Study Of FSCN1 Promoting Esophageal Carcinoma Progression Through Downregulating PTK6 Via Its RNA-binding Protein Effect

Objective:Esophageal cancer has the seventh highest incidence rate and the sixth highest mortality rate in the world in 2020.Histologically there are two main types: esophageal adenocarcinoma(EAC)and esophageal squamous cell carcinoma(ESCC).Among them,ESCC is the most common esophageal cancer worldwide,and its incidence is highest in Asian/Oriental countries.Early diagnosis of esophageal cancer can improve the chance of cure.Despite the great development of various means such as surgery,targeted drugs and neoadjuvant therapy in recent years,the specific causative mechanism of esophageal cancer is very complex and still unclear,and its late prognosis is still very poor.Therefore,it is very important to explore the pathogenesis of esophageal cancer for early diagnosis and development of specific drugs or treatments.The actin-bundling function of fascin actin-bundling protein 1(FSCN1)has been studied in the development of cancer,but the function of its newly discovered RNA-binding protein has not been reported.In this study,pre-experiments revealed that FSCN1 does have an RNA-binding protein function and can influence the development of esophageal cancer,and this new finding is completely different from the previous actin-binding function of FSCN1.Thus we propose the scientific hypothesis that FSCN1 plays a role in the development of esophageal carcinogenesis through its RNA-binding protein function.In this study,this scientific hypothesis was explored by whole-genome expression sequencing,RIP,in vivo and in vitro experiments.Method:(1)Whole-genome expression sequencing and RNA immunoprecipitation were used to study the transcriptome changes after interfering with FSCN1 in ESCC cell lines,and the RNAs binding to FSCN1 to screen the target genes.(2)Real-time fluorescence quantitative PCR and Western Blot were used to validate the screening results.(3)To interfere with FSCN1 and the corresponding target genes in ESCC cell lines cultured in vitro using overexpression plasmids and small interfering RNAs in combination with CCK8,Ed U staining,TUNEL staining,real-time fluorescence quantitative PCR,Western Blot,scratch assay,Transwell assay,clone formation assay and other techniques to explore the regulatory mechanism.(4)To verify the results of the in vitro experiments by using nude mice for the tumor-bearing experiment.Results:(1)Whole-genome expression sequencing and RNA immunoprecipitation revealed that FSCN1 was able to bind to PTK6 pre-m RNA and inhibit PTK6 protein expression.Validation of FSCN1 small interfering RNA and overexpression plasmids in two cell lines of ESCC revealed that interfering FSCN1 could promote PTK6 protein expression,while overexpressing FSCN1 did the opposite.(2)The corresponding intron region stage mutants of the pre-m RNA of PTK6 were constructed,and the T2 region was found to be the region where the pre-m RNA of PTK6 binds to FSCN1.In an in vitro cultured ECA-109 cell line,expression of PTK6-T2 in the cell line was found to block the inhibitory effect of FSCN1 on PTK6 protein expression.(3)In an in vitro cultured ECA-109 cell line,expression of PTK6-T2 was found to block the enhancement of cell proliferation,reduction of apoptosis,enhancement of migration and enhancement of invasion caused by FSCN1 overexpression.(4)FSCN1 was found to cause elevation of p AKT and p GSK3?downstream of PTK6 by inhibiting PTK6 expression.(5)In nude mice tumor experiments,FSCN1 overexpression was found to cause a significant increase in tumor volume and weight,which was significantly blocked by expression of PTK6-T2.(6)The tumor tissues from the tumor-bearing assay showed that FSCN1 overexpression significantly reduced PTK6 protein levels,kept apoptosis and promoted cell proliferation,while PTK6-T2 expression significantly blocked this phenomenon.Conclusion:This study confirmed a novel role of FSCN1: acts as an RNA-binding protein that binds to the T2 position of the pre-m RNA of PTK6 and inhibits the protein expression of PTK6,thus enhancing the downstream AKT/GSK3?signaling pathway,which plays an important role in the progression of esophageal cancer.