The Effects Of Long Non-coding RNA NRON On Triple Negative Breast Cancer And The Underlying Mechanisms

BackgroundBreast cancer is the most common malignancy in women.Compared with other molecular pathological types,triple-negative breast cancer(TNBC)has the characteristics of high aggressiveness,early onset age,high recurrence and metastasis rate,short survival time and poor clinical prognosis.Although with the development of modern diagnosis and treatment techniques,the early diagnosis rate of breast cancer is significantly improved and the 5-year survival is significantly improved,the treatment of TNBC in the middle and late stages has not made more significant progress.However,due to its molecular characteristics,so far,targeted therapy has not significantly improved the survival rate of TNBC patients,and chemotherapy is still the standard of treatment.While many patients with early TNBC can be cured with chemotherapy,the median overall survival of those who have metastasized is only 13 to 18 months.LncRNA NRON is a length of about 2.7 KB,consists of three exons of long chain non-coding RNA,alternative splicing of size range from 0.8 KB to 3.7 KB transcription,its enrichment in muscle(cardiac muscle),placenta,spleen,thymus and lymph nodes,due to its influence through activation of T cell nuclear transfer,is considered to be one of the activation of T cell nucleus factor inhibiting factor,identified as the regulation of T cell nuclear transfer,is considered in liver cancer,prostate cancer,lymphoma cells invasion,migration and angiogenesis plays an important role,Studies showed that the proliferation and invasion ability of tumor cells were significantly enhanced after LncRNA NRON was knocked out.However,the expression of LncRNA NRON in breast cancer has not been reported in studies at home and abroad.This study attempted to preliminarily explore the study of LncRNA NRON in triple-negative breast cancer,and verified the expression of LncRNA NRON in triple-negative breast cancer and its possible internal mechanism through clinical analysis,experimental verification and mechanism research,so as to provide reference for clinical diagnosis and treatment of triple-negative breast cancer.Purposes1.To detect the expression level of LncRNA NRON in triple negative breast cancer tissues,analyze the relationship between its expression level and the prognostic factors and its impact on overall survival,and clarify the significance of LncRNA NRON expression in triple negative breast cancer;2.The effect of overexpression of LncRNA NRON on the proliferation,invasion and migration of triple-negative the triple negative breast cancer cells;3.To preliminarily analysis the mechanism of overexpression of LncRNA NRON on triple negative the triple negative breast cancer cells.Part ?:Expression and significance of LncRNA NRON in triple negative.breast cancer tissuesMethods:1.Hematoxylin-eosin staining(HE staining)and immunohistochemical examination were performed on pathological specimens and adjacent tissues of patients after puncture in Henan tumor hospital to screen the pathological specimens of triple-negative breast cancer;2.RT-qPCR was used to detect the expression differences of LncRNA NRON in triple negative breast cancer tissues and adjacent tissues;3.The relationship between LncRNA NRON and prognostic factors and its impact on overall survival were clarified by one-way ANOVA and COX risk regression analysis of prognostic factors,and its predictive significance for prognosis of triple-negative breast cancer was discussed.Results:1.HE staining was used to identify benign and malignant tumors,and immunohistochemistry was used to detect the expression of ER,PR,HER2 and Ki67,and a total of 70 patients with ER<1%,PR<1%,and HER2 negative were screened;2.RT-qPCR was applied to the detection results of triple-negative breast cancer tissues and adjacant tissues.Compared with adjacant tissues,LncRNA NRON in triple-negative breast cancer tissues was significantly down-regulated,with statistically significant difference(P<0.0001);3.The median follow-up time of enrolled patients was 29.5 months(1-54 months),after which a total of 21 patients died(all of them from breast cancer)and 49 survived.Overall survival of patients with triple-negative breast cancer was related to clinical stage,whether they had visceral metastasis,and LncRNA NRON.The expression level of LncRNA NRON in triple negative breast cancer patients was negatively correlated with clinical stage,and the expression decreased with the increase of clinical stage.The higher the expression of LncRNA NRON,the longer the survival time.Part ?:The effect of upregulated LncRNA NRON expression on the proliferation,invasion and metastasis of breast cancer cellsMethods:1.Lentivirus expression vector of LncRNA NRON was constructed and purified to obtain lentivirus expressing LncRNA NRON;2.Establishment of upregulation LncRNA NRON breast cancer cell lines;The expression level of LncRNA NRON was detected by RT-qPCR,and the up-regulated LncRNA NRON breast cancer cell lines was identified successfully;3.The effect of up-regulated LncRNA NRON expression on the proliferation of the triple negative breast cancer cells was determined by CCK-8 experiment assay;4.The effect of up-regulated LncRNA NRON expression on breast cancer cell invasion and migration was detected by Transwell;5.Expression levels of TIMP2 and MMP2 were detected by Western-blot,and the effect of up-regulated LncRNA NRON expression on breast cancer cell invasion and migration was observed;6.Xenograft tumor assay in nude mice was conducted to determine the effect of up-regulated LncRNA NRON expression on breast cancer cell proliferation in vivo.RT-qPCR was performed to determine the expression level of LncRNA NRON in xenograft tumor.Results:1.Human genome DNA was extracted as the template,PCR amplification was carried out with the set primers,and the amplification product was 2730bp,which was cloned into lentivirus expression vector LV6.Sequencing analysis showed that the inserted fragment sequence was consistent with the target gene;2.The expression of LncRNA NRON was detected by rt-qpcr,and the expression level of LncRNA NRON was significantly increased in the overexpression group of Hs578T and BT-549 cell lines(P<0.001),indicating that the stable overexpression cell line was successfully constructed;3.CCK-8 results showed that the overexpression of LncRNA NRON in Hs578T and BT-549 cell lines significantly inhibited cell proliferation(P<0.001);4.Transwell assay showed that LncRNA NRON overexpression in Hs578T and BT-549 cell lines significantly inhibited cell invasion and migration(P<0.001);5.Western blot results showed that in Hs578T and BT-549 cell lines,the level of TIMP2 in the LncRNA NRON overexpression group significantly increased(P<0.05),and the expression level of MMP2 significantly decreased(P<0.001),suggesting that LncRNA NRON overexpression inhibited the invasion and migration of triple negative breast cancer cells;6.Upregulation of LncRNA NRON expression resulted in decreased tumor-forming volume and weight of triple negative breast cancer cells in nude mice(P<0.05),and significantly increased expression of LncRNA NRON in the transplanted tumor group(P<0.001),suggesting that upregulation of LncRNA NRON expression significantly inhibited tumor formation in nude mice.Part ?:The preliminary analysis of the mechanism of upregulation of LncRNA NRON expression in breast cancer cellsMethods:1.According to bioinformatics analysis results "there is an adsorption site binding to miR-3658in the LncRNA NRON sequence",their combination was detected by double luciferase experiment,and RT-qPCR detection of the effect of upregulation of LncRNA NRON on miR-3658in breast cancer cell lines;2.miR-3658 Inhibitor was synthesized and transfected into breast cancer cell lines,the effect of down-regulation of miR-3658expression on the proliferation,invasion and migration of the triple negative breast cancer cells was detected by CCK-8 experiment and Transwell experiment;3.The effect of miR-3658 on TIMP2 expression were detected by double luciferase and Western-blot experiment;4.Synthesis of si-TIMP2(silencing the expression of TIMP2),silencing the response of TIMP2 expression on the up-regulation of LncRNA NRON and down-regulation of miR-3658and si-TIMP2was detected by CCK-8 experiment and Transwell experiment.Results:1.We predicted the potential binding sites between LncRNA NRON and miR-3658based on biological information.In the double luciferase reporting experiment,luciferase expression activity was decreased in the cells transfected with both miR-3658and wild-type LncRNA NRON at the potential binding sites(P<0.001).RT-qPCR detected a significant decrease in the expression level of miR-3658in the overexpression group of Hs578T and BT-549 cell line LncRNA NRON(P<0.001).Together,this part of the experiment indicated that LncRNA NRON could bind to the miR-3658binding site through the fragment in its sequence,which has a negative regulatory effect on the expression level of miR-3658,confirming the correct analysis of biological information.2.CCK-8 experiment results showed that down-regulation of miR-3658expression in Hs578T and BT-549 cell lines significantly inhibited cell proliferation(P<0.001).Transwell assay showed that down-regulation of miR-3658expression in Hs578T and BT-549 cell lines significantly inhibited cell invasion and migration(P<0.001).That's a hint that the effect of the proliferation,invasion and metastasis in breast cancer cell lines was inhibited by down-regulation of miR-3658expression.3.Biological information analysis:there is a common binding site between TIMP2 and miR-3658;In the double luciferase reporting experiment,luciferase expression activity was decreased in cells transfected with both miR-3658and wild-type TIMP2 binding sites(P<0.001).Western blot analysis showed that in Hs578T and BT-549 cell lines,the level of TIMP2 significantly increased in the group with down-regulated miR-3658expression(P<0.001).These experiments jointly confirmed that TIMP2 is a target gene regulated by miR-3658.4.The inhibition of cell proliferation and invasion can be partly relieved by upgrading LncRNA NRON expression and silencing TIMP2 expression;The inhibition of cell proliferation and invasion can be partly relieved by down-regulating miR-3658expressionand and silencing TIMP2 expression.This part of the experiment showed that the up-regulation of LncRNA NRON and down-regulation of miR-3658could inhibit the proliferation and invasion of tumor cells through the link of TIMP2.Conclusion:1.Compared with paracancer tissues,LncRNA NRON in triple-negative breast cancer tissues was significantly down-regulated.The expression level of LncRNA NRON in triple negative breast cancer patients was negatively correlated with clinical stage,and the expression decreased with the increase of clinical stage.The higher the expression of LncRNA NRON,the longer the survival time;2.The overexpression of LncRNA NRON can significantly inhibit the proliferation,invasion and migration of the triple negative breast cancer cells,can significantly inhibit the growth of transplanted tumor in nude mice;3.The mechanism of LncRNA NRON inhibiting triple negative breast cancer may be realized by the adsorption of miR-3658by ceRNA,thus regulating the expression of TIMP2;4.The overexpression of LncRNA NRON can inhibit the development of triple negative breast,which may provide a new target for the treatment of triple negative breast cancer.