| Objectives: Leptomeningeal metastasis(LM)is a lethal complication of cancer,which characterized by tumor cells infiltration into leptomeninges and proliferation in cerebrospinal fluid(CSF).Due to limited access to leptomeningeal lesions,its diagnosis and disease course monitoring are still challenging.CSF miRNAs are attaching major interest as potential biomarkers for central nervous system malignancies.This study aimed to identify specific miRNAs in CSF for diagnosing and monitoring LM with lung adenocarcinoma,as well as explore the biological function the specific miRNAs.Methods: 1.MiRNA microarray was used to detect the CSF miRNA expression in patients with lung adenocarcinoma LM,brain metastasis of lung adenocarcinoma,and non-neoplastic diseases.Meanwhile,changes in the miRNA expression profile of CSF samples in LM patients with lung adenocarcinoma were detected at initial diagnosis and after LM-directed treatment,so as to screen LM-related CSF miRNAs.2.Quantive real-time PCR was used to detect whether the changes in the expression of candidate CSF miRNAs were consistent with the results of miRNA microarray screening in an independent cohort including LM patients with lung adenocarcinoma,brain metastasis patients with lung adenocarcinoma and non-tumor diseases controls.3.Quantive real-time PCR and receiver-operating characteristic curve were used to detecte the diagnostic sensitive and specificity of CSF miRNAs in suspected LM patients with lung adenocarcinoma.Then this method was compared with the conventional CSF cytology examination and neuroimaging examination.4.Quantive real-time PCR was used to detect the changes in the expression levels of candidate miRNAs in LM patients with lung adenocarcinoma at the initial diagnosis,after treatment,at recurrence and after salvage treatment,so as to determine whether specific CSF miRNAs have the potential to be used as indicators for LM disease status monitoring indicators.5.Mi R-7977 mimics/inhibitors were transfected to up regulate or down regulate the expression of miR-7977 in cells,and the effects of miR-7977 expression on the proliferation,migration and invasion of lung adenocarcinoma cells were detected by MTT assay,scratch assay and transwell invasion assay.6.Using three databases including miRDB(http://www.mirdb.org/),miRWalk(http://mirwalk.umm.uni-Heidelberg.DE),Target Scan(http://www.targetscan.org/)combined with the previous literature reports,we screened the predicted target genes of miR-7977,and through the transfection of miR-7977 mimics/inhibitors,the effect of miR-7977 on the expression of target genes was detected by q RT-PCR and western bloting.Results: 1.In the screening phase,compared with brain metastasis patients with lung adenocarcinoma and non-cancer controls,51 miRNAs with different expressions were found in the CSF of LM patients with lung adenocarcinoma patients,among which 36 were up-regulated and 15 were down-regulated,including miR-7641 with the highest fold-change value(58.3).In the comparison of CSF samples of patients with lung adenocarcinoma before treatment and after effective treatment,there were 3 miRNAs with significant expression differences,namely miR-7975,miR-7977 and miR-4800-5p,all of which were down-regulated after treatment with LM.We selected miR-7975,miR-7977,miR-7641 and miR-4800-5p as candidate miRNAs for subsequent studies.2.In the validation phase,compared with brain metastasis patients with lung adenocarcinoma and non-cancer controls,the expressions of miR-7975,miR-7977 and miR-7641 in CSF of LM patients with lung adenocarcinoma were significantly increased,which was consistent with the results of miRNA microarray detection.Compared with patients with brain metastasis patients with lung adenocarcinoma and non-cancer controls,there was no statistically significant difference in miR-4800-5p in CSF of LM patients with lung adenocarcinoma,which was inconsistent with the results of microarray detection.3.In suspected LM patients with lung adenocarcinoma,CSF cytology and imaging sensitivity at initial diagnosis were 64.4% and 68.9%,respectively.In contrast,the diagnostic sensitivity of miR-7975,miR-7977 and miR-7641 at the initial CSF detection was 88.9%,84.4% and 68.9%,respectively.The specificity was 85.7%,82.1% and 67.9%,respectively.The area under the curve(AUC)was 0.891,0.883 and 0.750,respectively.Combined miR-7975 and 7977 showed an AUC value of 0.923 with a sensitivity of 91.1% and a specificity of 82.1%.The AUC value,sensitivity and specificity of the combined diagnosis were 0.960,93.3% and 85.7%,respectively.4.Compared with the expression level of miRNAs at diagnosis,miR-7975 and miR-7977 in the CSF of LM patients were significantly reduced after initial treatment,while the expression level of miR-7641 was not significantly different.Among the 8 LM patients who relapsed and completed the salvage treatment,miR-7975 and miR-7977 in the CSF were increased again at recurrence,and then decreased again after the effective salvage treatment.5.Overexpressed of miR-7977 in NCI-H1650 and PC9 cells can promote cell proliferation,accelerate cell migration,and improve the ability of cell transmembrane invasion.However,miR-7977 with low expression can inhibit the proliferation of lung adenocarcinoma cells,slow down cell migration,and reduce the ability of cell transmembrane invasion.6.Both Target Scan and miRDB showed that PCBP1 was the predicted target gene of miR-7977.In combination with previous literatures,we selected PCBP1 as the target gene of miR-7977 for subsequent studies.We found that overexpression of miR-7977 significantly decreased the expression of PCBP1 mRNA and protein in NCI-H1650 and PC9 cells,while inhibition of miR-7977 significantly increased the expression of PCBP1 mRNA and protein in NCI-H1650 and PC9 cells.Conclusions: 1.Through screening and verification in two separate sample groups,it was found that the expression levles of miR-7977,miR-7975 and miR-7641 were obviously higher in CSF of LM paitents with lung adenocarcinoma patients compared with brain metastasis patients and non-cancer controls.2.Through verification and evaluation in patients with suspected LM in lung adenocarcinoma,it was found that miR-7977,miR-7975 and miR-7641 had higher sensitivity and specificity in the diagnosis of LM,and the combination of the three could further increase the diagnostic efficiency of LM in lung adenocarcinoma.This demonstrated that CSF miRNAs had the potential as biomarkers for the diagnosis of LM.3.Through the study on the changes in the expression levels of miRNAs in LM patients with lung adenocarcinoma at the initial diagnosis,after treatment,at recurrence and after rescue treatment,it was found that the expression levels of miR-7977 and miR-7975 were closely related to the development of LM.It suggested that the specific CSF miRNAs had the potential role in monitoring disease status of LM.4.Preliminary studies on the biological functions of miR-7977 have found that the changes in the expression of miR-7977 in cells can affect the biological behaviors of lung adenocarcinoma cells,which may be one of the molecular mechanisms to promote the development of LM.5.Through bioinformatics software analysis and experimental verification,it was found that the expression of miR-7977 in lung adenocarcinoma cells was negatively correlated with the expression of PCBP1,suggesting that miR-7977 may promote the occurrence of lung adenocarcinoma LM by regulating PCBP1. |
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