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. 2017 Feb 8:7:42226.
doi: 10.1038/srep42226.

CXCL12/CXCR4 axis induced miR-125b promotes invasion and confers 5-fluorouracil resistance through enhancing autophagy in colorectal cancer

Xinfeng Yu  1 , Wenna Shi  1 , Yuhang Zhang  1 , Xiaohui Wang  2 , Shiyue Sun  1 , Zhiyu Song  1 , Man Liu  1 , Qiao Zeng  1 , Shuxiang Cui  3 , Xianjun Qu  1
Affiliations

CXCL12/CXCR4 axis induced miR-125b promotes invasion and confers 5-fluorouracil resistance through enhancing autophagy in colorectal cancer

Xinfeng Yu et al. Sci Rep. .

Abstract

The activation of CXCL12/CXCR4 axis is associated with potential progression of cancer, such as invasion, metastasis and chemoresistance. However, the underlying mechanisms of CXCL12/CXCR4 axis and cancer progression have been poorly explored. We hypothesized that miRNAs might be critical downstream mediators of CXCL12/CXCR4 axis involved in cancer invasion and chemoresistance in CRC. In human CRC cells, we found that the activation of CXCL12/CXCR4 axis promoted epithelial-mesenchymal transition (EMT) and concurrent upregulation of miR-125b. Overexpression of miR-125b robustly triggered EMT and cancer invasion, which in turn enhanced the expression of CXCR4. Importantly, the reciprocal positive feedback loop between CXCR4 and miR-125b further activated the Wnt/β-catenin signaling by targeting Adenomatous polyposis coli (APC) gene. There was a negative correlation of the expression of miR-125b with APC mRNA in paired human colorectal tissue specimens. Further experiments indicated a role of miR-125b in conferring 5-fluorouracil (5-FU) resistance in CRC probably through increasing autophagy both in vitro and in vivo. MiR-125b functions as an important downstream mediator upon the activation of CXCL12/CXCR4 axis that involved in EMT, invasion and 5-FU resistance of CRC. These findings shed a new insight into the role of miR-125b and provide a potential therapeutic target in CRC.

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Conflict of interest statement

The authors declare no competing financial interests.

Figures

Figure 1
Figure 1. CXCL12 induced upregulation of miR-125b and concurrent EMT in CRC cells.
(A) HCT116 and SW620 cells were exposed to 100 ng/μl CXCL12 for the indicated time. RT-qPCR was performed to determine the expression of miR-125b. (B) HCT116 cells were transfected with 200 nM siRNA of CXCR4 or expression plasmid together with corresponding controls for 48 h. RT-qPCR was performed to determine the expression of miR-125b. (C–E) The cells were treated as indicated above by CXCL12, the mRNA levels of E-cadherin, vimentin and CXCR4 were determined by RT-qPCR assay. *P < 0.05 vs. control. (F) The protein level of E-cadherin, vimentin and CXCR4 was examined by Western blot assay.
Figure 2
Figure 2. Overexpression of miR-125b promoted EMT in CRC cells.
(A,B) HCT116 and SW620 cells were transfected with 100 nM miR-125b mimics (125 m) or inhibitors (125i) for 48 h. The levels of miR-125b were determined by RT-qPCR assay. (C,D) The cells were treated as indicated above, the mRNA levels of E-cadherin, vimentin and CXCR4 were determined by RT-qPCR assay. (E) The levels of ZEB1, Snail, vimentin and E-cadherin were determined by Western blot assay. Bar graphs indicated the relative levels of the proteins normalized to β-actin. *P < 0.05 vs. negative control. (F) HCT116 and SW620 cells were transfected with 100 nM miR-125b mimics (125 m) or inhibitor (125i) for 24 h, then were treated with or without 100 ng/μl CXCL12 for 24 h. The capacity of cell invasion was evaluated by transwell assay. *P < 0.05 vs. negative control (NC). #P < 0.05 vs. NC and 125i groups, respectively.
Figure 3
Figure 3. MiR-125b enhanced cell invasion by targeting APC gene.
(A) HCT116 and SW620 cells were transfected with 100 nM miR-125b mimics (125 m) or inhibitors (125i) for 48 h. The mRNA level of APC was determined by RT-qPCR assay. (B) The level of p-β-catenin/β-catenin was examined by Western blot assay. (C) HCT116 cells were transfected with luciferase constructs and miR-125b mimics. The comparison of luciferase activity of wild-type (WT) and mutant (MUT) APC-3′UTR constructs was performed 36 h after transfection. Data was normalized to renilla activity. *P < 0.05 vs. negative control (NC). (D) Western blotting was performed to determine the expression of APC, active-β-catenin, β-catenin, c-Myc and TCF1. Bar graphs indicated the relative levels of these proteins normalized to β-actin. *P < 0.05 vs. negative control. (E, F) RT-qPCR was performed to examine the expression of miR-125b and APC mRNA in 14 pairs of CRC tissues and adjacent normal colorectal tissue. *P < 0.05 vs. normal group.
Figure 4
Figure 4. MiR-125b reduced the 5-FU-induced apoptosis in CRC cells.
(A) HCT116 and SW620 cells were transfected with 100 nM miR-125b mimics (125 m) for 24 h, then were treated with 5, 10 μM 5-FU or 25, 50 μM 5-FU for 72 h respectively. Cell viability was estimated by the MTT assay. (B) HCT116 and SW620 cells were transfected with 100 nM miR-125b mimics (125 m) or 200 nM siRNA of CXCR4 for 24 h, then the cells were treated with or without 25 μM or 50 μM 5-FU for 48 h respectively. Annexin V-PI double staining assay was performed to determine apoptotic cells using flow cytometry. Bar graphs indicated the percentage of apoptotic cells. Data represent mean ± SD of three experiments. *P < 0.05 vs. negative control (NC). (C) HCT116 and SW620 cells were treated as above, Western blotting was performed to analyze the expression of apoptotic proteins Bax, Bcl-2, PARP, caspase-3 and autophagic protein LC3 cleavage. Bar graphs indicated the relative levels of Bax/Bcl-2, PARP, cleaved-caspase 3/pro-caspase 3 and LC3-II/LC3-I normalized to β-actin. *P < 0.05 vs. vehicle control, #P < 0.05 vs. negative control (NC).
Figure 5
Figure 5. MiR-125b activated autophagic activity in CRC cells.
(A) HCT116 and SW620 cells were transfected with 100 nM miR-125b mimics (125 m) or inhibitors (125i) for 48 h. The expression of beclin-1 and cleaved LC3-II was examined by Western blot assay. Bar graphs indicated relative levels of LC3-II and Beclin-1normalized to β-actin. Data represent mean ± SD of three experiments. *P < 0.05 vs. negative control. (B) HCT116 and SW620 cells were co-transfected with GFP-LC3 plasmid and 100 nM miR-125b mimics for 48 h in the presence or absence of 5 mM 3-MA. Representative photographs were taken using a confocal microscopy (scale bar = 25 μm). Numbers of GFP-LC3 puncta per cell were counted (*P < 0.05 vs. negative control, #P < 0.05 vs. 125 m group, n = 10). Data represent mean ± SD of three experiments.
Figure 6
Figure 6. MiR-125b inhibited the 5-FU-induced apoptosis in the xenograft model in vivo.
HCT116 cells infected with lentiviral miR-125b (125 m) and negative control (NC) were injected subcutaneously into nude mice. When tumor volume reached 100 mm3, 5-FU (20 mg/kg) was injected intraperitoneally five times per week for two weeks. (A) Tumor volumes were measured twice per week and the dynamic changes were shown in curves. Data represent mean ± SD. CON represents a short form of vehicle control. (B) Tumor images were taken at the end of the experiments. RNA was extracted from xenograft tumors of NC-CON and 125m-CON groups to determine the expression of miR-125b expression by RT-qPCR assay. (C) Tumor weight and volume were determined after removal. Data represent mean ± SD, *P < 0.05 vs. negative control. (D) Representative images were shown to indicate the expression of E-cadherin, PCNA and Beclin-1 in different groups through immunohistochemistry assay(400 ×). Results were semi-quantified by image-pro plus 6 software and statistical analyses were performed. *P < 0.05 vs. negative control. (E) Protein lysates were extracted from xenograft tumor tissues to determine the expression of Bax, PARP, caspases and autophagic proteins beclin-1 and LC3-II by Western blot assay. Bar graphs indicated the relative levels of cleaved PARP, cleaved caspase-9, caspase-3, LC3-II and Beclin-1 normalized to β-actin. Data represent mean ± SD of three experiments. *P < 0.05 vs. negative control. CON represents a short form of vehicle control, 5-FU is a short form of 5-fluororacil.
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