| Purposes:Colorectal cancer(CRC)is one of the most common malignant tumors around the world,and its mortality rate is at the forefront of the tumor.More and more studies have found that long non-coding RNA(IncRNA)plays an important role in tumor progression.Long non-coding RNA KCNQ10T1(IncRNA KCNQ10T1)is highly expressed in various tumors such as liver cancer,lung cancer,and tongue cancer,promoting tumor growth and metastasis.The purpose of this study was to explore the expression and clinical significance of IncRNA KCNQ10T1 in CRC,and further analyze molecular mechanism of IncRNA KCNQ10T1 in the biological function phenotypes such as proliferation,migration,invasion,apoptosis and cycle of CRC.Methods:1.Online data predicted the expression and clinical relevance of IncRNA KCNQIOT1 in CRC using the bioinformatics website GEPIA(http://gepia.cancer-pku.cn/).Postoperative specimens of 28 patients with CRC were collected.Real-time quantitative PCR(QRT-PCR)was used to detect the differential expression of IncRNA KCNQ1OT1 in CRC tissues and adjacent tissues,as well as five kinds of CRC cells(SW620,RKO,LOVO,HCT116 and COLO320)and normal intestinal epithelial cells(NCM460).Also,lncRNA KCNQ10T1 expression was analyzed by QRT-PCR in tumor size(<5 cm and>5 cm)and tumor stage(Ⅰ+Ⅱ andⅢ+Ⅳ).2.High-expression cells(SW620 and RKO)were transfected with lentivirus to construct stable transgenic cells.Both cell stable transgenic cells were divided into control group(sh-NC)and down-regulated group(sh-KCNQ1OT1-1 and sh-KCNQ1OT1-2).QRT-PCR was used to verify the efficiency of down-regulation.CCK8 and colony formation assays were used to detect cell proliferation in vitro,and the subcutaneous tumor formation assay in nude mice were used to detect the proliferative ability in vivo.The wound healing and transwell assays were used to detect CRC cell migration and invasion.Flow cytometry was used to detect apoptosis and cell-cycle in cells.3.Western blot analysis was used to detect the relative expression of apoptosis-related proteins(BAX and BCL2)in SW620 and RKO cells,and to further detect PI3K/AKT pathway-related proteins(P-PI3K,PI3K,P-AKT,AKT).Results:1.LncRNA KCNQ1OT1 expression in CRC tissues and cells was upregulated,and was associated with poor clinical prognosis.The difference was statistically significant(P<0.05).(1)The GEPIA website predicted that the expression of IncRNA KCNQ1OT1 was higher in colorectal cancer tissues than in normal tissues,and the expression increased with the raise of stage.The overall survival rate and disease-free survival rate of the high expression group were lower compared with those of the low expression group.(2)The QRT-PCR results of 28 clinical samples showed that the IncRNA KCNQ1OT1 expression in cancer tissues was higher than that in adjacent tissues,and the IncRNA KCNQ1OT1 expression in tumor size≥5cm was higher than tumor size<5cm,as well as the expression of KCNQ10T1 in high stage(Ⅲ+ Ⅳ)was higher than that of low tumor stage(Ⅰ+Ⅱ).(3)The expression of IncRNAKCNQ1OT1 in CRC cancer cells lines was higher than that in normal intestinal epithelial cells NCM460.2.Down-regulation of lncRNA KCNQ10T1 expression inhibited the proliferation of CRC cells in vitro and in vivo,inhibited cell migration,invasion,promoted apoptosis,and arrested cell cycle.The differences were statistically significant(P<0.05).(1)The results of CCK8 and colony formation assay showed that the proliferation ability of the down-regulated group was significantly lower than that of the control group in vitro.The results of subcutaneous tumor formation in nude mice showed that the proliferation ability of the down-regulated group was also significantly lower than that of the control group.(2)The results of wound healing and transwell assays showed that the cell migration and invasion ability of the down-regulated group was significantly lower than that of the control group;(3)Flow cytometry results showed that the apoptosis rate of the down-regulated group was significantly higher than that of the control group.Compared with the control group,the G0/G1 phase was increased significantly in the down-regulation group,in contrast the S phase decreased.3.Down-regulation of IncRNA KCNQIOT expression inhibited the activation of PI3K/AKT signaling pathway and altered the expression of apoptosis-related proteins.The differences were statistically significant(P<0.05).(1)Western blot analysis showed that the relative expression of BAX in the down-regulated group was increased in both cells,and the relative expression of BCL2 was decreased.(2)Western blot analysis showed that the relative expression of P-PI3K and P-AKT in the down-regulated group was significantly lower than that in the control group,but there was no significant difference in PI3K and AKT.Conclusion:The lncRNA KCNQ1OT expression was up-regulated in CRC tissues and cells.The high expression of IncRNA KCNQ1OT was significantly associated with tumor size,tumor stage and poor prognosis in CRC patients.Knocking down IncRNA KCNQ1OT could suppress proliferation,migration and invasion of CRC cells,promote apoptosis and adjust cell cycle via attenuating PI3K/AKT signaling pathway. |
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