Study On The Effect Of CDK12 On Biological Behavior Of Osteosarcoma And Its Clinical Application Value

BackgroundOsteosarcoma is the most common primary mesenchymal tissue-derived highly malignant bone tumor,accounting for 35%of primary bone malignancies.Its main characteristic is the formation of immature bone-like tissue structure by tumor cells.The global annual incidence of osteosarcoma is 3.4 per 1 million people.It has a bimodal age distribution,with the highest incidence in adolescents aged 15-19 years,followed by older adults over 60 years of age.The incidence is slightly higher in males than in females,with a ratio of 1.4 males to 1 females.Osteosarcoma usually occurs in the blood-rich epiphysis of the long diaphysis of the extremities.The most common sites are the distal femur,proximal tibia,and proximal humerus.Prior to the 1970s,amputation was the predominant mode of treatment,but most patients developed lung metastases within 6-12 months after amputation.Therefore,the 5-year survival rate of osteosarcoma patients after simple amputation was only about 20%.With the widespread application of chemotherapy in patients with osteosarcoma,the current treatment mode of "neoadjuvant chemotherapy + surgery+postoperative adjuvant chemotherapy" is mainly used,and the 5-year survival rate of patients has also jumped from 20%to 60-70%.However,in the past 40 years,despite’ the continuous optimization of chemotherapy regimen and surgical techniques,the treatment of osteosarcoma has fallen into a slow plateau,and the 5-year survival rate of patients has been hovering around 60%-70%.The reason is mainly attributed to the high metastasis of osteosarcoma and the resistance to traditional chemotherapy drugs.Therefore,the research of novel targets of osteosarcoma and the development of targeted drugs have become a hot spot.Protein kinases are highly expressed and over-activated in malignant tumor cells because they are involved in various cellular processes,and play an indispensable role in the occurrence and development of tumors.Therefore,protein kinase as a promising molecular target for tumor therapy has attracted more and more attention.Among them,Cyclin-dependent kinases(CDKs)are an important component of antitumor drug research.Currently,CDKs are subdivided into two main categories:cell cycle related kinases,which regulate the various phases of the cell cycle(including CDK1,CDK2,CDK3,CDK4,CDK5,CDK6,CDK14,CDK15,CDK16,CDK17,and CDK18);And transcription-related kinases that regulate gene transcription(including CDK7,CDK8,CDK9,CDK10,CDK11,CDK12,CDK13,CDK19,and CDK20).Among them,a variety of CDK inhibitors targeting CDK4 and CDK6(such as Pabocinib,Rebocilil,abesili)have been widely used in clinical practice and achieved good efficacy in the treatment of a variety of malignant tumors.Studies have also shown that CDK12 can drive and maintain the growth of various tumor cells by regulating DNA damage repair and other signaling pathways.So,could CDK 12 be a new target for osteosarcoma treatment in the future?PurposeTherefore,this study firstly analyzed the expression of CDK 12 in different types of malignant tumors by big data bioinformatics,and explored the relationship between the expression level of CDK 12 and the prognosis of osteosarcoma patients,so as to evaluate its value as a prognostic indicator of osteosarcoma patients.Then,the effect of CDK12 expression level on the biological behavior of osteosarcoma cells and the possible molecular mechanism were analyzed.Finally,the correlation between the expression level of CDK12 and the metastasis,recurrence,drug resistance and prognosis of osteosarcoma patients was summarized,so as to discuss its clinical application value,and provide certain theoretical basis for the subsequent clinical treatment of osteosarcoma by targeting CDK12.MethodsPart ⅠFirstly,sequencing information and clinical data of all cancer patients were obtained from the TCGA database,and the correlation between CDK12 expression and prognosis of cancer patients was analyzed by using TIMER 2.0 database and GEPIA 2.0 database.Then,sequencing data and clinical information of 85 patients with osteosarcoma were downloaded from the TARGET database to explore the relationship between CDK 12 and the prognosis of patients with osteosarcoma,and to evaluate its value as a prognostic indicator for patients with osteosarcoma.Part ⅡFirstly,the expression of CDK12 in 2 human osteoblast cell lines and 6 human osteosarcoma cell lines was detected by western blotting.Secondly,the sublocalization of CDK12 in human osteosarcoma cells was detected by immunofluorescence assay.Then,specific small interfering RNA targeting CDK12 was constructed to detect the changes in biological behavior of human osteosarcoma cells after CDK12 gene silencing.Finally,Western blotting was used to detect the expression of related proteins after CDK12 gene was silenced.Part ⅢFirstly,CCK-8 method,scratch repair experiment,2D plate cloning experiment and 3D ball forming experiment were used to determine the changes of biological behavior of human osteosarcoma cells after small molecule compound THZ531 inhibited CDK12 activity.Then,Western blotting was used to detect the expression of related proteins after the inhibition of CDK12 functional activity.Part ⅣFirstly,the expression of CDK12 in 8 human osteosarcoma samples was detected by western blotting.Secondly,the expression abundance of CDK12 in tissue microarray chips constructed from tissue samples of 70 patients with osteosarcoma was detected by immunohistochemistry to analyze the relationship between the expression level of CDK12 and the clinical characteristics of malignant progression(metastasis,recurrence and drug resistance)and prognosis of patients,and to explore its clinical application value.Finally,the organoid model of osteosarcoma was constructed to simulate in vivo experiments to further verify the possibility of CDK12 as a potential new target for the treatment of osteosarcoma.ResultsPart Ⅰ(1)CDK12 is highly expressed in a variety of different types of malignant tumors,and patients with high expression of CDK12 have a poor prognosis.(2)The expression level of CDK12 was positively correlated with the expression of ATR and CHK1 in DNA damage repair pathways.(3)Osteosarcoma patients.with high expression of CDK12 also have a poor prognosis,and CDK12 may become a prognostic indicator for osteosarcoma patients.Part Ⅱ(1)After the silencing of CDK12 gene in human osteosarcoma cells,the growth and proliferation of osteosarcoma cells were inhibited,and the morphology of osteosarcoma cells was significantly changed.(2)The sublocalization of CDK12 in human osteosarcoma cell lines is mainly in the nucleus.(3)After CDK12 expression decreased,pATR and pCHK1 protein levels decreased,while yH2AX protein levels increased.Part Ⅲ(1)Small molecule compound THZ531 inhibited the growth,proliferation,migration,2D clonogenesis and 3D pellet formation of human osteosarcoma cells after inhibiting CDK12 activity.(2)After CDK12 was inhibited by THZ531,pATR and pCHK1 protein levels decreased,while yH2AX protein levels increased.Part Ⅳ(1)CDK12 was differentially expressed in clinical tissue samples of patients with osteosarcoma.(2)The expression level of CDK12 in primary osteosarcoma was significantly lower than that in metastatic and recurrent osteosarcoma.Patients with high CDK12 expression were more likely to develop drug resistance;Patients with high CDK12 expression had a poor prognosis for survival.(3)Osteosarcoma organoid model confirms that CDK12 may be a potential new target for future treatment of osteosarcoma patients.Conclusions(1)Osteosarcoma patients with high CDK12 expression have a poor prognosis for survival.(2)After inhibiting the functional activity of CDK12 at gene level and protein level,small interfering RNA and small molecule compound THZ531 may activate DNA damage repair response by participating in or affecting ATR/CHK1 signaling pathway,thereby reducing the DNA double strand break of osteosarcoma cells and maintaining the genomic stability of osteosarcoma cells.Which in turn affects the biological behavior of osteosarcoma cells.(3)The expression level of CDK12 was positively correlated with metastasis,recurrence,drug resistance and poor prognosis of osteosarcoma patients.(4)CDK12 may be a prognostic indicator for patients with osteosarcoma and a potential new target for future treatment of osteosarcoma.