miR-1247-3p regulation of CCND1 affects chemoresistance in colorectal cancer

Abstract

The effectiveness of chemotherapy involving 5-fluorouracil and cisplatin (DDP) for the treatment of colorectal cancer (CRC) is often limited due to the emergence of drug resistance. An increasing body of research highlights the crucial role of abnormally expressed microRNAs (miR/miRNAs) in fostering drug resistance in various types of cancer. The present study was the first to explore the potential roles and mechanisms of the small non-coding RNA miR-1247-3p in CRC, particularly its association with DDP resistance in CRC. The findings of the current study revealed a significant decrease in miR-1247-3p expression in CRC cells, especially those resistant to drugs. By contrast, there was a marked increase in the expression of cyclin D1 (CCND1), a known target gene of miR-1247-3p that is negatively regulated by this miRNA. By modulating CCND1, miR-1247-3p can effectively reduce drug resistance and promote apoptosis in CRC cells, suggesting that miR-1247-3p could potentially reduce chemotherapy resistance by targeting CCND1. These results highlight the pivotal role of miR-1247-3p in reducing chemotherapy resistance through the inhibition of CCND1, providing insight into a promising therapeutic strategy for overcoming CRC resistance.

Fig 1. miR-1247-3p was downregulated in tissues of patients with CRC and was associated with prognosis and drug resistance.

(A) and (B) miR-1247-3p expression levels in CRC tissues based on the University of ALabama at Birmingham CANcer data analysis portal and NCBI databases. (C) The effect of miR-1247-3p on overall survival of patients with CRC was analyzed based on the ENCORI database. (D) Expression of miR-1247-3p in tumor cells from patients with drug-resistant and -sensitive CRC according to the NCBI dataset GSE190951. CRC, colorectal cancer; miR, microRNA; NCBI, National Center for Biotechnology Information.

Fig 2. miR-1247-3p was downregulated in CRC resistant cells.

(A) and (B) MTT assay to verify the drug resistance of the cells. (C) and (D) Transcriptome sequencing was performed to detect differences in miR-1247-3p expression in HCT8, HCT8/5-Fu and HCT8/DDP cells. (E) RT-qPCR was performed to verify the differential expression of miR-1247-3p in drug-resistant and parental cells. *P<0.05, **P<0.01, ***P<0.001. miR, microRNA; CRC, colorectal cancer; 5-Fu, 5-fluorouracil; DDP, cisplatin.

Fig 3. Overexpression of miR-1247-3p promoted apoptosis and inhibited resistance in drug-resistant cells.

(A) HCT8/5-Fu and HCT8/DDP cells transfected with miR-1247-3p mimics or negative control. (B) An MTT assay was used to measure the proliferation of HCT8/5-Fu and HCT8/DDP cells after transfection. (C) and (D) Flow cytometry was used to analyze cell cycle and apoptosis. (E) Western blotting was used to detect the expression of apoptosis-related proteins in HCT8/5-Fu and HCT8/DDP cells after transfection. (F) An MTT assay was used to evaluate the sensitivity of HCT8/5-Fu and HCT8/DDP cells to chemotherapeutic drugs after transfection. *P<0.05, **P<0.01, ***P<0.001. miR, microRNA; CRC, colorectal cancer; 5-Fu, 5-fluorouracil; DDP, cisplatin.

Fig 4. Downregulation of miR-1247-3p inhibited apoptosis and reduced chemosensitivity in cells.

(A) HCT8 cells transfected with miR-1247-3p inhibitor or negative control. (B) An MTT assay was used to measure the proliferation of HCT8 cells. (C) Flow cytometry was used to analyze the apoptosis of HCT8 cells. (D) Western blotting was used to detect the expression of apoptosis-related proteins in HCT8 cells. (E) and (F) An MTT assay was used to evaluate the sensitivity of HCT8 cells to chemotherapeutic drugs. *P<0.05, **P<0.01, ***P<0.001. miR, microRNA.

Fig 5. CCND1 is a target gene of miR-1247-3p.

(A) and (B) According to the UALCAN and Kaplan-Meier Plotter databases, CCND1 is expressed at high levels in CRC tissues and is associated with prognosis. (C) According to the ENCORI database, there is a certain negative association between CCND1 and miR-1247-3p. (D) Based on the NCBI database, CCND1 is upregulated in tissues of patients with CRC with drug resistance. (E) Dual luciferase detection of miR-1247-3p binding to CCND1. (F) and (G) RT-qPCR and western blotting were used to detect the changes in CCND1 expression in HCT8/5-Fu and HCT8/DDP cells after transfection with miR-1247-3p mimics. (H) and (I) RT-qPCR and western blotting were used to detect the changes in the CCND1 expression in HCT8 cells after transfection with miR-1247-3p inhibitor. *P<0.05, **P<0.01, ***P<0.001. miR, microRNA; CRC, colorectal cancer; 5-Fu, 5-fluorouracil; DDP, cisplatin; CCND1, cyclin D1; RT-qPCR.

Fig 6. CCND1 promoted chemoresistance in CRC.

(A) and (B) RT-qPCR and western blotting were used to investigate expression after knockdown of CCND1. (C) Changes in cellular chemosensitivity after knockdown of CCND1. *P<0.05, **P<0.01, ***P<0.001. CRC, colorectal cancer; CCND1, cyclin D1.