THE EFFECT OF LNCRNA ON THE PROLIFERATION AND APOPTOSIS OF COLORECTAL CANCER CELLS BY REGULATING THE MIRNAARPP19 AXIS

Peng Li¹, Wen Zeng², Hongli Zhong^{3, *}

¹Anorectal, Jing Men NO.2 People's Hospital, Jingmen, 448000, China - ²Anesthesiology, Jing Men NO.2 People's Hospital, Jingmen, 448000, China - ³Ophthalmology, Jing Men NO.2 People's Hospital, Jingmen, 448000, China

Objective: To explore the influence of long non-coding RNA (lncRNA) on the proliferation and apoptosis of colorectal cancer cells (CRC) by regulating the Phosphoprotein 19 (ARPP19) axis regulated by cAMP, a microRNA. 

Method: CRC cell lines-Colo 320 and normal colorectal cell lines (NCM460, 293T) were obtained from the American Type Culture Collection. All cell lines were in a humid environment at 37°C and maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum. The experiment had a control group (Colo 320 for experimental control), MCM3AP-AS1 silent group (infecting Colo 320 cells with sh-MCM3AP-AS1 for silence expression), and ARPP19 transfection group (ARPP19 overexpression in this group of cells). The mRNA expressions of MCM3AP-AS1, miR-599, and ARPP19 in normal rectal system (NCM460, 293T) and rectal cancer cell line Colo 320 were analyzed by PCR in real time. CCK-8 and the 24h, 48h, and 96h OD values of the cells were used to reflect their proliferation ability. Cell apoptosis was compared by detecting cell caspase activity. Wound healing and Transwell analysis were used to detect cell migration and invasion ability. The Epithelial-mesenchymal transition (EMT) process and the expressions of N-cadherin and vimentin were analyzed by Western blot. The dual luciferase reporter gene assay was adopted to verify the MCM3AP-AS1-miR-599-ARPP19 interaction relationship. 

Results: Compared with (NCM460, 293T), the mRNA expressions of MCM3AP-AS1 and RPP19 in Colo 320 increased, and the expression of miR-599 mRNA in Colo 320 decreased (P<0.05). Compared with the control group and ARPP19 transfection group, the cell proliferation ability of the MCM3AP-AS1 silenced group was lower (P<0.05), and the cell proliferation of the ARPP19 transfection group was higher than that of the MCM3AP-AS1 silenced group (P<0.05). At 48h, the Caspase-3 activity of the MCM3AP-AS1 silenced group was higher than that of the control group and the ARPP19 transfection group (P<0.05), and there was no difference in the Caspase-3 activity of the control group and the ARPP19 transfection group (P>0.05). At 96h, the Caspase-3 activity of the MCM3AP-AS1 silenced group was higher than that of the control group and ARPP19 transfection group (P<0.05), and the Caspase-3 activity of the ARPP19 transfection group was lower than that of the control group (P<0.05). Compared with the control group and ARPP19 transfection group, the number of cell migration and invasion in the MCM3AP-AS1 silenced group was lower (P<0.05), and the number of cell migration and invasion in the ARPP19 transfection group was higher than that in the control group (P<0.05). Compared with the control group and the ARPP19 transfection group, the expression of E-cadherin in the MCM3AP-AS1 silenced group increased, and the expression of N-cadherin and vimentin decreased (P<0.05). Compared with the MCM3AP-AS1 silenced group, the expression of E-cadherin in the ARPP19 transfection group decreased, and the expression of N-cadherin and vimentin increased (P<0.05). In the co-culture of the ARPP19-WT and miR-599 transfection group, the enzyme activity decreased (P<0.05), and the ARPP19-Mut enzyme activity did not change (P>0.05). When miR-599 and MCM3AP-AS1 were co-transfected and overexpressed, the luciferase activity of co-cultured ARPP19-WT was higher than that of miR-599 transfected and co-cultured alone (P<0.05). 

Conclusion: lncRNA MCM3AP-AS1 promoted the proliferation and apoptosis of CRC cells by regulating the miR-599/ARPP19 axis, and therefore might be an effective therapeutic target for CRC.