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. 2022 Aug 30;54(8):1290–1305. doi: 10.1038/s12276-022-00837-6

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© The Author(s) 2022

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 3 — a Potential target genes were predicted using the miRsystem, miRWalk, StarBase, TargetScan and RNA22v2 databases. A total of 50 genes were found to be shared in the five databases. b The potential binding site of miR-375 with the 3′-UTR of PTPN4 was predicted using the TargetScan database. Complementary sequences consisting of 8 nt were aligned and presented. c A luciferase assay was conducted to validate the direct binding and modulating function of miR-375 to the 3′-UTR of PTPN4. PCDH-empty: HEK-293 cells transfected with the pCDH-CMV vector. PCDH-375: HEK-293 cells transfected with pCDH-CMV vector that was subcloned with a fragment of pri-miR-375. PTPN4 WT: HEK-293 cells cotransfected with the P-MIR-Report firefly luciferase vector inserted with a fragment of the wild-type 3’-UTR of PTPN4 mRNA. PTPN4 MUT: HEK-293 cells cotransfected with the P-MIR-Report firefly luciferase vector subcloned with the 3′-UTR of PTPN4 mRNA mutated at the complementary site. d The expression of PTPN4 in PCa tissues compared with normal prostate tissues from the UALCAN database. e The correlation between miR-375 level and PTPN4 in PCa, statistically processed by StarBase. r = −0.178, p = 6.77e-5. PTPN4 at the mRNA level (f) and protein level (g), as determined by qRT-PCR and western blot in DU145 and PC-3 cells after miR-375 was overexpressed or knocked down. h IHC illustrating the differential expression of PTPN4 between 30 PCa and 17 normal tissues. The results show a representative IHC image (left) and quantitative analysis calculated by Image-Pro Plus version 6.0 software based on the 47 IHCs (right). AOD: average integrated optical density.