Targeting LINC00665/miR-199b-5p/SERPINE1 axis to inhibit trastuzumab resistance and tumorigenesis of gastric cancer via PI3K/AKt pathway

Abstract

Long noncoding RNAs (lncRNAs) serve as critical mediators of tumor progression and drug resistance in cancer. Herein, we identified a lncRNA, LINC00665, associated with trastuzumab resistance and development in gastric cancer (GC). LINC00665 was highly expressed in GC tissues and high expression of LINC00665 was correlated with poor prognosis. LINC00665 knockdown was verified to suppress migration, invasion, and resistance to trastuzumab in GC. Furthermore, we found that LINC00665 participates in the infiltration of naive B cells, mast cells, and T follicular helper (Tfh) cells. Mechanistically, LINC00665 was confirmed to regulate tumorigenesis and trastuzumab resistance by activating PI3K/AKt pathway. LINC00665 sponged miR-199b-5p to interact with SERPINE1 expression, resulting in the increase of phosphorylation of AKt, thus participating in the PI3K/AKt pathway. To summarize, LINC00665 facilitated the tumorigenesis and trastuzumab resistance of GC by sponging miR-199b-5p and promoting SERPINE1 expression, which further activated PI3K/AKt signaling; this finding reveals a new mechanism by which LINC00665 modulates tumor development and drug resistance in GC.

Keywords: Drug resistance; Gastric cancer; Long non-coding RNA; PI3K/AKt signaling; Targeted therapy; ceRNA.

Graphical abstract

Fig. 1

Identification of LINC00665/miR-199b-5p/SERPINE1 pathway. A Schematic diagram of the process design. B PCA shows overall differences between normal tissue and GC in GSE95667. C PCA shows overall differences between normal tissue and GC in GSE109476. D Heatmap of DEGs from GSE95667. E Heatmap of DEGs from GSE109476. F Venn diagram of common DEGs from GSE109476 and GSE95667. G The lncRNA-miRNA network predicted by the miRcode database. H Venn diagram of target genes from GC-related genes, target mRNA, and TOP 50 most differential survival genes. I The correlation between the expression of target genes and overall survival rate of GC patients: SERPINE1 is negatively related to the overall survival rate of GC patients. J Venn diagram of upstream gene of SERPINE1 from the TargetScan and miRDB databases. K The potential binding site of SERPINE1 predicted by the RNAhybrid database.

Fig. 2

LINC00665 is upregulated in GC tissues. A LINC00665 is overexpressed in GC tissues analyzed by the GEPIA-STAD database. B The mRNA level of LINC00665 is higher in paired GC tumor (n = 30) than that in adjacent normal tissues (n = 30). C GO analysis shows that LINC00665 participated in multiply processes (Biological Process, BP; Cellular Component, CC; Molecular Function, MF). D KEGG analysis indicates LINC00665 exerted its function in GC tumorgenesis via various pathways. E Top 10 BP enrichment suggested that LINC00665 is involved in the drug resistance. F-H Correlation between the expression level of LINC00665 And the infiltration levels of naive B cells, Tfh cells, and mast cells. I KEGG analysis shows that SERPINE1 was involved in diverse pathways. ∗P < 0.05; ∗∗∗∗P < 0.0001.

Fig. 3

LINC00665 targets miR-199b-5p to induce SERPINE1 level. A The level of miR-199b-5p is lower in paired GC tumor (n = 30) than that in adjacent normal tissues (n = 30). B SERPINE1 is highly-expressed in GC tissues. C The biding site of LINC00665 and miR-199b-5p predicted by the RNAhybrid database. D Correlation of the expression of LINC00665 and miR-199b-5p in clinical GC tissues. E Correlation of the expression of LINC00665 and SERPINE1 in clinical GC tissues. F A dual-luciferase reporter assay was performed to verify that LINC00665 obviously inhibits the luciferase activity of the WT 3′-UTR but not the MUT 3′-UTR of miR-199b-5p. G A dual-luciferase reporter assay was conducted to prove that miR-199b-5p significantly suppresses the luciferase activity of the WT 3′-UTR but not the MUT 3′-UTR of SERPINE1. ∗P < 0.05; ∗∗P < 0.01; ∗∗∗P < 0.001; ∗∗∗∗P < 0.0001.

Fig. 4

LINC00665 contributed to the tumorgenesis of GC via SERPINE1. A Scratch assay (scale bar, 200 μm) and Transwell assay (scale bar, 150 μm) evaluate the migration and invasion ability of GC cells with LINC00665 knockdown. B WB assay show expression of tumour progression-related proteins (PCNA, p16, MMP-2, E-cadherin, N-cadherin, and Vimentin) and PI3K/AKt signaling-associated proteins (p-AKt and AKt) in GC cells with LINC00665 knockdown or NC-siRNA or control. C CCK-8 assay demonstrated miR-199b-5p inhibitor restore cell proliferation in LINC00665 knockdown cells. D Scratch assay indicated miR-199b-5p inhibitor restored cell migration ability in LINC00665 knockdown cells. Scale bar, 200 μm. E Transwell assay showed that miR-199b-5p inhibitor restore cell invasion capacity in LINC00665 knockdown cells. Scale bar, 150 μm. F WB assay revealed the expression of proteins mentioned above in cells transfected as indicated. ∗P < 0.05; ∗∗P < 0.01; ∗∗∗P < 0.001; ∗∗∗∗P < 0.0001.

Fig. 5

LINC00665 promotes trastuzumab resistance through PI3K/AKt pathway. A The cell viability of cells treated with trastuzumab with or without LINC00665-shRNA. B Schematic diagram of subcutaneous xenograft model and representative images of tumors from nude mice inoculated with NCI-N87/TR cells. C The volume of subcutaneously transplanted tumors in 2 groups: LINC00665-shRNA and NC-shRNA. D The weight of subcutaneously transplanted tumors in the 2 groups. E The protein level of the subcutaneously transplanted tumors in the 2 groups. ∗P < 0.05; ∗∗P < 0.01; ∗∗∗P < 0.001; ∗∗∗∗P < 0.0001.

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