NF-?B interaction long non-coding RNA inhibits migration, invasion and epithelial-mesenchymal transition of cervical cancer cells through inhibiting NF-?B signaling pathways.
ABSTRACT: The long non-coding RNA (lncRNA) NF-?B interaction lncRNA (NKILA) has been found to exert tumor suppressive effects in numerous types of carcinoma; however, the relationship between NKILA and cervical cancer (CC) remains largely unclear. The present study aimed to investigate the effects of NKILA on the proliferation and metastasis of CC cell lines, in addition to the related molecular mechanisms. Reverse transcription-quantitative PCR was used to detect the expression levels of NKILA in cancer tissues and cell lines. The constructed overexpression vector, pcDNA3.1NKILA, and its corresponding negative control sequence were transfected into CaSki cells and short hairpin RNA targeting NKILA and the corresponding negative control sequence were transfected into C-33A cells. Subsequently, the proliferative, migratory and invasive ability, as well as the process of epithelial-mesenchymal transition (EMT) of C-33 A and CaSki cells were analyzed by performing Cell Counting Kit-8, wound healing, Matrigel invasion and western blot assays, respectively. The expression levels of proteins were detected using western blot analysis. The expression levels of NKILA were decreased in CC tissues and CC cell lines (SiHa, C-33A, CaSki and HeLa) and the downregulation of NKILA expression using shRNA was observed to significantly increase the proliferation of CC cells. Conversely, the upregulation of NKILA inhibited the proliferation of CC cells, in addition to significantly inhibiting the migration and invasion of CaSki cells, whereas the knockdown of NKILA promoted the invasion of C-33A cells. Thus, it was hypothesized that NKILA may inhibit the migration and invasion of CC cells via regulation of EMT processes, which was reflected by the expression of ZO-1, E-cadherin, N-cadherin and Vimentin. Furthermore, the overexpression of NKILA significantly inhibited the activation of NF-?B in CaSki cells, whereas the knockdown of NKILA expression promoted the degradation of inhibitory protein-?B and promoted the transfer of p65 into the nucleus in C-33A cells. In conclusion, the results from the present study suggested that NKILA may be involved in the inhibition of migration and invasion in CC cells through regulating EMT processes, which may be related to its inhibition of NF-?B activation.
Project description:Background:Cervical cancer (CC) is one of the most common female malignancies over the world. Microtubule-associated protein 7 (MAP7) belongs to the family of microtubule-associated proteins (MAPs) which involve in microtubule dynamics and are critical in several important cellular and intracellular activities. This study aimed to investigate the expression and potential role of MAP7 in CC. Methods:The expression level of MAP7 in CC tissues and normal tissues were analyzed using the data obtained from The cancer genomes atlas (TCGA) and genotype-tissue expression (GTEx) databases. The prognostic value of MAP7 in patients with CC was analyzed by Kaplan-Meier analysis, Univariate and Multivariate analyses. Moreover, the influences of MAP7 expression alteration on the viability and motility of Caski, HeLa and C-33A cells was measured by CCK8 assay, colony formation assay, scratch assay, and transwell migration and invasion assays. Flow cytometry was conducted to determine cell apoptosis. Western blot was performed to evaluate the impact of MAP7 on the expression of apoptotic-related proteins as well as mitogen-activated protein kinase (MAPK) signaling pathway-related proteins. In vivo tumorigenicity assay was performed to explore the influence of MAP7 on tumor growth. Results:Up-regulation of MAP7 was observed in CC tissues and high MAP7 expression was positively correlated with worse prognosis. Multivariate analyses suggested that MAP7 expression can be served as an independent predictor for overall survival of patients with CC. Knockdown of MAP7 markedly suppressed Caski and HeLa cell viability, migration and invasion while notably induced cell apoptosis. Furthermore, depletion of MAP7 in Caski and HeLa cells elevated the expression levels of Active-caspase 3 and Bax, but declined the level of Bcl-2. Whilst, overexpression of MAP7 in C-33A cells presented the opposite outcomes. Additionally, knockdown of MAP7 significantly decreased the phosphorylation of mitogen-activated protein kinase kinase (MEK) and extracellular signal-regulated kinase (ERK) in Caski and HeLa cells, and overexpression of MAP7 increased their phosphorylation in C-33A cells, indicating that MAP7 may regulate the MAPK signaling pathway in CC cells. In vivo assays revealed that knockdown of MAP7 remarkably repressed the growth of CC tumors. Conclusion:The results of the present study suggest that MAP7 functions as a promoter during the occurrence and progression of CC, and that MAP7 may serve as a promising therapeutic target in CC.
Project description:Numerous studies have shown that long non-coding RNAs (lncRNAs) play key roles during multiple cancer processes, such as cell proliferation, apoptosis, migration and invasion. The previous studies found that NKILA interacted with and suppressed the nuclear translocation of NF-KappaB, which influenced metastasis and prognosis in breast cancer. However the clinical significance and biological role of NKILA in non-small cell lung cancer (NSCLC) remains unknown.We examined expression levels of NKILA in 106 pairs of NSCLC tissues and cell lines. The expression level of NKILA after TGF-?1 stimulation also was examined by qRT-PCR and validated by Chromatin immunoprecipitation (ChIP). Gain-of-function and loss-of-function assays were performed to examine the effect of NKILA on proliferation, migration and invasion of NSCLC cells. RNA immunoprecipitation (RIP), western blot and rescue experiments were carried out to reveal the interrelation between NKILA, NF-?B and EMT signal pathway.The expression of NKILA was down-regulated in NSCLC cancer tissues compared with matched adjacent noncancerous tissues, and lower NKILA expression in tumor tissues were significantly correlated with lymph node metastasis and advanced TNM stage. We found that the expression of NKILA was mainly regulated by classical TGF-? signal pathway in NSCLC cells rather than NF-?B pathway reported in breast cancer. Gain and loss of function assays found that NKILA inhibited migration, invasion and viability of NSCLC cells. Mechanistic study showed that NKILA attenuated Snail expression via inhibiting the phosphorylation of I?B? and NF-?B activation, subsequently suppressed the expression of markers of epithelial-mesenchymal transition process.The present study found that the expression of NKILA was downregulated in tumor tissues of NSCLC, which improved the metastasis of NSCLC patients. In vitro studies further clarified that the expression of NKILA was regulated through classical TGF-? signal pathway, which subsequently inhibited migration and invasion of NSCLC cells through interfering NF-?B/Snail signal pathway in NSCLC cells.
Project description:The metastasis of hepatocellular carcinoma (HCC) is one of the major obstacles hindering its therapeutic efficacy, leading to low surgical resection rate, high mortality and poor prognosis. Accumulating evidence has shown that both long noncoding RNA (lncRNA) and NF-?B play vital roles in the regulation of cancer metastasis. However, the clinical significance and biological function of NKILA (NF-?B interacting lncRNA) and its interaction with NF-?B in HCC remain unknown. In this study, we demonstrated that NKILA was down-regulated in HCC tissues and cell lines, and decreased NKILA expression was significantly associated with larger tumor size and positive vascular invasion in HCC patients. NKILA reduction was an independent risk factor of HCC patients' poor prognosis, and the 5-year overall survival (OS) rates of patients with low and high NKILA expression were 15.6% and 60.0%, respectively. Moreover, NKILA inhibits migration and invasion of HCC cells both in vitro and in vivo. Mechanistically, NKILA prevents Slug/epithelial to mesenchymal transition (EMT) pathway via suppressing phosphorylation of I?B?, p65 nuclear translocation and NF-?B activation. In conclusion, these results indicate that NKILA might serve as an effective prognostic biomarker and a promising therapeutic target against HCC metastasis.
Project description:OBJECTIVE:Emerging studies have identified that long non-coding RNAs (lncRNAs) play critical roles in cancer development. This study aims to explore the mechanism of NF-KappaB (NF-?B) interacting lncRNA (NKILA) in the pathological process of oral squamous cell carcinoma (OSCC). METHODS:NKILA expression in OSCC tissues, paracancerous tissues, and normal human oral keratinocytes and OSCC cell lines was detected using RT-qPCR. KB cells were selected for the follow-up experiments. The role of NKILA in cell proliferation, migration, invasion, and NF-?B signaling pathway was identified using the gain- and loss-of function of NKILA in OSCC cells. Additionally, the role of NKILA in vitro was determined by inducing xenograft tumors in nude mice. RESULTS:NKILA was poorly expressed in OSCC tissues and cells. Cell proliferation, invasion and migration, tumor volume and weight were significantly suppressed in cells with overexpressed NKILA, while silencing NKILA led to opposite trends. Moreover, the protein levels of p-I?B? and nuclear-p65 were markedly decreased, while the levels of I?B? and cytoplasm-p65 were enhanced in cells with overexpressed NKILA. CONCLUSION:This study provided evidence that NKILA could reduce proliferation, invasion and migration of OSCC cells through inhibiting the NF-?B signaling pathway. The findings may offer new insights for OSCC prevention and treatment.
Project description:The viral oncoprotein E7 from the "high-risk" Human Papillomavirus 16 (HPV16) strain is able, when expressed in human keratinocytes, to physically interact with the actin severing protein gelsolin (GSN). In a previous work it has been suggested that this protein-protein interaction can hinder GSN severing function, thus leading to actin network remodeling. In the present work we investigated the possible implications of this molecular interaction in cancer cell metastatic potential by analyzing two different human CC cell lines characterized by low or high expression levels of HPV16 DNA (SiHa and CaSki, respectively). In addition, a HPV-null CC cell line (C-33A), transfected in order to express the HPV16 E7 oncoprotein as well as two different deletion mutants, was also analyzed. We found that HPV16 E7 expression level was directly related with cervical cancer migration and invasion capabilities and that these HPV16 E7-related features were associated with Epithelial to Mesenchymal Transition (EMT) processes. These effects appeared as strictly attributable to the physical interaction of HPV16 E7 with GSN, since HPV16 E7 deletion mutants unable to bind to GSN were also unable to modify microfilament assembly dynamics and, therefore, cell movements and invasiveness. Altogether, these data profile the importance of the physical interaction between HPV16 E7 and GSN in the acquisition of the metastatic phenotype by CC cells, underscoring the role of HPV16 intracellular load as a risk factor in cancer.
Project description:STC1 is a glycoprotein hormone involved in calcium/phosphate (Pi) homeostasis. There is mounting evidence that STC1 is tightly associated with the development of cancer. But the function of STC1 in cancer is not fully understood. Here, we found that STC1 is down-regulated in Clinical tissues of cervical cancer compared to the adjacent normal cervical tissues (15 cases). Subsequently, the expression of STC1 was knocked down by RNA interference in cervical cancer CaSki cells and the low expression promoted cell growth, migration and invasion. We also found that STC1 overexpression inhibited cell proliferation and invasion of cervical cancer cells. Moreover, STC1 overexpression sensitized CaSki cells to drugs. Further, we showed that NF-?B p65 protein directly bound to STC1 promoter and activated the expression of STC1 in cervical cancer cells. Thus, these results provided evidence that STC1 inhibited cell proliferation and invasion through NF-?B p65 activation in cervical cancer.
Project description:Understanding the molecular mechanism by which epithelial mesenchymal transition (EMT)-mediated cancer metastasis and how microRNA (miRNA) regulates lung cancer progression via Twist1-activated EMT may provide potential therapeutic targets for cancer therapy. Here we found that miR-33a, an intronic miRNA located within the sterol regulatory element-binding protein 2 (SREBP-2) gene, is expressed at low levels in metastatic non-small cell lung cancer (NSCLC) cells and is inversely correlated with Twist1 expression. Conversely, miR-33a knockdown induces EMT and miR-33a overexpression blocks EMT by regulating of Twist1 expression in NSCLC cells. Bioinformatical prediction and luciferase reporter assay confirm that Twist1 is a direct target of miR-33a. Additionally, Twist1 knockdown blocks EMT-related metastasis and forced expression of miR-33a inhibits lung cancer metastasis in a xenograft animal model. Clinically, miR-33a is found to be at low levels in NSCLC patients and down-regulation of miR-33a predicts a poor prognosis. These findings suggest that miR-33a targets Twist1 and inhibits invasion and metastasis in NSCLC. Thus, miR-33a might be a potential prognostic marker and of therapeutic relevance for NSCLC metastasis intervention.
Project description:BACKGROUND:MicroRNAs (miRNAs) and Twist1-induced epithelial-mesenchymal transition (EMT) in cancer cell dissemination are well established, but the involvement of long noncoding RNAs (lncRNAs) in Twist1-mediated signaling remains largely unknown. METHODS:RT-qPCR and western blotting were conducted to detect the expression levels of lncRNA JPX and Twist1 in lung cancer cell lines and tissues. The impact of JPX on Twist1 expression, cell growth, invasion, apoptosis, and in vivo tumor growth were investigated in lung cancer cells by western blotting, rescue experiments, colony formation assay, flow cytometry, and xenograft animal experiment. RESULTS:We observed that lncRNA JPX was upregulated in lung cancer metastatic tissues and was closely correlated with tumor size and an advanced stage. Functionally, JPX promoted lung cancer cell proliferation in vitro and facilitated lung tumor growth in vivo. Additionally, JPX upregulated Twist1 by competitively sponging miR-33a-5p and subsequently induced EMT and lung cancer cell invasion. Interestingly, JPX and Twist1 were coordinately upregulated in lung cancer tissues and cells. Mechanically, the JPX/miR-33a-5p/Twist1 axis participated in EMT progression by activating Wnt/?-catenin signaling. CONCLUSIONS:These findings suggest that lncRNA JPX, a mediator of Twist1 signaling, could predispose lung cancer cells to metastasis and may serve as a potential target for targeted therapy.
Project description:Background: The reciprocal repressive loop between ZEB1 and miRNAs has been extensively reported to play an important role in tumor progression and metastasis of various human tumor types. The aim of this study was to elucidate the role and the underlying mechanism of the double-negative feedback loop between ZEB1and miR-33a-5p in bone metastasis of prostate cancer (PCa). Methods: miR-33a-5p expression was examined in 40 bone metastatic and 165 non-bone metastatic PCa tissues by real-time PCR. Statistical analysis was performed to evaluate the clinical correlation between miR-33a-5p expression and clinicopathological characteristics, and overall and bone metastasis-free survival in PCa patients. The biological roles of miR-33a-5p in bone metastasis of PCa were investigated both by EMT and the Transwell assay in vitro, and by a mouse model of left cardiac ventricle inoculation in vivo. siRNA library, real-time PCR and chromatin immunoprecipitation (ChIP) were used to identify the underlying mechanism responsible for the decreased expression of miR-33a-5p in PCa. Bioinformatics analysis, Western blotting and luciferase reporter analysis were employed to examine the relationship between miR-33a-5p and its potential targets. Clinical correlation of miR-33a-5p with its targets was examined in human PCa tissues and primary PCa cells. Results: miR-33a-5p expression was downregulated in PCa tissues with bone metastasis and bone-derived cells, and low expression of miR-33a-5p strongly and positively correlated with advanced clinicopathological characteristics, and shorter overall and bone metastasis-free survival in PCa patients. Upregulating miR-33a-5p inhibited, while silencing miR-33a-5p promoted EMT, invasion and migration of PCa cells. Importantly, upregulating miR-33a-5p significantly repressed bone metastasis of PC-3 cells in vivo. Our results further revealed that recurrent ZEB1 upregulation induced by copy number gains transcriptionally inhibited miR-33a-5p expression, contributing to the reduced expression of miR-33a-5p in bone metastatic PCa tissues. In turn, miR-33a-5p formed a double negative feedback loop with ZEB1 in target-independent manner, which was dependent on TGF-β signaling. Finally, the clinical negative correlations of miR-33a-5p with ZEB1 expression and TGF-β signaling activity were demonstrated in PCa tissues and primary PCa cells. Conclusion: Our findings elucidated that copy number gains of ZEB1-triggered a TGF-β signaling-dependent miR-33a-5p-mediated negative feedback loop was highly relevant to the bone metastasis of PCa.
Project description:The epithelial-mesenchymal transition (EMT) induced by EGF promotes cervical cancer progression; however, the mechanisms underlying the EGF-induced EMT remain unclear. In this study, we reported that miR155 overexpression suppressed EGF-induced EMT, decreased migration/invasion capacities, inhibited cell proliferation and increased the chemo-sensitivity to DDP in human Caski cervical cancer cells. Further, the overexpression of miR155 increased TP53 expression but reduced SMAD2, and CCND1 expression levels. These data suggest that miR155 negatively regulates EGF-induced EMT. We conclude that miR155 does not act as an oncogene but as a tumour suppressor in Caski cells.