Study On The Relationship Between CK2 Gene And Liver Cancer Development And Chemotherapeutic Drug Sensitivity

Background and Objective:Currently,chemotherapy is one of the major clinical therapies for advanced hepatocellular carcinoma(HCC)treatment.However,clinical chemotherapeutic agents are highly toxic and ineffective due to(1)weak or no targeting and(2)rapidly established multidrug resistance(MDR).The occurrence and development of HCC involves the mutation of many genes,especially cancer-related genes,are involved in Oncogenesis or the role and mechanism of cancer development remain unclear.In our laboratory,casein kinase 2(CK2)was predicted as a target molecule of HCSP4 during the screening of HCC specific peptide(HCSP4,12aa).Therefore,we aimed at investigating(1)whether the expression of CK2 is related to the development of HCC?(2)Is CK2 a target molecule of HCSP4?And what is its significance in HCC targeted therapy?CK2 is a protein kinase that is widely distributed in cells?and can phosphorylate a variety of substrates,and it plays important regulatory roles in gene expression,DNA damage repair,protein synthesis and degradation,cell cycle control,cell proliferation,differentiation,and apoptosis,CK2 affects cancer cell growth by regulating the energy metabolism of cancer cells and change the sensitivity of cancer cells to apoptosis by altering the phosphorylation level of relevant apoptotic factors.It is a promising drug target that may be potentially utilized as a cancer therapeutic target.Doxorubicin(DOX)is an anticancer drug that produces cytotoxic effects in cells by inserting into the DNA double helix so that block and interfere with the genomic transcription,while CK2a,the catalytic subunit of CK2,can be transferred to the nucleus of cancer cells,and may be involved in the chemoresistance of cancer cells by phosphorylating or dephosphorylating the activity of anti-apoptotic proteins.Based on the above considerations,this study used the gene editing technology Clustered Regularly Interspaced Short Palindromic Repeats(CRISPR)to achieve CK2 deletion in the HCC cell line HepG2 and reveal the roles played by CK2 in the development of HCC at first,and then investigated its effect as a target for chemotherapeutic drug delivery for HCC cells in vitro.Method:1.Western blot was utilized to detect CK2 expression in HepG2 cells,HEK293 cells and SGC-7901 cells.2.CK2a gene information(NCBI database)was analyzed to design sgRNAs for CK2a by online tools.3.Synthesis of sgRNAs and construction of a Cas9 vector targeting CK2? gene.4.Transfection of HepG2 cells for Cas9 targeting efficiency identification(T7E1 assay).5.Resistance screening of Cas9 high-activity transfected cells(Puromycin).6.The monoclonalization of cells after resistance screening(Limited dilution method)7.Screening of positive cell clones.(1)Sequencing of CRISPR/Cas9 targeting genomic sequence of CK2? in cell monoclines.(2)Western blot assay for further identification of the suspected CK2?+/-HepG2 and CK2?-/-HepG2 cell clones based on the sequencing analysis results.(3)CK2?+/-HepG2 and CK2?-/-HepG2 cell clones were stored for backup.8.Effects of CK2? expression on the malignant phenotype of HCC cells.(1)The detection on the effects of CK2? expression on the proliferation and motility of HCC cells by wound healing assay,Transwell chamber assay,MTT assay and plate clone formation assay.(2)To observe the changes in motility-related microstructure of HepG2 cells after deletion of CK2? by Komas Brilliant Blue staining and scanning electron microscopy.(3)The detection of changes in the apoptotic index of HepG2 cells after deletion of CK2? by flow cytometry,DAPI staining and Mitoview633 assay.9.Investigation of the relationship between CK2? and HCSP4 targeting.(1)Coincubation of HCSP4 phage clones with HepG2,CK2?+/-HepG2,and CK2?-/-HepG2 cells,respectively.(2)ELISA and cellular immunofluorescence assay were performed with anti-M13 antibody.10.Cytotoxicity assay of HCSP4-Lipo-DOX-miRNA101(1)Construction of Liposomal drug delivery system and confirmation of targeting specificity.(2)Detection of cell survival rate after cell transfection(MTT assay).11.Analysis of CK2? bioinformatics using the databases.Result:1.Western blot results showed a high expression of CK2 in hepatocellular carcinoma cells.2.CK2?+/-HepG2 and CK2?-/-HepG2 cell lines were successfully obtained.3.MTT and plate clone formation assay results showed that the proliferation of HepG2 cells was significantly inhibited after CK2? deletion.4.The results of damage repair assay,Transwell assay showed that the motility of HepG2 cells was significantly inhibited after deletion of CK2? expression5.Results from coomassie brilliant blue staining and electron microscopy observation showed that the development of mobility-related microstructures(pseudopods and filamentous pseudopods)was significantly inhibited and the cell contact inhibition was significantly restored in HepG2 cells after deletion of CK2?.6.Flow cytometry,DAPI and Mitoview633 results showed that HepG2 cells presented a certain trend of apoptosis after deletion of CK2?.7.The results of targeting affinity assay(ELISA and cellular immunofluorescence)between HCSP4 phage clone and cells showed that the affinity of HCSP4 phage to HepG2 cells was greatly diminished after deletion of CK2?.8.The HCSP4-Lipo-DOX-miRNA101 drug delivery system was specific for HepG2 cells,and the cytotoxic effect on HepG2 cells after deletion of CK2? was greatly diminished.9.The results of bioinformatics analysis suggested that:(1)CK2? gene expression was ectopic in a variety of tumors,which elevated in mRNA and protein levels in hepatocellular carcinoma.(2)Such changes resulted in a significant decrease in survival(prognosis)of patients with hepatocellular carcinoma.(3)A schematic network of the interaction and regulation of CK2? and its related genes was obtained,which laid a theoretical foundation for further studies of CK2?.Conclusion:CK2?-/-HepG2 cell lines were obtained after deletion of CK2? gene using CRISPR/Cas9 technology.Using the CK2?-/-HepG2 cell line as a platform,a series of experimental results obtained and can be summarized as follows:1.Elevated expression of CK2? in HCC significantly enhances the malignancy of the cells and the experimental results support that CK2? is a pro-oncogene at least in HCC.2.Elevated expression of CK2? accelerated the G1 phase of the HCC cell cycle,but its deletion did not significantly increase the trend of apoptosis in HCC cells.3.CK2 is a binding site for the HCC-targeted peptide HCSP4 in HCC cells;this specific binding may be important in the results of pre-laboratory HCC-targeted peptide-directed DOX,miRNA101 liposome delivery system studies.4.CK2? expression in HCC occurred with changes in the mRNA and protein levels of hepatocellular carcinoma,which significantly reduced the survival rate of hepatocellular carcinoma patients.