THUMPD3-AS1 Is Correlated With Non-Small Cell Lung Cancer And Regulates Self-Renewal Through miR-543 And ONECUT2

Figures & data

Table 1 Overall Patient Characteristics

Clinicopathological Factors	Number (%)/Median (IQR)
Total:
N	51
Histological type:
Adenocarcinoma	33 (64.7%)
Squamouscarcinoma	18 (35.3%)
Gender:
Male	36 (70.6%)
Female	15 (29.4%)
Age (years):
Mean	56.6 (25–73)
Smoking
Nonsmoker	20 (39.2%)
Smoker	31 (60.8%)
TNM Clinical stage, n (%)
I	7 (13.7%)
II	17 (33.3%)
III	19 (37.3%)
IV	8 (15.7%)

Table 2 Correlation Between The Expression Level Of THUMPD3-AS1 And The Clinicopathological Features Of NSCLC Patients

Clinicopathological Factors		Expression Level^a Of THUMPD3-AS1		χ^2 value (Bold values χ^2>7.815(ν=3) χ^2>3.841(ν=1))	P value (Bold values P<0.05)
		High	Low
Histological type	Adenocarcinoma	18	15	0.202	0.653
	Squamouscarcinoma	11	7
Gender	Male	20	16	0.085	0.771
	Female	9	6
Age (years)	<60	21	13	1.003	0.317
	≥60	8	9
Smoking	Nonsmoker	10	10	0.629	0.428
	Smoker	19	12
TNM Clinical stage	I	2	5	10.37	0.016
	II	6	11
	III	15	4
	IV	6	2
Relapse	0	6	11	4.845	0.028
	1	23	11

Notes: ^aExpression level of THUMPD3-AS1 was used the mRNA expression level as categorical variables, where THUMPD3-AS1 expression level was dichotomised and its categories represented as follows: cases with higher expression in primary tissues compared with adjacent tissues were designated as “high”, whereas cases with lower expression in primary tissues compared with adjacent tissues were designated as “low”.

Figure 1 THUMPD3-AS1 was abnormal upregulated in NSCLC and involved in the regulation of cell proliferation and self-renewal. (A) Scatter plots of expression levels of THUMPD3-AS1 in NC and C tissues of NSCLC. NC, adjacent carcinoma tissues; C, primary carcinoma tissues. (B) THUMPD3-AS1 expressions were increased after transfection of THUMPD3-AS1 plasmids and decreased after transfection of THUMPD3-AS1 shRNAs in A549 and H1299 cells. (C) Cell growth assay was used to observe the regulation effect of THUMPD3-AS1 on the cell growth in A549 and H1299 cells. (D) Colony formation assay was used to observe the regulation effect of THUMPD3-AS1 on the colony-formation capability in A549 and H1299 cells. (E) Sphere formation assay was used to observe the regulation effect of THUMPD3-AS1 on the size and number of spheres in A549 and H1299 cells. (*P < 0.05, error bar refers to SD).

Figure 2 THUMPD3-AS1 function as a ceRNA by interacting with miR-543. (A) The subcellular location of THUMPD3-AS1 was tested in A549 and H1299 cells which were fractionated into cytoplasmic and nuclear fractions. U6 and GAPDH served as nucleus and cytoplasm control, respectively. (B) THUMPD3-AS1 contains the binding site for miR-543, including wild type and mutant type. (C) THUMPD3-AS1 regulated the expression of miR-543 in A549 and H1299 cells. (D) Luciferase reporter assay was used to observe the binding effect of miR-543 on the THUMPD3-AS1. (E) RNA pull-down assay was used to observe the direct binding between THUMPD3-AS1 and miR-543. (*P < 0.05, error bar refers to SD).

Figure 3 ONECUT2 was the direct target gene of miR-543. (A) The 3ʹ untranslated region (3ʹUTR) of ONECUT2 contains the binding site for miR-543, including wild type and mutant type. (B) miR-543 regulated the protein and mRNA expression of ONECUT2 in A549 and H1299 cells. (C) Luciferase reporter assay was used to observe the binding and targeting effects of miR-543 on the 3ʹUTR of ONECUT2. (*P < 0.05, error bar refers to SD).

Figure 4 THUMPD3-AS1 act as endogenous sponge of miR-543 to regulate ONECUT2 indirectly. (A) THUMPD3-AS1 regulates the protein and mRNA expression of ONECUT2 through miR-543 in A549 and H1299 cells. (B) Luciferase reporter assay was used to confirm the regulating effects of THUMPD3-AS1 on the 3ʹUTR of ONECUT2 through miR-543. (*P < 0.05, error bar refers to SD).

Figure 5 THUMPD3-AS1 regulated cell proliferation and self-renewal by miR-543 and ONECUT2. (A) Individual or combined regulation of ONECUT2 and miR-543 influenced the regulation effect of THUMPD3-AS1 on the cell growth in A549 and H1299 cells. (B) Individual or combined regulation of ONECUT2 and miR-543 influenced the regulation effect of THUMPD3-AS1 on the colony-formation capability in A549 and H1299 cells. (C) Individual or combined regulation of ONECUT2 and miR-543 influenced the regulation effect of THUMPD3-AS1 on the size and number of spheres in A549 and H1299 cells. (*P < 0.05, error bar refers to SD).

Figure 6 A summary diagram of THUMPD3-AS1, miR-543 and ONECUT2 function on self-renewal in NSCLC.