Krüppel‐like factor 4 (KLF4) has a tumor suppressor role in the progression of gastric cancer (GC), and inhibition or loss of KLF4 expression was identified in GC. The aim of this study was to explore the new molecular mechanism of KLF4 inactivation in gastric cancer. Herein, we report that Helicobacter pylori infection or Cag pathogenicity island protein A (CagA) gene transduction resulted in KLF4 expression downregulation and promoted gastric epithelial cell and gastric cancel cell proliferation, migration, and colony formation. Mechanistically, we found that CagA gene transduction led to DNA methylation of the KLF4 promoter, an effect that was relevant to the significant downregulation of TET1 expression. Causally, knockdown of TET1 expression decreased KLF4 expression, whereas overexpression of TET1 had the opposite effect. Clinically, we found that KLF4 expression and the 5‐hmC levels were lower in GC cells with H pylori infection than in GC cells without H pylori infection. Thus, our study not only sheds new light on how H pylori infection promotes the progression of GC but also elucidates a novel mechanism of KLF4 inactivation in GC pathogenesis. During pathogenesis, an alteration in the H pylori /CagA‐TET1‐KLF4 signaling pathway plays a critical role, suggesting that this pathway may be a prospective target for gastric carcinoma intervention and therapy.

Purpose The aim of this study was to investigate the effects of gain-of-function (GOF) E76K-mutant Src homology-2 domain containing protein tyrosine phosphatase-2 (SHP2) on the biological behaviors of glioblastoma (GBM) cells, and explore the molecular mechanisms of GBM progression. Methods Firstly, a negative control vector and vectors overexpressing SHP2 and E76K-mutant SHP2 were transduced into GBM cells (U87 and A172) using a lentivirus. The effect of GOF-mutant SHP2 on proliferation was measured using the MTT assay, flow cytometry, colony formation assay, and soft agar assay. Moreover, the migration and invasion of GBM cells were determined through the transwell assay. Related proteins of the extracellular signal-regulated kinase/cAMP response element binding protein (ERK/CREB) pathway were detected by Western blotting analysis. A xenograft model was established to confirm the tumor-promoting effect of GOF-mutant SHP2 in vivo. Finally, ERK was inhibited using a mitogen-activated protein kinase/ERK kinase inhibitor (U0126) to further explore the molecular mechanism of GOF-mutant SHP2 affecting GBM cells. Results After transduction, the expression of SHP2 in the SHP2-mutant and SHP2-overexpression groups was higher than that observed in the control and normal groups. Our data indicated that GOF-mutant SHP2 enhanced the abilities of GBM cells for proliferation, migration, and invasion in vitro, and promoted tumor growth in vivo. Mechanistically, the ERK/CREB pathway was activated, and the levels of relevant proteins were increased in the SHP2-mutant group. Furthermore, following inhibition of ERK in the GOF-SHP2 mutant group, the activation of CREB was also depressed, and the malignant biological behaviors were weakened accordingly. Conclusion The GOF-mutant SHP2 promoted GBM cell proliferation, metastasis, and tumor growth through the ERK/CREB pathway, providing a promising target for the treatment of GBM.

The Helicobacter pylori (H. pylori) cytotoxin-associated gene A (CagA) and Kruppel-like transcription factor (KLF4) were both closely associated with the development and progression of gastric cancer (GC). However, the nature of the interactions between CagA and KLF4 in GC development has not been elucidated. Therefore, we focused on the CagA-mediated promotion of the malignant transformation of gastric epithelial cells. Herein, we first examined the expression of KLF4 in both human cancer and paracarcinoma tissues with or without H. pylori infection and found that KLF4 expression was significantly decreased in H. pylori-positive GC cells compared with the H. pylori-negative GC cells. Further functional studies revealed that the increased expression of CagA could suppress KLF4 expression and promote the malignant transformation of normal epithelial cells. Subsequently, we found that CagA could upregulate miR-155 and further restrict the expression of downstream KLF4. More importantly, the overexpression of miR-155 in GES-1 promoted epithelial-mesenchymal transition and eventually facilitated tumor growth in vivo. Overall, the identification of the CagA/miR-155/KLF4 signaling pathway provided a new insight into the development and treatment of GC.

目的 构建过表达miR-155-5p慢病毒表达载体,建立稳定过表达miR-155-5p的GES-1胃上皮细胞株.在细胞水平上观察细胞增殖能力的变化.方法 第一部分:PCR扩增获得miR-155-5p基因片段;将该基因片段与已线性化的病毒载体连接;将重组质粒转化到DH5α细胞,进行克隆分离、质粒DNA提取和DNA测序.第二部分:序列证实了的GV-369-has-miR-155-5p载体和pHelper 1.0以及pHelper2.0载体共同转染到293-T细胞,用于慢病毒的包装,然后进行慢病毒收获、浓缩与纯化.将纯化的miR-155-5p慢病毒用来感染细胞,经过嘌呤霉素筛选后建立稳定过表达miR-155-5p的GES-1细胞株.qRT-PCR检测细胞株中miR-155表达;Western blot检测KLF4蛋白表达及MTIT法检测稳定表达miR-155-5p细胞的增殖.结果 过表达miR-155-5p的重组慢病毒构建成功;miR-155在恶性程度较高的SGC-7901胃癌细胞株中高表达(P＜0.05);建立了miR-1556p稳定过表达的GES-1细胞株;过表达miR-155-5p抑制GES-1细胞中KLF4蛋白表达(P＜0.05),且促进细胞增殖.结论 miR-155可能通过负向调控KLF4表达,参与调控胃癌发生发展.

Objective To construct plasmid for knockout of Kruppel-like factor 4 (KLF4) gene by using CRISPR/ Cas9 gene editing method and examine its impact on biological behavior of human GES-1 cells.Methods Synthesized KLF4 gene targeting sgRNA oligos were inserted into CRISPR/Cas9 plasmid vector of pX459.After transformation,clone isolation,and amplification,DNA sequencing confirmed plasmid DNA,termed pX459-KLF4-sgRNA,was used to transfect human gastric epithelial GES-1 cells in vitro.After transfection,GES-1 cells were first selected in cell-cultured medium containing puromycin,and then the selected GES-1 cells were replated into 100 mm dishes for clonal cell culture.Protein samples extracted from individual cell clones were used for Western blot analysis of KLF4 expression.Biological assays of plate clone formation,MTT cell proliferation,and transwell cell migration were used to compare the cellular behavior changes among GES-1 cells of parental,control clone or clone with KLF4 gene knockdown.Results DNA sequence analysis confirmed that KLF4 targeting sgRNA sequences were successfully constructed into of pX459 vector.Transfection of the resultant vector of pX459-KLF4-sgRNA into GES1 cells led to KLF4 gene knockdown indicated by drastic reduction of KLF4 protein expression,significant biological behavior changes including increased cell proliferation,clone formation,and migration abilities.Conclusion We successfully constructed human KLF4 gene editing plasmid vector based on CRISPR/Cas9 technique,and the biological behavior changes of the established GES-1 cell line with drastic KLF4 knockdown confirmed the tumor suppressive function of KLF4.