M⁶A-mediated up-regulation of LncRNA LIFR-AS1 enhances the progression of pancreatic cancer via miRNA-150-5p/ VEGFA/Akt signaling

Figures & data

Table 1. The relationship between LIFR-AS1 expression and PC patient clinic-pathological features.

Clinical features	Total	LIFR-AS1 expression		p-value
		High(N = 70)	Low(N = 69)
Age (years)				.239
< 60	40	17	23
≥ 60	99	53	46
Gender				.145
Male	76	34	42
Female	63	36	27
Tumor location				0.343
Head	62	34	28
Body and Tail	77	36	41
Tumor size (cm)				0.031
< 5	54	21	33
≥ 5	85	49	36
Differentiation grade				0.063
Well	46	18	28
Moderate + Poor	93	52	41
TNM stage				0.001
I + II	86	34	52
III	53	36	17
Depth of invasion				0.199
T1 + T2	64	36	28
T3 + T4	75	34	41
Lymph node metastasis				0.011
No	86	36	50
Yes	53	34	19
Distant metastasis				0.582
No	106	52	54
Yes	33	18	15
Adjuvant chemotherapy				0.353
No	65	30	35
Yes	74	40	34
CA19-9, kU/L				0.884
< 40	104	52	52
≥ 40	35	18	17

CA19-9 carbohydrate antigen 19–9; Pearson chi-square test was used for comparison between subgroups.

Figure 1. PC samples exhibit the upregulation of LIFR-AS1.

(a) LIFR-AS1 levels in 139 pairs of PC tissues and adjacent normal tissues were quantified via qRT-PCR. *P < 0.05 vs. adjacent normal. (b) Expression of LIFR-AS1 was quantified in the AsPC-1, BxPC-3, Mia-capa2, HPAC, and PANC-1 PC cell lines and in the control HPDE cell line via qRT-PCR. *P < 0.05 vs. HPDE. *P < 0.05; **P < 0.01; ***P < 0.001; P > 0.05.

Figure 2. METTL3 induced LIFR-AS1 m⁶A to enhance ItsmRNA stability in PC.

(a) The correlation the METTL3 and LIFR-AS1 in PC based on GEPIA database. (b, c) qRT-PCR analysis of the level of METTL3 and LIFR-AS1 in indicated PC cell lines. (d) Identify the specific m⁶A methylation loci of LIFR-AS1 by SRAMP website. (e) m⁶A level in LIFR-AS1 in PANC1 and Mia-capa cells with sh-NC or sh-METTL3 line by m⁶A-qPCR. (f) qRT-PCR analysis of LIFR-AS1 mRNA relative to ACTIN in indicated treated PC. *P < 0.05; **P < 0.01; ***P < 0.001; P > 0.05.

Figure 3. Reduced levels of LIFR-AS1 impair the proliferation, invasion, and survival of PC cellsin vitro.

(a) Expression of LIFR-AS1 in PANC1 and Mia-capa2 cells that had been transfected with si-LIFR-AS1 or si-NC constructs was assessed via qRT-PCR. (b) A CCK-8 assay was used to gauge the proliferation of cells transfected as in A. (c, d) the migration (c) and invasive (d) activity of cells transfected as in A were assessed using a Transwell assay system. *P < 0.05; **P < 0.01; ***P < 0.001.

Figure 4. LIFR-AS1 interacts with and sequesters miR-150-5p within PC cells.

(a) miR-150-5p interaction with LIFR-AS1 was predicted via bioinformatic analyses. (b) The expression of miR-150-5p was quantified in PANC1 and Mia-capa2 cells that had undergone agomir-150-5p or agomir-NC transfection. (c) Wild type (WT)-LIFR-AS1 or mutant (MUT)-LIFR-AS1 were co-transfected into PANC1 and Mia-capa2 cells along with agomir-150-5p or agomir-NC, and after 48 h luciferase activity was measured. (d) Higher levels of LIFR-AS1 and miR-150-5p were detectable in immunoprecipitates containing Ago2 relative to those prepared using a control IgG. (e) Levels of miR-150-5p expression in 139 pairs of PC and paracancerous control tissues were quantified via qRT-PCR. (f) A Spearman’s correlation analysis of the relationship between miR-150-5p and LIFR-AS1 expression in PC tissue samples from E was conducted. (g) Levels of miR-150-5p were measured via qRT-PCR in PANC1 and Mia-capa2 cells in which LIFR-AS1 had been knocked down. *P < 0.05; **P < 0.01; ***P < 0.001; #P > 0.05.

Figure 5. LIFR-AS1 serves as a ceRNA that sequesters miR-150-5p and thus alters the expression of VEGFA in PC cells.

(a, b) VEGFA expression was quantified via qRT-PCR and Western blotting in PANC1 and Mia-capa2 cells transfected using si-LIFR-AS1 or control constructs. (c) Following transfection with antagomir-150-5p or antagomir-NC, miR-150-5p levels in PC cell lines were confirmed via qRT-PCR. (d, e) PC cell lines were co-transfected with si-LIFR-AS and antagomir-150-5p or antagomir-NC after which VEGFA levels were measured via qRT-PCR and Western blotting. *P < 0.05; **P < 0.01; ***P < 0.001; #P > 0.05.

Figure 6. LIFR-AS1 targets miR-150-5p to activate VEGFA/Akt/mTOR Signaling.

Western blot was used to measure the expression of VEGFA, VEGFR2, p-VEGFR2, Akt, p-Akt, mTOR, p-mTOR in transfected PANC1 and Mia-capa2 cells.