‐type; mut, mutant; METTL3, methyltransferase‐like 3, an m6A methyltransferase enzyme; m6A RIP, m6A‐specific RNA immunoprecipitation.

Figure 8

TMEM99 ASO combined with PTX synergistically inhibits tumor growth in vivo. A) H1299 cells infected with sh‐CTL or sh1‐TMEM99 were subcutaneously injected into BALB/C nude mice for xenograft assays; tumors shown. B) Tumor growth curve from (A) (± SEM; ***p < 0.001). C) Tumor weight from (A) (± SEM; *p < 0.05). D) Bioluminescence imaging of nude mice intravenously injected with H1299 cells expressing luciferase and transduced with sh‐CTL or sh1‐TMEM99. Imaging was performed using the IVIS system at week 3 post‐injection. Representative images are shown. E) Quantification of in vivo bioluminescence signal from mice injected with sh‐CTL or sh1‐TMEM99 H1299 cells (n = 5 per group) (± SEM; ***p < 0.01). F) H1299 cells infected with sh‐CTL or sh1‐TMEM99, transfected with PCDH or p21 vector, were subcutaneously injected into BALB/C nude mice for xenograft assays; tumors shown. G) Tumor growth curve from (E) (± SEM; *p < 0.05; **p < 0.01; n.s. not significant). H) Tumor weight from (E) (± SEM; **p < 0.01; n.s. not significant). I) Representative immunohistochemistry micrographs of FUBP3, IGF2BP2, and p21 in tumor tissues from (E). Scale bar = 100 µm. J‐L) BALB/c nude mice (n = 20) inoculated with H1299 cells, randomized, and treated with ASO‐TMEM99 (5 nmol, every 3 days) or PTX (10 mg kg⁻¹, every 5 days) alone or combined. Treatment design shown (H); tumors excised, photographed (I), and weighed (J) (± SEM; **p < 0.01; ***p < 0.001). The data are shown as the mean ± SD (n = 5). *p < 0.05, **p < 0.01, ***p < 0.001, by two‐tailed unpaired Student's t test (B, C, E) and one‐way ANOVA followed by Dunnett's post hoc test (G, H, L). ASO, antisense oligonucleotide; PTX, paclitaxel, a chemotherapy drug; BALB/C nude mice, an immunodeficient mouse strain lacking T cells; IVIS, In Vivo Imaging System, for bioluminescence detection.