## 2 Results  
### MCPIP1 Promotes the Apoptosis of Cervical Cancer Cells  
MCPIP1 has been identified to function as a tumor suppressor or promoter in multiple cancers, but its function in cervical cancer remains unexplored. To investigate the impact of MCPIP1 on cervical cancer development, we first addressed whether MCPIP1's overexpression affects cervical cancer cells' growth. We infected the HeLa and SiHa cells with lentivirus of pLYX-TetOne-Puro (control) or pLYX-TetOne-MCPIP1 (MCPIP1) and induced the expression of MCPIP1 with 1 ug/mL doxycycline (Dox). Our cell counting assay and CCK-8 assay demonstrated that compared to the control group, MCPIP1 overexpression impeded the proliferation of both HeLa and SiHa cells (Figure 1,B). Notably, compared to 0 and 24 h, the cell number of HeLa cells decreased at 48 and 72 h after Dox-induced expression of MCPIP1. Therefore, we speculate that MCPIP1 leads to HeLa cell apoptosis. Figure 1: MCPIP1 promotes the apoptosis of cervical cancer cells. (**A**,**B**) The overexpression of MCPIP1 suppressed the proliferation of HeLa (**A**) and SiHa (**B**) cells, as assessed by the cell counting assay and CCK-8 assay. Data have been represented as mean \(\pm\) SD. n = 3. (**C**,**D**) Flow cytometry assay for apoptosis detection of HeLa (**C**) and SiHa (**D**) cells infected with lentivirus of control or MCPIP1 and treated with 1 ug/mL doxycycline for 24 h and 48 h, respectively. Representative images were shown in the left panel. The apoptosis rates from three independent experiments were shown in the right panel. (**E**,**F**) Protein expression levels of MCPIP1, pro-caspase-3, cleaved caspase-3, total PARP1,  
and cleaved PARP1 were detected by Western blotting in HeLa cells (**E**) infected with lentivirus of control or MCPIP1 and treated with 1 ug/mL doxycycline for 12 and 24 h, respectively, or in SiHa cells (**F**) infected with lentivirus of control or MCPIP1 and treated with 1 ug/mL doxycycline for 24 and 48 h, respectively. \(\beta\)-actin was used as a loading control. ***\(p<0.001\). Apoptosis is a pivotal pathway in the pathogenesis of many diseases, including cervical cancer [24]. Pro-caspase in cells is cleaved into active forms of caspase during apoptosis, such as cleaved caspase-3, which functions as the executor of apoptosis. PARP1 is a substrate of activated caspases-3 in caspase-dependent apoptosis [25]. Cleavage of PARP1 protein by caspase enzymes leads to its inactivation, which prevents DNA damage repair and promotes caspase-mediated DNA fragmentation in apoptosis. To further ascertain our hypothesis that MCPIP1 regulates cervical cancer cell apoptosis, we induced the expression of MCPIP1 in HeLa and SiHa cells with doxycycline after lentivirus-mediated infection of the control or MCPIP1 and performed the flow cytometry assay. We found that compared to the control group, MCPIP1 overexpression results in a significantly higher apoptosis rate of cervical cells (Figure 1C,D). Furthermore, our Western blot analysis showed that the protein level of pro-caspase-3 decreased, but cleaved caspase-3 and cleaved PARP1 increased in MCPIP1-overexpressing HeLa and SiHa cells (Figure 1E,F), which further validated the pro-apoptotic role of MCPIP1 in cervical cancer cells. The Apoptosis-Promoting Effect of MCPIP1 on Cervical Cancer Cells Depends on Its Endoribonuclease Activity  
Utilizing its endoribonuclease activity, MCPIP1 plays multiple roles, such as the regulation of inflammation, apoptosis, adipogenesis, angiogenesis, and tumor progression [26; 27]. To determine whether the endonuclease activity contributes to the apoptosis-promoting effect of MCPIP1 in cervical cancer cells, we mutated the pLVX-TetOne-MCPIP1 plasmid by changing the aspartic acid (D) at 141 of the PIN domain into asparagine (N), generating pLVX-TetOne-MCPIP1-D141N (D141N), which expresses mutant MCPIP1 that almost lost the endonuclease activity [7].