Effects Of Vitamin D₃ On Colitis-associated Colorectal Cancer In Mice And Its Mechanism

Part One:The effect of 1,25-Dihydroxyvitamin D3 on colon cancer cellsObjective:To explore the effect of 1,25(OH)2D2 on E-cadherin?PLEKHA7?miR-24?miR-30a?miR-30b and miR-19a.A]so,to explore whether PLEKHA7 affect the expression levels of microRNAs.Method:SW480 cells were treated with either 100nmol/L 1,25(OH)2D3 or DMSO for 48h,and then the mRNA and protein expression levels of E-cadherin and PLEKHA7,as well as the expression levels of miR-24?miR-30a?miR-30b and miR-19a were measured by qRT-PCR or Western blotting.SW480 cells were subjected to transfecting siPLEKHA7 to knock down PLEKHA7,after which the expression levels of miR-24?miR-30a?miR-30b and miR-19a were measured by qRT-PCR.Results:(1)The protein expression level of E-cadherin and PLEKHA7 were significantly elevated in SW480 cells treated with 1,25(OH)2D3 for 48h;(2)The expression levels of miR-24?miR-30a?miR-30b and miR-19a were significantly elevated in SW480 cells treated with 1,25(OH)2D3;(3)Knockdown of PLEKHA7 decreased the level of miR-24.Conclusion:1,25(OH)2D3 can increase the protein expression levels of E-cadherin and PLEKHA7,as well as the expression levels of miR-24?miR-30a?miR-30b and miR-19a.PLEKHA7 can af'f'ect the level of miR-24 in SW480 cells.Part Two:Effects of Vitamin D3 on Colitis-associated Colorectal Cancer in MiceObjective:To explore the therapeutic effect of vitamin D,(VitD3)on azoxymethane(AOM)and dextran sulfate sodium(DSS)induced colitis-associated colorectal cancer(CAC)in mice and its mechanism.Method:C57BL/6 male mice were randomly divided into 6 groups.Group 1 served as control with non-treatment.Group 2 to 6 established CAC model.Group 2 served as model control without gastric lavage.Group 4 to 6 served as low dose,medium dose and high dose intervention groups which received 15,30,60IU/g/w VitD3 respectively by gastric lavage(3 times per week)after model established,while group 3 served as saline control group,which was given the same amount of normal saline at the same time.Mice were sacrificed by the end of the 12th week.Tumor number(TN)and tumor load(TL)as well as pathologic characteristics was evaluated.The mRNA and protein expression levels of E-cadherin and PLEKHA7 in colorectal tissues were measured by qRT-PCR or immunohistochemistry.The expression levels of miR-24?miR-30a?miR-30b and miR-19a in colorectal tissues were measured by qRT-PCR.Results:(1)The tumor numbers(TN)and tumor loads(TL)of the medium dose group(n=9,TN=4.11±0.68,TL=6.94±1.69mm)and the high dose group(n=10,TN=4.40±0.64,TL=8.10±1.95mm)were significantly lower than which of the saline control group(n=9,TN=7.44±0.53,TL=14.61 ±0.98mm)(p<0.05).(2)According to the result of immunohistochemistry,the protein level of E-cadherin in the cell membranes of Colorectal epithelium were elevated in groups treated with VitD3.In terms of the scores of immunohistochemistry,the medium dose group(3.89±0.42)and the high dose group(3.80±0.39)were significantly higher than the saline control group(2.44±0.44)(p<0.05).(3)According to the result of immunohistochemistry,the protein level of PLEKHA7 in the cell membranes of Colorectal epithelium were elevated in groups treated with VitD3 In terms of the scores of immunohistochemistry,the high dose group(4.80±0.29)were significantly higher than the saline control group(3.111±0.31)(p<0.05).Also,the scores of immunohistochemistry of E-cadherin and PLEKHA7 were related with a correlation coefficient value of 0.741(P<0.0001).(4)In term of the expression levels ofmiR-19a,the model control group were significantly lower than the control group.Also,the middle dose group were significantly higher than the saline control group(p<0.05).Conclusion:VitD3 treatment suppresses AOM/DSS induced CAC in mice,which may be attributed to the elevating of the protein levels of E-cadherin and PLEKHA7 in the cell membranes of Colorectal epithelium.