Pseudogene HMGA1P6 Promotes Ovarian Cancer Malignancy Via Augmenting The Oncogenic HMGA1/2

Ovarian cancer is one of the common malignant tumors in female reproductive system.Its incidence is on the third place,but its case fatality rate is the highest in gynecological malignancies.In the past ten years,the incidence of ovarian cancer in China has been increased by 30%,and the mortality rate has been increased by 18%.Because the early symptoms of ovarian cancer are unconspicuous and there is no effective means for early screening and diagnosis,more than 70%of the patients are at advanced stage at the time of consultation.Because of the high malignancy,easy to metastasize,easy to resist,and easy to relapse property of ovarian cancer,the 3-year relapse rate is 70%,and the 5-year survival rate is only 39%.Therefore,it has become urgent problems to clarify the mechanism of the occurrence and development of ovarian cancer especially High Grade Serous Ovarian Cancer(HGSOC),and screen early diagnostic markers and treatment targets.HMGA1/2(High Mobility Group A 1/2)is a family of non-histone proteins expressed in the nucleus,which is named for its high migration speed in polyacrylamide gel electrophoresis.The HMGA1/2 protein has an AT-hook region,which binds to the minor groove of the DNA double helix,thereby changing the chromatin conformation and playing a regulatory role.HMGA1/2 is highly expressed in embryonic and stem cells,and is generally low-expressed in adults,but it is abnormally highly expressed in malignant tumors.As an important oncogene family in ovarian cancer,the mechanism of HMGA1/2 overexpression in tumors has not been fully elucidated,and the discovery of the pseudogene of HMGA1 provides new ideas for this.Pseudogenes are a class of genes that have a high degree of similarity to their parental genes but lose their protein-coding functions.Therefore,pseudogenes have been considered as "junk DNA" in the genome for a long time.However,in recent years,studies have found that pseudogenes have important biological functions.For example,pseudogenes can competitively combine with microRNA to regulate the expression of functional genes.Pseudogenes can produce antisense RNA to inhibit the expression of functional genes.Some pseudogenes can also encode functional proteins.Pseudogenes are abnormally expressed and play an important role in diseases including tumors.The pseudogene PTENP1 protects PTEN expression by competitively binding miRNA to inhibit tumor growth.The pseudogene Oct4-pg1 encodes a protein of 359 amino acids to promote tumors cell proliferation.HMGA1P6 is one of the eight pseudogenes of HMGA1.which is located on chromosome 13.It has a mutation at the stop codon so it generates a long non-coding RNA that cannot encode a protein.Long non-coding RNA(lncRNA)is a class of RNA with transcripts longer than 200 bp.Sequencing results show that more than 90%of the human genome is transcribed,but only 1%-2%are protein-encoding genes.Those multitudinous non-coding RNA participate in a variety of cellular activities and perform multiple functions in the form of RNA.Studies have found that HMGA1P6 plays an oncogenetic role in pituitary tumors and thyroid cancer.But whether HMGA1P6 plays a role in ovarian cancer is still unknown.This research is mainly divided into the following three parts:Part 1:Study of the Expression,Clinical Significance and Biological Function of HMGA1P6 in Serous Ovarian CancerObjectiveTo clarify the expression pattern of one of the pseudogenes,HMGA1P6,in ovarian cancer,its correlation with clinical information,and its biological function.MethodsFirstly,we used microarray technology,using the fallopian tube umbrella as a control,to analyze lncRNA expression profile of low grade and high grade serous carcinomas,to screen for differentially expressed pseudogenes and to analyze pseudogene expression patterns.Secondly,we used lentivirus to construct stable HMGA1P6 overexpression and knockdown cell lines to study the biological function of HMGA1P6.Finally,we explored the effects of HMGA1P6 on the proliferation of ovarian cancer cells in vitro through EdU incorporation,clonogenic assay and MTT assay.We observed the effects of HMGA1P6 on ovarian cancer proliferation in vivo through nude mice subcutaneous tumor formation experiments.3D cell culture assay and stem cell marker test were used to verify the effect of HMGA1P6 on the malignancy of tumor-cells.We tested the effect of HMGA1P6 on cell invasion and migration through matrigel invasion assay,high-content bright field migration experiment and the verification of EMT-related markers.The effects of HMGA1P6 on tumor cell metabolism was tested by Seahorse cell metabolism experiment and ATP concentration detection.We also tested the effect of HMGA1P6 on apoptosis by flow cytometryResults1.HMGA1P6 was highly expressed in serous ovarian cancer and correlated with poor prognosisMicroarray sequencing results showed that 577 pseudogenes were aberrant expressed in ovarian cancer(log2 fold change>l or<-1)and most of the them were up-regulated in ovarian cancer(538 of 577).Cluster analysis and correlation matrix analysis indicated that HGSOC has a unique pseudogene expression pattern.Further analysis of ovarian cancer tissue samples revealed that high expression of HMGA1P6 was associated with poor prognosis.2.HMGA1P6 overexpression promoted the proliferation of ovarian cancer cells both in vitro and in vivoWe used stable HMGA1P6 overexpression and knockdown cell lines to explore its biological function.EdU,clonogenic and MTT assays all confirmed that the cells proliferated faster and formed more clones after over-expressing HMGA1P6,and the cells with HMGA1P6 knockdown had more slowly proliferation rate and formed less clones than the control.In addition,subcutaneous tumor formation experiments in nude mice showed that the over-expressing cells of HMGA1P6 had larger tumor mass,and showed high expression of HMGA1 and Ki-67 in tumor immunohistochemistry.The tumorigenic ability of HMGA1P6 knockdown cells was significantly lower than that of control cells3.HMGA1P6 overexpression promoted the malignancy of ovarian cancer cellsThree-dimensional culture can simulate the micro-environment of cells in vivo more closely.We used the HMGA1P6 overexpression and knockdown cell lines for three-dimensional culture experiments.3D cell culture assay showed that cells with HMGA1P6 overexpression had higher spheroid formation efficiency,while HMGA1P6 knockdown significantly reduced the spheroid formation efficiency compared to the control.The stem cell markers also decreased after HMGA1P6 knockdown.4.HMGA1P6 overexpression promoted cell invasion and migrationMatrigel invasion assay showed that the cell invasion ability increased after overexpression of HMGA1P6,while the cell invasion ability decreased after HMGA1P6 knockdown.At the same time,EMT markers also showed corresponding changes.Cells turned to mesenchymal direction after overexpression of HMGA1P6,and cells turned to epithelial direction after HMGA1P6 was knocked down.High-content bright field migration experiments showed that cell displacement decreased after HMGA1P6 knockdown indicating that cell migration were weakened5.HMGA1P6 overexpression promoted cell metabolismWe used Seahorse system to investigate the function of HMGA1P6 in cell metabolism.After over-expression of HMGA1P6,the overall metabolic phenotype of the cells turned to an active state,while after HMGA1P6 knockdown,the cells turned to a silent state.In addition,the glycolytic function of the cells was enhanced after over-expression of HMGA1P6,and the glycolytic function as well as the ATP level of the cells was reduced after HMGA1P6 knockdown.6.HMGA1P6 knockdown promoted cell apoptosisWe used Tet-on system to knock down HMGA1P6.After 48-72 hours of DOX induction,we used flow cytometry to detect early apoptosis and found that apoptosis was increased significantly after HMGA1P6 knockdown.Western blot showed elevated levels of Bax and cleaved-Caspase 3,decreased level of Bcl-2 after HMGA1P6 knockdown.Part 2:HMGA1P6 Regulated HMGA1/2 Expression through ceRNA MechanismObjectiveIn the first part,we have identified the high expression of HMGA1P6 in ovarian cancer and it promoted the malignant behavior of ovarian cancer.In this part,we tried to explore the mechanism of HMGA1P6 in promoting cancer progression.MethodsWe found that the lncRNA produced by pseudogenes can protect the parental mRNA through competitive binding through a review of the studies.So we investigated whether HMGA1P6 works through this mechanism.Firstly,we observed the correlationship between HMGA1P6 and HMGA1/2 at expression level.Then we performed sequence comparison between HMGA1P6 and HMGA1/2.Secondly,we locate the HMGA1P6 in cell using nuclear-plasma separation experiment.Then,we verified the regulation of HMGA1P6 and HMGA1/2 expression by microRNA.We constructed HMGA1/2 3'UTR-dual-luciferase vectors,wild-type and microRNA binding site-mutant dual luciferase vector of HMGA1P6 to verify whether there is a direct binding relationship between microRNA and these three genes.Co-transfection experiments verified the ceRNA protection network between these three genes.We verified the binding relationship between HMGA1P6 and microRNA through RIP experiments and RNA pull-down experiments.Finally,we verified the upstream and downstream relationship between HMGA1P6 and HMGA1 through rescue experiments.Results1.HMGA1P6 regulated HMGA1/2 expressionTo explore the mechanism by which HMGA1P6 promotes the malignant behavior of ovarian cancer,we speculated that HMGA1P6 may regulate the expression of HMGA1/2.We found that both HMGA1 and HMGA2 decreased after HMGA1P6 knockdown.When we over-express HMGA1P6 with a concentration gradient,HMGA1 and HMGA2 also showed a concentration gradient change.TCGA data showed that HMGA1 and HMGA2 were also over-expressed in ovarian cancer,and tissue sample qPCR results also showed a correlation between HMGA1P6,HMGA1 and HMGA22.HMGA1P6 regulated HMGA1/2 expression by ceRNA mechanismTo further explore the biological function of HMGA1P6,we next analyzed the localization of HMGA1P6 in the cell,nuclear-plasma separation experiment found that HMGA1P6 was expressed both in the nucleus and cytoplasm.TargetScan and sequence comparison showed that microRNA which targeted on HMGA1/2 also had binding sites on HMGA1P6.We selected several microRNA and found that hsa-let-7c-5p,hsa-miR-106a-5p and hsa-miR-103a-3p can all down-regulate the expression of HMGA1P6 and HMGA1/2,and their inhibitors can up-regulate the expression of HMGA1P6 and HMGA1/2.The luciferase experiment confirmed the direct binding between hsa-let-7c-5p,hsa-miR-106a-5p and hsa-miR-103a-3p and HMGA1/2-3'UTR and HMGA1P6.HMGA1P6 and HMGA2 were down-regulated after HMGA 1 knockdown.HMGA1P6 and HMGA2 were up-regulated after over-expression of HMGA 1-3'UTR.The results of HMGA2 were accordant with HMGA1.The co-transfection rescue experiment also confirmed the ceRNA network between HMGA1P6 and HMGA 1/2.We detected a higher abundance of HMGA1P6 in the AG02-RIP group,which proved the binding of HMGA1P6 to microRNA and RISC complexes.The reverse RNA pull-down experiment also found that HMGA1P6 can bind to AG02.Above all,we demonstrated that HMGA1P6 regulated HMGA1/2 expression by ceRNA mechanism.3.HMGA1 was needed in HMGA1P6 promoting ovarian cancer invasionTo explore the biological significance of HMGA1P6 regulating HMGA1,we performed rescue experiment.It showed that over-expressing HMGA1P6 when we knock down HMGA1 at the same time in ovarian cancer cells could not rescue its reduced invasion ability.Part 3:MYC Regulated the Expression of HMGA1P6 in Serous Ovarian CancerObjectiveTo explore the upstream regulatory mechanism of HMGA1P6 overexpression in ovarian cancerMethodsFirstly,we conducted the bioinformatics analysis of transcription factors binding sites on HMGA1P6 promoter region.Secondly,ChIP and luciferase experiments were used to prove the binding relationship between transcription factors and the promoter region.Finally,the expression regulation relationship was verified.Results1.MYC bound to HMGA1P6 promoterWe used JASPER to analyse HMGA1P6 promoter region and we found that MYC has two binding sites in the promoter region of HMGA1P6.The results of the ChIP experiment showed that the MYC-ChIP group had a higher enrichment of HMGA1P6 promoter,but only on site 2,not site 1.We used JQ-1(an inhibitor of MYC)to treat cells,and then we performed ChIP experiments.It was found that enrichment of site 2 was reduced after JQ-1 treatment,but not site 1.Wild and site 2-mutant type HMGA1P6 promoter luciferase reporter experiments demonstrated that MYC could bound to HMGA1P6 promoter through site 2 and transcriptionally activated HMGA1P6.2.MYC regulated HMGA1P6 and HMGA1/2 expressionAfter over-expression of MYC,the expressions of HMGA1P6,HMGA1/2 were all increased,while the expression of HMGA1P6,HMGA1/2 were all decreased after MYC knockdown.Conclusions1.HMGA1P6 was over-expressed in serous ovarian cancer and associated with poor prognosis.2.Abnormally overexpression of HMGA1P6 promotes malignant behaviors such as ovarian cancer proliferation,invasion and migration.3.HMGA1P6 regulates the expression of HMGA1/2 through the ceRNA mechanism.4.MYC transcriptionally activated the expression of HMGA1P6 in ovarian cancer cells.