Long Non-Coding RNA USP30-AS1 Promotes The Malignant Progression Of Cervical Cancer Through Wnt Pathway Mediated By USP30

Part Ⅰ:LncRNA USP30-AS1 is highly expressed in cervical cancerExperimental purpose:The high expression of lncRNA in cervical cancer was searched through bioinformatics website and its influence on cervical cancer cell phenotype was evaluatedMaterials and methods:(1)Screening lncRNA associated with cervical carcinomaLncRNA USP30-AS1 was predicted through GEPIA database and UCSC-GTEx database,which is highly expressed in cervical carcinoma tissues.The expression of lncRNA USP30-AS1 in human cervical cancer tissues and adjacent tissues were detected by qRTPCR.The expression of lncRNA USP30-AS 1 in normal cervical epithelial cells(HcerEpic)and cervical cancer cells(C33A,CaSki,SiHa,HeLa)were detected by qRT-PCR to determine the expression of lncRNA USP30-AS1 were significantly high in cervical carcinoma cells.(2)Establishment of cervical carcinoma cell line model with stably knockdown of lncRNA USP30-AS1Firstly,two different sh-RNA sequences of lncRNA USP30-AS1 were designed according to lncRNA USP30-AS1 sequence in NCBI.Then the lentiviral vectors sh/USP30AS1#1,sh/USP30-AS1#2 and the blank control sh/NC with puromycin resistance were constructed.After transfecting those vectors into SiHa and HeLa cell lines,cells with stable puromycin resistance were screened and lncRNA USP30-AS 1 expression were also detected with qRT-PCR.(3)lncRNA USP30-AS1 regulates proliferation,metastasis,invasion,EMT and apoptosis of cervical carcinoma cells in vitroEdU staining,colony formation,TUNEL staining,flow cytometry,transwell and western bolt assays were conducted to test the proliferation,apoptosis,metastasis,invasion and EMT of cervical carcinoma cells which were transfected with sh/NC,sh/USP30-AS1#1 and sh/USP30-AS1#2,respectively.(4)Establishment of subcutaneous xenograft model of cervical carcinoma in nude miceSiHa cells with normal expression of lncRNA USP30-AS1 and stable knockdown of lncRNA USP30-AS1 were diluted in 0.2 mL DMEM medium without serum.The cells were mixed and injected into the right side of the armpit of nude mice with a 1 ml syringe,respectively.Tumor growth was observed every day after inoculation,and the time of Tumor formation was recorded.Tumor volume of nude mice was measured the next day after Tumor growth.Tumor volume=Tumor length×Tumor width×Tumor width/2.Experimental result:(1)GEPIA、UCSC-GTEx showed that the expression of lncRNA USP30-AS1 in cervical carcinoma tissues was significantly higher than that in normal cervical tissues,and qRT PCR detection results showed that the expression of lncRNA USP30-AS1 in cervical carcinoma cells was significantly higher than that in normal cervical epithelial cells.(2)EdU experiment and clone formation experiment showed that interference with lncRNA USP30-AS1 in SiHa and HeLa cells inhibited the proliferation of cervical carcinoma cells.(3)The TUNEL assay and flow cytometry assay showed that interference with lncRNA USP30-AS1 in SiHa and HeLa cells promoted apoptosis of cervical carcinoma cells.(4)Transwell results showed that interference with lncRNA USP30-AS 1 in SiHa and HeLa cells inhibited the migration and invasion of cervical carcinoma cells.(5)Western blot analysis of EMT-related proteins showed that interference with lncRNA USP30-AS1 in SiHa and HeLa cells inhibited epithelial to mesenchymal transformation of cervical carcinoma cells.(6)Tumor formation experiments in nude mice showed that lncRNA USP30-AS1 promoted the growth of cervical carcinoma tumors.Experimental conclusion:Bioinformatics prediction and experiments confirmed that lncRNA USP30-AS1 was highly expressed in cervical carcinoma,promoting the proliferation,migration,invasion,epithelialmesenchymal transformation and inhibiting apoptosis of cervical carcinoma cells,and lncRNA USP30-AS1 played a role as an oncogene in cervical carcinoma.Part Ⅱ:USP30-AS1 functions by activating the Wnt pathwayExperimental purpose:The signaling pathways involved in the regulation of lncRNA USP30-AS1 were explored and the specific mechanisms of the regulatory signaling pathways were explored.Materials and methods:(1)Screen the signaling pathway related to lncRNA USP30-AS 1 in cervical carcinomaSignaling pathway kits were used to detect which signaling pathway lncRNA USP30AS1 is involved in cervical carcinoma cells transfected with sh/NC and sh/USP30-AS1#1,respectively.(2)lncRNA USP30-AS 1 activates the Wnt/β-catenin signaling pathwayTo determine the influence of lncRNA USP30-AS1 on Wnt/β-catenin pathway in cervical carcinoma cells,Top/FOP Flash assay was used in cervical carcinoma cells transfected with sh/NC,sh/USP30-AS1#1 and sh/USP30-AS1#2,respectively.Then,the expression of key genes of Wnt/β-catenin signaling pathway,β-catenin,MMP2,Cyclin D1 and c-MYC,were detected by Western blot assay in cervical carcinoma cells which were transfected with sh/NC,sh/USP30-AS1#1 and sh/USP30-AS1#2.(3)lncRNA USP30-AS1 regulates the expression of β-cateninsh/NC and sh/USP30-AS1#1 were respectively transfected into SiHa cells.The protein synthesis inhibitor actinobenone(CHX)was used to detect the expression of p-catenin protein in each group of cell lines before under different treatment time(0h,4h,8h,12h)by Western blot assay to verify the effect of lncRNA USP30-AS1 on β-catenin protein synthesis.After treatment with protein degradation inhibitor MG 132 or not,the expression of β-catenin protein was detected by Western blot.Finally,the ubiquitination degradation of β-catenin protein before and after lncRNA USP30-AS1 interference was detected by in vitro ubiquitination assay to verify that lncRNA USP30-AS 1 can stabilize β-catenin protein.Experimental result(1)The detection results of signaling pathway kit showed that lncRNA USP30-AS1 was involved in Wnt/β-catenin signaling pathway.(2)The TOP/FOP Flash experimental results showed that lncRNA USP30-AS1 activated Wnt/β-catenin signaling pathway.(3)The results of qRT-PCR and Western blot showed that lncRNA USP30-AS1 regulated the protein level of-catenin without affecting its mRNA level.(4)After CHX treatment,lncRNAUSP30-AS1 interfered with the expression of β-catenin,and the results showed that lncRNA USP30-AS1 did not affect the synthesis of β-catenin.(5)After MG132 treatment,lncRNA USP30-AS1 interfered with the expression of βcatenin,and the results showed that lncRNA USP30-AS1 regulated the degradation of βcatenin.(6)The results of ubiquitination showed that lncRNA USP30-AS1 promoted the stability of deubiquitination of β-catenin.Experimental conclusion:LncRNA USP30-AS1 regulates the development of cervical carcinoma by stabilizing βcatenin protein and activating Wnt/β-catenin pathway.(7)The results of ubiquitination showed that lncRNA USP30-AS1 promoted the stability of deubiquitination of-catenin protein.Experimental conclusion:LncRNA USP30-AS1 regulates the development of cervical carcinoma by stabilizing βcatenin protein and activating Wnt/β-catenin pathway.Part Ⅲ:USP30 mediates β-catenin to activate Wnt pathwayExperimental purpose:The study explores the stable activation of Wnt signaling pathway by usP30-AS1 regulation of USP30 to promote the deubiquitination of-catenin protein.Materials and methods:(1)Detect effect of lncRNA USP30-AS1 on USP30Firstly,sh/NC,sh/USP30-AS1#and sh/USP30-AS1#2 were transfected into Siha cells and HeLa cells,respectively.Then,the expression of USP30 mRNA and protein in each group were detected by qRT-PCR and Western blot.(2)Construction of USP30 interference and overexpression vectorsBased on the analysis of USP30 sequence information in NCBI RefSeq gene database,the interference sh-RNA plasmid sequence was designed.sh/NC,sh/USP30-AS1#and sh/USP30-AS1#2 were transfected into Siha cells and HeLa cells,respectively.The interference efficiency of sh/USP30 was verified by qRT-PCR.Based on the analysis of USP30 sequence information in NCBI RefSeq gene database,pcDNA3.1/USP30 vector was constructed and transfected into Siha and HeLa cell lines.The overexpression efficiency of pcDNA3.1/USP30 was verified by qRT-PCR.(3)Cell function assay was used to detect the regulatory effect of USP30-AS1/USP30 on cervical cancerCell function rescue experiments were performed in Siha cells and HeLa cells.sh/NC,sh/USP30-AS1,sh/USP30-AS1+pcDNA3.1/USP30 were transfected,respectively.EdU,Clone formation,TUNEL,Flow cytometry,Transwell and Western blot assays were used to detect the regulation effect of lncRNA USP30-AS1/USP30 on proliferation,metastasis,invasion,EMT and apoptosis of cervical cancer cells.(4)USP30 Involvement in the activation of Wnt/β-catenin signaling pathwaysh/NC,sh1USP30#1,sh/USP30#2 were transfected into SiHA cells,respectively.The changes of Wnt/β-catenin pathway activity after USP30 interference were analyzed by TOP/FOP FLASH assay to verify the regulatory effect of USP30 on Wnt/β-catenin pathway.Western blot was used to detect the expression of some key proteins in Wnt/β-catenin signaling pathway——β-catenin,Cyclin D1,MMP2,and c-MYC in SiHa cells of different groups,and it was used to verify that USP30 promote the expression of key gene protein of Wnt/β-catenin signaling pathway.(5)The expression of β-catenin protein was regulated by USP30sh/NC,sh/USP30#1 were respectively transfected into SiHa cells to construct stable USP30 low expression cell lines and then the protein synthesis inhibitor actinomycetes(CHX)was used to treat two groups of cell lines.Western blot was used to detect β-catenin protein expression under different treatment time(0h,4h,8h,12h).After treatment with protein degradation inhibitor MG132 or not,the expression of β-catenin protein was detected by Western blot to verify the regulatory effect of USP30 on β-catenin protein degradation.Finally,the vitro ubiquitination assay was used to detect the degradation of β-catenin protein before and after USP30 interference.Experimental result:(1)TOP/FOP Flash results showed that USP30 promoted the activation of Wnt/β-catenin signaling pathway.(2)qRT-PCR and Western blot results showed that USP30 regulated the protein level of βcatenin without affecting its mRNA level.(3)After CHX treatment,the expression of β-catenin protein was detected with or without USP30 inference,and the results showed that USP30 did not affect the synthesis of β-catenin.(4)After MG132 treatment,the expression of β-catenin protein was detected with or without USP30 inference,and the results showed that USP30 regulated the degradation ofβ-catenin.(5)The results of ubiquitination showed that USP30 promoted the stability of deubiquitination of β-catenin protein.(6)Rescue experimental results showed that overexpression of USP30 could reverse the regulatory effect of interference with lncRNA USP30-AS1 on cervical carcinoma.Experimental conclusion:LncRNA USP30-AS1 promotes deubiquitination and stabilization of β-catenin protein through USP30,thereby activating the Wnt signaling pathway.Part Ⅳ:lncRNA USP30-AS1 upregulates USP30 by relieving the inhibition of Mir-2467-3P on USP30Experimental purpose:Explore the specific mechanism by which lncRNA USP30-AS1 regulates the expression of USP30.Materials and methods:(1)Detect the subcellular localization of lncRNA USP30-AS1 in cervical carcinoma cellsThe subcellular localization of lncRNA USP30-AS1 in SiHa and HeLa cells were detected by FISH and cytoplasmic separation assay.Firstly,According to the lncRNA USP30-AS1 sequence,RNA probes labeled with green fluorescence was designed to detecte the subcellular localization of lncRNA USP30-AS1 in SiHa and HeLa cells.Then,Cytoplasmic&Nuclear RNA Purification Kit was used to separate and extract the nuclear and cytoplasmic of SiHa or HeLa cells.The expression of lncRNA USP30-AS1 in the nucleus and cytoplasm was detected by qRT-PCR assay,and the proportion of lncRNA USP30-AS1 expression distribution in the nucleus and cytoplasm was calculated.(2)lncRNA USP30-AS1 effect on USP30 promoterTo clarify the transcriptional regulation effect of lncRNA USP30-AS1 on USP30,luciferase reporter gene vector containing USP30 promoter sequence was constructed and cotransfected into Siha and HeLa cells with sh/NC,sh/USP30-AS1#1,sh/USP30-AS1#2 to verify the transcriptional regulation effect of lncRNA USP30-AS1 on USP30.(3)lncRNA USP30-AS1 specifically binds to the RISC complexThe RNA which binds to AGO2 protein was obtained in Siha cells and HeLa cells by AGO2-RIP assay and the adsorption capacity of lncRNA USP30-AS1 in the RNA products was detected by qRT-PCR assay to verify whether lncRNA USP30-AS1 was captured by RISC complex and whether lncRNA USP30-AS1 was bound to RISC complex.(4)Screen miRNA which may bind to both lncRNA USP30-AS1 and USP30The Starbase database was used to obtain possible miRNAs that could bind to lncRNA USP30-AS1 and USP30,and the common miRNAs were obtained by comparison,confirming that miR-2467-3p was the possible miRNA of lncRNA USP30-AS1 and USP30.(5)miR-2467-3p stably binds to lncRNA USP30-AS 1 USP30RNA pulldown test was performed by lncRNA USP30-AS 1 and USP30 specific probes.After RNA extraction and purification of the product,the content of miR-2467-3p was detected to verify the direct binding relationship between miR-2467-3p and lncRNA USP30AS1 and USP30.Bioinformatics methods were used to predict possible binding sites of miR2467-3p on lncRNA USP30-AS1,USP30 sequence respectively,build USP30 AS1,USP30 wild-type luciferase reporter gene vector and mutation of miR-2467-3p binding sites mutant luciferase reporter gene vector,the luciferase reporter gene vector respectively with miR2467-3p mimics and NC mimics transfection,validation of miR-2467-3p affect USP30AS1,USP30 activity.(6)Cell function assay was used to detect the regulatory effect of lncRNA USP30AS1/miR-2467-3p on cervical cancerCell function rescue experiments were performed in Siha cells and HeLa cells.sh/NC,sh/USP30-AS1,sh/USP30-AS1+miR-2467-3p inhibitors were transfected,respectively.EdU,Clone formation,TUNEL,Flow cytometry,Transwell and Western blot assays were used to detect the regulation effect of lncRNA USP30-AS1/miR-2467-3p on proliferation,metastasis,invasion,EMT and apoptosis of cervical cancer cells.Experimental result:(1)The results of FISH and subcell separation showed that lncRNA USP30-AS1 mainly existed in cytoplasm.(2)The results of luciferase assay showed the lncRNA USP30-AS1 had no significant effect on USP30 promoter.(3)The RIP experiment results showed that lncRNA USP30-AS1 was enriched in a large amount in AGO precipitation.(4)The prediction results of Starbase database showed that mir-2467-3p was the shared miRNA of lncRNA USP30-AS1 and USP30.(5)The results of RNA pulldown luciferase report showed that mir-2467-3p was the miRNA of lncRNA USP30-AS1 and USP30.(6)RNA Pulldown results showed that the accumulation of USP30 in the compound pulled by the biotin labeled probe of Mir-2467-3p increased after the interference of lncRNA USP30-AS1.The results showed that the interference of mir-2467-3P partial rescue interference of lncRNA USP30-AS1 affected the expression of USP30.Experimental conclusion:LncRNA USP30-AS1 regulates USP30 expression by competitive adsorption of mir-24673P as ceRNA.Part Ⅴ:lncRNA USP30-AS1 recruits FUS to stabilize USP30 mRNAExperimental purpose:Explore how lncRNA USP30-AS1 regulates USP.30 expression by recruiting RBPMaterials and methods:(1)The RBP of lncRNA USP30-AS1 and USP30 associated with cervical cancer were screenedStarbase database was used to predict the RBP that lncRNA USP30-AS1 and USP30 might combine and identified that FUS was the common RBP of lncRNA USP30-AS1 and USP30.(2)The binding effect of lncRNA USP30-AS1 with FUSRNA pulldown assay was used to verify the binding effect of lncRNA USP30-AS 1 with FUS in SiHa cells and HeLa cells.Then,to rigorously prove the combination between lncRNA USP30-AS1 and FUS,RIP assay was carried out in SiHa cells and HeLa cells and qRT-PCR assay was used to detect the enrichment of lncRNA USP30-AS1 in RNA products.(3)The binding effect of USP30 and FUSRNA pulldown assay was used to verify the binding effect of USP30 with FUS in SiHa cells and HeLa cells.Then,to rigorously prove the combination between USP30 and FUS,RIP assay was carried out in SiHa cells and HeLa cells and qRT-PCR assay was used to detect the enrichment of USP30 in RNA products.(4)Effects of lncRNA USP30-AS1 on binding of USP30 to FUSsh/NC and sh/USP30-AS1#1 were transfected respectively into Siha cells and HeLa cells.RIP assay was used to obtain FUS-bound RNA before and after interfering with lncRNA USP30-AS1 in the cells and qRT-PCR assay was used to detect the enrichment of USP30 mRNA in RNA products to verify the regulatory effect of lncRNA USP30-AS1 on USP30-FUS binding.(5)Regulation of USP30 by FUSInterference vectors of FUS were constructed and transfected into SiHa cells and HeLa cells.The expression level of USP30 in cells with stable expression interference of FUS was detected by qRT-PCR and Western Blot assay to verify the regulatory effect of FUS on USP30 expression.(6)Effect of lncRNA USP30-AS1 FUS on USP30 mRNA stabilityTo verify lncRNA USP30-AS1 had an impact on on USP30 mRNA stability,2μg/mlactinomycetes D was used to treate SiHa cells and HeLa cells with low expression of lncRNA USP30-AS 1,and then the expression level of USP30 was detected under different treatment time(0h、4h、8h)by qRT-PCR.Furthermore,to verify FUS had an impact on on USP30 mRNA stability,2μg/ml actinomycetes D was used to treate SiHa cells and HeLa cells with low expression of FUS,and then the expression level of USP30 was detected under different treatment time(0h、4h、8h)by qRT-PCR.Experimental result:(1)According to the prediction results of Bioinformatics site Starbase,FUS is the RBP combined with lncRNA USP30-AS1 and USP30.(2)RNA Pulldown and RIP results showed that lncRNA USP30-AS1 could bind FUS.(3)RNA Pulldown results showed that USP30 can bind to FUS.(4)The results of qRT-PCR and Western blot assay showed the expression of FUS was positively correlated with USP30 expression.(5)RIP experimental results showed that the binding ability of USP30 and FUS was weakened after interference with lncRNA USP30-AS1.(6)qRT-PCR results after actinomycin D treatment showed that lncRNA USP30-AS1 and FUS promoted mRNA stability of USP30.Experimental conclusion:LncRNA USP30-AS1 stabilized the mRNA of USP30 by recruiting FUS.