| Primary livercancer(PLC)is a common malignant tumor of the digestive system in China.Hepatocellular carcinoma(HCC)is the most common pathological type of primary liver cancer,accounting for 80%of the total cases of primary liver cancer.Hepatocellular carcinoma is highly invasive and easy to metastasis.At present,surgical treatment is the preferred treatment in clinic.However,because the early symptoms of liver cancer are not obvious,the majority of patients have developed to the middle and late stage when seeking treatment and lose the opportunity of surgery.Although the comprehensive treatment represented by chemotherapy and radiotherapy can relieve symptoms to a certain extent,it has little benefit in improving 5-year survival rate and quality of life of patients.Therefore,it is still the focus of research in this field at the present stage to deeply study the malignant biological behavior and internal mechanism of liver cancer,such as proliferation and invasion,search for new therapeutic targets and improve the therapeutic effect of liver cancer.The occurrence and development of liver cancer is the sum of sequential events involving a series of related gene transcription,protein expression,oncogene activation.and tumor suppressor gene inactivation.Its internal mechanism of action has not been fully studied.DHX9 is a NTP-based helicase containing the conserved core domain of helicase,whose functions include the regulation of DNA replication,transcription and translation,microRNA synthesis,RNA processing and transport,and the maintenance of genome stability.In recent years,a number of studies have found that DHX9 is highly expressed in cancer tissues and plays a role as an oncogene in cancer cells.For example,DHX9 combines with SOX4 in prostate cancer cells to promote the invasion of prostate cancer cells.DHX9 interacts with EGFR in breast cancer cells to promote metastasis of breast cancer cells.DHX9 has also been found to act as a tumor suppressor gene in neuroblastoma.At present,the expression of DHX9 in HCC,its effect on the malignant biological behavior of HCC cells and its mechanism,as well as its relationship with the prognosis of HCC patients have not been systematically studied and reported.In order to clarify the above problems,this study conducted research from the following four aspects in order to clarify the regulatory mechanism of DHX9 on malignant biological behavior of HCC cells,and provide a potential target for clinical treatment of HCC.Part 1:Proteomic analysis of DHX9 in HCC tissue and its correlation with prognosis of patients.Part 2:The expression of DHX9 in HCC and its correlation with clinical factors.Part 3:The study of the regulation effect of DHX9 on the proliferation,migration and invasion of hepatoma cells.Part 4:The study on the mechanism of DHX9 on malignant biological behavior of hepatoma cells.Part 1 Proteomic analysis of DHX9 in HCC tissue and its correlation with prognosis of patientsObjectiveProteomics technology was used to analyze and detect the expression level of DHX9 in HCC tissues and adjacent non-tumor liver tissues,and the relationship between DHX9 and prognosis of HCC patients was analyzed using GEPIA online database.Methods1.Tissue specimens of 40 cases of HCC and corresponding adjacent non-tumor liver tissues were collected.2.Proteomics experiments such as mass spectrometry,protein clustering and protein interaction analysis were performed after extraction,purification and quantification of tissue proteins.3.GEPIA database was used to analyze online the expression of DHX9 in HCC tissues and non-tumor liver diseases tissues,and the correlation between DHX9 expression and prognosis of HCC patients.Results1.A total of 288 differentially expressed proteins were found in 40 HCC tissues and their corresponding adjacent non-tumor liver tissues,and the expression levels of these proteins were significantly different between HCC tissues and adjacent non-tumor liver tissues.2.DHX9 was differentially expressed in 40 HCC tissues and its corresponding adjacent non-tumor liver tissues,and the expression of DHX9 in HCC tissues was significantly higher than that in adjacent non-tumor liver tissues(P<0.001).3.PPI showed that DHX9 may interact with BCL-2,BAX,MMP2,MMP9,MAPK1,STAT3 through CTNNB1 in HCC cells.4.TCGA and GTEx database analysis showed that the transcription level of DHX9 gene(mRNA)in HCC tissues was significantly higher than that in non-tumor liver tissues.5.Patients with high DHX9 expression had shorter Overall Survival(OS)than those with low DHX9 expression(P=0.0087).Meanwhile,patients with high DHX9 expression had a shorter Disease Free Survival(DFS)than those with low DHX9 expression(P=0.034).Conclusions1.The proteomics experiments showed that the expression level of DHX9 in HCC tissues was higher than that in adjacent non-tumor liver tissues.2.TCGA and GTEx database analysis showed that the transcription level of DHX9 in HCC tissues was higher than that in adjacent non-tumor liver tissues.3.TCGA and GTEx database analysis showed that the expression level of DHX9 was negatively correlated with the Overall Survival and Disease Free Survival of HCC patients.Part 2 The expression of DHX9 in HCC and its correlation with clinical factorsObjectiveThe expression levels of DHX9 gene and protein in HCC tissues,adjacent non-tumor liver tissues,liver cancer cell lines and liver cell lines were detected.The correlation between DHX9 expression levels and related clinical factors was statistically analyzed to explore the clinical significance of DHX9.Methods1.The tissues of 69 cases of HCC tissues and corresponding adjacent non-tumor liver tissues were collected.2.mRNA and protein expression levels of DHX9 in HCC tissues and adjacent non-tumor tissues were detected by qRT-PCR and Western-blot,respectively.3.Immunohistochemistry(ICH)was used to detect the protein expression level of DHX9 in HCC tissues and corresponding adjacent non-tumor liver tissues.4.The correlation analysis,univariate analysis and multivariate analysis of DHX9 expression level and clinical factors such as age,gender,tumor size,TNM stage,tumor number,vascular invasion,lymph node metastasis and metastasis were performed.5.Six liver cancer cell lines,including Huh-7,Hep3B,SMMC-7721,MHCC97H,HepG2,BEL7402,and normal liver cell lines L02 were cultured.6.The mRNA and protein expression levels of DHX9 in the above 6 liver cancer cell lines and normal liver cell lines L02 were detected by qRT-PCR and Western-blot,respectively.Results1.qRT-PCR and Western-blot tests showed that the expression levels of DHX9 mRNA and protein in HCC tissues were significantly higher than those in adjacent non-tumor liver tissues,with statistical significance(P<0.05).2.Immnohistochemical staining and positive comprehensive score were performed on 69 cases of HCC tissues and corresponding adjacent non-tumor liver tissues.The results showed that DHX9 was expressed in both HCC tissues and adjacent non-tumor liver tissues,and mainly expressed in the cytoplasm,but hardly expressed in the nucleus.The expression of DHX9 in HCC tissues was significantly higher than that in adjacent non-tumor liver tissues(P<0.05).This experimental result further confirmed the obvious differential expression of DHX9 in HCC tissues and adjacent non-tumor liver tissues detected by western-blot above(P<0.05).3.Statistical analysis of clinical factors of 69 patients with HCC showed that the expression level of DHX9 was not correlated with gender,age,tumor number,tumor size,HBV infection,AFP,degree of differentiation,liver cirrhosis,lymph node metastasis and recurrence(P>0.05).However,it was related to TNM stage,vascular invasion and metastasis these three clinical factors(P<0.05).The expression level of DHX9 in liver cancer TNM stage(III+IV)was significantly higher than that in TNM stage(Ⅰ+Ⅱ)(P=0.007).The expression level of DHX9 in HCC tissues with vascular invasion was significantly higher than that in HCC tissues without vascular invasion(P=0.021).The expression level of DHX9 in HCC tissues with metastasis was significantly higher than that in’ HCC tissues without metastasis(P=0.042).4.Kaplan-Meier analysis was performed and survival curves were drawn according to the immunohistochemical positive comprehensive score results of 69 cases of HCC tissues and corresponding adjacent non-tumor liver tissues.Results showed that the overall survival of patients with high DHX9 expression(n=39)was shorter than that of patients with low DHX9 expression(n=30)(P=0.030);HCC patients with high DHX9 expression had shorter progression free survival than those with low DHX9 expression(P=0.048).This result further verifies the analysis results of biological information databases such as TCGA and GTEx in the first part.5.Log-rank test was used to conduct univariate analysis of the overall survival and progression free survival of the included clinical factors.The results showed that tumor size(P=0.011),degree of differentiation(P=0.047),lymph node metastasis(P=0.004),vascular invasion(P=0.001),metastasis(P=0.003)and DHX9 expression level(P=0.032)were correlated with overall survival.Tumor size(P=0.024),vascular invasion(P=0.009),and metastasis(P=0.001)were associated with progression free survival.Multivariate Cox analysis showed that tumor size(P=0.043),vascular invasion(P=0.038)and DHX9 expression level(P=0.045)were independent prognostic factors affecting overall survival.Tumor size(P=0.001)and vascular invasion(P=0.027)were independent prognostic factors for progression free survival.6.qRT-PCR and Western-blot analysis showed that the expression levels of DHX9 mRNA and protein in liver cancer cell lines(Huh-7,Hep3B,SMMC-7721,MHCC97H,HepG2,BEL7402)were significantly higher than those in normal liver cell lines L02(P<0.05).Conclusions1.By molecular biology experiments the expression level of DHX9 in HCC tissues was significantly higher than that in adjacent non-tumor liver tissues.The expression level of DHX9 in HCC cells significantly higher than that in normal liver cells2.The expression level of DHX9 is related to TNM stage,vascular invasion and metastasis in patients with HCC.The expression level of DHX9 was negatively correlated with OS and PFS in patients with HCC.3.Univariate and multivariate analysis showed that tumor size,vascular invasion and DHX9 expression are independent factors affecting the prognosis of HCC patients.Part 3 The study of the regulation effect of DHX9 on the proliferation,migration and invasion of hepatoma cellsObjectiveThe effects of DHX9 on the proliferation,migration,invasion of hepatoma cells were detected by a series of cell experiments in vitro,to explore the regulatory role of DHX9 on malignant biological behavior.Methods1.MHCC97H liver cancer cell line was selected as the research object in vitro cell experiment of this part,because of the highest DHX9 mRNA and protein expression level in the previous part.2.The expression of DHX9 in HCC cells was knocked down by siRNA transient transfection technique,and 72h after the transfection,the transfection effect was detected by Western-blot.When the DHX9 expression in liver cancer cells was successfully knocked down,cells were divided into blank control group(CON,liver cancer cell),nonspecific control group(NC,plus GP-transfect-Mate only but no siRNA)and experimental group(siRNA,DHX9 knoched down)for the following experiments.3.CCK-8 cell proliferation test was used to detect the effect of DHX9 on the proliferation of liver cancer cells,and the cell growth curve was drawn.4.Wound Healing test was used to test the effect of DHX9 on the migration ability of liver cancer cells.5.Transwell cell invasion and migration assay was used to detect the effect of DHX9 on the invasion and migration ability of liver cancer cells.Results1.Compared with blank control group and non-specific control group,Western-blot results showed that the expression of DHX9 was significantly reduced in the experimental group(liver cancer cells with DHX9 expression knocked down by transfected compound reagent at different concentrations),and the knockdown effect of DHX9 expression was more obvious in the transfected compound reagent concentration of 125nM than that in the transfected compound reagent concentration of 75nM and 100nM,and the expression of DHX9 was significantly down-regulated.125nM was used as the final transfection concentration of the transfection compound reagent for liver cancer cells in the experimental group.2.Knocking down the expression of DHX9 can inhibit the proliferation of liver cancer cells.According to the OD value measured in the CCK-8 cell proliferation experiment,the cell growth curve was drawn.Compared with the blank control group and the non-specific control group,the proliferation rate of liver cancer cells in the experimental group was significantly slower.Proliferation inhibition was most obvious between 36h and 48h(P<0.01),and slowed down between 48h and 96h(P<0.01).3.Knocking down the expression of DHX9 can inhibit the migration of liver cancer cells.According to the blank area measured in the Wound Healing test,compared with the blank control group and non-specific control group,the migration rate of liver cancer cells in the experimental group was significantly slower,and the inhibition intensity of migration ability was more significant at 48h(P<0.01)than at 24h(P<0.05).4.Knocking down the expression of DHX9 can inhibit the invasion and migration ability of liver cancer cells.According to the results of Transwell cell invasion and migration assay,compared with the blank control group and the non-specific control group,the number of liver cancer cells passing through the polycarbonate membrane and matrix gel in the experimental group was significantly reduced(P<0.05).ConclusionsDHX9 can promote the proliferation,migration and invasion of liver cancer cells.Part 4 The study on the mechanism of DHX9 on malignant biological behavior of hepatoma cellsObjectiveThe effects of DHX9 on the apoptosis,EMT and related signaling pathways of liver cancer cells were detected by molecular biological methods to explore the mechanism of DHX9 promoting the proliferation,migration and invasion of liver cancer cells.Methods1.MHCC97H liver cancer cell line was selected as the research object in vitro cell experiment of this part,because of the highest DHX9 mRNA and protein expression level in the previous part.Cells were divided into blank control group(CON,liver cancer cell),nonspecific control group(NC,plus GP-transfect-Mate only but no siRNA)and experimental group(siRNA,DHX9 knoched down)for the following experiments.2.Flow cytometry and AnnexinV Apoptosis Kit were used to detect the effect of DHX9 knockdown on apoptosis of liver cancer cells.3.The effect of DHX9 knockdown on the expression of apoptosis-related proteins in liver cancer cells was detected by Western-blot technique.4.The effect of DHX9 knockdown on the expression of EMT-related proteins in liver cancer cells was detected by Western-blot technique.5.The effects of DHX9 knockdown on the expression and phosphorylation levels of key proteins in JAK/STAT3,PI3K/Akt,MAPK/ERK and NF-κB signaling pathways in liver cancer cells were detected by Western-blot technique.Results1.Knocking down the expression of DHX9 can promote the apoptosis of liver cancer cells.Flow cytometry showed that compared with blank control group and non-specific control group,the apoptosis rate of experimental group was significantly increased,and the difference was statistically significant(P<0.01).Western-blot results showed that compared with blank control group and non-specific control group,the expressions of pro-apoptotic protein Caspase3 and Bax in experimental group were significantly increased(P<0.05),and the expression of anti-apoptotic protein Bcl-2 was significantly decreased(P<0.05).2.DHX9 knockdown can inhibit the occurrence of EMT in liver cancer cells.Western-blot results showed that compared with blank control group and non-specific control group,the expression of epithelial phenotype specific protein E-cadherin in the experimental group was increased,and the expression of stromal phenotype specific protein MMP2,MMP9,and Vimentin was decreased(P<0.05).3.Knocking down DHX9 expression can increase p-ERK expression.Western-blot analysis showed that the expression levels of key proteins in JAK/STAT3,PI3K/Akt and NF-κB 3 signaling pathways and their phosphorylated form proteins were not statistically significant among all groups(P>0.05).Compared with blank control group and non-specific control group,p-ERK expression level in experimental group was significantly increased(P<0.05).Conclusions1.Molecular biology experiment showed that DHX9 can promote the proliferation of liver cancer cells by inhibiting apoptosis.2.Molecular biology experiment showed that DHX9 can promote EMT of liver cancer cells and promote their invasion and migration ability.3.DHX9 can regulate the proliferation,apoptosis,migration,invasion and other malignant biological behaviors of liver cancer cells through MAPK/ERK signaling pathway. |
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