The Study Of Effects And Mechanisms Of Long Non-coding RNA LINC01116 In Endometriosis

Objective:Endometriosis（EMs）,refers to the infiltration and growth of functional endometrial tissue（interstitium and glands）in other parts outside the uterine cavity,accompanied by shedding and bleeding according to sexual cycles.The common symptoms of endometriosis are:dysmenorrhea,chronic pelvic pain and infertility.As a common disease in women of childbearing age,the incidence of endometriosis is about6%-10%,and in women with infertility or chronic pelvic pain,the incidence is as high as 35%-50%.Although endometriosis is a benign disease,it has some biological behaviors similar to malignant tumors such as tissue invasion,local spread,remote metastasis,and recurrence,which seriously threaten women’s health.Therefore,in-depth research on the pathophysiological basis of endometriosis is the key to improve EMs’diagnosis and treatment.There are many theorys on the pathogenesis of endometriosis,genetic factors are among them.Only 1%-2%of the genes in the human genome can be transcribed and translated into proteins,and the remaining 98%are non-protein coding sequences,whose transcription products form a large and complex family of non-coding RNAs（nc RNAs）.In the past scholars tended to interpret these transcription products as meaningless transcription fragments,but in recent years,more and more studies have found that these nc RNAs are involved in many human pathophysiological processes.According to the length of nc RNAs,we can divide them into short non-coding RNA（snc RNA）and long non-coding RNA（lncRNA）.The length of short non-coding RNA generally does not exceed 40 nucleotides,such as micro RNA（micro RNA,miRNA）and the length of lncRNA generally exceeds 200 nucleotides.In recent years,non-coding RNA（nc RNA）has become a new focus of research due to its important regulatory role in many diseases and its ability as a potential molecular biomarker.Long non-coding RNA（Lnc RNA）refers to nc RNA with a length of more than 200 nucleotides but does not encode a protein.Current research has found that lncRNA plays an important role in malignant tumors,cardiovascular diseases,immune system diseases and many other diseases.In 2011,Salmena et al.first proposed the theory of“competitive endogenous RNA（ceRNA）”,ceRNA refers to transcripts which have miRNA response elements（miRNA response element,MRE）,including lncRNA and circular RNA（circ RNA）.All of those transcripts can regulate each other’s expression by competitively binding to one common miRNA,which is to play the role of"molecular sponge".This theory provides an important theoretical basis for the mechanism of lncRNAs to perform regulatory functions in living organisms.Up to now,there are few researches about the relationship between lncRNA and endometriosis at home and abroad,especially the research on the mechanism of lncRNA.Ovarian endometriosis is the most common type of endometriosis,and ovarian endometriosis often affects the ovarian reserve function of women of childbearing age,leading to infertility.Therefore,this study will start with ovarian endometriosis,high-throughput sequencing technology was utilized to screen out the differential expression profiles of lncRNAs in ectopic endometrial tissue and eutopic endometrial tissue in patients with ovarian endometriosis,and primary endometrial stromal cells（ESCs）are used as a cell model to explore the effect of LINC01116 on the biological behavior of EMs and its mechanism of action,in order to provide a new breakthrough for the pathogenesis of endometriosis and molecular targeted therapy point.Method:Part 1:High-throughput sequencing technology was utilized to detect the expression profiles of lncRNAs in 6 pairs ectopic endometrial and eutopic endometrial tissue samples of endometriosis patients,then we selected several lncRNAs which were obviously differentially expressed for validation using q RT-PCR in an expanded number of tissue samples.The ectopic endometrial stromal cells（ESCs）were extracted from the ectopic endometrial tissue and subjected by immunofluorescence identification as a nice cell model for in vitro experiments of endometriosis.Further through the construction of knockdown lentivirus and overexpression plasmids,we downregulated or upregulated the expression of LINC01116 in ESCs,CCK-8,Annexin V-FITC/PI double staining method,scratch experiment and transwell experiment were used to explore the biological effects of LINC01116 on ESCs.Part 2:Bioinformatics analysis was used to predict the miRNAs that could bind with LINC01116,q RT-PCR detected the effect of LINC01116 on the expression of target miRNAs.QRT-PCR was performed to measure the expression of miR-9-5p in endometriotic tissues and dual luciferase experiment was used to verify whether miR-9-5p has targeted binding with LINC01116.Subsequently,miR-9-5p mimics and inhibitor were used to upregulated or downregulated the expression of miR-9-5p in ESCs.CCK-8,scratch test and transwell test were used to explore the impact of miR-9-5p on proliferation and migration ability of ESCs.Part 3:Firstly,we used bioinformatics websites to predict the target gene of miR-9-5p,and we used q RT-PCR to detect the expression of FOXP1 in endometriosis tissues,and dual luciferase experiment was performed to verify whether FOXP1 is targeted binding with miR-9-5p.QRT-PCR and western Blot were used to detect the effect of miR-9-5p on FOXP1 m RNA and protein expression,and then we co-transfected sh-LINC01116 and miR-9-5p inhibitor,oe-LINC01116 and miR-9-5p mimics to see the effect of both LINC01116 and miR-9-5p on the expression of FOXP1.Finally,CCK-8,scratch test and transwell test were used to verify whether miR-9-5p can rescue the effect of LINC01116 on the biological behavior of ESCs.Part 4:The eutopic and ectopic endometrial tissues were used to construct an allograft model of endometriosis,and HE staining and immunohistochemistry were used to detect the expression level of FOXP1 in the lesions of nude mice.LINC01116expression was knocked down to construct a stable transgenic cell line of Ishikawa.And we conduct tumor formation experiments in nude mice using Ishikawa to verify the effect of LINC01116 on in vivo lesions by measuring the tumor growth curve.Results:Part 1:High-throughput sequencing technology was used to detect the expression profiles of lncRNAs in the ectopic endometrial and eutopic endometrial tissue samples of patients with endometriosis.The results showed 92 down-regulated and 50 up-regulated lncRNAs（∣log₂FC∣≥2,P<0.01）,among which LINC01116 was significantly up-regulated in ectopic endometrial tissue（log₂FC=3.668,P<0.01）.We further used q RT-PCR for validation in enlarged number of tissue samples,and the experiment results were consistent with the sequencing results.Primary endometrial stromal cells（ESCs）were extracted from endometriosis ectopic endometrial tissue,immunofluorescence experiments showed that the cell purity was more than 90%.The LINC01116 knockdown lentivirus and overexpression plasmid were constructed and transfected into ESCs.The q RT-PCR experiment showed that the knockdown or overexpression effect was obvious（P<0.05）.CCK-8 experiment showed that the knockdown of LINC01116 inhibited the proliferation ability of ESCs,and overexpression of LINC01116 promoted the proliferation ability of ESCs;Annexin V-FITC/PI double staining method showed that knockdown of LINC01116 had no significant effect on the apoptosis of ESCs;scratch test and transwell experiment proved knockdown of LINC01116 inhibits the migration ability of ESCs,and overexpression of LINC01116 promotes the migration ability of ESCs.Part 2:According to the"ceRNA"hypothesis,we used bioinformatic analysis to predict the potential miRNAs that can bind to LINC01116,and subsequent q RT-PCR experiments showed that miR-9-5p was up-regulated after knockdowning the upstream LINC01116,and exerted the opposite effects after overexpressing LINC01116.The expression of miR-9-5p was downregulated in ectopic endometrium compared to eutopic endometrium（P<0.05）.The dual luciferase experiment verified the targeted binding of miR-9-5p and LINC01116.Subsequently,miRNA mimics and inhibitors were used to knockdown or overexpress the expression of miR-9-5p in ESCs,CCK-8experiments showed that knockdown of miR-9-5p promotes the proliferation ability of ESCs,and overexpression of miR-9-5p inhibits the proliferation ability of ESCs;Scratch experiment and transwell experiment proved that knockdown of miR-9-5p promotes the migration ability of ESCs,and overexpression of miR-9-5p inhibits the migration ability of ESCs.Part 3:4 bioinformatics websites were utilized to predict the potential target genes of miR-9-5p,it is found that FOXP1 was among the intersection of the 4 results.The QRT-PCR experiment proved that the expression of FOXP1 was higher in the ectopic endometrium compared to eutopic endometrium（P<0.05）.The dual luciferase experiment further verified that the targerted binding between FOXP1 and miR-9-5p.QRT-PCR and western blot experiments showed that overexpression of miR-9-5p inhibited the expression of FOXP1 m RNA and protein,and knockdown of miR-9-5p promoted the expression of FOXP1 m RNA and protein.We used q RT-PCR and western blot experiments to detect the expression levels of FOXP1 m RNA and protein in the sh-NC group,sh-LINC01116 group,miR-9-5p inhibitor group,or sh-LINC01116+miR-9-5p inhibitor group.The results It shows that down-regulated LINC01116 can reduce the expression of FOXP1 m RNA and protein in ESCs,while inhibiting the expression of miR-9-5p can reverse the down-regulated LINC01116 effect on FOXP1 expression.CCK-8 experiments show that miR-9-5p inhibbitor can resue the inhibitory effect of sh-LINC01116 on the proliferation ability of ESCs,and miR-9-5p mimics can resue the promotion effect of oe-LINC01116 on the proliferation ability of ESCs.Scratch experiments and transwell experiments showed that miR-9-5p inhibbitor can rescue the inhibitory effect of sh-LINC01116 on the migration ability of ESCs,and miR-9-5p mimics can rescue the promotion effect of oe-LINC01116 on the migration ability of ESCs.Part 4:Transplantation of human endometrial tissue under the skin of nude mice can successfully construct lesions,and the growth of the ectopic endometrium is better than that of the ectopic endometrium.The expression of FOXP1 protein in ectopic endometrial lesions was higher than that in eutopic endometrial lesions（P<0.05）.The growth rate of ishikawa subcutaneous tumor in the sh-LINC01116 group was significantly slower than that of the blank group and sh-NC group,and the tumor volume was significantly smaller than the blank group and sh-NC group（P<0.01）,also the expression of FOXP1 in the tumor tissue of the sh-LINC01116 group was significant lower than sh-NC group and blank group.Conclusion:1.Hgh-throughput sequencing technology was performed to screen the expression of lncRNAs in 6 pairs ectopic endometrium and eutopic endometrium of endometriosis,92 down-regulated and 50 up-regulated lncRNAs were obtained（∣log₂FC∣≥2,P<0.01）.LINC01116 is highly expressed in ectopic endometrium tissues and can promote the proliferation and migration ability of ESCs.2.MiR-9-5p is lowly expressed in ectopic endometrium tissues,and the expression of miR-9-5p is negatively regulated by LINC01116.LINC01116 can sponge miR-9-5p and the down-regulation of miR-9-5p can promote the proliferation and migration ability of ESCs.3.FOXP1 is highly expressed in ectopic endometrium tissues.The expression of FOXP1 is negatively regulated by miR-9-5p,and FOXP1 is the target gene of miR-9-5p.LINC01116 can promote the proliferation and migration ability of ESCs through the LINC01116/miR-9-5p/FOXP1 axis.4.The growth state of ectopic endometrium under the skin of nude mice is better than that of eutopic endometrium,and the expression of FOXP1 in ectopic endometrium lesions is higher than that of eutopic endometrium lesions.Down-regulating the expression of LINC01116 can inhibit the growth of ishikawa nude mice tumorigenesis and at the same time inhibit the expression of FOXP1.