Sox9 Induces Sorafenib Resistance Of Hepatocellular Carcinoma Through AKT Pathway

Research purpose and background Hepatocellular carcinoma(HCC)is a type of liver cancer,which is a common malignant tumor.Its biological behavior is malignant,yet because of the strong compensatory ability of the liver,there is no significant symptom at the early stage of HCC.The disease is often found when the tumor is already in progress.Radical surgical treatment,including liver transplantation,hepatectomy or radiofrequency ablation are only applicable to patients at early stage.Advanced HCC patients are lack of effective treatment,so the mortality rate of HCC is high.In China,most HCC patients are companioned with hepatitis B virus infection.In recent years,the proportion of hepatocellular carcinoma caused by hepatitis C infection has increased.According to reports,for patients with viral hepatitis,the incidence of HCC is 20 times more than the normal population.Loss of surgical opportunities and lack of effective treatment of advanced HCC patients cause dissatisfying prognosis.Sorafenib is a new class of drugs for the treatment of HCC,which inhibits tumor growth directly by inhibiting the serine / threonine kinase activity of RAF-1 and B-RAF.And by regulating the activity of FLT-3,VGFR-2,VEGF-3,PDGF-?,KIT and other receptors of tyrosine kinase,sorafenib inhibits tumor angiogenesis and thus indirectly achieves the purpose of inhibiting the tumor growth.However,regardless of sorafenib or chemotherapy drugs,the chemoresistance has been an important factor hindering their efficacy,many reports reported that chemo-resistance of HCC is related to the existence of tumor stem cells.For example,Ep CAM-positive tumor stem cells can mediate HCC resistance to sorafenib,and Oct4-positive HCC cells can produce resistance to doxorubicin.In recent years,Sox9(sex-determining region SRY box-9)has been recognized as one of the markers of tumor stem cells in HCC.Sox9 is one of the most important markers of tumor stem cells in HCC,including Ep CAM,CD90,CD133,Oct4,Nanog and so on.Sox9 was found to be involved in sex formation,trunk development as well as organ maturation,and has recently been found to be elevated in a variety of tumors.Sox9-positive cells show the characteristics of tumor stem cells,which promote tumor proliferation,enhance cell epithelial-mesenchymal transition,promote cancer cell metastasis,and promote self-renewal of liver cell stem cell.In addition,Sox9 affects the occurrence and differentiation of tumor by Wnt,Notch,TGF-? pathway [2-3],and promotes the self-renewal and proliferation of HCC stem cells through Notch pathway and Numb protein.The study found that cancerous tissues with high expression of Sox9 had worse prognosis [17].Therefore,we hope to explore whether Sox9 is involved in chemo-resistance to sorafenib in HCC and to figure out the specific biological mechanism of Sox9 regulating HCC resistance to sorafenib so as to provide a potential way to address HCC's resistance to sorafenib,and to improve the prognosis of clinical patients.Research methods and results Part One: Explores the expression of SOX9 in hepatocellular carcinoma cells after the application of sorafenib.In this part,we used SMMC-7721 and LM3 hepatocellular carcinoma cell lines for in vitro culture using sorafenib at a concentration of 4 ?M.Digest and fix the cells after incubating for 0 hour,12 hours,24 hours,36 hours,48 hours,60hours and 72 hours.The m RNA expression of Sox9 is examined through q PCR.The protein expression of Sox9 was detected by immunoblotting using Sox9-specific antibody.In addition,immunofluorescence and flow cytometry were performed on cells cultured with 4 ?M sorafenib for 0 hour,24 hours and 48 hours.The trend of expression of Sox9 in two kinds of hepatocellular carcinoma cell lines increased along with the incubation time at both transcription level and protein level.Immunofluorescence assay showed that Sox9 expression in nucleus in 7721 and LM3 cell lines increased as the incubation time increased.Flow cytometry showed that the number of Sox9 positive cells increased significantly as the culture time increased.Part Two: Investigate the effect of Sox9 over-expression and interference on the resistance to sorafinib in HCC We first constructed liver cancer cell lines with Sox9 overexpression and interference,then verify Sox9 expression by quantitative PCR and immunoblot.We culture those cell lines under a concentration of 4?M sorafenib in vitro,then we measure the cell viability using CCK-8 kit at indicated time.In addition,after incubated in vitro for 48 hours,the tumor cells with specific lentivirus infected were digested and passage at a low cell density of 1000/6cm culture dish in order to observe the clone formation ability of tumor cell.Then we examine cell apoptosis through the use of flow cytometry.After the use of sorafenib,the viability of HCC cells overexpressing Sox9 was higher than that of the control group,and the viability of HCC cells with Sox9 expression interference was lower than that of the control group.The clone formation ability of HCC cells with Sox9 over-expression was higher than that of the control group while cells with Sox9 knocked down was lower,the apoptosis level was higher than that of the control group.In the cell lines constructed above,lentivirus containing genes programming luciferase was transfected.And 7721 cells with Sox9 overexpression,Sox9 interference and empty virus infected were used to construct subcutaneous tumor and caudal vein injection model.After the construction of two models,sorafenib was given every 72 hours by 20 mg / kg body weight through intragastric treatment.The fluorescence signal of nude mice was recorded with different observation nodes(0w,1w,3w,5w,7w).The tumor cells were taken out after 7w and tumors' weight were recorded so that we can evaluate the growth of tumor cells of different groups.The fluorescence intensity of the subcutaneous tumor in the nude mice overexpressing Sox9 was higher than that in the control group.After 7 weeks,the tumor weight of the subcutaneous tumor was higher than that of the control group,and the tumor fluorescence value of the nude mice injected by cells with Sox9 knocked down was lower than that of the control group.And the tumor weight was lower than that of the control group.Part Three: Sox9 affects the hepatocellular carcinoma resistance to sorafenib through the Akt pathway The 7721 cell line with Sox9 interference was used to detect the expression of differential gene by using empty virus 7721 cell line as the control gene.And Pathway analysis was used to observe the enrichment of the gene in specific signal pathway.And the expression of the differential gene was verified using immunoblotting.Results: 1.The expression profile showed that the down-regulated genes were enriched in the pathways of TGF-?,m TOR,VEGF,and the Akt pathway,which was closely related to drug resistance.BCRP,programming a usual drug pump,which is in the downstream of Akt pathway,was also down-regulated.According to the literature,HCC can produce resistance to chemotherapeutic drugs through Oct4-Akt-BCRP pathway.Therefore,we used immunoblotting to demonstrate that Akt / BCRP pathway was upregulated in Sox9 overexpressing cell lines and downregulated in Sox9-interfering cell lines.The results of immunofluorescence showed that the expression of Sox9 and BCRP was significantly increased in hepatocarcinoma cell lines treated with 4?M sorafenib for 72 hours.Part Four:Sox9 affects the sorafenib resistance of the HCC through BCRP expression The 7721 cell lines of Sox9 + / BCRP + / Sox9 + BCRP-/ NC were constructed using the corresponding lentivirus,Then Brdu,clone formation and TUNEL experiment were performed after using 4 ?M sorafenib in vitro.We constructed four of those cell lines with Luciferase then build nude mice model of spleen injection of tumor cells.Then we give them sorafenib intragastrically every 72 h.Then we use live imaging instrument to record tumor fluorescence signal.The results showed that: 1.After the use of sorafenib,cell proliferation and clonogenic ability of Sox9 + and BCRP + cells were higher than those in the control group,while the percentage of apoptotic and necrotic cells was lower than that of the control group.In addition,clone formation ability of Sox9 + BCRP-cells were weaker than the control group,and the proportion of cells with apoptosis and necrosis was higher than that of the control group.The fluorescence of Sox9 + and BCRP + cells in nude mice was higher than that in the control group,while the fluorescence of Sox9 + BCRP-cells in nude mice was lower than that in the control group.Conclusion Based on the above results in four parts,we can conclude that: 1.The expression of Sox9 whether on m RNA level or protein level in HCC cell lines is gradually increased after the use of sorafenib,and the percentage of Sox9+ cells increases as the same way.Hence it can be speculated that Sox9 increased chemo-resistence of liver cancer cells,and thus resist the killing effect of sorafenib.2.Overexpression of Sox9 in hepatocellular carcinoma cell lines under the pressure of sorafenib,compared with the control group,expressed higher cell viability,stronger clone formation ability and lower level of cell apoptosis.In the nude mice model of subcutaneous tumor and caudal vein injection,the signal of tumor was stronger in Sox9 overexpression group,whereas the Sox9 interference group was the opposite.These results can confirm from the other side that Sox9 elevation can enhance the resistance of hepatocellular carcinoma to sorafenib.3.Microarray gene expression profile of Sox9 interfered 7721 cells suggested that Sox9 can affect the expression of Akt/BCRP pathway,which is closely related to sorafenib resistance.So Sox9 may affect the resistance to sorafenib of hepatocellular carcinoma through the regulation of Akt/BCRP pathway.And the following rescue experiment manifested that HCC cells with BCRP knocked down can reverse the effect of Sox9 overexpression on sorafenib resistance of HCC,thus verified previous hypothesis.