MiR-3150b-3p inhibits the progression of colorectal cancer cells via targeting GOLPH3

The aim of this study was to investigate the function of miR-3150b-3p in malignant behaviors of colorectal cancer (CRC). The tumor-inhibitive effect of miR-3150b-3p was determined by cell viability, invasion, and migration assays. The influence of miR-3150b-3p on the expression of Golgi phosphoprotein 3 (GOLPH3) and Janus kinase 2 (JAK2)/signal transducer and activator of transcription 3 (STAT3) pathway was evaluated by luciferase reporter, qRT-PCR and western blot analysis. MiR-3150b-3p was markedly decreased in CRC cell lines compared with colonic mucosal epithelial cell line (FHC). Furthermore, miR-3150b-3p inhibited malignant biological behaviors by targeting GOLPH3, an oncogene in CRC. Additionally, we suggested that miR-3150b-3p ameliorated CRC tumorigenesis in vitro through GOLPH3-mediated JAK2/STAT3 pathway. MiR-3150b-3p might function as a promising tumor suppressor in CRC.


Significance of this study
What is already known about this subject? ► MiR-3150b-3p expression was significantly decreased in colorectal cancer (CRC) tissues. ► The activation of Janus kinase 2/signal transducer and activator of transcription 3 (JAK2/STAT3) was implicated in CRC progression. ► Golgi phosphoprotein 3 (GOLPH3) is a novel oncogene in CRC.
How might these results change the focus of research or clinical practice? ► MiR-3150b-3p might be a valuable target for developing therapeutic strategy against CRC.

AbSTrACT
The aim of this study was to investigate the function of miR-3150b-3p in malignant behaviors of colorectal cancer (CRC). The tumor-inhibitive effect of miR-3150b-3p was determined by cell viability, invasion, and migration assays. The influence of miR-3150b-3p on the expression of Golgi phosphoprotein 3 (GOLPH3) and Janus kinase 2 (JAK2)/signal transducer and activator of transcription 3 (STAT3) pathway was evaluated by luciferase reporter, qRT-PCR and western blot analysis. MiR-3150b-3p was markedly decreased in CRC cell lines compared with colonic mucosal epithelial cell line (FHC). Furthermore, miR-3150b-3p inhibited malignant biological behaviors by targeting GOLPH3, an oncogene in CRC. Additionally, we suggested that miR-3150b-3p ameliorated CRC tumorigenesis in vitro through GOLPH3-mediated JAK2/STAT3 pathway. MiR-3150b-3p might function as a promising tumor suppressor in CRC.

InTrOduCTIOn
Colorectal cancer (CRC) remains one of the most common malignancies worldwide. 1 2 Previous studies have highlighted the aberrant activation of various cellular pathways in CRC progression. 3 4 However, the mechanism of CRC remains unclear. MicroRNAs (miRNA) play crucial roles in a variety of biological processes, 5-7 by regulating expression of multiple protein. 8 9 MiR-3150b-3p is located at 8q22.1 and belongs to the miR-3150b family. Heller et al have observed higher levels of methylated miR-3150b in non-small-cell lung cancer tissues. 10 In addition, miR-3150b-5p, another member of miR-3150b family, was identified as the most significantly downregulated miRNA in laryngeal squamous cell carcinoma cells after paclitaxel treatment. 11 Moreover, miR-3150b-5p has been found to increase the risk of death from CRC in cases diagnosed with rectal cancer when its expression increased in carcinoma tissues. 12 Nevertheless, until now, the expression and the potential function of miR-3150b-3p in CRC remain unknown. Our study provided evidence that miR-3150b-3p suppressed CRC progression through the Janus kinase 2/signal transducer and activator of transcription 3 (2JAK2/ STAT3) signaling by directly targeting Golgi phosphoprotein 3 (GOLPH3).

Cell transfection
HCT116 and SW480 cells in the logarithmic growth phase were seeded in 6-well plates. When these cells reached 30%-50% confluence, they were transfected with miR-3150b-3p mimic/inhibitor or their negative controls using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA).

Migration assay
Briefly, transfected cells were wounded using a sterile micropipette tip, incubated in serum-free RPMI-1640 medium, and photographed under a microscope (Olympus, Tokyo, Japan) at 0 and 48 hours after wounding.

Transwell assay
Cell invasion ability was assessed using transwell chambers coated with 40 µL Matrigel as previously described. 14 HCT116 and SW480 cells (1×10 5 cells per well) were added to the upper chamber, while serum-supplemented culture medium was added to the lower chamber. Following 48 hours of incubation, the number of stained cells was calculated under a microscope.

luciferase reporter assay
The indicated luciferase plasmids (Promega, Madison, WI, USA) along with mimic NC or miR-3150b-3p mimic were co-transfected into HEK293T cells. Luciferase activities were analyzed 24 hours after transfection.

rnA isolation and real-time PCr
Following standard quantitative PCR procedure, quantitative PCR was carried out for detecting miR-3150b-3p and GOLPH3 mRNA expression levels using U6 and β-actin as the internal controls.

Statistical analysis
All data were analyzed by one-way analysis of variance. Significant differences were indicated as p<0.05 or p<0.01.

Mir-3150b-3p was downregulated in CrC cell lines
As shown in figure 1, miR-3150b-3p was significantly downregulated in 4 CRC cell lines compared with FHC cells. Since overexpression and downregulation of miR-3150b-3p was more evidently observed in HCT116 and SW480 cells, respectively, these 2 cell lines were chosen for the following experiments.

Mir-3150b-3p directly targeted GOlPH3 in CrC cells
We then performed the luciferase assay. The results revealed that miR-3150b-3p mimic could significantly decrease the luciferase activity of wild-type GOLPH3 3′-UTR vector in HEK293T cells (figure 4A). Figure 4B,C showed that overexpression of miR-3150b-3p in HCT116 cells memorably downregulated GOLPH3 mRNA and protein expression levels, while suppression of miR-3150b-3p expression led to an opposite effect.

upregulation of GOlPH3 reversed the antitumor effect of mir-3150b-3p in CrC
The results indicated that ectopic expression of GOLPH3 (figure 5A) could partially overturn miR-3150b-3p-induced inhibition of HCT116 cell proliferation (figure 5B), migration (figure 5C) and invasion (figure 5D), which were confirmed in the online supplementary figure S1.

dISCuSSIOn
In the present study, we first found that miR-3150b-3p was frequently downregulated in human CRC cells. The overexpression of miR-3150b-3p inactivates the JAK2-STAT3 axis by downregulating the target gene GOLPH3, thereby inhibiting CRC tumorigenesis. In recent years, abundant studies provide strong evidence that miRNAs act as tumor suppressor genes in CRC. For example, Huang et al showed that miR-4319 overexpression suppressed CRC carcinogenesis by regulating cell cycle distribution. 15   MiR-3150b-3p inhibited Janus kinase 2/signal transducer and activator of transcription 3 (JAK2/STAT3) signaling through downregulating Golgi phosphoprotein 3 (GOLPH3) expression. GOLPH3 protein levels, total and phosphorylated proteins of JAK2 and STAT3, and the protein levels of survivin, c-Myc, matrix metalloproteinase (MMP)-2/9 in HCT116 cells cotransfected with miR-3150b-3p mimic or mimic NC along with pcDNA3.1-GOLPH3 or blank vector. Significant differences were indicated as **p<0.01.
of let-7e significantly delayed cell proliferation, migration, epithelial-mesenchymal transition process and stemness, and promoted cell apoptosis in CRC cells. 16 In this study, decreased expression of miR-3150b-3p was found in CRC cell lines. Further studies demonstrated that miR-3150b-3p overexpression could suppress CRC cell proliferation, migration and invasion, revealing that the aberrant expression of miR-3150b-3p might be crucial for CRC progression.
GOLPH3 is a well-known oncogene in several solid tumors, such as hepatocellular carcinoma, 17 ovarian cancer 18 and CRC. 19 Increasing number of studies revealed the fact that miRNAs result in target mRNA degradation or translational inhibition. 20 To date, a series of tumor-suppressor miRNAs have been confirmed to target GOLPH3. For instance, Li et al found that miR-134 might directly target GOLPH3, thereby inhibiting cell proliferation in gastric cancer. 21 Herein, miR-3150b-3p could reduce the expression of GOLPH3. Moreover, the rescue experiments indicated that GOLPH3 overexpression abrogated the effects mediated by miR-3150b-3p overexpression in CRC cells.
Several lines of evidence suggest that abnormal activation of the JAK2/STAT3 signaling pathway is critical for the development and progression of various cancers, including CRC. 22 GOLPH3 was shown to be engaged in JAK2/STAT3 signaling pathway in glioma progression. 23 Our study in vitro demonstrated that miR-3150b-3p by decreasing the expression of GOLPH3, inactivated JAK2/STAT3 signaling pathway in CRC cells.
In conclusion, miR-3150b-3p might be the potential target for treatment of CRC. Several limitations were included in our study. First, the in vivo experiments were excluded. Second, the other molecular mechanisms may be involved need to further investigation.
Contributors WZ and XC designed the study, conducted most of the experiments and wrote the manuscript. JJ conducted the experiments and analyzed the data.
Funding The authors have not declared a specific grant for this research from any funding agency in the public, commercial or not-for-profit sectors.
Competing interests None declared.

Patient consent for publication Not required.
Provenance and peer review Not commissioned; externally peer reviewed. data availability statement Data are available upon reasonable request.