HOXD-AS1 functions as an oncogenic ceRNA to promote NSCLC cell progression by sequestering miR-147a

Figures & data

Figure 1 HOXD-AS1 is overexpressed in NSCLC and correlates with patient outcome.

Notes: (A) qRT-PCR evaluation of HOXD-AS1 expression in NSCLC samples compared with NCTs. (B) Expression levels of HOXD-AS1 expression in different NSCLC cell lines, including A549, H1703, SK-MES-1, and NCI-H1299 cells. (C) Overall survival Kaplan–Meier analysis of NSCLC patients. ***P<0.001.
Abbreviations: HOXD-AS1, HOXD cluster antisense RNA 1; NCTs, noncancerous tissues; NSCLC, non-small cell lung cancer; qRT-PCR, quantitative reverse-transcription polymerase chain reaction.

Table 1 Correlation of the expression of HOXD-AS1 with NSCLC clinical pathologic features

Characteristic	Number of patients	HOXD-AS1 mRNA expression
		High	Low	P-value
Gender				0.714
Male	50	29	21
Female	37	20	17
Age (years)				0.570
<65	35	21	14
≥65	52	28	24
Size of tumor (cm)				0.006*
≤ 3	45	19	26
>3	42	30	12
TNM stage				0.044*
I	14	4	10
II	26	14	12
III	47	31	16
Recurrence status				0.031*
Positive	48	32	16
Negative	39	17	22

Note:

* Statistically significant difference (P<0.05).

Abbreviations: HOXD-AS1, HOXD cluster antisense RNA 1; NSCLC, non-small cell lung cancer.

Figure 2 The expression of HOXD-AS1 affects NSCLC cell proliferation, cell cycle and apoptosis.

Notes: (A) qRT-PCR evaluation of HOXD-AS1 expression in HOXD-AS1–knockdown A549 and HOXD-AS1–overexpressing SK-MES-1 cells. (B) Cell proliferation assay was performed in HOXD-AS1–knockdown A549 and HOXD-AS1–overexpressing SK-MES-1 cells. Cell cycle and apoptosis analysis were performed in (C) HOXD-AS1–knockdown A549 and (D) HOXD-AS1–overexpression. *P<0.05; **P<0.01; ***P<0.001.
Abbreviations: CTRL, control; HOXD-AS1, HOXD cluster antisense RNA 1; NC, negative control; NSCLC, non-small cell lung cancer; qRT-PCR, quantitative reverse-transcription polymerase chain reaction; FL, fluorescence; PI, propidium iodide.

Figure 3 HOXD-AS1 negatively regulates the expression of miR-147a in NSCLC cells.

Notes: (A) Binding site was predicted between HOXD-AS1 and miR-147a using DIANA tools. (B) Correlation analysis was performed to detect the relationship between HOXD-AS1 and miR-147a in NSCLC tissues. The expression level of miR-147a was detected in (C) HOXD-AS1–knockdown A549 and (D) HOXD-AS1–overexpressing cells. (E) Luciferase activity of reporters containing HOXD-AS1-WT and HOXD-AS1-MUT sequences in 293T cells transfected with miR-147a mimic or control. ***P<0.001.
Abbreviations: HOXD-AS1, HOXD cluster antisense RNA 1; HOXD-AS1-MUT, HOXD-AS1 sequence with mutational miR-147a binding site, which was cloned into pGL3 vector; HOXD-AS1-WT, HOXD-AS1 sequence with miR-147a wild-type binding site, which was cloned into pGL3 vector; NC, negative control; NSCLC, non-small cell lung cancer.

Figure 4 miR-147a inhibition abrogates the effect of HOXD-AS1 knockdown on NSCLC cell proliferation by regulating pRB expression.

Notes: A549 cells were transfected with si-NC, si-HOXD-AS1, si-HOXD-AS1+miR-147a inhibitor, and miR-147a inhibitor. (A) The expression of miR-147a in these cells. (B) CCK-8 assay was performed to examine cell proliferation. (C) Cell cycle status was determined. (D) Cell apoptosis was detected by flow cytometry. (E) HOXD-AS1 positively correlated with pRB expression in NSCLC (GDS3627). mRNA (F) and protein (G) levels of pRB were examined in cells transfected with si-NC, si-HOXD-AS1, and si-HOXD-AS1+miR-147a inhibitor. *P<0.05; **P<0.01; ***P<0.001.
Abbreviations: CCK-8, cell counting kit-8; HOXD-AS1, HOXD cluster antisense RNA 1; NC, negative control; NSCLC, non-small cell lung cancer; pRB, retinoblastoma protein; FL, fluorescence; PI, propidium iodide.