Long non-coding RNA B4GALT1-Antisense RNA 1/microRNA-30e/SRY-box transcription factor 9 signaling axis contributes to non-small cell lung cancer cell growth

Abstract

Long non-coding (lnc) RNAs serve crucial functions in human cancers. However, the involvement of the lncRNA B4GALT1-antisense RNA 1 (AS1) in non-small cell lung cancer (NSCLC) has not been extensively studied. Reverse transcription-quantitative PCR was performed to detect B4GALT1-AS1 levels in NSCLC tissues and cell lines. Potential influences of B4GALT1-AS1 on biological functions of NSCLC were assessed through a series of in vitro experiments, and the molecular mechanism was determined via RNA immunoprecipitation (RIP) and bioinformatics analyses. The results of the present study demonstrated that knockdown of B4GALT1-AS1 significantly attenuated the proliferative ability and clonality of H1299 and A549 cells. In the present study, B4GALT1-AS1 competed as an endogenous RNA by sequestering microRNA-30e (miR-30e) leading to an enhanced expression of SRY-box transcription factor 9 (SOX9). The effects of silencing B4GALT1-AS1 on NSCLC cells proliferation could be ameliorated by inhibiting miR-30e or restoring SOX9. Hence, B4GALT1-AS1 acted as a lncRNA that drives tumor progression in NSCLC via the regulation of the miR-30e/SOX9 axis. The findings of the present study indicated that the B4GALT1-AS1/miR-30e/SOX9 axis maybe an effective target for NSCLC treatment and management.

Keywords: B4GALT1-antisense RNA 1; SRY-box transcription factor 9; microRNA-30e; non-small cell lung cancer.

Figure 1.

B4GALT1-AS1 expression is enhanced in NSCLC. (A) B4GALT1- AS1 expression was estimated by RT-qPCR in NSCLC and control adjacent healthy tissues (n=56). (B) Total RNA was extracted from 3 NSCLC cell lines (PG49, A549 and H1299) and a human healthy lung cell line (MRC-5) and was analyzed by RT-qPCR to evaluate the mRNA expression of B4GALT1-AS1. ***P<0.001, **P<0.01, and *P<0.05. T, tumor; N, normal; AS1, anti-sense RNA; RT-q, reverse-transcription quantitative; NSCLC, non-small cell lung carcinoma; OD, optical density.

Figure 2.

An in vitro decline in B4GALT1-AS1 level inhibited H1299 and A549 cell proliferation. (A) Expression analysis of B4GALT1-AS1 using RT-qPCR in A549 and H1299 cells post-transfection (by either si-B4GALT1-AS1 or si-NC). (B) Detection of the proliferation of H1299 and A549 cells deficient in B4GALT1-AS1 by the CCK-8 assay. (C and D) Following transfection of si-B4GALT1-AS1 or si-NC, H1299 and A549 cells were assayed for colony formation. ***P<0.001, **P<0.01, and *P<0.05. AS1, antisense RNA; RT-q, reverse-transcription quantitative; NC, negative control; si, small interfering.

Figure 3.

A direct interaction occurs between B4GALT1-AS1 and miR-30e to sequester the level of miR-30e expression in NSCLC cells. (A) Bioinformatics evaluation presenting the bindings sites of WT and MUT miR-30e on B4GALT1-AS1. (B) Assessment of miR-30e level in agomiR-30e or agomir-NC transfected A549 and H1299 cells. (C) WT-B4GALT1-AS1 or MUT-B4GALT1-AS1 was transfected along with agomir-NC or agomiR-30e in A549 and H1299 cells. Detection of luciferase activity 48 h following transfection. (D) B4GALT1-AS1 and miR-30e were enriched in immunoprecipitate containing AGO2 and compared with the control (IgG). (E) RT-qPCR to estimate miR-30e level in B4GALT1-AS1-silenced A549 and H1299 cells. ***P<0.001 and *P<0.05. AS1, antisense RNA; RT-q, reverse-transcription quantitative; NC, negative control; si, small interfering; NSCLC, non-small cell lung carcinoma; AGO2, protein arganaute-2; miR, micro RNA; WT, wild-type; MUT, mutant.

Figure 4.

Role of B4GALT1-AS1 as a competing endogenous RNA which sequestered miR-30e to regulate the expression level of SOX9. (A) After transfecting si-B4GALT1-AS1 or si-NC in A549 and H1299 cells, SOX9 transcript level was quantified by RT-qPCR. (B) The level of miR-30e was estimated by RT-qPCR after the transfection of H1299 and A549 cells with antagomiR-30e or antagomir-NC. (C) Transfection of H1299 and A549 cells with antagomiR-30e or antagomir-NC in si-B4GALT1-AS1 presence. miR-30e expression was investigated via RT-qPCR assay. (D) B4GALT1-AS1 and SOX9 expression was positively correlated in NSCLC tissue. (E) Co-transfection of A549 and H1299 cells with si-B4GALT1-AS1 and either antagomiR-30e, or antagomir-NC and assessment of cell proliferation by the CCK-8 assay. (F) RT-qPCR assay to estimate SOX9 level in LV-SOX9 or LV-NC transfected A549 and H1299 cells. (G) Co-transfection of H1299 and A549 cells with si-B4GALT1-AS1 along with either pc-SOX9 or pcDNA3.1. CCK-8 assay was performed to estimate cellular proliferation. ***P<0.001 and *P<0.05. AS1, antisense RNA; RT-q, reverse-transcription quantitative; NC, negative control; si, small interfering; NSCLC, non-small cell lung carcinoma; miR, micro RNA; LV, lentiviral; OD, optical density; SOX 9, SRY-box transcription factor 9.