| Ⅰ.ObjectiveHepatocellular carcinoma(HCC)accounts for 80%of primary hepatocellular carcinoma,which is the main cause of cancer-related death.China has a large number of pateients of liver cancer,where new cases and deaths account for more than 50%of the world.Less than 20%of HCC patients can receive radical resection clinically.Recurrence and metastasis are important factors affecting the prognosis of HCC.At present,there is still a lack of specific therapeutic drugs for the patients after liver carcinomectomy.HCC is devoid of sensitivity to radiotherapy and/or chemotherapy,and the therapeutic effect is not ideal.HCC patients with liver cirrhosis have poor tolerance to drugs.Sorafenib as a representative targeted drug is expected to improve the prognosis of HCC patients,but only suitable for non-surgical patients.Furthermore,the long-term efficacy of Sorafenib needs to be further verified by a large number of clinical research data.Therefore,there is an urgent need to find effective novel treatment targets for liver cancer.Alpha fetoprotein(AFP)is currently the most widely used diagnostic marker for liver cancer,but the positive rate in patients with liver cancer is still low,and there is still a lack of new and effective early diagnosis and prognostic markers.Tumor testis antigen is an antigen specifically expressed in testis and tumor,which may promote tumorigenesis and development by regulating cell signal transduction.Abnormal expression of antigen protein in testis can induce cellular and/or humoral immune response and is considered as the target of immunotherapyZinc finger protein 165(ZNF165)is a tumor-testicular antigen,which belongs to the SCAN family and is specifically expressed in testicular and tumor tissues.It has been reported that ZNF165 may be an oncogene to affect the proliferation and migration of breast cancer cells by regulating TGF signaling pathway.In addition,some researchers have detected abnormal expression of ZNF165 in liver cancer tissues,but the effect of ZNF165 on liver cancer and the specific mechanism of action have not been reported.This study aims to reveal the correlation between ZNF165 and the clinical prognosis of HCC,and to explore the role of ZNF165 in HCC tumorigenesis and development.Our study will provide important theoretical basis and novel strategies for the early diagnosis and targeted treatment of HCC.Ⅱ.Methods1.There were 20 tumor tissue samples and 20 paracancerous tissue samples for RNA and protein detection.Gene chip of liver cancer tissue was provided by Shanghai xinchao biotechnology co.,LTD.,with complete clinical data,including 93 cases of liver cancer tissue and 87 cases of adjacent normal tissue.A total of 180 points were detected and analyzed.The expression of ZNF165 in all pathological specimens was determined by immunohistochemistry,and all cases were followed up.The correlation was analyzed between the expression of ZNF165 and the clinicopathological features and prognosis of HCC patients.The SPSS 19.0 statistical software was used for data analysis,with p<0.05 was considered significant.The correlation between the two indicators and the major clinical characteristics of HCC was analyzed by chi-square test.Kaplan-meier curve was used for univariate analysis of prognosis,and log-rank test was used for comparison between groups.Multivariate analysis of prognosis was performed using COX regression risk model.2.The recombinant lentiviruses expressing ZNF165 were transfected into HCC cell lines(Be17402/HCCLM3)by liposome transfection reagent.Stable transfectants were selected with 1～3μg/ml puromycin,and individual clones were isolated.3.The mRNA levels of ZNF165 and tryptophan pathway related genes(IDO1,IDO2,TDO2,AhR.CYP1A1,CYP1B1)were detected by RT-PCR(two step:degeneration within 95℃.5 minutes;degeneration within 95℃.30 seconds:annealing within 60℃.60 seconds;40 cycles).4.Western blot assay was used to detect the expression levels of ZNF165 and tryptophan-related proteins(IDO1.IDO2,TDO2,AhR,CYP1A1,CYP1B1).5.A double luciferase assay kit was used to detect the transcriptional activity of AhR.6.The intracellular localization of ZNF165,p-akt and AhR was detected by indirect immunofluorescence assay.7.The consentrations of tryptophan and kynurenine in cell supernatant,tissue homogenate and urine were detected by Elisa kit.8.The effect of ZNF165 on the activation of kinases was detected using a phospho-kinase Array kit.9.The effect of ZNF165/CYP1A1 on the growth of HCC cells was detected by MTT/Brdu and clone formation assays,and the migration/invasion ability of HCC cells was analyzed by anoikis/scratch/transwell assays.Cell cycle and apoptosis were detected by flow cytometry.10.The subcutaneously implanted tumor model was established.Nude mice aged 6 weeks without thymus were reared in SPF independent cages.5×106 cells were inoculated under the dorsal lateral skin of nude mice with a volume of 150μl.The experiments were conducted.11.Establishment of a diethyl nitrosamine(DEN)-induced primary liver cancer model.Male SD rats with 200g around were selected,and the DEN group was given an intraperitoneal injection of DEN(150 mg/kg)for the first time,and 0.01%DEN was added to drinking water for the following 8 weeks.After intraperitoneal injection of DEN(150 mg/kg)for the first time in the inhibitor group,LY294002(10mg/kg,)was added into the water containing 0.01%DEN for 12 weeks.DEN was replaced by sterilized PBS in the blank control group and relevant experiments were conducted 20 weeks later.Ⅲ.Results(Ⅰ)The correlation between the expression of ZNF165 and prognosis in HCC1.The expression of ZNF165 in liver cancer tissues was significantly higher than that in adjacent normal tissues.The expression of ZNF165 was detected by IHC staining and Western blot in 20 pairs of fresh hepatocellular carcinoma and adjacent normal tissues.ZNF165 was expressed to varying degrees in hepatocellular carcinoma(13/20)and adjacent normal tissues(2/20).ZNF165 is highly expressed in patients with age 50,tumor 5cm,and clinical stage Ⅲ+Ⅳ,with no significant difference between male and female.The results of gene microarray showed that ZNF165 was mainly located in the nucleus,highly expressed in the cancer tissues,and low expressed in the adjacent normal tissues.In the paired 84 cancer tissues,the expression rate was 27.4%(23/84).The expression rate of ZNF165 was 3.6%(3/84)in para-cancer normal tissues.TCGA database was used to analyze and compare 369 cases of liver cancer tissues and 50 cases of adjacent normal tissues.The results showed that the expression of ZNF 165 in liver cancer tissues was significantly higher than that in adjacent normal tissues.2.The expression of ZNF165 in HCC tissues was significantly higher than that in adjacent normal tissues(P<0.01).The expression rate of ZNF 165 in T1/T2 stage patients was 23.4%(10/43),while that in T3/T4 stage patients was 43.9%(18/41),P<0.045.3.The expression level of ZNF 165 was significantly correlated with T stage in HCC patients.In this study,84 patients with hepatocellular carcinoma with complete follow-up data were analyzed for survival.Kaplan-meier analysis showed that compared with the low expression group,the overall survival rate of ZNF 165 in liver cancer tissues with high expression was significantly reduced,and the survival rates were 20.0%and 43.9%,respectively,P<0.05.The mean survival time of the ZNF165 high-expression group was 28.44±5.426 months,and the median survival time was 17 months.The mean survival time of the low expression group of ZNF 165 was 45.212±3.982 months,and the median survival time was 35 months.4.Related factors affecting prognosis of patients with hepatocellular carcinoma.Single factor survival analysis showed that ZNF 165 was highly expressed(HR:2.519;95%CI:1.486-4.268;P=0.001),tissue differentiation(HR:1.975;95%CI:1.135-3.437;P=0.016),T staging(HR:2.053;95%CI:1.369-3.079;P=0.001),M staging(HR:41.166;95%ci:3.733-453.988;P=0.002)and TNM staging(HR:2.148;95%CI:1.392-3.313;P=0.001)was significantly correlated with the prognosis of patients with hepatocellular carcinoma(see table 3).Multivariate survival analysis showed that ZNF165 was highly expressed(HR:2.000;95%CI:1.113-3.592;P=0.001),M staging(HR:21.085;95%CI:1.043-426.125;P=0.047)is an independent prognostic factor for patients with HCC.(Ⅱ)ZNF165 regulates IDO-Kyn-AhR pathway targeting CYP1A1 and promotes the growth of HCC cells via Akt1.Knockdown/overexpression of ZNF165 in HCC cells(Bel7402/HCCLM3)can inhibit/promote proliferation,migration and invasion,and resistance of Sorafenib,respectively.2.RNAseq array results showed that Tryptophan pathway and expression of CYP1A1 were significantly down-regulated in ZNF165 knockdown cells.qPCR and Western blot assays confirmed that knockdown/overexpression of ZNF165 reduced/increased the expression of IDO,AhR,CYP1A1 and the transcriptional activity of AhR in HCC cells,respectively.Elisa assay showed that knockdown/overexpression of ZNF165 reduced/increased the content of Kynurenine/Tryptophan in the supernatant of cells,respectively.3.ZNF165 affects the expression of CYP1A1 through AhR.Overexpression of ZNF165 increased the transcriptional activity of AhR,while knockdown of ZNF165 decreased the transcriptional activity of AhR.Confocal results showed that overexpression of ZNF165 could increase AhR enucleation,while knockdown of ZNF165 could decrease AhR enucleation.After ZNF165 overexpression,AhR expression increased in the nucleus and decreased in the cytoplasm.After ZNF165 knockdown,AhR decreased in the nucleus and increased in the cytoplasm.In cells treated with knockdown AhR or AhR inhibitor,overexpression of ZNF165 could not increase the expression of CYP1A1.4.ZNF165 regulates the kyn-ahr-cyplal pathway through IDO1.Kynurenine can increase the transcriptional activity of AhR and increase the expression of CYP1A1.Overexpression of ZNF165 did not increase the content of Kynurenine in the supernatant of cells treated with IDO1 inhibitors or knockdown IDO1.The application of inhibitors of IDO1 and AhR or knockdown of AhR and IDO1 can block ZNF165 and increase the expression of CYP1A1.5.ZNF165 promotes the idol-kyn-ahr pathway through Akt.Overexpression of ZNF165 increased p-akt protein levels and promoted p-akt localization on cell membranes.The co-localization of ZNF165 and Akt on the nuclear surface was observed by confocal microscopy.Exogenous and endogenous interactions between ZNF165 and Akt were detected by co-ip experiment.ZNF165 promotes the secretion of FGF9 and indirectly increases the expression of p-akt.IDO1 and AhR were treated with overexpression of activated Akt and Akt activator.The effect of ZNF165 on CYP1A1 expression was blocked by the inhibitors of Akt,IDO1 and AhR.DEN induced HCC model was established in SD rats.The expressions of ZNF165,p-akt,IDO and CYP1A1 in tumor tissues were detected by IHC and Western blot and the expression levels of these molecules were significantly increased and positively correlated.6.PI3K inhibitor LY294002 inhibited the expression of ZNF165 After treatment with PI3K inhibitor,inhibitor LY294002 can significantly reduce the expression of ZNF165 in a dose-dependent and time-dependent manner.Activated Akt and Akt activator SC79 reverted to ly294002-induced reduction in ZNF165 expression.IP results showed that LY294002 could degrade ZNF165 by ubiquitination,mainly acting on K48Ub.7.LY294002 inhibited DEN to induce hepatocellular carcinoma.DEN induced liver cancer model in SD rats was established,and the expressions of ZNF165,p-akt,IDO and CYP1A1 in liver cancer tissues were detected to be significantly increased by IHC and Western blot.The contents of Kynurenine in liver homogenates and urine of den-induced SD rats were significantly increased compared with the control group.DEN can induce the expression of ZNF165 in normal hepatocytes.which can be blocked by LY294002.Ⅳ.Conclution1.Overexpression of ZNF165 predicts poor prognosis in HCC.2.ZNF165 promotes the proliferation,migration,and invasion of HCC cells through targeted regulate CYP1A1.3.ZNF165 up-regulates IDO-Kyn-AhR pathway via activates Akt.4.DEN induces HCC tumorigenesis and promotes the expression of ZNF165.5.LY294002 degrades ZNF165 through ubiquitination and inhibits DEN-induced HCC tumorigenesis. |
