| Research backgroundColorectal cancer(CRC)has the third highest incidence and second highest mortality of cancer all over the world.KRAS is a common mutated gene in CRC,with a mutation rate of about 40%.If patients with KRAS mutated CRC and distant metastasis can not be treated by tumor resection,they also limited to the treatment with molecular targeted drug cetuximab.Based on this clinical problem,in part one of this paper,we aim to looking for some new therapeutic targets for patients with KRAS mutated CRC and distant metastasis.Circular RNAs(circRNAs)are a special kind of circular non-coding RNAs that affect regulation of mRNA via competitively combined with miRNA,this also named competitively endogenous RNA(ceRNA).The ceRNA network of circRNA has been reported in a variety of cancers,but less in gastric cancer.In part two of this paper,we also aim to building the ceRNA network of circRNA in gastric cancer by database analysis.Research methods1.The TCGA database is used to found out genes whose expression increased with the increasing level of tumor metastasis in patients with KRAS mutated CRC at mRNA level,however,the expression of these genes was independent of the degree of tumor metastasis in patients with KRAS wild type CRC.It is verified again in the CPTAC database.2.Transwell assay was used to investigate the effect of ZBTB20 knockdown or overexpression on metastatic ability of colorectal cancer cells in vitro.The effect of ZBTB20 knockdown on the metastatic ability of colorectal cancer cells in nude mice was evaluated in vivo model of liver metastasis induced by spleen injection.3.Transcriptome high-throughput sequencing(RNA-seq)and chromatin immunoprecipitation high-throughput sequencing(ChIP-seq)data were used to identify potential target genes of ZBTB20.Luciferase reporter assay and electrophoretic mobility shift assay(EMSA)were used to identify the binding region of the ZBTB20 target gene promoter.4.The ZBTB20 interacting protein was identified by immunoprecipitation assay(IP)and mass spectrometry.5.GEO database was used to analyze the circRNA specifically up-regulated or down-regulated in gastric cancer.Combined with TCGA database,miRNAs and mRNAs specifically up-regulated or down-regulated in gastric cancer were obtained,and the target gene prediction database was used to construct a ceRNA network.Research results1.TCGA analysises show that the mRNA expression of ZBTB20 increased with the degree of tumor metastasis in patients with KRAS mutation,but there is no correlation in patients with KRAS wild type colorectal cancer.The analysis results of CPTAC are also consistent with TCGA.2.ZBTB20 knockdown colorectal cancer cell lines show decreased metastatic ability in the Transwell assay,while ZBTB20 overexpression of colorectal cancer cells show increased metastatic ability.Liver metastasis model of spleen injection in nude mice also confirms that the metastatic ability of colorectal cancer cells decreases after ZBTB20 knockdown.3.The combination of RNA-seq and ChIP-seq reveals that TGFBR2 is the target gene of ZBTB20,and the binding region is located at the promoter-810 to-720 and-600 to-480.4.Immunoprecipitation and mass spectrometry show that ZBTB20 interact with phosphorylated ERK1/2,and phosphorylated ERK1/2 phosphorylate ZBTB20 threonine at sites 138,142 and 232,and the phosphorylation of ZBTB20 promote TGFBR2 expression.5.The ceRNA network of circRNA specifically expressed in gastric cancer was successfully established.Research conclusionsIn colorectal cancer,the transcription factor ZBTB20 promotes the expression of TGFBR2,which activates the TGF-β signaling pathway and EMT process,and promotes the metastatic ability of tumor cells.Phosphorylation of ERK 1/2 catalyze the phosphorylation of threonine of ZBTB20 and promote the regulation of TGFBR2 expression by ZBTB20.In gastric cancer,ceRNA networks with 6 specific circRNAs,6 specific miRNAs and 36 specific mRNAs were successfully established. |
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