LncRNA MIR210HG promotes proliferation and invasion of non-small cell lung cancer by upregulating methylation of CACNA2D2 promoter via binding to DNMT1

Figures & data

Table 1 Correlation between MIR210HG expression and clinicopathological characteristics of NSCLC patients

Parameters	MIR210HG expression		p-value
	High (n=50)	Low (n=50)
Age (years)			0.688
<65	28	26
≥65	22	24
Gender			0.689
Male	25	27
Female	25	23
Histological subtype			0.313
Squamous cell carcinoma	31	26
Adenocarcinoma	19	24
TNM stage			0.001
Ia+Ib	8	22
IIa+IIb	18	19
IIIa	24	9
Tumor size (cm)
≤5	23	28	0.317
>5	27	22
Lymph node metastasis			0.003
No	15	30
Yes	35	20
Smoking history			0.680
No	18	20
Yes	32	30

Note: Bold values indicate p<0.05. 

Abbreviation: NSCLC, non-small cell lung cancer.

Table 2 Univariate and multivariate analysis of survival in NSCLC patients

Variables	Univariate analysis			Multivariate analysis
	HR	95% CI	p-value	HR	95% CI	p-value
Univariate and multivariate analysis of disease-free survival in NSCLC patients (n=100)
Age	1.187	0.612–1.689	0.644
Gender	0.881	0.576–1.511	0.567
Smoker	0.699	0.465–1.243	0.216
Histological subtype	0.533	0.312–0.866	0.016	0.566	0.352–1.032	0.054
Tumor size	1.923	1.087–2.876	0.017	1.234	0.878–2.066	0.265
Lymphatic metastasis	1.988	1.089–3.007	0.006	1.473	0.946–2.471	0.107
TNM stage (I versus II or IIIa)	2.601	1.645–3.167	0.002	1.758	1.216–2.725	0.002
MIR210HG expression	2.209	1.196–2.098	0.002	3.169	1.764–5.926	<0.001
Univariate and multivariate analysis of overall survival in NSCLC patients (n=100)
Age	1.098	0.619–1.814	0.744
Gender	0.799	0.473–1.494	0.575
Smoker	0.703	0.379–1.195	0.228
Histological subtype	0.712	0.376–1.245	0.164
Tumor size	1.899	1.075–3.216	0.013	1.576	0.938–3.273	0.088
Lymphatic metastasis	2.896	1.516–5.465	0.002	1.634	0.835–3.679	0.083
TNM stage	3.217	2.492–5.748	<0.001	2.649	1.547–5.003	<0.001
MIR210HG expression (low versus high)	5.476	2.439–9.806	0.002	2.642	1.219–5.268	0.008

Abbreviation: NSCLC, non-small cell lung cancer.

Figure 1 MIR210HG was highly expressed in NSCLC. (A) The heatmap of differentially expressed lncRNAs. (B, C) The microarray data showed that DFS (B) and OS (C) in NSCLC patients with higher expression of MIR210HG were shorter than those with lower expression. (D) MIR210HG was highly expressed in collected NSCLC tissues. (E, F) DFS (E) and OS (F) in collected NSCLC tissues with higher expression of MIR210HG were shorter than those with lower expression. (GAPDH is used as the internal control of MI2210HG.)

Abbreviation: NSCLC, non-small cell lung cancer.

Figure 2 The effect of sh-RNA and plasmid of MIR210HG in NSCLC cell lines. (A) MIR210HG was overexpressed in lung cancer cells compared with that of normal pulmonary epithelial cells. (B) Transfection efficacies of sh-MIR210HG-1 and sh-MIR210HG-2 were verified by qRT-PCR. (C) The MIR210HG expression level was up-regulated when transfected with pcDNA-MIR210HG. (GAPDH is used as the internal control of MI2210HG.)

Abbreviation: NSCLC, non-small cell lung cancer.

Figure 3 Low expression of MIR210HG inhibited proliferation and migration of NSCLC cells. (A, B) MIR210HG knockdown remarkably inhibited proliferation of NSCLC cells. (C) MIR210HG knockdown remarkably inhibited colony formation of NSCLC cells. (D, E) Transwell assay results showed that knockdown of MIR210HG inhibited invasion and migration of NSCLC cells.

Abbreviation: NSCLC, non-small cell lung cancer.

Figure 4 MIR210HG inhibited CACNA2D2 expression by binding to DNMT1. (A) CACNA2D2 expression was negatively correlated with MIR210HG in GSE30219 dataset. (B) CACNA2D2 expression was negatively correlated with MIR210HG in collected NSCLC samples. (GAPDH is used as the internal control of MI2210HG and CACNA2D2.) (C, D) MIR210HG knockdown upregulated mRNA (C) and protein (D) levels of CACNA2D2. (E) Expression level of the CACNA2D2 was negatively correlated to methylation level of its promoter (GAPDH is used as the internal control of MI2210HG). (F) RIP results confirmed that the MIR210HG could bind to DNMT1. (G) CACNA2D2 expression was significantly up-regulated after DNMT1 knockdown (GAPDH is used as the internal control of MI2210HG). (H) DNMT1 inhibitor treatment up-regulated CACNA2D2 expression (GAPDH is used as the internal control of MI2210HG). (I) DNMT1 bound to the CACNA2D2 promoter. (J) MIR210HG knockdown remarkably decreased the binding kurtosis of DNMT1 and CACNA2D2.

Abbreviation: NSCLC, non-small cell lung cancer.

Figure 5 High expression of CACNA2D2 can reverse the effect of MIR210HG on NSCLC. (A) Construction of the overexpressed plasmid of CACNA2D2. (B, C) CACNA2D2 overexpression remarkably inhibited proliferation of NSCLC cells. (D, E) Co-transfection of si-CACNA2D2 and sh-MIR210HG in NSCLC cells remarkably improved the proliferation and colony formation abilities than those transfected with sh-MIR210HG individually.

Abbreviation: NSCLC, non-small cell lung cancer.

Figure 6 Proposed model in which MIR210HG mediates the proliferation and invasion progression of NSCLC.

Abbreviation: NSCLC, non-small cell lung cancer.

Figure S1 (A) The protein level of CACNA2D2 in normal and tumor tissues. (B and C) The distribution of MIR210HG in H1975 and H1299.