Investigating The Mechanism Of STAT3 In RNA Polymerase Ⅰ/Ⅲ-directed Gene Transcription And Tumor Cell Proliferation

Background:Cancer is one of the most significance diseases threatening human health.One of the reasons why cancer is difficult to cure is that we do not understand the causes of carcinogenesis.Therefore,it is of great significance to uncover the pathogenic mechanisms of various cancers for cancer treatment.Signal transducer and activator of transcription 3(STAT3)is a member of the STAT family that regulates numerous biological processes,including cell proliferation and migration,apoptosis,angiogenesis,immunosuppression and cancer stem cell maintenance;whereas the abnormal activation of STAT3 leads to tumorigenesis and malignant transformation.The transcription products of RNA polymerase I and Ⅲ(Pol Ⅰ and Pol Ⅲ)participate in the assembly of ribosomes and protein synthesis,regulate the metabolism and growth of cells.Dysregulation of Pol Ⅰ and Pol Ⅲ transcription is closely related to tumorigenesis.Givn that STAT3,Pol Ⅰ and Pol Ⅲ are all related to carcinogenesis,whether STAT3 is conneted with transcription directed by Pol Ⅰ and Pol Ⅲ has not been investigated.Previous studies have confirmed that cytoskeletal Filamin A(FLNA)can suppress Pol Ⅰ and Pol Ⅲ dependent transcription.When studying the mechanism by which FLNA regulates Pol Ⅰ-directed transcription,we found that FLNA silencing severely affects the expression of STAT3 in tumor cell lines,suggesting that STAT3 may participate in Pol Ⅰ and Ⅲ dependent transcription.Therefore,the aim of this project is to investigate the role of STAT3 in Pol Ⅰ and Ⅲ-directed gene transcription and its regulatory mechanisms;moreover,explore the effects of Pol Ⅰ and Pol Ⅲ products alteration induced by STAT3 on the proliferation of tumor cells and the potential application effect of anti-tumor.Methods:STAT3 knockdown and overexpression lentiviral expression vectors were constructed by PCR and gene cloning.STAT3 knockdown and overexpression stable cell lines such as HeLa and HepG2 were established by transfection,packaging lentivirus,infecting cells and etc.STAT3 knockdown or overexpression and Pol Ⅰ and Ⅲ-directed transcription were detected by Western Blot and RT-qPCR respectively.The effects of STAT3 alterations on Pol Ⅰ-directed gene transcription were verified using EU assay.Cell counting,CCK-8 and EdU were used to detect whether STAT3 and Pol Ⅰ alterations affect the proliferative activity of cancer cells;Clonogenic and nude mice xenograft assays were utilized to verify the effect of STAT3 knockdown on cell tumorigenic ability in vivo and in vitro.STAT3 inhibitors and Pol Ⅰ inhibitors were utilized to investigate whether inhibition of STAT3 activity and Pol Ⅰ transcription has a effect on anti-tumor.The regulatory mechanism of Pol Ⅰ and Ⅲ-directed gene transcription mediated by STAT3 was investigated by RNA-seq,chromatin immunoprecipitation assay,rescue assay,and immunoprecipitation.Results:(1)Role and regulatory mechanism of STAT3 in Pol Ⅰ-directed transcription and tumor cell proliferation: To establish cell lines with STAT3 silencing,we first examined the expression of STAT3 and Pol Ⅰ products in HeLa and HepG2 cells by Western Blot,the results showed that STAT3,pSTAT3,and Pol Ⅰ products are highly expressed in these cancer cells.Afterwards,lentiviral expression systems were used to establish the cell lines with STAT3 silencing and overexpression,the effects of STAT3 expression alteration on Pol Ⅰ transcription and cell proliferation were also analyzed.The results showed that STAT3 can promote Pol Ⅰ-directed gene transcription,cancer cell proliferation and tumor growth.The effect of anti-tumor study using a STAT3 inhibitor(STAT3-IN-3)and a Pol Ⅰ inhibitor(CX-5461)showed that both inhibitors can inhibit tumor growth and the combination of the two drugs exihibits a better effect on the inhibition of tumor growth.Mechanistic studies revealed that STAT3 increases the recruitment of Pol Ⅰ machinery components to rDNA promoter by promoting the expression of Pol Ⅰ subunit RPA34,thereby promoting Pol Ⅰ transcription.Further studies on the mechanism by which STAT3 regulates Rpa34 gene expression revealed that STAT3 increases the binding of Pol Ⅱ transcription machinery factors to the Rpa34 promoter via assocating with Rpa34 promoter,which in turn activates the transcription of Rpa34 gene.(2)Role and regulatory mechanism of STAT3 in Pol Ⅲ-directed transcription:Expression of Pol Ⅲ products were anlyzed by RT-qPCR using established cell lines with STAT3 silencing and overexpression,and the results showed that STAT3 promotes Pol Ⅲ-directed gene transcription.By mining the RNA-seq data and RT-qPCR experiments,STAT3 can activate miR-106a-5p expression and inhibit TP73 expression.The assays by silencing TP73 expression showed that TP73 can not only participate in the transcription regulation of Pol Ⅲ mediated by STAT3,but also independently inhibit the gene transcription directed by Pol Ⅲ.Moreover,TP73 mainly affects the assembly of Pol Ⅲ machinery through disrupting the assembly of TFⅢB subunit.Targetscan analysis indicated that TP73 mRNA is a potential target of miR-106a-5p.Indeed,miR-106a-5p mimics transfection showed that miR-106a-5p can inhibit TP73 expression by targeting TP73 and promote Pol Ⅲ-directed gene transcription.Promoter sequence analysis and reporter assays showed that STAT3 is likely to bind to the miR-106a-5p promoter to activate its expression.These results indicated that STAT3 regulates the expression of TP73 through miR-106a-5p,and then affects the transcription of Pol Ⅲ.Conclusion:STAT3 enhances Pol Ⅰ-directed gene transcription and tumor growth by activating RPA34;STAT3 promotes Pol Ⅲ-directed transcription by controlling the miR-106a-5p/TP73 axis.