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Knockout of STK10 Promotes the Migration and Invasion of Cervical Cancer Cells

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Knockout of STK10 Promotes the Migration and Invasion of Cervical Cancer Cells 7090 Original Article Knockout of STK10 promotes the migration and invasion of cervical cancer cells Lu Zhang1, Shun-Yuan Lu1, Rui Guo1, Jin-Xia Ma1, Ling-Yun Tang1, Yan Shen1, Chun-Ling Shen1, Li-Ming Lu2, Zhu-Gang Wang1, Jie Liu3, Hong-Xin Zhang1 1Research Center for Experimental Medicine, State Key Laboratory of Medical Genomics, Shanghai Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China; 2Shanghai Institute of Immunology, Shanghai Jiao Tong University School of Medicine, Shanghai, China; 3Shanghai Institute of Orthopaedics and Traumatology, Shanghai Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China Contributions: (I) Conception and design: HX Zhang, J Liu; (II) Administrative support: ZG Wang, LM Lu; (III) Provision of study materials or patients: L Zhang, Y Shen, CL Shen; (IV) Collection and assembly of data: L Zhang, R Guo, SY Lu, JX Ma, LY Tang; (V) Data analysis and interpretation: LM Lu, ZG Wang, J Liu, HX Zhang; (VI) Manuscript writing: All authors; (VII) Final approval of manuscript: All authors. Correspondence to: Dr. Hong-Xin Zhang. Research Center for Experimental Medicine, State Key Laboratory of Medical Genomics, Shanghai Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, 197 Rui Jin Road II, Shanghai, 200025, China. Email: [email protected]; Dr. Jie Liu. Shanghai Institute of Orthopaedics and Traumatology, Shanghai Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, 197 Rui Jin Road II, Shanghai 200025, China. Email: [email protected]. Background: Serine threonine kinase 10 (STK10) is an ERM kinase involved in the activation of ERM proteins and plays an essential role in the aggregation and adhesion of lymphocytes. STK10 is expressed in about 17 cancer types, including cervical cancer. Cervical cancer is the fourth most common cancer that seriously threatens women’s health worldwide. Previous studies have shown that STK10 may affect LFA-1- mediated cell adhesion. Other studies reported a mutation (R634H) of STK10 detected in peripheral T-cell lymphoma. This study aimed to evaluate the functional roles of STK10 in the pathogenesis of cervical cancer. Methods: We generated STK10 knockout cervical cancer cell lines using the CRISPR-Cas9 gene- editing system, and further analyzed the effects of STK10 deficiency on tumor biological behaviors. The proliferation, apoptosis, migration and invasive activity of these cells were respectively detected by BrdU incorporation, AnnexinV/propidium iodide (PI) staining, wound healing assay and Transwell assays without and with Matrigel. The phosphorylation and expression level of indicated proteins were analyzed by Western blot. The differential expression genes between STK10 knockout and control cells were identified by RNA- seq analysis and further confirmed using qRT-PCR. Results: Our data revealed that target deletion of STK10 does not affect cell proliferation and apoptosis, but promotes the adhesion, migration, and invasion of cervical cancer cells. Most strikingly, the phosphorylation and expression level of ezrin and other ERM proteins in STK10 knockout cells was comparable with that in the control cells. Further, RNA-seq analysis indicated that the knockout of STK10 resulted in a profound alteration of gene expression in cervical cancer cells. Conclusions: This is the first study to provide evidence that STK10 executes various physiological functions in addition to phosphorylation of ERM proteins, and plays a vital role in the migration and invasion of cervical cancer cells. Keywords: Cervical cancer; ERM proteins; invasion; migration; STK10 Submitted Mar 20, 2020. Accepted for publication Sep 12, 2020. doi: 10.21037/tcr-20-1601 View this article at: http://dx.doi.org/10.21037/tcr-20-1601 © Translational Cancer Research. All rights reserved. Transl Cancer Res 2020;9(11):7079-7090 | http://dx.doi.org/10.21037/tcr-20-1601 7080 Zhang et al. Knockout of STK10 promotes the migration and invasion of cervical cancer cells Introduction Methods According to data from the World Health Organization, Cell culture cervical cancer is the fourth most common cancer in Cervical cancer cell lines (HeLa, Caski) were purchased women, with about 570,000 new cases in 2018, representing from the American Type Culture Collection (USA) and 6.6% of female cancers (https://www.who.int/cancer/ cultured in DMEM and RPMI, respectively, with 10% (vol/ prevention/diagnosis-screening/cervical-cancer/en/). It vol) fetal bovine serum (FBS, Gibco) at 37 in a 5% CO2 is the second leading cause of cancer-associated deaths in incubator. women, severely endangers women’s health, and imposes ℃ a substantial economic burden worldwide (1). Although significant advances in the diagnostic and therapeutic Plasmid construction strategies of cervical cancer have been achieved in the The guide-RNA (gRNA, 5-GGCGGACGTGCTCATA past several decades, the overall 5-year survival rate for TTCG-3′) was designed by the online CRISPR patients remains poor (2,3). The principal causes of the design tool (https://zlab.bio/guide-design-resources). high mortality are metastasis and recurrence of cervical Two partially complementary oligonucleotides cancer (4). Therefore, it is necessary to clarify the (5'-CACCGGCGGACGTGCTCATATTCG-3' and underlying pathogenic mechanism and identify novel factors 5'-AAACCGAATATGAGCACGTCCGCC-3') were involved in the progression of cervical cancer, which is the annealed and cloned into the PX459 plasmid (addgene, only way to find new, effective methods for the prevention, #62988) which was digested by the BbsI restriction enzyme. diagnosis, and treatment of this disease. This construct was called the STK10 KO plasmid, which STK10 (serine-threonine kinase 10), also called a could guide hSpCas9 to the genomic target site in STK10. lymphocyte-oriented kinase (LOK), is a serine/threonine kinase predominantly expressed in lymphoid organs. It has been demonstrated that STK10 can phosphorylate the Transfection and puromycin selection conserved threonine residues at the C-terminal domain A total of 4 μg STK10 KO plasmids and PX459 of ERM proteins (T567, T564, and T558, for ezrin, plasmids (control) were transfected into 8×105 cells radixin, and moesin, respectively), which is essential for the with Lipofectamine 3000 reagent (Thermo Fisher), and activation of these proteins (5,6). ERM proteins are linking the supernatant was exchanged for fresh medium 12 h proteins that mediate the interaction between the actin later. After 2 days, the cells (including the control) were cytoskeleton and the plasma membrane and are involved incubated in fresh medium with 1 μg/mL puromycin. in several physiological processes such as maintenance of After 7 days, cells resistant to puromycin could be selected. the cytoskeletal structure, cell motility, cell adhesion, cell Western blot analysis was used to identify the effect of movement, and signal transduction (7-9). Activated ERM knockout. The HeLa STK10 KO and Caski STK10 KO proteins also play essential roles in the pathogenesis of cell lines that were not the single clone were established, various cancers via regulation of cytoskeletal rearrangement, and DNA sequencing by a specific sequencing primer cell migration, and invasion (10-12). STK10 is expressed in (5'-GTGCTCCGAAACAGGGC-3') identified the about 17 cancer types, including cervical cancer, according genomic changes. to the “Human Protein Atlas” (https://www.proteinatlas. org/ENSG00000072786-STK10). However, the biological Western blot analysis functions of STK10 in the pathogenesis of cervical cancer remain undetermined. HeLa and Caski cell lysates were prepared using RIPA In the present study, to investigate the function of buffer [1% Nonidet P-40, 0.5% sodium deoxycholate, STK10 in cervical cancer, we generated STK10 knockout 0.1% SDS in phosphate-buffered saline (PBS)] with freshly (KO) HeLa and Caski cell lines using the CRISPR-Cas9 supplemented protease inhibitors cocktail (Roche). Next, the gene-editing system. We analyzed the effects of STK10 KO protein concentration was assayed by BCA (bicinchoninic on tumor biological behaviors such as cell proliferation, acid) method, and 30 μg protein was separated on 10% apoptosis, adhesion, migration, and invasion. SDS-PAGE membranes. Standard protocols were used for © Translational Cancer Research. All rights reserved. Transl Cancer Res 2020;9(11):7079-7090 | http://dx.doi.org/10.21037/tcr-20-1601 Translational Cancer Research, Vol 9, No 11 November 2020 7081 electrophoresis and immunoblotting analyses. Membranes albumin at 37 in cell incubator for 1 h. After washing were blocked with 5% milk in PBS. An anti-STK10 three times with PBS, 2×104 cells in 100 μL medium with ℃ antibody (abcam, ab70484), anti-GAPDH rabbit polyclonal 1% FBS were seeded to the well. Cells were incubated at antibody (BBI, D110016), recombinant anti-ezrin antibody 37 in a cell incubator for the appropriate time (HeLa: (abcam, ab40839), recombinant anti-moesin antibody 30 min, Caski: 1 h). The wells were washed twice with PBS ℃ (abcam, ab52490), recombinant anti-radixin antibody to remove unbound cells, and 100 μL medium with 1% (abcam, ab52495) and Anti-ezrin (pThr567)/radixin FBS containing 10% CCK8 was added to each well, and (pThr564)/moesin (pThr558) antibody (abcam, ab76247) the plate was incubated at 37 for 2 h. Absorbance was were used as the primary antibodies. The membranes were detected at 450 nm using a microplate reader (BioTek). ℃ then incubated with IRDyeCW800-conjugated
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