| Colorectal cancer(CRC) is one of the most common malignancies around the world, and World Cancer Report 2014 showes that the number of patients newly indentified and cancer-induced mortality is the most among the world, and the incidence of patients with gastrointestinal malignancies, including CRC, has grown rapidly. Therefore, investigation into the main underylying molecular mechanism of tumorigenesis and progression is crucial for the development of new anti-cancer strategies.Recently, studies on cancer stem cell have provided new understanding of colorectal cancer therapy. Our previous studies have found that semaphorins-3F(SEMA3F), an axon guidance molecular in the development of central nervous system, inhibited the growth and metastasis of CRC, however, a possible role of SEMA3 F in regulating cancer cell stemness remains unknown. Plexin A1, as a receptor of SEMA3 F, which has a split Ras-GTPase activating protein domain in the cytoplasmic region. It plays a crucial role in regulating cell cytoskeleton dynamics, cell adhesion and migration elicited by SEMA3 F. Moreover, recent evidence has demonstrated that GTPase is a pivotal player in the regulation of stemness. Addithonally, we found that the expression of SEMA3 F is low in colorectal cancer stem cells in our pre-experments, and is negatively correlated with tumor grade. All these results suggest that SEMA3 F may play an important and undefined role in the initation of cancer.In this study, we hypothezed that SEMA3 F might inhibite colorectal cancer cell stemness by inactivating Rac1. We first detected the effect of SEMA3 F on colorectal cancer cell HCT116 self-renewal and tumorigenesis in vivo and associated Wnt signaling pathway. We then explored the role of GTP-Rac1 in the function mediated by SEMA3 F. Finally, we detected the expression distribution of SEMA3 F, GTP-Rac1 and LGR5, and explored the clinical significance of SEMA3 F and GTP-Rac1.Main results and conclusions are as follows1. SEMA3 F inhibits colorectal cancer stemness(1) Overexpression of SEMA3 F could significantly inhibit the expression of stemness associated genes, such as Nanog, Sox2 and Oct4.(2) Reduced expression of SEMA3 F could significantly promote the expression of stemness associated genes, such as Nanog, Sox2 and Oct4.(3)and(4) SEMA3 F could significantly inhibit the expression level of LGR5 and the ratio of LGR5 positive subpopulation(3.31%±0.33%, 4.0%±0.83% VS 0.75%±0.15%,p<0.05).(5) Overexpression of SEMA3 F could reduce the colony formation capacity of CRC cells(21.67±1.63%, 21±1.70% VS 12.0±0.82%, p<0.05), and sphere formation ability of CRC cells(22.33±2.78%, 20.67±1.72% VS 8±1.47%,p<0.05).(6) Knock-down expression of SEMA3 F could significantly cause a increase of expression of LGR5 and LGR5 positive subpopulation(3.31%±0.33%, 4.0%±0.83% VS 8.92%±1.15%,p<0.05).(7) Reduced expression of SEMA3 F lead to an increase in the ability of colony formation(20.83±0.76%, 20.33±2.02% VS 26.83±1.26%, p<0.05) and sphere formation(28.0±2.64%, 23.67±2.06% VS 47.67±2.74%, p<0.05).(8) The tumorigenicity of CRC cells in xenograft model was enhanced by SEMA3 F knocking down, the incidence and weight of xenograft tumor formed by reduced expression of SEMA3 F was superior to the control. Moreover, mean tumor volumes post subcutaneous injection at 1×106, the volume(347.872±90.66mm3, 277.35±54.166 mm3 VS 1025.96±169.5 mm3, p<0.05) and weight(0.0299±0.029 g, 0.068±0.006 g VS 0.4426±0.1279 g, p<0.05) formed by Sh-SEMA3 F was enhanced compared with controls.2. SEMA3 F inhibits the activity of Wnt signaling pathway.(1) TOP flash assay showed that overexpression of SEMA3 F could inhibit activity of Wnt sighaling pathway(0.489±0.06, 0.465±0.08 VS 0.15±0.005, p<0.05), in the meantime, the protein expression of β-catenin could be inhibited significantly.(2) TOP flash assay showed that reduced expression of SEMA3 F could promote the activity of Wnt signaling pathway(1.59±0.06, 1.19±0.09 VS 6.56±0.79, p<0.05).(3) Western blotting showed that the β-catenin in the fraction of nuclear was elevated after reduced expression of SEMA3 F, but without change in the fraction of cytoplasm.3. SEMA3 F inhibits the stemness of CRC cells in a Rac1 dependent manner.(1) and(2) Expression of ARHGEF7 and GTPBP4 could be upregulated or reduced when SEMA3 F knockdown or overexpression.(3) Rac1 could be inactivated by overexpression of SEMA3 F.(4) Knockdown expression of SEMA3 F could promote activation of Rac1.(5) when treated HCT116 cells with Rac1 activation inhibitor, NSC23766, the protein expression level could be reduced with the increase of NSC23766, and 75 um is optimal concentration.(6) treated CRC cells with 75 u M NSC23766 could abrogated the increasement of LGR5, β-catenin, Nanog, Sox2, Oct4 modulated by knockdown expression of SEMA3 F.(7) the promotive effects of SEMA3 F on colony formation and sphere formation by CRC cells could be abolished by NSC23766.(8) The suppressive effects of SEMA3 F on colony formation could be enhance by NSC23766 with 75 um.(9) When treated cell with Si RNA, increase of LGR5, β-catenin, Nanog, Sox2, Oct4 modulated by knockdown expression of SEMA3 F could be abolished.4. Expression status and clinical significance of SEMA3 F, LGR5 and GTP-Rac1 in human colorectal cancer specimens.(1) SEMA3 F was expressed in the cytoplasm of glandular cells of normal colorectal mucosa and in the early stage CRC cells; the expression of SEMA3 F was decreased with TNM stages increased. Moreover, SEMA3 F expression was correlated with tumor recurrence, lymph nodes, distant metastasis and TNM stages significantly. And the patients with higher expression of SEMA3 F had a good prognosis.(2) GTP-Rac1 was highly expressed in colorectal cancer cells, and GTP-Rac1 stainig was positive correlated with tumor TNM stages. Moreover, GTP-Rac1 expression was positively correlated with tumor recurrence, distant metastasis, lymph nodes metastasis and TNM stages, and the patients with higher expression of GTP-Rac1 had a poor prognosis.(3) LGR5 was highly expressed in colorectal cancer cells and tumor invasion edge, and its expression was positively correlated with tumor invasion depth, lymph node metastasis, recurrence, and TNM stages. The patients with higher expression of LGR5 had a poor prognosis. Immunofluorescence staning showed that SEMA3 F and LGR5 hadn’t no significantly co-location.(4) SEMA3 F expression was negatively correlated with GTP-Rac1, the patients with higher expression SEMA3 F had a lower expression of GTP-Rac1, and the patients with SEMA3FlowGTP-Rac1 high had a most poor prognosis. Moreover, univariate and multivariate analyses showed that the combination of SEMA3 F and GTP-Rac1 expression in CRC was an independent prognostic indicator of overall survival of patients.In summary, these results suggest that SEMA3 F played an negative regulators on colorectal cancer cell stemness, and knockdown expression of SEMA3 F could promote the tumorigenicity of colorectal cancer cells; SEMA3 F could inhibit activity of Wnt signaling pathways; these biological effects mediated by SEMA3 F is modulated by inactivating Rac1 in APC-independent manner; expression of SEMA3 F was negatively correlated with clinical pathologic parameters, GTP-Rac1 expression was positively correlated with clinical pathological parameters and negatively correlated with survival times, combination of SEMA3 F and GTP-Rac1 is an independent prognostic indicator of overall survival of patients with CRC. |
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