Autotaxin is a novel target of microRNA-101-3p

Abstract

Autotaxin (ATX), a vital enzyme that generates lysophosphatidic acid (LPA), affects many biological processes, including tumorigenesis, via the ATX-LPA axis. In this study, we demonstrate that microRNA-101-3p (miR-101-3p), a well-known tumor suppressor, downregulates ATX expression at the posttranscriptional level. We found that miR-101-3p inhibits ATX regulation by directly targeting a conserved sequence in the ATX mRNA 3'UTR. Moreover, we observed an inverse correlation between ATX and miR-101-3p levels in various types of cancer cells. ATX is highly expressed in several human cancers. Here, we verified that ATX expression is significantly inhibited by miR-101-3p in U87 and HCT116 cells. ATX downregulation contributed to the suppression of migration, invasion, and proliferation mediated by miR-101-3p; furthermore, the tumor-suppressing activity of miR-101-3p was partially reduced by the addition of LPA in U87 cells. Our data suggest that ATX is a novel target of miR-101-3p.

Keywords: autotaxin; cancer cell; invasion; miR‐101‐3p; migration; proliferation.

Figure 1

Identification of candidate miRNAs targeting ATX. (A) HT29 and MCF7 cells were transfected with siDicer duplexes or negative control siRNA (NC). RNA was isolated from the cells 48 h after transfection and then subjected to RT‐qPCR to assess ATX mRNA levels (left) and Dicer mRNA levels (right). (B) HeLa cells were cotransfected with pTRE‐d2EGFP‐ATX 3′UTR reporter plasmid and Dicer siRNA, an unrelated gene Pgrp siRNA, or a nonspecific siRNA (NC) as indicated. At 48 h after transfection, RT‐qPCR was performed to assess the levels of reporter EGFP mRNA (left), Dicer mRNA, and Pgrp mRNA (right). (C) HEK293 cells were cotransfected with the pRLuc‐ATX‐3′UTR plasmid and the indicated miRNA mimic or negative control (miR‐NC). A luciferase activity assay was conducted 48 h after transfection. Data are representative of three independent experiments. The error bars represent ± SEM. Statistical significance was determined using Student's t‐test. *P < 0.05, **P < 0.01.

Figure 2

miR‐101‐3p targets the ATX mRNA 3′UTR at a conserved site. (A) A schematic diagram indicating a miR‐101‐3p binding site in the ATX mRNA 3′UTR sequence, as predicted by targetscan. Mutations were introduced to eliminate base‐pairing with the miR‐101‐3p seed sequence. (B, C) HEK293 cells were cotransfected with miR‐101‐3p mimics (miR‐101‐3p) and pRLuc (Ept), pRLuc‐ATX‐3′UTR (WT), or pRLuc‐ATX‐3′UTR‐mut (Mut). Luciferase mRNA levels (B) and luciferase activities (C) were detected 48 h after transfection. The error bars represent ± SEM. Statistical significance was determined using Student's t‐test. **P < 0.01. (D) Conservation of the 8‐mer seed sequence of the miR‐101‐3p binding site (gray) within the 3′UTR of ATX mRNA was analyzed among different species. The sequences are from Homo sapiens (NM_001040092.2), Pan troglodytes (XM_009455833), Gorilla gorilla (XM_004047471.1), Macaca mulatta (XM_015145919.1), Sus scrofa (XM_013996524.1), Equus caballus (XM_014728071.1), Canis lupus familiaris (XM_014118597.1), Mus musculus (NM_015744.2), and Rattus norvegicus (NM_057104.2).

Figure 3

miR‐101‐3p downregulates ATX expression in cancer cells at the posttranscriptional level. (A) The expression levels of ATX mRNA and miR‐101‐3p in various cancer cell lines were detected by RT‐qPCR. (B) U87 and HCT116 cells were transfected with miR‐101‐3p mimics (miR‐101‐3p) or a negative control (miR‐NC); (C) U87 cells were transfected with the plasmid expressing pre‐miR‐101 or an empty plasmid (shNC). (D) U87 and HCT116 cells were cotransfected with the indicated luciferase reporter plasmid and miR‐101‐3p mimics or miR‐NC. Luciferase activities were detected 48 h after transfection. (E) MCF7 and HT29 cells were transfected with miR‐101‐3p inhibitor (anti‐miR‐101‐3p) or microRNA inhibitor NC (anti‐miR‐NC). Protein levels of secreted ATX in the conditional medium (CM) and EZH2 in the cells were detected by western blot, and ATX mRNA levels were detected by RT‐qPCR (B, C, E). Data are representative of three independent experiments. The error bars represent ± SEM. Statistical significance was determined using Student's t‐test. *P < 0.05, **P < 0.01.

Figure 4

ATX downregulation contributes to the suppression of U87 cell migration, invasion, and proliferation by miR‐101‐3p. (A) Transwell migration assay. U87 cells treated with the indicated RNA duplexes in the absence or presence of LPA (2 μm) as indicated. Images of the cells on the upper chamber's lower surface were taken 24 h after the cells were seeded into the upper chamber. The relative migration was calculated by counting the cell number in the upper chamber's lower surface, and data were obtained from three randomly chosen fields. (B) Wound‐healing assay. U87 cells were treated as described in (A) and were subjected to the wound‐healing assay with the incucyte zoom longtime live cell image monitoring system. The percentage of migrated cells covering a scratch was enumerated at the indicated time points. (C) Transwell invasion assay was performed as described in (A), except that the polycarbonate membrane was coated by Matrigel before adding U87 cells to upper chamber. (D) Proliferation assay. U87 cells were treated as indicated in (A) and were subjected to the proliferation assay with the incucyte zoom longtime live cell image monitoring system. The percentage of cell growth/confluence was counted at the indicated time points. Data are representative of three independent experiments. The error bars represent ± SEM. Statistical significance was determined using Student's t‐test. *P < 0.05, **P < 0.01.