Long non-coding RNA LINC01270 is an onco-promotor in lung adenocarcinoma by upregulating LARP1 via sponging miR-326

Figures & data

Table 1. The sequences used in this study.

Name	Sequences
LncRNA 01270 forward primer	5’-AACCACCAGCACAATCAAGACA-3’
LncRNA 01270 reverse primer	5’-AACTAATCCACAGCCGCAGAA-3’
miR-326 forward primer	5’-CGCCTCTGGGCCCTTC-3’
miR-326 reverse primer	5’-AGTGCAGGGTCCGAGGTATT-3’
miR-326 inhibitor	5’-CUGGAGGAAGGGCCCAGAGG-3’
inhibitor NC miR-326 mimics mimics NC si-LncRNA 01270 si-NC	5’-CAGUACUUUUGUGUAGUACAA-3’ 5’-CCUCUGGGCCCUUCCUCCAG-3’ 5’-UUGUACUACACAAAAGUACUG-3’ 5’-ACUAGUUAGUGAAGAUCAA-3’ 5’-UUCUCCGAACGUGUCACGU-3’

Figure 1. LINC01270 was highly expressed in LUAD tissues and cell lines. Upregulation of LINC01270 was found in LUAD tissues (a) and LUAD cell lines (b). The 5-year overall survival rate in patients with LINC01270 overexpression was lower versus those with LINC01270 downregulation (c). **p < 0.01, ***p < 0.001.

Table 2. The clinicopathological characteristics analysis of LINC01270 expression in LUAD patients.

Feature	LINC01270 expression		No	P value
	High (40)	Low (40)
Age (years)
≤60	29	25	54	0.34
>60	11	15	26
Gender
Male	22	23	45	0.82
Female	18	17	35
TNM stage
I+ II	22	27	49	0.25
III	18	13	31
Invasion range
T1–T2	26	33	59	0.075
T3–T4	14	7	21
Lymph node metastasis
Yes	29	20	49	0.039*
No	11	20	31

Figure 2. LINC01270 knockdown resulted in impaired LUAD cell growth and invasion. (a) LINC01270 knockdown in A549 was detected via qRT-PCR. The depressed cell growth ability in LINC01270 knockdown cells was detected via CCK-8 (b), EdU assay (c), and colony formation assay (d). Transwell assay explored the inhibited cell invasion (e) and migration (f) of LINC01270 knockdown cells. **p < 0.01, ***p < 0.001.

Figure 3. Aberrant overexpression of LINC01270 led to LUAD progression. (a) Upregulation of LINC01270 in PC-9 cells was detected via qRT-PCR. The enhanced cell growth ability in LINC01270 upregulation cells was detected via CCK-8 (b), EdU assay (c), and colony formation assay (d). Transwell assay showed the promoted cell invasion (e) and migration (f) of LINC01270 upregulation cells. *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 4. In vivo study of LINC01270 on LUAD was observed. Tumor size of LUAD with si-LINC01270 transfection (a) was smaller while LINC01270 ov transfection group showed bigger tumor size (b). The average tumor volume of LUAD with si-LINC01270 or LINC01270 ov transfection was continuously measured in 21-days. The average tumor weight of LUAD si-LINC01270 or LINC01270 ov transfection was scored after 21-days.

Figure 5. Mir-326 interacted with LINC01270, that is a participant in regulating LUAD. (a) Bioinformatics analysis showed the existence of a complementary sequence between miR-326 and LINC01270. (b) The interaction of miR-326 and LINC01270 was proved by dual-luciferase activity assay. (c) Mir-326 was downregulated in LUAD tissues. (d) Negative correlation between LINC01270 and miR-326 expression. (e) Mir-326 was downregulated in LUAD cell lines. **p < 0.01, ***p < 0.001.

Table 3. The clinicopathological characteristics analysis of miR-326 expression in LUAD patients.

Feature	miR-326 expression		No	P value
	High (40)	Low (40)
Age (years)
≤60	26	28	54	0.63
>60	14	12	26
Gender
Male	25	20	45	0.26
Female	15	20	35
TNM stage
I+ II	21	28	49	0.11
III	19	12	31
Invasion range
T1–T2	34	25	59	0.022*
T3–T4	6	15	21
Lymph node metastasis
Yes	20	29	49	0.039*
No	20	11	31

Figure 6. Inhibition of mir-326 restored cell growth and invasion, which was depressed via LINC01270 knockdown. (a) Mir-326 expression was detected via qRT-PCR. The depressed cell growth ability in LINC01270 knockdown cells was restored when giving miR-326 inhibition, which was detected via CCK-8 (b), EdU assay (c), and colony formation assay (d). Transwell assay explored the inhibited cell invasion (e) and migration (f) in LINC01270 silencing cells but augmented invasion when treated with miR-326 inhibitor. *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 7. LARP1 interacted with miR-326. (a-b) RIP experiment showed that miR-326 directly targeted LARP1. (c) The complementary sequence was found in miR-326 and LARP1. (d) The existence of interaction between miR-326 and LARP1 was confirmed by the dual-luciferase assay. (e) Negative correlation between LARP1 and miR-326 expression. (f) Aberrant upregulation of LARP1 was shown in LUAD tissues by western blot. **p < 0.01.

Table 4. The clinicopathological characteristics analysis of LARP1 mRNA expression in LUAD patients.

Feature	mRNA expression		No	P value
	High (40)	Low (40)
Age (years)
≤60	30	24	54	0.56
>60	10	16	26
Gender
Male	25	20	45	0.26
Female	15	20	35
TNM stage
I+ II	16	33	49	0.012*
III	24	7	31
Invasion range
T1–T2	27	32	59	0.20
T3–T4	13	8	21
Lymph node metastasis
Yes	32	17	49	0.035*
No	8	13	31

Figure 8. Overexpression of LARP1 conferred LUAD progression. (a) The expression of LARP1 was examined in the different treated groups. The depressed cell growth ability in mir-326 mimics treated cells was restored when upregulating LARP1, which was detected via CCK-8 (b), EdU assay (c), and colony formation assay (d). Transwell assay explored the inhibited cell invasion (e) and migration (f) in mir-326 mimics treated cells yet enhanced invasion when LARP1 was overexpressed. *p < 0.05, **p < 0.01.

Figure 9. Western blot results demonstrated up-regulation of LARP1, p-PI3K, p-AKT, Bcl-2, and CyclinD1 in the LINC01270 overexpression group, while the proteins were down-regulated in the si-LINC01270 group.