| Prostate cancer(PCa)is one of the most common tumors in urinary system.According to the latest statistics from the United States Independent Institutes of Health,the incidence and mortality of prostate cancer in the United States in 2020 are predicted to be 248,530 / year and 34,130/ year respectively.Despite the rapid development of the diagnosis and treatment of prostate cancer,the poor treatment effect and high prevalence rate are still serious clinical challenges.Therefore,the identification of new potential biomarkers and therapeutic targets is crucial to the improvement of alternative therapies.Researchers have found that non-coding RNAs(nc RNAs)are also an important part of the complex regulatory network of the body,and the localization and function of a large number of nc RNAs in the body have been revealed in subsequent studies and reports.Lnc RNA,a class of RNA with a length greater than 200 nts,does not encode proteins,but regulates the expression levels of genes at various levels in the form of RNA.Researchers have recognized that functional lnc RNA participate in a variety of physiological and pathological processes,particularly oncogenesis,due to those lnc RNA can affect gene expression by sponging of micro RNAs and m RNA in various tumor types.For example,PCA3,PCGEM1 and Plnc RNA-1 are known to be overexpressed in prostate cancer and to promote cancer progression.In the early stage,we extracted 3 pairs of RNA samples from prostate cancer and adjacent prostate tissues,performed lnc RNA transcriptome sequencing through RNA-seq,and screened out lnc RNA(LINC00467)with obvious differential expression by combining bioinformatics analysis.LINC00467 plays an important role in many malignant tumors.However,the role of LINC00467 in prostate cancer remains unclear.In this study,we aimed to explore how LINC00467 regulates the progression of prostate cancer.On this basis,qRT-PCR assay was used to detect the expression level of LINC00467 in clinical tissue samples and prostate cancer cell lines of prostate cancer patients,and it was found that the RNA level of LINC00467 in prostate cancer tissue samples and cell samples was significantly higher than that in para-cancer tissue samples and normal cell samples.In the study of biological phenotype,si RNA transfection technology was used to knock down LINC00467 in the in vitro experiment of prostate cancer cell lines,and it was found that LINC00467 could promote the proliferation,survival,migration and invasion of prostate cancer cells.LINC00467 was shown to promote EMT transformation in prostate cancer cells by Western Blot.Subsequently,we used the constructed lentivirus to stabilize and interfere with the prostate cancer cell line LINC00467 to verify the above function of promoting the proliferation of prostate cancer at the cellular and animal levels.Proliferative inflammatory atrophy(PIA)may be the bridge between prostatic inflammation and prostate cancer,and PIA tissue is often accompanied by a large number of macrophage infiltration,so we speculate that LINC00467 may be related to the polarization of macrophages.Through si RNA transfection,it was found that LINC00467 can promote the polarization of macrophages of type M2,thus promoting the migration and invasion of prostate cancer.Molecular mechanism study,aiming at LINC00467 role in the cytoplasm,using open database forecast targeting the lnc RNA micro RNAs,the use of qRT-PCR correlation with the expression of the two after the preliminary verification screening miR-494-3p,then through the dual luciferase reporter gene detection experiments found LINC00467 as competition combined with endogenous RNA and inhibit the activity of miR-494-3p,Furthermore,the role of STAT3 was enhanced to promote EMT transformation,migration and invasion of prostate cancer cells.Part ? Screening and identification of long non-coding RNA LINC00467Methods:(1)The differential Lnc RNA expression was obtained by bioinformatics analysis of RNA-seq sequencing results.(2)The expression levels of target lnc RNA in the online database samples were analyzed by bioinformatics.(3)The expression levels of target lnc RNA in the prostate cancer tissue samples and cell lines were detected by qRT-PCR.(4)ROC curve was used to analyze the diagnostic value of target lnc RNA in prostate cancer.(5)Cell localization of target lnc RNA was detected by nucleocytoplasmic separation assay.Results:(1)Lnc RNA LINC00467 was selected as the target gene according to the criteria of significant differential expression(differential multiple ?2,FDR ?0.05)in the prostate cancer tissues,para-carcinoma PIA tissues and para-carcinoma normal tissues with the same trend of change in the three groups assisted by open database analysis.(2)LINC00467 was highly expressed in most cancer tissues.(3)LINC00467 was highly expressed in prostate cancer tissues and cells.(4)ROC curve analysis showed that LINC00467 had certain diagnostic value for prostate cancer.(5)The localization of LINC00467 in cells was mainly concentrated in the cytoplasm.Conclusion: By RNA-seq and bioinformatics analysis,an abnormal lnc RNA with certain diagnostic value was found in prostate cancer,and the cell localization was mainly located in the cytoplasm.Part 2: LINC00467 enhances the proliferation,migration and invasion of prostate cancer cellsMethods:(1)CCK8 and EDU cell proliferative activity kit were used to detect the effect of LINC00467 knockdown on the proliferation of prostate cancer cells.(2)Clonal formation assay was used to detect the effect of LINC00467 knockdown on the survival ability of prostate cancer cells.(3)The cycle kit was used to detect the effect of LINC00467 knockdown on the cycle ability of prostate cancer cells.(4)Transwell chamber experiment was used to detect the effect of LINC00467 knockdown on the migration and invasion ability of prostate cancer cells.(5)Western Blot was used to detect the effect of LINC00467 knockdown on EMT-related markers.(6)The effect of LINC00467 knockdown on prostate cancer tumor growth ability was studied by transfecting LINC00467 interference lentivirus in DU145 cell line to conduct in vivo tumor-bearing model experiment.Results:(1)The proliferation activity of CCK8 and EDU cells showed that LINC00467 could promote the proliferation of prostate cancer cells.(2)Clonal formation experiments showed that LINC00467 could promote the survival of prostate cancer cells.(3)Flow cytometry showed that LINC00467 could block the prostate cancer cell cycle in the G0/G1 phase.(4)Transwell laboratory experiment confirmed that LINC00467 could promote the migration and invasion of prostate cancer cells.(5)Western Blot showed that after LINC00467 knockdown,the expression of E-cadherin in EMT-related markers was up-regulated,and the expression of N-cadherin and Vimentin was down-regulated.It was confirmed that LINC00467 can promote the EMT process of prostate cancer cells.(6)It was found that LINC00467 could promote the growth of prostate cancer in vivo in subcutaneous tumor-bearing mouse models.Conclusion: LINC00467 was found to promote the proliferation,migration and invasion of prostate cancer cells in vitro cell line model,and it was found to promote the growth of prostate cancer cells in vivo in animal model test.The results showed that LINC00467 is a carcinogenic gene of prostate cancer.Part ?: LINC00467 promotes malignant progression of prostate cancer through M2 macrophage polarizationMethods:(1)Induction and polarization of macrophages.(2)The expression level of LINC00467 in various types of macrophages.(3)Linc00467 was knocked down in M2 macrophages to observe the changes of cytokine markers.(4)LINC00467was knocked down in prostate cancer cells,and the supernatant was co-cultured with macrophages to observe the changes of cytokines.(5)LINC00467 was knocked down in M2 macrophages,and the supernatant was co-cultured with DU145 to observe the changes in its migration and invasion abilities.Results:(1)The induction and polarization of macrophages were successfully completed,and the cytokine markers of various types of macrophages were verified by q PCR.(2)LINC00467 was more expressed in M2 macrophages.(3)LINC00467knockdown can inhibit the level of markers of M2 macrophages.(4)LINC00467knockdown of prostate cancer cells can inhibit the level of M2 macrophage markers.(5)LINC00467 knockdown by M2 macrophages can inhibit the migration and invasion of prostate cancer cells.Conclusion: LINC00467 can promote the polarization of M2 macrophages and promote the malignant progression of prostate cancer.Part ?: LINC00467 promotes malignant progression of prostate cancer through miR-494-3p/STAT3 signaling axisMethods:(1)Open database was used for miRNA prediction;(2)qRT-PCR and Western Blot were used to verify that miRNA could target LINC00467 and STAT3 at the same time;(3)dual luciferase reporter gene assay was used to verify whether there were binding sites between miR-494-3p and LINC00467 and STAT3,respectively.(4)The expression of miR-494-3p in prostate cancer tissues and cell lines was detected by qRT-PCR.(5)miR-494-3p can inhibit the progression of prostate cancer by targeting STAT3.(6)Rescue experiment verified that LINC00467 promotes the progression of prostate cancer through miR-494-3p/STAT3 signaling pathway.Results:(1)miR-494-3p could target both LINC00467 and STAT3.(2)qRT-PCR showed that knockdown of LINC00467 could increase the expression of miR-494-3p.(3)Dual luciferase reporter gene assay confirmed that miR-494-3p had binding sites with LINC00467 and STAT3,respectively.(4)qRT-PCR detection revealed that miR-494-3p was highly expressed in prostate cancer tissues and cell lines,and was correlated with LINC00467 to a certain extent.(5)miR-494-3p can inhibit the progression of prostate cancer through the STAT3 signaling pathway.(6)Rescue experiment found that miR-494-3p could partially reverse the inhibitory effect of LINC00467 on prostate cancer cells.Conclusion: LINC00467 promotes the malignant progression of prostate cancer through miR-494-3p/STAT3 signaling pathway... |
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