TET2 Suppresses HCC Stemness And Metastasis Through MiR-22-3p Regulation

[Backgrounds] Hepatocellular carcinoma(HCC)has a high incidence in malignant tumors and mainly concentrated in Asia.HCC is the second highest in the mortality rate of malignant tumors in China.HCC has the characteristics of high malignant degree,easy recurrence and metastasis,which leads to poor clinical prognosis of HCC patients.However,the mechanism that causes high recurrence rate of HCC and high metastasis rate is still not very clear.Recent studies have found that cancer stem cells(CSCs)have the ability of self-renewal and differentiation and are closely related to malignant proliferation,metastasis and recurrence of tumors.TET2 is a methyl transferase that catalyzes the transformation of 5 methyl cytosine into 5 hydroxymethyl cytosine,allowing DNA to be methylated.DNA methylation is closely related to the occurrence of tumors.TET2 has been reported to act as a tumor suppressor gene in many tumors and to be involved in regulating the self-renewal and differentiation of CSCs.The abnormal expression of micro RNA in epigenetics is closely related to the development of various tumors,which can regulate cancer cells and CSCs by influencing multiple signaling pathways.This study will explore the regulating effect of TET2 and mi R-22-3p on Liver cancer stem cells(LCSCs)and then reveal the new mechanism of metastasis and recurrence of HCC.[Methods](1)Through the collection of clinical HCC tissue specimens and case data,the expression level of TET2 in HCC tissue was detected,and the relationship between the expression level of TET2 and the prognosis of clinical patients was statistically analyzed.(2)In order to study the regulating effect of TET2 on the function of LCSCs and cell metastasis of HCC,we constructed the TET2 Knock out(KO)and TET2 Activation(ACT)stable HCC cell lines through CRISPR/CAS9 technology.(3)To detect the regulating effect of TET2 on LCSCs through stem cell related experiments,to detect the effect of TET2 on the migration and invasion of HCC cells through migration and invasion experiments,and to detect the effect of TET2 on tumorigenicity of nude mice in HCC cells through the experiment of nude mice tumorigenicity assay.(4)The correlation between TET2 and mi R-22-3p was detected by the Dual Luciferase Reporter Gene assay.(5)In order to study the regulating effect of mi R-22-3p on the function of LCSCs and cell metastasis of HCC,we constructed mi R-22-3p up-regulated and down-regulated stable cell lines by lentivirus infection.(6)To detect the regulating effect of mi R-22-3p on LCSCs through stem cell related experiments,to detect the effect of mi R-22-3p on the migration and invasion of HCC cells through migration and invasion experiments,and to detect the effect of mi R-22-3p on tumorigenicity of nude mice in HCC cells through the experiment of nude mice tumorigenicity assay.(7)Through the co-transfection of TET2 and mi R-22-3p and related behavioral experiments to complete the rescue experiment.(8)The correlation between ?-catenin and mi R-22-3p was detected by transfection of ?-catenin inhibitors and activators.[Results](1)TET2 was down-regulated in clinical HCC tissues and HCC cells,and the low expression of TET2 lead to poor clinical prognosis of HCC patients.(2)TET2 played the role of tumor suppressor gene in HCC: Knockout TET2 lead to malignant differentiation of HCC cells in 3D culture,increased formation of tumorspheres,increased proportion of LCSCs,accelerated tumor growth in nude mice;TET2 knockout HCC cells had stronger migration and invasion ability.Activating the expression of the TET2 gene rescued these malignant transformations.(3)TET2 was a direct downstream target gene of mi R-22-3p.The expression level of TET2 and mi R-22-3p in clinical HCC tissues were negatively correlated.(4)mi R-22-3p was highly expressed in clinical HCC tissues and HCC cells.(5)mi R-22-3p played the role of oncogene which opposite TET2 in HCC: Overexpression mi R-22-3p caused malignant differentiation of HCC cells in 3D culture,increased formation of tumorspheres,increased proportion of LCSCs,and accelerated tumor growth in nude mice.The HCC cells highly expressed mi R-22-3p had stronger migration and invasion ability.However,inhibiting the expression level of mi R-22-3p would rescued these malignant transformations.(6)The inhibitory effect of TET2 gene in HCC required mi R-22-3p regulation.(7)?-catenin was an upstream activator of mi R-22-3p.[Conclusion](1)TET2 gene acted as a tumor suppressor in HCC,inhibited the stemness and metastasis of HCC cells.(2)mi R-22-3p was the upstream regulator of TET2 and negatively regulate TET2.mi R-22-3p played the role of oncogene in HCC.(3)The Wnt-?-catenin/mi R-22-3p/TET2 axis was involved in regulating the stemness and metastasis of HCC cells.