The miR-1269a/PCDHGA9/CXCR4/β-catenin pathway promotes colorectal cancer invasion and metastasis

Abstract

Background: Colorectal cancer (CRC) is the third most common cancer worldwide and the second leading cause of cancer-related death. This research focuses on investigating the impact and underlying molecular mechanisms of protocadherin gamma subfamily A, 9 (PCDHGA9) on the invasion and metastasis of CRC, aiming to identify more precise molecular markers for the diagnosis and prognosis of CRC.

Methods: PCDHGA9 expression was detected using quantitative real-time quantitative polymerase chain reaction (RT-qPCR) in 63 pairs of colorectal cancer tissues. Differential gene expression from high-throughput sequencing was analyzed using ingenuity pathway analysis (IPA) to explore the biological functions of PCDHGA9 and its potential regulated genes. Bioinformatics tools were employed to explore potential upstream regulatory microRNAs of PCDHGA9. Dual-luciferase assays were performed to demonstrate the regulation between PCDHGA9 and miR-1269a. Protein mass spectrometry suggested an interaction between PCDHGA9 and HOXA1. JASPAR predicted that HOXA1 may act as a transcription factor of CXCR4. Coimmunoprecipitation, dual-luciferase assays, and nuclear-cytoplasmic fractionation experiments confirmed the molecular mechanism involving PCDHGA9, CXCR4, HOXA1, and β-catenin. Transwell, wound healing, and western blot assays were conducted to confirm the impact of PCDHGA9, miR-1269a, and CXCR4 on the invasion, metastasis, and epithelial-mesenchymal transition (EMT) functions of CRC cells in in vitro experiments. A whole-body fluorescence imaging system was used to evaluate the combined impact of miR-1269a and PCDHGA9 on the invasion and metastasis of CRC in in vivo experiments.

Results: The expression of PCDHGA9 was found to be lower in CRC tissues compared with their corresponding adjacent tissues. Low expression of PCDHGA9 potentially correlated with worse prognosis and increased chances of invasion and metastasis in CRC. miR-1269a was highly expressed in CRC tissues and acted as a negative regulator for PCDHGA9, promoting invasion, migration, and EMT of CRC cells. PCDHGA9's interaction with HOXA1 downregulated CXCR4, a transcription factor, leading to accumulation of β-catenin and further promoting invasion, migration, and EMT of CRC cells.

Conclusions: PCDHGA9, acting as a tumor suppressor, is downregulated by miR-1269a. The low level of PCDHGA9 activates the Wnt/β-catenin pathway by releasing its interaction with HOXA1, promoting the expression of CXCR4, and causing invasion, migration, and EMT in CRC.

Keywords: CRC; CXCR4; HOXA1; PCDHGA9; miR-1269a; β-Catenin.

Fig. 1

Comprehensive investigation of PCDHGA9 in CRC: expression in tissue, survival analysis, cell line manipulation, and high-throughput sequencing results. a Real-time quantitative PCR (qPCR) revealed decreased PCDHGA9 expression in 42 CRC tissues among 63 paired samples, compared with adjacent normal tissues. b, c Patients with low PCDHGA9 expression exhibited shorter overall survival and disease-free survival rates. d, f Western blot analysis assessed PCDHGA9 expression in HCT116, RKO, HCT8, and SW620 cells, as well as the impact of overexpressing or knocking down PCDHGA9 on PCDHGA9 and β-catenin expression. e Immunohistochemistry exhibits the expression of PCDHGA9 in CRC tissues with low, moderate, and high differentiation. g High-throughput sequencing identified differential gene expression between vector and PCDHGA9-overexpressing cells, with downregulated genes listed on the right

Fig. 2

PCDHGA9 suppresses invasion, metastasis, and EMT of CRC cells. a Wound healing assays were conducted on CRC cells with overexpressed or downregulated PCDHGA9 and their respective controls. The width of wounds was measured at 0 and 48 h, and the percentage of wound closure was calculated and analyzed using the Student’s t-test. b Transwell assays were performed on CRC cells with manipulated PCDHGA9 expression and their controls, and the number of cells that had migrated through the chamber was observed and quantified. c Western blot analysis showing changes in E-cadherin, N-cadherin, vimentin, and Snail expression following the overexpression or downregulation of PCDHGA9. Notably, E-cadherin was not detected in RKO cells from our laboratory, resulting in the absence of an E-cadherin blot

Fig. 3

miR-1269a targets PCDHGA9 and downregulates PCDHGA9. a Venn diagram illustrating the microRNAs predicted to interact with PCDHGA9 across three databases (starBase, Targetscan, and miRBD). Among these, hsa-miR-1269a, hsa-miR-5586-5p, hsa-miR-580-3p, hsa-miR-760, hsa-miR-625-5p, and hsa-miR-3179 were identified as commonly predicted microRNAs. b PCDHGA9 expression was assessed via qPCR after transfection of miR-1269a and miR-3179 mimics into 293T cells. c Four groups of 293T cells were transfected with different combinations of vectors (NC or miR-1269a mimic) and luciferase reporter plasmids (wild type or mutant type PCDHGA9). The luciferase activity was measured using the dual luciferase assay system. d The relative expression of PCDHGA9 was examined by qPCR following the transfection of miR-1269a inhibitor or mimic into CRC cells. e The protein levels of PCDHGA9 and β-catenin were analyzed by western blotting in CRC cells transfected with miR-1269a inhibitor or mimic. f The interacting region between PCDHGA9 and miR-1269a is shown

Fig. 4

MiR-1269a promotes invasion, metastasis, and EMT of CRC cells. a Wound healing assays were conducted in CRC cells transfected with either miR-1269a inhibitor or mimic, along with their respective controls. The widths of wounds were measured at 0 h and 48 h, and the percentages of wound closure were calculated and analyzed. b Transwell assays were performed in CRC cells transfected with either miR-1269a inhibitor or mimic, along with their respective controls. The cells that had migrated out of the chamber were observed and counted. c Western blotting results demonstrated changes in the protein levels of E-cadherin, N-cadherin, vimentin, and Snail after transfection with either miR-1269a inhibitor or mimics into cells

Fig. 5

CXCR4 is downregulated by PCDHGA9 interacting with HOXA1. a Co-IP assays revealed the presence of the FLAG and HOXA1 proteins in both the input and immunoprecipitated samples. b Dual luciferase assays showing the relative activity of the wild-type (WT) CXCR4 luciferase reporter plasmid after either overexpressing or downregulating PCDHGA9. c Predicted binding sites of HOXA1 within the CXCR4 promoter region at positions 1437, 400, and 77. Two mutant constructs, MUT1 and MUT2, were generated for the three predicted binding sites. Dual luciferase assays were conducted to assess the relative activity of WT, MUT1, and MUT2 CXCR4 luciferase reporter plasmids in the presence or absence of HOXA1. d Western blot analysis depicting the protein expression of CXCR4, PCDHGA9, and β-catenin in CRC cells with altered CXCR4 expression

Fig. 6

CXCR4 promotes invasion, metastasis, and EMT of CRC cells. Wound healing (a), Transwell assays (b), and WB (c) showing the effects of CXCR4 on promoting the invasion, metastasis, and EMT of CRC cells

Fig. 7

The regulatory relationship among PCDHGA9, miR-1269a, CXCR4, and β-catenin. a Quantitative PCR (qPCR) analysis demonstrated the expression levels of miR-1269a following the downregulation or overexpression of CXCR4. b Western blotting (WB) revealed the protein expression of CXCR4 and β-catenin in the presence or absence of the HOXA1 overexpressing plasmid. c WB showed alterations in the protein levels of HOXA1, CXCR4, and β-catenin upon PCDHGA9 overexpression and the introduction of the miR-1269a mimic. d WB depicted changes in the HOXA1 distribution between the cytoplasm and nucleus in the presence or absence of PCDHGA9 overexpression and the miR-1269a mimic. e WB analysis demonstrated that (E)-ferulic acid, a β-catenin inhibitor, affected β-catenin expression without impacting PCDHGA9 and CXCR4. Similarly, CHIR-99021, a β-catenin activator, influenced β-catenin expression while leaving PCDHGA9 and CXCR4 unaffected. f A schematic diagram illustrating the regulatory network involving miR-1269a, PCDHGA9, HOXA1, CXCR4, and β-catenin

Fig. 8

miR-1269a and PCDHGA9 together affect the invasion and metastasis of CRC in vivo. a Whole-body fluorescence images showing lung metastases across varying conditions of miR-1269a and PCDHGA9 expression. Subsequent photos depicted lung metastases following the sacrifice of these mice. b Immunohistochemical staining of lung metastases was carried out for E-cadherin and vimentin. The immunohistochemical staining highlighted alterations in E-cadherin and vimentin expression in response to different miR-1269a and PCDHGA9 expression conditions. miR-1269a reduced the expression of E-cadherin, but this result was reversed in the condition of high expression of PCDHGA9. Vimentin showed the opposite of E-cadherin