MiR-27a Inhibits Tumor Proliferation And Invasion Through Targeting KRAS In Esophageal Squamous Cell Carcinoma

Esophageal cancer(EC) is a clinical common malignancy of the Digestivediseases. Through high-throughput synergy screening, such as miRNA array,researchers found that there are significant difference of miRNA expression inesophageal cancerous tissues when compared with that in normal esophageal tissues.Some miRNAs have been reported to regulate the carcinogenesis of esophagealcancer, But to date, there is no report about the regulation of miR-27a in thecarcinogenesis of esophageal squamous cell carcinoma(ESCC). Considering thosereports about the role of miR-27a in other type cancers, In the present study, we firstanalyzed the correlation between miR-27a and ESCC. We also demonstrated thatmiR-27a regulates Ras/Raf/MEK/ERK signal pathway through targeting KRAS. It issuggested that the downregulation of miR-27a may promote the tumor growth andnodal involvement in ESCC, and miR-27a may be used as an important therapeutictarget to treat esophageal cancer.Part I：MiR-27a expression and clinical significance in ESCCMethods1. Search GEO datasets for the data about miRNA array in ESCCs. The correlationanalysis was conducted to analyze the expression of miR-27a and miR-27b in44ESCC patients and32ADC patients. 2. RT-PCR was used to detect the miR-27a expression in25pairs of tissues, whichhave nodal involvement.3. RT-PCR was used to detect the miR-27a expression in cultured normalesophageal epithelial cells and6ESCC cell lines.Results1. Datasets published in GEO reference series GSE13937was used for miRNAexpression analysis of hsa-miR-27a and has-miR-27b in ESCC compared withadjacent normal esophageal tissues. The expression of hsa-miR-27a wasdecreased in tumors when compared with that in adjacent normal tissues frompatients with nodal involvement (n=20, p=0.041). However, in ESCC patientswithout nodal involvement, this difference was not significant (n=24, p=0.785).The expression of miR-27a and miR-27b in ADC tissues (n=32) did notsignificantly differ with that in control, regardless of nodal involvement.2. miR-27a was significantly downregulated in ESCC tissues of patients with nodalinvolvement when compared with the paired adjacent normal tissues.3. In six esophageal carcinoma cell lines, the expression of miR-27a was decreased,particularly in TE-1cells.ConclusionsMiR-27a was downregulated in ESCC with nodal involvement, and theexpression of miR-27a was downregulated in6ESCC cell lines. It is suggested thatthe downregulation of miR-27a may promote the carcinogenesis and migration ofESCC.Part II：Study of miR-27a target gene in ESCC tissues and cellsMethods1. A bioinformatic search (Targetscan) was performed to predict the miR-27atargeting KRAS gene.2. The3’-UTR of human KRAS gene and its mutant control were cloned into pimrGLO. HEK293cells were co-transfected with wt-pmirGLO or mut-mirGLOwith miR-27a or scramble, separately. Dual-luciferase reporter assay wasconducted to validate miR-27a target role to KRAS gene.3. TE-1cells were transfected miR-27a precursor, scramble, miR-27a inhibitor, andinhibitor control, separately. RT-PCR and western blot were used to detect mRNAand protein expression of KRAS.4. RT-PCR and western blot were used to detect mRNA and protein expression ofKRAS in25pairs of cancer tissues and adjacent normal tissues from ESCCpatients with nodal involvement.Results1. MiR-27a was found to have two seed regions that matched the3’-UTR of humanKRAS:nucleotide343-349and4583-4589.2. Luciferase test demonstrated that co-transfection of miR-27a with the wt KRAS3’-UTR construct in HEK293cells resulted in a significant inhibition of luciferaseactivity compared with the negative control. Mutagenesis of the miR-27a bindingsite within the KRAS3’-UTR abolished the ability of miR-27a to regulate theluciferase expression.3. Overexpression of miR-27a strongly reduced the endogenous protein and mRNAlevels of KRAS when compared with these levels in the control. miR-27ainhibition promoted the mRNA and protein expression of KRAS.4. In clinical ESCC specimens, there was an inverse correlation between themRNA/protein levels of KRAS and the level of miR-27a expression.ConclusionsMiR-27a can directly inhibit the expression of endogenous mRNA and protein ofKRAS in ESCC. There was an inverse correlation between the mRNA/protein levelsof KRAS and the level of miR-27a expression in clinical ESCC specimens, and thedownregulation of miR-27a may promote the expression of KRAS in ESCC. Part III：The mechanism of miR-27a targeting KRAS gene in TE-1cellsMethods1. TE-1cells were transfected miR-27a precursor, scramble, miR-27a inhibitor, andinhibitor control, separately. RT-PCR and western blot were used to detect mRNAand protein expression of KRAS, phosphorylated ERK and c-Fos.2. TE-1cells were transfected miR-27a precursor, scramble, miR-27a inhibitor,inhibitor control, and full length KRAS cDNA, separately. MTT assay was usedto detect the cell proliferation.3. Scratch assay was applied to detect the invasion ability in TE-1cells transfectedwith miR-27a precursor, scramble, miR-27a inhibitor or inhibitor control.4. Nude mouse transplantation tumor experiment was conducted to detect theoncogenicity of TE-1cells transfected with miR-27a precursor, scramble,miR-27a inhibitor or inhibitor control. Tumor volume of nude mice weremeasured in10d、15d、20d、25d and30d after transplantation. The tumor wasseparated and weighted in30d after transplantation.Results1. Overexpressing of miR-27a inhibited the expression of ERK phosphorylation andits downstream c-Fos in TE-1cells; while knockdown of miR-27a promoted theexpression of ERK phosphorylation and its downstream c-Fos. Co-transfectedwith miR-27a and KRAS, the expression of exogenous KRAS weakened ERKphosphorylation.2. Overexpression of miR-27a markedly attenuated cell proliferation andknockdown of miR-27a promoted cell proliferation in TE-1cells.Forcedexpression of KRAS rescued the cell growth inhibition of miR-27a partially.3. Overexpression of miR-27a significantly inhibited the ability of TE-1cells to healscratch assays. However, knockdown of miR-27a significantly promoted thisbehavior.4. In nude mice xenograft models, TE-1cells with miR-27a overexpression developed smaller tumors compared with TE-1cells with normal miR-27a.Accordingly, miR-27a knockdown in TE-1cells promoted tumor formation.ConclusionsMiR-27a regulated Ras/Raf/MEK/ERK signal pathway and tumor biologicalprocess, such as tumor cell growth, cell proliferation and invasion, through targetingKRAS. It is suggested that the tumor growth and nodal involvement in ESCCpartially owe to the downregulation of miR-27a.