MIB1 Upregulates IQGAP1 And Promotes Pancreatic Cancer Progression By Inducing ST7 Degradation

Objective: Pancreatic cancer is a malignant tumor of digestive tract with insipid onset and rapid development.At present,there is no effective treatment for pancreatic cancer except radical resection.The genetic heterogeneity of pancreatic cancer is closely related to its highly malignant biological behavior.MIB1(E3 Ubiquitin Ligase Mind Bomb 1)is an E3 ubiquitin ligase that is involved in a variety of cellular biological processes.Our previous studies have confirmed that MIB1 is highly expressed in pancreatic cancer cells and tissues,but the role of MIB1 in the progression of pancreatic cancer is still unclear.The purpose of this study was to investigate the role of MIB1 in regulating the development and progression of pancreatic cancer and to clarify its mechanism.Methods:(1)The mRNA level of MIB1 in pancreatic cancer and normal pancreatic tissues was evaluated by TCGA database.(2)The protein level of MIB1 in pancreatic cancer specimens was detected by tissue microarray and IHC.(3)The tissue samples of pancreatic cancer in our hospital were collected and the level of MIB1 protein in pancreatic cancer tissue was analyzed by Western Blot.(4)Silencing the expression of MIB1 with two different sh RNA in Bx PC-3 and SW1990 cells.The effects of knockdown of MIB1 gene on the proliferation and invasion of pancreatic cancer cells were investigated by MTS test,clone formation test and tumor formation test in nude mice.(5)To explore the effect of overexpression of MIB1 on the proliferation and invasion of Bx PC-3 and SW1990 pancreatic cancer ells.(6)Immunoprecipitation(Co-IP)technology and protein profilometry were used to identify and verify the protein ST7 interacting with MIB1.(7)The adjacent junction test(Proximity Ligation Assay,PLA)was used to confirm the interaction between MIB1 and ST7 in Bx PC-3 cells.(8)After MIB1 and ST7 were silenced by si RNA/sh RNA or overexpressed by plasmids,the protein and transcription levels of MIB1 and ST7 were detected by Western Blot and RT-PCR,respectively.(9)The immunoprecipitation technique of protein ubiquitination was used to verify whether MIB1 could directly participate in ST7 ubiquitination and to detect the change of the half-life of ST7 protein after regulation.(10)The RING structure mutant of MIB1 was constructed and the E3 ligase activity of RING structure was verified.(11)The relationship between MIB1 and ST7 was determined by pancreatic cancer tissue microarray.(12)To investigate the effects of silence or non-silence and overexpression of ST7 on the proliferation and invasion of pancreatic cancer cells.(13)After silencing MIB1 and ST7 alone or in combination,their effects on the proliferation of pancreatic cancer cells were investigated in vivo and in vitro.(14)RNAseq analysis was carried out by using Bx PC-3 with or without silencing ST7,and KEGG pathway enrichment analysis of differentially expressed genes was performed to find significant regulatory pathways and genes.(15)After silencing IQGAP1 and ST7 alone or in combination,Western Blot and RTPCR assays were used to detect the changes of protein and m RAN levels,and MTS assay was used to explore the growth and proliferation ability of pancreatic cancer cells.Results:(1)The mRNA level of MIB1 in pancreatic cancer and normal pancreatic tissues was evaluated by TCGA database,and it was found that the expression of MIB1 was up-regulated in 16% of pancreatic cancer.(2)The protein level of MIB1 in pancreatic cancer was detected by tissue microarray(25 cases of normal pancreatic tissue and 35 cases of PDAC)and IHC.It was found that the level of MIB1 protein in pancreatic cancer tissue was significantly higher than that in normal pancreatic tissue(P < 0.001).(3)Western blot analysis of samples from our hospital confirmed that the level of MIB1 protein in pancreatic cancer tissue(n=12)was higher than that in adjacent non-malignant tissues(n=12).(4)In vivo and in vitro experiments confirmed that knockdown of MIB1 gene inhibited the proliferation and invasion of pancreatic cancer cells(PANC-1 and Bx PC-3).(5)Overexpression of MIB1 gene promoted the proliferation and invasion of pancreatic cancer cells(PANC-1 and Bx PC-3)in vivo and in vitro.(6)The interesting interaction protein ST7,was found by protein spectrum analysis,and the interaction between MIB1 and ST7 was further confirmed by co-IP.(7)PLA confirmed the direct interaction between MIB1 and ST7 in Bx PC-3 cells.(8)It was found that MIB1 gene knockout increased the protein level of ST7 in pancreatic cancer cells Bx PC-3,SW1990 and ASPC-1,but did not increase the mRNA level of ST7.On the contrary,the overexpression of MIB1 decreased the level of ST7 protein in pancreatic cancer cells,and the mRNA level of ST7 remained unchanged.(9)Using protein half-life assay,it was found that the inhibition of MIB1 prolonged the half-life of ST7 protein in Bx PC-3 cells.(10)The expression of MIB1-Δ RING mutant did not change the ubiquitination level of ST7 in Bx PC-3 cells,but overexpression of wild type MIB1 increased the ubiquitination level of ST7.(11)It was found that there was a negative correlation between the protein levels of ST7 and MIB1 in pancreatic cancer tissue microarray(n = 35).(12)MTS and clone formation experiments showed that two different kinds of sh RNA silencing ST7,significantly promoted the growth and invasion ability of Bx PC-3 and SW1990 cells in vitro.(13)ST7 silencing promotes tumor growth,while MIB1 silencing inhibits the proliferation of Bx PC-3 cells.The combined silencing of ST7 and MIB1 attenuated the down-regulated anti-proliferation effect of MIB1 in vitro and in vivo.(14)RNA-seq analysis and KEGG pathway enrichment analysis of differentially expressed genes showed that ST7 gene knockout upregulated many genes involved in actin cytoskeleton pathway.Among them,the silence of ST7 significantly upregulated the expression of IQGAP1.(15)Western blotting and q RT-PCR analysis confirmed that ST7 gene knockout increased the mRNA and protein levels of IQGAP1.Conclusion: MIB1 is highly expressed in pancreatic cancer tissues and cells,and the high expression of MIB1 indicates a poor prognosis in pancreatic cancer patients.MIB1 can promote the growth and invasion of pancreatic cancer cells in vivo and in vitro.The N-terminal related domain of MIB1 is responsible for the interaction between MIB1-ST7,and its RING structure has E3 ligase activity to degrade ST7 protein.MIB1 promotes pancreatic cancer progression by targeting ST7 degradation.IQGAP1 plays an important role in the anti-tumor effect of ST7.Therefore,this study suggests that MIB1 targets ST7 for degradation,thereby upregulating the expression level of IQGAP1 and promoting the progression of pancreatic cancer.