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. 2019 Sep 6:17:563-577.
doi: 10.1016/j.omtn.2019.06.003. Epub 2019 Jun 15.

LINC00472 Acts as a Tumor Suppressor in NSCLC through KLLN-Mediated p53-Signaling Pathway via MicroRNA-149-3p and MicroRNA-4270

Aimei Zou  1 , Xingli Liu  1 , Zongjiong Mai  2 , Junke Zhang  1 , Zhuohuan Liu  1 , Qilu Huang  3 , Aibing Wu  4 , Chenyu Zhou  5
Affiliations

LINC00472 Acts as a Tumor Suppressor in NSCLC through KLLN-Mediated p53-Signaling Pathway via MicroRNA-149-3p and MicroRNA-4270

Aimei Zou et al. Mol Ther Nucleic Acids. .

Retraction in

Abstract

Long non-coding RNAs and microRNAs (miRNAs) have been reported to participate in the progression of non-small-cell lung cancer (NSCLC). Long intergenic non-protein-coding RNA 472 (LINC00472), miR-149-3p, and miR-4270 were found to be involved in tumor activities, suggesting potential roles in NSCLC. Thus, this study aimed to examine the ability of LINC00472 to influence the progression of NSCLC with the involvement of miR-149-3p and miR-4270. Initially, differentially expressed long non-coding RNAs (lncRNAs), downstream regulatory miRNAs, and genes related to NSCLC were identified. Next, the interaction among LINC00472, miR-149-3p and miR-4270, and KLLN and the p53-signaling pathway was determined. The effect of LINC00472 on the expression of E-cadherin, N-cadherin, and Vimentin was examined through gain-of-function and loss-of-function experiments. Lastly, the effects of LINC00472 on NSCLC tumor growth were assessed in vivo. LINC00472 and KLLN were found to exhibit low levels, while miR-149-3p and miR-4270 were highly expressed in NSCLC. In addition, the overexpression of LINC00472 was observed to upregulate KLLN and activate the p53-signaling pathway, which ultimately inhibited the invasion, migration, and EMT of NSCLC cells via miR-149-3p and miR-4270, corresponding to decreased N-cadherin and Vimentin and increased E-cadherin. The overexpression of LINC00472 exerted an inhibitory effect on tumor growth in vivo. Taken together, the key evidence suggests that the overexpression of LINC00472 can downregulate miR-149-3p and miR-4270 to upregulate KLLN and activate the p53-signaling pathway, thus inhibiting the development of NSCLC. This study highlights the potential of LINC00472 as a promising therapeutic target for NSCLC treatment.

Keywords: KLLN; epithelial-mesenchymal transition; invasion; long intergenic non-protein-coding RNA 472; microRNA-149-3p; microRNA-4270; migration; non-small-cell lung cancer; p53-signaling pathway.

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Figures

Figure 1
Figure 1
LINC00472 Is Lowly Expressed in NSCLC Cells and Mainly Expressed in the Cytoplasm and Nucleus (A) The fold changes of differentially expressed LINC00472 and three other lncRNAs in GEO: GSE44077. (B) The expressions of four lncRNAs in the NSCLC-related microarray data, GEO: GSE44077. The abscissa represents the name of the lncRNA, while the ordinate is representative of the lncRNA expression in the microarray data; the green box diagram represents the normal sample, and the red box diagram represents the tumor sample. *p < 0.005, ***p < 0.0001. (C) The expression of LINC00472 in 119 cases of human NSCLC tissues and adjacent normal tissues detected by qRT-PCR, normalized by GAPDH. *p < 0.05 versus the adjacent normal tissues. (D) The relative expression of LINC00472 in 5 NSCLC cell lines and human normal lung epithelial cell line (BEAS-2B) detected by qRT-PCR, normalized by GAPDH. *p < 0.05 versus BEAS-2B cells. (E) Kaplan-Meier curves demonstrating the correlation between LINC00472 and disease prognosis in NSCLC patients. (F) Localization of the expression of LINC00472 in NCI-H1299 cells as detected by RNA-FISH (original magnification, ×400; scale bar, 25 μm). The representative measurement data were expressed as mean ± SD. Comparisons between NSCLC tissues and adjacent normal tissues were analyzed by paired t test. Comparisons among multiple groups were analyzed by one-way ANOVA. The experiment was repeated three times. LINC00472, long intergenic non-protein-coding RNA 472; NSCLC, non-small-cell lung cancer; RNA-FISH, RNA-fluorescence in situ hybridization.
Figure 2
Figure 2
Overexpression of LINC00472 Represses the Proliferation, Migration, Invasion, and EMT of NCI-H1299 Cells NCI-H1299 cells were transfected with oe-LINC00472 or oe-NC. (A) The transfection efficiency of the overexpression of LINC00472 detected by qRT-PCR, normalized by GAPDH. (B) The expressions of EMT-related markers detected by western blot analysis. (C) Cell proliferation of each group detected by EdU assay (the original magnification is ×200). (D) Cell migration and invasion in each group detected by Transwell assay (the original magnification is ×200). *p < 0.05 versus the oe-NC group. The representative measurement data were expressed as mean ± SD. Comparisons between two groups were analyzed by unpaired t test. Comparisons among multiple groups were analyzed by one-way ANOVA. The experiment was repeated three times. LINC00472, long intergenic non-protein-coding RNA 472; NSCLC, non-small-cell lung cancer; EMT, epithelial-mesenchymal transition; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.
Figure 3
Figure 3
Silencing of LINC00472 Facilitates the Proliferation, Migration, Invasion, and EMT of NCI-H1299 Cells NCI-H1299 cells were transfected with sh-LINC00472 or sh-NC. (A) The transfection efficiency of the silencing of LINC00472 detected by qRT-PCR, normalized by GAPDH. (B) The expressions of EMT-related markers detected by western blot analysis, normalized by GAPDH. (C) Cell proliferation of each group detected by EdU assay (the original magnification is ×200). (D) Cell migration and invasion in each group detected by Transwell assay (the original magnification is ×200). *p < 0.05 versus the sh-NC group. The representative measurement data were expressed as mean ± SD. Comparisons between two groups were analyzed by unpaired t test. Comparisons among multiple groups were analyzed by one-way ANOVA. The experiment was repeated three times. LINC00472, long intergenic non-protein-coding RNA 472; NSCLC, non-small-cell lung cancer; EMT, epithelial-mesenchymal transition; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.
Figure 4
Figure 4
Overexpression of LINC00472 Upregulates the KLLN Expression through Modulating the miR-149-3p and miR-4270 Expressions in NCI-H1299 Cells NCI-H1299 cells were transfected with miR-149-3p mimic or miR-4270 mimic alone or with oe-LINC00472. (A) Prediction of LINC00472 downstream regulatory miRNAs. The left circle indicates prediction results of the RNA22 database, the right circle indicates the GEO: GSE53882 chip analysis results, and the overlapping part indicates the intersection of the two sets of data. (B) Binding sites predicted by the RNA22 database of miR-149-3p and miR-4270, respectively, with LINC00472 and KLLN, and binding sites predicted by TargetScan of miR-149-3p and miR-4270, respectively, with LINC00472 and KLLN. (C) The qRT-PCR demonstrating the expression of miR-149-3p and miR-4270 in tissues and cells, normalized by U6. *p < 0.05 versus the adjacent normal tissues or the BEAS-2B cells. (D) Correlation analysis of miR-149-3p and miR-4270, respectively, with LINC00472. (E) The qRT-PCR demonstrating the effect of overexpression or silencing of LINC00472 on miR-149-3p, miR-4270, and KLLN, normalized by U6 or GAPDH. *p < 0.05 versus oe-NC group, #p < 0.05 versus the sh-NC group. (F) The qRT-PCR demonstrating the effect of overexpression or silencing of miR-149-3p and miR-4270, respectively, on the expression of KLLN, normalized by U6 or GAPDH. *p < 0.05 versus the NC mimic group, #p < 0.05 versus the NC inhibitor group. (G) The dual-luciferase reporter assay showing the fluorescence intensity of NC mimic, miR-149-3p mimic, and miR-4270 mimic separately co-transfected with WT-LINC00472, mut-LINC00472, WT-KLLN, and mut-KLLN. *p < 0.05 versus NC mimic group. (H) RIP assay showing miR-149-3p and miR-4270, respectively, binding to LINC00472 or KLLN. *p < 0.05 versus the anti-IgG group. (I) The qRT-PCR and western blot analysis showing the expression of KLLN. *p < 0.05 versus the NC mimic group, #p < 0.05 versus the miR-149-3p mimic + oe-NC group or the miR-4270 mimic + oe-NC group. The representative measurement data were expressed as mean ± SD. Comparisons between the NSCLC tissues and adjacent normal tissues were analyzed by paired t test, and other comparisons between two groups were analyzed by unpaired t test. Comparisons among multiple groups were analyzed by one-way ANOVA. Pearson correlation analysis was performed between miR-149-3p, miR-4270, respectively, and LINC00472. The experiment was repeated three times. LINC00472, long intergenic non-protein-coding RNA 472; NSCLC, non-small-cell lung cancer; miR-149-3p, microRNA-149-3p; miR-4270, microRNA-4270; RIP, RNA-binding protein immunoprecipitation.
Figure 5
Figure 5
Overexpression of LINC00472 Inhibits the EMT, Migration, and Invasion of NCI-H1299 Cells through Inhibiting miR-149-3p and miR-4270 Expressions NCI-H1299 cells were transfected with miR-149-3p mimic or miR-4270 mimic alone or with oe-LINC00472. (A) The qRT-PCR and western blot analysis showing the expressions of EMT-related epithelial marker (E-cadherin) and interstitial markers (N-cadherin and Vimentin), normalized by GAPDH. (B) EdU assay showing the proliferation of cells in each group (the original magnification is ×200). (C and D) Transwell assay showing the migration and invasion in each group (the original magnification is ×200). *p < 0.05 versus the NC mimic group, #p < 0.05 versus the miR-149-3p mimic + oe-NC group or the miR-4270 mimic + oe-NC group. The representative measurement data were expressed as mean ± SD. Comparisons between two groups were analyzed by unpaired t test. The experiment was repeated three times. LINC00472, long intergenic non-protein-coding RNA 472; NSCLC, non-small-cell lung cancer; miR-149-3p, microRNA-149-3p; miR-4270, microRNA-4270; EMT, epithelial-mesenchymal transition.
Figure 6
Figure 6
Overexpressed LINC00472 Activates the p53-Signaling Pathway by Upregulating the Expression of KLLN to Suppress the EMT, Migration, and Invasion of NCI-H1299 Cells NCI-H1299 cells were co-transfected with oe-LINC00472 and sh-NC or sh-KLLN, with oe-NC or sh-NC as the control. (A) Correlation analysis of core target genes and NSCLC-related known genes. Each ellipse in the figure represents a gene, and the line between the ellipses indicates the interaction between the two genes. (B) The immunohistochemistry showing the expression of KLLN in NSCLC tissues (the original magnification is ×400). *p < 0.05 versus the adjacent normal tissues. (C) The qRT-PCR showing the expression of KLLN in NSCLC cells, normalized by GAPDH. *p < 0.05 versus the BEAS-2B cells. (D) Correlation analysis of LINC00472 and KLLN expression. (E) The qRT-PCR showing the transfection efficiency of each group. (F) The qRT-PCR and western blot analysis showing the expression of p53 and epithelial marker (E-cadherin) and interstitial markers (N-cadherin and Vimentin) related to the EMT, normalized by GAPDH. (G) The EdU assay showing cell proliferation (the original magnification is ×200). (H) The Transwell assay showing cell migration and invasion (the original magnification is ×200). *p < 0.05 versus the oe-NC + sh-NC group (E–H). The representative measurement data were expressed as mean ± SD. Comparisons between the NSCLC tissues and adjacent normal tissues were analyzed by paired t test, and other comparisons between two groups were analyzed by unpaired t test. Comparisons among multiple groups were analyzed by one-way ANOVA. Pearson correlation analysis was performed between LINC00472 and KLLN. The experiment was repeated three times. LINC00472, long intergenic non-protein-coding RNA 472; NSCLC, non-small-cell lung cancer; EMT, epithelial-mesenchymal transition; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.
Figure 7
Figure 7
Overexpressed LINC00472 Suppresses Tumor Growth of NCI-H1299 Cells In Vivo The nude mice were injected with NCI-H1299 cells harboring oe-LINC00472 or oe-NC. (A) The images and mean volume of the tumor xenografts in the two groups of NCI-H1299 cells with stable transfection of oe-NC and oe-LINC00472 in nude mice. (B) The qRT-PCR demonstrating the expressions of LINC00472, miR-149-3p, and miR-4270 of tumor xenografts in the two groups, normalized by U6 or GAPDH. (C) Immunohistochemistry showing the expressions of related factors in tumors of the two groups (the original magnification is × 400). *p < 0.05 versus the oe-NC group. The representative measurement data were expressed as mean ± SD. Comparisons between two groups were analyzed by unpaired t test. Data at different time points were compared by repeated-measurement ANOVA. LINC00472, long intergenic non-protein-coding RNA 472; miR-149-3p, microRNA-149-3p; miR-4270, microRNA-4270; NSCLC, non-small-cell lung cancer.
Figure 8
Figure 8
Schematic Representation of the Role of LINC00472 in NSCLC Overexpression of LINC00472 upregulated the expression of KLLN and further activated the p53-signaling pathway to suppress the migration, invasion, and EMT of NSCLC cells by inhibiting miR-149-3p and miR-4270 expressions.
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