LncRNA-AC002454.1 Promotes The Progression Of Endometriosis Patients By Upregulating CDK6 Expression In The Endometrial Cell Cycle Dysregulation In Endometrial Stromal Cells

Objective: Endometriosis(EMS)is an estrogen-dependent chronic gynecological disease which is refractory and associated with cell dysfunction.The main clinical symptoms are chronic pelvic pain,infertility and other symptoms.The typical features of EMS are pelvic mass and dysmenorrhea,which are associated with extensive maturation,invasion and a growth ability similar to a malignant tumor.Studies have shown that patients with EMS in the lining of the endometrium,especially during the secretory phase,appear different in terms of endometrial molecular function,such as migration,invasion,proliferation,angiogenesis,apoptotic ability and the expression of certain genes.In recent years,the abnormal expression of genes associated with the cell cycle of eutopic endometrium(EU)has also attracted attention.Previous preliminary work by our group used long non-coding RNA(lnc RNA)chip screening and bioinformatic analysis to investigate the endometrium in EMS and normal subjects.This work found that the AC002454.1 gene is one of the most highly expressed genes.AC002454.1 is a natural antisense lnc RNA.The classical mode in which antisense lnc RNAs function is to form RNA-RNA dimers with the m RNAs of adjacent genes,improve the stability of the m RNAs of neighboring genes,and thereby up-regulate the expression of adjacent genes.Antisense lnc RNA is divided into three main forms: head-to-head,tail-to-tail and complete overlap,according to the overlapping direction and length of the gene encoding the sense strand.In our previous chip analysis,we found that AC002454.1 is related to its adjacent gene,cyclin-dependent kinase-6(CDK6).They are both located at chromosome 7 and have overlapping regions of approximately 139 nucleotides in length.The positional relationship between AC002454.1 and CDK6 is head-to-head;that is the 5' end sequence of the antisense strand is complementary to the 5' end sequence of the sense strand.Quantitative real-time polymerase chain reaction(qRT-PCR)confirmed that the relative expression of AC002454.1 m RNA in EU of patients with EMS was significantly increased and correlated with CDK6.CDK6 is an important regulatory molecule in the G1 phase in the cell cycle.This binds to cyclin D and phosphorylates downstream p Rb and releases binding to the transcription factor E2 F,which initiates transcription of the genes necessary for cell cycle and cell proliferation.CDK6 is the m RNA with the highest fold difference in the cell cycle pathway and was ranked first in our pathway analysis.Gene ontology(GO)analysis suggested that differential m RNA transcripts are primarily associated with the cell cycle.In addition to promoting cell proliferation,CDK6 also enhances vascular endothelial growth factor A(VEGF-A)expression,thereby inducing angiogenesis and endothelial cell growth,enhancing vascular permeability;this action is independent of its kinase activity.This function is consistent with the function of endometrial cells which needs to pass through the process of adhesion,invasion and angiogenesis,and then survive in an ectopic site in order to form EMS lesions.Therefore,it is reasonable to speculate that: the regulation of CDK6 by AC002454.1may causes cell cycle disorder in endometrial cells,then escaping immune clearance,causing changes in their proliferative capacity,angiogenic ability;and participates in the disease progression of EMS.In this study,we investigate the expression,biological function and regulatory relationships of AC002454.1 and CDK6 in EU in the pathogenesis and development of endometriosis from the tissue level and in vitro cell level.Thus,our study might provide a novel insight into understanding the molecular mechanisms of endometriosis occurrence and development.Methods: qRT-PCR was used to analysis the AC002454.1 and CDK6 m RNA expression level and correlation,and their protein expression level of CDK6 was evaluated by immunohistochemistry(IHC)and western blot.Primary culture of eutopic endometrium cells(ESC)and normal endometrium cells(NESC).qRT-PCR was used to detect the m RNA expression of AC002454.1 and CDK6 in ESC and NESC,and their protein expression levels were evaluated by western blot.ESC was transfected with AC002454.1 and CDK6 silencing virus to knockdown the AC002454.1 and CDK6 gene,NESC was transfected with AC002454.1 and CDK6 overexpressing virus to overexpress the AC002454.1 and CDK6 gene.The transfection efficiency of AC002454.1 and CDK6 was detected by qRT-PCR and western blot,and the expression of p Rb,cyclin D1 and E2 F in ESC and NESC were also detected.CCK-8 assay was used to detect the alteration of cell proliferation ability after transfected with AC002454.1 and CDK6 of ESC and NESC.Wound Healing assay was used to detect the alteration of cell migration ability after transfected with AC002454.1 and CDK6 of ESC and NESC.The alteration of cell invasion ability after transfected with AC002454.1 and CDK6 of ESC and NESC was detected by transwell cell invasion assay.Flow cytometry assay was used to detect the alteration of cell cycle after transfected with AC002454.1 and CDK6 of ESC and NESC.Chromatin isolation by RNA purification assay-Mass spectrometry(Ch IRP-MS)was used to detect the RNA-specific binding proteins with AC002454.1.qRT-PCR was used to detect the m RNA expression of the RNA-specific binding proteins in ESC and NESC,and the correlation with AC002454.1 and CDK6.Results: 1.qRT-PCR results suggested that the relative expression of AC002454.1and CDK6 m RNA in the EU was significantly higher than that of the normal endometrium(NE)(P < 0.05),while the difference between the ectopic endometrium(EC)and EU was not significant(P > 0.05).In addition,the expression of AC002454.1 and CDK6 m RNA in the EU and NE was positive correlated(Pearson correlation coefficient: 0.691,P < 0.001).Western blot suggested the relative expression of CDK6 protein in EC and EU of patients with EMS was significantly higher than that in NE(P < 0.05).There was no difference between EC and the EU(P > 0.05).Immunohistochemistry suggested CDK6 protein was mainly expressed in the cytoplasm and nucleus of stromal cells.The positive rate of CDK6 protein expression was 15.0% in normal endometrial tissue,82.0% in eutopic endometrial tissue of patients with EMS and 68% in EC of patients with EMS.Results showed that the positive expression rate of CDK6 protein in the EU of patients with EMS were significantly higher than that of NE(P < 0.05),while there was no significant difference between EC and EU(P > 0.05).2.In vitro cell experiment,qRT-PCR results suggested that the relative expression levels of AC002454.1 m RNA in the AC002454.1 virus-infected silencing group were significantly lower than those in the negative control group and blank control group(P< 0.05).The relative expression levels of AC002454.1 m RNA in the AC002454.1virus-infected overexpression group were significantly higher than those in the negative control group group and the blank control group(P < 0.05).The relative expression levels of CDK6 m RNA in the CDK6 virus-infected silencing group were significantly lower than those in the negative control group and blank control group(P< 0.05).The relative expression levels of CDK6 m RNA in the CDK6 virus-infected overexpression group were significantly higher than those in the negative control group group and the blank control group(P < 0.05).Compared to the negative control group and blank control group,the relative expression levels of CDK6 m RNA and protein in the virus-infected silencing group of AC002454.1 were significantly decreased(P < 0.05)and increased in the virus-infected overexpression group(P < 0.05).The relative expression levels of p Rb,cyclin D1 and E2 F protein in the virus-infected silencing group of AC002454.1 were significantly decreased(P < 0.05)and increased in the virus-infected overexpression group(P < 0.05).Compared to the negative control group and blank control group,the proliferative ability was significantly decreased in the virus infection silencing group of AC002454.1 and CDK6(P < 0.05)and increased in the virus-infected overexpression group(P < 0.05)through CCK8 assays.The migration ability was significantly decreased in the virus infection silencing group of AC002454.1 and CDK6(P < 0.01)and increased in the virus-infected overexpression group(P < 0.01)through cell scratch assays.The invasive ability was significantly decreased in the virus infection silencing group of AC002454.1 and CDK6(P < 0.01)and increased in the virus-infected overexpression group(P < 0.01)through transwell invasion assay.The proportion of cells in G0/G1 phase increased significantly while the proportion of cells in S phase decreased significantly in the virus infection silencing group of AC002454.1 and CDK6(P < 0.01),in G0/G1 phase,the cell pecent was significantly decreased,while in S phase was significantly increased in the virus-infected overexpression group(P < 0.01).3.Ch IRP-MS results identified a total of 37 RNA-specific binding proteins combined with AC002454.1,including heat shock protein 90 k Da alpha,class B member1(HSP90AB1).Based on the STRING database,we found that there was an interaction between HSP90AB1 and CDK6 with a predicted score of 0.472.The Hit Predict database shows that the interaction score is 0.665.qRT-PCR results suggested that compared with NESC,HSP90AB1 m RNA expression was increased in ESC(P < 0.05).The expression of HSP90AB1 m RNA was positively correlated with the expression of AC002454.1 m RNA and CDK6 m RNA(Pearson correlation coefficient was 0.980 and 0.895,P<0.01).Conclusion: 1.The expression of AC002454.1 and CDK6 in the eutopic endometrium tissues and cells of EMS was significantly increased and correlated.Both AC002454.1 and CDK6 can change the biological behavior,including cell proliferation,migration and invasion of ESC.AC002454.1 and CDK6 may plays an important role in the process of EMS by affecting the basic biological behavior of eutopic endometrium.2.After regulated the expression of AC002454.1,the expression of CDK6 m RNA and protein have changed and the cell cycle related proteins,including p Rb,cyclin D1,and E2 F changed.The proportion of cells in G0/G1 phase and S phase has transformed.The expression of CDK6 regulating by AC002454.1 may be the molecular mechanism of endometrial cell cycle disorder in endometriosis.3.The protein HSP90AB1,which was shown to specifically interact with AC002454.1 was found to exhibit protein interactions with CDK6 protein.In addition,the expression of HSP90AB1 m RNA was positively correlated with the expression of AC002454.1 m RNA and CDK6 m RNA in ESC.Consequently,AC002454.1 may enhance the expression of CDK6 by acting on HSP90AB1 and then promote the progression of EMS by affecting the basic biological behavior of eutopic endometrium thus have an effect on EMS.