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Gastrokine 2 Regulates the Antitumor Effect of JAK2/STAT3 Pathway in Gastric Cancer

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Gastrokine 2 Regulates the Antitumor Effect of JAK2/STAT3 Pathway in Gastric Cancer Hindawi Evidence-Based Complementary and Alternative Medicine Volume 2021, Article ID 1343808, 9 pages https://doi.org/10.1155/2021/1343808 Research Article Gastrokine 2 Regulates the Antitumor Effect of JAK2/STAT3 Pathway in Gastric Cancer Yu Zhou,1,2 Shan Xu,3 Jiao Liu,3,4 Yaping Zhu,1,2 Yaxin Zhu,1 Wei Li,3 and Hui Ling 3 1 e First Affiliated Hospital of Shaoyang University, Shaoyang, Hunan 422000, China 2Shaoyang University, Shaoyang, Hunan 422000, China 3Key Laboratory of Tumor Cellular & Molecular Pathology (University of South China), College of Hunan Province, Cancer Research Institute, Hengyang Medical College, University of South China, Hengyang, Hunan 421001, China 4Department of Pathology, Changsha Fourth People’s Hospital, Changsha, Hunan 410006, China Correspondence should be addressed to Hui Ling; [email protected] Received 25 June 2021; Accepted 21 July 2021; Published 2 August 2021 Academic Editor: Songwen Tan Copyright © 2021 Yu Zhou et al. ,is is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. GKN2 (gastrokine 2) mainly plays a regulatory role in gastric mucosal defense and cell protection mechanisms, and its role in gastric cancer has not been thoroughly elucidated. Immunohistochemistry was used to detect GKN2 and TFF1 expressions in 90 gastric cancer tissues, 48 neoplastic resection margins, and 22 normal gastric mucosa epithelia. It showed that the downregulation of GKN2 and TFF1 expressions in gastric cancer tissues was significantly different from that in adjacent normal gastric tissues and distal gastric mucosal tissues. Nevertheless, correlation analysis showed that GKN2 expression in gastric cancer tissues was independent of TFF1 expression. After overexpression of GKN2 was constructed in human gastric cancer cell line MKN28 with the Ad-GFP-GKN2 transfected, cell viability was measured by CCK-8 assay, and migration and invasion ability were analyzed by transwell migration assay and transwell invasion assay. It indicated that overexpression of GKN2 significantly reduced the viability of MKN28 and SGC7901 cells. Overexpression of GKN2 could also inhibit the migration and invasion ability in MKN28 and SGC7901 cells. In addition, upregulation of GKN2 can inactivate the JAK2/STAT3 pathway. Our data suggest that GKN2 and TFF1 play the antitumor role in gastric carcinoma, and TFF1 may not interact or cooperate with GKN2. GKN2 overexpression can inhibit the growth and metastasis by downregulating the JAK2/STAT3 pathway in gastric cancer cells. 1. Introduction cells and reduce their migration and invasion ability [5]. In gastric cancer, GKN2 (gastrokine-2) also has the same effect Gastric cancer (GC) not only has a high incidence in the as GKN [6–9]. GKN2 is downregulation or loss in gastric population of our country but also ranks high in mortality cancer tissues. GKN2 is an obviously expressed gene after among many cancers [1]. ,e development of gastric cancer the elimination of Helicobacter pylori [10]. It shows that the is a complex process of multistage and multistep. In this regulatory pattern to cytokines is the same as those of the process, there are many genes involved, including the reg- TFF gene [11]. It is reported that GKN2 affects the growth of ulation of cell proliferation, apoptosis, differentiation, in- gastric cancer cells in a trefoil factor 1- (TFF1-) dependent vasion, and metastasis. However, there are few molecules manner [12]. GKN2 forms disulfide-linked heterodimers specifically expressed in gastric mucosa. GKN is a new family with TFF1 [13, 14]. of gastrin-specific proteins, which is almost completely Although the downregulation of GKN2 expression in expressed and secreted by gastric mucosal epithelial cells. gastric cancer has been reported [15], the potential rela- GKNs gene family is considered as a specific tumor sup- tionship between GKN2 and TFF1 in regulating the pro- pressor gene of gastrointestinal tissue [2–4]. According to gression of gastric cancer and improving the biological reports, GKN1 can inhibit the metastasis of gastric cancer behavior of cells remains unclear. ,e role of JAK2/STAT3 2 Evidence-Based Complementary and Alternative Medicine pathway in tumors is gradually discovered, and it also has a were preserved from our institute. Cells were stored in the certain impact on the occurrence and development of gastric RPMI1640 complete medium (Hangzhou Sijiqing Tech- cancer [16–18]. In this study, we refer to the method in the nology Co., Ltd., China), supplemented with 10% fetal previous study such as by Ling et al. [19]. We first examine bovine serum and placed in an incubator at 37°C and 5% the expressions of GKN2 and TFF1 in gastric cancer tissues, CO2. MKN28 and SGC7901 cells were transfected with the adjacent gastric mucosa, and distal gastric mucosa tissues Ad-GFP-GKN2 vector and Ad-GFP-empty vector. Blank, using tissue chip and analyze its role in gastric carcino- cells without any treatment; NC, negative control, cells genesis. ,en, the constructed GKN2 expression vector was transfected with Ad-GFP-empty vector negative control; transfected into human gastric cancer MKN28 cells, and a GKN2, cells transfected with Ad-GFP-GKN2. stable transfection cell line was established. ,e prolifera- tion, invasion, and migration of the cells were analyzed in order to explain the inhibitory effect of GKN2 on the growth 2.1.4. qRT-PCR. Total RNA was extracted from cells or of human gastric cancer MKN28 and SGC7901 cells through tissues using Trizol (catalog number 15596-018, Invitrogen), inhibiting the activation of the JAK2/STAT3 pathway. ,is reverse transcribed to generate cDNA, and then, primers study provides a good basis for the further study of the were amplified. Gene primers were synthesized by Invi- function of GKN2 in gastric cancer. trogen, as given in Table 1. β-Actin was used as a control for detecting the target gene. 2−ΔΔCt was used to calculate the 2. Materials and Methods relative expression of the detected gene. 2.1. Experimental Method 2.1.5. CCK-8 Experiment. 4 ×103 cells were seeded in a 96- 2.1.1. Materials. Gastric cancer tissue microarrays including well plate, and a medium containing CCK-8 was added. 90 gastric cancers, 48 neoplastic resection margins, and 22 CCK-8 accounted for 1/10 of the volume of the cell culture normal gastric mucosa epithelium >5 cm from the tumor medium. After the color of the culture medium changes, edge were provided by the Department of Pathology, First took out the 96-well plate and put it into a microplate reader Affiliated Hospital of University of South China. Clinico- for detection and set the wavelength to 570 nm. pathological data include tumor stage, degree of differen- tiation, age range, and gender. ,e study was approved by the Clinical review Committee of First Affiliated Hospital of 2.1.6. Transwell Experiment. Cell migration was guided as University of South China. previously described [7]. Briefly, MKN28 and SGC7901 cells Matrigel was provided by BD Company. Transwell were added to the upper chamber of the transwell chamber. chambers (8 μm; Corning) were provided by Corning. MTT ,e lower chamber was a culture medium containing 50 mg/ was provided by Beijing Kangwei Century Co., Ltd., China. L VEGF. After 1 day, it was rinsed with PBS, and 1 mL 4% ,e Ad-GFP-GKN2 expression vector and Ad-GFP-empty paraformaldehyde was added to the lower chamber for vector were constructed by Han Heng Biotechnology Co., fixation. 30 min, after proper drying, Giemsa staining for Ltd. (Shanghai, China). 30 min, rinsed with PBS, took out the filter cover, and 5 fields of view were randomly selected for counting. Cell invasion: MKN28 and SGC7901 cells were inoculated onto the 2.1.2. Immunohistochemistry. ,e 4 μm paraffin-embedded Matrigel-coated filters. ,e cells were GKN2-transfected and tissue sections were immunostained with citrate buffer untransfected for 24 h. Random use of hematoxylin staining antigen extraction and the UltrasensitiveTM SP streptavidin is done. Invasion rates were represented as ratios between peroxidase signal amplification detection system (catalog GKN2-transfected and control group values. number KIT-9710, Maixin Biotech. Co., Ltd. Fuzhou, China). ,e primary antisera were diluted 1 : 2000. ,e monoclonal anti-pS2 antibody (catalog number ab92377, 2.1.7. Western Blot Analysis. Total protein was then Abcam, Carlsbad, CA) and the monoclonal anti-GKN2 extracted and quantified with a BCA protein assay kit. ,e antibody (catalog number ab184163, Abcam, Carlsbad, CA) total protein was separated by SDS-PAGE and transferred to were at a dilution of 1 : 50. DAB dye, hematoxylin staining, the solid phase carrier. Anti-GKN2 (LSBio, 1 :1000), anti- and neutral gum sealing slices were done. Criteria: the JAK2 (Abcam, 1 :1000), anti-p-JAK2 (MilliporeSigma, 1 : positive staining cells were found to be yellow or brown 1000), anti-STAT3 (Abcam, 1 :1000), anti-p-STAT3 (Cell colored in the cytoplasm of the cells in the tissue microarray. Signaling Technology, 1 :1000), and anti-β-actin (Santa Cruz ,e results were as follows: the number of stained cells less Biotechnology, 1 :1000) were added and incubated over- than 5 was negative in each high-power microscope. ,e night at 4°C. β-Actin is an internal control. After washing number of stained cells more than 5 was positive in each and removing the unbound primary antibody, the NC high-power microscope. membrane treated with the primary antibody is treated with the labeled secondary antibody for 1 hour. After the 2.1.3. Cell Culture and Establishment of GKN2 Over- treatment is completed, wash 3 times, 10 minutes each time. expressing Cell Lines. Human gastric cancer cell lines ,en, use a chemiluminescence kit to develop and evaluate MKN28 and SGC7901 (Shanghai Gefan Biological Co., Ltd.) the OD of the band density.
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