Abstract:

Objective To discuss the expression levels of long non-coding RNA paired box 8 antisense RNA 1 （PAX8-AS1） in the human colorectal cancer （CRC） tissue and its effects on the proliferation， apoptosis and invasion of the CRC cells，and to charify its mechanism. Methods The expression levels of PAX8-AS1 mRNA and miR-22-3p in cancer tissue and cancer cells of the CRC patients were detected by real-time fluorescence quantitative PCR （RT-qPCR） method.The PAX8-AS10 siRNA small molecule sequences and miR-22-3p inhibitors were transfected alone or co-transfected into the CRC cells， and the cells after transfection were divided into si-NC group （transfected with negative sequences）， si-PAX8-AS1 group （transfected with PAX8-AS1 siRNA）， si-PAX8-AS1+inhibitors NC group （co-transfected with PAX8-AS1 siRNA and inhibitors NC）， and si-PAX8-AS1+ inhibitors group （co-transfected with PAX8-AS1 siRNA and miR-22-3p inhibitors）， and the SW480 cells without any transfection were regarded as control group. The expression levels of PAX8-AS1 mRNA and miR-22-3p in the cells in various groups after transfection were measured by RT-qPCR method； the proliferation activities of the cells in various groups were detected by MTT method； the migration and invasion rates of the cells in various groups were measured by Transwell chamber assay； the apoptotic rates of cells in various groups were measured by flow cytometry； the expression levels of B-cell lymphoma-2（Bcl-2）， Bcl-2 associated X protein（Bax）， and cleaved Caspase-3 proteins in the cells in various groups were measured by Western blotting method； the target relationship between PAX8-AS1 and miR-22-3p was verified by dual luciferase reporter gene assay. Results The RT-qPCR results showed that compared with adjacent tissue，the expression level of PAX8-AS1 mRNA in cancer tissue was significantly increased（P<0.01）， and the expression level of miR-22-3p was significantly decreased（P<0.01）； compared with the human normal colonic epithelial cells NCM460，the PAX8-AS1 mRNA expression levels in the SW480， SW620， HT-29 and LoVo cells were all significantly increased （P<0.01）， while the miR-22-3p expression levels were all significantly decreased （P<0.05 or P<0.01）. Compared with control group and si-NC group， the expression level of PAX8-AS1 mRNA in the cells in si-PAX8-AS1 group was decreased（P<0.01）， and the expression level of miR-22-3p was significantly increased（P<0.01）； compared with si-NC group，the expression level of miR-22-3p in the cells in si-PAX8-AS1+inhibitors group was increased（P<0.01）.The MTT assay results showed that compared with control group，the proliferation activities of the cells in si-PAX8-AS1 group，si-PAX8-AS1+inhibitors NC group，and si-PAX8-AS1+inhibitors group were significantly decreased after culture for 48 and 72 h （P<0.01）； compared with si-NC group， the proliferation activities of the cells in si-PAX8-AS1+ inhibitors NC group were significantly decreased after culture for 48 and 72 h （P<0.01）.The flow cytometry results showed that compared with control group， the apoptotic rate of cells in si-PAX8-AS1 group was significantly increased （P<0.01）；compared with si-NC group，the apoptotic rate of cells in si-PAX8-AS1+ inhibitors NC group was significantly increased （P<0.01）.The Transwell chamber assay results showed that compared with control group， the rates of migration and invasion cells in si-PAX8-AS1 group were significantly decreased（P<0.01）； compared with si-NC group，the rates of migration and invasion cells in si-PAX8-AS1 group and si-PAX8-AS1+ inhibitors NC group were significantly decreased（P<0.01）.The Western blotting results showed that compared with control group and NC group， the expression levels of Bax and cleaved Caspase-3 proteins in the cells in si-PAX8-AS1 group were significantly increased（P<0.01）， and expression level of the Bcl-2 protein was significantly decreased（P<0.05）.The TargetScan predicted that there were target binding sites between PAX8-AS1 and miR22-3p.The dual luciferase reporter gene assay results showed that compared with the cells co-transfected with mimics NC， the luciferase activity in the PAX8-AS1-WT cells was significantly decreased after co-transfected with miR-22-3p mimics （P<0.01）. Conclusion The PAX8-AS1 is highly expressed in the human CRC tissue， and silencing the PAX8-AS1 expression can inhibit the proliferation， migration and invasion of the CRC cells and induce the apoptosis，and its mechanism may be ralated to up-regulation the miR-22-3p expression.

Key words: Long non-coding RNA, Paired box 8 antisense RNA 1, Micro RNA, Colorectal neoplasm, Apoptosis