CADM1 impairs the effect of miR-1246 on promoting cell cycle progression in chemo-resistant leukemia cells

Abstract

The interruption of normal cell cycle execution acts as an important part to the development of leukemia. It was reported that microRNAs (miRNAs) were closely related to tumorigenesis and progression, and their aberrant expression had been demonstrated to play a crucial role in numerous types of cancer. Our previous study showed that miR-1246 was preferentially overexpressed in chemo-resistant leukemia cell lines, and participated in process of cell cycle progression and multidrug resistant regulation. However, the underlying mechanism remains unclear. In present study, bioinformatics prediction and dual luciferase reporter assay indicated that CADM1 was a direct target of miR-1246. Evidently decreased expression of CADM1 was observed in relapsed primary leukemia patients and chemo-resistant cell lines. Our results furtherly proved that inhibition of miR-1246 could significantly enhance drug sensitivity to Adriamycin (ADM), induce cell cycle arrest at G0/G1 phase, promote cell apoptosis, and relieve its suppression on CADM1 in K562/ADM and HL-60/RS cells. Interference with CADM1 could reduce the increased drug sensitivity induced by miR-1246 inhibition, and notably restore drug resistance by promoting cell cycle progression and cell survival via regulating CDKs/Cyclins complexes in chemo-resistant leukemia cells. Above all, our results demonstrated that CADM1 attenuated the role of miR-1246 in promoting cell cycle progression and cell survival, thus influencing multidrug resistance within chemo-resistant leukemia cells via CDKs/Cyclins. Higher expression of miR-1246 and lower expression of CADM1 might be risk factors for leukemia.

Keywords: CADM1; CDKs/Cyclins axis; Cell cycle; Chemo-resistant leukemia cells; miR-1246.

Fig. 1

CADM1 was a direct target of miR-1246. (A) The relative miR-1246 mRNA expression level in samples from donors and patients were checked by RT-qPCR. ns, not significant; *p < 0.05 vs. the first diagnosed leukemia patients. (B) RT-qPCR was used to examine the mRNA expression level of miR-1246 in chemo-resistant leukemia cells and their parental leukemia cells. **p < 0.01 and ^##p < 0.01 vs. their parental leukemia cells. (C) miRNA target prediction tools inferred that CADM1 was a potential target of miR-1246. (D) Dual luciferase reporter assay was carried out to detect the targeting relationship between miR-1246 and CADM1. *p < 0.05 vs. the NC mimics + CADM1 sense group. (E) Western blot was carried out to compare the CADM1 expression differences in chemo-resistant leukemia cells transfected with miR-1246 inhibitor or NC inhibitor and in parental sensitive leukemia cells transfected with miR-1246 mimics or NC mimics. *p < 0.05 and ^#p < 0.05 vs. its NC mimics or NC inhibitor group

Fig. 2

CADM1 expression was inhibited in relapsed leukemia patients and chemo-resistant leukemia cell lines. (A) The expression of CADM1 in tumor and normal samples was analyzed across TCGA cancers. BLCA, bladder urothelial carcinoma. BRCA, breast invasive carcinoma. CESC, cervical & endocervical cancer. CHOL, cholangiocarcinoma. COAD, colon adenocarcinoma. ESCA, esophageal carcinoma. GBM, glioblastoma multiforme. HNSC, head & neck squamous cell carcinoma. KICH, kidney chromophobe. KIRC, kidney renal clear cell carcinoma. KIRP, kidney renal papillary cell carcinoma. LIHC, liver hepatocellular carcinoma. LUAD, lung adenocarcinoma. LUSC, lung squamous cell carcinoma. PAAD, pancreatic adenocarcinoma. PRAD, prostate adenocarcinoma. PCPG, pheochromocytoma & paraganglioma. READ, rectum adenocarcinoma. SARC, sarcoma. SKCM, skin cutaneous melanoma. THCA, thyroid carcinoma. THYM, thymoma. STAD, stomach adenocarcinoma. UCEC, uterine corpus endometrioid carcinoma. (B) The expression of CADM1 was extracted from TCGA tumors. ACC, adrenocortical cancer. DLBC, diffuse large B-cell lymphoma. LGG, brain lower grade glioma. OV, ovarian serous cystadenocarcinoma. MESO, mesothelioma. LAML, acute myeloid leukemia. TGCT, testicular germ cell tumor. UCS, uterine carcinosarcoma. UVM, uveal melanoma. (C) The relative CADM1 mRNA expression level was detected by RT-qPCR in samples from donors and patients. ns, not significant; *p < 0.05 vs. the first diagnosed leukemia patients. (D) The protein expression level of CADM1 was examined by Western blot. **p < 0.01 vs. the first diagnosed leukemia patients. (E) RT-qPCR was carried out to test the CADM1 mRNA expression level in chemo-resistant K562/ADM and HL-60/RS cells and their parental leukemia cells. **p < 0.01 vs. K562; ^##p < 0.01 vs. HL-60. (F) Western blot was used to measure the protein expression levels of CADM1 in chemo-resistant leukemia cells and their parental leukemia cells. *p < 0.05 vs. K562

Fig. 3

CADM1 mediated viability, drug sensitivity, proliferation, apoptosis and cell cycle distribution of chemo-resistant leukemia cells. (A) The cell viability was measured using MTT assay in chemo-resistant K562/ADM and HL-60/RS cells transfected with in-NC + si-NC, in-miR-1246 + si-NC, or in-miR-1246 + si-CADM1. (B) MTT assay was performed to detect the IC₅₀ of K562/ADM and HL-60/RS cells transfected with in-NC + si-NC, in-miR-1246 + si-NC, or in-miR-1246 + si-CADM1 to ADM. (C) EdU incorporation assay was used to examine the cell proliferation ability of K562/ADM and HL-60/RS cells transfected with in-NC + si-NC, in-miR-1246 + si-NC, or in-miR-1246 + si-CADM1. (D) The cells were stained with Annexin V-FITC and PI, and the cell apoptotic rate was tested by flow cytometry. (E) The relative c-PARP expression level was detected by Western blot. (F) After the cells were fixed and stained, flow cytometry was used to check the cell cycle distribution in K562/ADM transfected with in-NC + si-NC, in-miR-1246 + si-NC, or in-miR-1246 + si-CADM1. *p < 0.05 and **p < 0.01 vs. the in-NC + si-NC group; ^#p < 0.05 and ^##p < 0.01 vs. the in-miR-1246 + si-NC group

Fig. 4

miR-1246/CADM1 axis regulated cell cycle progression by tuning up CDKs. (A) and (B) The protein levels of CDK2, CDK4, Cyclin E and Cyclin D1 were detected in chemo-resistant leukemia cells, their parental sensitive leukemia cells, and clinical samples. *p < 0.05 and **p < 0.01 vs. their parental leukemia cells or the first diagnosed leukemia patients. (C) Comparing to NC-inhibitor transfected chemo-resistant leukemia cells, the protein levels of CDK2, CDK4, Cyclin E and Cyclin D1 in miR-1246 inhibitor transfected chemo-resistant leukemia cells were analyzed. *p < 0.05 vs. the in-NC group. (D) The protein immunoblotting was applied to test the protein expression of CADM1, CDK2, CDK4, Cyclin E, Cyclin D1 in chemo-resistant leukemia cells transfected with in-NC + si-NC, in-miR-1246 + si-NC, or in-miR-1246 + si-CADM1. *p < 0.05 and **p < 0.01 vs. the in-NC + si-NC group; ^#p < 0.05 and ^##p < 0.01 vs. the in-miR-1246 + si-NC group. (E) Western blot assay was used to detect the protein levels of cell cycle related proteins CDK2, CKD4, Cyclin E, Cyclin D1. *p < 0.05 vs. the in-miR-1246 + si-CADM1 group. (F) Flow cytometer combined PI was used to examine the cell cycle distribution of K562/ADM cells transfected with in-miR-1246 + si-CADM1 with or without flavopiridol. **p < 0.01 and ^##p < 0.01 vs. the in-miR-1246 + si-CADM1 group