The Effects Of Wnt6 On Tumorigenesis And Development Of Colon Cancer

Colon cancer,one of the most common gastrointestinal cancer,is the third global largest cancer and has the fourth largest global mortality rate.In china,the incidence of colon cancer ranked fourth and the mortality rate ranked fifth,only less to lung cancer and gastric cancer。Colon cancer has low 5-year survival rate,poor treatment and prognosis and has seriously bad impacts on human health.Wnt pathway plays an important role in growth,development and differentiation.And it is reported that the aberrant of Wnt pathway was closely related with many diseases and cancer,including colon cancer.The occurrence and devolpment of colon cancer are associated with many genetic factors and environmental factors.In the colon cancer,many gene mutations has been found including APC,β-catenin,KRAS,and P53 genes,as well as the abnormal expressions of a variety of Wnt proteins.It is reported that the polymorphism of Wnt6 gene is closely related to colon cancer,however the effects of Wnt6 on colon cancer is rarely reported.The aim of our study was to compare the mRNA and protein expressions of Wnt6 in normal and cancerous tissues of colorectal cancer patients,investigate the effects of Wnt6 on colorectal cancer cell lines and carcinogenesis and development of colorectal cancer and further to study the molecular mechanism of Wnt6 on the occurrence and development of colon cancer.Part one:The expression levels of Wnt6 in colon cancer tissuesObjective:To detect the expression levels of Wnt6 in adjacent normal colonic and colon cancer tissues of colon cancer patients and colon tissue of normal people,and to explore the role of Wnt6 on colon cancer.Methods:We have collected colon tissues of 45 primary colon cancer cases from our hospital in July 2015 to July 2016 while at the same time of surgery,20 cases of normal adjacent colon cancer tissue were randomly collected.Another 20 cases of normal colon cancer tissue from healty people,deteted by gastrointestinaloscopy and pathological examination,were colleted.There were 23 males and 22 females in 45 colon cancer patients(aged from 39 to 68 years,mean age:54.44 ± 7.03 year).And there were 11 males and 9 females in healty people(aged from 41 to 62 years,mean age:52.15 ± 5.91years).The t-test and chi-square test were used to analyze the differences of sex and age in the colon cancer patients and healty people.Tissues detected were divided into three groups:normal colonic tissue,paracancerous tissue and colon cancer tissue.The mRNA expression of Wnt6 was measured by real-time quantitative PCR(qPCR).Eight samples were randomly selected from the three groups,and then the protein expression of Wnt6 was detected by Western blotting.Results:There was no significant difference between the colon cancer patients and the healthy people in age(t = 1.272,p = 0.208)and sex(F = 0.084,p = 0.772).The mRNA expression of Wnt6 in colon cancer tissue was significantly higher than that in normal colonic tissue and paracancerous tissue(P<0.01).And there was no significant difference in the m RNA expression of Wnt6 between paracancerous tissue and normal colonic tissue.8 samples of each group were randomly selected for western blotting.The protein expression of Wnt6 in colon cancer tissue was higher than that in paracancerous tissue and normal colonic tissue.Conclusions:Highly expressed Wnt6 was associated with colon cancer,and Wnt6 might play important roles in the development and progression of colon cancer.Part two:Construction of expression and interference vectors andscreening of Wnt6 highly expressed colon cancer cellsObjective:To construct the Wnt6 expression and interference vectors,and screen Wnt6 highly expressed colon cancer cell lines from 5 colon cancer cell lines for plasmid transfection to obtain transfected colon cancer cell lines.Methods:Real-time PCR was used to detect the mRNA expression of Wnt6 in five colon cancer cell lines,and Wnt6 highly expressed cell lines were chose for transfection of shRNA interference and expression vectors.The expression of green fluorescence in each group(CON,shNC,shWnt6,EV and pWnt6)was observed under fluorescence microscope 24 hours after transfection.And then real-time PCR was used to detect the expression of Wnt6 in transfected cells.Results:The mRNA expression of Wnt6 in SW620,HCT116 and SW480 cells was the highest in the five colon cancer cell lines,which were significantly higher than those in LoVo cell lines(P<0.01).And there was no significant difference in the mRNA expression of Wnt6 between LoVo and HT29 cell lines.The expressions of green fluorescent protein were observed in each group under fluorescence microscope 24 hours after the transfection of shRNA interference vector and overexpressing vector.And the Wnt6 expression were downregulated when cell lines were tranfected with shRNA interference vectors.But the results in expression vector-transfected cell lines were Just the opposite.Conclusions:HCT116 and SW480 cell lines have the highest mRNA expression of Wnt6 among the five colon cancer cell lines.shRNA interference plasmid were able to silence the Wnt6 while expression plasmid overexpressed Wnt6.Part three:The effects of Wnt6 on the growth of HCT116 and SW480 cell linesObjective:To investigate the effect of Wnt6 on growth of the two colon cancer cells.Methods:The HCT116 and SW480 cells were transfected with shRNA interference and expression vectors.And then cell proliferation of transfected cells(CON,shNC,shWnt6,EV and shWnt6 + pWnt6)was measured by MTT assay.Cell cycle and apoptosis were determined on flow cytometer.Cell migration was determined though transwell asssy and western blotting were used to detecte the protein expressions of Bax,Caspase-3 and MMP2 genes.Results:The transfection of shRNA interference vector of Wnt6 have reduced the cell proliferation,cell migration,promoted the cell apoptosis,increased cell number at G0-G1 stage,reduced cell number at S stage,improved the protein expressions of Bax and decreased the pretion expression of Caspase-3 and MMP2 in HCT116 and SW480 cells.And the results of co-transfection of shRNA interference and overexpression vecters of Wnt6 were the opposite.Conclusions:Wnt6 enhances the cell growth and cell migration of colon cancer cells,which might be work though the inhibition of cell apoptosis and the increasing expression of oncogenes.