Down-Regulation of TMPO-AS1 Induces Apoptosis in Lung Carcinoma Cells by Regulating miR-143-3p/CDK1 Axis

Abstract

Evidence has shown that long non-coding RNAs (lncRNA) play pivotal roles in cancer promotion as well as suppression. But the molecular mechanism of lncRNA TMPO antisense transcript 1 (TMPO-AS1) in lung cancer (LC) remains unclear. This study mainly investigated the effect of TMPO-AS1 in LC treatment. TMPO-AS1 was tested by Kaplan-Meier method. Quantitative real time polymerase chain reaction (qRT-PCR) was employed to assess the expressions of TMPO-AS1, miR-143-3p, and CDK1 respectively in LC tissues and cell lines. TMPO-AS1, miR-143-3p and CDK1 expressions in LC cells were regulated through cell transfection, followed by MTT for cell viability detection. Besides, dual-luciferase reporter assays were performed to verify the interrelated microRNA of TMPO-AS1 and the target of miR-143-3p. Western blot experiments were used to examine the expressions of apoptosis-related proteins, and flow cytometry tested the cell apoptosis in treated cells. TMPO-AS1 and CDK1 were overexpressed in LC tissues and cells, while miR-143-3p level was suppressed. The decrease of TMPO-AS1 led to the increase of miR-143-3p, which further resulted in the reduction of CDK1. Down-regulating TMPO-AS1 reduced LC cell viability, motivated cell apoptosis, as well as promoted the expressions of Bcl and CCND1 and restrained Caspase-3 level, but all these consequences were abrogated by miR-143-3p inhibitor. Simultaneously, siCDK1 could reverse the anti-apoptosis and pro-activity functions of miR-143-3p inhibitor in LC cells. Down-regulation of TMPO-AS1 has the effects of pro-apoptosis in LC by manipulating miR-143-3p/CDK1, which is hopeful to be a novel therapy for LC patients.

Keywords: TMPO antisense transcript 1; cyclin-dependent kinase 1; lung cancer; miR-143-3p.

Figure 1.

Survival-related TMPO-AS1 was up-regulated in LC tissues and LC cells, together with the down-regulation of miR-143-3p. (A) Box-plots showed the up-regulation of TMPO-AS1 and down-regulation of hsa-miR-143-3p levels in LUAD and LUSC samples. (B, C) TMPO-AS1 and miR-143-3p expression levels in 50 pairs of LC tissues were compared with adjacent normal tissues through qantitative real-time polymerase chain reaction (qRT-PCR) assay. (D) Kaplan-Meier method and log-rank test were used to test how TMPO-AS1 expression level affected survival rate among LC patients. Low: TMPO-AS1 expression in cancer tissue was lower than that in corresponding adjacent normal lung tissues; High: TMPO-AS1 expression in cancer tissue was higher than that in corresponding adjacent normal lung tissues. (E, F) TMPO-AS1 and miR-143-3p expression levels in human bronchial epithelial cells (16HBE) and LC cell lines (H1299, A549, 95D, and H125) were tested through qRT-PCR. ^** P < 0.001 vs. normal, ^# P < 0.05 and ^## P < 0.001 vs. 16HBE, n = 3.

Figure 2.

miR-143-3p was the target of TMPO-AS1. (A) Putative binding site of miR-143-3p on TMPO-AS1 was predicted by starBase v3.0. (B, C) Dual-luciferase reporter assays were performed to detect the H1299 cells and A549 cells co-transfected with wild-type TMPO-AS1 (TMPO-AS1-WT) or mutant-type TMPO-AS1 (TMPO-AS1-MUT), as well as miR-143-p inhibitor, miR-143-3p inhibitor control, miR-143-3p mimic, or miR-143-3p mimic control. (D, E) In LC cells, qantitative real-time polymerase chain reaction (qRT-PCR) assays were used to measure the expressions of GAPDH, U6 and TMPO-AS1 in nuclear and in cytoplasm respectively. (F, G) LC cells were transfected with siNC, siTMPO-AS1, (H, I) miR-143-3p inhibitor or miR-143-3p inhibitor control, followed by the respective detection of genetic expression for miR-143-3p and TMPO-AS1 through qRT-PCR. ^* P < 0.05 vs. inhibitor control, ^^P < 0.001 vs. mimic control, ^## P < 0.001 vs. cytoplasm, ^△△ P < 0.001 vs. siNC, n = 3.

Figure 3.

TMPO-AS1 silencing in LC cells regulated cell viability and cell apoptosis, which could be reversed by miR-143-3p inhibitor. (A, B) TMPO-AS1 expression levels were assayed through qantitative real-time polymerase chain reaction (qRT-PCR) assays in LC cells transfected with siNC or siTMPO-AS1. After siNC, siTMPO-AS1, siTMPO-AS1 plus inhibitor, and siTMPO-AS1 plus inhibitor control were singly transfected in LC cells, (C, D) cell viability was tested through MTT, (E, F, G) apoptosis capability was measured through flow cytometry, (H, I, J) and apoptosis-related proteins were detected by western blot experiments. ^△△ P < 0.001 vs. siNC, ^* P < 0.05 and ^** P < 0.001 vs. siTMPO-AS1, ^# P < 0.05 and ^## P < 0.001 vs. siTMPO-AS1+Inhibitor control, n = 3.

Figure 4.

CDK1 was the target of miR-143-3p. (A) Position 281-288 of CDK1 3’ UTR was predicted as consequential pairing target region of hsa-miR-143-3p by TargetScan 7.2. (B, C) LC cells transfected with wild-type CDK1 (CDK1-WT) or mutant-type CDK1 (CDK1-MUT), together with inhibitor control, inhibitor, mimic control or mimic were then undergone dual-luciferase reporter assay. ^** P < 0.001 vs. inhibitor control, ^{^^} P < 0.001 vs. mimic control, n = 3.

Figure 5.

TMPO-AS1 affected gene expressions of CDK1, but the effects were abrogate by miR-143-3p. LC cells were transfected with siNC, siTMPO-AS1, siTMPO-AS1 plus inhibitor control, or siTMPO-AS1 plus inhibitor, (A, B, C) followed by the measurement of apoptosis-related protein through western blot, (D, E) and the detection of CDK1 expression level through qantitative real-time polymerase chain reaction (qRT-PCR) assays. (F) CDK1 expression level was also tested through qRT-PCR in 50 pairs of LC and normal tissues respectively. ^△ P < 0.05 and ^△△ P < 0.001 vs. siNC, ^** P < 0.001 vs. siTMPO-AS1, ^## P < 0.001 vs. siTMPO-AS1+Inhibitor control, ^‡‡ P < 0.001 vs. normal, n = 3.

Figure 6.

miR-143-3p inhibitor increased cell activity and decreased apoptosis rate in LC cells, which could be reversed by silencing CDK1. LC cells were transfected with siNC or siCDK1, (A, B, C) then apoptotic proteins were measured through western blot assays, (D, E) followed by testing CDK1 expression levels through qantitative real-time polymerase chain reaction (qRT-PCR) assays. Plasmids inhibitor control, inhibitor, inhibitor plus siCDK1, or inhibitor plus siNC were respectively transfected into LC cells, (F, G) after which cell activity was assayed through MTT, (H, I, J) while cell apoptosis was measured through flow cytometry. ^* P < 0.05 vs. inhibitor control, ^△△ P < 0.001 vs. siNC, ^# P < 0.05 and ^## P < 0.001 vs. inhibitor, ^{^} P < 0.05 and ^{^^} P < 0.001 vs. inhibitor+siNC, n = 3.