## 2 Results
### MCPIP1 Promotes the Apoptosis of Cervical Cancer Cells via Suppressing XIAP Expression  
Subsequently, we explore the underlying mechanism by which MCPIP1 regulates cervical cancer cell apoptosis. Inhibitors of apoptosis proteins (IAPs) are a family of proteins mainly known for their ability to bind and directly inhibit caspase activity [28]. As a result, IAPs have been identified to play extensive roles in cancer cell death, proliferation, invasion, and metastasis. Consequently, targeting IAP proteins has been considered an attractive strategy for cancer therapy. Among human IAP proteins, XIAP, IAP-1, and IAP-2 have well-characterized anti-apoptotic activity [29]. To examine whether MCPIP1 exerts its apoptosis-promoting effect by regulating the expression of IAP family members, we first induced the expression of MCPIP1 in HeLa and SiHa cells. Our RT-qPCR analysis showed  
Figure 2: MCPIP1-mediated apoptosis in cervical cancer cells depends on its endonuclease activity. (**A,B**) Protein expression levels of MCPIP1, pro-caspase-3, cleaved caspase-3, total PARP1, and cleaved PARP1 were detected by Western blotting in HeLa (**A**) or SiHa (**B**) cells infected with lentivirus of control, MCPIP1, or D141N and treated with 1 \(\mathrm{\SIUnitSymbolMicro g}\)/mL doxycycline for 24 and 48 h, respectively. \(\mathrm{\SIUnitSymbolMicro g}\)-actin was used as a loading control. (**C,D**) The number of floating cells (dead cells) were counted from the HeLa (**C**) and SiHa (**D**) cells after infection with control, MCPIP1, or D141N lentivirus for 24, 48, 72, and 96 h. (**E,F**) A flow cytometry assay was performed for apoptosis detection of HeLa (**E**) and SiHa (**F**) cells infected with lentivirus of control, MCPIP1, or D141N and treated with 1 \(\mathrm{\SIUnitSymbolMicro g}\)/mL doxycycline for 24 and 48 h, respectively. Representative images and statistical analysis based on three independent experiments are shown. ***\(p<0.001\). that the overexpression of MCPIP1, but not D141N, leads to downregulating XIAP but not IAP-1 or IAP-2 mRNA levels, as well as decreased XIAP and increased cleaved PARP1 protein levels (Figures 3A,B and S2A-D). Then, we addressed the function of XIAP in cervical cancer cell apoptosis. Figure 3: MCPIP1 facilitates cervical cancer cell apoptosis via XIAP. (**A**,**B**) Protein expression levels were detected by Western blotting in HeLa (**A**) or SiHa (**B**) cells, which were infected with a lentivirusus of MCPIP1 or D141N and treated with or without 1 \(\mu\)g/mL doxycycline for 24 and 48 h, respectively. \(\beta\)-actin was used as a loading control. (**C**,**D**) HeLa (**C**) or SiHa (**D**) cells were co-infected with a lentivirus of MCPIP1 and pLVML-3\(\times\)HA-MCS-IRES-Puro (EV) or pLVML-3 xHA-XIAP (XIAP) and treated with or without 1 ug/mL doxycycline for 24 and 48 h, respectively. Then, Western blotting was performed with primary antibodies to MCPIP1, HA tag (HA-XIAP), pro-caspase-3, cleaved caspase-3, total PARP1, and cleaved PARP1. \(\beta\)-actin was used as a loading control. (**E**,**F**) A flow cytometry assay was utilized for the apoptosis assessment of HeLa cells co-infected with a lentivirus of MCPIP1 and XIAP and treated with or without 1 ug/mL doxycycline for 24 h. Representative images (**E**) and statistical analysis based on three independent experiments (**F**) were shown. (**G**,**H**) The floating dead cells were counted from the HeLa (**G**) and SiHa (**H**) cells after co-infection of the MCPIP1 and XIAP lentivirus and treated with or without 1 ug/mL doxycycline for 24, 48, and 72 h. *** \(p<0.001\). As an IAP family member, XIAP is a highly conserved and widely expressed protein consisting of 497 amino acid residues and containing three BIR (baculovirus IAP repeat) domains, a UBA (ubiquitin-associated) domain, and a RING domain [30]. BIR2 and BIR3 domains of XIAP can directly inhibit the activation of caspase-3, -7, and -9, thus exerting its anti-apoptotic activity [29; 30; 31].