The Role Of UCA1-miR-204-SPDEF Axis In Gastric Carcinogenesis

Objective:MicroRNAs(miRNAs)are small RNA molecules of about 20-24 nucleotides in length that are widely found in eukaryotes.miRNAs can play an important role in regulating gene expression,cell cycle and organism development by binding to the 3'-untranslated region of the target genes mRNA,thereby inhibiting post-transcriptional expression of the gene.By regulating tumor suppressors or oncogenes respectively,miRNA can participate in all stages of tumorigenesis and development.At the same time,as the endogenous regulatory molecules,their role in early diagnosis and treatment of cancers is also important.MiRNAs have been determined to play important roles in gastric carcinoma.The previous study has found that the transcription factor SPDEF,which is specifically expressed in epithelial cells,is highly expressed in human gastric cancer tissues,and participates in gastric carcinoma through interaction with FoxMl,the key positive regulator of cell cycle.Bioinformatics predictions and previous studies prompted that miR-204 could directly bind to the 3'-UTR region of SPDEF to regulate its expression and participate in disease progress of prostate cancer.The results of miRNA microarray in the previous study also showed that the expression of miR-204 gradually decreased from the stage of superficial gastritis to the stage of gastric cancer in gastric mucosal epithelial tissues.All the above results showed the possibility of the negative correlation between miR-204 and SPDEF expression,which suggested that SPDEF might be negatively regulated by miR-204 in the process of gastric carcinoma.At the same time,miR-204 might be directly inhibited by long non-coding RNA(IncRNA)UCA1.UCA1 is a recently discovered IncRNA whose aberrant expansion is considered to be one of the hallmarks of tumors malignant progression.The result of tissue expression profiling showed that UCA1 was significantly overexpressed in human gastric cancer tissues compared with normal tissues,suggesting that UCA1 might play an important role in gastric carcinoma.LncRNA can be used as an endogenous competitive RNA to inhibit the expression of miRNAs,which is to play a role as the specific miRNAs "sponge" in long-chain non-coding RNA.Our team predicted through bioinformatics that the 3'-UTR region of the UCA1 gene might contain the recognition and binding site of miR-204.Therefore,in this research,it was investigated from different levels of human gastric cancer tissue specimens,cells and animal models to determine the role of UCA1?miR-204?SPDEF axis in gastric carcinoma and the relative mechanism.Methods1.Human gastric cancer tissue species levelThe expression of UCA1,miR-204 and SPDEF in human gastric cancer tissue samples,the correlation of genes expression and the relationship between genes expression and prognosis were analyzed using bioinformatics technology combined with database information.Fresh gastric cancer tissue specimens and adjacent normal tissue specimens from five gastric cancer patients were collected respectively.The mRNA level expression of UCA1,miR-204 and SPDEF in the samples was analyzed by real-time quantitative gene amplification fluorescence detection(QRT-PCR).The collected human tissues were also embedded in paraffin and the expression of SPDEF was detected by immunohistochemistry(IHC).Preliminary analysis showed the differential expression of UCA1,miR-204 and SPDEF in cancer tissues compared with adjacent normal tissues.2.Cells and molecular levelThe expression of SPDEF both at the mR:NA and protein levels were detected by QRT-PCR and western blotting after the overexpression or knockdown of miR-204 in human gastric mucosal epithelial-derived cell lines respectively,which was used to prove the negative regulation of miR-204 on SPDEF.Correspondingly,both the mRNA and protein expressions of miR-204 and SPDEF were detected by QRT-PCR and western blotting after inhibition of UCA1 or UCA1 overexpression in the gastric cancer cell lines.This can illustrate the regulation of UCA1 on miR-204 and SPDEF.And the recovery experiment verified that the effect of UCA1 on SPDEF was achieved at least partially by miR-204.The dual luciferase reporter plasmid that contains UCA1 3'-UTR with the binding site of miR-204 was constructed and the effect of miR-204 binding to the 3'-UTR of UCA1 was verified by detection of fluorescence intensity.The colony formation assay was used to observe the effect of UCA1?miR-204?SPDEF axis on the proliferation of gastric cancer cells.3.Animal models levelThe effect of SPDEF expression regulated by UCA1?miR-204?SPDEF axis on the.tumorigenesis of the gastric cancer cells was further verified in vivo by constructing nude mice tumor models.The mRNA expression level of UCA1,miR-204 and SPDEF was detected by QRT-PCR.Results1.Human gastric cancer tissue species levelBased on bioinformatics analysis data and QRT-PCR results of human gastric cancer and adjacent normal tissues,it was found that in human gastric cancer tissues,the mRNA level expression of SPDEF and UCA1 were significantly increased while expression of miR-204 was significantly decreased compared with the corresponding adjacent normal tissues.IHC results showed that SPDEF was highly expressed in human gastric cancer tissue species compared with the adjacent normal tissues at the protein level.At the same time,the expression of UCA1 was negatively correlated with miR-204 in gastric cancer tissue samples,and negatively correlated with the prognosis of patients.The expression of miR-204 and SPDEF was also negatively correlated.2.Cells and molecular levelsBoth the Knockdown or high expression of miR-204 in gastric cancer cell lines could negatively regulate SPDEF expression.While high expression of UCA1 or interference of UCA1 in gastric cancer cell lines could positively regulate SPDEF expression by antagonizing endogenous miR-204.It was confirmed by the recovery experiments that the regulation of SPDEF by UCA1 was at least partially mediated by miR-204.The dual luciferase activity assay demonstrated that miR-204 acted directly on the 3'-UTR of UCA1,and therefore,UCA1 exerted a "sponge absorption" effect on miR-204 to regulate the expression of SPDEF.The results of colony formation showed that UCA1?miR-204?SPDEF axis was involved in the proliferation of gastric cancer cells.3.Animal model levelThe subcutaneous tumorigenic ability of nude mice was inhibited with suppressed expression of UCA1in the gastric cancer cell line.After obtaining RNA from the tumors respectively,the QRT-PCR results showed that the expression of miR-204 was increased,while the expression of SPDEF was decreased at the same time.Similarly,the subcutaneous tumorigenic ability of the gastric cancer cell line with high expression of miR-204 also showed a certain degree of inhibition.The expression of UCA1 and SPDEF also decreased by QRT-PCR analysis.ConclusionBoth SPDEF and UCA1 were significantly highly expressed in gastric cancer tissue samples.While the expression of miR-204 in gastric cancer species was relatively lower compared with the adjacent normal tissues.LncRNA UCA1 could play a role of "sponge absorption" to antagonize the endogenous tumor suppressor molecule miR-204,which thus promoted the expression of SPDEF for gastric carcinoma.This study investigated the role of UCA1?miR-204?SPDEF axis in gastric carcinoma and the relative mechanism.It might explore certain new theoretical basis for the diagnosis and treatment of gastric cancer.