Cervical Squamous Cell Carcinoma Cells Research Articles

N6-methyladenosine methylation on FSCN1 mediated by METTL14/IGF2BP3 contributes to human papillomavirus type 16-infected cervical squamous cell carcinoma.

Human papillomavirus (HPV) infection has been reported to be associated with N6-methyladenosine (m6A) modification in cancers. However, the underlying mechanism by which m6A methylation participates in HPV-related cervical squamous cell carcinoma (CSCC) remains largely unclear. In this study, we observed that m6A regulators methyltransferase like protein (METTL14) and insulin like growth factor 2 mRNA binding protein 3 (IGF2BP3) were upregulated in HPV-positive CSCC tissues and cell lines, and their high expression predicted poor prognosis for HPV-infected CSCC patients. Cellular functional experiments verified that HPV16 oncogenes E6/E7 upregulated the expression of METTL14 and IGF2BP3 to promote cell proliferation and epithelial mesenchymal transition of CSCC cells. Next, we found that E6/E7 stabilized fascin actin-bundling protein 1 (FSCN1) mRNA and elevated FSCN1 expression in CSCC cells through upregulating METTL14/IGF2BP3-mediated m6A modification, and FSCN1 expression was also validated to be positively associated with worse outcomes of HPV-positive CSCC patients. Finally, HPV16-positive CSCC cell lines SiHa and CaSki were transfected with knockdown vector for E6/E7 or METTL14/IGF2BP3 and overexpressing vector for FSCN1, and functional verification experiments were performed through using MTT assay, flow cytometry, wound healing assay and tumour formation assay. Results indicated that knockdown of E6/E7 or METTL14/IGF2BP3 suppressed cell proliferation, migration and tumorigenesis, and accelerated cell apoptosis of HPV-positive CSCC cells. Their tumour-suppressive effects were abolished through overexpressing FSCN1. Overall, HPV E6/E7 advanced CSCC development through upregulating METTL14/IGF2BP3-mediated FSCN1 m6A modification.

Cyanidin-3-O-Glucoside Combined with PGC1α Inhibitor Promotes Cancer Cell Death by Inducing Excessive Levels of ROS in Cervical Squamous Cell Carcinoma

Cervical cancer is a global public health problem, particularly in the low-income and middle-income countries. Natural products, such as cyanidin and its derivative cyanidin-3-O-glucoside (C3G), are considered safe and nontoxic food additives, potent therapeutic options for cancer. The present study aims to evaluate the beneficial effects of C3G on cervical squamous cell carcinoma (CSCC) in vitro and explore more potential therapeutic strategies for CSCC. C3G significantly inhibited the cell growth of CSCC cells and induced cancer cell apoptosis in a dose-dependent manner. Meanwhile, C3G promoted cellular reactive oxygen species (ROS) accumulation and decreased mitochondrial membrane potential and mitochondrial mass. The antioxidant regent N-acetyl-L-cysteine (NAC) rescued the effect of C3G on CSCC cells, further demonstrating ROS’s important role in C3G treatment. To explore the underlying mechanism, autophagy-related signaling pathways were investigated, and the results showed that C3G induced cell autophagy but not mitophagy. More importantly, C3G caused a significant activation of mitochondria biogenesis through the peroxisome proliferator-activated receptor gamma coactivator 1 alpha (PGC-1α) signaling pathway. C3G synergized with PGC-1α inhibitor SR-18292 induced more severe ROS accumulation and showed more potent inhibition of cell proliferation and mitochondrial membrane potential than C3G treatment alone. Thus, the present study suggests a new potential therapeutic strategy for CSCC based on the synergistic effect of C3G and PGC1α inhibitors.

LncRNA WWTR1-AS1 upregulates Notch3 through miR-136 to increase cancer cell stemness in cervical squamous cell carcinoma

BackgroundThis Study investigated the role of WWTR1-AS1 in cervical squamous cell carcinoma (CSCC).ResultsWWTR1-AS1 expression was upregulated in CSCC tissues. WWTR1-AS1 was predicted to interact with miR-136, whereas correlation analysis revealed that there was no close correlation between WWTR1-AS1 and miR-136 across CSCC samples. Moreover, WWTR1-AS1 and miR-136 did not regulate the expression of each other. In addition, overexpression of WWTR1-AS1 increased the expression levels of Notch3, which could be targeted by miR-136. Cell stemness analysis indicated that the overexpression of WWTR1-AS1 and Notch3 increased CSCC cell stemness and the capacity of CSCC cell to grow as spheroids. Overexpression of miR-136 decreased CSCC cell stemness and reversed the effects of overexpression of WWTR1-AS1 on Notch3 in CSCC cells.ConclusionTherefore, WWTR1-AS1 may upregulate Notch3 through miR-136 to increase cancer cell stemness in CSCC.

Hypoxia-induced ZEB1 promotes cervical cancer immune evasion by strengthening the CD47-SIRPα axis

BackgroundThe dynamic interaction between cancer cells and tumour-associated macrophages (TAMs) in the hypoxic tumour microenvironment (TME) is an active barrier to the effector arm of the antitumour immune response. Cancer-secreted exosomes are emerging mediators of this cancer-stromal cross-talk in the TME; however, the mechanisms underlying this interaction remain unclear.MethodsExosomes were isolated with ExoQuick exosome precipitation solution. The polarizing effect of TAMs was evaluated by flow cytometry, western blot analysis, immunofluorescence staining and in vitro phagocytosis assays. Clinical cervical cancer specimens and an in vivo xenograft model were also employed.ResultsOur previous study showed that hypoxia increased the expression of ZEB1 in cervical squamous cell carcinoma (CSCC) cells, which resulted in increased infiltration of TAMs. Here, we found that hypoxia-induced ZEB1 expression is closely correlated with CD47-SIRPα axis activity in CSCC, which enables cancer cells to evade phagocytosis by macrophages and promotes tumour progression. ZEB1 was found to directly activate the transcription of the CD47 gene in hypoxic CSCC cells. We further showed that endogenous ZEB1 was characteristically enriched in hypoxic CSCC cell-derived exosomes and transferred into macrophages via these exosomes to promote SIRPα+ TAM polarization. Intriguingly, exosomal ZEB1 retained transcriptional activity and reprogrammed SIRPα+ TAMs via activation of the STAT3 signalling pathway in vitro and in vivo. STAT3 inhibition reduced the polarizing effect induced by exosomal ZEB1. Knockdown of ZEB1 increased the phagocytosis of CSCC cells by macrophages via decreasing CD47 and SIRPα expression.ConclusionsOur results suggest that hypoxia-induced ZEB1 promotes immune evasion in CSCC by strengthening the CD47-SIRPα axis. ZEB1-targeted therapy in combination with CD47-SIRPα checkpoint immunotherapy may improve the outcomes of CSCC patients in part by disinhibiting innate immunity.

Effect of GLIS1 on the Lymph Node Metastasis of Cervical Squamous Carcinoma Based on Transcriptome Analysis.

Cervical cancer remains the leading cause of cancer-related mortality among female reproductive malignancies, and lymph node metastasis (LNM) represents the major reason for its poor prognosis. In this study, we aimed to identify transcriptome differences in patients with cervical squamous cell carcinoma (CSCC) who developed LNM or not and to outline the function of GLIS1 in determining metastatic fate in CSCC. In The Cancer Genome Atlas-endocervical adenocarcinoma project, patients with LNM had shorter overall survival than those without. Transcriptome data from CSCC patients with and without LNM were analyzed to screen for differentially expressed genes (DEGs). DEGs were enriched in metastasis-related pathways, such as extracellular matrix organization, cell-cell adhesion, and regulation of tissue remodeling. GLIS1 was overexpressed in tumor tissues of patients with LNM. COMP and ITGA11 were screened as downstream targets of GLIS1. GLIS1 promoted their transcription by binding to the promoter regions of COMP and ITGA11. GLIS1 enhanced the migration, invasion, and epithelial-mesenchymal transition in CSCC cells, while the knockdown of COMP or ITGA11 reversed the promotion of GLIS1 on CSCC cell malignant phenotype. Together, our results demonstrate that GLIS1 might be related to the LNM of CSCC patients via COMP and ITGA11.

Cancer susceptibility 18 positively regulates NUAK Family Kinase 1 expression to promote migration and invasion via sponging of miR-5586-5p in cervical cancer cells.

Cervical squamous cell carcinoma (CESC) is the most common gynecological malignancy worldwide. Although the cancer susceptibility 18 (CASC18) gene was involved in the regulation of cancer biology, its specific role in CESC is not well characterized. CASC18-related axis was predicted by bioinformatic analyses, and the competing endogenous RNA (ceRNA) interaction was further validated using quantitative real-time PCR, western blotting, RNA pulldown, and luciferase reporter assays. Transwell and wound healing assays were performed to verify the effect of CASC18 on SiHa and HeLa cell motility. We found that CASC18 was upregulated in CESC tissues. Moreover, interference with CASC18 attenuated NUAK1-mediated epithelial-mesenchymal transition (EMT) and thus suppressed cancer cell motility. Furthermore, the effects of CASC18 knockdown on CESC cells were partly rescued by transfection with the miR-5586-5p inhibitor. Additionally, our findings indicated that CASC18 acts as a ceRNA to enhance NUAK1 expression by sponging miR-5586-5p. Our study showed a novel CASC18/miR-5586-5p/NUAK1 ceRNA axis that could regulate cell invasion and migration by modulating EMT in CESC. These findings suggest that CASC18 may potentially serve as a novel therapeutic target in CESC treatment.

USP39 interacts with SIRT7 to promote cervical squamous cell carcinoma by modulating autophagy and oxidative stress via FOXM1

BackgroundSirtuin 7 (SIRT7) is an oncogene that promotes tumor progression in various malignancies, however, its role and regulatory mechanism in cervical squamous cell carcinoma (CSCC) is unknown. Herein, we attempted to investigate the functional role and molecular mechanism of SIRT7 underlying CSCC progression.MethodsSIRT7 expression was evaluated in CSCC cells using various assays. We then used a series of function gain-and-loss experiments to determine the role of SIRT7 in CSCC progression. Furthermore, mechanism experiments were conducted to assess the interaction between SIRT7/USP39/FOXM1 in CSCC cells. Additionally, rescue assays were conducted to explore the regulatory function of USP39/FOXM1 in CSCC cellular processes.ResultsSIRT7 was highly expressed in CSCC patient tissues and cell lines. SIRT7 deficiency showed significant repression on the proliferation, and autophagy of CSCC cells in vitro and tumorigenesis in vivo. Similarly, apoptosis and ROS production in CSCC cells were accelerated after the SIRT7 knockdown. Moreover, SIRT7 and USP39 were found colocalized in the cell nucleus. Interestingly, SIRT7 was revealed to deacetylate USP39 to promote its protein stability in CSCC cells. USP39 protein was also verified to be upregulated in CSCC tissues and cells. USP39 silencing showed suppressive effects on CSCC cell growth. Mechanistically, USP39 was revealed to upregulate SIRT7 by promoting the transcriptional activity of FOXM1. Rescue assays also indicated that SIRT7 promoted autophagy and inhibited ROS production in CSCC cells by regulating USP39/FOXM1.ConclusionThe SIRT7/USP39/FOXM1 positive feedback network regulates autophagy and oxidative stress in CSCC, thus providing a new direction for CSCC-targeted therapy.Graphical

NPHS2-6 drives cervical squamous cell carcinoma (CSCC) progression via hsa-miR-1323/SMC1B axis to activate PI3K-Akt pathway.

A substantial amount of evidence demonstrates suggests that long non-coding RNAs (lncRNAs) play a key role in the progression of various malignancies, cervical squamous cell carcinoma (CSCC) included. In our study, we deeply investigated the role and molecular mechanism of lncRNA NPHS2-6 in CSCC. The expression level of gene and protein expression were measured by qRT-PCR and western blot. To test the cell proliferation and cell metastasis ability, we carried out the CCK-8 experiment, clone formation assay, transwell assay and wound healing, respectively. The interactivity among NPHS2-6, miR-1323 and SMC1B were co demonstrated using the bioinformatics tool, dual-luciferase reporter system, and RNA pulldown assay. The subcutaneous tumor model of nude mice was established to verify the results of previous studies at the in vivo. NPHS2-6 was upregulated in CSCC tissues and cells. NPHS2-6 deficiency significantly inhibited CSCC cell growth and EMT in vitro. In addition, NPHS2-6 deficiency also inhibited the growth of CSCC xenograft tumors in mice in vivo. Importantly, NPHS2-6 was a competing endogenous RNA (ceRNA) to increases SMC1B levels by binding to miR-1323, leading to activate the PI3K/Akt pathway, thereby exacerbating tumorigenesis of CSCC. In conclusion, NPHS2-6/miR-1323/SMC1B/PI3K/Akt signaling accelerates the progression of CSCC, providing a new direction for the treatment strategy of CSCC.

ALKBH5 inhibits the SIRT3/ACC1 axis to regulate fatty acid metabolism via an m6A-IGF2BP1-dependent manner in cervical squamous cell carcinoma.

Cervical cancer (CC) is the most common malignancy of the female reproductive system, among which cervical squamous cell carcinoma (CESC) is the most common type. The demethylase ALKBH5 has been previously revealed to be downregulated in CC tissue. N6 methyladenine (m6A) is the most common modification in eukaryotic RNAs and is involved in modulating tumour progression. Therefore, we attempted to clarify the ALKBH5 role and mechanism underlying CESC progression. In CESC, patient tissue and control tissue m6A levels were measured. Reverse transcription quantitative real-time polymerase chain reaction, western blotting and immunochemistry were used to measure ALKBH5 levels. A correlation between CESC patient survival and ALKBH5 levels was evaluated. Wound healing, transwell and colony formation assays were used to detect CESC cellular behaviours. Corresponding kits and BODIPY staining were used to detect CESC lipid metabolism. Bioinformatics, immunoprecipitation, RNA pulldown and RNA immunoprecipitation assays as well as half-life measurements were used to assess the association and mechanism of ALKBH5 with silent mating type information regulation 2 homologue 3 (SIRT3), acetyl-CoA carboxylase 1 (ACC1) and insulin-like growth factor 2 mRNA binding protein 1 (IGF2BP1). The m6A demethylase ALKBH5 was depleted in CESC tissue and cells, and a low level of ALKBH5 predicted an unfavourable prognosis in CESC patients. ALKBH5 overexpression suppressed CESC growth and lipid metabolism in vitro and CESC tumour growth in vivo, and ACC1 overexpression rescued these changes. ALKBH5 downregulated ACC1 levels in CESC cells by facilitating SIRT3 methylation to repress ACC1 deacetylation. ALKBH5 destabilized SIRT3 to downregulate SIRT3 levels in CESCs in an m6A-IGF2BP1-dependent manner. ALKBH5 demethylates and destabilizes SIRT3 in an m6A-IGF2BP1-dependent manner, repressing CESC growth, lipid metabolism and tumorigenesis by downregulating ACC1.

Circular RNA ARHGAP5 inhibits cisplatin resistance in cervical squamous cell carcinoma by interacting with AUF1.

Cervical squamous cell carcinoma (CSCC) is one of the leading causes of cancer death in women worldwide. Patients with advanced cervical carcinoma always have a poor prognosis once resistant to cisplatin due to the lack of effective treatment. It is urgent to investigate the molecular mechanisms of cisplatin resistance. Circular RNAs (circRNAs) are known to exert their regulatory functions in a series of malignancies. However, their effects on CSCC remain to be elucidated. Here, we found that cytoplasmic circARHGAP5, derived from second and third exons of the ARHGAP5 gene, was downregulated in cisplatin-resistant tissues compared with normal cervix tissues and untreated cervical cancer tissues. In addition, experiments from overexpression/knockdown cell lines revealed that circARHGAP5 could inhibit cisplatin-mediated cell apoptosis in CSCC cells both in vitro and in vivo. Mechanistically, circARHGAP5 interacted with AU-rich element RNA-binding protein (AUF1) directly. Overexpression of AUF1 could also inhibit cell apoptosis mediated by cisplatin. Furthermore, we detected the potential targets of AUF1 related to the apoptotic pathway and found that bcl-2-like protein 11 (BIM) was not only negatively regulated by AUF1 but positively regulated by circARHGAP5, which indicated that BIM mRNA might be degraded by AUF1 and thereby inhibited tumor cell apoptosis. Collectively, our data indicated that circARHGAP5 directly bound to AUF1 and prevented AUF1 from interacting with BIM mRNA, thereby playing a pivotal role in cisplatin resistance in CSCC. Our study provides insights into overcoming cancer resistance to cisplatin treatment.

New insights on the interaction between m6A modification and non-coding RNA in cervical squamous cell carcinoma

BackgroundN6-Methyladenosine (m6A) and long non-coding RNAs (lncRNAs) are both crucial regulators in human cancer growth and metastasis. However, their regulation on cervical squamous cell carcinoma (CSCC) is largely unclear. The present study aimed to explore the role of m6A-associated lncRNAs in CSCC.MethodsWe screened the expression of methylation modification-related enzymes in CECC samples from TCGA. The qRT-PCR was used to detect METTL3 and lncRNA METTL4-2 expression. The biological activities of METTL3 in CSCC cells were evaluated by CCK-8, colony formation, transwell, wound healing, and xenograft tumor assays, respectively. The SRAMP tool was used to screen m6A modification sites of METTL4-2. Finally, the quantitative analysis of m6A modification was carried out by MeRIP.ResultsMETTL3 expression was upregulated in CSCC cells and tissues. Biological function and function loss analysis indicated that METTL3 promoted the migration and proliferation of CSCC cells. In addition, METTL3 promoted CSCC tumor growth in vivo. Mechanically, METTL3 installed the m6A modification and enhanced METTL4-2 transcript stability to increase its expression. Meanwhile, the m6A “reader” YTHDF1 recognized METTL4-2 installed by METTL3 and facilitated the translation of METTL4-2.ConclusionsIn conclusion, our study highlights the function and mechanism of METTL3-induced METTL4-2 in CSCC. These findings support that METTL3-stabilized METTL4-2 promoted CSCC progression via a m6A-dependent modality, which provides new insights into therapeutic strategies for CSCC.

HOXA5 inhibits the proliferation and metastasis of cervical squamous cell carcinoma by suppressing the β-catenin/Snail signaling.

HOXA5, as a transcription factor, plays an important role in a variety of malignant tumors. Nevertheless, its biological role in cervical squamous cell carcinoma (CSCC) is largely unknown. In our study, we aimed to explore the function of HOXA5 in CSCC and its molecular mechanism. Immunohistochemistry showed that HOXA5 expression was downregulated in human CSCC tissues and HOXA5 staining was negatively correlated with tumor size and histological grade of CSCC. Ectopic expression of HOXA5 inhibited proliferative and metastatic abilities of CSCC cells in vitro and in vivo. Furthermore, overexpression of HOXA5 inhibited the cell cycle by arresting the S/G2 phase by flow cytometry and that was related to the downregulation of Cyclin A. Further study showed that HOXA5 suppressed EMT by inhibiting the β-catenin/Snail signaling resulting in reduced metastasis of CSCC cells. Altogether, our results suggested that HOXA5 inhibited the proliferation and metastasis via repression of the β-catenin/Snail pathway, proposing the potential role of HOXA5 in the prevention and treatment of CSCC.

E2F Transcription Factor 1 Activates FKBP Prolyl Isomerase 4 to Promote Angiogenesis in Cervical Squamous Cell Carcinoma Via the PI3K/AKT Signaling Pathway.

Angiogenesis, namely the formation of blood vessels, is crucial for tumor growth, metastasis and development. E2F transcription factor 1 (E2F1) has been linked to tumorigenesis in several human cancers. This work examines the role of E2F1 and its downstream targets in angiogenesis in cervical squamous cell carcinoma (CSCC). E2F1 was predicted as a candidate oncogene in CSCC using a GSE63514 dataset. Increased E2F1 expression was detected in CSCC tumor samples and cell lines by RT-qPCR, immunohistochemistry, and western blot assays. E2F1 downregulation reduced the angiogenesis activity of HUVECs and the invasiveness of CSCC cells. In vivo, E2F1 knockdown also reduced the xenograft tumor growth and promoted tumor necrosis in mice. FKBP prolyl isomerase 4 (FKBP4) was identified as a target of E2F1. E2F1 bound to FKBP4 promoter for transcriptional activation. Further upregulation of FKBP4 blocked the tumor-suppressive role of E2F1 silencing. FKBP4 was enriched in the PI3K/AKT signaling. In cells and xenograft tumors, the E2F1/FKBP4 axis promoted PI3K and AKT phosphorylation. Activation of the PI3K/AKT signaling restored the angiogenesis activity in cells blocked by E2F1 silencing. In summary, this work demonstrates that E2F1 promotes FKBP4 transcription to activate the PI3K/AKT pathway, which augments the angiogenesis and invasiveness of CSCC.

Use of Single Cell Transcriptomic Techniques to Study the Role of High-Risk Human Papillomavirus Infection in Cervical Cancer.

High-risk human papillomavirus (hrHPV) infection has been associated with a higher probability of progression to cervical cancer. However, several extensive studies have reported that the presence of hrHPV can lead to a better prognosis, but the mechanism of how this occurs is unclear. In this study, microbiological analysis was used to identify HPV infection as a factor for the prognosis of patients with cervical squamous cell carcinoma (CSCC). Comparing the interactions of HPV+ and HPV- malignant cells with immune cells as well as the trajectory of malignant cells either with or without HPV, we found that most of the HPV+ cells are well differentiated while HPV- cells appear to be hypo-fractionated. Using transcriptomic and immunostaining data, we validated a set of unfavourable molecules in the HPV- CSCC cells, including KRT16, ITGB1, CXCR1, VEGFA, CRCT1 and TNFRSF10B/DR5. This study provides a basis for the development of a rational post-operative follow-up programme and the development of an appropriate treatment plan for patients with cervical cancer.

MiR-99a-5p Constrains Epithelial-Mesenchymal Transition of Cervical Squamous Cell Carcinoma Via Targeting CDC25A/IL6.

MiR-99a-5p participates in processes and pathogenesis of varying diseases. However, the molecular mechanism of miR-99a-5p in human cervical squamous cell carcinoma (CSCC) remains unclear. Here, we found that miR-99a-5p was lowly expressed in CSCC cells and negatively associated with overall survival. In addition, cellular experiments including CCK8, wound healing, Transwell and flow cytometry assays disclosed that transfection of miR-99a-5p mimic could suppress the cell activity, cell migratory, and invasive abilities, and promote cell apoptosis, thus inhibiting the tumor progression of CSCC cells. Luciferase reporter gene assay indicated that miR-99a-5p targeted 3'-UTR of CDC25A. Also, enforced CDC25A level rescued the impact of miR-99a-5p on CSCC progression. Silencing CDC25A could restrain the mRNA and protein levels of IL-6 in CSCC. CDC25A overexpression or IL-6 treatment could attenuate inhibiting impact of miR-99a-5p overexpression on epithelial-mesenchymal transition (EMT). These findings suggested that miR-99a-5p may play an anti-tumor role in tumor metastasis by targeting CDC25A/IL6 to hamper EMT process, which revealed a novel molecular mechanism in CSCC.

MiR-125a attenuates the malignant biological behaviors of cervical squamous cell carcinoma cells through Rad51

ABSTRACT Cervical squamous cell carcinoma (CSCC), the most common cervical malignancy, is more likely to invade and metastasize than other cervical cancers. miR-125a, a tumor suppressor gene, has been confirmed to be associated with cancer metastasis. However, the role of miR-125a in CSCC and the underlying mechanism are unknown. miR-125a expression was confirmed by real-time quantitative PCR (RT–qPCR), and the Rad51 expression level was measured by western blotting analysis. CSCC cell proliferation, migration and invasion were assessed with functional assays, including CCK-8, colony formation, wound healing and Transwell assays. Our data confirmed that miR-125a is expressed at low levels in CSCC tissues and cells. Functionally, the overexpression of miR-125a greatly prevented the proliferation, migration and invasion of CSCC cells, and the inhibition of miR-125a expression strongly enhanced these behaviors in CSCC cells. Moreover, the expression of Rad51, a miR-125a target gene, greatly reversed the miR-125-mediated inhibition of CSCC cell proliferation, migration and invasion. In addition, we discovered that miR-125a downregulated the levels of phosphorylated PI3K, AKT and mTOR through Rad51 in CSCC cells. miR-125a, a tumor suppressor, can attenuate the malignant behaviors of CSCC cells by targeting Rad51. Therefore, the miR-125a/Rad51 axis might be a target for CSCC therapy.

TUSC3 inhibits cell proliferation and invasion in cervical squamous cell carcinoma via suppression of the AKT signalling pathway.

The decreased expression of tumour suppressor candidate 3 (TUSC3) is associated with proliferation in several types of cancer, leading to an unfavourable prognosis. The present study aimed to assess the cellular and molecular function of TUSC3 in patients with cervical squamous cell carcinoma (CSCC). Levels of mRNA expressions of TUSC3 were analysed in CSCC tissues and six cell lines using qRT‐PCR. Immunohistochemistry(IHC) was used to evaluate the protein expression level of TUSC3 in four paired specimens, 220 paraffin‐embedded CSCC specimens and 60 cases of normal cervical tissues(NCTs), respectively. Short hairpin RNA interference was employed for TUSC3 knockdown. Cell proliferation, migration and invasion were evaluated using growth curve, MTT assay, wound healing, transwell assay and xenograft tumour model, respectively. The results demonstrated that TUSC3 mRNA and protein expression levels were downregulated in CSCC samples. Multivariate and univariate analyses indicated that TUSC3 was an independent prognostic factor for patients with CSCC. Decreased TUSC3 expression levels were significantly associated with proliferation and an aggressive phenotype of cervical cancer cells both in vitro and in vivo. Moreover, the knockdown of TUSC3 promoted migration and invasion of cancer cells, while the increased expression of TUSC3 exhibited the opposite effects. The downregulation of TUSC3 facilitated proliferation and invasion of CSCC cells through the activation of the AKT signalling pathway. Our data demonstrated that the downregulation of TUSC3 promoted CSCC cell metastasis via the AKT signalling pathway. Therefore, TUSC3 may serve as a novel prognostic marker and potential target for CSCC.

Upregulation of NOD1 and NOD2 contribute to cancer progression through the positive regulation of tumorigenicity and metastasis in human squamous cervical cancer

BackgroundMetastatic cervical squamous cell carcinoma (CSCC) has poor prognosis and is recalcitrant to the current treatment strategies, which warrants the necessity to identify novel prognostic markers and therapeutic targets. Given that CSCC is a virus-induced malignancy, we hypothesized that the pattern recognition receptors (PRRs) involved in the innate immune response likely play a critical role in tumor development.MethodsA bioinformatics analysis, qPCR, IHC, immunofluorescence, and WB were performed to determine the expression of NOD1/NOD2. The biological characteristics of overexpression NOD1 or NOD2 CSCC cells were compared to parental cells: proliferation, migration/invasion and cytokines secretion were examined in vitro through CCK8/colony formation/cell cycle profiling/cell counting, wound healing/transwell, and ELISA assays, respectively. The proliferative and metastatic capacity of overexpression NOD1 or NOD2 CSCC cells were also evaluated in vivo. FCM, mRNA and protein arrays, ELISA, and WB were used to identify the mechanisms involved, while novel pharmacological treatment were evaluated in vitro and in vivo. Quantitative variables between two groups were compared by Student’s t test (normal distribution) or Mann-Whitney U test (non-normal distribution), and one-way or two-way ANOVA was used for comparing multiple groups. Pearson χ2 test or Fisher’s exact test was used to compare qualitative variables. Survival curves were plotted by the Kaplan-Meier method and compared by the log-rank test. P values of < 0.05 were considered statistically significant.ResultsNOD1 was highly expressed in CSCC with lymph-vascular space invasion (LVSI, P < 0.01) and lymph node metastasis (LM, P < 0.01) and related to worse overall survival (OS, P = 0.016). In vitro and in vivo functional assays revealed that the upregulation of NOD1 or NOD2 in CSCC cells promoted proliferation, invasion, and migration. Mechanistically, NOD1 and NOD2 exerted their oncogenic effects by activating NF-κb and ERK signaling pathways and enhancing IL-8 secretion. Inhibition of the IL-8 receptor partially abrogated the effects of NOD1/2 on CSCC cells.ConclusionsNOD1/2-NF-κb/ERK and IL-8 axis may be involved in the progression of CSCC; the NOD1 significantly enhanced the progression of proliferation and metastasis, which leads to a poor prognosis. Anti-IL-8 was identified as a potential therapeutic target for patients with NOD1high tumor.

LncRNA MCM3AP-AS1 Regulates miR-21/PTEN Axis to Affect Cervical Squamous Cell Carcinoma Cell Proliferation and Apoptosis.

LncRNA MCM3AP-AS1 has been reported to be upregulated and plays an oncogenic role in papillary thyroid cancer. However, analysis of the Cancer Genome Atlas (TCGA) dataset revealed MCM3AP-AS1 downregulation in cervical squamous cell carcinoma (CSCC). This observation encouraged us to analyze the function of MCM3AP-AS1 in CSCC. The research subjects of the present study were 62 CSCC patients (42 to 68 years; 53.7 ± 6.8 years). Based on medical records, there were 51 HPV-positive cases of and 11 HPV-negative cases. Gene expression was analyzed by RT-qPCR. The interactions among MCM3AP-AS1, miR-21, and PTEN were explored by overexpression assays followed by RT-qPCR and Western blot. CCK-8 cell proliferation analysis and cell apoptosis analysis were applied to study the roles of MCM3AP-AS1, miR-21, and PTEN in regulating cell proliferation and apoptosis in CSCC. We found that MC-M3AP-AS1 was downregulated in CSCC patients, and its low level was closely correlated with patients' poor survival. MCM3AP-AS1 could directly interact with miR-21. However, miR-21 overexpression failed to affect MCM3AP-AS1 expression. Interestingly, MCM3AP-AS1 overexpression decreased the expression of PTEN, which is a target of miR-21. Cell proliferation and apoptosis analysis showed that MCM3AP-AS1 and PTEN overexpression increased apoptosis but decreased proliferation of CSCC cells. MiR-21 overexpression played an opposite role and attenuated the effects of MCM3AP-AS1 overexpression. Therefore, MCM3AP-AS1 may regulate the miR-21/PTEN axis to regulate CSCC cell proliferation and apoptosis.

MiR-140-Modified Bone Marrow Mesenchymal Stem Cells Enhance Chemotherapy Sensitization in Cervical Squamous Cell Carcinoma Cells via Targeting Microtubule Depolymerization Protein 1 (STMN1)

Effect of bone marrow mesenchymal stem cells (BMSCs) on the sensitivity of chemotherapy drugs and microRNAs (miRNAs) is still unclear. This study explored the role of miR-140 modified BMSCs in enhancing paclitaxel sensitivity of cervical squamous cell carcinoma (CSCC). Hela cells, BMSCs cells, and miR-140 modified BMSCs were transfected with miR-140 mimic, miR-140 inhibitor, and miR-140 NC, respectively. After transfection, they were co-cultured with Hela cells and paclitaxel to set up miR-140 mimic group, miR-140 inhibitor group, and miR-140 NC group (without paclitaxel treatment) followed by analysis of cell proliferation, apoptosis, ROS generation, expression of miR-140, STMN1, STAT3, p-STAT3, and survivin mRNA and protein. miR-140 inhibitor group showed lowest cell proliferation number and expressions of miR-140, STMN1, STAT3, p-STAT3, and survivin mRNA and protein with highest number of apoptotic cells, which were all reversed in miR-140 mimic group. There was a positive correlation between STMN1 level and miR-140 expression (r = 0.449, P = 0.108). BMSCs modified with miR-140 inhibitor can target STMN1, enhance the sensitivity of chemotherapy drugs, and exert an inhibitory effect on CSCC cell proliferation, suggesting that STMN1 might be a therapy target for treating CSCC.