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  • br Hongda Pan and Jingxin Pan contributed equally to this

    2020-08-28


    1 Hongda Pan and Jingxin Pan contributed equally to this article.
    patients are far from ideal. Therefore, it is critical to explore the regula-tory mechanisms of CRC to promote the development of promising di-agnostic biomarkers as well as optimal therapeutic targets. Carboxypeptidase A4 (CPA4) is a member of the metallocarboxypeptidase family. CPA4 functions in neuropeptide pro-cessing and regulation in the extracellular environment, which are closely related to cancer progression [3]. Previous studies have sug-gested that CPA4 is an imprinted gene and it may become a strong can-didate gene for predicting the aggressiveness of prostate cancer [4]. CPA4 is secreted as an N-glycan and elevated levels of CPA4 can be de-tected in the media of breast cancer cell lines, and may be useful as a glycan-based biomarker for the prognosis of breast cancer [5]. Altered HG-9-91-01 of CPA4 has also been detected in head and neck squamous cell carcinoma tissues, and higher expression of CPA4 is related to poor survival [6]. Several studies have demonstrated that CPA4 is aberrantly expressed in a wide variety of cancers, and elevated CPA4 expression levels are associated with advanced tumor stage, metastasis, and poor prognosis [7–12]. However, the function and mechanism by which CPA4 contributes to cancer development and progression are unclear.
    In our study, we demonstrated a significant correlation between high CPA4 expression and poor prognosis in CRC patients. The onco-genic function of CPA4 was examined by in vitro and in vivo
    experiments. Moreover, we found that CPA4 promoted CRC progression via STAT3 and ERK signaling pathways. This study thus reports the on-cogenic and prognostic roles of CPA4 in CRC.
    Fig. 1. CPA4 was overexpressed in colorectal cancer (CRC) tissues and cells, and correlated with survival in HCC patients. (A) and (B) CPA4 mRNA levels in 42 pairs of tumor samples and matched normal tissues were determined by qRT-PCR. Compared to matched normal tissues, CPA4 expression was upregulated in 76.2% (32/42) of CRC samples. (C) and (D) mRNA and protein levels of CPA4 in a human colon cell line and five CRC cell lines as measured by qRT-PCR and western blot, respectively. (E) Representative IHC staining for CPA4 in CRC and normal tissues (scale bar: 200 μm and 50 μm). (F) High CPA4 expression correlated with poorer overall survival in CRC patients. (G) and (H) CPA4 expression was increased in tumor tissues compared with normal colorectal tissues in TCGA-COAD and TCGA-READ datasets.
    2. Methods
    2.1. Patients and specimens
    In total, 42 pairs of matched CRC and adjacent normal tissues were collected from the Department of Gastrointestinal Surgery, Beijing Hos-pital (BJH). All tissues were frozen immediately in liquid nitrogen after surgical excision and stored at −80 °C. Archived formalin fixed, paraffin-embedded specimens of 120 CRC patients who underwent sur-gical resection from July 2011 to May 2013 at BJH were retrieved from the Department of Pathology. Clinicopathological information was re-trieved from the hospital database, which was updated until May 2018. Written informed consent was obtained from all patients and the study was approved by the ethics committees of BJH.
    2.2. Cell lines and culture
    The human colon cell line NCM460 and CRC cell lines HCT116, LS123, SW480, SW620, and RKO were purchased from the American Type Culture Collection (Manassas, VA, USA). Cell lines were cultured in Dulbecco's modified Eagle's medium (DMEM) (Gibco) containing 10% fetal bovine serum (FBS). The STAT3 inhibi-tor cryptotanshinone and ERK inhibitor U0126 (Selleck Chemicals, USA) were dissolved in DMSO prior to use. All cells were grown at 37 °C in a 5% CO2, humidified atmosphere.
    2.3. Lentivirus constructs and transfection
    CPA4 short hairpin RNAs (shCPA4), the CPA4 overexpression plas-mid, and the scramble shRNA control were purchased from GeneChem Company (Shanghai, China). The target sequences of the shRNAs were as follows: shCPA4#1: 5′-GTATGACAACGGCATCAAA-3′ and shCPA4#2: 5′-GGAAATCTCCCTCCTCCTTCA-3′. HCT116 and LS123 cells were transfected with the shCPA4 plasmid, and SW620 and RKO cells were transfected with the CPA4 overexpression plasmid. Cells transfected with scramble vector were used as controls. The shRNAs and plasmids were transfected into cells using Lipofectamine 2000 (Invitrogen) following the manufacturer's protocol. Levels of CPA4 were measured by western blot.
    2.4. RNA extraction and qRT-PCR analysis 
    transferred to a 0.45-μm PVDF membrane (Millipore, USA). The mem-brane was then incubated with the appropriate monoclonal antibody at 4 °C for 24 h. CPA4 was detected with a mouse polyclonal anti-CPA4 antibody (Abcam, USA). GAPDH (CST, USA) expression was used as an equal loading control. The secondary antibody used was goat anti-mouse IgG-HRP (Abcam, USA). The signal detection from each blot was performed using a visual imaging system (Bio-Rad, USA). The grey values of western blot bands were measured using Image J soft-ware (National Institutes of Health, USA) to quantify relative expression levels.