Long noncoding RNA lnc-SNAPC5-3:4 inhibits malignancy by directly upregulating miR-224-3p in non-small cell lung cancer

Abstract

The mounting body of evidence demonstrates the growing importance of long noncoding RNAs in the advancement of tumors. This study aimed to investigate the molecular mechanism of lnc-SNAPC5-3:4 in non-small cell lung cancer (NSCLC). We investigated the expression of miR-224-3p and lnc-SNAPC5-3:4 in clinical NSCLC samples and NSCLC cell lines using reverse transcription polymerase chain reaction (RT-PCR). In vitro studies, A549 cell growth was estimated using Cell Counting Kit-8 (CCK-8), 5-ethynyl-2'-deoxyuridine (EDU), and flow cytometry assays. In vivo studies, NSCLC tumorigenesis was determined using xenograft tumor mouse models, tumor growth was evaluated using antigen Kiel 67 (Ki67) staining, and tumor apoptosis was detected through terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining. The relationship between lnc-SNAPC5-3:4 and miR-224-3p was determined by luciferase reporter gene assay. Results indicated that the expression of lnc-SNAPC5-3:4 was observed to be downregulated in NSCLC tissues and cell lines. After overexpression of lnc-SNAPC5-3:4 in cultured A549 cells, proliferation decreased and apoptosis increased. Furthermore, the expression of miR-224-3p was targeted and negatively regulated by lnc-SNAPC5-3:4. The lnc-SNAPC5-3:4 upregulation inhibited cell proliferation and promoted apoptosis, which was partially blocked by miR-224-3p overexpression in A549 cells. In addition, we constructed a subcutaneous inoculation model using BALB/c nude mice, and the results indicated that lnc-SNAPC5-3:4 overexpression restrained the growth of subcutaneous tumors, decreased Ki67 expression levels, and increased apoptosis, as indicated by TUNEL staining in nude mice. However, miR-224-3p transfection resulted in the reversal of the inhibitory effect of lnc-SNAPC5-3:4 on tumor growth. In conclusion, our study revealed that lnc-SNAPC5-3:4 inhibits tumor progression in NSCLC by targeting miR-224-3p. This study provides a potential therapeutic target for inhibiting NSCLC progression.

Keywords: Non-small cell lung cancer; lnc-SNAPC5-34; miR-224-3p.

Fig. 1

Expression of lnc-SNAPC5-3:4 in non-small cell lung cancer (NSCLC) cells A. Expression of lnc-SNAPC5-3:4 in NSCLC (n = 5) and paracancerous tissues (n = 5). B. Expression of lnc-SNAPC5-3:4 in NSCLC cell lines. Data are shown as mean ± SD, **P < 0.01.

Fig. 2

Upregulation of lnc-SNAPC5-3:4 inhibited the proliferation of non-small cell lung cancer (NSCLC) cells and promoted apoptosis. Efficiency of lnc-SNAPC5-3:4 overexpression in A549 cells. B. The activity of A549 cells was determined using a Cell Counting Kit-8 (CCK-8) assay. C. The proliferation of A549 cells was detected using the 5-ethynyl-2′-deoxyuridine (EdU) assay. D. The apoptosis of A549 cells was detected using fluorescence-activated cell sorting (FACS). E. The expression levels of Bax and Caspase3 in A549 cells were determined using western blotting. Data are shown as mean ± SD, **P < 0.01, ***P < 0.001.

Fig. 3

The regulation of lnc-SNAPC5-3:4 and miR-224-3p expression A. The binding sites of lnc-SNAPC5-3:4 and miR-224-3p. B. Expression of miR-224-3p in non-small cell lung cancer (NSCLC) and paracancerous tissues. C. Expression of lnc-SNAPC5-3:4 in NSCLC cell lines. D. Efficiency of miR-224-3p overexpression in A549 cells. E. Dual-luciferase assay was used to detect the binding of lnc SNAPC5-3:4 to miR-224-3p. F. Effect of SNAPC5-3:4 overexpression on miR-224-3p expression. G. Spearman's correlation analysis of miR-224-3p expression correlating with SNAPC5-3:4. Data are shown as mean ± SD, **P < 0.01, ***P < 0.001.

Fig. 4

The lnc-SNAPC5-3:4 inhibits proliferation and promotes apoptosis of non-small cell lung cancer (NSCLC) cells by sponge adsorption of miR-224-3p. A. Efficiency of lnc-SNAPC5-3:4 overexpression in A549 cells. B. The activity of A549 cells was determined using a Cell Counting Kit-8 (CCK-8) assay. C. The proliferation of A549 cells was detected using the 5-ethynyl-2′-deoxyuridine (EdU) assay. D. The apoptosis of A549 cells was detected using fluorescence-activated cell sorting (FACS). E. The expression levels of Bax and Caspase3 in A549 cells were determined using western blotting. Data are shown as mean ± SD, *P < 0.05, **P < 0.01, ***P < 0.001.

Fig. 5

The lnc-SNAPC5-3:4 inhibits the growth of non-small cell lung cancer (NSCLC) tumors by sponge adsorption of miR-224-3p in vivo. A. Expression of lnc-SNAPC5-3:4 in tumor tissues of nude mice. B. Expression of miR-224-3p in tumor tissues of nude mice. C. Images of nude mice and tumors. D. Tumor weight. E. Tumor growth curve. ***P < 0.001.

Fig. 6

Signaling pathway changes in tumors. A. The expression level of the proliferation-related protein antigen Kiel 67 (Ki67) was detected using immunohistochemistry (IHC). B. Apoptosis was detected using terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining. C. The expression levels of Bax and Caspase3 in the tumors were determined using western blotting. Data are shown as mean ± SD, *P < 0.05, **P < 0.01.