Examination of the functions and mechanism associated with the involvement of KCP in mediating paclitaxel resistance in cervical squamous cell carcinoma cells
Authors: He Yue , Xu Jian-Qing , Zhang Jing-Jing , Ji Chen , Liu Yang , Wang Yun-Fan , Wang Ming , Wang Yan , Wu Yu-Mei 
Published: June 2023

Objective
To elucidate the mechanism of Kielin/chordin-like protein (KCP) involved in the resistance of cervical cancer cells to paclitaxel and to provide a new target for the precise treatment of patients with cervical cancer resistant to paclitaxel.
Method
A cervical squamous carcinoma cell line (SiHa) with KCP knockout was constructed, treated with paclitaxel, and then compared with normal control SiHa cells (NC cells). Cell function experiments, such as plate imaging time, 3-(4, 5-Dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) cell proliferation, and FACS-based single staining to detect apoptosis were performed. Then, the downstream mechanism of KCP-mediated resistance to paclitaxel was examined using human gene chip detection and IPA bioinformatics analysis. qPCR analysis was used to validate its downstream genes.
Results
① Functional studies on SiHa cells after KCP knockout (sgRNA) showed that the knockout inhibited cloning and proliferation of SiHa cells after treatment with paclitaxel, reduced drug resistance of SiHa cells to paclitaxel, and decreased the resistance to paclitaxel (P < 0.05). ② Using the whole human genome microarray, a total of 491 differential genes were identified upon a comparison between the KCP knockout group and the NC group in SiHa cells. IPA-based bioinformatics analysis of classical signalling pathways showed that the interferon signalling pathway was significantly activated, and four differential genes, namely, IFIT1, OAS1, G1P2, and IFIT3, were important factors associated with this pathway. IPA-based bioinformatics analysis of upstream regulators showed that SPI1 was strongly activated and that SPI1 inhibited CCND1 and activated PML and CEBPA, which was consistent with gene chips showing CCND1, PML, and CEBPA expression after KCP knockout. IPA-based bioinformatics analysis of the changes in differential genes associated with disease and function showed that genes, such as PML and SERPINB3/4, were key genes associated with significant activation of apoptosis of the fibroblast cell lines. Analysis of the top-ranked regulatory network in terms of regulatory effects demonstrated that the dataset could be attributed to the inhibitory effect of differential genes, such as ACKR2 and SPI1, which was mediated by genes such as PML, on regulators such as replication of murine herpesvirus 4 and replication of vesicular stomatitis virus, thereby affecting the functions of KCP. ③ A total of 30 differential genes associated with tumour cell proliferation during the examination of diseases and functions were identified based on the differential gene results of the gene microarray and IPA analysis. qPCR was used to verify changes in the aforementioned gene after KCP knockout. Here, SERPINB3 and CEBPA expression was significantly lower and higher compared to that in the control group. SERPINB3 and CEBPA might be the downstream genes of KCP.
Conclusion
We observed KCP could act as an upstream gene via SPI1 on downstream genes such as CCND1, PML, and CEBPA, to participate in the regulatory network of replication of murine herpesvirus 4 and replication of vesicular stomatitis virus. KCP could also act positively on the downstream gene of SERPINB3 and negatively on the downstream gene of CEBPA to participate in the proliferative function of cervical squamous carcinoma and affect the resistance of cervical carcinoma to paclitaxel.