KIF20A Promotes The Development Of Fibrosarcoma Via The PI3K-AKT Signaling Pathway

Soft tissue sarcoma（STS）is a rare tumor group that accounts for approximately1%of adult cancers and 10%of pediatric tumor.There are approximately 50 STS subtypes showing heterogeneity in clinical manifestations,histopathological features and molecular signature.At present,surgery plus chemotherapy is still the primary treatment of soft tissue sarcoma.However,there are some problems existed such as postoperative recurrence and metastasis,poor prognosis and poor effect of chemotherapy.With the development of molecular pathology,multiple genes have been confirmed to be associated with the progression of soft tissue sarcomas.However,no clinical biomarkers have been used to guide the prognosis.WGCNA（Weighted Gene Co-expression Network Analysis）classifies genes with similar expression into the same module by dynamic tree cut method.Through the correlation analysis between clinical traits and gene modules,the correlation between modules and clinical traits was explored.Through the network analysis and functional analysis of modules,the functions of gene modules are explained and the hub genes with biological significance are screened out.WGCNA has been used to find gene modules and core genes related to clinical traits or prognosis in a variety of solid tumors,and has been well verified in experiments and clinical practice.However,the application of WGCNA in soft tissue sarcoma is rarely reported.KIF20A（Kinesin Family Member 20A）plays a role in cell mitosis,cell migration,and intracellular transport.A large number of studies have proved that KIF20A is abnormally high expressed in a variety of tumors and shows poor prognosis.KIF20A is closely related to cell cycle and apoptosis,etc.In glioma,downregulation KIF20A causes cell cycle arrest and apoptosis by inhibiting PI3k-AKT signaling pathway.KIF20A antibodies in advanced pancreatic cancer brought the good curative effect,has now entered the stage of clinicalⅡresearch.Previous studies have shown that the expression of KIF20A is closely related to the occurrence,development and prognosis of tumors,but the expression and role of KIF20A in soft tissue sarcomas are still unclear.Based on the background,we used the WGCNA to screen out the hub gene KIF20A,which is associated with the occurrence and development of soft tissue sarcomas.In vitro experiments,we used KIF20A knockdown fibrosarcoma cells as the research object.The effect of KIF20A on gene expression in fibrosarcoma cells was detected by transcriptome sequencing.The effects of KIF20A on cell growth were detected by CCK-8,Western blot,flow cytometry and clonogenesis.The effects of KIF20A on apoptosis were detected by Western blot and flow cytometry.The effects of KIF20A on cell migration and invasion ability were tested by scratch,Transwell migration and invasion experiments.The molecular mechanism was studied by Western blot.In vivo,we used KIF20A knockdown fibrosarcoma cells to construct tumor-bearing mice.The effect of KIF20A on tumor growth was measured by recording tumor size and weight.The effect of KIF20A on proliferation was detected by immunohistochemical staining with Ki67.The effect of KIF20A on tumor metastasis was detected by small animal imaging and HE staining.The research contents of this study include the following aspects:1.Bioinformatics analysis of soft tissue sarcomaWe constructed a gene co-expression network based on gene expression data and using the method of WGCNA.The four hub genes（RRM2,BUB1B,CENPF,KIF20A）that are in the center of the network and highly correlated with prognosis were screened by network structure and function analysis.2.Analysis of the expression of hub genes in fibrosarcomaWe detected the expression of hub genes in human fibrosarcoma cell line HT-1080 by RT-qPCR and Western blot,and found that KIF20A was significantly higher than the control cell line in both gene level and protein level.In the following experiment,we took KIF20A as the target gene to further explore its role and mechanism in fibrosarcoma.3.Effects of KIF20A on gene expression in human fibrosarcoma cellsWe constructed and transcriptome sequenced the KIF20A knockdown HT-1080cell line.The results showed that the expression of a total of 792 genes changed significantly after KIF20A gene was knocked down,and the programmed cell death,apoptosis process,response to organic compounds（chemotherapy drugs,etc.）,nuclear division and other biological processes,as well as PI3K-AKT,mTOR,p53,MAPK,cell cycle and other signaling pathways were affected.These results suggested that KIF20A might affect the occurrence and development of human fibrosarcoma through PI3K-AKT,mTOR,p53,MAPK and other signaling pathways.4.The role and mechanism of KIF20A in human fibrosarcoma（1）In order to determine the effect of KIF20A on the proliferation of human fibrosarcoma cell line HT-1080,we took the knocked down HT-1080 cell line of KIF20A and the control cell line as research objects to detect the changes of cell cycle,cell vitality and cloning ability.The results showed that the cells were arrest in G2/M phase after the down-regulation of KIF20A,the cell vitality was significantly decreased,p21^Waf/Cip1 was significant upregualated,Cyclin A2 and Cyclin E1 were significant upregualated,the ability of cloning formation was significantly reduced,suggesting that down-regulation of KIF20A could inhibit the proliferation of HT-1080cells.（2）We tested the apoptosis of KIF20A knocked down HT-1080 cell line and control cell line.The results showed that the apoptosis of KIF20A knocked down HT-1080 cells was significantly increased,Bcl-2 was significant downregulated,Bax,Cleaved Caspase 9 and Cleaved Caspase 3 were significant upregulated,suggesting that down-regulating KIF20A could promote the apoptosis of HT-1080 cells.（3）In the migration and invasion experiments.It was found that the migration and invasion ability were significantly reduced after the knock down of KIF20A,suggesting that downregulation of KIF20A could inhibit the migration and invasion of HT-1080 cells.（4）Tumor-bearing mice were constructed from KIF20A knockdown HT-1080 and control cell lines,and their tumor size and metastasis were measured.The results showed that the tumor significantly decreased after KIF20A knockdown,the expression of Ki67 in tumor tissues decreased,and the incidence of liver metastasis decreased,suggesting that down-regulating KIF20A could inhibit the growth and metastasis of fibrosarcoma.（5）In order to explore the mechanism of down-regulating KIF20A to inhibit the occurrence and development of human fibrosarcoma,we detected the changes of PI3K-AKT signaling pathway,NF-κB signaling pathway in the KIF20A knockdown HT-1080 cell line and control cell line,and found that the expressions of p-PI3K^Tyr453and p-AKT^ser473 in the PI3K-AKT signaling pathway were decreased after the down-regulation of KIF20A.The expressions of p-IKKα/βand p-p65 in the NF-κB signaling pathway were decreased.These results indicated that the PI3K-AKT signaling pathway was inhibited and the downstream NF-κB signaling pathway was also inhibited after KIF20A was knocked down.5.The effect of Kif20a on fibrosarcoma in miceTo clarify the role of Kif20a in mouse fibrosarcoma,we first examined its expression in mouse fibrosarcoma cell lines WEHI164,MCA101 and MCA207,and found that Kif20a was highly expressed.The mouse soft tissue sarcoma cell line with Kif20a knockdown was successfully constructed by transfection,and the cell proliferation,apoptosis,invasion and migration were detected.Soft tissue sarcoma cell lines of Kif20a knockdown mice were used to construct tumor-bearing mice,and the tumor size and metastasis were observed.The results showed that after the down-regulation of Kif20a,the vitality of mouse fibrosarcoma cells was decreased,cell cycle arrested in G2/M phase,the ability of of clone formation was significantly reduced,apoptosis was increased,invasion and migration ability was reduced,tumor size was decreased,and the expression of Ki67 in tumor tissues was decreased.It suggested that in mouse fibrosarcoma,the down-regulation of Kif20a could still inhibit cell proliferation,migration and invasion,promote cell apoptosis,and inhibit tumor growth.In summary,this study showed that KIF20A was the hub gene in the biological regulatory network of soft tissue sarcoma,which was closely related to the prognosis of soft tissue sarcoma.In the most common histologic subtype,fibrosarcoma.Transcriptome sequencing found that KIF20A was down-regulated,and a variety of genes related to biological processes and signaling pathways were changed.Further studies showed that the low expression of KIF20A in fibrosarcoma inhibited the activity of PI3K-AKT,and other signaling pathways such as downstream NF-κB,cell cycle and apoptosis were also altered,thus inhibiting tumor proliferation,migration,invasion and metastasis,and promoting tumor apoptosis.This study clarified the expression of KIF20A in fibrosarcomas,and discussed its role in fibrosarcomas and related mechanisms,suggesting that KIF20A was a potential molecular marker and therapeutic target for the treatment of fibrosarcomas,providing new ideas for the treatment of fibrosarcoma.