The Role Of Musashi-1 In The Pathogenesis Of Colon Cancer And Its Significance

Objective:Colon cancer is one of the most common digestive system tumors in our country,and its incidence is increasing in recent years.Due to the tumor early nonspecific symptoms,and diagnosis is late.Colon cancer can not be completely removed by surgery.The effect of chemotherapy drugs available for the treatment is poor.The low postoperative survival rate and poor prognosis are common.In order to explore a more effective treatment of colon cancer,we need to research the molecular development mechanism in colon cancer deeply.Recent studies show that there is a small amount of stem cells of stem characteristics in colon cancer,these stem cells express a number of stem cell markers on the surface,such as CD133,CD44,Musashi-1,EpCAM and so on.Based on our previous studies,Musashi-1 protein overexpressed in colon cancer.In addition,there has been no sensitivity to targeted drugs in colon cancer treatment in recent years,it affect the recovery of the patients after surgery.So the study of role of Musashi-1 in the pathogenesis of colorectal cancer will provide new ideas for the treatment of patients with colon cancer.This study was performed to detect the expression of Musashi-1 and its related molecules protein(P21,Hes-1)in human colon cancer tissue,and we treat human colon cancer cell line SW48 by using siRNA and EGFR(epidermal growth factor receptor)targeted therapy drugs cetuximab(cetuximab,CTX).Our aim is to investigate whether Musashi-1 can participate in the pathogenesis of colon cancer by regulating the Notch signaling pathway and whether the expression of Musashi-1 gene can improve the efficacy of cetuximab.Methods:1.Immunohistochemical(EnVision)analysis was performed to investigate the expression of Musashi-1?P21 and Hes-1 protein in 110 colon cancer tissures microarray,and we compared it with normal colon mucosa protein expression rate in 10 tissures microarray.The correlation between the protein expression of Musashi-1,P21,Hes-1 and clinicopathological parameters was analyzed;2.According to the different treated factors,the experiment was divided into three experimental groups and one control group,the experimental group included cetuximab group,Musashi-1 group,Musashi-1 siRNA interference interference plus cetuximab combined treatment group;3.First of all,we applied MTT method to detect cell proliferation activity of SW48 which was dealed with different concentrations(0-1000ug/ml),and then the proliferative activity of the above four groups was detected;4.The four groups cells were treated for 48 hours,Musashi-1,P21,CyclinB1 mRNA expression levels were detected in different groups by Q-PCR;5.The four groups cells were treated for 48 hours,Musashi-1,P21,Notch1,EGFR protein expression levels were detected by Q-PCR in different groups;6.Cell apoptosis was detected in flow cytometry instrument in above four groups in 24 h and 48 h;7.SPSS19.0 statistical software was used to analyze the data,and the data were expressed by mean ± standard deviation(Mean ± SD).Statistical methods included variance analysis,T test,Spearman rank correlation analysis,chi square test and Fisher exact probability method.Result:1.Immunohistochemistry showed that the Musashi-1 expressed in the cytoplasm or nuclear of colon carcinoma cells and normal colon mucosa cells.The P21 protein was nuclear expression.Hes-1 protein expressed in cytoplasm or nuclear.In 110 cases of colon adenocarcinoma,the rate of Musashi-1 protein positive expression was78.2%(86/110),the positive rate of Hes-1 protein was 68.2%(75/110),and the positive rate of P21 protein was 21.8%(24/110).In 10 cases of normal colorectal mucosa,the rate of Musashi-1,P21,and Hes-1 proteinpositive expressionwas 40%(4/10),60%(6/10),30%(3/10),respectively.Compared with normal colon tissues,Musashi-1 and Hes-1 protein positive expression ratesignificantly increased,P21 protein positive expression rate decreased,the difference was statistically significant(P<0.05).Spearman correlation analysis showed that Musashi-1 was positively correlated with Hes-1 expression(r=0.372,P=0.000).Musashi-1 was negatively correlated with P21(r=-0.221,P=0.021).High expression of Musashi-1,Hes-1 and low P21 expression was not correlated with clinicopathological parameters of colon adenocarcinoma(P>0.05);2.MTT results showed the growth inhibition rate of Musashi-1 siRNA interference group(42 + 1.25%)was higher than that of negative control group(P<0.05),the inhibition rate of colon cancer cells increased with theincrease of cetuximab concentration(P<0.05).When the Musashi-1 siRNA interference combined with cetuximab acted on cells,the growth inhibition rate(64.10 + 3.13%)was significantly higher than that of the negative control group(15.63 + 1.51%),cetuximab treatment group(52.09 + 2.64%)and Musashi-1 siRNA interference group(42 + 1.25%),the growth rate of SW48 cells decreased,the proliferation of SW48 cell line rate was inhibited,the difference was statistically significant(P<0.05);3.According to the Q-PCR results,Musashi-1 mRNA and CyclinBl mRNA expression were significantly decreased in three treatment groups compared to the negative control group(P<0.05).The expression of the combined group was lower than the other three groups(P<0.05).The expression of P21 mRNA in the combined gruop was significantly increased,compared to the other three groups(P<0.05).The P21 mRNA expression of the combined effects of Musashi-1 siRNA interference combined cetuximab group was higher than the other three groups(P<0.05);4.Western results showed that the expression levels of Musashi-1,P21,Notch1 and EGFR protein in the three experimental groups were significantly lower than those in the negative control group(P<0.05).Compared to the single factor group,in Musashi-1 siRNA plus cetuximab combined groupthe Musashi-1 and Notch1 protein expression fell(P<0.05),P21 protein expression went up(P<0.05),the difference was statistically significant;5.The testing results of flow cytometry showed that SW48 colon cancer cells treated for 48 hours by Musashi-1 siRNA combined cetuximab can obviously promote the apoptosis of cells,compared to the other three groups(P<0.05).Conclusion:1.Musashi-1 and Hes-1 overexpressed in colon adenocarcinoma.Musashi-1 may be involved in the pathogenesis of colon adenocarcinoma by up-regulating the expression of Hes-1 protein and decreasing the expression of P21 protein.2.The interference of Musashi-1 siRNA down-regulated the expression of Musashi-1,Notch1,EGFR,andpromoted the expression of P21 in human colon cancer cell line SW48,inhitbitingthe slow growth of cells.3.Cetuximab can inhibit the proliferation of SW48 colon cancer cells.High concentration of cetuximab can reduce the expression of Musashi-1.4.Combined application of Musashi-1 small interfering RNA and cetuximab inhibited the growth of SW48 colon cancer cells and promote the apoptosis of tumor cells.5.Musashi-1 can be used as a candidate target gene for colon cancer targeted therapy,and Musashi-1 targeted therapy combined with cetuximab may improve the therapeutic effect.