| Liver cancer is one of the most common malignancies,its occurrence and development is a complex process related to the abnormal changes of various genes,and the specific molecular mechanism has not yet been fully understood.The human abnormal spindle-like microcephaly-associated gene(ASPM)is located on chromosome 1q31,and has a total length of 62528 bp and a coding region of 10434 bp.It consists of 28 exons.ASPM was initially found to play a key role in embryonic neural formation,controlling the brain’s capacity,and its genetic mutations are the primary cause of neurodevelopmental defects in autosomal recessive genetic microcephaly.As the study progressed,ASPM was found to be highly expressed in proliferating tissues and tumor tissues.Our previous experiments found that the expression level of ASPM in liver cancer tissue was significantly up-regulated.Therefore,we speculated that ASPM may be involved in the occurrence and development of liver cancer.It is necessary to have a further study to clarify the mechanism of its action in liver cancer.The classic Wnt/β-catenin signal pathway is abnormally activated in most liver cancers.Our previous study also found that the expression level of ASPM in liver cancer was significantly correlated with the expression level of β-catenin.Based on this,we proposed that ASPM may affect the occurrence and development of liver cancer by regulating Wnt/β-catenin signaling pathway.The subject are mainly studied from two aspects:(1)The expression level of ASPM in in liver cancer and paracancerous tissues,clinical pathological significance and its relationship with important switch molecules in Wnt/β-catenin signaling pathway;(2)Effect of ASPM on malignant biological behavior of Hepatoma Cells and its mechanisms.Methods 1.Expression and clinical pathological significance of ASPM in human liver tissues 1.1 The RT-qPCR method is used to detect the mRNA expression levels of ASPM in cancerous and paraneoplastic tissues,analyzing the expression level and clinical pathological significance of ASPM in liver cancer and paracancerous tissue.1.2 Application of Western Blotting and immunohistochemistry are used to detect the protein levels of ASPM in liver cancer and adjacent tissue,analyzing the differences of ASPM protein levels of liver cancer and paracancer tissue.1.3 The RT-qPCR method is used to detect the mRNA expression levels of important switch molecules in the Wnt/β-catenin signaling pathways in liver cancer and paracancerous tissue.The correlation between mRNA expression levels of ASPM and the important switch molecules in Wnt/β-catenin signaling pathways is analyzed.2.The Effect and Mechanism of ASPM on Malignant Biological Behavior of Hepatoma Cells 2.1 The RT-qPCR method is used to detect ASPM the expression level of 2 Hepatoma cell lines,and the best shASPM sequence with the best interference effect is screened by RT-qPCR method to construct lentivirus mediated ASPM-shRNA Hepatoma cell lines.2.2 The effects of ASPM on the proliferation,migration and invasive ability of hepatoma cells are detected by CCK-8,wound healing experiment,and Transwell assay.2.3 Western Blotting technology is used to detect the protein expression levels of DVL2,β-catenin,TCF4,and LEF1 of Wnt/β-catenin signaling pathway in different groups of Hepatoma cell lines(Scrambled Control、shASPM).Results 1.Expression and clinical pathological significance of ASPM in human liver tissues 1.1 The mRNA expression level of ASPM in human liver tissues and its clinicopathological significanceUsing RT-qPCR method,102 cases of human liver specimens ASPM mRNA are quantified,wherein cancerous and paracancerous tissues in 90 pairs of cases.The results show that the expression level of ASPM mRNA in liver cancer tissue is significantly higher than that in para-tumor tissues(P<0.00001),the ASPM mRNA expression is that 69 cases of tumor tissues are up-regulation in 90 paired samples,and high expression rate is 76.7%.According to the tumor size,liver cirrhosis and AFP expression levels in 102 cases of liver cancer tissues and paracancerous tissues,the difference of ASPM mRNA expression is analyzed.The results show that the cancer tissues in each subgroup are significantly higher than those in paracancerous tissues(P<0.05).Similarly,according to tumor thrombus degree,vascular invasion degree,and differentiated degree,the expression of ASPM mRNA is significantly higher in the tumor tissue with tumor thrombus,vascular invasion,moderately differentiated,and poorly differentiated group than in the paracancer tissue(P≤0.01).It is noteworthy that,the patients whose AFP in the normal range(AFP≤20ng/mL),the expression of ASPM mRNA in the tumor tissue is still significantly higher than that in the paracancer tissue(P=0.048).The analysis of 96 cases of liver cancer tissue show that the expression of ASPM mRNA in severe cirrhosis group is significantly higher than that in moderate cirrhosis group(P=0.011),and the expression level of ASPM mRNA in poorly differentiated group is also significantly higher than that in highly differentiated group(P=0.017).1.2 The mRNA expression level of ASPM in different types of liver cancer patientsAccording to the clinical diagnosis,102 cases of human liver specimens are divided into 5 groups: HBV-related primary hepatocellular carcinoma(HBV-HCC,74 cases),recurrent HBV-related primary hepatocellular carcinoma(HBV-RHCC,10 cases),hepatocellular carcinoma(HCC,4 cases),primary cholangiocellular carcinoma(ICC,7 cases),and metastatic liver cancer(MCC,7 cases).The analysis found that in 74 cases of HBV-HCC hepatoma tissue,the expression of ASPM mRNA was significantly higher than that in paracancer tissues(P<0.00001),71 pairs of matched samples,52 cases of high expression of HCC,the high expression rate is 73.2%.The expression of ASPM mRNA in HBV-RHCC tissues is significantly higher than that in paracancer tissues(P=0.003).All of the 9 pairs matched samples have high expression of tumor tissue;compared with paracancer tissues,the expression of ASPM mRNA in ICC tissues is significantly increased(P=0.02);the expression levels of ASPM mRNA in paracancerous and cancer tissues of HCC and MCC are not statistical difference(P>0.05).1.3 The mRNA expression level of ASPM of HBV-HCC and its clinicopathological significanceAnalysis of the expression of ASPM mRNA in 74 cases of HBV-HCC and paracancerous tissues revealed that the ASPM mRNA expression level in the tumor tissue,which with a diameter > 5cm,tumor thrombus,vascular invasion,AFP > 20ng/mL,mild or moderate cirrhosis,poorly differentiated or moderately differentiated group,is significantly higher than the paracancerous tissues(P ≤ 0.01).Analysis of the expression level of ASPM mRNA in HBV-HCC tissue show that the expression level of ASPM mRNA in severe cirrhosis group is significantly higher than that in moderate cirrhosis group(P=0.041).In summary,ASPM is highly expressed in liver cancer tissues.And the expression of ASPM mRNA is closely related to the degree of differentiation of liver cancer,the degree of liver cirrhosis,and the recurrence of HBV-HCC after surgery.1.4 The protein expression level of ASPM in liver cancer tissues is highly expressedImmunohistochemistry method is used to quantify the protein level of ASPM in 32 pairs of matched liver carcinoma and paracancerous tissues.The result shows that 23 cases are higher of ASPM protein in tumor tissue than paracancerous tissues,and the high expression rate is 71.9%.Western blotting is used to detecte the protein levels of ASPM in 65 pairs of matched hepatocellular carcinoma and paracancerous tissues.The result shows that 48 cases are higher of ASPM protein in tumor tissue than paracancerous tissues,and the high expression rate is 73.8%.1.5 The correlation between mRNA level of ASPM and DVL2,β-catenin,TCF4,LEF1The RT-qPCR method is used to quantitate the expression levels of DVL2,β-catenin,TCF4,and LEF1 mRNA in 102 human liver specimens.The result shows that there is a non-normal distribution.Through the Spearman correlation analysis,we found that The mRNA expression levels of ASPM and DVL2,β-catenin,TCF4 and LEF1 are significantly correlated(P<0.001).2.Effect and Mechanism of ASPM on Malignant Biological Behavior of Hepatoma Cells 2.1 Constructing lentivirus mediated ASPM-shRNA Hepatoma cell linesFor a further study about ASPM and its mechanisms of malignant biological behavior of hepatoma cells,we selected 2 strains of hepatoma cell lines HepG2,SMMC-7721 to establish lentivirus interference ASPM liver cancer cell lines.we use the lentivirus interference ASPM of HepG2 and SMMC-7721 to explore the biological behavior of malignant liver cancer cells and the mechanisms.2.2 ASPM promotes the proliferation,migration and invasion of Hepatoma CellsThrough the CCK-8 to check the proliferationa ability of HepG2 and SMMC-7721(Scrambled Control group,shASPM group),we found that the proliferationa ability of HepG2 and SMMC-7721 with a stable interference ASPM is decreased significantly.The scratch repair experiment is used to detect t the migration ability of HepG2 and SMMC-7721(Scrambled Control group,shASPM group).The result shows that after stable interference ASPM,the migration ability of HepG2 and SMMC-7721 cells is decreased significantly.Transwell assay is used to detect the invasion ability of HepG2 and SMMC-7721(Scrambled Control group,shASPM group),the result shows that,after a stable interference ASPM,the invasion ability of HepG2 and SMMC-7721 is decreased significantly.2.3 The protein expression levels of DVL2,β-catenin,TCF4,and LEF1 in Hepatoma cell linesWestern blotting is used to detect the expression levels of DVL2,β-catenin,TCF4 and LEF1 in different groups(Scambled Control group,shASPM group)of HepG2 and SMMC-7721.The result shows that the Wnt/β-catenin signaling pathway is silenced after ASPM silenced.The DVL2,TCF4 and LEF1 expression levels of downstream important switch molecules are down-regulated significantly,but there is no significant changes with the expression level of β-catenin.Conclusions 1.ASPM is highly expressed in liver cancer compared with paracancer tissues..2.ASPM mRNA has prognostic value for liver cancer with normal AFP level,and has better diagnostic complementarity with AFP federation.The expression level of ASPM mRNA is closely related to the degree of hepatic tumor differentiation, liver cirrhosis,and postoperative recurrence of HBV-HCC,so it can be used to be an indicator of poor prognosis of liver cancer.ASPM may be used as a therapeutic target for broad-spectrum anti-liver cancer drugs.3.ASPM promotes the malignant biological behaviors such as proliferation, migration,and invasion of hepatoma cells.4.ASPM elevates the expression level of Dvl-2,TCF4 and LEF1,and ASPM may promote the progression of liver cancer through Wnt/β-catenin signaling pathway. |
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