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. 2019 Sep 5;38(1):390.
doi: 10.1186/s13046-019-1379-5.

Long Non-Coding RNA PXN-AS1 Suppresses Pancreatic Cancer Progression by Acting as a Competing Endogenous RNA of miR-3064 to Upregulate PIP4K2B Expression

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Long Non-Coding RNA PXN-AS1 Suppresses Pancreatic Cancer Progression by Acting as a Competing Endogenous RNA of miR-3064 to Upregulate PIP4K2B Expression

Jiayan Yan et al. J Exp Clin Cancer Res. .
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Abstract

Background: Dysregulation of microRNAs (miRNAs) play critical roles in cancerous processes. Although miR-3064 was reported to be an important tumor suppressor in ovarian cancer, the cellular impact of miR-3064 on pancreatic cancer (PC) progression, its downstream target genes and upstream mechanisms that control the expression of miR-3064 remain to be fully clarified.

Methods: We compared miRNA expression profiles between PC tissues compared with normal tissues using a miRNA microarray analysis of clinical samples, and screened the identified miRNAs for their influence on cell proliferation. We measured the expression of miR-3064 in PC tissues and PC cell lines using quantitative real-time PCR assays. Gain- and loss-of-function experiments were conducted to explore the biologic significance of miR-3064 in PC progression both in vitro and in vivo. The interactions between miR-3064 and long noncoding RNA (lncRNA) PXN-AS1 was verified using the luciferase reporter assay and RNA immunoprecipitation assay.

Results: We showed that miR-3064 was significantly overexpressed in PC tissues compared to normal tissues. High miR-3064 was associated with worse prognosis in patients with PC. Functionally, ectopic expression of miR-3064 promoted the proliferation, invasion, clone formation and sphere formation of PC cells in vitro and stimulated PC growth in vivo, while specific knockdown of miR-3064 or CRISPR/Cas9-mediated knockout of miR-3064 resulted in opposite phenotypes. Further investigation revealed that miR-3064 directly targeted PIP4K2B, which was reduced in PC tissues and attenuated PC cell proliferation, invasion and sphere formation induced by miR-3064. Importantly, lncRNA PXN-AS1 expression was downregulated in PC samples, and it directly interacted with miR-3064 and suppressed its levels in PC cells. Enforced expression of PXN-AS1 remarkably decreased cell proliferation, invasion and sphere formation, while re-expression of miR-3064 abrogated these effects of PXN-AS1.

Conclusions: MiR-3064, a key oncogenic miRNA, could promote PC cell growth, invasion and sphere formation via downregulating the levels of tumor suppressor PIP4K2B. PXN-AS1 functioned as a sponge to suppress the expression of miR-3064. These observations offer fresh insight into the mechanisms through which miR-3064 modulates the development of PC.

Keywords: PIP4K2B; PXN-AS1; Pancreatic cancer; miR-3064.

Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Fig. 1
Fig. 1
Identification of miR-3064 as a potential oncogenic miRNA in PC. (a) Heatmap of miRNAs expression in human PC tissues (n = 5) compared with normal tissues (n = 5) measured by miRNA microarray. (b) qRT-PCR analysis of indicated miRNAs in PC tissues (n = 5) and normal tissues (n = 5). (c) Effects of transfection with 11 upregulated miRNAs on PC cell proliferation. (D) Expression of miR-3064 in PC tissues and normal tissues using qRT-PCR analysis (n = 50). (e) Levels of miR-3064 in PCs with lymph node metastasis tissues and PCs without node metastasis. (f) The expression of miR-3064 was examined in five PC cell lines and a normal pancreatic cell line HPDE6-C7 using qRT-PCR analysis. (g) Kaplan-Meier curves for overall survival of PC patients with low or high miR-3064 levels. (h) Kaplan–Meier analysis for overall survival based on low or high miR-3064 levels (the KMPlotter database). *P < 0.05
Fig. 2
Fig. 2
MiR-3064 increases the proliferation, clone formation, invasion and sphere formation of PC cells in vitro. (a) Verification of miR-3064 overexpression or knockdown in PC cells using qRT-PCR analysis. (b-e) CCK-8 assay (b), clone formation assays (c), cell invasion assays (d) and sphere formation assays (e) in AsPC-1 cells after overexpression of miR-3064 and in PaCa-2 cells after knockdown of miR-3064. *P < 0.05
Fig. 3
Fig. 3
Depletion of miR-3064 suppresses the aggressive phenotypes of PC cells in vitro and inhibits tumor growth in xenograft mouse models. (a) Schematic diagram of gRNAs targeting at miR-3064 locus (upper). DNA sequencing confirmed the deletions generated by CRISPR/Cas9 system in the miR-3064 locus (bottom). (b) CRISPR/Cas9 with designed gRNAs significantly reduced the expression of miR-3064, and suppressed the proliferation, invasion and sphere formation of PaCa-2 cells. (c, d) AsPC-1 cells were transfected with or without miR-3064 mimic, and injected into nude mice. Tumor growth rates (c) and images (d) of xenograft tumors were shown. (e, f) The miR-3064 knockout or control PaCa-2 cells were injected into nude mice, and tumor growth rate (e) and images (f) of xenograft tumors were shown. (g, h) Immunohistochemical staining of Ki-67 in tumors derived from (c and e). *P < 0.05
Fig. 4
Fig. 4
MiR-3064 targets PIP4K2B and represses its expression in PC cells. (a) The predicted binding sites between miR-3064 and PIP4K2B 3′-UTR. (b) qRT-PCR analysis of miR-3064 expression in PC tissues and normal tissues. (c) Analysis of PIP4K2B expression in PC tissues and normal tissues using the TCGA data obtained from the UALCAN database. (d, e) miR-3064 expression in PCs classified by tumor stages (d) and grades of differentiation (e) according to UALCAN database. (f) Association of PIP4K2B expression with overall survival in PC patients using the KMplotter database. (g) Examination of PIP4K2B expression in PC cell lines and a normal pancreatic cell line HPDE6-C7 using qRT-PCR assays. (h) The luciferase activity in AsPC-1 cells cotransfected with reporter vectors containing wild type (WT) or mutant (MUT) PIP4K2B 3′-UTR, together with or without miR-3064 mimic, and in PaCa-2 cells cotransfected with reporter vectors containing wild type or mutant PIP4K2B 3′-UTR, together with or without miR-3064 inhibitor. (i) The protein levels of PIP4K2B in AsPC-1 cells transfected with or without miR-3064 mimic, and in PaCa-2 cells transfected with or without miR-3064 inhibitor. *P < 0.05
Fig. 5
Fig. 5
Restoration of PIP4K2B reverses the tumor-promoting effects of miR-3064 in PC cells. (a) PIP4K2B protein expression in AsPC-1 cells transfected with miR-3064 mimic with (or without) PIP4K2B expression vector, and in PaCa-2 cells transfected with miR-3064 inhibitor with (or without) PIP4K2B siRNAs. (b) Cell proliferation, invasion and sphere formation assays in AsPC-1 cells transfected with miR-3064 mimic with (or without) PIP4K2B expression vector. (c, d, e) Cell proliferation (c), invasion (d) and sphere formation assays (e) in PaCa-2 cells transfected with miR-3064 inhibitor with (or without) PIP4K2B siRNA-1 and siRNA-2. *P < 0.05
Fig. 6
Fig. 6
PXN-AS1 acts as a sponge for miR-3064 to repress its expression in PC cells. (a) Putative miR-3064 binding site in PXN-AS1 sequence. (b) The expression level of PXN-AS1 in PC tissues and normal tissues from the TCGA PC datasets (the lncRNAtor database). (c) The expression of PXN-AS1 was measured in PC cell lines and a normal pancreatic cell line HPDE6-C7 using qRT-PCR analysis. (d) Luciferase reporter assays were conducted to confirm the direct binding between miR-3064 and PXN-AS1. (e) qRT-PCR analysis of miR-3064 expression in PC cells following knockdown or overexpression of PXN-AS1. (f) RIP assays were performed in PC cells transfected with or without miR-3064 mimic. The expression of PXN-AS1 was examined using qRT-PCR analysis. (g) Analysis of PXN-AS1 expression in PC tissues and normal tissues using qRT-PCR assays. (h) Kaplan-Meier curves for overall survival of PC patients with low or high PXN-AS1 levels. (i) Kaplan-Meier curves for overall survival of PC patients with high or low levels of PXN-AS1 (the GEPIA database). (j) Kaplan-Meier curves for overall survival of PC patients with high or low levels of PXN-AS1 (the KMPlotter database). *P < 0.05
Fig. 7
Fig. 7
PXN-AS1 suppresses PC cell proliferation, invasion and sphere formation partly through inhibiting miR-3064 expression. (a, b) Cell proliferation, invasion and sphere formation assays in AsPC-1 cells transfected with PXN-AS1 siRNA-1 (a), or PXN-AS1 siRNA-2 (b), along with (or without) miR-3064 inhibitor. (c) Cell proliferation, invasion and sphere formation assays in PaCa-2 cells transfected with PXN-AS1 expression vector, along with (or without) miR-3064 mimic. (d) PC cells were transfected with PXN-AS1 siRNA-1 or PXN-AS1 expression vector as indicated, and then injected into nude mice. Tumor growth rates of xenograft tumor were shown. (e) Immunohistochemical staining of Ki-67 in tumors derived from (d). *P < 0.05
Fig. 8
Fig. 8
A schematic diagram depicting a potential mechanism by which the lncRNA PXN-AS1/miR-3064/PIP4K2B axis regulates the proliferation, invasion and sphere formation of PC cells

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