lncRNA PCAT6 promotes non-small cell lung cancer cell proliferation, migration and invasion through regulating miR-330-5p

Abstract

Background: Investigating the roles of lncRNA prostate cancer-associated transcript 6 (PCAT6) in modulating the growth and aggressiveness of non-small-cell lung carcinoma (NSCLC) cell.

Method: The levels of PCAT6 in NSCLC tissues and cell lines were determined by quantitative real-time PCR assay. MTT as well as colony formation assays were applied to explore the effect of PCAT6 on the growth of NSCLC cell in vitro. Wound healing and Transwell assays were utilized to analyze the impact of PCAT6 on the migration and invasion of NSCLC cell. Bioinformatics analysis and luciferase reporter assay were used to prove that miR-330-5p was the target of PCAT6. Colony formation, wound healing, and Transwell invasion assays were applied to demonstrate that PCAT6 promoted NSCLC cell growth, migration, and invasion through binding miR-330-5p. Finally, xenograft model was used to explore the role of PCAT6 in the tumor growth of NSCLC cell in vivo.

Results: PCAT6 was highly overexpressed in NSCLC tissues and cells compared with normal tissues and non-tumorigenic bronchial epithelial cell line, BEAS-2B. Downregulation of PCAT6 markedly reduced the proliferation, migration, and invasion of NSCLC cell. Moreover, down-expression of PCAT6 significantly increased the level of miR-330-5p in NSCLC cell. Further functional experiments indicated that down-expression of miR-330-5p reversed the inhibitory effect of PCAT6 on NSCLC cell growth, migration, and invasion.

Conclusion: Our results reveal that lncRNA PCAT6 facilitates the proliferation, migration, and invasion of NSCLC cell via competitively binding to miR-330-5p.

Keywords: MiR-330-5p; NSCLC; PCAT6; growth; invasion; migration.

Figure 1

PCAT6 is overexpressed in NSCLC. Notes: (A) Heatmap: the dysexpression pattern of lncRNAs in NSCLC tissues compared with non-cancer tissues. (B) The level of PCAT6 was measured in NSCLC tissues and adjacent normal tissues using qRT-PCR. **P<0.01 compared with normal, Student’s t-test. (C) PCAT6 was overexpressed in advanced NSCLC tissues. **P<0.01 compared with stage I–II, Student’s t-test. (D) PCAT6 was overexpressed in patients with metastasis compared with patients with non-metastasis. **P<0.01 compared with non-metastasis, Student’s t-test. (E) The level of PCAT6 in four NSCLC cell lines A549, H1650, HCC827, and H1975, and BEAS-2B cell was detected by qRT-PCR. **P<0.01 compared with BEAS-2B, Student’s t-test. Abbreviations: NSCLC, non-small-cell lung carcinoma; PCAT6, prostate cancer-associated transcript 6; qRT-PCR, quantitative real-time polymerase chain reaction.

Figure 2

Knockdown of PCAT6 inhibits NSCLC cell growth. Notes: (A) si-PCAT6 or si-Con was transfected into A549 and H1975 cells. The levels of PCAT6 were determined by qRT-PCR assay. **P<0.01 compared with control, Student’s t-test. (B, C) The cell proliferation of A549 and H1975 cells that was transfected with si-PCAT6 was determined by MTT assay, one-way ANOVA. (D, E) The colony formation analysis of si-Con or si-PCAT6 transfected A549 and H1975 cells. **P<0.01 compared with si-Con, Student’s t-test. (F) Knockdown of PCAT6 significantly inhibits A549 cell growth in xenograft model, one-way ANOVA. **P<0.01 compared with si-Con. (G) Tumor weight, **P<0.05 compared with si-Con, Student’s t-test. (H) The levels of Ki-67 in tumor that was formed by si-PCAT6 or si-Con-transfected A549 cells were detected by immunohistochemical staining. **P<0.01 compared with si-Con, Student’s t-test. Abbreviations: NSCLC, non-small-cell lung carcinoma; PCAT6, prostate cancer-associated transcript 6; qRT-PCR, quantitative real-time polymerase chain reaction.

Figure 3

Down-expression of PCAT6 inhibits the migration and invasion of NSCLC cell. Notes: (A, B) Knockdown of PCAT6 inhibited the migration of A549 and H1975 cells. (C, D) Knockdown of PCAT6 suppressed the invasion of NSCLC cell. **P<0.01 compared with control, Student’s t-test. Abbreviations: NSCLC, non-small-cell lung carcinoma; PCAT6, prostate cancer-associated transcript 6.

Figure 4

PCAT6 is the target of miR-330-5p. Notes: (A) Bioinformatics analysis using online tool showed the putative binding sites between miR-330-5p and PCAT6. (B) The level of miR-330-5p was detected using qRT-PCR assay in A549 and H1975 cells. **P<0.01 compared with control, Student’s t-test. (C) The levels of miR-330-5p in miR-330-5p overexpressing A549 and H1975 cells were determined by qRT-PCR. **P<0.01 compared with control, Student’s t-test. (D) The expressions of PCAT6 in miR-330-5p overexpressing A549 and H1975 cells were determined by qRT-PCR. **P<0.01 compared with control, Student’s t-test. (E) wt-PCAT6 or mut-PCAT6 was transfected into 293 T cell combination with miR-330–5p. The luciferase activity was measured. **P<0.01 compared with miR-ctr+wt-PCAT6, Student’s t-test. (F) The association between PCAT6 and miR-330-5p in NSCLC tissues was analyzed using qRT-PCR assay. Abbreviations: NSCLC, non-small-cell lung carcinoma; PCAT6, prostate cancer-associated transcript 6; qRT-PCR, quantitative real-time polymerase chain reaction.

Figure 5

Regulation of NSCLC cell growth, migration, and invasion by PCAT6 requires miR-330-5p. Notes: (A) The level of miR-330-5p was measured using qRT-PCR in A549 cell that was transfected with si-PCAT6 alone, or si-PCAT6 combination with miR-330-5p inhibitor. (B) Cell proliferation assay was conducted in A549 cell, one-way ANOVA. (C) Cell colony formation assay was conducted in A549 cell. (D, E) The migration and invasion of A549 cell that was transfected with si-PCAT6 alone, or si-PCAT6 combination with miR-330–5 p inhibitor was determined using wound healing and Transwell assays. **P<0.01 compared with control, ^##P<0.01 compared with si-PCAT6, Student’s t-test. Abbreviations: NSCLC, non-small-cell lung carcinoma; PCAT6, prostate cancer-associated transcript 6; qRT-PCR, quantitative real-time polymerase chain reaction.