Paracancerous Specimens Research Articles

Role of COX-2/PGE2/EP4 Axis-induced Macrophage Functional Activation in NSCLC Development

Tumor microenvironment (TME) is one of the important factors in tumorigenesis and progression, in which tumor-associated macrophages (TAMs) play an important role in non-small cell lung cancer (NSCLC) progression. However, the mechanism of TAMs in NSCLC progression remains unclear, so this study aimed to investigate the role of TAMs in NSCLC progression and to find potential therapeutic targets. Gene Expression Profiling Interactive Analysis (GEPIA) database was used to analyze the expression of prostaglandin E2 receptor 4 (EP4) mRNA in NSCLC and normal lung tissues; the protein expression levels of cyclooxygenase-2 (COX-2), EP4, cluster of differentiation 86 (CD86), CD163 and CD31 were detected by immunohistochemistry (IHC) in 120 NSCLC tissues and 24 paracancerous tissues specimens. The nude mouse lung adenocarcinoma cell A549 and macrophage RAW264.7 co-transplanted tumor model was established. And the samples were collected by gavage with EP4 inhibitor E7046, and then stained with hematoxylin-eosin (HE), IHC, and immunofluorescence (IF), and then detected by Western blot for the epithelial mesenchymal transformation (EMT) of the tumor tissues of the nude mice in each group. Western blot was used to detect the expressions of EMT related protiens in each group of nude mice; full-length transcriptome sequencing was used to screen the key genes causing liver metastasis and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis was performed. EP4 mRNA expression level in NSCLC tissues was generally lower than that in normal lung tissues (P<0.05); COX-2, EP4, CD163, CD31 proteins were differentially expressed in NSCLC tissues and adjacent tissues, and differences were observed in many clinicopathological parameters of NSCLC patients; RAW264.7 shortened the latency period of tumorigenesis of A549 and promoted the proliferation of tumors and liver metastasis of tumors, and E7046 could reduce tumor cell proliferation activity, tumor tissue vascular density and M2-type macrophage infiltration in nude mice; IF staining showed that macrophages were mainly distributed around the metastatic foci of tumors; Western blot results showed that compared with A549 alone transplantation group, the relative expression of E-cadherin protein in tumor tissues of mice in A549 and RAW264.7 co-transplantation group was significantly decreased, and the difference was statistically significant (P<0.05), while the relative expression of N-cadherin protein was up-regulated, but the difference was not statistically significant (P>0.05); the main pathways enriched in the differential genes of the full-length transcriptome were the PI3K-AKT and MAPK signaling pathways. During NSCLC development, the COX-2/PGE2/EP4 axis may promote tumor progression by inducing macrophage functional activation, and EP4 may be a potential new target for tumor immunotherapy. This study provides new perspectives and ideas for in-depth exploration of the mechanisms of NSCLC development, as well as a theoretical basis for the development of new therapeutic strategies for NSCLC.

Connexin43 is associated with the progression of clear cell renal carcinoma and is regulated by tangeretin to sygergize with tyrosine kinase inhibitors

BackgroundThe roles of Connexin43 (Cx43) in clear cell renal cell carcinoma (ccRCC) microenviroment remains to be poorly defined. MethodsThe expression profile, prognosis and immune analysis of Cx43 in various cancers, particularly in ccRCC were performed using TCGA database, and various biological function assays were applied to explore the physiological role of Cx43 and tangeretin in ccRCC. Western blot were applied to examine the protein expression and Kunming mice were used to evaluate preliminary safety or anti-tumor activity of tangeretin and sunitinib. ResultsCompared with the normal group, higher expression levels of Cx43 in ccRCC, and distinct associations between Cx43 expression and ccRCC prognosis or immune infiltration, were found. Notably, the expression of Cx43 was found to be highly correlated with that of receptor tyrosine kinases (RTKs), particularly with VEGFR1, VEGFR2 and VEGFR3. The expression of Cx43 and EGFR was also found to be higher in ccRCC than that in the para-cancerous specimens. Knocking down Cx43 expression decreased RCC cell viability, cell migration, p-EGFR, MMP-9 and survivin expression. Using 14 Chinese medicine monomers, tangeretin was screened and found to inhibit tumor cell viability and Cx43 expression. Tangeretin also enhanced the sensitivity of RCC cells to tyrosine kinase inhibitors (TKIs) sunitinib and sorafenib. However, the same concentration of tangeretin exerted a less prominent effect on normal renal cell viability. ConclusionsCx43 is strongly associated with RTK expression and ccRCC progression, while tangeretin can inhibit RCC cell malignancy by inhibiting Cx43 expression and enhance the sensitivity of RCC cells to TKIs.

Chloride Intracellular Channel 1 is a Potential Biomarker for Breast Cancer.

PurposeMultiple reports have demonstrated that highly expressed chloride intracellular channel 1 (CLIC1) exists in a range of malignant tumors and is involved in proliferation, invasion, and migration of cancer cells. There are few studies on CLIC1 and breast cancer (BC). The purpose of this research was to evaluate the expression level of CLIC1 in BC and its impact on prognosis of BC patients.Patients and MethodsDifferences in CLIC1 expression levels in 25 pairs of BC and corresponding paracancerous specimens were tested by quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot (WB). Immunohistochemistry (IHC) was performed to discuss the relevance between CLIC1 expression in BC tissue chips and clinicopathological parameters of BC patients. The effect of CLIC1 expression on patient prognosis was evaluated by Kaplan–Meier survival curve and Cox regression analysis. Receiver operating characteristic (ROC) curve assessed the diagnostic performance of CLIC1 for BC.ResultsThe experimental results of qRT-PCR and WB demonstrated that CLIC1 was highly expressed in BC tissues. IHC results showed that overexpression of CLIC1 was strictly correlated with tumor size, TNM classification, pathological grade, lymph node metastasis and Ki67. Patients with lower CLIC1 expression had longer overall survival (OS) and progression-free survival (PFS). Cox regression analysis and ROC curve confirmed that CLIC1 could independently influence the prognosis of BC patients and might have diagnostic efficiency.ConclusionOverexpressed CLIC1 is closely related to the progression of BC and the poor prognosis of the patients, suggesting that it may act as a potential biological diagnostic index for BC.

Overexpressed lncRNA FTX promotes the cell viability, proliferation, migration and invasion of renal cell carcinoma via FTX/miR‑4429/UBE2C axis.

The present study aimed to explore the role of long non-coding (lnc)RNA FTX and ubiquitin-conjugating enzyme E2C (UBE2C) in promoting the progression of renal cell carcinoma (RCC) and the underlying regulatory mechanism. Relative levels of lncRNA FTX, UBE2C, AKT, CDK1 and CDK6 in RCC cell lines were detected by reverse transcription-quantitative (RT-q). Expression levels of UBE2C, phosphorylated (p)-AKT/AKT, p-CDK1/CDK1 and p-CDK6/CDK6 in RCC and paracancerous specimens and RCC cells were measured by western blot or immunohistochemistry assay. In addition, the proliferative rate, cell viability, cell cycle progression, migratory rate and invasive rate of RCC cells overexpressing lncRNA FTX by lentivirus transfection were determined by a series of functional experiments, including the colony formation assay, MTT assay, flow cytometry, Transwell assay and wound healing assay. The targeted binding relationship in the lncRNA FTX/miR-4429/UBE2C axis was validated by dual-luciferase reporter assay. By intervening microRNA (miR)-4492 and UBE2C by the transfection of miR-4429-mimics or short interfering UBE2C-2, the regulatory effect of lncRNA FTX/miR-4429/UBE2C axis on the progression of RCC was evaluated. Finally, a xenograft model of RCC in nude mice was established by subcutaneous implantation, thus evaluating the in vivo function of lncRNA FTX in the progression of RCC. The results showed that lncRNA FTX and UBE2C were upregulated in RCC specimens and cell lines. The overexpression of lncRNA FTX in RCC cells upregulated UBE2C. In addition, the overexpression of lncRNA FTX promoted the cell viability and proliferative, migratory and invasive capacities of RCC cells and accelerated the cell cycle progression. A dual-luciferase reporter assay validated that lncRNA FTX exerted the miRNA sponge effect on miR-4429, which was bound to UBE2C 3′UTR. Knockdown of UBE2C effectively reversed the regulatory effects of overexpressed lncRNA FTX on the abovementioned phenotypes of RCC cells. In the xenograft model of RCC, the mice implanted with RCC cells overexpressing lncRNA FTX showed a larger tumor size and higher tumor weight than those of controls, while the in vivo knockdown of UBE2C significantly reduced the size of RCC lesions, indicating the reversed cancer-promoting effect of lncRNA FTX. Overall, the present study showed that lncRNA FTX was upregulated in RCC and could significantly promote the proliferative, migratory and invasive capacities, enhancing the viability and accelerating the cell cycle progression of RCC cells by exerting the miRNA sponge effect on miR-4429 and thus upregulating UBE2C. lncRNA FTX and UBE2C are potential molecular biomarkers and therapeutic targets of RCC.

Distribution and Clinical Significance of IL-17A in Tumor-Infiltrating Lymphocytes of Non-Small Cell Lung Cancer Patients.

Objective: To investigate the distribution of IL-17A and its clinical significance in tumor infiltrating lymphocytes (TILs) of patients with non-small cell lung cancer (NSCLC). Methods: Expression level of IL-17A in TILs of 3 paired NSCLC and paracancerous specimens was measured by qRT-PCR. The distribution of IL-17A in immune cell subsets of 15 paired NSCLC and paracancerous specimens was examined by flow cytometry. The correlation between IL-17A and clinical features of NSCLC was identified. Results: IL-17A was significantly upregulated in TILs of NSCLC specimens than those of paracancerous ones (p < 0.0001). Meanwhile, T helper 17 cells (Th17 cells, p < 0.001), IL-17-secreting CD8+ T cells (Tc17 cells, p < 0.001) and IL-17-producing cells (γδT17 cells, p < 0.0001) were significantly abundant in TILs of NSCLC specimens than those of controls, and the higher abundance of the latter was much pronounced than that of the former two. Moreover, γδT17 cells in TILs were significantly correlated with lymphatic metastasis and CYFRA 21-1 level of NSCLC patients (p < 0.05). Conclusion: Tumor infiltrated γδT cells are the main source of IL-17 in early-stage NSCLC, and IL-17 may be a vital regulator involved in the development of NSCLC.

Histone methyltransferase KMT2D cooperates with MEF2A to promote the stem-like properties of oral squamous cell carcinoma

BackgroundEpigenetic reprogramming is involved in multiple steps of human cancer evolution and is mediated by a variety of chromatin-modifying enzymes. Specifically, the histone lysine methyltransferase KMT2D is among the most frequently mutated genes in oral squamous cell carcinoma (OSCC). However, the mechanisms by which KMT2D affects the development of OSCC remain unclear.ResultsIn the present study, we found that the expression of KMT2D was elevated in OSCC compared to paracancerous specimens and was correlated with a more advanced tumor grade. More importantly, knockdown of KMT2D impaired their reconstitution in patient-derived organoids and decreased the expression of CD133 and β-catenin in OSCC cells. In in vitro and in vivo models, knockdown of KMT2D reduced the colony formation, migration and invasion abilities of OSCC cells and delayed tumor growth. Mechanistically, the dual-luciferase reporter and co-immunoprecipitation assays in two individual OSCC cell lines indicated that KMT2D may cooperate with MEF2A to promote the transcription activity of CTNNB1, thereby enhancing WNT signaling.ConclusionThe upregulation of KMT2D contributes to stem-like properties in OSCC cells by sustaining the MEF2A-mediated transcriptional activity of CTNNB1.

Circular RNA hsa_circ_0004543 Aggravates Cervical Cancer Development by Sponging MicroRNA hsa-miR-217 to Upregulate Hypoxia-Inducible Factor.

Cervical cancer (CC) is the 4th principal source of cancer death in females with 604,000 new patients and 342,000 deaths in 2020 worldwide. It has been extensively shown that circRNAs are involved in regulating CC development. Nevertheless, the function and mechanisms of hsa_circ_0004543 in regulating CC need to be clearly elucidated. Herein, hsa_circ_0004543 expressions were compared between 40 paired paracancerous and cancerous specimens from CC patients and between 6 CC cell lines and a normal human cervical epithelial cell line based on qRT-PCR. Potential complementary binding sites between hsa-miR-217 and hsa_circ_0004543 were predicted using the interactome, while binding sites for the hypoxia-inducible factor-1a (HIF-1a) were predicted by TargetScan. The function and mechanism of hsa_circ_0004543 in the development of CC were estimated by silencing hsa_circ_0004543 with/without hsa-miR-217 or HIF-1a overexpression. The association between gene expressions was evaluated with Pearson's correlation analysis. Molecular mechanisms were explored by ribonucleic acid (RNA) pulldown, dual-luciferase activity, and rescue experimental assays. Our results revealed that the hsa_circ_0004543 expression was considerably increased in CC tissues and cells. Its silencing repressed proliferation and metastasis, while it increased apoptosis of CC cells. The investigation of the mechanism showed that hsa-miR-217 silencing or HIF-1a overexpression rescued hsa_circ_0004543, and silencing inhibited malignant phenotypes of CC cells. hsa_circ_0004543 upregulated the HIF-1α expression by sponging hsa-miR-217 in CC development. Therefore, the hsa_circ_0004543 functioned as a competing endogenous RNA (ceRNA) of hsa-miR-217 to increase CC oncogenesis and metastasis by the upregulation of the HIF-1α expression. Consequently, targeting the hsa_circ_0004543/hsa-miR-217/HIF-1α axis might be a potential treatment approach for CC.

Expression of CXCR4 and CXCR7 in papillary thyroid carcinoma and adjacent tissues and their relationship with pathologic indicators of tumor aggressiveness

The receptors of chemokines play a significance role in the aggressiveness of tumor. CXCR4/CXCR7 promote metastasis of papillary thyroid carcinoma (PTC). This study examined the expresssion of chemokine receptors CXCR4/CXCR7 in human tissue specimens of PTC and peritumoral nonmalignant tissues. The correlation between CXCR4/CXCR7 and the clinicopathological factors in PTC was also determined. CXCR4/CXCR7 were examined in 115 PTC tissues from 115 patients using immunohistochemistry. Staining intensity was compared with patients and tumor characteristics including gender, age, tumor size, capsule invasion, multifocality, lymph node metastasis, and nature of paracancerous tissue. [Statistics: rank sum test, Spearman rank order correlation test; p < 0.05]. Higher expression rates of CXCR4/CXCR7 exhibited in PTC compared with peritumoral nonmalignant tissues. The expression of them was correlated in cancer and paracancerous specimens. A trend toward higher CXCR4/CXCR7 expression was found among tumors showing positive lymph nodes and capsule invasion, while no association with sex, age, tumor size, and nature of paracancerous tissue. Number of lymph nodes was associated with higher intensity IHC staining for CXCR4/CXCR7. Intense staining for CXCR4 was also associated with multifocality. Expression of CXCR4/CXCR7 by PTCs was correlated with lymph node metastasis and capsule invasion. Although multiple bias, they were thought to play a significance role in the aggressiveness of PTC, which provides potential targets for therapeutic interventions.

A novel ferroptosis-related genes model for prognosis prediction of lung adenocarcinoma

BackgroundFerroptosis is a newly discovered form of cell death characterized by iron-dependent lipid peroxidation. This study aims to investigate the potential correlation between ferroptosis and the prognosis of lung adenocarcinoma (LUAD).MethodsRNA-seq data were collected from the LUAD dataset of The Cancer Genome Atlas (TCGA) database. Based on ferroptosis-related genes, differentially expressed genes (DEGs) between LUAD and paracancerous specimens were identified. The univariate Cox regression analysis was performed to screen key genes associated with the prognosis of LUAD. LUAD patients were divided into the training set and validation set. Then, we screened out key genes and built a prognostic prediction model involving 5 genes using the least absolute shrinkage and selection operator (LASSO) regression with tenfold cross-validation and the multivariate Cox regression analysis. After dividing LUAD patients based on the median level of risk score as cut-off value, the generated prognostic prediction model was validated in the validation set. Moreover, we analyzed the somatic mutations, and estimated the scores of immune infiltration in the high-risk and low-risk groups. Functional enrichment analysis of DEGs was performed as well.ResultsHigh-risk scores indicated the worse prognosis of LUAD. The maximum area under curve (AUC) of the training set and the validation set in this study was 0.7 and 0.69, respectively. Moreover, we integrated the age, gender, and tumor stage to construct the composite nomogram. The charts indicated that the AUC of LUAD cases with the survival time of 1, 3 and 5 years was 0.698, 0.71 and 0.73, respectively. In addition, the mutation frequency of LUAD patients in the high-risk group was significantly higher than that in the low-risk group. Simultaneously, DEGs were mainly enriched in ferroptosis-related pathways by analyzing the functional results.ConclusionsThis study constructs a novel LUAD prognosis prediction model involving 5 ferroptosis-related genes, which can be used as a promising tool for decision-making of clinical therapeutic strategies of LUAD.

Construction and validation of an immune-related lncRNA prognostic model for rectal adenocarcinomas*

Abstract Objective This study aimed to construct a prognostic model for rectal adenocarcinomas based on immune-related long noncoding RNAs (lncRNAs) and verify its prediction efficiency. Methods Transcript data and clinical data of rectal adenocarcinomas were downloaded from The Cancer Genome Atlas (TCGA) database. Perl software (strawberry version) and R language (version 3.6.1) were used to analyze the immune-related genes and immune-related lncRNAs of rectal adenocarcinomas, and the differentially expressed immune-related lncRNAs were screened according to the criteria |log2FC| &gt; 1 and P &lt; 0.05. The key immune-related lncRNAs were screened using single-factor Cox regression analysis and lasso regression analysis. Multivariate Cox regression analysis was performed to construct an immune-related lncRNA prognostic model using the risk scores. Next, we evaluated the effectiveness of the model through Kaplan-Meier (K-M) survival analysis, ROC curve analysis, and independent prognostic analysis of clinical features. In addition, prognostic biomarkers of immune-related lncRNAs in the model were analyzed by K-M survival analysis. Results In this study, we obtained gene expression profile matrices of 89 rectal adenocarcinomas and 2 paracancerous specimens from TCGA database and applied immunologic signatures to these transcripts. Through R and Perl software analysis, we obtained 847 immune-related lncRNAs and 331 protein-encoded immune-related genes in rectal adenocarcinomas. Eight important immune-related lncRNAs related to the prognosis of rectal adenocarcinomas were identified using univariate Cox regression and lasso regression analysis. Furthermore, four immune-related lncRNAs were identified as prognostic markers of rectal adenocarcinomas via multivariate Cox regression analysis. The prognostic risk model was as follows: risk score = (-4.084) * expression LINC01871 + (3.112) * expression AL158152.2 + (7.616) * expression PXN-AS1 + (-0.867) * expression HCP5. The independent prognostic effect of the rectal adenocarcinoma risk score model was revealed through K-M analysis, ROC curve analysis, and univariate, and multivariate Cox regression analysis (P = 0.035). LINC01871 (P = 0.006), PXN-AS1 (P = 0.008), and AL158152.2 (P = 0.0386) were closely correlated with the prognosis of rectal adenocarcinomas through the K-M survival analysis. Conclusion We constructed a prognostic model of rectal adenocarcinomas based on four immune-related lncRNAs by analyzing the data based on TCGA database, with high prediction accuracy. We also identified two biomarkers with poor prognosis (PXN-AS1 and AL158152.2) and one biomarker with good prognosis (LINC01871).

TRIM56 suppresses the malignant development of hepatocellular carcinoma via targeting RBM24 and inactivating the Wnt signaling.

The aim of this study was to explore the expression pattern of TRIM56 in Hepatocellular carcinoma (HCC) patients and its influence on the prognosis, and to illustrate the molecular mechanisms of TRIM56 in regulating HCC cell behaviors. TRIM56 levels in HCC specimens and paracancerous specimens were detected. Then, the influences of TRIM56 on clinical data and prognosis in HCC patients were assessed. Next, the regulatory effects of TRIM56 on proliferative potential in Huh7 and Bel-7402 cells were determined, and the role of TRIM56 on the Wnt signaling was examined. Finally, biological characteristics between TRIM56 and RBM24 in HCC development were illustrated by Luciferase assay and rescue experiments. TRIM56 was lowly expressed in HCC tissues and cell lines. HCC patients expressing a low level of TRIM56 suffered advanced T stage and poor survival. Besides, overexpression of TRIM56 inhibited proliferative potential of Huh7 cells, while knockdown of TRIM56 in Bel-7402 yielded the opposite result. TRIM56 was able to negatively regulate key genes in the Wnt signaling. In addition, RBM24 was proven to be the downstream target of TRIM56, which was involved in TRIM56-influenced HCC development. Downregulated TRIM56 in HCC samples is closely linked to pathological staging and prognosis. TRIM56 alleviates the malignant development of HCC by inactivating the Wnt signaling and targeting RBM24.

MiR-329-3p inhibits hepatocellular carcinoma cell proliferation and migration through USP22-Wnt/β-Catenin pathway.

MicroRNA-329-3p (miR-329-3p) has been shown to be involved in tumor development. But its role in hepatocellular carcinoma has not been explored. Our study aims to explore the effect and mechanism of miR-329-3p on hepatocellular carcinoma development. Hepatocellular carcinoma tissues and paired paracancerous specimens from 31 hepatocellular carcinoma patients undergoing surgery were collected. Quantitative real-time polymerase chain reaction and Western blot were employed to measure genes expression at mRNA and protein level. CCK-8 and transwell assays were performed to evaluate hepatocellular carcinoma cells proliferation and migration. Dual-Luciferase reporter gene assay was designed to validate the target gene of miR-329-3p. Our study showed miR-329-3p expression was significantly lower in hepatocellular carcinoma tissue. MiR-329-3p mimic inhibits proliferation and migration of HepG2 cells. By using Dual-Luciferase reporter gene assay, we proved that miR-329-3p inhibited HepG2 cell proliferation and migration by targeting USP22 directly. By up- and downregulation of USP22 expression, we also proved that USP22 can activate the Wnt/β-Catenin pathway, which in turn affected the proliferation and migration of HepG2 cells. We demonstrated that miR-329-3p can inhibit HepG2 cell proliferation and migration by inhibiting USP22-Wnt/β-Catenin pathway. Our study provides novel insights into the aetiology and potential treatment of hepatocellular carcinoma.

MiR-4262 inhibits the development of esophageal cancer by negatively regulating KLF6 level

ObjectiveTo uncover the expression pattern and the prognosis of miR-4262 in these patients with esophageal cancer, and its potential mechanism. MethodsMiR-4262 levels in 57 esophageal cancer and paracancerous specimens were detected. The relationship between miR-4262 level and clinical features of esophageal cancer was analyzed. After overexpression of miR-4262 in OE19 and EC-109 cells, changes in proliferative potential and apoptosis were examined. The interaction between miR-4262 and KLF6 was explored by dual-luciferase reporter assay. Their involvement in the development of esophageal cancer was finally determined. ResultsMiR-4262 was downregulated in esophageal cancer specimens and cell lines. Low level of miR-4262 predicted advanced pathological staging and poor prognosis in esophageal cancer patients. Overexpression of miR-4262 reduced proliferative potential and enhanced apoptosis in esophageal cancer cells. KLF6 was the downstream gene binding to miR-4262. The interaction between miR-4262 and KLF6 was responsible for alleviating the malignant development of esophageal cancer. ConclusionsMiR-4262 is downregulated in esophageal cancer and linked to its pathological staging and prognosis. MiR-4262 inhibits the malignant development of esophageal cancer by down-regulating KLF6.

MiR-193 promotes cell proliferation and invasion by ING5/PI3K/AKT pathway of triple-negative breast cancer.

Triple-negative breast cancers (TNBC) are a subtype of breast cancer lacking of estrogen receptor (ER), progesterone receptor (PR), and human EGF-like receptor 2 (HER2). MiR-193 always acted as an oncogene and promoted toxic aldehyde accumulation and tyrosine hydroxylase dysfunction. The purpose of this study is to explore the function of miR-193 in triple-negative breast cancer. Quantitative Real Time-Polymerase Chain Reaction (qRT-PCR) was performed to examine the mRNA level of miR-193 expression in 50 cases of TNBC tissues and para-cancerous specimens. Also, the relation between miR-193 level and the overall survival of TNBC patient was analyzed. MiR-193 mimic and miR-193 inhibitor oligos, as well as the corresponding negative control, were synthesized from RiboBio (Guangzhou, China). MiR-193 expression was higher in triple-negative breast cancer tissues and cell lines than the corresponding adjacent non-tumor tissues and normal cell lines. Upregulation of miR-193 predicted poor prognosis of TNBC patients. Overexpression of miR-193 promoted cell proliferation and invasion, while that was suppressed by the knockdown of miR-193. MiR-193 binds to the 3'-UTR of an inhibitor of growth family member 5 (ING5) mRNA to mediate the expression of ING5 in TNBC cells. The knockdown of miR-193 inhibited cell invasion-mediated epithelial-mesenchymal transition (EMT). Furthermore, the knockdown of miR-193 suppressed cell proliferation through the ING5/phosphatidylinositol 3-hydroxy kinase/protein kinase B (PI3K/AKT) signal pathway. MiR-193 enhanced cell invasion-mediated EMT and improved cell proliferation through the ING5/PI3K/AKT signal pathway in triple-negative breast cancer. The newly identified miR-193/ING5/PI3K/AKT axis provides novel insight into the pathogenesis of triple-negative breast cancer.

HOXA10 promotes the development of bladder cancer through regulating FOSL1.

This study was aimed to investigate the expression characteristics of HOXA10 in bladder cancer (BCa), and to further study whether it can promote the development of BCa via regulating FOSL1. The expression of HOXA10 was examined by quantitative Real Time-PCR (qRT-PCR) in 37 pairs of tumor tissue and paracancerous specimens of BCa patients; meanwhile, in BCa cell lines, the expression of HOXA10 was also verified using qRT-PCR. Subsequently, after HOXA10 knockdown model was constructed in BCa cell lines (EJ and J82) using lentivirus transfection, transwell, as well as wound healing assays, were performed to analyze the influence of the downregulation of HOXA10 on the biological function of BCa cells. Finally, Luciferase reporting assay and cell reverse experiment were applied to explore the specific interaction between HOXA10 and FOSL1. The results of qRT-PCR indicated that the expression level of HOXA10 in BCa tissue samples was remarkably higher than that in adjacent normal ones, with a statistically significant difference. At the same time, the overall survival rate of patients with high expression of HOXA10 was found to be lower than those with low expression. Meanwhile, compared with cells in sh-NC group, the metastasis ability of BCa cells in sh-HOXA10 group was remarkably weakened. In addition, it was found that the levels of FOSL1 and HOXA10 were negatively correlated in BCa tissues. The result of the Luciferase reporter gene assay revealed that HOXA10 could be targeted by FOSL1 through certain binding sites between them. In addition, HOXA10 was found to be capable of further regulating the malignant progression of BCa by modulating FOSL1. HOXA10 expression is remarkably elevated in BCa tissues and cell lines, which is closely relevant to the poor prognosis of BCa patients. In addition, HOXA10 may be able to accelerate BCa metastasis via modulating FOSL1 expression.

Targeting EZH2 by microRNA-449a inhibits osteosarcoma cell proliferation, invasion and migration via regulation of PI3K/AKT signaling pathway and epithelial-mesenchymal transition.

Osteosarcoma (OS) is one common bone malignant tumor prevailing in young adults and children. It is increasingly recognized microRNA 449a (miR 449a) as an anti-tumor factor in various tumours. However, little is known about the biological significance of miR 449a in OS. The intent of our study was to seek the prognostic values of miR-449a in OS. Quantitative real-time polymerase chain reaction (qRT-PCR) was performed to examine the level of miR-449a expression in 48 pairs of OS tissues and para-cancerous specimens, and the relationship between miR-449a level and clinical features of OS patient prognosis was analyzed. Moreover, we measured the miR-449a expression levels in OS cells. Transwell assay was further performed to investigate whether miR-449a influenced MG63 cell migration and invasion, which was important for malignant metastases. Quantitative real-time polymerase chain reaction (qRT-PCR) analysis demonstrated a notable decrease of miR-449a expressions in OS. The declined miR-449a expression was relevant with the poor prognosis and malignant clinicopathologic characteristics of OS patients. Thereafter, the functional assay was performed to determine the role of miR-449a in OS progression. Results of MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assays and transwell assays indicated that miR-449a overexpression significantly repressed OS cell proliferation, invasion, and migration. Furthermore, luciferase reporter assay showed that enhancer of zeste homolog 2 (EZH2) was a downstream target of miR-449a in OS cells. Additionally, Western blot analysis demonstrated that miR-449a exerted anti-OS functions via the regulation of phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT) signaling pathway and epithelial-mesenchymal transition. We also indicated that miR-449a restoration could inhibit in vivo tumor growth. These results manifested that miR-449a may thus be used as a therapeutic target in OS treatments.

MiR-15b facilitates breast cancer progression via repressing tumor suppressor PAQR3.

Recently, breast cancer (BC) has become a common tumor that threatens the physical and mental health of women. Microribonucleic acids (miRNAs) have been chosen as a study object because of their roles in various cancers, including BC. Here, we mainly study the role of miR-15b in BC progression and its underlying mechanism. Quantitative Real Time-Polymerase Chain Reaction (qRT-PCR) was performed to examine the level of MiR-15b expression in 60 pairs of BC tissues and para-cancerous specimens, and the relationship between MiR-15b level and clinical features of BC patient prognosis was analyzed. MiR-15b and PAQR3 level in BC tissues and cells was tested by Western blot. The results showed that miR-15b expression was higher and PAQR3 level was lower in BC. The identification of PAQR3 as a target of miR-15b in BC was carried out by Luciferase reporter assay and the results stated that the Luciferase activity was reduced by miR-15b mimic, indicating PAQR3 being a target of miR-15b in BC. Transwell assay was used for examining BC cell migration and invasion and found that miR-15b could promote BC cell migration and invasion, while the effect of PAQR3 was inhibition. Furthermore, PAQR3 could reverse the promotion effect of miR-15b on BC cell migratory and invasive ability. In addition, miR-15b expression was negatively correlated with PAQR3 performed by regression analysis. Our data stated that miR-15b could facilitate BC progression via repressing tumor suppressor PAQR3, indicating that miR-15b/PAQR3 axis provided a therapeutic target for treating BC.

MicroRNA-149 suppresses the malignant phenotypes of ovarian cancer via downregulation of MSI2 and inhibition of PI3K/AKT pathway.

Ovarian cancer (OC) is one of the most lethal gynecologic malignant tumors. Emerging evidence has indicated that the dysregulation of microRNAs (miRNAs/miRs) participates in the OC progression. It has been revealed that miR-149 acts either as an oncogene or a tumour suppressor in various human tumors. The current study focused on the biological roles and potential mechanism of miR-149 in OC. Quantitative Real Time-Polymerase Chain Reaction (qRT-PCR) was performed to examine the level of miR-149 expression in 72 pairs of OC tissues and para-cancerous specimens. We further measured the miR-149 levels in OC cells. As we indicated that miR-149 inhibited OC cell viability, we further explored the roles of miR-149 in OC cell invasion and migration by performing the transwell assays. As we suggested that MSI2 was one target for miR-149 in OC cell lines, the expressions and clinical significance of MSI2 in OC were further investigated. We first detected miR-149 expressions in the OC tissues using quantitative Real Time-Polymerase Chain Reaction (qRT-PCR) and the data showed that miR-149 was dramatically downregulated in the OC tissue samples in comparison to matched normal tissue samples. Additionally, the downregulation of miR-149 in OC was found to be related to the poor prognosis and malignant clinicopathologic characteristics of patients with OC. MiR-149 overexpression significantly suppressed the OC cell proliferation, invasion, and migration as determined by functional assays, including MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assays and transwell assays. Furthermore, the dual-luciferase reporter assay demonstrated that MSI2 was an efficient target of miR-149 in OC cells. Finally, some findings also revealed that miR-149 exerted its biological function in OC cells via direct regulation of phosphatidylinositol 3-kinase/protein kinase B (PI3K/AKT). Collectively, miR-149 exerted anti-OC roles at least partially by regulating MSI2 via PI3K/AKT. The findings of this study suggested that miR-149 might be a promising target in the diagnosis and prognosis for OC patients.

Bioinformatic analysis identifies potentially key differentially expressed genes in oncogenesis and progression of clear cell renal cell carcinoma.

Clear cell renal cell carcinoma (ccRCC) is one of the most common and lethal types of cancer within the urinary system. Great efforts have been made to elucidate the pathogeny. However, the molecular mechanism of ccRCC is still not well understood. The aim of this study is to identify key genes in the carcinogenesis and progression of ccRCC. The mRNA microarray dataset GSE53757 was downloaded from the Gene Expression Omnibus database. The GSE53757 dataset contains tumor and matched paracancerous specimens from 72 ccRCC patients with clinical stage I to IV. The linear model of microarray data (limma) package in R language was used to identify differentially expressed genes (DEGs). The protein–protein interaction (PPI) network of the DEGs was constructed using the search tool for the retrieval of interacting genes (STRING). Subsequently, we visualized molecular interaction networks by Cytoscape software and analyzed modules with MCODE. A total of 1,284, 1,416, 1,610 and 1,185 up-regulated genes, and 932, 1,236, 1,006 and 929 down-regulated genes were identified from clinical stage I to IV ccRCC patients, respectively. The overlapping DEGs among the four clinical stages contain 870 up-regulated and 645 down-regulated genes. The enrichment analysis of DEGs in the top module was carried out with DAVID. The results showed the DEGs of the top module were mainly enriched in microtubule-based movement, mitotic cytokinesis and mitotic chromosome condensation. Eleven up-regulated genes and one down-regulated gene were identified as hub genes. Survival analysis showed the high expression of CENPE, KIF20A, KIF4A, MELK, NCAPG, NDC80, NUF2, TOP2A, TPX2 and UBE2C, and low expression of ACADM gene could be involved in the carcinogenesis, invasion or recurrence of ccRCC. Literature retrieval results showed the hub gene NDC80, CENPE and ACADM might be novel targets for the diagnosis, clinical treatment and prognosis of ccRCC. In conclusion, the findings of present study may help us understand the molecular mechanisms underlying the carcinogenesis and progression of ccRCC, and provide potential diagnostic, therapeutic and prognostic biomarkers.

MiR-491 suppresses migration and invasion via directly targeting TPX2 in breast cancer.

Breast cancer (BC) is one of the primary causes of tumor-related female mortalities. Although in recent years, we have made great progress in the systemic therapy and earlier diagnosis for BC patients, recurrence or distant metastasis remains leading obstacles for the successful therapy of BC. Therefore, a comprehensive understanding of the molecular mechanism underlying the progression may be crucial in developing an effective strategy against BC. The current research aimed to explore the expressions, functions and molecular mechanism of microRNA-491 (miR-491) in BC. Quantitative Real Time-Polymerase Chain Reaction (qRT-PCR) was performed to examine the level of miR-491 expression in 52 pairs of BC tissues and para-cancerous specimens, and the relation between miR-491 level and the clinical features of BC patient prognosis was analyzed. Transwell invasion and migration assays were conducted to determine whether miR-491 had effects on the regulation of BC metastasis. Potential target genes of miR-491 were found out using TargetScan to explore the molecular functions of miR-491 in inhibiting breast cancer cell invasion and migration. To elucidate the mechanism of TPX2 in suppressing cell invasion and migration medicated by miR-491in breast cancer, we further transfected TPX2 siRNAs into MCF-7 cells to delete endogenous TPX2, along with the transfections with miR-491 inhibitor into MCF-7 cell lines. The findings demonstrated that miR-491 expressions were significantly decreased in BC tissues and cells. The miR-491 restoration suppressed the invasion and migration of BC cells. In addition, we identified the targeting protein for Xklp2 (TPX2) as a direct target of miR-491 in BC. The knockdown of TPX2 markedly reversed miR-491-medicated inhibition of cell invasion and migration in BC cell lines. In short, all the results suggested that miR-491 functioned as a tumor suppressor by targeting TPX2 in BC and the miR-491 restoration may be an effective therapy for the BC treatment in the future.