## 2 Results
### The Regulation of XIAP by MCPIP1 Depends on Its ZF Domain  
Due to the CCCH zinc finger domain of MCPIP1 being usually responsible for its binding to mRNAs [9], we then tested whether the regulatory function of MCPIP1 to XIAP's  
Figure 4: MCPIP1 attenuates XIAP mRNA’s stability, leading to its downregulated mRNA and protein expression. (**A**,**B**) HeLa (**A**) and SiHa (**B**) cells were infected with a lentivirusirus of MCPIP1 or D141N and treated with or without 1 ug/mL doxycycline for 24 and 48 h, respectively. Then, the XIAP mRNA levels were determined by RT-qPCR analysis. (**C**,**D**) HeLa (**C**) and SiHa (**D**) cells were infected with MCPIP1 or D141N lentivirus and treated with or without 1 ug/mL doxycycline for 24 h and 48 h, respectively. Then, cells were treated with 5 ug/mL actinomycin D for 0, 1, 2, and 4 h and collected to detect the remaining mRNAs of XIAP by RT-qPCR. (**E**,**F**) HeLa (**E**) and SiHa (**F**) cells were infected with the lentivirusirus of NTC or shRNAs to MCPIP1 (shMCPIP1#1 and shMCPIP1#2) for 48 h, and then the XIAP mRNA levels were determined by RT-qPCR analysis. (**G**,**H**) HeLa (**G**) and SiHa (**H**) cells were infected with the lentivirusirus of NTC, shMCPIP1#1, or shMCPIP1#2 for 48 h. Then, cells were treated with 5 ug/mL actinomycin D for 0, 1, 2, and 4 h and collected to determine the remaining mRNAs of XIAP by RT-qPCR. (**I**) Protein expression levels of MCPIP1, XIAP, pro-caspase-3, cleaved caspase-3, total PARP1, and cleaved PARP1 were detected by Western blotting in HeLa or SiHa cells, which were infected with the lentivirusirus of NTC, shMCPIP1#1, or shMCPIP1#2 for 48 h. \(\beta\)-actin was used as a loading control. **\({}^{**}p<0.01\)**.  
mRNA stability relies on its zinc finger domain. We infected HeLa and SiHa cells with the lentivirus of wild-type MCPIP1 or its mutant C306R that lost the ability to bind the XIAP mRNA and then treated the cells with or without doxycycline. The Western blotting results showed that MCPIP1 but not C306R overexpression reduced the protein levels of XIAP but increased cleaved caspase-3 and cleaved PARP1 in cervical cancer cells (Figure 5A). Similarly, the XIAP mRNA levels decreased in HeLa and SiHa cells with the induction of MCPIP1 but not C306R overexpression (Figure 5B). Consequently, C306R does not affect the mRNA or the protein expression level of XIAP in cervical cancer cells, which differs from MCPIP1.  
Figure 5: The ZF domain of MCPIP1 mediates its binding to XIAP mRNA and contributes to XIAP mRNA decay. HeLa and SiHa cells were infected with MCPIP1 or C306R lentivirus and treated with MCPIP1.  
or without 1 ug/mL doxycycline for 24 and 48 h, respectively. (**A**) Protein expression levels of MCPIP1, XIAP, pro-caspase-3, cleaved caspase-3, total PARP1, and cleaved PARP1 were detected by Western blotting. \(\beta\)-actin was used as a loading control. (**B**) The XIAP mRNA expression was determined by RT-qPCR analysis. (**C**) HeLa or SiHa cells were further treated with 5 ug/mL actinomycin D for 0, 1, 2, and 4 h and collected to determine the remaining mRNAs of XIAP by RT-qPCR. (**D**) After an RIP assay with an antibody to MCPIP1 or IgG control, the enrichment of XIAP mRNA by MCPIP1 or C306R protein was examined by RT-qPCR. **\(p<0.01\).  
Furthermore, the RNA stability detection assay showed that overexpressed MCPIP1 but not C306R leads to lower mRNA stability of XIAP (Figure 5C). Our RIP assay demonstrated that the XIAP mRNA was enriched by MCPIP1 but not its ZF domain mutant C306R (Figures 5D and S6A,B), suggesting that the ZF domain contributes to the physical association between MCPIP1 and XIAP mRNA. Additionally, the flow cytometry assay showed that MCPIP1 but not C306R overexpression increased the apoptosis rate of HeLa and SiHa cells (Supplementary Figure S7A,B). Collectively, our findings indicate that the CCCH ZF domain of MCPIP1 is indispensable for its negative regulatory role in XIAP's mRNA stability and its anti-apoptotic function.