Targeted Silencing Of CNN2 By SiRNA Influences The Cellular Behavior Of Hepatocellular Carcinoma Cells And Tumorigenesis In Nude Mice

OBJECTIVE:Establish a stable transfection of CNN2 siRNA cell line of liver cancer, to observe the effect of CNN2 protein on cellular behavior and tumorigenesis in nude mice.METHODS:Lentiviral vector encoding small interfering RNA targeting CNN2 and negative control lentiviral vector were transfected into SK-hep-1 and screened by puromycin. (1) CNN2 expression was monitored by quantitative real-time polymerase chain reaction (qRT-PCR) and western blot. (2) Cell invasion and migration were examined by transwell migration/invasion analysis; Cell proliferation was examined by methyl thiazol tetrazolium (MTT). Cell cycle were examined by flow cytometry (FCM). (3) Subcutaneous xenotransplanted tumor models of nude mice with SK-hep-1-siRNA, SK-hep-1-LV3, and SK-hep-1 were constructed (12 nude mice of each group, half male mice and half female mice). Mice living conditions were observed everyday. Tumor volume and mice weight were measured every week. After four weeks, the mice were sacrificed and then tumors volume and weight were measured. The mice were dissected to observe tumor metastasis. Tumor tissues were embedded in paraffin. Hematoxylin & Eosin (H&E) staining was performed. Apoptosis was detected by TUNEL method. Immunohistochemistry was used to detect the expression of CNN2, phosphorylated MEK, phosphorylated ERK, phosphorylated AKT, ERK, MEK and AKT protein.RESULTS:(1) SK-hep-1-siRNA cell line and SK-hep-1-LV3 cell line were observed under the fluorescence microscope after 24 h of infection. The transfection was confirmed successful if there were cells with green fluorescence. Almost all the cells have green fluorescence when treated with puromycin for 72 h. siRNA efficiently downregulated the expression of CNN2 in SK-hep-1 cells at both mRNA and protein levels (P<0.001). The inhibitory rate of CNN2 expression in the transcription level was about 68.3% and in the translation level was about 63.9%. (2) CNN2-siRNA can inhibited cell proliferation and reduced cell invasion (P<0.01) and migration (P<0.001). Furthermore, knockdown of CNN2 slowed down the cell population at S phase (P<0.01). (3) The xenotransplanted tumor models were successfully established. The tumors growth rate were slower and the tumors were smaller in the SK-hep-1-siRNA group. After 4 weeks, the SK-hep-1-siRNA group has lighter tumor weight and smaller tumor volume than the negative contol and blank group (P<0.001). No tumor metastasis was observed and there was no significant difference among the three groups in the weight of the nude mice. The result of H&E staining showed the three groups of nude mice all had typical features of tumor tissue:larger nucleus, deeper dyeing and cells arranged closely. The expression of CNN2, phosphorylated MEK and phosphorylated ERK were down regulated in SK-hep-1-siRNA group (P<0.01, P<0.05, P<0.01), while the expression of phosphorylated AKT, MEK, ERK, AKT had no significant difference among the three groups.Conclusion:(1) SK-hep-1-siRNA cell line and SK-hep-1-LV3 cell line were successfully established. (2) CNN2-siRNA can inhibited cell proliferation and reduced cell invasion, migration. Knockdown of CNN2 slowed down the cell population at S phase. (3) CNN2-siRNA can inhibit the tumor development and CNN2 may play a role in promoting the development of tumors by increasing the phosphorylation level of MEK and ERK.