| BACKGROUND&OBJECTIVEHepatocellular carcinoma(HCC),is the fifth commom and the third deadly cancer in the world,frequent in East Asia and Sub-Saharan Africa.The development of the Hepatocellular carcinoma is a multi-factor,multi-step,multi-stage process.Viral hepatitis,obesity and type ? diabetes were known as the key factors for Hepatocellular carcinoma.Due to the lack of early diagnosis and effective treatment,the prognosis of patients with hepatocellular carcinoma is poor,and five-year survival rate is very low.However,the middle of the process from pathogenic factors to the comeout of hepatocellular carcinoma,was less known.Therefore,need more in-depth research for understanding the molecular pathogenesis of hepatocellular carcinoma occurrence and development,to find an early diagnostic marker for hepatocellular carcinoma,an effective target for treatment of hepatocellular carcinoma.Erbin is an important member of the LAP(1eucine-rich repeat and PDZ domain)proteins family,belongs to polar protein molecule.For its molecular structure,Erbin contains 1412 amino acids,includes a LRR Structure in the N-term and a C-terminal PDZ domain.In the present study,Erbin get more and more attentionfor its important of the regulation of cell proliferation and differentiation,organs development,inflammation,tumorigenesis and metastasis.In this paper,We are interested to invest the regulation effect of Erbin in the occurence and development of hepatocellular carcinoma and its medchanisms.Our research through multiple levels,included clinical samples analysis,biological characteristics of liver cancer cell lines and hepatocellular carcinoma model of the Erbin transgenic mice.Methods1.Detection expression levels and correlation analysis of Erbin in clinical specimens of hepatitis,cirrhosis and hepatocellular carcinoma.1.1 Analyze the expression of Erbin in hepatitis and cirrhosis by tissue microarray.1.2 Analyze the expression of Erbin in 214 cases of HCC specimens through immunohistochemical staining,and the relationship of Erbin expression level and the relevant clinical parameters.1.3 Analyze the relationship between Erbin expression levels and the survival time of hepatocellular carcinoma patients.1.4 Statistical analysis:By using the SPSS 16.0 software.Multiple comparisons(LSD)Erbin expression of normal liver tissue,hepatitis,cirrhosis was analyzed in microchip of LV20812 by Multiple comparisons(LSD and Dunnett T3).Paired-Samples T test analyzed expression of Erbin in hepatocellular carcinoma and adjacent tissue,Multiple comparisons(LSD)analyzed expression of Erbin in high,medium,poorly difference hepatocellular carcinoma;Chi-Square Tests analyzed the relationship between Erbin expression groups of low and high.Kaplan-Meier analyzed the relationship between Erbin expression with their survival prognosis.2.The effect of biological characteristics in vivo and vitro when Erbin was stably knockdowned in hepatocellular carcinoma cells.2.1 The mRNA,protein levels of Erbin were detected in human liver cancer cell lines,included MHCC97H,MHCC97L,HCCLM6,SMMC7721,BEL7402,QGY7703,Huh7,HepG2 and immortalized liver cell line of L02.2.2 Human liver cancer cell lines of SMMC7721 and BEL7402 were stably knockdowned of Erbin.2.3 Colony formation assay,CCK-8 cell proliferation assay,cell scratch test,Transwell chamber cell migration assay and FN cell adhesion experiments were carried out in Erbin stably knockdown cell lines.2.4 Tumor formation in nude mice experiments were carried out by using Erbin stably knockdowned cell lines.2.5 Datas were analyzed using SPSS 16.0 software.CCK-8 cell proliferation assay results was analyzed using factorial analysis of variance.subcutaneous tumor experiments were analyzed using Repeated measures analysis of variance.Colony formation,Transwell chamber experiments FN migration and adhesion experiment were analyzed by One-way.ANOVA.3?Using Erbin?C/?C gene mutation mice to establish and observe inflammation and tumor models.3.1 Acute hepatitis and cirrhosis models of C57 mice were established by CCL4 induce,and detected expression of Erbin in these models..3.2 Erbin?C/?C gene mutation mice was identified by PCR.3.3 Acute hepatitis and cirrhosis models of Erbin?C/?C mice were established by CCL4 induce,and alanine aminotransferase and aspartate aminotransferase was detected.3.4 DEN-induced liver cancer model was established and observed in Erbin?C/?C mice.3.5 DEN-CCL4.-induced liver cancer model was established and observed in Erbin?C/?C mice to investigate the mechanism of the development of liver cancer.3.6 Datas were analyzed using SPSS 16.0 software.One-way ANOVA analyzed the changes of ALT and AST in mice with acute inflammation and cirrhosis.Independent T test analyzed the difference of tumor size and number induced by CCL4 + DEN and DEN-.Chi-Square Tests analyzed the differences of tumor rates.Induced by CCL4 + DEN and DEN.4.The regulation effects of Erbin in the occurrence and development of Hepatocellular carcinoma.4.1.Erbin PDZ domain-specific antibodies were identified and detected.4.2 Erbin was knockdowned to detecte its effects on the cell cycle in HCC cells.4.3 Erbin was knockdowned to detecte relative cancer stem cell makers of HCC4.4 Erbin promotes the growth of hepatocellular carcinoma by affecting ERa signaling.4.5 Using mass spectrometry to find new possibilities interacting proteins of Erbin in hepatocellular carcinoma.4.5 Using RNA-seq analysis of the impact effects of related genes and signaling pathways when erbin was knockdowned.4.6 Datas were analyzed using SPSS 16.0 software.Independent T test analyzed the cell cycle datas and real-time PCR results.Result:1.Detection expression levels and correlation analysis of Erbin in clinical specimens of hepatitis,cirrhosis and hepatocellular carcinoma.1.1 Detection expression level of Erbin in clinical specimens of hepatitis,cirrhosis.Cytoplasmic staining score:Erbin expression score of normal tissue(n = 16)is 2.75 ± 1.17,hepatitis tissues score is 4.21 ? 2.17,score of the cirrhosis is 5.58 ±2.22.Multiple comparisons(LSD)analysis concluded that the difference between hepatitis liver tissue and normal liver tissue was statistically significant(p = 0.024),cirrhotic tissue scores were significantly higher than normal liver tissue scores(p<0.001),and cirrhotic scores compared with hepatitis had a statistical difference significance(p = 0.011).Nuclear staining score:Erbin expression score of normal tissue(n = 16)is 0.594 ± 0.712,score of hepatitis is 1.313 ± 1.092,score of the cirrhosis is 1.641 ± 1.116;Normal liver tissue has a 50%positive nuclei coloring,hepatitis tissue has a 70.83%positive nuclei coloring,and cirrhosis tissue has a 81.25%positive nuclei coloring.Multiple comparisons(Dunnett T3)analysis concluded that the difference of nuclear coloring score between hepatitis and normal liver tissue was statistically significant((p = 0.035),cirrhotic nuclear coloring score was significantly higher than normal liver tissue score(p = 0.001),and cirrhotic score compared with hepatitis had a statistical difference significance(p = 0.244).1.2 The analysis of Erbin expression in hepatocellular carcinoma.By Paired-Samples T test analysis,the difference was statistically significant between carcinoma(5.266 ± 2.613)and adjective tissue(4.528 ± 1.900)(t = 3.356,df=213,p = 0.001).Multiple comparisons(LSD)analysis of Erbin expression in well,medium and poorly differentiated hepatocellular carcinoma:well-differentiated hepatocellular carcinoma score was 3.654 ± 1.832(n = 26),moderately differentiated hepatocellular carcinoma score 4.939 ±2.609(n = 130),poorly differentiated hepatocellular carcinoma score 6.724 ± 2.246(n = 58);among the three groups have a statistically significant(F = 17.296,P<0.001);The difference between well group and medium group was statistically significance(p = 0.015),the difference between well group and medium group was statistically significance(p<0.001),and the difference between well group and medium group was statistically significance(p<0.001).Chi-Square Tests analysis of the relationship between Erbin high and low expression groups compared to age,sex,tumor size,number of tumors,differentiation,AJCC staging and other clinical indicators.The results show that:the expression level of Erbin between HCC well,medium and pool differentiation was significant difference(p = 0.000).1.3 Analysis of the expression level of Erbin in hepatocellular carcinoma associated with survival time of patients.Kaplan-Meier analyzed the differences between Erbin low expression and high expression in HCC patients' survival time.According to immunohistochemical scoring of 131 cases of hepatocellular carcinoma with the average score of 5.266 ±2.613,patients were divided into Erbin low expression group(n = 63)and high expression group(n = 68).Erbin low expression group(n = 63)has an average survival time of 34.199 months,and a median survival time of 23.6 months.Erbin high expression group has an average survival time of 15.196 months,and a median survival time month of 7.567.The difference between two groups was significant(Log Rank Chi-Square = 19.100,p<0.001).Chi-Square Tests analysis of Erbin expression correlated with clinical relationship indicators.The results showed that:difference between Erbin expression levels and clinical HCC well,medium and poor differentiation was significant(p = 0.022);there was a significant(p = 0.042)in the CLIP stage of HCC patients.2.The effect of biological characteristics in vivo and vitro when Erbin was stably knockdowned in hepatocellular carcinoma cells.2.1 Erbin expression was detected in HCC cell lines and immortalized liver cell line of LO2.Quantitative analysis of the results of Western blot showed Erbin expression in hepatoma cell lines was higher than immortalized liver cell line of L02.At the same time,hepatoma cell lines of MHCC97L,HCCLM6,SMMC7721 BEL7402,HepG2 etc.were relatively high expression of Erbin,hepatoma cell lines of MHCC97H,QGY7703 and Huh7 were relatively low expression of Erbin.Quantitative PCR results showed that the majority of hepatocellular carcinoma cell lines(except QGY7703)expressed higher level of Erbin than the immortalized liver cell line of L02.Relatively,BEL7402,SMMC7721,HCCLM6 and HepG2 cell lines were relatively higher expression of Erbin mRNA,and cell lines of MHCC97H,MHCC97L,QGY7703,Huh7 were relatively lower expression of Erbin mRNA.2.2 Human liver cancer cell lines were stably knockdowned of Erbin.According Erbin expression levels in HCC cell lines,SMMC7721 and BEL7402 were selected to build Erbin stably knockdowned cell lines.After 48h of Cell lines transfected with slow virus,GFP fluorescence was successfully observed by using fluorescence microscope.Reverse transcription-quantitative PCR Results analyzed by One-way ANOVA shows,cell lines of SMMC7721 stably knockdowned of Erbin(SMMC7721/shRNA)and control cells(SMMC7721/NC)have a significant difference(F = 68.196,p<0.001);Cell lines of BEL7402 stably knockdowned of Erbin(BEL7402/shRNA)and control cells(BEL7402/NC)have a significant difference(F = 86.723,p<0.001).2.3 Results of CCK-8 cell proliferation test analyzed by two-Way ANOVA showed that:The difference between SMMC7721/shRNA and control group was significant(F = 489.849,p<0.001);The difference between points of time in cell proliferation was significant(F = 1519.548,p<0.001);Interaction of two factors in cell groups and time point groups have a significantly difference(F = 36.868,p<0.001);Except day 1(F = 0.079,p = 0.786),cell groups of rest time points have a significant difference.SMMC7721 cell proliferated slowly after Erbin stably knockdowned.The difference between BEL7402/shRNA and control group was significant(F = 201.595,p<0.001);The difference between points of time in cell proliferation was significant(F = 1897.042,p<0.001);Interaction of two factors in cell groups and time point groups have a significantly difference(F = 27.434,p<0.001);Except day 1((F = 1.732,p = 0.225),cell groups of rest time points have a significant difference.BEL7402 cell proliferated slowly after Erbin stably knockdowned.2.4 Colony formation assay.By One-way ANOVA analyzed,colony formation assay showed that:The capacity of colony formation decreased in SMMC7721/shRNA cells compared with control cells(SMMC7721/NC),and the difference was significant(F = 143.685,p<0.001);The capacity of colony formation decreased in BEL7402/shRNA cells compared with control cells(BEL7402/NC),and the difference was significant(F = 273.014,p<0.001).2.5 FN adhesion test.By One-way ANOVA analyzed,results of FN adhesion show:The capacity of colony formation decreased in SMMC7721/shRNA cells compared with control cells(SMMC7721/NC),and the difference was significant(F=19.434,p = 0.002);The capacity of colony formation decreased in BEL7402/shRNA cells compared with control cells(BEL7402/NC),and the difference was significant(F = 14.620,p = 0.005).2.6 Transwell migration assay.By One-way ANOVA analyzed,results of FN adhesion show:The capacity of cell migration increased in SMMC7721/shRNA cells compared with control cells(SMMC7721/NC),and the difference was significant(F = 66.17,p<0.001);The capacity of cell migration increased in BEL7402/shRNA cells compared with control cells(BEL7402/NC),and the difference was significant(F = 409.301,p<0.001)2.7 Tumor xenograft model.The resuilts of Repeated Measures analysis showed:The capacity of tumor formation in nude mice decreased in SMMC7721/shRNA cells group compared with the group of SMMC7721/NC,and the difference was significant(F= 31.648,P<0.001);The difference between points of time in groups was significant(F= 25.478,P<0.001);Interaction of two factors in cell groups and time point groups have a significantly difference(F=22.269,P<0.001);Except day 7((F = 1.220,p = 0.228),cell groups of rest time points have a significant difference.Tumor formation of SMMC7721 cell proliferated slowly after Erbin stably knockdowned.The difference between BEL7402/shRNA and control group was significant(F=26.637,p<0.001);The difference between points of time in cell proliferation was significant(F=42.629,P<0.001);Interaction of two factors in cell groups and time point groups have a significantly difference(F= 12.967,P<0.001);Except day 7((F = 2.004,p = 0.195),cell groups of rest time points have a significant difference.Tumor formation of BEL7402 cell proliferated slowly after Erbin stably knockdowned.3.Using Erbin?C/?C gene mutation mice to establish and observe inflammation and tumor models..3.1 Acute hepatitis model of C57 mice was established by CCL4 induce.Liver tissues were embedded in paraffin,sliced,HE staining and observed under the microscope:When 12h,liver cells degeneration and edema,hepatic vascular congestion expansion;when 24h,liver cells degeneration and edema,necrosis and vascular hyperemia;when 48h,large areas of necrosis and hemorrhage were observed.Results of Western blot detection show that:in the time points of 24h and 48h,the expression of Erbin protein level declined sharply in mouse hepatocytes.3.2 Cirrhosis model of C57 mice was established by CCL4 induce.Liver tissues were embedded in paraffin,sliced and HE staining and observed under the microscope:16 weeks CCL4 induce and stop a week after CCL4.induce,lobular surrounding tissue was fibrous and hyperplasia,lobular division boundaries clear and liver blood vessels was hyperemia.Results of Western blot detection show that:the expression of Erbin protein level increased in mouse hepatocytes of CCL4.induce compared to Oil injected mice.3.3 PCR identification of Erbin?C/?C mice.wild type(WT)On an agarose gel,a size of the amplified between 400bp-500bp band was observed in wild type(WT)Erbin?C/?C mice,a size of the amplified between 300bp-400bp band was observed in homozygous Erbin?C/?C mice and two amplified bands between 300bp-400bp and 400-500bp was observed in heterozygous Erbin?C/?C mice.3.4 Erbin?C/?C mouse model of acute inflammation induced by CCL4.H&E staining showed:compared with wild-type mice,Eroin?C-/?C homozygous mice have more inflammatory cell infiltration on the sixth day of CCL4 induced.On the sixth day of acute hepatitis,serum ALT and AST were significant difference in homozygous mice compared with wild-type mice(ALT:F = 7.200,p = 0.028;AST:F = 35.478,p<0.001).3.5 Erbin?C/?C mouse model of liver cirrhosis induced by CCL4.H&E staining showed:compared with wild-type mice,fibrous tissue proliferated more seriously in Erbin?C/?C homozygous mice.The difference of ALT between WT and Ho mice was not statistically significant(ALT:F = 0.890,p = 0.342),and there was significant difference in serum AST(AST:F = 5.279,p = 0.040).The ratio of AST/ALT in WT mice was 0.364,and 0.569 in Ho mice.3.6 DEN-induced liver cancer model of Erbin?C/?C mice.14-15 days after birth,male Erbin?C/?C mice was injected of DEN with 25mg/kg dose.After 36 weeks,mice were sacrificed by cervical dislocation.Tumor formation rate,number and size were recorded in detail.The results show as follows:by Chi-Square Tests analysis,the difference of tumor formation rate was statistically significant(WT? 92.31%,Ho?50%,F = 11.58,p = 0.002);by T-Test analysis,the difference of tumor size was statistically significant(WT?4.0769 ± 3.17393,Ho?1.7143 ± 1.89852,df = 25/13,p<0.001);by T-Test analysis,the difference of tumor number was as statistically significant(WT?11.6538 ± 9.624,Ho?3.4286 ± 5.3452,df=25/13,p<0.001).3.7 CCL4 and DEN-induced liver cancer model of Erbin?C/?C mice.14-15 days after birth,male Erbin?C/?C mice was injected of DEN with 25mg/kg dose,and were given intraperitoneal injection once 0.25ul/g body weight of 20%CCL4,at the points of the 12,20,28 week.After 32 weeks,mice were sacrificed by cervical dislocation.Tumor formation rate,number and size were recorded in detail.The results show as follows:by Chi-Square Tests analysis,the difference of tumor formation rate was not statistically significant(WT? 94.12%,Ho?100%,F = 0.401,p = 0.533);by T-Test analysis,the difference of tumor size was statistically significant(WT?3.0882 ± 2.10828,Ho?2.2857 ± 1.38013,df=16/6,p<0.001);by T-Test analysis,the difference of tumor number was as statistically significant(WT?15.3125 ± 5.12144,Ho?6.4286 ± 3.82349,df= 16/6,p<0.001).4.The regulation effects of Erbin in the occurrence and development of Hepatocellular carcinoma.4.1 Erbin PDZ domain-specific antibodies were identified and detected.The antibody can be used for western blot,immunofluorescence in human and mice.Morever,Erbin PDZ antibodies can be located in the cytoplasm and nucleus.4.2 Erbin was knockdowned to detecte its effects on the cell cycle in HCC cells.Erbin was instantaneous knockdowned in SMMC7721 for flow cytometry,cell percentage of the control group compared with the interference group was 61.7933 ±2.2377 and 75,6833 ± 3.41791 in G1 phase,18.9300 ± 3.2261 and 17.1700 ± 2.3383 in S phase,19.2400 ± 1.3479 and 7.1500 ± 1.0739 in G2 phase.The results showed that G1 phase prolongated,S and G2 phase shortened.4.3 Erbin was knockdowned to detecte relative cancer stem cell makers of HCCThe result of RT-PCR showed the mRNA level of CD13and CD90 increased,the mRNA level of CD24 and CD 133 decreased.4.4 Erbin promotes the growth of hepatocellular carcinoma by affecting ER?signaling.4.4.1 Confocal immunofluorescence detected the co-location of Erbin and ER?.In liver cancer cells of SMMC7721,Erbin N-term antibody expressed a red fluorescence and localized in the cytoplasm,but Erbin PDZ antibody expressed a red d fluorescence and localized in the cytoplasm and nucleus.ERa antibody localized in the cytoplasm and the nucleus(where the signal is strong nucleus)with a GFP fluorescence.The results showed that:the red fluorescence(Erbin PDZ)and the GFP Fluorescence(ERa)colocalizated in nucleus.In liver cancer cells of BEL7402,Erbin N-term antibody expressed a red fluorescence and localized in the cytoplasm,but Erbin PDZ antibody expressed a red d fluorescence and localized in the cytoplasm and nucleus.ERa antibody localized in the cytoplasm and the nucleus(where the signal is strong nucleus)with a GFP fluorescence.The results showed that:the red fluorescence(Erbin PDZ)and the GFP Fluorescence(ERa)colocalizated in nucleus.4.4.2 Western Blot the detected expression of ERa in hepatoma cell lines through Erbin stable and instantaneous interferenced.After Erbin stabley knockdowned,ERa expression levels were significantly increased.And the same result come out,when Erbin was instantaneously knockdowned witn siRNA interference frag:ment 1 and 2.The results showed that Erbin suppressed ER? signal in liver cancer cells.4.4.3 ERa protein interaction network was search through protein interaction prediction website STRING.4.4.4 The result of RT-PCR showed:the expression of ErbB4,EP300,SRC,ER?,CCND1,SP1,NRIP1 and HDAC1 ecreased through Erbin knockdowned.4.5 Possible interaction protein of Erbin was get by mass spectrometry of protein co-immunoprecipitation samples.4.6 RNA-seq analyzed the sample of Erbin transient interference in HCC cell of SMMC7721.Results show that 5474 genes were significant downregulation and 2022 genes were significant upregulation after Erbin kockdowned.Pathway analysis results showed the downregulation of Erbin contributed to the pathways of Spliceosome,RNA degradation,B cell receptor signaling pathway,Proteasome,pylori infection,Apoptosis,P53 pathway,cell cycle pathway,Metabolic pathway,Epithelial cell signaling in Helicobacter and other signaling pathways.Conclusions:1,Erbin expression increased in hepatocellular carcinoma,closely related to the differentiation of hepatocellular carcinoma,and patients with high expression level of Erbin haved a poor prognosis.2,Knocking down Erbin inhibited the growth of hepatocellular carcinoma in vivo and vitro.3,Mutation of the PDZ domain of Erbin inhibited the development of liver cancer.4,Erbin downregulated,caused cell cycle arrest in G1 phase in liver cancer cells and may inhibit the growth of liver cancer by upregulating ERa signaling.Erbin downregulation inhibited cell proliferation in HCC possibly worked by metabolism and apoptosis signaling pathways. |
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