IGF2 Induces Human Immortalized Hepatocytes To Produce The Stemness Of Liver Cancer Stem Cells And Self-renewal Ability

Objective: Insulin-like growth factor 2(IGF2)can promote the proliferation,invasion and metastasis of liver cancer cells by activating PI3K/AKT,MAPK,JAK1/2-Stat3 and other signaling pathways.However,there is still a lack of systematic research on whether IGF2 can also activate the expression of reprogramming factor to induce the formation of liver cancer stem cells,thus promoting the invasion and metastasis of hepatocellular carcinoma(HCC).This research through the experimental observation of IGF2 on the expression of reprogramming factors(Oct4,Nanog,Sox2,c-myc),stemness markers [CD44,CD24,CD133,epithelial cell adhesion molecule(Ep CAM)],E-cadherin,vimentin protein in human immortalized hepatocyte THLE-2,discusses its influence on liver cancer stem cell formation,which will shed new light on the targeted therapy of HCC.Methods:(1)The total RNA of human liver cancer cell line Huh7 was reverse-transcribed into c DNA,amplified the protein coding region of IGF2 gene,and clones it into the lentivirus expression vector plvx-cmv-zsgreen-puro to construct the lentivirus expression vector plvx-cmv-zsgreen-IGF2,which can stably express IGF2.Plvx-cmv-zsgreen-IGF2 and the control empty vector plvx-cmv-zsgreen-puro were packaged into lentivirus,then infected human immortalized liver cells THLE-2 were constructed into THLE-2-IGF2 and THLE-2-con.(2)Thle-2-IGF2 and THLE-2-con cells were cultured in vitro for 21 days,and then detect the m RNA and protein expression levels of AFP and reprogramming factors(Oct4,Nanog,Sox2 and c-myc)by real-time fluorescent quantitative PCR and Western blot.(3)the expression of liver cancer stem cells stemness markers(CD24,CD44,CD133,Ep CAM),E-cadherin,and vimentin of the THLE-2-IGF2 and THLE-2-con were tested by immunofluorescence assay after 21 days in vitro culture.(4)Proliferative and tumorigenicity of the THLE-2-IGF2 and THLE-2-con were tested by soft agarose assay after 21 days in vitro culture.(5)Observe the effect of stable expression of IGF2 on the stemness of carcinoma stem cells(CSCs)and self-renewal ability,via sphere formation assay.Results:(1)The expression level of IGF2 m RNA in THLE-2-IGF2 cell line was 98.6 times higher than that in THLE-2-con cell line,and the expression level of IGF2 protein in THLE-2-IGF2 cells was also significantly higher than that of thle-2-con cells.(2)The m RNA and protein expression levels of AFP,Oct4,Nanog,Sox2 and c-myc in THLE-2-IGF2 cells were significantly higher than THLE-2-con cells(P<0.01).(3)The expressions levels of CD24,CD44,CD133,Ep CAM and vimentin in THLE-2-IGF2 cells were significantly higher than THLE-2-con cells,while the expression of e-cadherin was lower.(4)Theclone formation rate of THLE-2-IGF2 cells was significantly higher than that of THLE-2-con cells(P<0.01).(5)The sphere formation rate of THLE-2-IGF2 cells was significantly higher than that of THLE-2-con cells(P<0.01).Conclusion: High level of IGF2 expression induces human immortalized hepatocytes to produce the stemness of liver cancer stem cells and self-renewal ability by up-regulating the expression of Oct4,Nanog,Sox2 and c-myc.