Journal of Cellular Biochemistry

Long noncoding RNA CHL1‐AS1 promotes cell proliferation and migration by sponging miR‐6076 to regulate CHL1 expression in endometrial cancer

AbstractEndometrial cancer (EC) is deemed to be the most typical gynecologic malignant tumor. Despite the incidence of EC being lower in Asia than that in western countries, substantial increased incidence has been observed in the past few decades in Asia. Although various molecular testing methods and genomic science have developed, the overall prognosis is still disappointing. LncRNAs have been found to influence the progression of various cancers. CHL1‐AS1 has been found to be upregulated in ovarian endometriosis, nevertheless, the molecular mechanism and biological function of CHL1‐AS1 in EC have not been explored. In our exploration, both CHL1‐AS1 and CHL1 were upregulated in EC cells. Knockdown of CHL1‐AS1 or CHL1 inhibited cell proliferation and migration in EC. Furthermore, microRNA‐6076 (miR‐6076) could bind with CHL1‐AS1 or CHL1, and regulate the expression of CHL1. Finally, absence of miR‐6076 or overexpression of CHL1 can partially rescue the effect of CHL1‐AS1 knockdown or miR‐6076 upregulation on cell proliferation and migration, respectively. All in all, our research was the first endeavor to study the underlying mechanism of CHL1‐AS1 in EC and confirmed that CHL1‐AS1 regulated EC progression via targeting the miR‐6076/CHL1 axis, offering new insight into treating EC.

Retracted : Long noncoding RNA PCAT‐1 knockdown prevents the development of ovarian cancer cells via microRNA‐124‐3p

Abstract Long noncoding RNA prostate cancer‐associated transcript 1 (PCAT‐1) is overexpressed in human malignancies and its silence abates the exaggeration of cancers. Whereas, the activity of PCAT‐1 silence in ovarian cancer (OC) remains elusive. Here, our study was designed to corroborate the function of PCAT‐1 silence in cellular activities and the molecular mechanisms. PCAT‐1 in human ovarian tumor tissue specimens and cell lines (A2780 and SKOV3) were quantified by real‐time quantitative reverse polymerase chain reaction (qRT‐PCR). Reinforced silence of PCAT‐1 and microRNA (miR)‐124‐3p was established by transfection and identified by qRT‐PCR. The viability, apoptosis as well as migration and invasion were examined. Western blot was exploited for analysis of proteins involved in proliferation, apoptosis, migration and invasion, and signaling transduction. OC tissues showed the accumulation of PCAT‐1. Silencing PCAT‐1 caused the impediment of proliferation, migration, and invasion with the increase in apoptosis. PCAT‐1 knockdown repressed the expression of cyclin D1, CDK6, p53, Bax, cleaved caspase‐3, metallopeptidases, and vimentin with the restoration of miR‐124‐3p. However, the roles of PCAT‐1 silence were weakened in the absence of miR‐124‐3p. PCAT‐1 silence caused decrease in Wnt3a, β‐catenin, and phosphorylation of protein kinase B and mechanistic target of rapamycin was abolished by miR‐124‐3p inhibitor. The tumor‐suppressive role of PCAT‐1 silence was mediated by miR‐124‐3p.

miR‐200c overexpression inhibits the invasion and tumorigenicity of epithelial ovarian cancer cells by suppressing lncRNA HOTAIR in mice

Abstract Epithelial ovarian cancer (EOC) is a common ovarian cancer in gynecological cancers today. It has been found that microRNAs and long‐chain noncoding RNA (lncRNA) regulate the gene transcriptional expression in cells. However, it is not well understood that the upstream and downstream regulatory molecules of lncRNA HOX antisense intergenic RNA ( HOTAIR ). The effects of miR‐200c overexpression on the invasion and nude mouse tumorigenicity, as well as lncRNA HOTAIR and snail expression of EOC SKOV3 cells, should be further explored. The expression of miR‐200c and lncRNA HOTAIR was detected by reverse transcription PCR (RT‐PCR) in EOC SKOV3 cells. The whole miR‐200c gene fragment was cloned into a lentiviral plasmid vector. The miR‐200c expression in transducted SKOV3 cells with reconstructed miR‐200c lentivirus was significantly higher than the negative control ( P  < .01). The lentivirus‐miR‐200c‐SKOV3 cells show that the invasion ability was significantly decreased compared with the negative control ( P  < .01). The nude mouse tumorigenicity was significantly decreased compared with that of the control group ( P  < .01). The snail protein expression in lentivirus‐miR‐200c‐SKOV3 xenograft tumor was significantly decreased compared with the negative control lentivirus‐SKOV3 group ( P  < .05). The miR‐200c overexpression significantly decreased the expressions of lncRNA HOTAIR and snail, but increased E‐cadherin expression in the lentivirus‐miR‐200c transducted SKOV3 cells of xenograft tumor, compared with the negative control ( P  < .05). The miR‐200c overexpression in SKOV3 cells with transducted lentivirus‐miR‐200c can inhibit lncRNA HOTAIR expression, decrease snail, increase E‐cadherin and significantly reduce the invasion and tumorigenicity of EOC SKOV3 cells. These results suggest that the miR‐200c and lncRNA HOTAIR could be effective therapeutic targets for human epithelial ovarian cancer treatment.

A six‐CpG‐based methylation markers for the diagnosis of ovarian cancer in blood

Abstract DNA methylation markers in the peripheral blood are able to be applied to treat epithelial cancer. Nevertheless, the diagnostic potential value of it for ovarian cancer (OV) has not been studied. The study aimed to explore the difference of DNA methylation in peripheral blood between OV patients and healthy women. Firstly, the whole blood of DNA methylation data was provided by the Gene Expression Omnibus (GEO) database. The linear model was applied to the identification of significantly differentially expressed methylated CpG sites (differentially methylation sites [DMP]), and the further screen of co‐expression CpG sites (Co‐DMP). A total of 2812 DMPs were identified, and weighted gene co‐expression network analysis helped to obtain seven co‐expression modules. Among them, the yellow module was the most related to OV. Co‐DMPs (167) in the yellow module were mainly distributed near the transcription start sites. However, most of them were not in the CpG island. Least absolute shrinkage and selection operator (LASSO) regression analysis was applied to the identification of stable OV‐related blood biomarkers that six Co‐DMPs (cg00134539, cg00226923, cg25268718, cg25697314, cg25839227, cg26574610) with the highest frequency were found as potential biomarkers. Finally, the diagnostic classifier was established using the support vector machine (SVM) with the accuracy rate of 87.1% and 74.5% in training data set and validation data set, respectively. To sum up, a new feature was provided here for the diagnosis of OV, which is helpful for the diagnosis and individualized treatments of early OV.

KLF8 is activated by TGF‐β1 via Smad2 and contributes to ovarian cancer progression

AbstractKrüppel‐like factor 8 (KLF8) is a transcription factor expressed abnormally in various cancer types and promotes oncogenic transformation. However, the role of KLF8 in ovarian cancer (OC) progression remains unclear. This study reports that transforming growth factor‐β1 (TGF‐β1)/Smad2/KLF8 axis regulates epithelial–mesenchymal transition (EMT) and contributes to OC progression. We analyzed the KLF8 expression in OC cells and tissues, wherein a significant overexpression of KLF8 was observed. Increased KLF8 expressions were correlated with higher cell proliferation, EMT, migration, and invasion and conferred poor clinical outcomes in OC patients. Overexpressed KLF8 increases F‐actin polymerization and induces cytoskeleton remodeling of OC cells. Furthermore, a dissection of the molecular mechanism defined that TGF‐β1 triggers KLF8 through the Smad2 pathway and regulates EMT. Pharmacological and genetic inhibition of Smad2 followed by TGF‐β1 treatment failed to activate KLF8 expression and induction of EMT. Using promoter‐luciferase reporter assays, we defined that upon TGF‐β1 activation, phosphorylated Smad2 binds and promotes the KLF8 promoter activity, and knockdown of Smad2 inhibits KLF8 promoter activation. Together, these results demonstrate that TGF‐β1 activates KLF8 expression by the Smad2 pathway, and KLF8 contributes to OC progression and may serve as a potential therapeutic strategy for treating OC patients.

SP1‐induced lncRNA FOXD3‐AS1 contributes to tumorigenesis of cervical cancer by modulating the miR‐296‐5p/HMGA1 pathway

AbstractLong noncoding RNAs (lncRNAs) have drawn growing attention due to their regulatory roles in various diseases, including tumors. Recently, lncRNA FOXD3 antisense RNA 1 (FOXD3‐AS1) was shown to be overexpressed in colon adenocarcinoma and glioma, exerting oncogenic functions. However, its expression and effects in cervical cancer (CC) remained unknown. In this research, our group first reported that the levels of FOXD3‐AS1 were distinctly elevated in CC samples and cell lines. The distinct upregulation of FOXD3‐AS1 was associated with lymphatic invasion, distant metastasis, and International Federation of Gynecology and Obstetrics stage, and also predicted poor clinical results of CC patients. Next, transcription factor SP1 was demonstrated to resulting in the upregulation of FOXD3‐AS1 in CC. Functional assays indicated that knockdown of FOXD3‐AS1 distinctly suppressed CC progression via affecting cell proliferation, cell apoptosis, and metastasis. Moreover, mechanistic studies suggested that FOXD3‐AS1 acted as an endogenous sponge by directly binding miR‐296‐5p, resulting in the suppression of miR‐296‐5p. In addition, we also reported that high mobility group A, a direct target of miR‐296‐5p, could mediate the tumor‐promotive effects that FOXD3‐AS1 displayed. Overall, our present study might help to lead a better understanding of the pathogenesis of CC, provide a novel possible tumor biomarker, and probe the feasibility of lncRNA‐directed treatments for CC.

Hsa_circ_0031288/hsa‐miR‐139‐3p/Bcl‐6 regulatory feedback circuit influences the invasion and migration of cervical cancer HeLa cells

AbstractCircular RNA (circRNA) molecules contain microRNA (miRNA) response elements that are able to competitively bind miRNAs as well as function as miRNA sponges within cells, which can reduce miRNA inhibition of target genes, thereby increasing their expression. TargetScan and miRanda bioinformatic tools were used to analyze the binding sites between genes. The relative levels of gene expression in tissues and cells were verified using quantitative reverse transcription‐polymerase chain reaction. Inhibition of cell proliferation was detected using a WST‐8 method. Cell invasion ability and migration ability were assessed using a Transwell migration assay and a scratch assay, respectively. The binding of miRNA and circRNA was detected using an RNA pull‐down assay. Bifluorescence reporter gene vectors were constructed to verify the binding of miRNA to messenger RNA. A tumor model of cervical cancer cell transplantation in mice was constructed to observe the effect of the genes on tumor growth. hsa_circ_0031288 and B‐cell CLL/lymphoma 6 (Bcl‐6) exhibited high expression in cervical cancer cells and tissue, while hsa‐miR‐139‐3p exhibited low expression. Reducing hsa_circ_0031288 and Bcl‐6 expression or increasing hsa‐miR‐139‐3p expression significantly inhibited the migration, invasion, proliferation, and growth of xenograft and HeLa cells. hsa_circ_0031288 had a regulatory effect on hsa‐miR‐139‐3p, and hsa‐miR‐139‐3p targeted the 3′ untranslated region of Bcl‐6. Reducing hsa_circ_0031288 expression promoted hsa‐miR‐139‐3p expression, while overexpressing miR‐139‐3p inhibited the transcription of Bcl‐6. In the cervical cancer HeLa cell line, the hsa_circ_0031288/hsa‐miR‐139‐3p/Bcl‐6 regulatory axis affects cell migration and proliferation, and its mechanism may involve hsa_circ_0031288 acting as a sponge for hsa‐miR‐139‐3p, thereby relieving the transcriptional inhibition of Bcl‐6. This suggests an approach for elucidating the pathogenesis of cervical cancer while offering new intervention targets for cervical cancer treatment.

LncRNA SNHG8 accelerates proliferation and inhibits apoptosis in HPV‐induced cervical cancer through recruiting EZH2 to epigenetically silence RECK expression

AbstractInfection of human papillomaviruses (HPVs), such as subtypes HPV16 and HPV18 is carcinogenic to human and is prominent cause of HPV‐positive cervical carcinoma (CC). A closer investigation into the mechanism of HPV‐induced CC may stimulate the generation of an improved therapy treating cervical cancer. Our study herein interrogated the function of a small nucleolar RNA host gene 8 (SNHG8) in HPV‐induced CC. As a result, a notable increase of SNHG8 in HPV‐induced CC cells was found compared with HPV‐negative CC cells. Functionally, it identified that SNHG8 aggravated the cell proliferation and migration in Cell Counting Kit‐8 and transwell assays. Besides, flow cytometry apoptosis assay displayed that blockade of SNHG8 exacerbated apoptosis of HPV‐positive CC cells. As detected by fluorescence in situ hybridization analysis and subcellular fractionation assay, SNHG8 was primarily expressed in the nucleus and exerted suppressive role on reversion inducing cysteine‐rich protein with kazal motifs (RECK) expression, which implied a potential transcriptional regulation of SNHG8 on RECK level. Mechanically, SNHG8 was disclosed to interact with enhancer of zeste homolog 2 (EZH2) based on RNA immunoprecipitation assay. ChIP assay further unveiled the occupancy of EZH2 in the promoter region of RECK. An additional chromatin immunoprecipitation assay highlighted that SNHG8 intensified the enrichment of EZH2 and H3K27me3 in RECK promoter region. Altogether, it reflected that SNHG8 recruited EZH2 to downregulate RECK expression, leading to HPV‐induced CC aggravation.

LINC00958 facilitates cervical cancer cell proliferation and metastasis by sponging miR‐625‐5p to upregulate LRRC8E expression

AbstractAccepted as a malignant tumor worldwide, cervical cancer (CC) has attracted much attention for its high incidence and mortality rates. Previous studies have elucidated the critical regulatory function that long noncoding RNAs (lncRNAs) exert on the tumorigenesis and progression of diverse tumors. Although multiple investigations have depicted that LINC00958 has a great impact on the complex biological process of many cancers, knowledge concerning the regulatory role of LINC00958 in CC remains limited and needs to be further explored. In our study, LINC00958 expression was evidently overexpressed in CC tissues and cells. Besides this, LINC00958 negatively regulated miR‐625‐5p expression and was verified to bind with miR‐625‐5p in CC. Subsequently, it was testified by a series of experiments that LINC00958 promotes CC cell proliferation and metastasis by sponging miR‐625‐5p. Furthermore, the leucine‐rich repeat containing the eight family member E (LRRC8E) could bind with miR‐625‐5p, and its expression was negatively modulated by miR‐625‐5p, whereas positively regulated by LINC00958 in CC. Final rescue assays verified the effects of LINC0095/LRRC8E interaction and miR‐625‐5p/LRRC8E interaction on CC cell proliferation and metastasis. Collectively, LINC00958 facilitates CC cell proliferation and metastasis via the miR‐625‐5p/LRRC8E axis.

Impact of modulation of telomerase and cancer stem‐cell marker OCT4 axis in cervical cancer pathogenesis with underlying HPV16 infection

AbstractLacunae exist in the molecular event(s) specificity associated with cervical cancer (CaCx) pathogenesis. The present study aimed to evaluate the significance of telomerase‐cervical cancer stem cells (CSCs) modulation in CaCx pathogenesis with underlying HPV16 infection. The study included HPV16 positive cases only (N = 65) of the total enrolled cases from Northeast India. The analysis of viral load and the differential messenger RNA expression of E6, E7, hTERT, hTR, and cancer stem‐cell markers was studied by real‐time polymerase chain reaction. Further the protein and colocalization study for E6, hTERT, and oct4 was performed by immunofluorescence. The real‐time polymerase chain reaction based analysis showed an upregulation of HPV16 viral oncoprotein E6 and E7, and telomerase component hTERT and hTR expression and their correlation in CaCx susceptibility and severity. The hTERT expression correlated with viral load; while the E6 and telomerase protein expression colocalized in the nucleus. The CSCs marker octamer‐binding transcription factor 4 (OCT4) was significantly upregulated in CaCx cases, was associated with CaCx susceptibility and severity, and colocalized with E6 expression in the nucleus as revealed from the immunofluorescence studies. To conclude, the telomerase‐OCT4 axis modulation holds key in HPV16 CaCx pathogenesis mediated by HPV16 E6 viral oncoprotein expression, and underlines its potential for therapeutic targeting.

LINC00116 enhances cervical cancer tumorigenesis through miR‐106a/c‐Jun pathway

AbstractSome studies imply that LINC00116 is involved in cervical cancer progression; however, the molecular mechanism by which LINC00116 modulating tumorigenesis of cervical cancer remains not clear. Reverse transcription‐quantitative PCR (RT‐qPCR) and the Western blot approaches were employed to probe genes expression levels. To examine the tumorigenic abilities of cervical cancer cells, MTT assay, Transwell assay, and wound‐healing assay were used to investigate proliferation, invasion, and migration of HeLa or C‐33A cells. LINC00116 knockdown attenuates cell proliferation, invasion, and migration of cervical cancer cells. miR‐106a directly binds LINC00116 and regulate each other. Moreover, miR‐106a inhibitor remarkably enhanced tumorigenesis of shLINC00116 HeLa cells. Through bioinformatic and dual‐luciferase reporter assay, the results showed that miR‐106a mimic directly targeted and downregulated the c‐Jun. c‐Jun overexpression could greatly rescue miR‐106a mimic‐modulated cervical cancer tumorigenesis. LINC00116 knockdown and miR‐106a mimic‐modulated programmed cell death ligand 1 (PD‐L1) expression, which could be reverted by c‐Jun introduction. LINC00116, PD‐L1, and JUN were both upregulated in cervical cancer tumors compared to normal tissues. Lower expression levels of LINC00116 and JUN, as well as higher level of miR‐106a were closely associated with higher overall survival of cervical cancer patients. Here, we report a novel role for LINC00116 in tumorigenesis of cervical cancer by regulating miR‐106a/c‐Jun axis. Our findings provide a foundation for understanding cervical cancer and facilitate the development of therapeutical approaches by targeting LINC00116.

DNA methylation data–based molecular subtype classification related to the prognosis of patients with cervical cancer

AbstractCervical cancer is one of the leading female health‐killers among all types of malignancies globally. Human papillomavirus infection combined with genetic and epigenetic alterations have been indicated to be closely associated with the pathogenesis, progression, and malignant transformation of cervical cancer. Notably, during the complex tumorigenesis process, a series of DNA methylations occurs early and is the most frequent molecular behavior. In this study, to exploit the specific DNA methylation sites influencing the prognosis of patients with cervical cancer, 275 samples were downloaded from The Cancer Genome Atlas database and further analyzed. As a result, 1253 CpGs were found to have a significant correlation with patient prognosis and were further selected for the consistent clustering of samples into six subgroups. Specifically, the samples in every subgroup were different regarding the following: race, age, tumor stage, receptor status, histological type, metastasis status, and patient prognosis. In addition, we calculated the levels of methylation sites in all subgroups, with 79 methylation sites (corresponding to 81 genes) screened as the intrasubgroup‐specific methylation sites. Moreover, signaling pathway enrichment analysis was conducted on the genes of the corresponding promoter regions of the above‐described specific methylation sites, revealing that these genes were enriched in biological pathways closely associated with tumors, such as the cyclic guanosine monophosphate–dependent protein kinase and focal adhesion signaling pathways. Finally, the least absolute shrinkage and selection operator algorithm was employed to establish a prognostic prediction model for cervical cancer patients, with training and test sets used for testing and validation, respectively. In summary, the specific DNA methylation site–based classification is able to reflect the heterogeneity of cervical cancer tissue, contributing to the development of personalized therapy and the accurate prediction of patient prognosis.

Peripheral blood circulating microRNA‐4636/−143 for the prognosis of cervical cancer

AbstractCervical cancer is the third leading cause of female death in the world. Serum microRNAs (miRNAs) are currently considered to be valuable as noninvasive cancer biomarkers, but their role in the prognosis of cervical cancer has not been elucidated. We aimed to find serum miRNAs that can be used as prognostic factors for cervical cancer. A traumatic pathological biopsy is the only reliable method for determining the severity of cervical cancer currently. Thus, noninvasive diagnostic markers are needed. The serological expression of candidate miRNAs were measured in 90 participants, including 60 patients with cervical cancer and 50 patients with cervical intraepithelial neoplasia. Two patients with cervical cancer were excluded from the study because of lack of data. miRNAs were evaluated by quantitative reverse transcription polymerase chain reaction. miR‐143/−4636 appeared specific for cervical cancer compared with cervical intraepithelial neoplasia (P < .001). The classification performance of validated miRNAs for cervical cancer [Area under the receiver operating characteristic curve (AUC) = 0.942] was better than that reached by squamous cell carcinoma antigen (SCC‐Ag; AUC = 0.727). Poor‐differentiation group has lower miR‐143/−4636 levels in serum (P < .05). miR‐4636 level was correlated gross tumor volume and the depth of invasion (P < .0001). In our study, we found a combination of miR‐143 and miR‐4636 that is independently and strongly associated with cervical cancer prognosis and can be used as a clinically prognostic factor.

LncRNA TP73‐AS1 is a novel regulator in cervical cancer via miR‐329‐3p/ARF1 axis

AbstractCervical cancer holds one of the highest morbidity and mortality in various types of cancers. It even leads to the most number of cancer‐related deaths of women. A lot of research has indicated that the anomalous expression of long noncoding RNAs (lncRNAs) would induce carcinogenesis and is associated with poor prognosis of patients with cancer. However, the function and mechanism of many lncRNAs still call for further research. Tumor Protein P73 Antisense RNA 1 (TP73‐AS1) is no exception. LncRNA TP73‐AS1 has been found to promote cancer progressions in various cancers. It is upregulated in cervical cancer cells. The proliferation and migration ability of cervical cancer cells can also be boosted by TP73‐AS1 in return. Meanwhile, miRNA‐329‐3p is downregulated in cervical cancer cells and could bind with both TP73‐AS1 and ADP Ribosylation Factor 1 (ARF1). TP73‐AS1 inhibited miR‐329‐3p expression while miR‐329‐3p inhibited ARF1 expression. More importantly, TP73‐AS1 can positively regulate ARF1 expression. Based on all these experiments, TP73‐AS1 regulates ARF1 expression by competitively binding with miR‐329‐3p, thus regulating cervical cancer progression. Further rescue assays confirmed TP73‐AS1 regulates cervical cell proliferation and migration via miR‐329‐3p/ARF1. TP73‐AS1 might serve as a novel regulator in cervical cancer.

Identifying prognostic biomarkers in endometrial carcinoma based on ceRNA network

AbstractPurposeEndometrial carcinoma (EC), a common gynecological malignancy with high incidence, affects the mental and physical health of women. Mounting evidence shows that long noncoding RNAs (lncRNAs), messenger RNAs (mRNAs), and microRNAs (miRNAs) have instrumental roles in various biological processes associated with the pathogenesis of EC. In this research, we intend to further study the mechanism of EC and the potential predictive markers of EC.MethodsFirst, we obtained original data of EC RNA transcripts from The Cancer Genome Atlas database and performed differential analysis. Subsequently, according to the miRcode online software, relationship pairs of lncRNA‐miRNA were constructed, and miRNA‐mRNA pairs were established based on miRDB, TargetScan, and miRTarBase. Then, we constructed the competing endogenous RNA (ceRNA) network based on lncRNA‐miRNA and miRNA‐mRNA pairs. To further explain the function of the ceRNA network and explore the potential prognostic markers, functional enrichment analysis, and survival analysis were carried out.ResultsThe research showed that there were 744 differential expression lncRNAs (DElncRNAs), 164 differential expression miRNAs (DEmiRNAs), and 2447 differential expression mRNAs (DEmRNAs) between EC tissues and normal tissues. Subsequently, we built 103 DEmiRNA‐DEmRNA interaction pairs and 369 DElncRNA‐DEmiRNA pairs. Then, we established the ceRNA network of EC, including 62 DElncRNAs, 26 DEmiRNAs, and 70 DEmRNAs. Moreover, 10 of 62 lncRNAs, 19 of 70 mRNAs, and 4 of 26 miRNAs that closely related to the survival of EC with P < .05 were obtained. Notably, based on this network, it was found that LINC00261‐hsa‐mir‐31 pair and LINC00261‐hsa‐mir‐211 target pairs could be used as the potential prognostic markers of EC.ConclusionThis research recommended an available basis for the molecular mechanism of EC and prognosis prediction, which could help guide the subsequent treatments and predict the prognosis for patients with EC.

Mesenchymal stem cells and ovarian cancer: Is there promising news?

AbstractOvarian cancer (OC) is described as a heterogeneous complex condition with high mortality, weak prognosis, and late‐stage presentation. OC has several subgroups based on different indices, like the origin and histopathology. The current treatments against OC include surgery followed by chemotherapy and radiotherapy; however, these methods have represented diverse side effects without enough effectiveness on OC. Recently, mesenchymal stem cell (MSC)‐based therapy has acquired particular attention for treating diverse problems, such as cancer. These multipotent stem cells can be obtained from different sources, such as the umbilical cord, adipose tissues, bone marrow, and placenta, and their efficacy has been investigated against OC. Hence, in this narrative review, we aimed to review and discuss the present studies about the effects of various sources of MSCs on OC with a special focus on involved mechanisms.

Oncogene‐mediated nuclear accumulation of lactate promotes epigenetic alterations to induce cancer cell proliferation

AbstractHomeobox gene families are associated with embryonic development and organogenesis. Pieces of evidence suggest that these Homeobox genes are also crucial in facilitating oncogenesis when mutated or overexpressed. Paired homeodomain transcription factor‐2 (PITX2), one of the members of this family, is involved in oncogenic regulation apart from its different development regulatory functions. PITX2 has been earlier shown to induce ovarian cancer cell proliferation through the activation of different signaling cascades. Increased cancer cell proliferation requires a constant supply of nutrients for both adenosine triphosphate and biomass synthesis, which is facilitated by altered cancer cell metabolism that includes enhanced glucose uptake and increased glycolytic rate. This present study highlights the involvement of PITX2 in enhancing the cellular glycolysis pathway in ovarian cancer cells through protein kinase B‐phosphorylation (phospho‐AKT). PITX2 expression correlates positively with that of the glycolytic rate‐determining enzyme, lactate dehydrogenase‐A (LDHA), in both high‐grade serous ovarian cancer tissues and common ovarian cancer cell lines. Interestingly, transient localization of enzymatically active LDHA in the nucleus was observed in PITX2‐overexpressed ovarian cancer cells. This nuclear LDHA produces higher concentrations of the glycolytic end product, lactate, which accumulates in the nuclear compartment resulting in decreased histone deacetylase (HDAC1/2) expression and increased histone acetylation at H3/H4. However, the mechanistic details of lactate–HDAC interaction are still elusive in the earlier reports. Our in silico studies elaborated on the interaction dynamics of lactate in the HDAC catalytic core through ligand‐binding studies and molecular dynamics simulation approaches. Blocking lactate production by silencing LDHA reduced cancer cell proliferation. Thus, PITX2‐induced epigenetic changes can lead to high cellular proliferation and increase the size of tumors in syngeneic mice as well. Taken together, this is the first report of its kind to show that the developmental regulatory homeobox gene PITX2 could enhance oncogenesis through enhanced glycolysis of tumor cells followed by epigenetic modifications.

VEGFR3 suppression through miR‐1236 inhibits proliferation and induces apoptosis in ovarian cancer via ERK1/2 and AKT signaling pathways

AbstractVascular endothelial growth factor receptor 3 (VEGFR3) is expressed in cancer cell lines and exerts a critical role in cancer progression. However, the signaling pathways of VEGFR3 in ovarian cancer cell proliferation remain unclear. This study aimed to demonstrate the signaling pathways of VEGFR3 through the upregulated expression of miR‐1236 in ovarian cancer cells. We found that the messenger RNA and protein of VEGFR3 were expressed in the ovarian cancer cell lines, but downregulated after microRNA‐1236 (miR‐1236) transfection. The inhibition of VEGFR3, using miR‐1236, significantly reduced cell proliferation, clonogenic survival, migration, and invasion ability in SKOV3 and OVCAR3 cells (p < 0.01). The flow cytometry results indicated that the rate of apoptotic cells in SKOV3 (38.65%) and OVCAR3 (41.95%) cells increased following VEGFR3 inhibition. Moreover, VEGFR3 stimulation (using a specific ligand, VEGF‐CS) significantly increased extracellular signal‐regulated kinase 1/2 (ERK1/2) and protein kinase B (AKT) phosphorylation (p < 0.01), whereas VEGFR3 suppression reduced p‐ERK1/2 (67.94% in SKOV3 and 93.52% in OVCAR3) and p‐AKT (59.56% in SKOV3 and 78.73% in OVCAR3) compared to the VEGF‐CS treated group. This finding demonstrated that miR‐1236 may act as an endogenous regulator of ERK1/2 and AKT signaling by blocking the upstream regulator of VEGFR3. Overall, we demonstrated the important role of the miR‐1236/VEGFR3 axis in ovarian cancer cell proliferation by regulating the ERK1/2 and AKT signaling that might be an effective strategy against ovarian cancer.

The lncRNA SNHG15/miR‐18a‐5p axis promotes cell proliferation in ovarian cancer through activating Akt/mTOR signaling pathway

AbstractHere, we report the expression pattern, function and regulatory mechanism of SNHG15 together with miR‐18a‐5p micro RNA in ovarian cancer (OC) for the first time. We recruited 20 patients and took normal ovarian tissues and ovarian tumor tissues from them. We used cell culture, transfection, in vivo tumor xenograft assay, and multiple types of detection assays to investigate the expression and regulation of long noncoding RNA (lncRNA) SNHG15/miR‐18a‐5p in ovarian tissues and cells. Results: We found that the messenger RNA expression level of SNHG15 was significantly higher and miR‐18 was decreased in ovarian cancer tissues and in OC cells. Functional experiments showed that SNHG15 overexpression potentiated the migration and invasion of OC cells, while SNHG15 inhibition reduced the tumor proliferation, which was restored via overexpression of miR‐18a. SNHG15 was found to directly target and suppress the expression of miR‐18a. Our results illustrate the possible molecular mechanism of lncRNA SNHG15/miR‐18a‐5p functions in cell proliferation in OC. SNHG15/miR‐18a promoted the progression of OC cells via the protein kinase B/mammalian target of rapamycin signaling pathway.

The role of nucleolar spindle‐associated protein 1 in human ovarian cancer

AbstractOvarian cancer (OC) is one of the deadliest malignancies of the female reproductive system. The present study focused on the role of Nucleolar spindle‐associated protein 1 (NuSAP1) in OC. Relative expression of NuSAP1 was detected in OC tissues as well as cells. After knocking down NuSAP1 with lentivirus‐mediated shRNA and verifying the knockdown efficiency via quantitative real‐time polymerase chain reaction and Western blot assays, the cell proliferation, apoptosis, and cell cycle were determined using 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide, colony formation, and flow cytometry, respectively. Transwell assay was conducted to detect the migration and invasion of OC cells. It was showed that NuSAP1 was abundantly expressed in OC tissues and cell lines. After knocking down NuSAP1 in OC cells, in addition to significantly inhibiting proliferation and colony forming ability, it also promotes apoptosis and affects cell cycle distribution. Moreover, cells in the shNuSAP1 group showed significantly suppressed migration and invasion ability compared with that in the shCtrl group. In conclusion, NuSAP1 may act as an oncogenic factor in OC and therefore might serve as an indicator for prognosis and therapeutic target for OC treatment.

NCALD affects drug resistance and prognosis by acting as a ceRNA of CX3CL1 in ovarian cancer

AbstractDrug resistance, an impenetrable barrier in the treatment of ovarian cancer (OC), is often associated with poor outcomes. Hence, it is urgent to discover new factors controlling drug resistance and survival. The association between neurocalcin delta (NCALD) and cancer drug resistance is poorly understood. Here, we reveal that NCALD messenger RNA expression, probably regulated by DNA methylation and microRNAs, was significantly downregulated in at least three independent microarrays covering 633 ovarian carcinomas and 16 normal controls, which includes the Cancer Genome Atlas (TCGA) ovarian cohort. In the sub‐groups of the TCGA cohort, NCALD was suppressed in 90 platinum‐resistant tissues vs in 197 sensitive tissues. It is consistent with the quantitative reverse transcription polymerase chain reaction results revealing gene downregulation in carboplatin‐resistant SKOV3 and HeyA8 OC cells as compared with that in controls. Low expression of NCALD predicted poor overall survival (OS) in sub‐groups of 1656 patients, progression‐free survival (PFS) in 1435 patients, and post‐progression survival (PPS) in 782 patients according to Kaplan‐Meier plotter covering 1815 OC patients. Comprehensive bioinformatic analyses strongly implicated NCALD in the regulation of drug resistance, probably via competing for endogenous RNA (ceRNA) interactions with CX3CL1 and tumor immune‐microenvironment. NCALD acted as a ceRNA for CX3CL1 in 21 different cancers includes OC according to Starbase. These two genes negatively correlated with tumor purity and positively correlated with infiltration levels of neutrophils and dendritic cells in OC. The combined low expression of NCALD and CX3CL1 showed better prognosis potential for OS, PFS, and PPS in the 1815 OC patients than any of the individually tested genes. In summary, NCALD acts as a ceRNA for CX3CL1, and its downregulation may affect drug resistance and prognosis in OC. Thus, NCALD could be a new therapeutic target for anticancer therapy and a new biomarker for survival prediction in OC.

Interference with SMO increases chemotherapy drug sensitivity of A2780/DDP cells by inhibiting the Hh/Gli signaling pathway

AbstractAberrant activation of the Hedgehog (Hh)/Gli pathway contributes to the tumorigenesis of several human cancers, including ovarian cancers. We investigated the function of SMO on cell growth, drug resistance, and invasive ability in A2780/DDP cells. Moreover, we also tested the levels of the downstream target genes of the Hh/Gli pathway in SMO short hairpin RNA (shRNA) lentivirus‐infected A2780/DDP cells. Western blot analysis results revealed that the Hh/Gli pathway was activated in cisplatin‐resistant A2780/DDP cells. After infection by SMO shRNA lentivirus, the colony formation rate and invasive rate of cisplatin‐resistant A2780/DDP cells were decreased. 3‐(4,5‐Dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide assay showed that upon transfection with SMO shRNA, cell growth was decreased and drug sensitivity to cisplatin was upregulated. Moreover, interference with SMO decreased the expression of MMP‐2, MMP‐9, VEGF, and Snail in cisplatin‐resistant cells. Thus, the Hh/Gli signaling pathway was aberrantly activated in A2780/DDP cells. The colony formation rate and invasive rate were decreased in SMO shRNA lentivirus–infected A2780/DDP cells. All results showed that inhibiting Hh/Gli signaling may negatively regulate the proliferation, invasion, and metastasis of cisplatin‐resistant A2780/DDP cells, as well as increase the sensitivity of A2780/DDP to the chemotherapeutic drug of cisplatin.

A single‐nucleotide polymorphism in lnc‐LAMC2‐1:1 interferes with its interaction with miR‐128 to alter the expression of deleted in colorectal cancer and its effect on the survival rate of subjects with ovarian cancer

AbstractThis study aimed to identify the association between lnc‐LAMC2‐1:1 polymorphism rs2147578 and the recurrence of ovary cancer, as well as to study the underlying mechanism of rs2147578 in ovary cancer. Real‐time polymerase chain reaction, Western blot analysis, immunohistochemistry, 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide assay, Logrank test, and Kaplan‐Meier analysis were carried out to explore the role of rs2147578 in ovary cancer. No obvious difference was observed concerning all clinical characteristics among 90 patients genotyped as CC (N = 28), CG (N = 38), and GG (N = 24) in their rs2147578 polymorphism. In addition, the subjects carrying the CC genotype had longer recurrence‐free survival time and showed a lower level of malignancy compared with those carrying CG and GG genotypes. Lnc‐LAMC2‐1:1 and miR‐128 were lowly expressed in the CC group, while deleted in colorectal cancer (DCC) was highly expressed in the CC group. Furthermore, DCC was identified as a target gene of miR‐128, and miR‐128 mimics decreased the luciferase activity of cells cotransfected with wild‐type DCC 3′‐untranslated region. Lnc‐LAMC2:1‐1 directly targeted and affected miR‐128 expression, and the G allele in lnc‐LAMC2‐1:1 rs2147578 upregulated miR‐128 expression. Transfection with a miR‐128 precursor evidently downregulated the expression of lnc‐LAMC2‐1:1, miR‐128, and DCC expression, but did not affect the expression of ABCC5 and body mass index. Finally, miR‐128 precursor promoted cell proliferation and inhibited cell apoptosis. Compared with lnc‐LAMC2‐1:1 rs2147578C allele, the G allele increases the risk of ovarian cancer by reducing the binding between lnc‐LAMC2‐1:1 and miR‐128‐3p, which in turn further decreases the expression of DCC and inhibits cell apoptosis.

The activation of M2 muscarinic receptor inhibits cell growth and survival in human epithelial ovarian carcinoma

AbstractOvarian cancer is the fifth leading cause of cancer‐related deaths in females. Many ovarian tumor cell lines express muscarinic receptors (mAChRs), and their expression is correlated with reduced survival of patients. We have characterized the expression of mAChRs in two human ovarian carcinoma cell lines (SKOV‐3, TOV‐21G) and two immortalized ovarian surface epithelium cell lines (iOSE‐120, iOSE‐398). Among the five subtypes of mAChRs (M1–M5 receptors), we focused our attention on the M2 receptor, which is involved in the inhibition of tumor cell proliferation. Western blot analysis and real‐time PCR analyses indicated that the levels of M2 are statistically downregulated in cancer cells. Therefore, we investigated the effect of arecaidine propargyl ester hydrobromide (APE), a preferential M2 agonist, on cell growth and survival. APE treatment decreased cell number in a dose and time‐dependent manner by decreasing cell proliferation and increasing cell death. FACS and immunocytochemistry analysis have also demonstrated the ability of APE to accumulate the cells in G2/M phase of the cell cycle and to increase the percentage of abnormal mitosis. The higher level of M2 receptors in the iOSE cells rendered these cells more sensitive to APE treatment than cancer cells. The data here reported suggest that M2 has a negative role in cell growth/survival of ovarian cell lines, and its downregulation may favor tumor progression.