LncRNA ZFPM2-AS1 promotes lung adenocarcinoma progression by interacting with UPF1 to destabilize ZFPM2

Abstract

Lung adenocarcinoma (LUAD), a histological subclass of non-small-cell lung cancer, is globally the leading cause of cancer-related deaths. Long noncoding RNAs (lncRNAs) are emerging as cancer regulators. Zinc finger protein multitype 2 antisense RNA 1 (ZFPM2-AS1) is an oncogene in gastric cancer, but its functions have not been investigated in LUAD. We showed that ZFPM2-AS1 expression is high in LUAD samples based on GEPIA database (http://gepia.cancer-pku.cn/) and validated ZFPM2-AS1 upregulation in LUAD cell lines. Functionally, ZFPM2-AS1 facilitated proliferation, invasion, and epithelial-to-mesenchymal transition of LUAD cells. Thereafter, we found that ZFPM2 was negatively regulated by ZFPM2-AS1, and identified the suppressive effect of ZFPM2 regulation by ZFPM2-AS1 on LUAD progression. Mechanistically, we showed that ZFPM2-AS1 interacted with up-frameshift 1 (UPF1) to regulate mRNA decay of ZFPM2. Rescue assays in vitro and in vivo confirmed that ZFPM2-AS1 regulated LUAD progression and tumor growth through ZFPM2. Taken together, our findings demonstrate a role for the ZFPM2-AS1-UPF1-ZFPM2 axis in LUAD progression, suggesting ZFPM2-AS1 as a new potential target for LUAD treatment.

Keywords: UPF1; ZFPM2; ZFPM2-AS1; lung adenocarcinoma; mRNA decay.

Figure 1

ZFPM2‐AS1 was upregulated in LUAD, promoting proliferation in LUAD cells. (A) Upregulation of ZFPM2‐AS1 in LUAD samples was obtained in GEPIA database. (B) RT‐qPCR results of the upregulation of ZFPM2‐AS1 in LUAD cell lines (mean ± SD; n = 6; one‐way ANOVA). (C) RT‐qPCR results of the overexpression of ZFPM2‐AS1 by pcDNA3.1/ZFPM2‐AS1 in A549 cells and the knockdown of ZFPM2‐AS1 by si‐ZFPM2‐AS1#1/2/3 in SPC‐A1 cells (mean ± SD; n = 6; Student’s t‐test and one‐way ANOVA). (D, E) CCK‐8 (mean ± SD; n = 6; two‐way ANOVA) and EdU assays (scale bar = 100 μm; mean ± SD; n = 6; Student’s t‐test and one‐way ANOVA) were used to assess proliferation of LUAD cells upon ZFPM2‐AS1 overexpression and knockdown. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 2

ZFPM2‐AS1 promoted invasion and EMT in LUAD. (A) Transwell invasion assay was used to assess LUAD cell invasive ability upon ZFPM2‐AS1 overexpression and knockdown (scale bar = 50 μm; mean ± SD; n = 6; Student’s t‐test and one‐way ANOVA). (B, C) IF (scale bar = 20 μm; n = 6) and western blot assays were used to evaluate the expressions of E‐cadherin and N‐cadherin upon ZFPM2‐AS1 overexpression and knockdown in LUAD cells. **P < 0.01.

Figure 3

ZFPM2, negatively regulated by ZFPM2‐AS1, was downregulated in LUAD and inhibited proliferation, invasion, and EMT. (A) UCSC database showed that ZFPM2 was a nearby gene for ZFPM2‐AS1. (B) The downregulation of ZFPM2 in LUAD samples was obtained in TCGA database. (C, D) RT‐qPCR (mean ± SD; n = 6; Student’s t‐test and one‐way ANOVA) and western blot results of ZFPM2 expression upon ZFPM2‐AS1 overexpression and knockdown in LUAD cells. (E) Downregulation of ZFPM2 expression in LUAD cell lines was detected by RT‐qPCR results (mean ± SD; n = 6; one‐way ANOVA). (F) Knockdown of ZFPM2 by si‐ZFPM2#1/2/3 in A549 cells was confirmed by RT‐qPCR (mean ± SD; n = 6; one‐way ANOVA). (G–I) CCK‐8 (mean ± SD; n = 6; two‐way ANOVA), EdU (scale bar = 100 μm; mean ± SD; n = 6; one‐way ANOVA), and Transwell assays (scale bar = 50 μm; mean ± SD; n = 6; one‐way ANOVA) were used to evaluate the proliferative and invasive abilities of LUAD cells upon ZFPM2 silence. (J, K) IF staining (scale bar = 20 μm; n = 6) and western blot analyses were used to evaluate the expressions of E‐cadherin and N‐cadherin upon ZFPM2 knockdown in LUAD cells. *P < 0.05, **P < 0.01.

Figure 4

ZFPM2‐AS1 potentially regulated ZFPM2 through interacting with UPF1. (A, B) FISH (scale bar = 10 μm; n = 3) and subcellular fractionation assays were used to determine the cellular localization of ZFPM2‐AS1 in LUAD cells. (C) Luciferase reporter assay (mean ± SD; n = 6; Student’s t‐test and one‐way ANOVA) was used to assess the effect of ZFPM2‐AS1 on promoter transcription of ZFPM2. (D) Pull‐down assay followed by MS was used to identify the interacting partner for ZFPM2‐AS1. (E, F) RIP (mean ± SD; n = 6; Student’s t‐test) and western blot assay after pull‐down assays confirmed the interaction between ZFPM2‐AS1 and UPF1. (G) FISH assay (scale bar = 10 μm; n = 3) confirmed the overlapped expression of ZFPM2‐AS1 in cytoplasm of LUAD cells. ***P < 0.001, n.s: no significance.

Figure 5

UPF1 was upregulated in LUAD and cofunctioned with ZFPM2‐AS1 to interact with ZFPM2. (A) RT‐qPCR results confirmed the upregulation of UPF1 in LUAD cell lines (mean ± SD; n = 6; one‐way ANOVA). (B) The overexpression and knockdown of UPF1 were confirmed by RT‐qPCR analysis (mean ± SD; n = 6; Student’s t‐test and one‐way ANOVA). (C, D) The effect of UPF1 on ZFPM2 expression was determined by RT‐qPCR (mean ± SD; n = 6; Student’s t‐test and one‐way ANOVA) and western blot analyses. (E, F) RIP (mean ± SD; n = 6; Student’s t‐test) and pull‐down assays (mean ± SD; n = 6; Student’s t‐test and one‐way ANOVA) were used to evaluate the interaction between UPF1 and ZFPM2 mRNA. (G, H) RIP assay (mean ± SD; n = 6; Student’s t‐test and one‐way ANOVA) and pull‐down assay (mean ± SD; n = 6; Student’s t‐test and one‐way ANOVA) showed that silencing ZFPM2‐AS1 interfered with the interaction of UPF1 with ZFPM2 mRNA and vice versa. Western blot and RT‐qPCR analyses confirmed that UPF1 had no effect on ZFPM2‐AS1 expression and vice versa. *P < 0.05, **P < 0.01, ***P < 0.001, n.s: no significance.

Figure 6

ZFPM2‐AS1 cofunctioned with UPF1 to destabilize ZFPM2 mRNA. (A) The binding motif of UPF1 and potential binding sites for UPF1 on ZFPM2 mRNA at 3′UTR region were obtained from Starbase. (B) The construction of MS2 vector containing ZFPM2 3′UTR region (mean ± SD; n = 6; Student’s t‐test). (C‐D) MS2‐RIP and MS2‐CoIP assays confirmed the interaction of UPF1 and ZFPM2‐AS1 with ZFPM2 mRNA at the binding sites in 3′UTR region. (E, F) LUAD cells were treated with actinomycin D to block the mRNA generation. RT‐qPCR analysis was used to evaluate the mRNA stability of ZFPM2 upon the overexpression and knockdown of UPF1 and ZFPM2‐AS1 (mean ± SD; n = 6; Student’s t‐test and one‐way ANOVA). *P < 0.05, **P < 0.01.

Figure 7

ZFPM2‐AS1 regulated LUAD progression in vitro and in vivo through UPF1/ZFPM2 axis. A549 cells were transfected with pcDNA3.1, pcDNA3.1/ZFPM2‐AS1, or pcDNA3.1/ZFPM2‐AS1 + ZFPM2, for in vitro and in vivo assays. (A–C) CCK‐8 (mean ± SD; n = 6; two‐way ANOVA), EdU (scale bar = 100 μm; mean ± SD; n = 6; one‐way ANOVA), and Transwell invasion assays (scale bar = 50 μm; mean ± SD; n = 6; one‐way ANOVA) were used to assess cell proliferation and invasion of A549 cells in each group. (D) Western blot analysis was used to evaluate the expressions of E‐cadherin and N‐cadherin in each group. (E) A549 cells with different transfection were injected into nude mice, and the growth of xenografts was evaluated over time. Pictures of xenografts from each group were taken. (F, G) The growth curve (mean ± SD; n = 6; two‐way ANOVA) and final tumor weight of the xenografts (mean ± SD; n = 6; one‐way ANOVA) in each group. (H) ZFPM2 expression from the xenografts in each group was evaluated by RT‐qPCR analysis (mean ± SD; n = 6; one‐way ANOVA). (I) Western blot analysis was used to evaluate the expression of ZFPM2, E‐cadherin, and N‐cadherin from the tumors of mice in each group. *P < 0.05, **P < 0.01.