Molecular Mechanism Of MYH9 Promoting Cell Invasion And Migration In Esophageal Squamous Carcinoma Cells

Esophageal squamous cell carcinoma(ESCC)is one of the common malignancies in China,and its incidence and mortality rate ranked third and fourth in malignant tumors,respectively.Invasion and metastasis of tumors is one of the important causes of treatment failure and death of patients with ESCC.Therefore,it is urgent to study the molecular mechanism of invasion and metastasis of esophageal cancer cells and improve the classification and treatment of the disease.In the previous work,we found that protein tyrosine phosphatase 1B(PTP1B)interacted with myosin heavy chain 9(MYH9)to dephosphorylate the Y1408 site of MYH9 tail domain(TD),which promoted the transfer of transcription factor SP1 from cytoplasm to nucleus and upregulated the expression of epidermal growth factor receptor(EGFR),thus enhancing the invasion and migration of ESCC cells.However,whether there is a direct interaction between PTP1B and MYH9 tail domain,and what are the downstream molecules affected by MYH9 tail domain are still unclear.In this study.MYH9 siRNA was first used to verify the effect of MYH9 on the invasion and migration of ESCC cells.Transwell assay and wound healing assay showed that knockdown of MYH9 significantly inhibited the invasion,migration and wound healing of KYSE510 cells.Furthermore,the coding sequence(CDS)of MYH9 tail domain was amplified by reverse transcription PCR from cDNA of KYSE510 cells.The recombinant plasmid p3×FLAG-MYH9(TD)-Y1408(F/D)of the Y1408 mutant(Y1408F and Y1408D)was constructed and transformed into human embryonic kidney 293FT cells.Recombinant protein 3×FLAG-MYH9(TD)-Y 1408(F/D)was expressed in 293FT cells and purified by FLAG tag affinity purification.The direct interaction between PTP1B and MYH9 was further verified by using active tyrosine phosphatase PTP1B and the aforementioned recombinant protein.In order to further study the molecular mechanism of MYH9 promoting invasion and migration of ESCC cells,we used co-immunoprecipitation(Co-IP)and immunofluorescence co-localization technology to verify that there was no interaction between MYH9 and the downstream transcription factor SP1 in KYSE510 cells,suggesting that MYH9 may promote the transfer of SP1 from cytoplasm to nucleus through other interacting molecules.Subsequently,we identified the interacting proteins of MYH9 tail domain(aa837-aa1960)in KYSE510 using GST pull-down combined with liquid chromatography-tandem mass spectrometry(LC-MS/MS)technology.The results showed that there were 72 potential proteins interacting directly or indirectly with MYH9 tail domain in KYSE510.Gene Ontology(GO)enrichment analysis showed that potential interacting proteins are involved in a variety of biological processes,including JAK-STAT signaling pathway,cell adhesion regulation,NIK/NF-kappa B signaling pathway,cell cycle checkpoint,membrane lipid raft formation,DNA transcription initiation,and RNA processing.Co-IP analysis further confirmed that the PC4 and SFRS1 interacting protein 1(PSIP1)and BRG1(encoded by SWI/SNF related,matrix associated,actin dependent regulator of chromatin,subfamily a,member 4[SMARCA4])interact with MYH9.The present work indicates that knockdown of MYH9 remarkably inhibit the invasion and migration of ESCC cells.The tail domain of MYH9 TD mediates various interactions with cell movement-related proteins,among which the interaction of MYH9 with PSIP1 and BRG1 has been verified.These results provide candidate molecules for further studying the mechanism of invasion and migration of ESCC cells,and provide potential targets for the treatment of esophageal squamous carcinoma.