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Background An increasing number of studies have demonstrated that long non-coding RNAs (lncRNAs) play pivotal roles in cancer onset and development. LncRNA AFAP1-AS1 has been validated to be abnormally upregulated and play oncogenic roles in various malignant tumors. The biological role and mechanism of AFAP1-AS1 in OS (osteosarcoma) remains unclear. Methods Quantitative reverse transcription PCR (qRT-PCR) is applied to examine AFAP1-AS1 expression in OS tissues and OS cell lines. The function of AFAP1-AS1 in OS cells is investigated via in-vitro and in-vivo assays. Western bolt and rescue experiments are applied to detect the expression changes of key molecules including epithelial-mesenchymal transition markers and identify the underlying molecular mechanism. RNA immunoprecipitation is performed to reveal the interaction between AFAP1-AS1 and RhoC. Results AFAP1-AS1 expression is upregulated in human OS tissues and cell lines. AFAP1-AS1 knockdown induces OS cell apoptosis and cell cycle G0/G1 arrest, suppresses OS cells growth, migration, invasion, vasculogenic mimicry formation and epithelial-mesenchymal transition (EMT), and affects actin filament integrity. AFAP1-AS1 knockdown suppresses OS tumor formation and growth in nude mice. AFAP1-AS1 knockdown elicits a signaling inhibition including decreased levels of RhoC, ROCK1, p38MAPK and Twist1. Moreover, AFAP1-AS1 interacts with RhoC. Overexpression of RhoC can partly reverse AFAP1-AS1 downregulation-induced cell EMT inhibition. Conclusions AFAP1-AS1 is overexpressed in osteosarcoma and plays an oncogenic role in osteosarcoma through RhoC/ROCK1/p38MAPK/Twist1 signaling pathway, in which RhoC acts as the interaction target of AFAP1-AS1. Our findings indicated a novel molecular mechanism underlying the tumorigenesis and progression of osteosarcoma. AFAP1-AS1 could serve as a promising therapeutic target in OS treatment.

This study aimed to explore the role of circular RNAs (circRNAs) in M2 macrophage (M2M)-derived small extracellular vesicles (SEVs) in myocardial fibrosis development. : The regulatory role of M2M-derived extracellular vesicles (EVs) was evaluated in a mouse model of acute myocardial infarction. Immunofluorescence, quantitative real-time PCR (RT-qPCR), nanoparticle tracking analysis, Western blot analysis and electron microscopy were used to identify macrophages, large extracellular vesicles (LEVs) and SEVs. The circRNA expression profiles of M0 macrophages (M0Ms) and M2Ms were determined by microarray analysis. Bioinformatic analysis, cell coculture and cell proliferation assays were performed to investigate the expression, function, and regulatory mechanisms of circUbe3a . qPCR, RNA immunoprecipitation (RIP), dual-luciferase reporter assays, RNA fluorescence hybridization (RNA-FISH), Western blot analysis and a series of rescue experiments were used to verify the correlation among circUbe3a, miR-138-5p and RhoC. CircUbe3a from M2M-derived SEVs triggered functional changes in cardiac fibroblasts (CFs). CircUbe3a was synthesized and loaded into SEVs during increased M2M infiltration after myocardial infarction. The fusion of the released SEVs with the plasma membrane likely caused the release of circUbe3a into the cytosol of CFs. Silencing or overexpressing circUbe3a altered CF proliferation, migration, and phenotypic transformation . We confirmed that circUbe3a plays a crucial role in enhancing functional changes in CFs by sponging miR-138-5p and then translationally repressing RhoC . , the addition of M2M-derived SEVs or overexpression of circUbe3a significantly exacerbated myocardial fibrosis after acute myocardial infarction, and these effects were partially abolished by circUbe3a-specific shRNA. Our findings suggest that M2M-derived circUbe3a-containing SEVs promote the proliferation, migration, and phenotypic transformation of CFs by directly targeting the miR-138-5p/RhoC axis, which may also exacerbate myocardial fibrosis after acute myocardial infarction.

Background Radioresistance remains a challenge to the successful treatment of various tumors. Intrinsic factors like alterations in signaling pathways regulate response to radiation. RhoC, which has been shown to modulate several tumor phenotypes has been investigated in this report for its role in radioresistance. In vitro and clinical sample-based studies have been performed to understand its contribution to radiation response in cervical cancer and this is the first report to establish the role of RhoC and its effector ROCK2 in cervical cancer radiation response. Methods Biochemical, transcriptomic and immunological approaches including flow cytometry and immunofluorescence were used to understand the role of RhoC and ROCK2. RhoC variants, siRNA and chemical inhibitors were used to alter the function of RhoC and ROCK2. Transcriptomic profiling was performed to understand the gene expression pattern of the cells. Live sorting using an intracellular antigen has been developed to isolate the cells for transcriptomic studies. Results Enhanced expression of RhoC conferred radioprotection on the tumor cells while inhibition of RhoC resulted in sensitization of cells to radiation. The RhoC overexpressing cells had a better DNA repair machinery as observed using transcriptomic analysis. Similarly, overexpression of ROCK2, protected tumor cells against radiation while its inhibition increased radiosensitivity in vitro. Further investigations revealed that ROCK2 inhibition abolished the radioresistance phenotype, conferred by RhoC on SiHa cells, confirming that it is a downstream effector of RhoC in this context. Additionally, transcriptional analysis of the live sorted ROCK2 high and ROCK2 low expressing SiHa cells revealed an upregulation of the DNA repair pathway proteins. Consequently, inhibition of ROCK2 resulted in reduced expression of pH2Ax and MRN complex proteins, critical to repair of double strand breaks. Clinical sample-based studies also demonstrated that ROCK2 inhibition sensitizes tumor cells to irradiation. Conclusions Our data primarily indicates that RhoC and ROCK2 signaling is important for the radioresistance phenotype in cervical cancer tumor cells and is regulated via association of ROCK2 with the proteins of DNA repair pathway involving pH2Ax, MRE11 and RAD50 proteins, partly offering insights into the mechanism of radioresistance in tumor cells. These findings highlight RhoC-ROCK2 signaling involvement in DNA repair and urge the need for development of these molecules as targets to alleviate the non-responsiveness of cervical cancer tumor cells to irradiation treatment.

Rho GTPases are highly conserved proteins that play critical roles in many cellular processes including actin dynamics, vesicular trafficking, gene transcription, cell-cycle progression, and cell adhesion. The main mode of regulation of Rho GTPases is through guanine nucleotide binding (cycling between an active GTP-bound form and an inactive GDP-bound form), but transcriptional, post-transcriptional, and post-translational modes of Rho regulation have also been described. In the present review, we summarize recent progress on the mechanisms that control the expression of the three members of the Rho-like subfamily (RhoA, RhoB, and RhoC) at the level of gene transcription as well as their post-transcriptional regulation by microRNAs. We also discuss the progress made in deciphering the mechanisms of cross-talk between Rho proteins and the transforming growth factor β signaling pathway and their implications for the pathogenesis of human diseases such as cancer metastasis and fibrosis.

Background/Aim: Aggressive breast cancer (BC) cells show high expression of Rho GTPase activating protein 29 (ARHGAP29), a negative regulator of RhoA. In breast cancer cells in which mesenchymal transformation was induced, ARHGAP29 was the only one of 32 GTPase-activating enzymes whose expression increased significantly. Therefore, we investigated whether there is a correlation between expression of ARHGAP29 and tumor progression in BC. Since tamoxifen-resistant BC cells exhibit increased mesenchymal properties and invasiveness, we additionally investigated the relationship between ARHGAP29 and increased invasion rate in tamoxifen resistance. The question arises as to whether ARHGAP29 is a suitable prognostic marker for the progression of BC. Materials and Methods: Tissue microarrays were used to investigate expression of ARHGAP29 in BC and adjacent normal breast tissues. Knockdown experiments using siRNA were performed to investigate the influence of ARHGAP29 and the possible downstream actors RhoC and pAKT1 on invasive growth of tamoxifen-resistant BC spheroids in vitro. Results: Expression of ARHGAP29 was frequently increased in BC tissues compared to adjacent normal breast tissues. In addition, there was evidence of a correlation between high ARHGAP29 expression and advanced clinical tumor stage. Tamoxifen-resistant BC cells show a significantly higher expression of ARHGAP29 compared to their parental wild-type cells. After knockdown of ARHGAP29 in tamoxifen-resistant BC cells, expression of RhoC was significantly reduced. Further, expression of pAKT1 decreased significantly. Invasive growth of three-dimensional tamoxifen-resistant BC spheroids was reduced after knockdown of ARHGAP29. This could be partially reversed by AKT1 activator SC79. Conclusion: Expression of ARHGAP29 correlates with the clinical tumor parameters of BC patients. In addition, ARHGAP29 is involved in increased invasiveness of tamoxifen-resistant BC cells. ARHGAP29 alone or in combination with its downstream partners RhoC and pAKT1 could be suitable prognostic markers for BC progression.

Tumor heterogeneity results in differential response to therapy due to the existence of plastic tumor cells, called cancer stem cells (CSCs), which exhibit the property of resistance to therapy, invasion and metastasis. These cells have a distinct, signaling network active at every stage of progression. It is difficult to envisage that the CSCs will have a unique set of signaling pathways regulating every stage of disease progression. Rather, it would be easier to believe that a single pivotal pathway having significant contribution at every stage, which can further turn on a battery of signaling mechanisms specific to that stage, would be instrumental in regulating the signaling network, enabling easy transition from one state to another. In this context, we discuss the role of RhoC which has contributed to several phenotypes during tumor progression. RhoC (Ras homolog gene family member C) has been widely reported to regulate actin organization. It has been shown to impact the motility of cancer cells, resultantly affecting invasion and metastasis, and has contributed to carcinoma progression of the breast, pancreas, lung, ovaries and cervix, among several others. The most interesting finding has been its indispensable role in metastasis. Also, it has the ability to modulate various other phenotypes like angiogenesis, motility, invasion, metastasis, and anoikis resistance. These observations suggest that RhoC imparts the plasticity required by tumor cells to exhibit such diverse functions based on microenvironmental cues. This was further confirmed by recent reports which show that it regulates cancer stem cells in breast, ovary and head and neck cancers. Studies also suggest that the inhibition of RhoC results in abolition of advanced tumor phenotypes. Our review throws light on how RhoC, which is capable of modulating various phenotypes may be the apt core signaling candidate regulating disease progression. Additionally, mice studies show that RhoC is not essential for embryogenesis, giving scope for its development as a possible therapeutic target. This review thus stresses on the need to understand the protein and its functioning in greater detail to enable its development as a stem cell marker and a possible therapeutic target .

HOXD10, a key regulator of cell-differentiated phenotype maintainence, has been demonstrated to be involved in the tumorigenesis of many human malignacies. However, the status of HOXD10 expression and its biological function in cholangiocellular carcinoma (CCC) remain to be clarified. In the present study, we investigated the clinical significance and biological functions of HOXD10 in CCC and found that the expression of HOXD10 and its downstream effector RHOC was significantly different in well-differentiated CCC tissues compared with poorly-differentiated lesions. We also observed a significant correlation between low HOXD10 and high RHOC expression levels and worse prognosis. The stable overexpression of HOXD10 by lentivirus vector significantly inhibited cell invasion partly by downregulating the expression of MMP2 and MMP9, and significantly increased early apoptosis in CCC cell lines and induced G1 phase cell cycle arrest, contributing to the inhibition of cell proliferation in vitro. Additionally, we demonstrated that the inactivation of the RHOC/AKT/MAPK pathway was involved in the tumor-suppressive functions of HOXD10 in CCC. These results suggested that HOXD10 may be a putative suppressor gene and can act as a prognostic marker and potentially a novel therapeutic target for CCC.

Copy number gains in genes coding for Rho activating exchange factors as well as losses affecting genes coding for RhoGAP proteins are common in breast cancer (BC), suggesting that elevated Rho signaling may play an important role. Extra copies and overexpression of RHOC also occur, although a role for RhoC overexpression in driving tumor formation has not been assessed in vivo. To this end, we report on the development of a Rosa26 (R26)-targeted Cre- conditional RhoC overexpression mouse (R26 RhoC ). This mouse was crossed to two models for ERBB2/NEU + breast cancer: one based on expression of an oncogenic ErbB2/Neu cDNA downstream of the endogenous ErbB2 promoter ( FloxNeoNeu NT ), the other, a metastatic model that is based on high-level expression from MMTV regulatory elements ( NIC ). RhoC overexpression dramatically enhanced mammary tumor formation in FloxNeoNeu NT mice but showed a more subtle effect in the NIC line, which forms multiple mammary tumors after a very short latency. RhoC overexpression also enhanced mammary tumor formation in an activated Pik3ca model for breast cancer ( Pik3ca H1047R ) . The transforming effect of RhoC was associated with epithelial/mesenchymal transition (EMT) in ErbB2/Neu NT and Pik3ca H1047R systems. Thus, our study reveals the importance of elevated wildtype Rho protein expression as a driver of breast tumor formation and highlights the significance of Copy Number Abberations that affect Rho signalling.

The study was undertaken to determine the mechanism of miR-372-3p activating hepatic stellate cell (HSC). Transforming growth factor-β1 (TGF-β1) induced LX-2 cells were transfected with miR-372-3p mimics and/or RhoC overexpression vector (oe-RhoC), after which the miR-372–3 and RhoC expressions were detected and the biological functions of transfected cells were assessed. The relation between miR-372-3p and RhoC predicted online was validated using the dual-luciferase assay. Protein level of Collagen I (COL I), α-smooth muscle actin (α-SMA), and key proteins in the RhoC/ROCK pathway were determined using western blot. Activated LX-2 cells had decreased miR-372-3p and increased RhoC expression. Overexpression of miR-372-3p led to inhibited LX-2 cell proliferation, accelerated apoptosis, and decreased protein level of COL I and α-SMA, while such an expression pattern can be partially reversed by RhoC overexpression. miR-372-3p can bind and target RhoC expression. miR-372-3p inhibited RhoC expression to block the activation of the Rho/ROCK pathway and thus mediate LX-2 cell proliferation and apoptosis. miR-372-3p mediated RhoC/ROCK pathway to inhibit HSC activation.

Metastasis of bladder cancer is a complex process and has been associated with poor clinical outcomes. However, the mechanisms of bladder cancer metastasis remain largely unknown. The present study found that the long noncoding RNA lnc00892 was significantly downregulated in bladder cancer tissues, with low lnc00892 expression associated with poor prognosis of bladder cancer patients. Lnc00892 significantly inhibited the migration, invasion, and metastasis of bladder cancer cells in vitro and in vivo. In-depth analysis showed that RhoA/C acted downstream of lnc00892 to inhibit bladder cancer metastasis. Mechanistically, lnc00892 reduces nucleolin gene transcription by competitively binding the promoter of nucleolin with c-Jun, thereby inhibiting nucleolin-mediated stabilization of RhoA/RhoC mRNA. Taken together, these findings provide novel insights into understanding the mechanisms of bladder cancer metastasis and suggest that lnc00892 can serve as a potential therapeutic target in patients with invasive bladder cancer.