| MicroRNAs(miRNAs)are one of the most studied epigenetic regulatory mechanisms,and are small(20-40 nt)non-coding RNA molecules that are ubiquitous in eukaryotes,usually clustered on the genome.They are highly conserved,temporally and tissue-specific,and can bind to the complementary sequences in the 3’-UTR region of the target gene,degrade mRNAs or inhibit the translation processes,and thereby participate in almost all fundamental biological functions in cells,including cell growth,apoptosis,cell cycle,proliferation,metabolism,etc.Thus,it has attracted much attention in the fields of the growth and development of biological individuals and the occurrence and progress of body diseases.In fact,similar to protein-coding genes,the expression of miRNAs is also controlled by epigenetic mechanisms.This effect is often reciprocal.On the one hand,miRNAs can be epigenetically regulated through DNA methylation and/or specific histone modifications.miRNAs,on the other hand,can bind to complementary sequences in 3’-UTR region of the target gene,act to inhibit key enzymes that drive epigenetic remodeling,or recruit specific protein complexes that regulate chromatin structure and gene expression,thereby affecting the transcriptional environment of cells.The miR-371-373 gene cluster is found on chromosome 19q13.4,and can be transcribed into Pre-miR-371,Pre-miR-372,and Pre-miR-373,resulting in four mature mRNAs including miR-371,miR-372,miR-373,and miR-373*.Among them,microRNA-372(miR-372)has been studied relatively more and is involved in regulating the occurrence and progression of various tumors.In the context of tumor cells,it exhibits dual functionality either as a tumor suppressor or an oncogenic factor,depending upon the specific gene being targeted.According to existing study findings,there is a lack of documented evidence about the influence of miR-372 on enzymes responsible for controlling chromatin structure.The human INO80 chromatin remodeling complex,which consists of 15 subunits,facilitates the ATP-dependent movement of nucleosomes along DNA.The eight core subunits,including Arp5,Arp8,TIP49a/b,Ies2,Ies6,Arp4,and YY1,exhibit a high degree of evolutionary conservation from yeast to humans.These subunits together constitute an enzymatic core,which encompasses the HSA and SNF2 modules.Hence,the impairment of any one catalytic subunit within the INO80 complex or its enzyme core has the potential to impact the overall biological functionality of the INO80 complex.YY1 is a ubiquitous transcription factor in cells and a member of the human INO80 chromatin remodeling complex.In our prior investigation,our study team confirmed that YY1,functioning as a transcription factor,has the capability to recruit the INO80 complex to the promoter region of its target genes and initiate the transcriptional activity of such genes.Additionally,prior studies have identified two YY 1 binding sites within the promoter region of miR-372.Suggesting that YY1 may recruit the INO80 complex to the promoter region of miR-372 and regulate its transcriptional activation.This paper takes HeLa and HCT116 cells as the research objects and constructs pSingle-shINO80 and pSingle-shArp8 to induce gene silencing cell lines,pLVX-shRNA gene silencing plasmids,pmR-mCherry-miR-372 expression plasmids,and pMIR-INO80-3’-UTRLuc and pMIR-Arp8-3’-UTR-Luc dual-luciferase reporter gene plasmids,combined with DNA chip multi-omics analysis,network database bioinformatics analysis,and a sequence of biological experimental techniques to elucidate the INO80 complex interaction with miR-372 provides an experimental and theoretical foundation for understanding the mode of action of the INO80 complex.The following are the main findings and results of this paper:1.In this thesis,in order to facilitate the subsequent study of the mode of action of INO80 chromatin remodeling in cells,Firstly,INO80 and Arp8 inducible gene knockdown cell lines were constructed using pSingle-tTS-shRNA Vector,and the silencing of INO80 and Arp8 were confirmed by doxycycline induction.At the same time,it was found that after the induction of INO80 silencing,the intercellular adhesion was enhanced,and aggregated growth was observed over time;Secondly,stable silent cell lines of INO80,Arp8 and YY 1 were constructed using the pLVX-shRNA lentivirus system,and their knockdown effects were confirmed by Western blotting.The establishment of the above cell lines and plasmid construction laid foundation for the follow-up study of the interplay between miR-372 and the INO80 chromatin remodeling complex.2.To explore the biological function of INO80 complex in cells,we used siINO80,siIes2,siIes6,siArp5,and siArp8 to silence related genes,respectively,and then performed transcriptome high-throughput sequencing analysis on the total RNA.Gene Ontology(GO)functional annotation analysis showed that the INO80 complex is closely related to cell proliferation,growth,adhesion,and cycle division.In addition,we found in the gene expression profiling that miR-372 and miR-373 could be co-regulated by the above five subunits.Furthermore,through UALCAN website analysis,it was found that multiple subunits of the INO80 complex including the INO80 catalytic subunit,YY1,Arp8,INO80D,and MCRS1,all their 3’-UTR region contained the complementary sequence of miR-372,suggesting a correlation between the INO80 chromatin remodeling complex and miR-372.3.We used siRNA,pLVX-shRNA or pSingle-shRNA inducible gene silencing cell lines to silence INO80,Arp8,YY1 or Ies2 and found that the expression level of miR-372 in the cells were up-regulated,suggesting that the INO80/YY1 complex may bind to miR-372 upstream of the transcription start site and transcriptionally regulate the expression of miR-372.At the same time,we transiently transfected the constructed pri-miR-372 expression plasmid into HCT116 colon cancer cells,the expression levels of multiple mRNAs and proteins that may be targeted by miR-372,including INO80 and Arp8,were detected after 48 hours.As expected,overexpression of pri-miR-372 significantly inhibited the expression levels of INO80 and Arp8,suggesting that miR-372 could potentially influence the functions of INO80 complex by targeting and regulating the expression levels of different subunits of the INO80 chromatin remodeling complex.4.To confirm the above results,we predicted and two binding sites were identified in the INO80-3’-UTR(153-159 and 4644-4650)and Arp8-3’-UTR(457-463 and 1594-1601)regions through the UALCAN website.We used the pMIR-REPORTLuc vector to construct INO80-3’-UTR and Arp8-3’-UTR luciferase reporter plasmids containing these target sequences(or mutated target sequences),and then these plasmids were co-transfected with miR-372 or empty vector,respectively,The effect of miR-372 on dual-luciferase activity was detected in HCT116 colon cancer cells.The results showed that miR-372 significantly inhibited the luciferase activity of pMIR-INO80-3’-UTR-Luc/pMIR-Arp8-3’-UTR-Luc.However,when the miR-372 binding site in the INO80-3’-UTR region was mutated,the protein expression level of INO80 was no longer affected by miR-372,demonstrating that miR-372 directly binds to the 3’-UTR of INO80 and Arp8 region,and posttranscriptionally regulates the expression level of the INO80 chromatin remodeling complex.5.Finally,when we added doxycycline to the pSingle-shINO80 inducible cell line to induce INO80 silencing,it was found that the protein expression levels of p53 and p21 increased.In HCT116 cells,overexpression of miR-372 not only caused a decrease in the expression level of INO80 protein,but also increased the expression levels of p53 and p21 proteins.Therefore,we speculate that miR-372 may induce the expression of p53 and p21 by inhibiting the INO80 complex,thereby inhibiting cell proliferation.In addition,research data on tissue samples from primary colon cancer patients also confirmed that the expression of proliferating cell-related antigen Ki67 was significantly increased in colon cancer tissues with low expression of miR-372,suggesting that miR-372 is regulating the proliferation process of colon cancer cells.Colony formation using HCT116 cells confirmed this speculation.As expected,both silencing INO80 and overexpressing miR-372 can significantly inhibit the number of colony formation,and when miR372 and its inhibitor are used together,the inhibitory effect of miR-372 on cell colony formation is antagonized,indicating that miR-372/INO80 complex may synergistically participate in the proliferation process of colon cancer cells.In conclusion,the empirical data provided in this work reveal a mutual regulatory interaction between miR-372 and the INO80 chromatin remodeling complex.MiR-372 may clearly interact with the target sequence located in the 3’-UTR region of INO80 or Arp8,exerting post-transcriptional control on the expression and stability of the INO80 complex.This,in turn,has an impact on the INO80 complex’s cellular activities.The findings of this study provide a basis for understanding the operational mechanism of the INO80 chromatin complex and provide unique insights for the development of future cancer therapy approaches.Furthermore,miR-372 has the potential to be used as a biomarker for early cancer identification. |
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