| Background and AimIntrahepatic cholangiocarcinoma(iCCA)is primary liver malignant tumor,accounting for 10%-15% of all liver tumors.It is believed that iCCA originates from bile duct epithelial cells in the liver.Clinically,patients with iCCA have no obvious symptoms in the early stage and poor survival after the operation.Therefore,combined with multi-dimensional data to establish an accurate iCCA prognosis prediction model,Delicacy management based on the prognosis model is expected to improve the overall survival of iCCA patients with a lower economic cost.A large number of studies have revealed the roles of lncRNA MNX1-AS1 in promoting tumorigenesis and progression in a variety of tumors.However,the role of MNX1-AS1 in iCCA has not been reported.It has been revealed that MNX1 is a transcription factor involved in the occurrence and development of many malignant tumors.Published literature has also demonstrated that there exists a highly positive correlation between the expression of MNX1-AS1 and MNX1.However,the correlation between MNX1-AS1 and MNX1 in iCCA has not been confirmed.In this study,bioinformatics analysis was used to determine the correlation between the expression of MNX1-AS1 and MNX1 in iCCA tissues.in vivo and in vitro experiments were used to verify the biological function of MNX1-AS1 in the occurrence and development of the iCCA.Current studies have demonstrated that there were differences in the carcinogenic mechanism of MNX1-AS1 in different tumors.Previous studies have revealed that MNX1-AS1 can play the role of "molecular sponge",competitively bind micro RNAs in the cytoplasm and indirectly participate in the regulation of tumor progression.Other studies have revealed that MNX1-AS1 can also regulate the classic tumor signal transduction pathway to promote tumorigenesis and progression.Another study on gastric cancer has revealed that MNX1-AS1 can inhibit BTG2 transcription via recruiting polycomb repressive complex 2 into the nucleus and binding to the BTG2 promoter region and finally promote the progression of gastric cancer.Our previous studies have also uncovered that MNX1-AS1 is mainly located in the nucleus in iCCA cells.Based on our previous research and current literature,we intend to make more in-depth research on the mechanism of MNX1-AS1 in the nucleus of iCCA.Method1.Based on the transcriptome sequencing data of iCCA cancer tissues and paracancerous tissues,the differentially expressed lnc RNA(DElnc RNA)was screened out by the DESeq2 package in R software.2.Combined with survival data,univariate COX regression was used to screen DElnc RNA which had a significant effect on the prognosis of the iCCA patients.3.The DElnc RNA which had a significant impact on prognosis was Further compressed by the LASSO regression.We then calculated the risk score based on the results of the LASSO regression formula.4.The iCCA transcriptome data were downloaded from the public database as external validation data and verify the established lnc RNA-related risk scores.5.Combined with the established lnc RNA-related risk scores and clinical data,univariate and multivariate COX regression was used to screen the independent risk factors affecting the prognosis of iCCA patients.6.Based on the established multivariate COX regression,a Nomogram diagram was established to predict the prognosis of the iCCA patients.7.The iCCA transcriptome sequencing data and the transcriptome data downloaded from the public database were employed to verify whether there was a correlation between the expression of MNX1-AS1 and MNX1.Furthermore,the q RT-PCR assay using RNA extracted from paired iCCA tissues was employed to verify the correlation as well.8.qRT-PCR was used to verify the expression of MNX1-AS1 in several cholangiocarcinoma cell lines,and fluorescence in situ hybridization assay was used to determine the location of MNX1-AS1 in the cells.9.The MNX1-AS1 knockdown and overexpression cell lines were established in the iCCA cell line,and the effects of MNX1-AS1 expression on iCCA cell proliferation,invasion and migration,and angiogenesis were verified by cell proliferation assay,cell clone formation assay,transwell assay,and HUVECs angiogenesis assay.10.The effect of MNX1-AS1 on the growth and metastasis of iCCA was validated via the subcutaneous transplantation tumor model and lung metastasis model in nude mice.11.Whether the expression of MNX1-AS1 can regulate the expression of MNX1 protein was determined by the Western blotting assay.12.The MNX1-binding transcription factors were retrieved in the database,and then verified by the Ch IP assay,and the transcription factors binding to the upstream promoter region of MNX1 were screened.13.The public database was used to predict whether the transcription factors screened in the above steps might bind to MNX1-AS1 and then to the potential binding sites were predicted.14.The binding relationship between the transcription factors and MNX1-AS1 was verified by the RIP assays.15.SiRNA was used to interfere with the expression of the above transcription factors in the iCCA cell lines,and Western blotting was used to verify the expression of MNX1 protein to reveal the regulatory effect of the selected transcription factors on the expression of MNX1.16.The Ch IP assay of MNX1 and Ch IP-seq was carried out to detect the genes to which MNX1 can bind.17.GO and KEGG enrichment analysis was employed to identify the potential downstream tumor signal pathways that MNX1 may be involved.18.Based on the results from GO and KEGG enrichment analysis,the key genes in the downstream tumor signal pathway affected by MNX1 were identified.Then,the Ch IP assay was used to verify whether MNX1 could bind to the upstream promoter region of the key genes.19.Based on the established MNX1 overexpression and knockdown cell lines,the Western blotting assay was used to verify the effect of MNX1 on the expression of key genes in the tumor signal pathway and other key genes in the tumor signal pathway.ResultThe first part of our project is the bioinformatics analysis of iCCA transcriptome sequencing data.Using transcriptome sequencing data from iCCA cancer and paracancerous tissues,a total of 1253 DElnc RNA were screened.Then combined with the survival data of patients with iCCA,a total of 22 DElnc RNA with significant impact on the prognosis of patients with iCCA were screened.Furthermore,the number of DElnc RNA which had a significant impact on the prognosis of iCCA patients was further compressed by LASSO regression,and the lnc RNA-related risk score of each sample was calculated.The verification results of public data uncover that the lnc RNA-related risk score we established is stable.Finally,eight risk factors were screened out by COX regression: multiple tumors,lnc RNA-related risk score,NLR,tumor diameter,CEA,ALP,GGT,CA199.Furthermore,we established a multivariate COX regression model,and the Nomogram diagram of the prognosis of patients with iCCA was established.The second part of our project is the function of lnc RNA MNX1-AS1 in promoting the development and metastasis of iCCA.First of all,according to the analysis of transcriptome sequencing data,we determined that there was a highly positive correlation between the expression of MNX1-AS1 and MNX1.The q RT-PCR of paired iCCA tissues uncovered that there was a highly positive correlation between the two expression levels as well.The FISH assay revealed that MNX1-AS1 was mainly located in the nucleus.In the aspect of cell experiment,a cell line with low expression of MNX1-AS1 was established in the FRH0201 cell line and a cell line with overexpression of MNX1-AS1 was established in the RBE cell line.Cell function assays revealed that the proliferation,invasion and metastasis ability of the RBE cells were significantly enhanced after overexpression of MNX1-AS1,and the supernatant of the culture medium could significantly enhance the angiogenesis ability of the HUVECs.Knocking down the expression of MNX1-AS1 in the FRH0201 cells elicited the opposite trends.As for in vivo experiments,the subcutaneously transplanted tumor model and lung metastasis model in nude mice demonstrated that knocking down the expression of MNX1-AS1 could significantly inhibit the growth and metastasis of the iCCA tumor.The third part of this study is the mechanism of lncRNA MNX1-AS1 promoting the proliferation and metastasis of intrahepatic cholangiocarcinoma.First of all,we proved that MNX1-AS1 can positively regulate the expression of MNX1 protein by Western blotting assay.Then the database is used to predict the transcription factors that can bind to the upstream promoter region of MNX1.Ch IP assay uncovered that only c-Myc and MAZ can bind to the upstream promoter region of MNX1,and then RIP assay show that c-Myc and MAZ can indeed combine with MNX1-AS1.Si RNA was used to interfere with the expression of c-Myc and MAZ in the iCCA cell line.Western blotting demonstrated that the expression of MNX1 protein decreased significantly after interfering with the expression of c-Myc or MAZ.Then the Ch IP and Ch IP-seq of MNX1 are conducted.GO and KEGG enrichment analysis of Ch IP-seq results determined that MNX1 may be involved in the regulation of the Hippo pathway via the Ajuba gene.The Ch IP of MNX1 determined that MNX1 could bind to the upstream promoter region of the Ajuba gene.Western blotting revealed that the expression of the Ajuba was significantly increased and the expression of MST1,MST2,p-Mob,p-Last1,and p-YAP1 in the Hippo pathway were significantly decreased in MNX1-AS1 overexpression or MNX1 overexpression cell,which resulting in a significant increase in YAP1 expression.The opposite results were obtained in MNX1-AS1 or MNX1 knock down cells established in the FRH0201 cell line.In summary,our results uncovered that MNX1 can up-regulate the expression of the Ajuba gene,and then inhibit the Hippo pathway,resulting in the overexpression of YAP1 to play a role in promoting cancer.ConclusionIn this study,based on the iCCA transcriptome sequencing data and public data we screened out the lnc RNA,which had a significant impact on the prognosis of the iCCA patients,and then the lnc RNA-related risk score by LASSO regression was established.The external data set verification results determined that the lnc RNA-related risk score we established is stable.Combined with clinical variables,a Nomogram was established to predict the prognosis of the iCCA patients.The Nomogram diagram we established lays a foundation for the clinical application of our research.Then,by analyzing the transcriptome sequencing data and the q RT-PCR results of iCCA tissues,we found that there was a highly positive correlation between the expression of lnc RNA MNX1-AS1 and MNX1.In the functional study,we first revealed that MNX1-AS1 can promote cell proliferation,metastasis and invasion,and HUVEC angiogenesis in iCCA cells.The subcutaneous transplantation tumor model and lung metastasis model in nude mice further confirm that MNX1-AS1 can promote the growth and metastasis of iCCA tumor in vivo.As for the mechanism,we proved that MNX1-AS1 can positively regulate the expression of MNX1 protein by recruiting transcription factors c-Myc and MAZ to bind to the upstream promoter region of the MNX1 gene.Then,MNX1 protein inhibits the Hippo pathway via the Ajuba gene,which eventually leads to the increase of YAP1 entry into the nucleus and plays a role in promoting cancer.The MNX1-AS1/c-Myc & MAZ/MNX1/Ajuba/Hippo signaling pathway is a potential therapeutic target for iCCA. |
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