Su Bingsen, Zhong Meifeng, Zhang Yuzhao, Wu Kunhe, Huang Qiyuan, Zhu Chuiyu, Zeng Tao
Zhongshan Torch Development Zone People's Hospital, Zhongshan, Guangdong 528437, China.
Department of Medical Laboratory, Affiliated Hospital of Guangdong Medical University, Zhanjiang, Guangdong 524000, China.
J Oncol. 2022 Jul 20;2022:3156968. doi: 10.1155/2022/3156968. eCollection 2022.
The treatment of cervical cancer in the late stage is still quite challenging, because of nonspecificity in conventional therapies and the lack of molecular targeted drugs. It is necessary to find novel biomarkers for cervical cancer treatment.
In the present study, cervical cell lines HeLa and SiHa with kin17 knockdown were constructed by transfection of the recombinant lentiviral vector carrying KIN17 siRNA and screened by puromycin. The established cells with kin17 knockdown were determined by fluorescence observation and western blotting. Cell apoptosis and the mitochondrial membrane potential (MMP) were detected by flow cytometry. The activity of caspase 3 enzyme was tested by spectrophotometry. The expression profile of apoptosis-associated proteins was analyzed by western blotting. Finally, we used bioinformatics and proteomic data to analyze KIN-related genes in cervical cancer.
The results showed high fluorescent positive rates (>90%) and high gene silencing efficiency (>65%) in HeLa and SiHa cells transfected with gene silencing vectors. Moreover, kin17 deficiency decreased the MMP and increased the apoptosis rates in HeLa and SiHa cells, respectively. Furthermore, knockdown of kin17 enhanced the activity of caspase 3 enzyme, increased the expression of cleaved PARP and Bim, while decreasing the expression of Bcl-xL and phosphorylated BAD in HeLa and SiHa cells. Identification of KIN-related prognostic genes in cervical cancer revealed that a total of 5 genes (FZR1, IMPDH1, GPKOW, XPA, and DDX39A) were constructed for this risk score, and the results showed that CTLA4 expressions were negatively correlated with the risk score.
Our findings demonstrated that kin17 knockdown facilitates apoptosis of cervical cancer cells by targeting caspase 3, PARP, and Bcl-2 family proteins. Besides, kin17 could regulate cancer cell apoptosis through the mitochondrial pathway and could be used as a novel therapeutic target for the regulation of cell apoptosis in cervical cancer.
晚期宫颈癌的治疗仍然颇具挑战性，这是由于传统疗法缺乏特异性且缺乏分子靶向药物。因此，有必要寻找用于宫颈癌治疗的新型生物标志物。
在本研究中，通过转染携带KIN17小干扰RNA的重组慢病毒载体构建了kin17基因敲低的宫颈癌细胞系HeLa和SiHa，并使用嘌呤霉素进行筛选。通过荧光观察和蛋白质印迹法对构建成功的kin17基因敲低细胞进行鉴定。采用流式细胞术检测细胞凋亡和线粒体膜电位（MMP）。通过分光光度法检测半胱天冬酶3酶的活性。利用蛋白质印迹法分析凋亡相关蛋白的表达谱。最后，我们使用生物信息学和蛋白质组学数据来分析宫颈癌中与KIN相关的基因。
结果显示，转染基因沉默载体的HeLa和SiHa细胞具有较高的荧光阳性率（>90%）和较高的基因沉默效率（>65%）。此外，kin17基因缺失分别降低了HeLa和SiHa细胞的MMP并增加了细胞凋亡率。此外，kin17基因敲低增强了HeLa和SiHa细胞中半胱天冬酶3酶的活性，增加了裂解的PARP和Bim的表达，同时降低了Bcl-xL和磷酸化BAD的表达。对宫颈癌中与KIN相关的预后基因进行鉴定发现，共构建了5个基因（FZR1、IMPDH1、GPKOW、XPA和DDX39A）用于该风险评分，结果显示CTLA4表达与风险评分呈负相关。
我们的研究结果表明，kin17基因敲低通过靶向半胱天冬酶3、PARP和Bcl-2家族蛋白促进宫颈癌细胞凋亡。此外，kin17可通过线粒体途径调节癌细胞凋亡，并可作为调节宫颈癌细胞凋亡的新型治疗靶点。
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