Qiang Fu1, Jing Cheng2, Jindai Zhang1, Yonglei Zhang1, Xiaobing Chen3, Jianguo Xie4, Suxia Luo3. 1. Gastrointestinal Surgery Center, Henan Cancer Hospital (Affiliated Cancer Hospital of Zhengzhou University), Zhengzhou 450008, China. 2. Department of Medical Oncology, Zhengzhou Central Hospital (Zhengzhou Central Hospital Affiliated to Zhengzhou University), Zhengzhou 450002, China. 3. Department of Gastroenterology and Medical Oncology, Henan Cancer Hospital (Affiliated Cancer Hospital of Zhengzhou University), Zhengzhou 450008, China. 4. Gastrointestinal Surgery Center, Henan Cancer Hospital (Affiliated Cancer Hospital of Zhengzhou University), Zhengzhou 450008, China. huanghai598@163.com.
Abstract
OBJECTIVE: To predict and identify the target gene of miR-145, and to explore the underlying mechanism of the inhibition of miR-145 on drug resistance to Oxaliplatin (L-OHP) in human colorectal cancer cells. METHODS: L-OHP-resistant human colorectal cancer cell line (HCT116/L-OHP) was established in vitro by exposing to increased concentrations of L-OHP in cell culture medium. MiR-145-mimics and its negative control (NC-miRNA) were transfected into HCT116/L-OHP cells using liposome to establish HCT116/L-OHPmimics over-expressing miR-145 and HCT116/L-OHPNC. The target genes of miR-145 were predicted by bioinformatic analysis, and validated by dual luciferase activity assay. After determination of G protein coupled receptor 98(GPR98) as target gene, corresponding plasmids were constructed and transfected to establish HCT116/L-OHPGPR98 over-expressing GPR98 and HCT116/L-OHPcontrol. HCT116/L-OHP cells over-expressing both GPR98 and miR-145 (HCT116/L-OHPmimics+GPR98) were acquired through modification of the binding sites of GPR98 cDNA with miR-145. CCK-8 assay was used to assess the proliferation (A value) and sensitivity to L-OHP (the lower the IC50, the stronger the sensitivity) in HCT116/L-OHP cells. Real-time quantitative PCR was used to measure the mRNA expression of miR-145 and GPR98. Western blot was used to examine the protein expression of GPR98 and drug-resistant associated protein, such as P-glycoprotein (gp), multiple drug-resistance protein 1(MRP1), cancer-inhibition gene PTEN. RESULTS: HCT116/L-OHP cell line was successfully established with IC50 of (42.34±1.05) mg/L and miR-145 mRNA expression of 0.27±0.04, which was higher than (9.81±0.95) mg/L (t=39.784, P=0.000) and lower than 1.00±0.09 (t=13.021, P=0.000) in HCT116 cells. Based on HCT116/L-OHP cells, HCT116/L-OHPmimics cells were established successfully, with relative miR-145 expression of 10.01±1.05, which was higher than 1.06±0.14 in HCT116/L-OHPNC and 1.00±0.16 in HCT116/L-OHP (F=161.797, P=0.000). GPR98 was identified to be the target gene of miR-145. The relative mRNA and protein expressions of GPR98 in HCT116/L-OHPGPR98 cells were 8.48±0.46 and 1.71±0.09, respectively, which were higher than those in HCT116/L-OHPcontrol (mRNA: 3.65±0.40, protein: 1.21±0.10) and HCT116/L-OHP (mRNA: 3.49±0.35, protein: 1.22±0.08; all P<0.05). The A value was 1.31±0.10, and the relative protein expressions of P-gp and MRP1 were 1.53±0.18 and 1.49±0.20 in HCT116/L-OHPGPR98 cells, which were higher than those in HCT116/L-OHP (A value: 0.82±0.08, relative protein expression: 1.00±0.06 and 1.21±0.13, all P<0.05). The A value was 0.89±0.08, and the relative protein expressions of P-gp and MRP were 1.02±0.24 and 1.38±0.25 in HCT116/L-OHPmimics+GPR98 cells, which were higher than those in HCT116/L-OHPmimics(A value: 0.20±0.05, relative protein expression: 0.20±0.07, 0.55±0.10, all P<0.05). The relative protein expression of PTEN in HCT116/L-OHPGPR98 cells was 0.12±0.03, which was lower than 1.25±0.14 in HCT116/L-OHP cells(P<0.05). In addition, relative protein expressions of P-gp and MRP1 were 1.02±0.24 and 1.38±0.25 in HCT116/L-OHPmimics+GPR98 cells, which were higher than those in HCT116/L-OHPmimics cells (0.20±0.07 and 0.55±0.10), while PTEN expression in HCT116/L-OHPmimics+GPR98 cells was lower as compared to HCT116/L-OHPmimics cells (1.41±0.16 vs. 1.98±0.13, P<0.05). CONCLUSION: MiR-145 inhibits drug resistance to L-OHP of HCT116 cells through suppressing the expression of target gene GPR98.
OBJECTIVE: To predict and identify the target gene of miR-145, and to explore the underlying mechanism of the inhibition of miR-145 on drug resistance to Oxaliplatin (L-OHP) in humancolorectal cancer cells. METHODS:L-OHP-resistant humancolorectal cancer cell line (HCT116/L-OHP) was established in vitro by exposing to increased concentrations of L-OHP in cell culture medium. MiR-145-mimics and its negative control (NC-miRNA) were transfected into HCT116/L-OHP cells using liposome to establish HCT116/L-OHPmimics over-expressing miR-145 and HCT116/L-OHPNC. The target genes of miR-145 were predicted by bioinformatic analysis, and validated by dual luciferase activity assay. After determination of G protein coupled receptor 98(GPR98) as target gene, corresponding plasmids were constructed and transfected to establish HCT116/L-OHPGPR98 over-expressing GPR98 and HCT116/L-OHPcontrol. HCT116/L-OHP cells over-expressing both GPR98 and miR-145 (HCT116/L-OHPmimics+GPR98) were acquired through modification of the binding sites of GPR98 cDNA with miR-145. CCK-8 assay was used to assess the proliferation (A value) and sensitivity to L-OHP (the lower the IC50, the stronger the sensitivity) in HCT116/L-OHP cells. Real-time quantitative PCR was used to measure the mRNA expression of miR-145 and GPR98. Western blot was used to examine the protein expression of GPR98 and drug-resistant associated protein, such as P-glycoprotein (gp), multiple drug-resistance protein 1(MRP1), cancer-inhibition gene PTEN. RESULTS: HCT116/L-OHP cell line was successfully established with IC50 of (42.34±1.05) mg/L and miR-145 mRNA expression of 0.27±0.04, which was higher than (9.81±0.95) mg/L (t=39.784, P=0.000) and lower than 1.00±0.09 (t=13.021, P=0.000) in HCT116 cells. Based on HCT116/L-OHP cells, HCT116/L-OHPmimics cells were established successfully, with relative miR-145 expression of 10.01±1.05, which was higher than 1.06±0.14 in HCT116/L-OHPNC and 1.00±0.16 in HCT116/L-OHP (F=161.797, P=0.000). GPR98 was identified to be the target gene of miR-145. The relative mRNA and protein expressions of GPR98 in HCT116/L-OHPGPR98 cells were 8.48±0.46 and 1.71±0.09, respectively, which were higher than those in HCT116/L-OHPcontrol (mRNA: 3.65±0.40, protein: 1.21±0.10) and HCT116/L-OHP (mRNA: 3.49±0.35, protein: 1.22±0.08; all P<0.05). The A value was 1.31±0.10, and the relative protein expressions of P-gp and MRP1 were 1.53±0.18 and 1.49±0.20 in HCT116/L-OHPGPR98 cells, which were higher than those in HCT116/L-OHP (A value: 0.82±0.08, relative protein expression: 1.00±0.06 and 1.21±0.13, all P<0.05). The A value was 0.89±0.08, and the relative protein expressions of P-gp and MRP were 1.02±0.24 and 1.38±0.25 in HCT116/L-OHPmimics+GPR98 cells, which were higher than those in HCT116/L-OHPmimics(A value: 0.20±0.05, relative protein expression: 0.20±0.07, 0.55±0.10, all P<0.05). The relative protein expression of PTEN in HCT116/L-OHPGPR98 cells was 0.12±0.03, which was lower than 1.25±0.14 in HCT116/L-OHP cells(P<0.05). In addition, relative protein expressions of P-gp and MRP1 were 1.02±0.24 and 1.38±0.25 in HCT116/L-OHPmimics+GPR98 cells, which were higher than those in HCT116/L-OHPmimics cells (0.20±0.07 and 0.55±0.10), while PTEN expression in HCT116/L-OHPmimics+GPR98 cells was lower as compared to HCT116/L-OHPmimics cells (1.41±0.16 vs. 1.98±0.13, P<0.05). CONCLUSION:MiR-145 inhibits drug resistance to L-OHP of HCT116 cells through suppressing the expression of target gene GPR98.
Authors: Eman A Toraih; Manal S Fawzy; Bo Ning; Mourad Zerfaoui; Youssef Errami; Emmanuelle M Ruiz; Mohammad H Hussein; Muhib Haidari; Melyssa Bratton; Giovane G Tortelote; Sylvia Hilliard; Naris Nilubol; Jonathon O Russell; Mohamed A Shama; Samir S El-Dahr; Krzysztof Moroz; Tony Hu; Emad Kandil Journal: Cancers (Basel) Date: 2022-08-26 Impact factor: 6.575