Hsa-miR-1290 Regulating The Characteristics And Metastasis Of Prostate Cancer Stem Cells Through RORA/NF-κB Pathway

Purpose:Prostatic carcinoma(PCa)is the most common cancer in men in more than half(112/185)of the world’s countries,with approximately 1.4million new cases in 2020.Pca is also the leading cause of cancer-related death in men in about a quarter(48/185)of the world’s countries,with an estimated 375,000 deaths in 2020,posing a serious threat to patients’ lives and health.hsa-miR-1290 has been proved to be dysregulated in a variety of malignant tumors,and plays an important role in the occurrence and development of malignant tumors.The expression level of hsa-miR-1290 in the serum of patients with prostate cancer is significantly up-regulated,but its biological role and molecular mechanism in prostate cancer have not been fully elucidated,so more in-depth studies are needed to explore the function and clinical value of hsa-miR-1290 in prostate cancer.The aim is to provide a theoretical basis for Pca to find new diagnosis and treatment targets.Methods:Firstly,bioinformatics software was used to analyze the expression of hsa-miR-1290 in the GSE112264 dataset,and q RT-PCR was used to detect the expression level of hsa-miR-1290 in PCa tissues and cells.The correlation between hsa-miR-1290 and clinicopathological features of prostate cancer patients was statistically analyzed.To clarify the regulatory role of hsa-miR-1290 in prostate cancer,we knocked down the expression of hsa-miR-1290 in prostate cancer.CCK-8 assay,clonogenesis assay,Ed U assay,Transwell assay and tumor pellet formation assay were used to detect the effects of knockdown of hsa-miR-1290 on the proliferation,migration and invasion of prostate cancer cells as well as the ability of pellet formation.In addition,q RT-PCR and Western blot assay were used to detect the effects of knockdown of hsa-miR-1290 on the expression levels of stem cell specific markers and epithelial interstitium transformation(EMT)markers of prostate cancer cells,so as to initially clarify the biological function of hsa-miR-1290 in prostate cancer.Secondly,we transfected hsa-miR-1290 overexpression plasmid into prostate cancer DU145 cells.The effects of hsa-miR-1290 overexpression on the proliferation,migration and invasion of prostate cancer cells and the ability of pellet formation were detected by CCK-8 assay,clonogenesis assay,Ed U assay,Transwell assay and tumor pellet formation assay.In addition,the effects of hsa-miR-1290 overexpression on the expression levels of stem cell characteristic markers and EMT markers of prostate cancer cells were detected by q RT-PCR and Western blot experiments,so as to determine whether hsa-miR-1290 overexpression can promote the disease process of prostate cancer.Finally,the downstream target genes of hsa-miR-1290 were predicted by bioinformatics software and verified by double luciferase reporter gene assay.RORA expression levels in PCa tissues and PCa cells were detected by q RT-PCR and Western blot assay,and the effect of hsa-miR-1290 knockdown on RORA protein expression was detected.Subsequently,according to different intervention conditions,the cells were divided into NC,hsa-miR-1290-antagomir and hsa-miR-1290-antagomir+sh-RORA groups,and the proliferation,migration and invasion ability of the cells in each group were detected by CCK-8 assay,Ed U assay and Transwell assay.The expression of key molecules in NF-κB pathway was detected by Western blot assay.In addition,we also constructed a nude mouse transplanted tumor model to determine whether hsa-miR-1290 affects the volume and weight of tumor growth in nude mice by regulating RORA/NF-κB.The expression levels of RORA,NF-κB pathway and tumor stem cell specific markers in tumor tissues of each group were detected by Western blot and immunohistochemistry.Results:(1)hsa-miR-1290 was significantly overexpressed in serum,tissues and cells of patients with prostate cancer,and its high expression was closely related to the poor prognosis of patients;In addition,knockdown of hsa-miR-1290 can inhibit the proliferation,clonogenesis,tumor globularization,migration and invasion of prostate cancer cells.In addition,knockdown of hsa-miR-1290 significantly decreased the expression of CD133,Oct4,Sox2,Nanog,c-Kit and Notch3,while significantly effect the expression levels of EMT-related markers E-cadherin,Vimentin and N-cadherin.(2)Overexpression of hsa-miR-1290 can significantly promote the proliferative ability,clonal formation ability,tumor globularization ability,migration and invasion ability of prostate cancer DU145.In addition,overexpression of hsa-miR-1290 can also significantly increase the expression of CD133,Oct4,Sox2,Nanog,c-Kit and Notch3 in prostate cancer DU145 cells,and effect the EMT-related marker E-cadherin.Expression levels of Vimentin and N-cadherin.(3)Bioinformatics software predicted that the downstream target gene of hsa-miR-1290 might be RORA,and double luciferase reporter gene experiment confirmed that RORA was the downstream target of hsa-miR-1290.RORA expression is abnormally low in prostate cancer tissues and cells,and knockdown of hsa-miR-1290 can significantly up-regulate the expression of RORA.In addition,knockdown of hsa-miR-1290 significantly inhibited the proliferation,clonogenesis,migration and invasion of LNCa P and PC-3 cells,and significantly inhibited the NF-κB pathway activity.After co-transfection with sh-RORA,the proliferation,clonogenesis,migration and invasion of LNCa P and PC-3 cells were partially improved,while the NF-κB pathway activity was partially restored.The results of in vivo experiments showed that knocking down hsa-miR-1290 can significantly inhibit the growth rate of tumors,promote the expression level of RORA in tumor tissues,and inhibit the activity of NF-κB pathway and the characteristics of tumor stem cells.Conclusion:(1)hsa-miR-1290 was significantly highly expressed in serum,tissues and cells of prostate cancer patients,and its high expression was closely related to the malignancy degree of prostate cancer patients.(2)Knockdown or overexpression of hsa-miR-1290 can regulate the proliferation,migration and invasion of prostate cancer cells and the ability of tumor pellet formation,and affect the stem cell characteristics and EMT of prostate cancer cells.(3)hsa-miR-1290 regulates the proliferation,migration,invasion,stem cell characterization,and EMT of prostate cancer by targeting the RORA/NF-κB pathway,thereby promoting the disease progression of prostate cancer.