TWIST2 Promotes Lymph Node Metastasis In Cervical Cancer By Directly Targeting MiR-221-3p/THBS2 Pathway

BackgroundCervical cancer is one of the most prevalent malignancies in women worldwide and is the leading cause of cancer death for women in developing countries.Although widespread vaccination against human papilloma virus,periodic cancer screening and prompt surgical treatment have resulted in a significant decrease in the incidence of cervical cancer,it remains one of the most common diseases causing mortality in women.Lymph node metastasis is the main metastatic pathway of cervical cancer and an independent indicator affecting the treatment and prognosis of cervical cancer.To strengthen the basic research and elucidate the molecular mechanism of lymph node metastasis for cervical cancer,and early prediction,diagnosis,targeted prevention as well as prognosis evaluation for lymph node metastasis are important parts of study for cervical cancer,which are important for improving the cure rate and reducing mortality for cervical cancer with lymph node metastasis.TWIST2 belongs to the class B member of the basic helix-loop-helix protein(bHLH)family,in which the member protein of class B is easy to form an allogeneic two polymer with protein of class A.Transcription factors in bHLH family can regulate the differentiation of multiple cells via binding the E-box consensus sequence(CANNTG)in the gene control region to achieve the transcriptional regulation of the target genes.Therefore,TWIST2 can act as the transcription factor to bind the promoter region of downstream target genes and exert the transcriptional inhibition function through combining with the CANNTG sequence.Many studies have shown that TWIST2 plays a central role in the process of tumor metastasis.It mainly affects the migration and invasion of tumor cells by regulating the expression of Epithelial-Mesenchymal Transition(EMT)related proteins.However,report focuses on TWIST2 in cervical cancer is less compared with that in other cancer,and the mechanism and pathway that TWIST2 involves in are also less studied.In 2014,Wang et al.found that TWIST2 was up-regulated in cervical cancer and was positively related to FIGO staging and lymph node metastasis of cervical cancer.In addition,compared with TWIST1,knockdown expression of TWIST2 can significantly upregulate E-Cadherin expression and downregulate N-Cadherin expression,thereby inhibiting migration and invasion of cervical cancer cells.However,the molecular mechanism and signaling pathway of TWIST2 for lymph node metastasis of cervical cancer remains to be further explored.microRNAs(miRNAs)is a class of endogenous non-coding small RNA,which complementary base pair with the target gene’s 3’-untranslated region(UTR)of messenger RNA(mRNA)and trigger translation repression or RNA degradation.Thus,miRNAs are considered as master regulators of many important physiological processes,including cell proliferation,differentiation,development,and apoptosis.Recently,genome-wide analyses indicated that 50%of miRNA genes are located in cancer-associated genomic regions or in fragile sites.Therefore,the dysregulation of miRNAs is closely related to the progression of multiple cancers.miRNAs can alter transcription and expression of downstream target gene via binding to its 3’-UTR,thus playing a biological role.At the same time,miRNAs are also regulated by the upstream transcription factors.Therefore,the transcriptional factor-miRNAs-target gene forms a complete regulatory network to further participate in the development of tumor,including cervical cancer.Considerable experimental results showed that miRNAs can be used as a diagnostic marker and therapeutic target for tumor because of its stability and conservatism.Various studies reported TWIST2 had relationship with miRNAs.And TWIST2,as a transcription factor,can the bind the promotor region of miRNAs then inhibits or promotes its transcription.Therefore,we performed Agilent miRNAs array and detected more than 36 miRNAs.Then qRT-PCR confirmed miR-221-3p was upregulated after overexpression TWIST2.Meanwhile,we screened the upstream promoter region of miR-221-3p through Jaspar database,and found 85 potential transcription factors that regulated miR-221-3p and promote tumor metastasis,including TWIST2.Therefore,we speculate that TWIST2,as a transcription factor,may promote its transcriptional expression by directly binding to the promoter region of miR-221-3p.The main content of this paper is to investigate whether TWIST2 can mediate the EMT of cervical cancer and promote lymph node metastasis through targeting miR-221-3p.At the same time,the molecular mechanism of miR-221-3p for regulation EMT of cervical cancer was also explored.This provides us a theoretical basis to understand the EMT and metastasis in cervical cancer,and helps us to find potential therapeutic target genes for cervical cancer.Content and methods1.TWIST2 regulated EMT of cervical cancer cells and then promoted lymph node metastasis(1)The stable overexpressed TWITS2 lentivirus vector was transfected into cervical cancer cells after design and construction.The transfection efficiency was detected by qRT-PCR and Western blot.Cervical cancer cells with stable overexpression of TWIST2 were selected for subsequent experiment.(2)Wound healing and Transwell chamber invasion assay were used to detect the effect of TWIST2 on the migration and invasion ability of cervical cancer cells in vitro.(3)Popliteal lymph node metastasis model was used to verify the effect of TWIST2 on lymph node metastasis of cervical cancer cells.(4)The effect of TWIST2 on the expression of EMT related protein in cervical cancer cells was detected by qRT-PCR and Western Blot.2.TWIST2 promoted the migration and invasion of cervical cancer cells through promoting the transcription of miR-221-3p(1)miRNAs array was used to detect the change of miRNAs in TWIST2 overexpression Siha cell and further qRT-PCR confirmed that miR-221-3p was upregulation after TWIST2 overexpression.(2)Bioinformatics analysis identified TWIST2 could bind the promoter of miR-221-3p.Dual-luciferase reporter assay was then performed to confirm TWIST2 bind the promoter of miR-221,3p and promote its transcription.(3)The miR-221-3p stable overexpression lentivirus vector transfected into cervical cancer cells was verified by qRT-PCR after design and construction.(4)Wound healing and Transwell chamber invasion assay were used to detect the effect of miR-221-3p on the migration and invasion ability of cervical cancer cells in vitro.(5)Popliteal lymph node metastasis model was used to verify the effect of miR-221-3p on lymph node metastasis of cervical cancer cells.(6)The effect of miR-221-3p on the expression of EMT related protein in cervical cancer cells was detected by qRT-PCR and Western Blot.(7)Wound healing and Transwell chamber invasion assay were applied to identify TWIST2 promoted cervical cancer cells migration and invasion through miR-221-3p.(8)qRT-PCR and Western Blot were applied to identify TWIST2 regulated the expression of EMT related protein in cervical cancer cells through miR-221-3p.3.miR-221-3p direct targeted THBS2 then promoted cervical cancer cell function(1)Bioinformatics analysis confirmed THBS2 might be a target gene of miR-221-3p,and further qRT-PCR and Western blot confirmed THBS2 was downregulation after miR-221-3p overexpression.Subsequently,dual-luciferase reporter assay was performed to confirm miR-221-3p can bind the 3’-UTR regions of THBS2.(2)Wound healing and Transwell chamber invasion assay were used to detect the effect of THBS2 on the migration and invasion ability of cervical cancer cells in vitro.(3)The effect of THBS2 on the expression of EMT related protein in cervical cancer cells was detected by qRT-PCR and Western Blot.(5)Wound healing and Transwell chamber invasion assay were applied to identify miR-221-3p promoted cervical cancer cells migration and invasion through THBS2.(6)qRT-PCR and Western Blot were applied to identify miR-221-3p regulated the expression of EMT related protein in cervical cancer cells through THBS2.4.The expression of TWIST2-miR-221-3p-THBS2 pathway in clinical samples(1)qRT-PCR and IHC detected TWIST2 and THBS2 expression in 10 normal cervical tissues,23 cervical cancer tissues without lymph node metastasis,22 cervical cancer tissues with lymph node metastasis.(2)qRT-PCR and ISH detected miR-221-3p expression in 10 normal cervical tissues,23 cervical cancer tissues without lymph node metastasis,22 cervical cancer tissues with lymph node metastasis.(3)The expressive relationship among TWIST2,miR-221-3p and THBS2 in cervical cancer clinical samples was evaluated by statistical analysis.Results1.TWIST2 regulated EMT of cervical cancer cells and then promoted lymph node metastasis.Lentivirus stably overexpressing TWIST2 was transfected into cervical cancer cells and transfection efficiency was detected by qRT-PCR and Western blot.The results showed Siha-TW2 and Hela-TW2 overexpressing TWIT2 were successfully constructed.Subsequently,wound healing and Transwell chamber invasion assay were applied to identify TWIST2 overexpression promoted cervical cancer cell migration and invasion.Wound healing assay showed the rapid increasing migration rate in Siha-TW2 and Hela-TW2 compared with Vector group(P=0.005;P=0.003).Transwell chamber invasion assay show more number of transmembrane cells after TWIST2 overexpression(P<0.001;P=0.003).Popliteal lymph node metastasis model was applied to identify TWIST2 overexpression promoted cervical cancer cell metastasis.The results showed the higher metastatic rate in Siha-TW2 and Hela-TW2 compared with Vector group(33.3%vs.83.3%;16.7%vs.66.7%).Ultimately,qRT-PCR and Western Blot were applied to identify the effect of TWIST2 on expression of EMT related protein in cervical cancer cells.The results suggested N-Cadherin and Vimentin were upregulated and E-Cadherin was downregulated after overexpression of TWIST2,whereas N-Cadherin and Vimentin were downregulated and E-Cadherin was upregulated after knockdown of TWIST2.2.TWIST2 promoted the migration and invasion of cervical cancer cells through promoting the transcription of miR-221-3pAgilent miRNAs array detected more than 36 changed miRNAs in TWIST2 overexpression Siha cell,and further qRT-PCR confirmed miR-221-3p was upregulation after overexpression of TWIST in Siha and Hela(P<0.001;P<0.001).Bioinformatics analysis identified TWIST2 could bind the promoter of miR-221-3p.Subsequently,dual-luciferase reporter assay confirmed that overexpression of TWIST2 could promote the binding between TWIST2 and the promoter of miR-221-3p(P<0.001),whereas knockdown of TWIST2 could inhibit the binding(P<0.001).Lentiviral vector that stably overexpression miR-221-3p was transfected into cervical cancer cells and qRT-PCR showed miR-221-3p was overexpressed more than 200 times and 120 times in Siha and Hela cells(P<0.001;P<0.001).Subsequently,wound healing and Transwell chamber invasion assay were applied to identify the change of migration and invasion.The results of two assays showed that the migration(P=0.003;P=0.001)and invasion(P=0.001;P=0.001)ability of Siha-221 and Hela-221 were significantly improved.In addition,popliteal lymph node metastasis model assay confirmed that the higher metastatic rate in Siha-221 and Hela-221 compared with Vector group(37.5%vs.87.5%;25%vs.75%).More importantly,qRT-PCR and Western Blot assay showed that N-Cadherin and Vimentin were upregulated and E-Cadherin was downregulated in Siha-221 and Hela-221.Furthermore,miR-221-3p inhibitor was transfected into Siha-221 and Hela-221.Subsequently,wound healing and Transwell chamber invasion assay were used to confirm TWIST2 promotes cell migration and invasion through miR-221-3p.The results of two assays both showed that miR-221-3p inhibitor could reverse the cell migration(P=0.004;P=0.005)and invasion promotion induced by TWIST2 overexpression(P<0.001;P=0.003).Ultimately,qRT-PCR and Western Blot were applied to identify the expression of EMT related protein in Siha-TW2,Siha-TW2/221 KN,Hela-TW2 and Hela-TW2/221 KN.qRT-PCR and Western Blot assay showed that miR-221-3p inhibitor could reverse the N-Cadherin and Vimentin promotion as well as E-Cadherin inhibition induced by TWIST2 overexpression.3.miR-221-3p direct targeted THBS2 then promoted cervical cancer cell functionBioinformation analysis from 3 databases confirmed THBS2 might be target gene of miR-221-3p,and further qRT-PCR and Western Blot confirmed THBS2 was downregulated after miR-221-3p overexpression(P<0.001),whereas THBS2 was upregulated after miR-221-3p knockdown(P<0.001).Dual-luciferase reporter assay confirmed miR-221-3p bound the wild 3’-UTR of THBS2(P<0.001),and miR-221-3p could not bound the mutative 3’-UTR of THBS2.Wound healing and Transwell chamber invasion assay were used to detect the migration and invasion of Siha and Hela after transfection with plasmid with THBS2 overexpression.The results of two assays both showed that the migration(P=0.001;P=0.002)and invasion(P=0.001;P=0.003)of Siha-TS2 and Hela-TS2 were significantly inhibited.qRT-PCR and Western Blot were applied to identify the effect of THBS2 on expression of EMT related protein in cervical cancer cells.qRT-PCR and Western Blot assay showed that N-Cadherin and Vimentin were downregulated and E-Cadherin was upregulated in Siha-TS2 and Hela-TS2.Furthermore,plasmid withTHBS2 overexpression was transfected into Siha-221 and Hela-221.Subsequently,wound healing and Trans well chamber invasion assay were used to confirm miR-221-3p promotes cell migration and invasion through THBS2.The results of two assays both showed that THBS2 overexpression could reverse the cell migration(P=0.003;P=0.002)and invasion promotion induced by miR-221-3p overexpression(P<0.001;P<0.001).Ultimately,qRT-PCR and Western Blot were applied to identify the expression of EMT related protein in Siha-221/TS2 and Hela-221/TS2.qRT-PCR and Western Blot assay showed that THBS2 overexpression could reverse the N-Cadherin and Vimentin promotion as well as E-Cadherin inhibition induced by miR-221-3p overexpression.4.The expression of TWIST2-miR-221-3p-THBS2 pathway in clinical samplesTWIST2,miR-221-3p and THBS2 expression in 10 normal cervical tissues,23 cervical cancer tissues without lymph node metastasis,22 cervical cancer tissues with lymph node metastasis were detected by qRT-PCR,IHC and ISH.The results showed that TWIST2 and miR-221-3p hardly expressed in normal cervical tissues,whereas THBS2 highly expressed in normal cervical cells.The expression of TWIST2 and miR-221-3p was moderately low in cervical cancer tissues without lymph node metastasis,whereas the expression of THBS2 was moderate.In cervical cancer tissues with lymph node metastasis,the expression of TWIST2 and miR-221-3p was high,but the expression of THBS2 was low.In addition,TWIST2 mainly expressed in nuclei,while miR-221-3p and THBS2 both mainly expressed in cytoplasm.ConclusionTWIST2,as a transcription factor,which could bind to the promoter region of miR-221-3p then promoted its transcription and enhanced the binding between miR-221-3p and 3’-UTR of THBS2,which upregulated N-Cadherin and Vimentin as well as downregulated E-Cadherin to promote migration,invasion,and metastasis of cervical cancer cell.In addition,TWIST2 and miR-221-3p both highly expressed in cervical cancer tissues with lymph node metastasis,whereas THBS2 lowly expressed.Moreover,the expressive relationship between TWSIT2 and miR-221-3p was positive,whereas the expressive relationship between miR-221-3p and THBS2 was negative.