The Roles Of FXYD3 Alternative Splicing In The Development Of Cervical Cancer

Background:Cervical cancer is one of the most common malignant diseases in women.Alternative splicing is implicated in the tumorigenesis and progression of cervical cancer.FXYD3,a small,single-span membrane protein,has been identified as specific regulator of Na-K-ATPase and involves in several important cellular activities.However,the roles of FXYD3,especially its alternative splicing,in cervical cancer is still unclear.Methods:1.Cervical cancer tissues and cancer-adjacent tissues samples were collected to perform transcriptome sequencing.The screened differentially expressed genes were verified by qRT-PCR and conventional PCR.2.FXYD3-L and FXYD3-S lentiviral overexpression plasmid and CRISPR-Cas9 knockout plasmid were constructed.Then,cervical cancer cell was infected with overexpression lentiviral supernatant or knockout lentiviral supernatant.3.MTT assay and plate cloning experiment were used to detect the proliferation ability of cervical cancer cell.Scratch wound-healing assay was used to detect the migration ability of cervical cancer cell.Subcutaneous xenograft tumor model was used for in vivo tumor growth assays.4.RNA sequencing was applied to analyze the different effects of FXYD3-L and FXYD3-S overexpression on the expression of genes in cervical cancer cell.Intracellular iron was quantified using the iron assay kit,and expression of iron regulatory proteins were detected by Western blotting.5.Immunohistochemistry was conducted to evaluate the expression of FXYD3 and FXYD3-L in human cervical cancer samples.Result:1.We identified that FXYD3 had two isoforms,FXYD3-L and FXYD3-S,in cervical cancer tissues.2.We constructed FXYD3-L and FXYD3-S overexpressing stable cervical cancer cell line and FXYD3-KO stable cervical cancer cell line.3.FXYD3-S promoted proliferation and invasion of cervical cancer cell,while FXYD3-L inhibited proliferation of cervical cancer cell.4.FXYD3-L regulated the proliferation of cervical cancer through NF-кB signaling pathway.FXYD3-S promoted tumor growth and cancer cell invasion through regulating extracellular matrix and angiogenesis.FXYD3-L can upregulate KEAP1 expression and downregulate SLC7A11,GPX4 and FTH1,subsequently inducing ferroptosis in cervical cancer cell.5.FXYD3 was high expression in high clinical stage,vascular invasion and tumor recurrence.However,FXYD3-L expression was inversely correlated with tumor aggressiveness and poor prognosis.Conclusion:1.FXYD3-S promotes the growth and invasion of cervical cancer,but FXYD3-L inhibits the growth of cervical cancer.2.The FXYD3-L overexpression induces ferroptosis in cervical cancer cell.3.High FXYD3 expression is related to the poor prognosis of the patients.4.High FXYD3-L expression is correlated with the favorable prognosis of the patients.