ADAMTS8 Targets ERK To Suppresses Cell Proliferation,Invasion And Metastasis Of Hepatocellular Carcinoma

Part One Study on the correlation between the expression of ADAMTS8 in tumor tissue and the clinical features of patients with HCCObjective: Hepatocellular carcinoma(HCC)is one of the most common malignant tumours of the digestive system.A disintegrin and metallopeptidase with thrombospondin motifs(ADAMTS)has been identified as a secreted metalloproteinase that participates in the inhibition of tumour cell growth and invasion.The aims of the present study in part one were to investigate the clinical significance of ADAMTS8 in patients with HCC.Methods:1.Tumor tissues of 61 patients with liver cancer and 29 cases of adjacent non-tumor tissues were collected.The expression of ADAMTS8 was tested by immunohistochemistry.The correlation of expression level of ADAMTS8 and sex,age,clinical stage,metastasis,liver cirrhosis,tumor number,size of tumor tissue and differentiation degree were analyzed,thereby analyzing the clinical significance of ADAMTS8 in HCC.2.The expression level of ADAMTS8 was tested by Western blotting technique in tumor tissues of patients with liver cancer and 29 cases of adjacent non-tumor tissues.Results:1.The expression of ADAMTS8 protein tested by immunohistochemical staining.The results of immunohistochemistry showed that the expression of ADAMTS8 was localized in cell membrane and cytoplasm.The expression rate of ADAMTS8 was 69.7%(20 / 29)in paracancerous tissues.However,the expression rate in cancer tissues was 29.5%(19 / 61),which was significantly lower than that of paracancerous tissues(P < 0.01).2.Further analysis showed that the expression level of ADAMTS8 in tumor tissue was significantly correlated with the clinical stage and tumor metastasis(P<0.05),but there was no correlation with sex,age,liver cirrhosis,tumor number,tumor size and differentiation degree(P>0.05).For patients in phase one,phase two and non-metastatic patients,the expression level of ADAMTS8 in tumor tissues was significantly higher than that of patients in phase three,four and metastasis(P>0.05).Therefore,low expression of ADAMTS8 in tumor tissue is often one of the predictors of poor clinical characteristics.3.The expression level of ADAMTS8 tested by Western blotting:The expression level of ADAMTS8 protein in 61 patients with HCC was significantly lower than that in the adjacent normal tissues tested by Western blotting(P<0.05).The expression level of protein is consistent with Result 1.Part Two Inhibition effect and the underlying mechanism of ADAMTS8 on the biological activity of HCC cellsObjective: The aim of the present study was to characterize the inhibition effect and the underlying mechanism of ADAMTS8 on proliferation,migration and invasion,and chemosensitivity of the HCC cells.Methods:1.Western blotting assays were used to detect the expression of ADAMTS8 in the HCC cells lines(SMMC-7721,HepG2,Lm-3)and the normal liver cell line(LO-2).2.ADAMTS8 overexpression plasmids targeting human ADAMTS8 gene were designed and transcribed in vitro and carried purine mycin resistance screening markers.ADAMTS8 overex-pression plasmid was transfected into HepG2 cells by liposome(Lipofectamine TM2000).The overexpression of ADAMTS8 in HepG2 cells was detected by Western blot.3.MTS assays were employed to detecte the effect of ADAMTS8 transfection on proliferation and chemosensitivity of the HepG2 cells.4.Transwell and wound scratch assays were used to evaluate the effect of ADAMTS8 transfection on migratory and invasion ability of tumor cells.5.Western blotting assays were used to detect the effect of ADAMTS8 on the expression of ERK、p-ERK、Stat3、p-Stat3,Akt,p-Akt,P53,P21 proteins in HCC cells.Results:1.Western blotting analysis found that the expression of ADAMTS8 at protein level was lost in the HepG2 cells,while the expression of ADAMTS8 were found in the SMMC-7721,Lm-3 and LO-2 cells.In the following studies,we make HepG2 as study subject.2.After transfection of ADAMTS8 overexpression plasmid into HepG2 cells,the transfection efficiency was 40%-50%,and the transfection efficiency was more than 90% after screening with 0.6ug/ml purine mycin.3.MTS assay showed that cell viability was markedly decrease(P<0.05)in HepG2 cells transfected with ADAMTS8 than that in their corresponding blank control or vector cells,and the chemosensitivity of the HCC cells to cisplatin was significantly enhanced(P<0.05).4.The results of FCM showed that the apoptosis level of HepG2 was significantly increased after transfection of ADAMTS8(6.03 ±1.92%vs.1.91 ±0.98;6.03 ±1.92%vs.1.83 ±0.95%),and the difference was statistically significant(P < 0.01).5.The results of Transwell chamber and cell scratch test showed that the invasion and migration ability of hepatoma cell line HepG2 were significantly decreased after transfection of ADAMTS8(P < 0.01).6.The results of Western blotting assay showed that after transfection of ADAMTS8,the expression of p-ERK in HepG2 of hepatoma cell lines decreased significantly,while the expression of p53 and P21 increased significantly(P<0.05).Part Three Inhibitory effect of ADAMTS8 on the growth of hepatoma xenografts in nude miceObjective: To investigate the effect of ADAMTS8 on viability of HCC in vivo,the HepG2 nude mice model was used to evaluate the effect of ADAMTS8 on HCC cells viability in vivo.Methods:1.The Balb-c/null model of nude mice bearing tumor was established by subcutaneous injection of hepatoma cell line HepG2.The model was divided into three groups.The control group was treated by injection of untransfected HepG2 cells(100 μl)around the scapula of nude mice.In the empty vector group,the empty vector HepG2 cells(100 μl)were injected into the parascapular of nude mice and the HepG2 cells stably transfected with ADAMTS8 were injected into the parascapular of nude mice(100 μl).Each nude mouse was injected with 1 × 106 cells.On the 28 th day,the nude mice were killed by neck drawing.2.Nude mice were anesthetized by intraperitoneal injection of 2% pentobarbital sodium(0.04ml).After the mice were breathed evenly and were not active,the transplanted tumor was placed on the bottom of the dark box.After setting the size and height of the shooting object,the shooting precheck is carried out,and the position of nude mouse is adjusted according to the image,which adapts to the lens coverage,and adjusts the setting of the size and height to make the focus accurate and clear.Then the exposure time is pre-checked by 475 nm wavelength excitation,and the reasonable exposure time is 2 seconds.Finally,the in-vivo imaging is taken.Evaluating xenograft activity by capturing the fluorescence luminescent energy value of xenografts(counts per second CPS).3.The measurement of tumor volume: the size of tumor tissue was observed once a week.The length and diameter of tumor were measured by caliper.The volume of tumor was calculated according to the formula and the growth curve was drawn.4.NightOwl in vivo imaging system is used to monitor tumor growth.Results:1.The Balb-c/null model of nude mice bearing tumor was established by subcutaneous injection of hepatoma cell line HepG2.The measurement of tumor volume: the size of tumor tissue was observed once a week.The growth of ADAMTS8 transfected tumors in mice was significantly slower than those in control and vector transfected groups(P<0.05).2.NightOwl in vivo imaging system is used to monitor tumor growth.The results of in-vivo imaging system showed that activity of HepG2 cells stably transfected with ADAMTS8 was significantly lower than that of control group and empty vector group in vivo.3.Meanwhile,according to Western blotting,the expression of p-ERK in the transplanted tumor tissues transfected with ADAMTS8 decreased significantly.However,the expression level of P53 increased significantly(P<0.05).Conclusion:1.The expression level of ADAMTS8 in tumor tissue was closely related to the clinical stage and metastasis of HCC.The patients with low expression level of ADAMTS8 were often late in clinical stage and more prone to tumor metastasis.Therefore,the low expression level of ADAMTS8 in tumor tissue can be used as a predictor of the progression of hepatocellular carcinoma.2.In the present study,we found that decreased p-ERK and increased P53 and P21 in ADAMTS8-transfected HCC cells,indicated that ERK activation blockade and upregulation expression of P53 and P21 might be the key factor affected by ADAMTS8 regulating the inhibition on proliferation,migration and invasion of HCC cells.3.In vivo,stable transfection of ADAMTS8 can significantly inhibit the growth and invasive activity of ESCC xenografts by regulating the ERK pathway and p53 expression,which further indicates that ADAMTS8 has the potential to be used as a new target for the comprehensive treatment of HCC.