LncRNA PRRT3-AS1 exerts oncogenic effects on nonsmall cell lung cancer by targeting microRNA-507/homeobox B5 axis

Abstract

Long noncoding RNAs (lncRNAs) act as key regulators controlling complex cellular behaviors in nonsmall cell lung cancer (NSCLC). We investigated the expression of lncRNA PRRT3 antisense RNA 1 (PRRT3-AS1) in paired samples of NSCLC and adjacent normal tissues from a patient cohort in our hospital using real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) and found that it was significantly higher in NSCLC tissue than in normal tissue, consistent with The Cancer Genome Atlas database. Furthermore, functional investigation revealed that lncRNA PRRT3-AS1 depletion inhibited NSCLC-cell proliferation, colony formation, invasion, and migration, whereas its overexpression exerted the opposite effects. Moreover, PRRT3-AS1 knockdown suppressed in vivo NSCLC growth. Investigation of downstream mechanisms using RNA immunoprecipitation and luciferase reporter assay revealed that lncRNA PRRT3-AS1 acted as a competing endogenous RNA by adsorbing microRNA-507 (miR-507) and enhanced the expression of its target gene, homeobox B5 (HOXB5), in NSCLC. Furthermore, miR-507 downregulation or HOXB5 upregulation eliminated the cancer-inhibiting effects of lncRNA PRRT3-AS1 depletion in NSCLC cells. To conclude, the lncRNA PRRT3-AS1/miR-507/HOXB5 pathway acts as a promoter of malignant characteristics in NSCLC, and this newly identified competing endogenous RNA pathway may be an effective diagnostic, prognostic, and therapeutic target in NSCLC.

Keywords: NSCLC; Targeted therapy; lncRNA; microRNA.

Figure 1. PRRT3-AS1 is upregulated in NSCLC. (A) PRRT3-AS1 level in all human cancer types from TCGA dataset (N = 3). (B) PRRT3-AS1 ranks the 47^th overexpressed lncRNA in LUAD. (C) PRRT3-AS1 level in LUAD and LUSC tissues from the TCGA database. (D) PRRT3-AS1 level in NSCLC tissues from our cohort (N = 3). (E) PRRT3-AS1 level in NSCLC cell lines (N = 3). **p < 0.001.

Figure 2. PRRT3-AS1 facilitates the malignant properties of NSCLC cells. (A) Inhibition efficiency of two siRNAs targeting PRRT3-AS1 in H460 cells. The efficiency of pc-PRRT3-AS1 was also explored in A549 cells (N = 3). (B) Cell proliferation of PRRT3-AS1-knockdown H460 cells and PRRT3-AS1-overexpressed A549 cells (N = 3). (C) The colony formation of si-PRRT3-AS1-transfected H460 cells and pc-PRRT3-AS1-transfected A549 cells (N = 3). (D) The migration of si-PRRT3-AS1-transfected H460 cells and pc-PRRT3-AS1-transfected A549 cells (N = 3) 100× magnification. (E) The invasion of si-PRRT3-AS1-transfected H460 cells and pc-PRRT3-AS1-transfected A549 cells (N = 3) 100× magnification. *p < 0.01 and **p < 0.001.

Figure 3. PRRT3-AS1 is an miR-507 sponge in NSCLC cells. (A) Predicted location of PRRT3-AS1 according to lncLocator. (B) Subcellular distribution of PRRT3-AS1 in NSCLC cells. (C) miRDB online database to predict potential miRNA-targeting PRRT3-AS1. (D) Levels of the six candidates in H460 cells after PRRT3-AS1 depletion. Also, the levels of six candidates in A549 cells after PRRT3-AS1 upregulation was also investigated (N = 3). (E) The WT and MUT binding sites of miR-507 within PRRT3-AS1. (F) Luciferase reporter assay implemented in NSCLC cells after cotransfection with the miR-507 mimic or miR-NC and WT-PRRT3-AS1 or MUT-PRRT3-AS1 (N = 3). (G) RIP assay to analyze the interaction between PRRT3-AS1 and miR-507 (N = 3). **p < 0.001.

Figure 4. Overexpressed miR-507 inhibits the malignant process of NSCLC cells. (A) The efficiency of miR-507 mimic transfection in NSCLC cells (N = 3). (B, C) Cell proliferation and colony formation of miR-507-overxressed NSCLC cells (N = 3). (D, E) The motility of miR-507-overexpressed NSCLC cells (N = 3) 100× magnification. **p < 0.001.

Figure 5. HOXB5 is under the control of PRRT3-AS1/miR-507 axis. (A) The WT and MUT binding sites between miR-507 and HOXB5 3′-UTR. (B) Luciferase reporter assay to verify binding interaction between miR-507 and HOXB5 3′-UTR in NSCLC cells (N = 3). (C and D) HOXB5 levels in miR-507-overxressed NSCLC cells (N = 3). (E–H) PRRT3-AS1-silenced H460 cells were cotransfected with miR-507 inhibitor or NC inhibitor, while miR-507 mimic or miR-NC was transfected into PRRT3-AS1-overexrssed A549 cells. After transfection, HOXB5 mRNA and protein levels were examined (N = 3). (I) RIP assay to certify the interaction among PRRT3-AS1, miR-507, and HOXB5 (N = 3). **p < 0.001 and ^##p < 0.001.

Figure 6. PRRT3-AS1 regulates NSCLC cell proliferation via targeting miR-507/HOXB5. (A) Inhibition efficiency of miR-507 inhibitor in NSCLC cells (N = 3). (B) The efficiency of pc-HOXB5 in H460 cells and si-HOXB5 in A549 cells (N = 3). (C) PRRT3-AS1-silenced H460 cells were cotransfected with miR-507 inhibitor or pc-HOXB5 while miR-507 mimic or si-HOXB5 was introduced into PRRT3-AS1-overexrssed A549 cells. After transfection, cell proliferation was determined via CCK-8 assay (N = 3). *p < 0.01, **p < 0.001, ^#p < 0.01, and ^##p < 0.001.

Figure 7. PRRT3-AS1 promotes NSCLC cell colony formation and motility through controlling miR-507/HOXB5. (A) The si-PRRT3-AS1-transfected H460 cells underwent cotransfection with miR-507 inhibitor or pc-HOXB5. Cell colony formation was then examined (N = 3). (B) miR-507 mimic or si-HOXB5 alongside pc-PRRT3-AS1 was cotransfected into A549 cells, and then subjected to colony formation determination (N = 3). (C) The si-PRRT3-AS1 together with miR-507 inhibitor or pc-HOXB5 was transfected into H460 cells. Cell motility was detected (N = 3) 100× magnification. (D) A549 cells were transfected with pc-PRRT3-AS1 alongside miR-507 mimic or si-HOXB5. Cell motility was also measured (N = 3) 100× magnification. **p < 0.001, ^##p < 0.001 and *^#p < 0.001.

Figure 8. PRRT3-AS1 depletion decreases tumor growth in vivo. (A) Representative photographs of harvested tumors. Each group contained four nude mice. (B) Growth curve of tumor xenografts. (C) The comparison of the weight in the tumor xenografts. (D, E) PRRT3-AS1 and HOXB5 expression in tumor xenografts. (F) miR-507 level in tumor xenografts. **p < 0.001.