The Study In The Expression Of ASPM And Its Correlation With Wnt Signal Pathway In Colonic Adenocarcinoma

Background:Colonic adenocarcinoma(CAC)is one of the most common malignant tumors.In recent years,the incidence of CAC has increased year by year.With the improvement of treatment,the prognosis of CAC has been greatly improved.However,recurrence and metastasis are still the main causes of death.At present,tumor stem cells were reported to play important roles in recurrence and metastasis of tumors.ASPM was an abnormal spindle-like microcephalic deformation-associated gene whose main function was to regulate cell mitosis.ASPM was considered to be a marker of CSCs,and its expression was up-regulated in many kinds of tumors,which was closely related to tumor progression and prognosis.In addition,studies have shown that ASPM was involved the development of tumors through the Wnt/β-catenin signal pathway in pancreatic and prostate cancer.More than 90%of CACs were caused by abnormal Wnt signal pathway.At present,the significance of ASPM in CAC and its mechanism were less expounded.Our preliminary study showed that the expression of ASPM in CAC was higher than that in normal colonic mucosa.Therefore,we infer that ASPM may be involved in the development of CAC through Wnt/β-catenin signal pathway.Objective:1.The expression of ASPM in CAC and the relationship between ASPM and clinicopathological parameters was detected by immunohistochemical and RNAscope.In addition,the expression of ASPM and mismatch repair protein PMS2,MLH1,MSH6 and MSH2 in CAC was detected by immunohistochemistry to evaluate the correlation between ASPM and MSI.2.The effects of ASPM on proliferation,invasion and apoptosis of colonic cancer cells were detected in vitro.3.The relationship between ASPM and FZD,DVL,β-catenin,c-myc and CyclinDl were analyzed respectively.Methods:1.Ninty nine cases of CAC companied by colonic adenoma confirmed by pathologists in PL A and 99 cases of adjacent normal colonic mucosa were studied.In CAC,adenoma and normal colonic mucosa,the expression of ASPM,β-catenin,c-myc,CyclinD1,PMS2,MLH1,MSH6 and MSH2 was analyzed by immunohistochemistry.The relationship between the expression of ASPM and clinicopathological parameters of CAC,and the expression between ASPM and β-catenin,c-myc and CyclinDl in CAC were also evaluated respectively.The correlation between repair protein(MLH1,MSH6,MSH2 and PMS2)and ASPM in CAC was explored.2.Twenty cases of primary CAC and liver metastases counterpart were selected to detect the expression of ASPM by RNAscope.3.ASPM gene was silenced by transient transfection technique in colonic cancer cell lines.The effects of transfection on proliferation,apoptosis and invasion of colonic cancer cells were observed.4.The relationship between ASPM and vital factors such as DVL,FZD,β-catenin and the downstream effector molecules(c-myc and CyclinD1)of Wnt singal pathway was observed before and after transfection by qRT-PCR and Western blot.The relationship between ASPM and Wnt signal pathway was further analyzed by rescue experimentsResults:1.The expression of ASPM in CAC was positively correlated with TNM stage and lymph node metastasis(P<0.05).The ASPM mRNA level in primary CAC was significantly lower than that in liver metastasis counterpart(P<0.05).The expression of ASPM was positively correlated with β-catenin,c-myc and CyclinD1(P<0.05).There was no correlation between the expression of ASPM and MSI in CAC.2.In colonic cancer cells,the proliferative ability was decreased after silencing the ASPM gene,the apoptosis rate increased,and the invasion ability decreased significantly.After silencing ASPM,the expression of Wnt signal transduction related protein β-catenin and downstream effectors c-myc and CyclinDl were significantly down-regulated(P<0.05).Adding Wnt pathway activator to colonic cancer cells after silencing ASPM gene,the mRNA and protein level ofβ-catenin,c-myc and CyclinDl were saved(P<0.05).Conclusions:1.ASPM was closely related to the TNM stage and lymph node metastasis of CAC,and could be used as an index to judge the invasion and metastasis of CAC.2.ASPM might promote the proliferation and invasion of colonic cancer cells.3.ASPM might involve the development of CAC through Wnt-FZD-DVL-β-catenin signal pathway.