| Objective: Endometriosis(EMs)refers to the presence of active endometrial tissue(glands and stroma)in a location other than the uterine cavity,which is pathologically benign and has a biological behavior similar to that of a malignant tumor.The clinical manifestations are infiltration,implant growth,destruction of surrounding tissues,distant metastasis and easy recurrence after treatment.The incidence of endometriosis is about 10%,which continues to plague about 190 million women of childbearing age worldwide.It mainly causes infertility,dysmenorrhea,chronic pelvic pain,etc.,and Malignant transformation of endometriosis.More attention has been paid to the problem of malignant change of endometriosis.Epidemiological studies have shown that the malignant transformation rate of endometriosis is as high as 1%,and the risk of malignant transformation is positively correlated with the course of disease,age and other factors.Endometriosis is mainly in the ovary,and the risk of direct malignant change in ovarian endometriosis increases by 0.7-1.6% approximately every 8 years.At present,the mechanism of malignant transformation of endometriosis is still unclear,and there is a lack of characteristic clinical symptoms and non-invasive detection methods.Therefore,it is urgent to find specific molecular markers for early diagnosis of malignant transformation of endometriosis.The topic of malignant transformation of endometriosis has always been a hot topic in gynecological research.At the same time,it is also a difficult field to carry out research because of the lack of good cell model and disease model for a long time.To closer to the different disease of disease states,generally take a compromise approach is the use of endometrial tissue extracts the original generation of endometrial cells in vitro,and the generation of endometrial cells before immortalized limited training time and the number of batches,at the same time,there are some individual differences and extraction operation error,primitive cells that are less than difficult to satisfy the roll out of the cells in the experiment.The long-term surgical endometriosis mouse model is only suitable for shortterm(1-2 months)feeding,and the short survival time of ectopic lesions is difficult to observe the typical malignant transformation trend,which hinders the modeling of endometriosis malignant transformation.In conclusion,the research on malignant transformation of endometriosis is relatively lagged behind,and the molecular mechanism of malignant transformation of endometriosis is very scarce.There are more studies in different disease with high risk of malignant change happen,and different disease frequently-occurring disease in young women,for its malignant transformation provides a window period is very long,so we need to in-depth study inside different disease progression related mechanism in order to identify possible early malignant transformation,so as to achieve the goal of early detection and early intervention.Only less than 2% of the gene in the human genome encode proteins,and only about 10% of the transcribed RNA molecules are ultimately directly involved in protein synthesis.The remaining 90% of the RNA molecules that cannot be translated into proteins are collectively referred to as non-coding RNA(nc RNA).They include long non-coding RNA(lnc RNA),micro RNA(mi RNA)and circular RNA(circ RNA).Through internal different disease related to ovarian cancer,not within different disease related to ovarian cancer,ovarian and benign ovarian ectopic endometrium tissue after testing found within different disease related to ovarian cancer molecular type is more close to the source of endometrial malignant tumor,and different disease related to ovarian cancer in the pathological type,type of pathogenic factors and molecular characteristics and Ⅰ endometrial cancer has many similarities.In recent years,many studies have reported that lncrnas are involved in various aspects of cell biology,which may promote the occurrence and development of tumors by inhibiting cell proliferation,apoptosis,differentiation,invasion and metastasis.Because it is difficult to obtain fresh tissues of endometriosis malignant transformation in a short time,our group conducted LncRNA/mRNA microarray screening on type Ⅰ endometrial cancer tissues in the early stage,and found that LncRNA BANCR and CPNE3 mRNA were significantly up-regulated among them,and there was a significant correlation.Bioinformatics analysis revealed that LncRNA BANCR,CPNE3 and mi RNA-612 had the same binding site.Therefore,it was speculated that LncRNA BANCR upregulated the expression of CPNE3 through competitive binding of mi RNA-612 to promote malignant transformation of endometriosis.This study will prove that LncRNA BANCR/ mi RNA-612 /CPNE3 regulatory axis plays a promoting role in the malignant transformation of endometriosis by promoting proliferation and migration through tissue,cell and in vivo levels.LncRNA BANCR may be a new molecular target of malignant transformation of endometriosis.This research idea and method may inject new vitality into the research field of malignant change of endometriosis.Methods: Part I: The Arraystar Human LncRNA/mRNA tissue microarray was used to detect three pairs of type Ⅰ endometrial carcinoma tissues and their control tissues,and the molecular expression profile was established to screen the target molecules combined with bioinformatics database analysis and the scientific design proposed by the preexperimental results.Twelve pairs of malignant tissues of ovarian endometriosis and their control tissues were collected.qRT-PCR and Western blot were used to verify the expression levels and correlation of LncRNA BANCR,mi RNA-612 and CPNE3 molecules in each tissue.Part Ⅱ: Fish experiment confirmed the intracellular expression localization of LncRNA BANCR,and double luciferase experiment was used to test the possibility of LncRNA BANCR/ mi RNA-612 /CPNE3 binding regulation.Using plasmid transfection technology,Eutopic endometrial stromal cells(EESCs),Normal endometrial stromal cells,NESCs was used to conduct molecular regulation of BANCR,mi RNA-612 and CPNE3,and CCK8,scratch and Transwell experiments were used to verify the influence of each gene on the biological behavior of cells.NESCs transfection and subcutaneous injection in nude mice were used to confirm the effect of BANCR,mi RNA-612 and CPNE3 genes on the subcutaneous viability of primary endometrial cells.The growth curve of Ishikawa cells was drawn by tumor measurement,and the expression of CPNE3 protein in each tumor-bearing tissue was detected by immunohistochemistry.The effect of abnormal expression of BANCR,mi RNA-612 and CPNE3 genes on the growth of subcutaneous tumors at the in vivo level was explored.Results: Part Ⅰ: Through microarray screening,we found 2283 up-regulated and 5801 down-regulated lncrnas,5557 up-regulated and 2374 down-regulated mrnas.LncRNA BANCR and CPNE3 were a pair of genes with increased expression and significant correlation.Analysis results of bioinformatics GEO database related data sets verified that LncRNA BANCR and CPNE3 were highly expressed in endometrial cancer,and further mining of other data sets found that CPNE3 expression was significantly higher in endometriosis than in normal endometrium.Then,in the detection of malignant tissues of endometriosis,we verified that LncRNA BANCR and CPNE3 were increased and positively correlated in cancer tissues.Immunohistochemistry results also showed that CPNE3 protein expression was higher in malignant tissues of endometriosis than in endometriosis.And endometriosis > normal endometrium.Part Ⅱ: Fish results confirmed that LncRNA BANCR was mainly located in the cytosol.The sea kidney and firefly double luciferase experiment confirmed that the fluorescence value of wild-type LncRNA BANCR or CPNE3 co-transfected with mi RNA-612 into 293 T cells was lower,while the fluorescence value of mutant LncRNA BANCR or CPNE3 co-transfected with mi RNA-612 did not change significantly compared with NC group.Compared with NC group,NESCs cells transfected with LncRNA BANCR or CPNE3 overexpression plasmid showed enhanced CCK8 cell activity,larger healing area,more Transwell transmembrane cells,and increased CPNE3 protein and mRNA expression at the same time.After co-transfection of NESCs cells with BANCR or CPNE3 overexpression plasmid and mi RNA-612 mimics,it was found that compared with single transfection of BANCR or CPNE3 overexpression group,cell proliferation and migration ability were weakened,cell function was restored,and CPNE3 protein and mRNA levels were also reduced.Compared with NC group,EESCs transfected with LncRNA BANCR or CPNE3 knockdown plasmid had decreased proliferation ability,smaller healing area,fewer Transwell transmembrane cells,and decreased CPNE3 protein and mRNA expression.After co-transfection with BANCR or CPNE3 knockdown plasmid and mi RNA-inhibitor,EESCs cells with BANCR or CPNE3 knockdown plasmid and mi RNAinhibitor also showed functional recovery,and CPNE3 protein and mRNA levels increased.NESCs cells overexpressing BANCR or CPNE3 had higher subcutaneous survival and higher CPNE3 protein expression than NC cells.However,the cell survival rate and CPNE3 protein expression of co-transfected BANCR+ mi RNA-612 mimics group and CPNE3+ mi RNA-612 mimics group were lower than those of single over-table group.After stable transformation of Ishikawa cells in nude mice,the growth rate of tumors in BANCR or CPNE3 overexpression group was faster than that in NC group,and the final tumor volume was larger and the expression of CPNE3 protein was increased.Compared with the single overexpression group,the co-transfected BANCR+ mi RNA-612 mimics group and CPNE3+ mi RNA-612 mimics group had a slower growth rate and a smaller final tumor volume.Immunohistochemical experiments confirmed that the CPNE3 protein expression in the tumors was reduced.Conclusion: 1.LncRNA BANCR and CPNE3 genes were highly expressed candidate molecules in microarray screening and database validation results of type Ⅰ endometrial cancer.According to the results of biogenic analysis,mi RNA-612 interacts with two molecules,which may be the regulatory molecules of each other.2.In the malignant tissues of endometropia,LncRNA BANCR expression was upregulated,mi RNA-612 expression was down-regulated,and CPNE3 gene expression was up-regulated.LncRNA BANCR was positively correlated with CPNE3 expression,while mi RNA-612 was negatively correlated with CPNE3 expression.At the same time,the different disease progression in ectopic,within different disease group and normal endometrium in ectopic,three molecules have the same gradient expression level features,provide good evidence base for further research.3.LncRNA BANCR can competitively bind with mi RNA-612,thereby reducing the inhibitory effect on CPNE3 gene expression and promoting cell proliferation and migration.4.The abnormal expression of LncRNA BANCR and CPNE3 genes can promote the subcutaneous ectopic survival ability of endometrial cells and the growth of implant tumors,while the abnormal expression of mi RNA-612 can inhibit the growth of endometrial cells,which is similar to the molecular regulation at the cell level. |
PDF Full Text Request