LINC00511 enhances LUAD malignancy by upregulating GCNT3 via miR-195-5p

Abstract

Background: Accumulating evidence suggests that LINC00511 acts as an oncogenic long non-coding RNA (lncRNA) in various cancers, including lung adenocarcinoma (LUAD). Hence, we attempted to elucidate the potential role of LINC00511 in LUAD.

Methods: LINC00511, miR-195-5p, and GCNT3 expression in LUAD was detected by qRT-PCR. Changes in the proliferation, migration, and invasion of LUAD cells after abnormal regulation of LINC00511, miR-195-5p, or GCNT3 were detected by CCK-8, BrdU, wound healing, and transwell assays. Bax and Bcl-2 protein expression was measured by western blotting. Additionally, we identified the targeting effects of LINC00511, miR-195-5p, and GCNT3 using luciferase and RNA immunoprecipitation (RIP) assays.

Results: LINC00511 and GCNT3 were found to be upregulated in LUAD, while miR-195-5p was downregulated. Silencing LINC00511 or GCNT3 decreased the proliferation, migration, invasion, and Bcl-2 protein content in LUAD cells and increased the expression of Bax. Interference with miR-195-5p promoted malignant proliferation of cancer cells. miR-195-5p expression was affected by LINC00511and targeted GCNT3.

Conclusion: Silencing LINC00511 promotes GCNT3 expression by inhibiting miR-195-5p and ultimately stimulates the malignant progression of LUAD.

Keywords: GCNT3; LINC00511; LUAD; miR-195-5p.

Fig. 1

LINC00511/miR-195-5p/GCNT3 axis was identified to be a potential regulator in lung cancer via a ceRNA network. A. Common upregulated lncRNAs in LUAD and LUSC compared with normal non-tumor samples were screened by GEPIA 2 with adj. P < 0.05 and logFC>1.5. LUAD: lung adenocarcinoma. LUSC: lung squamous cell carcinoma. B. The top 5 most significantly upregulated genes from GSE85841 were presented. GSE85841 is a mRNA microarray data of 8 LUAD and adjacent non-tumor samples to screen the upregulated genes with P < 0.05 and logFC> = 1.5. C-G. The expression of the 5 genes in LUAD and LUSC. Data obtained from GEPIA database. H. The candidate miRNAs that link LINC00511 and GCNT3 mRNA were overlapped by Venny 2.1.0. The targets of LINC00511 and GCNT3 mRNA were both predicted using starbase algorithm

Fig. 2

LINC00511 was upregulated in LUAD. A The expression of LINC00511 was detected by qRT-PCR assay in Human LUAD cell line (A549, Calu-3, DV-90 and PC-9) and human bronchial epithelial cell line BEAS-2B. **P < 0.001 compared with BEAS-2B. B The expression of LINC00511 was detected by qRT-PCR assay in cancer tissues (n = 40) and normal tissues (n = 40). C The subcellular localization assay for LINC00511. D The expression of LINC00511 was tested by qRT-PCR in A549 and PC9 cells transfected with si-NC and si-lnc for 48 h. **P < 0.001 compared with si-NC. si-lnc, siRNA of LINC00511. si-NC, negative control of si-RNA

Fig. 3

Interference with LINC00511 inhibited the malignant proliferation of LUAD ells. A Cell viability was detected by CCK-8 assay in A549 and PC9 cells transfected with si-NC and si-lnc for 0, 24, 48 or 72 h. B Cell proliferation was determined by BrdU assay in A549 and PC9 cells transfected with si-NC and si-lnc for 48 h. C Bax and Bl-2 protein expression detected by western blot assay in A549 and PC9 cells transfected with si-NC and si-lnc for 48 h. D Migration capacity was determined by wound healing assay in A549 and PC9 cells transfected with si-NC and si-lnc for 48 h. (E) Cell invasion ability was detected by transwell invasion assay in A549 and PC9 cells transfected with si-NC and si-lnc for 48 h. **P < 0.001 compared with si-NC. si-lnc, siRNA of LINC00511. si-NC, negative control of si-RNA

Fig. 4

LINC00511 targeted and negatively regulated miR-195-5p. A The binding site of LINC00511 with miR-195-5p was confirmed according to bioinformatics analysis. B Luciferase reporter assay confirmed the molecular binding. **P < 0.001 compared with mimic-NC. C RIP was conducted to measure the enrichment of LINC00511 in Ago2 immunoprecipitate and IgG-pellet. **P < 0.001. D The expression of miR-195-5p was detected by qRT-PCR assay in cancer tissues (n = 40) and normal tissues (n = 40). **P < 0.001. E pearson analysis was used to analyze the expression of LINC00511 and miR-195-5p in cancer tissues. F The expression of miR-195-5p was detected by qRT-PCR assay in A549, PC9 and BEAS-2B cells. **P < 0.001 compared with BEAS-2B. G The LINC00511 and miR-195-5p expression were detected by qRT-PCR assay in A549 and PC9 cells transfected with si-lnc or inhibitor for 48 h. **P < 0.001 compared with si-NC group. $$P < 0.001 compared with inhibitor-NC. ##P < 0.001 compared with si-lnc + inhibitor. si-lnc, siRNA of LINC00511. si-NC, negative control of siRNA. inhibitor, miR-195-5p inhibitor. inhibitor-NC, negative control of inhibitor. si-lnc + inhibitor, siRNA of LINC00511 + miR-195-5p inhibitor

Fig. 5

LINC00511 negatively regulated miR-195-5p to affect the proliferation and apoptosis of LUAD cells. A Cell viability was detected by CCK-8 assay in A549 and PC9 cells transfected with si-lnc or inhibitor for 0, 24, 48 or 72 h. B Cell proliferation was determined by BrdU assay in A549 and PC9 cells transfected with si-lnc or inhibitor for 48 h. C. Bax and Bl-2 protein expression detected by western blot assay in A549 and PC9 cells transfected with si-lnc or inhibitor for 48 h. **P < 0.001 compared with si-NC group. $P < 0.05, $$P < 0.001 compared with inhibitor-NC. #P < 0.05, ##P < 0.001 compared with si-lnc + inhibitor. si-lnc, siRNA of LINC00511. si-NC, negative control of siRNA. inhibitor, miR-195-5p inhibitor. inhibitor-NC, negative control of inhibitor. si-lnc + inhibitor, siRNA of LINC00511 + miR-195-5p inhibitor

Fig. 6

LINC00511 negatively regulated miR-195-5p to affect the metastasis of LUAD cells. A Migration capacity was determined by wound healing assay in A549 and PC9 cells transfected with si-lnc or inhibitor for 48 h. B Cell invasion ability was detected by transwell invasion assay in A549 and PC9 cells transfected with si-lnc or inhibitor for 48 h. *P < 0.05, **P < 0.001 compared with si-NC group. $$P < 0.001 compared with inhibitor-NC. #P < 0.05, ##P < 0.001 compared with si-lnc + inhibitor. si-lnc, siRNA of LINC00511. si-NC, negative control of siRNA. inhibitor, miR-195-5p inhibitor. inhibitor-NC, negative control of inhibitor. si-lnc + inhibitor, siRNA of LINC00511 + miR-195-5p inhibitor

Fig. 7

GCNT3 was the target gene of miR-195-5p. A The schematic diagram presents the complementary binding sites within GCNT3 and miR-195-5p. B Luciferase reporter assay confirmed the molecular binding. **P < 0.001 compared with WT + NC. #P < 0.05 compared with WT + mimic. C The expression of GCNT3 was detected by qRT-PCR assay in cancer tissues and normal tissues. **P < 0.001. D Pearson analysis was used to analyze the expression of GCNT3 and miR-195-5p in cancer tissues. E The expression of GCNT3 was detected by qRT-PCR assay in A549, PC9 and BEAS-2B cells. **P < 0.001 compared with BEAS-2B. F The GCNT3 mRNA expression were detected by qRT-PCR assay in A549 and PC9 cells transfected with si-lnc for 48 h. G The GCNT3 protein expression were detected by western blot assay in A549 and PC9 cells transfected with si-lnc for 48 h. H The GCNT3 protein expression were detected by western blot assay in A549 and PC9 cells transfected with si-GCNT3 or inhibitor for 48 h. **P < 0.001 compared with si-NC group. $$P < 0.001 compared with inhibitor-NC. ##P < 0.001 compared with si-GCNT3 + inhibitor. si-GCNT3, siRNA of GCNT3. si-NC, negative control of siRNA. inhibitor, miR-195-5p inhibitor. inhibitor-NC, negative control of inhibitor. si-GCNT3 + inhibitor, siRNA of GCNT3 + miR-195-5p inhibitor

Fig. 8

GCNT3 silencing reversed the effect of miR-195-5p on the proliferation and apoptosis of LUAD cells. A Cell viability was detected by CCK-8 assay in A549 and PC9 cells transfected with si-GCNT3 or inhibitor for 0, 24, 48 or 72 h. B Cell proliferation was determined by BrdU assay in A549 and PC9 cells transfected with si-GCNT3 or inhibitor for 48 h. C Bax and Bl-2 protein expression detected by western blot assay in A549 and PC9 cells transfected with si-GCNT3 or inhibitor for 48 h. *P < 0.05, **P < 0.001 compared with si-NC group. $P < 0.05, $$P < 0.001 compared with inhibitor-NC. #P < 0.05, ##P < 0.001 compared with si-GCNT3 + inhibitor. si-GCNT3, siRNA of GCNT3. si-NC, negative control of siRNA. inhibitor, miR-195-5p inhibitor. inhibitor-NC, negative control of inhibitor. si-GCNT3 + inhibitor, siRNA of GCNT3 + miR-195-5p inhibitor

Fig. 9

GCNT3 silencing reversed the effect of miR-195-5p on the p metastasis of LUAD cells. A Migration capacity was determined by wound healing assay in A549 and PC9 cells transfected with si-GCNT3 or inhibitor for 48 h. B Cell invasion ability was detected by transwell invasion assay in A549 and PC9 cells transfected with si-GCNT3 or inhibitor for 48 h. **P < 0.001 compared with si-NC group. $$P < 0.001 compared with inhibitor-NC. ##P < 0.001 compared with si-GCNT3 + inhibitor. si-GCNT3, siRNA of GCNT3. si-NC, negative control of siRNA. inhibitor, miR-195-5p inhibitor. inhibitor-NC, negative control of inhibitor. si-GCNT3 + inhibitor, siRNA of GCNT3 + miR-195-5p inhibitor