Semaphorin 4C regulates ovarian steroidogenesis through RHOA/ROCK1-mediated actin cytoskeleton rearrangement.

Semaphorins are a family of evolutionarily conserved morphogenetic molecules that were initially found to be associated with axonal guidance. Semaphorin 4C (Sema4C), a member of the fourth subfamily of semaphorins, has been demonstrated to play multifaceted and important roles in organ development, immune regulation, tumor growth, and metastasis. However, it is completely unknown whether Sema4C is involved in the regulation of ovarian function. We found that Sema4C was widely expressed in the stroma, follicles, and corpus luteum of mouse ovaries, and its expression was decreased at distinct foci in ovaries of mice of mid-to-advanced reproductive age. Inhibition of Sema4C by the ovarian intrabursal administration of recombinant adeno-associated virus-shRNA significantly reduced oestradiol, progesterone, and testosterone levels in vivo. Transcriptome sequencing analysis showed changes in pathways related to ovarian steroidogenesis and the actin cytoskeleton. Similarly, knockdown of Sema4C by siRNA interference in mouse primary ovarian granulosa cells or thecal interstitial cells significantly suppressed ovarian steroidogenesis and led to actin cytoskeleton disorganization. Importantly, the cytoskeleton-related pathway RHOA/ROCK1 was simultaneously inhibited after the downregulation of Sema4C. Furthermore, treatment with a ROCK1 agonist after siRNA interference stabilized the actin cytoskeleton and reversed the inhibitory effect on steroid hormones described above. In conclusion, Sema4C may play an important role in ovarian steroidogenesis through regulation of the actin cytoskeleton via the RHOA/ROCK1 signaling pathway. These findings shed new light on the identification of dominant factors involved in the endocrine physiology of female reproduction.

Hsa-miR-10a-5p promotes pancreatic cancer growth by BDNF/SEMA4C pathway.

This article aims to explore the expression and mechanism of miR-10a-5p in pancreatic cancer. MiR-10a-5p mimic, MiR-10a-5p inhibitor and negative control were transfected into human pancreatic cancer cell line SW1990. Real-time quantitative PCR technology was used to analyze the expression level of miR-10a-5p in pancreatic cancer tissues and cells. The proliferation, invasion and apoptosis of SW1990 cells in each group were detected by CCK-8 analysis, Transwell analysis, TUNEL method and flow cytometry. Targetscan7.2 was used to predict the target protein of MiR-10a-5p, and the expression of related proteins was detected by Western blot analysis. The results showed that the expression of miR- 10a-5p in cancer tissues of patients with pancreatic cancer was significantly higher than that in adjacent tissues (P <0.05). The expression of miR-10a-5p in cancer cells increased significantly, which could promote the proliferation and invasion of SW1990 cells and inhibit apoptosis (P <0.05). Overexpression of miR-10a-5p can regulate the expression of BDNF and SEMA4C. miR-10a-5p can promote the occurrence and development of pancreatic cancer by regulating the BDNF / SEMA4C pathway, and may become a molecular target for the diagnosis and treatment of pancreatic cancer in the future.

MiR-205 suppresses tumor growth, invasion, and epithelial-mesenchymal transition by targeting SEMA4C in hepatocellular carcinoma.

Growing evidence indicates that microRNAs are involved in tumorigenesis and progression of hepatocellular carcinoma (HCC). However, the functional mechanisms of miR-205 in HCC remain largely unknown. Here, we demonstrate that miR-205 expression was significantly down-regulated in HCC tissues and cell lines and was correlated with metastatic pathologic features and shorter disease-free and overall survival. Overexpression of miR-205 dramatically inhibited HCC cell proliferation, apoptosis, migration, invasion, epithelial-mesenchymal transition (EMT) in vitro, and tumor growth in vivo. We subsequently identified semaphorin 4C (SEMA4C) as a novel target of miR-205. Furthermore, high expression levels of SEMA4C were frequently found in HCC tissues and were associated with poor prognosis. Ectopic expression of SEMA4C restored the suppressive effect of overexpressed miR-205 on migration, invasion, and EMT. Taken together, our findings provide new insight into the critical role of miR-205 in regulating tumor growth, invasion, and EMT of HCC, suggesting miR-205 may serve as a promising therapeutic target and novel prognostic indicator for patients with HCC.-Lu, J., Lin, Y., Li, F., Ye, H., Zhou, R., Jin, Y., Li, B., Xiong, X., Cheng, N. MiR-205 suppresses tumor growth, invasion and epithelial-mesenchymal transition by targeting SEMA4C in hepatocellular carcinoma.

MiR-138 inhibits cell proliferation and reverses epithelial-mesenchymal transition in non-small cell lung cancer cells by targeting GIT1 and SEMA4C.

Non-small-cell lung cancer (NSCLC) is one of the most common and lethal malignant tumours worldwide with a poor 5-year survival rate. Recent studies indicated that miRNAs have been involved in the tumorigenic driver pathways in NSCLC, but the relevant molecular mechanisms are not well-understood. In this study, we investigated the biological functions and molecular mechanisms of miR-138 in human NSCLC. The effects of miR-138 on the NSCLC cell growth and epithelial-mesenchymal transition (EMT) were first examined. Then the targeting connections of miR-138 with G-protein-coupled receptor kinase-interacting protein 1 (GIT1) and semaphorin 4C (SEMA4C) were confirmed by dual luciferase reporter assays. Finally, the effects of GIT1 and SEMA4C on the NSCLC cell growth and EMT were investigated respectively. We found that the ectopic expression of miR-138 resulted in a significant inhibition of NSCLC growth and reversion of EMT. GIT1 and SEMA4C were identified as two novel targets of miR-138. Furthermore, GIT1 and SEMA4C knockdown inhibited the cell growth and reversed EMT, just like the effects of miR-138 overexpression on NSCLC cells, whereas ectopic expression of GIT1 and SEMA4C partly rescued the suppressive effects of miR-138 in NSCLC cells. These data represent a crucial step towards the understanding of the novel roles and molecular mechanism of miR-138, GIT1 and SEMA4C in NSCLC progression, which may provide some new targets or prognostic biomarkers for NSCLC treatment, thus having implications in translational oncology.

miR-129-2-3p binds SEMA4C to regulate HCC development and inhibit the EMT.

BACKGROUND: Among primary liver cancers, HCC is the most prevalent. Small noncoding RNAs called miRNAs control the expression of downstream target genes to take part in a variety of physiological and pathological processes, including those related to cancer. METHODS: miR-129-2-3p and SEMA4C expression levels were assessed using RT-qPCR. The CCK-8, invasion, and wound healing assays were used to confirm the capacity of HCC cells for proliferation, invasion and migration respectively. Serum SEMA4C levels were detected via ELISA. The RIP and dual-luciferase reporter assays were used to confirm the existence of intergenic binding sites. Cell apoptosis assay and cell cycle assay were performed to detect the apoptosis rate and cycle distribution of cells, and WB was performed to detect the protein expression of SEMA4C, RhoA, ROCK1, E-cadherin, N-cadherin, and vimentin. Furthermore, cancer-inhibiting role of miR-129-2-3p were further confirmed by animal tests. RESULTS: miR-129-2-3p expression was reduced in HCC tissues and cells. Overexpression of miR-129-2-3p decreased the proliferation, invasion, migration, and EMT in HCC cells, whereas inhibition of miR-129-2-3p had the opposite effects. Our research also showed that SEMA4C was increased in HCC tissues, serum and cells, and that SEMA4C knockdown prevented HCC cell invasion, migration, proliferation, and EMT. Overexpression of SEMA4C reversed the inhibitory effect of miR-129-2-3p on HCC. CONCLUSIONS: Overall, we discovered that through binding to SEMA4C, miR-129-2-3p regulates HCC cell proliferation, invasion, migration, and EMT.

Semaphorin 4C promotes motility and immunosuppressive activity of cancer cells via CRMP3 and PD-L1.

Semaphorins (SEMAs) are membrane-bound or soluble proteins that participate in organ development and cancer progression, however, the detailed role of SEMAs in carcinogenesis is not fully elucidated yet. Our in silico analysis showed among the differentially expressed SEMAs in colon cancer tissues, patients with higher SEMA4C expression tumors had worse survival. The migration and invasion of the HCT116 and CT26 colon cancer cells were significantly suppressed by SEMA4C neutralizing antibody treatment; while enhanced by ectopic expression of SEMA4C. Subsequently, RNA sequencing study revealed microtubule polymerization- and nucleation-related genes are highly enriched in SEMA4C overexpression HCT116 cells. Western blotting showed the negative correlation between the levels of SEMA4C expression and tubulin acetylation. Mechanistic study showed SEMA4C interacted with and stabilized collapsin response mediator protein 3 (CRMP3), a novel deacetylase, to increase α-tubulin deacetylation and cell motility, which could be effectively attenuated after HDAC inhibitors treatment. We also found that a tumor-suppressive miRNA let-7b can target SEMA4C and act synergistically with SEMA4C neutralizing antibody to suppress the motility of colon cancer cells. In addition, blockade of SEMA4C could attenuate the expression of program death ligand 1 (PD-L1). Collectively, our results highlight that SEMA4C may promote colon cancer progression through modulating CRMP3-mediated tubulin deacetylation and PD-L1-mediated immunosuppression.

Long non-coding RNA TDRG1 promotes hypoxia-induced glycolysis by targeting the miR-214-5p/SEMA4C axis in cervical cancer cells.

Long non-coding RNA (lncRNA) has been demonstrated as vital regulator in human cancer. However, the precise role of lnc-TDRG1 in cervical cancer (CC) remains unclear, so this study was aimed to clarify the role and underlying molecular mechanism of lnc-TDRG1 in CC. The real-time quantitative polymerase chain reaction (RT-qPCR) was conducted to assess the expression levels of lnc-TDRG1, miR-214-5p and Semaphorin 4C (SEMA4C). Under hypoxia condition, the biological behaviors of CC cell, including invasion and glycolysis were determined by transwell assay and Glucose Assay Kit and Lactate Assay Kit, respectively. The Western blot assay was employed to test the expression level of SEMA4C and hexokinase 2 (HK2) expression. The interaction relationship between miR-214-5p and lnc-TDRG1 or SEMA4C was analyzed bioinformatics database and confirmed by dual-luciferase reporter assay, respectively. A xenograft experiment in nude mice was established to clarify the functional role of lnc-TDRG1 in vivo. We found Lnc-TDRG1 was highly expressed in CC tissues and cells and it was upregulated in response to hypoxia. Loss-of-functional experiment suggested that knockdown of lnc-TDRG1 impede invasion, hypoxia-induced glycolysis in vitro and tumor growth in vivo, which was abolished by knockdown of miR-214-5p or overexpression of SEMA4C. Moreover, we confirmed that miR-214-5p specifically bound to SEMA4C and negatively correlated with SEMA4C expression. Collectively, lnc-TDRG1 regulated SEMA4C expression by sponging miR-214-5p in CC. Collectively, mechanistically, lnc-TDRG1 could act as a sponge of miR-214-5p to regulate the expression of SEMA4C, and further regulate invasion and hypoxia-glycolysis in CC cells.

miR-642 serves as a tumor suppressor in hepatocellular carcinoma by regulating SEMA4C and p38 MAPK signaling pathway.

Hepatocellular carcinoma (HCC) is a malignant tumor with high incidence and high risk. Study of the role and mechanism of miRNAs are a hot spot of research providing new treatment ideas in malignant tumors. The effect of miR-642a on HCC progression and the underlying molecular mechanism were investigated. Expression of miR-642a and SEMA4C was measured by western blot analysis and RT-PCR. miR-642a expression was elevated while SEMA4C expression was attenuated in HCC tissues and cells. Results of luciferase reporter and western blot analyses show that miR-642a modulated SEMA4C expression by binding to its 3'UTR. Moreover, miR-642a negatively regulated SEMA4C expression. HCC cell migration and invasion was tested by Transwell assays. The findings revealed that the number of migrated and invaded cells were reduced by miR-642a mimic and raised by miR-642a inhibitor, indicating that miR-642a showed a suppression effect on HCC cell migration and invasion. Additionally, the migration and invasion of HCC cells were inhibited by SEMA4C siRNA, and SEMA4C reversed miR-642a effect on HCC migration and invasion. Furthermore, p38 MAPK signaling pathway was proven to be inhibited by miR-642a mimic, whereas facilitated by miR-642a inhibitor and SEMA4C siRNA could overturn the promotion effect of miR-642a inhibitor. Briefly, miR-642a targeted SEMA4C to repress HCC cell migration and invasion through p38 MAPK signaling pathway providing a new strategy for treatment of HCC patients.

LncRNA FOXD2-AS1 Regulates miR-25-3p/Sema4c Axis To Promote The Invasion And Migration Of Colorectal Cancer Cells.

PURPOSE: Although the roles of lncRNA FOXD2-AS1 have been investigated in many types of cancers including colorectal cancer (CRC), its functionality remains to be further investigated. Analysis of the TCGA data set revealed that FOXD2-AS1 was up-regulated in CRC tissues. This study aimed to analyze the function of FOXD2-AS1 in CRC. METHODS: FOXD2-AS1 expression was detected by qPCR. A 5-year follow-up study was performed to analyze the prognostic value of FOXD2-AS1 for CRC. Overexpression experiments were performed to analyze the interactions among FOXD2-AS1, miR-25-3p and Sema4C. Transwell assays were performed to analyze cell invasion and migration. RESULTS: In this study, we further confirmed the up-regulation of FOXD2-AS1 in CRC patients and showed that high FOXD2-AS1 level predicted poor survival. Bioinformatics analysis showed that miR-25-3p may bind FOXD2-AS1, while over-expression experiments showed no effects on each other's expression. Instead, FOXD2-AS1 over-expression led to the up-regulate Sema4C, which is a target of miR-25-3p. Transwell assay showed that FOXD2-AS1 and Sema4C over-expression led to the increased invasion and migration rates of CRC cells. MiR-25-3p plays the opposite role and attenuated the effects of FOXD2-AS1 and Sema4C over-expression. CONCLUSION: FOXD2-AS1 may regulate the miR-25-3p/Sema4C axis to promote the invasion and migration of CRC cells.

miR-125b targets Sema4C regulating invasion and migration of Non-Hodgkin′s lymphoma by STAT3 signaling pathway involved / 中国免疫学杂志

Objective:To investigate the effect of miR-125b targeting Sema4C on STAT3 signaling pathway on invasion and metastasis of non-Hodgkin′s lymphoma.Methods: Expression of miR-125b in non-Hodgkin′s lymphoma tissues and cell lines was detected by QT-PCR.Expression of Sema4C in normal lymphocyte tissues and non-Hodgkin′s lymphoma tissues was detected by immunohistochemistry.Dual luciferase effect of miR-125b on the transcriptional activity of Sema4C was examined by the reporter gene system.Transwell invasion assay was used to detect the expression of miR-125b in the non-Hodgkin′s lymphoma cell line NK-92.Scratch test Western blot was used to detect the expression of Sema4C.Western blot was used to detect the protein expression of STAT3 signal pathway after silencing Sema4C.The protein expression of Sema4C was detected by Western blot.Results: Expression of miR-125b was significantly lower in non-Hodgkin′s lymphoma tissues than that in normal tissues[(0.48±0.05)% vs (1.59±0.38)%,P<0.05].Sema4C was highly expressed in non-Hodgkin′s lymphoma[(326.25±7.75)% vs (58.75±5.76)%].After overexpression of miR-125b,non-Hodgkin′s lymphoma cells expression level of Sema4C was down-regulated after silencing Sema4C[(326.25±7.75)% vs (58.75±5.76)%],and the expression of JAK and STAT3 protein were down-regulated after miR-125b overexpression[(85.26±6.94)% vs (12.61±4.32)%,P<0.05].The dual luciferase reporter gene system showed that miR-125b could directly regulate the transcriptional activity of Sema4C.Conclusion: miR-125b can regulate the invasion and migration of non-Hodgkin′s lymphoma cells by targets the expression of Sema4C.

Construction of Recombinant Adenovirus Expression Vector of Human Sema4C Gene and Its Expression in Mouse Myoblasts Cell Line C2C12 / 中国生物工程杂志

To generate recombinant adenovirus expression vector of human Sema4C gene and observe its expression in mouse myoblasts cell line C2C12 for ensuring easy access to investigate the role of Sema4C gene during myogenesis. The recombinant plasmid was packaged and amplified after being transfected in HEK293 cells through Lipofectamine. After infecting C2C12 myoblasts with recombinant adenovirus vector, the adenoviral infection efficiency was determined by confocal microscope which showed that the expression of green fluorescence could be detected at 12h and then reached peak at 24h after recombinant adenovirus infection. The infection efficiency was almost 100% confirmed by FACS examination. Detection of WB indicated that the expression of Sema4C in C2C12 of recombinant adenoviral infection group was significantly higher than that of the control group (P