Regulatory role of ZDHHC5 in IFN-Ⅰ Expression and cellular proliferation and migration in colorectal cancer cells
Objective To elucidate the regulatory role of zinc finger DHHC-type containing 5(ZDHHC5)in expression of type Ⅰ interferon(IFN-Ⅰ)expression and its consequent impact on the proliferation and migration of colorectal cancer cells.Methods The mouse colon cancer cell line MC38ova underwent ZDHHC5 knockdown and overexpression by using lentiviral infection and plasmid transient transfection.Simultaneous knockdown of ZDHHC5 and EZH2 was also conduc-ted.Quantitative real-time PCR(qRT-PCR)assay was used to assess IFN-Ⅰ expression in MC38ova cell line post-ZDH-HC5 knockdown or overexpression.Additionally,the expression level of IFN-Ⅰ following concurrent knockdown of ZDHHC5 and EZH2 was determined.The interaction between ZDHHC5 and EZH2 proteins was examined through im-munoprecipitation assays.Changes in cell proliferation and migration after ZDHHC5 knockdown were evaluated by using Cell Counting Kit 8(CCK 8)assays,colony formation assays,and scratching assays.Results qRT-PCR results showed that,compared with the IFN-a(1.00±0.05)and IFN-β(1.00±0.06)mRNA levels of NC group,the KD-ZDHHC5 #1-gfp group mRNA levels of IFN-a(5.72±0.27,t=29.56,P＜0.001)and IFN-β(2.59±0.03,t=42.90,P＜0.001)were increased.Similarly,the KD-ZDHHC5 # 2 group exhibited a marked increase in IFN-a(4.74±0.58,t=11.64,P=0.001)and IFN-p(3.85±0.34,t=13.97,P＜0.001)compared with the NC group,which had levels of 1.00±0.13 and 1.00±0.10,respectively.In contrast,the OE-ZDHHC5 group demonstrated significantly reduced mRNA lev-els of IFN-a(0.33±0.02,t=16.64,P＜0.001)and IFN-β(0.47±0.03,t=14.29,P＜0.001)as opposed to the OE-NC group,which had levels of 1.00±0.07 and 1.00±0.06,respectively.Furthermore,the KD-EZH2+KD-ZDHHC5 group showed an increase in IFN-a mRNA level(1.82±0.06,t=8.15,P=0.001),when assessed against the KD-EZH2 group,which had levels of 1.00±0.16,respectively.Immunoprecipitation assays demonstrated the FLAG-ZDH-HC5 bind to the HA-EZH2 protein.The CCK 8 results revealed that the cell proliferation in the KD-ZDHHC5 # 1-gfp group was significantly reduced compared with the NC-gfp group(48 h,t=29.89,P＜0.001;72 h,t=32.94,P＜0.001).Similarly,the KD-ZDHHC5 # 2 group showed significantly reduced proliferation compared to the NC group(48 h,t=20.95,P＜0.001;72 h,t=28.67,P＜0.001).The results of colony formation assays showed that,com-pared to the NC-gfp group and the NC group,the cell proliferation ability was significantly reduced in the KD-ZDHHC5# 1-gfp group(t=11.44,P＜0.001)and KD-ZDHHC5 # 2 group(t=6.02,P=0.004).The scratch assay results in-dicated that,compared to the NC-gfp group and NC group,the migration ability of KD-ZDHHC5 # 1-gfp(t=5.78,P=0.029)and KD-ZDHHC5 # 2 group(t=15.22,P=0.004)was significantly decreased,respectively.Conclusion ZDH-HC5 inhibits the expression of IFN-a and IFN-β through interaction with EZH2,thereby enhancing the proliferation and migration capabilities of colon cancer cells.
ZDHHC5enhancer of Zeste homolog 2type Ⅰ interferoncolon cancer
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