Paired Adjacent Non-tumor Tissues Research Articles

Pathway Mutations in Breast Cancer Using Whole-Exome Sequencing.

The genomic landscape of breast cancer (BC) is complex. The purpose of this study was to decipher the mutational profiles of Taiwanese patients with BC using next-generation sequencing. We performed whole-exome sequencing on DNA from 24 tumor tissue specimens from BC patients. Sanger sequencing was used to validate the identified variants. Sanger sequencing was also performed on paired adjacent nontumor tissues. After genotype calling and algorithmic annotations, we identified 49 deleterious variants in canonical cancer-related genes in our BC cohort. The most frequently mutated genes were PIK3CA (16.67%), FKBP9 (12.5%), TP53 (12.5%), ATM (8.33%), CHEK2 (8.33%), FOXO3 (8.33%), NTRK1 (8.33%), and NUTM2B (8.33%). Seven mutated variants (ATR p.V1581fs, CSF1R p.R579Q, GATA3 p.T356delinsTMKS, LRP5 p.W389*, MAP3K1 p.T918fs, MET p.K1161fs, and MTR p.P1178S) were novel variants that are not present in any gene mutation database. After grouping the samples according to molecular subtype, we found that the cell cycle, MAPK, and chemokine signaling pathways in the luminal A subtype of BC; the focal adhesion, axon guidance, and endocytosis pathways in the luminal B subtype; and amyotrophic lateral sclerosis in the basal-like subtype were exclusively altered. Survival curve analysis showed that the presence of the MAPK signaling pathway and endocytosis mutations were correlated with a poor prognosis. These survival data were consistent with cBioPortal analyses of 2,051 BC cases. We discovered novel mutations in patients with BC. These results have implications for developing strategic, adjuvant, and gene-targeted therapies.

Long noncoding RNA UCA1 promotes cell growth, migration, and invasion by targeting miR-143-3p in oral squamous cell carcinoma.

BackgroundThe long noncoding RNA (lncRNA) urothelial carcinoma‐associated 1 (UCA1) is dysregulated in many types of tumors; however, its role in oral squamous cell carcinoma (OSCC) remains unclear. This study aims to determine the effect of lncRNA UCA1 on OSCC.MethodsFifty‐six paired OSCC and adjacent nontumorous tissues were collected and the levels of UCA1, miR‐143‐3p, and MYO6 in the tissues were evaluated by qRT‐PCR. In in vitro experiments, cell viability, migration, and invasion were measured by, respectively, performing CCK‐8, wound healing, and transwell assays. The target relationships among UCA1, miR‐143‐3p, and MYO6 were verified by dual‐luciferase assay. Western blot and immunohistochemistry were carried out to determine the protein levels. Xenograft mouse model was established to explore the effects of UCA1 in vivo.ResultsLevels of UCA1 and MYO6 were increased significantly in OSCC, while the level of miR‐143‐3p was decreased compared with the adjacent nontumorous tissues. UCA1 promoted OSCC cell growth, migration, and invasion both in vitro and in vivo, while miR‐143‐3p reversed the progression. MYO6 was validated as a target for miR‐143‐3p, and MYO6 overexpression reversed the effects of miR‐143‐3p mimic on OSCC cells.ConclusionLncRNA UCA1 contributes to the proliferation and metastasis of OSCC cells by targeting miR‐143‐3p and upregulating its downstream gene MYO6. UCA1 could serve as a promising novel target therapy for treatment of OSCC.

Copy number variations of MMP-9 are prognostic biomarkers for hepatocellular carcinoma.

BackgroundThis study aimed to investigate the effect of matrix metalloproteinase-9 (MMP-9) copy number variations (CNVs) on hepatocellular carcinoma (HCC) poor prognosis and recurrence.MethodsA total of 35 patients were collected between January 2016 and December 2018. The copy number and expression level of MMP-9 were measured in 35 HCC tumor tissues and 35 paired adjacent non-tumor tissues using digital polymerase chain reaction (dPCR) and quantitative reverse transcription polymerase chain reaction (RT-qPCR), respectively.ResultsOur results showed that MMP-9 expression was significantly upregulated in HCC tumor tissues compared to adjacent non-tumor tissues (5.521±9.545 versus 1.000±0.000, P=0.0047). Interestingly, MMP-9 CNVs only existed in tumor tissues (15/35 versus 0/35, P=0.002). A breakdown analysis by the occurrence of CNVs in tumor tissues had shown that there were significant differences between CNVs group and non-CNVs group in the expression levels of tissue alpha-fetoprotein (AFP) (P=0.015), tumor size (P<0.001), differentiation (P<0.001), microvascular invasion (MVI) (P=0.009), and clinical stage (P<0.001). Receiver operating characteristic (ROC) curves showed that MMP-9 CNVs and expression were significant predictors of HCC [P<0.0001, area under the curve (AUC) =0.76].ConclusionsOur results demonstrated that MMP-9 CNVs were a promising diagnostic biomarker for HCC.

MicroRNA‐802 accelerates hepatocellular carcinoma growth by targeting RUNX3

Hepatocellular carcinoma (HCC) is one of the most common malignancies worldwide. Prognosis is often unfavorable. In this study, the effects of microRNA-802 (miR-802) on HCC progression were assessed in vivo and in vitro. miR-802 was found to be significantly upregulated in HCC tumor tissue compared to paired adjacent nontumor tissue. In vitro, transfection with a miR-802 mimic accelerated SMMC-7721 cellular proliferation, increased accumulation of the cell-cycle S-phase cell populations, as well as cell migration. In vivo injection of a miR-802 agomir promoted HCC proliferation in nude mice. Targets of miR-802 were predicted by miRWalk, miRanda, RNA22, and Targetscan. By luciferase reporter assay RUNX3 was identified as a direct target of miR-802. As judged by western blot analysis, RUNX3 was upregulated when miR-802 was inhibited. These data demonstrate increased miR-802 expression in patients with HCC and that miR-802 overexpression promotes tumor cell growth, in a RUNX3-dependent manner.

Clinicopathological and Prognostic Characteristics of RAD51 in Colorectal Cancer.

Background and Objectives: RAD51 plays an essential role in DNA repair via homologous recombination. RAD51 facilitates strand transfer between interrupted sequences and their undamaged homologies. Therefore, we studied the RAD51 mRNA expression levels in colorectal cancer (CRC), and evaluated the clinicopathological and prognostic significance of RAD51. Materials and Methods: The RAD51 expression was examined in 48 CRCs and paired adjacent non-tumor tissues. We further evaluated the survival to determine the prognostic value of RAD51 in our CRC and The Cancer Genome Atlas (TCGA) data. Results: We confirmed that the RAD51 expression in tumor tissues, compared with that of paired non-tumor tissues, was upregulated 2.5-fold. Additionally, the RAD51 expression was significantly associated with the T stage (p = 0.027). According to a higher T stage, the RAD51 expression showed an increasing trend. However, the RAD51 expression did not show a prognostic value statistically. Conclusions: We confirmed that RAD51 was upregulated in tumors and was significantly associated with the T stage. Although there was no statistically significant prognostic value found in our samples and TGCA data, our study will provide new insight for RAD51 in CRC.

MiR‐335 modulates Numb alternative splicing via targeting RBM10 in endometrial cancer

Numb is a conserved protein plays important roles in the development of cancer. Two Numb isoforms have been found produced by alternative splicing and play contrast roles in regulating cellular functions. It is reported that the expression of Numb long isoform (Numb-L) was increased in various kinds of cancers, but in endometrial cancer, the condition is still unknown. The level of two Numb transcripts and protein isoforms were detected by semiquantitative polymerase chain reaction and immunoblotting in 47 paired endometrial tumor and adjacent non-tumor control tissues. The level of three alternative splicing related proteins: RBM5, RBM6, and RBM10 was determined by immunoblotting. MiRNAs targeting RBM10 were predicted by bioinformatics tools and their interaction with RBM10 was confirmed by luciferase assay and immunoblotting. The function of miR-335 in endometrial cancer was examined in xenograft mouse model. Numb-L level was increased in tumors and negatively correlated with RBM10 protein level. RBM10 mRNA level was not significantly altered in endometrial tumors suggesting its expression may regulated by post transcriptional regulators such as miRNAs. We identified miR-133a, miR-133b, and miR-335 directly target RBM10, but only miR-335 level increased in tumors and negatively correlated with RBM10 protein level. miR-335 overexpression promoted tumor growth by downregulating RBM10 and upregulating Numb-L level in xenograft mouse model. miR-335 overexpression promoted Numb-L expression via targeting RBM10 in endometrial cancer, which may provide new biomarkers for EC diagnosis.

Up-regulation of peroxiredoxin-1 promotes cell proliferation and metastasis and inhibits apoptosis in cervical cancer.

Objective: To investigate the effect of peroxiredoxin 1 (PRDX1) on the biological behavior of cervical cancer cells and the possible mechanism.Materials and methods: The expression of PRDX1 in human cervical cancer tissues and adjacent non-tumor tissues were detected by immunohistochemistry (IHC). Lentivirus containing PRDX1-cDNA or shRNA against PRDX1 was constructed to overexpress or knockdown PRDX1 in SiHa cervical cancer cells. Cell proliferation was tested by CCK-8 and BrdU incorporation assay and cell apoptosis was evaluated by AnnexinV-PE /7AAD assay. Scratch wound and transwell invasion assay were used to test migration and invasion activity after PRDX1 was overexpressed or suppressed. Furthermore, the effect of PRDX1 on cell proliferation and apoptosis was also studied using a xenograft model of nude mice.Results: The expression of PRDX1 protein was significantly up-regulated in the tumor tissues compared with the paired adjacent non-tumor tissues. Meanwhile, PRDX1 overexpression was associated with tumor stage, lymphatic metastasis and differentiation. Overexpression of PRDX1 significantly promoted proliferation and inhibited apoptosis by increasing the expression of Nanog, proliferating cell nuclear antigen (PCNA), B-cell lymphoma-2 (Bcl-2) and downregulating the expression of Bcl2-associated X protein (BAX) in SiHa cervical cancer cells. Moreover, PRDX1 overexpression increased invasion and migration of SiHa cervical cancer cells via up-regulating the expression of Snail and matrix metalloprotein 9 (MMP-9) and down-regulating the expression of E-cadherin. Knockdown of PRDX1 resulted in the opposite results. The role of PRDX1 in promoting SiHa cervical cancer cell proliferation and inhibiting apoptosis has also been confirmed in vivo in a mouse xenograft model.Conclusions: PRDX1 promoted cell proliferation, migration, and invasion and suppressed apoptosis of cervical cancer possibly via regulating the expression of related protein.

Downregulating integrin subunit alpha 7 (ITGA7) promotes proliferation, invasion, and migration of papillary thyroid carcinoma cells through regulating epithelial-to-mesenchymal transition

Thyroid cancer is one of the common malignancies of the endocrine system and the number of thyroid cancer cases is increasing constantly. Significant work has focused on the molecular mechanisms of thyroid cancer, but many mechanisms remain undiscovered. In this study, we employed a comprehensive analysis of whole-transcriptome resequencing derived from paired papillary thyroid cancer (PTC) and normal thyroid tissues. We performed a massive parallel whole-transcriptome resequencing of matched PTC and normal thyroid tissues in 19 patients and found that integrin subunit alpha 7 (ITGA7) was downregulated in thyroid tumor tissues, but the function of ITGA7 in this cancer is still unclear. We also discovered that ITGA7 gene in thyroid cancer tissues was downregulated compared to paired adjacent non-tumor tissues by real-time quantitative polymerase chain reaction. After transfection with small interfering RNA to knock down ITGA7, the abilities of colony formation, proliferation, migration, and invasion were enhanced in PTC cell lines (TPC1 and KTC-1). Meanwhile, ITGA7 knockdown decreased apoptotic cell death in thyroid cells but promoted the expressions of N-cadherin and vimentin and decreased E-cadherin expression by epithelial-to-mesenchymal transition, which may induce invasion and migration. In conclusion, these results indicated that ITGA7 is involved in the progress of PTC and might act as a tumor suppressor gene.

A novel circFMN2 promotes tumor proliferation in CRC by regulating the miR-1182/hTERT signaling pathways.

Circular RNAs (circRNAs) are a class of non-coding RNAs broadly expressed in cells of various species. However, the molecular mechanisms that link circRNAs with colorectal cancer (CRC) are not well understood. In the present study, we attempted to provide novel basis for targeted therapy for CRC from the aspect of circRNA-microRNA (miRNA)-mRNA interaction. We investigated the expression of circRNAs in five paired CRC tissues and adjacent non-tumor tissues by microarray analysis. Differentially expressed circRNAs were identified between CRC tissues and non-cancerous matched tissues. We focused on hsa_circ_0005100, which is located on chromosome 1 and derived from FMN2, and thus we named it as circFMN2. The expression of circFMN2 was detected in 88 CRC tissues and cell lines by quantitative real-time PCR. Functional assays were performed to evaluate the effects of circFMN2 on proliferationin vitro, and on tumorigenesisin vivo. The relationship between circFMN2 and miR-1182 was confirmed by luciferase reporter assay. circFMN2 was found to be significantly up-regulated in CRC tissues and cell lines. Moreover, knockdown of circFMN2 significantly inhibited cell proliferation and migrationin vitro. Bioinformatics analysis predicted that there is a circFMN2/miR-1182/hTERT axis in CRC progression. Dual-luciferase reporter system validated the direct interaction of circFMN2, miR-1182, hTERT. Western blot verified that inhibition of circFMN2 decreased hTERT expression. Importantly, we demonstrated that circFMN2 was up-regulated in serum exosomes from CRC patients. In conclusion, circFMN2 is a central component linking circRNAs to progression of CRC via an miR-1182/hTERT axis.

Decreased Expression of Retinoblastoma Protein-Interacting Zinc-Finger Gene 1 Is Correlated With Poor Survival and Aggressiveness of Cervical Cancer Patients.

Background: Retinoblastoma protein-interacting zinc finger gene 1 (RIZ1) is a tumor suppressor deregulated in several human cancers. We aim to (1) explore RIZ1 expression in FIGO stages I–II cervical cancer tissues and its association with the clinical outcome of cervical cancer patients, (2) the role of RIZ1 in proliferation, apoptosis, migration, and invasion in cervical cancer cells.Methods: The expression of RIZ1 in 268 cervical cancer tissues and 30 paired adjacent non-tumor tissues were assessed by immunohistochemistry. We also examined RIZ1 at mRNA and protein level in 20 paired fresh frozen cervical cancer tissues and the adjacent non-tumor tissue using real-time PCR and western blot. We then examined proliferation, apoptosis, migration, and invasion in two human cervical cancer cells, HeLa and SiHa, with overexpression of RIZ1.Results: RIZ1 expression generally decreased in cervical cancer tissues. Decreased RIZ1 expression was significantly correlated with advanced FIGO stage (P = 0.005), deep stromal invasion (P = 0.001), lymphovascular space involvement (P = 0.041), pelvic lymph node metastasis (P = 0.005), and postoperative recurrence (P = 0.002). Kaplan-Meier analysis demonstrated that patients with low RIZ1 expression had shorter overall survival (OS) and disease-free survival (DFS) than those with high RIZ1 expression. Multivariate analysis showed that RIZ1 was an independent prognostic factor for DFS (HR = 2.184, 95% CI 1.365–3.496, P = 0.001) and OS (HR = 1.899, 95% CI 1.112–3.241, P = 0.019). In vitro analysis demonstrated that overexpression of RIZ1 inhibited cell proliferation, migration, and invasion, but promoted apoptosis in HeLa and SiHa cells.Conclusion: Down-regulation of RIZ1 may contribute to tumor migration, invasiveness, and poor survival of cervical cancer patients. RIZ1 may be a prognostic biomarker for cervical cancer patients.

FOXD3, frequently methylated in colorectal cancer, acts as a tumor suppressor and induces tumor cell apoptosis under ER stress via p53.

Forkhead box D3 (FOXD3), an important member of the forkhead box transcription factor family, has many biological functions. However, the role and signaling pathways of FOXD3 in colorectal cancer (CRC) are still unclear. We examined FOXD3 expression and methylation in normal colon mucosa, CRC cell lines and primary tumors by reverse transcription-polymerase chain reaction, methylation-specific PCR and bisulfite genomic sequencing. We also evaluated its tumor-suppressive function by examining its modulation of apoptosis under endoplasmic reticulum (ER) stress in CRC cells. The FOXD3 target signal pathway was identified by western blotting, immunofluorescence and chromatin immunoprecipitation. We found that FOXD3 was frequently methylated and silenced in CRC cell lines and was downregulated in CRC tissues compared with paired adjacent non-tumor tissues. Meanwhile, low FOXD3 protein expression was significantly correlated with poor histopathological grading, lymph node metastasis and poor prognosis of patients, indicating its potential as a tumor marker that may be of potential value as a therapeutic target for CRC. Moreover, restoration of FOXD3 expression inhibited the proliferation and migration of tumor cells. FOXD3 also increased mitochondrial apoptosis through the unfolded protein response under ER stress. Furthermore, we found that FOXD3 could bind directly to the promoter of p53 and enhance its expression. Knockdown of p53 impaired the effect of apoptosis induced by FOXD3. In conclusion, we showed for the first time that FOXD3, which is frequently methylated in CRC, acted as a tumor suppressor inducing tumor cell apoptosis under ER stress via p53.

MiR-383 inhibits cell growth and promotes cell apoptosis in hepatocellular carcinoma by targeting IL-17 via STAT3 signaling pathway

ObjectivesEmerging microRNAs (miRNAs) are validated to take part in pathological processes, including numerous carcinomas. Currently, we focused on the functional role of miR-383 and interleukin-17 (IL-17) in hepatocellular carcinoma (HCC), and the underlying molecular mechanisms were also the emphases in our research. MethodsWe used reverse transcription-quantitative polymerase chain reaction (RT-qPCR) to measure the expression levels of miR-383 in 45 paired tumor tissues and adjacent non-tumor tissues extracted from patients with hepatocellular carcinoma. These tissues were also stained for IL-17 using immunohistochemical staining. Western blot was performed to detect the protein expressions of following protein-coding genes, including p-Stat3, Stat3 and GAPDH. A dual-luciferase activity was carried out to determine whether IL-17 was the downstream gene of miR-383 in hepatocellular carcinoma development. The colony assay, CCK8 assay, and apoptosis assay were used to explore the detailed regulatory effects of miR-383/IL-17 axis in the cellular processes of hepatocellular carcinoma separately. ResultsmiR-383 was down-regulated significantly in tumor tissues, while IL-17 was up-regulated. IL-17 was certificated to act as the downstream gene of miR-383. Furthermore, overexpression of miR-383 suppressed cell proliferation and promoted apoptosis in hepatocellular carcinoma. However, the raised IL-17 attenuated the inhibition effect of miR-383 in hepatocellular carcinoma. In addition, we found that p-Stat3 was repressed by miR-383, and the up-regulation of IL-17 reversed the suppression effect in hepatocellular carcinoma. ConclusionsmiR-383 may play a anti-tumor role in the pathogenesis of hepatocellular carcinoma by targeting IL-17 through STAT3 signaling pathway. miR-383/IL-17 axis maybe a potent target for the clinical diagnosis and treatment of hepatocellular carcinoma.

Alternative splicing of VEGFA is regulated by RBM10 in endometrial cancer.

Vascular endothelial growth factor A (VEGFA) gene has three alternative exons which results in multiple isoforms. VEGFA has been found overexpressed in patients with endometrial cancer, but the VEGFA expression pattern and how it is regulated are still unknown. The level of VEGFA transcripts and protein isoforms were detected by semi-quantitative Polymerase chain reaction (PCR) and immunoblotting in 29 paired endometrial tumor and adjacent nontumor control tissues. The level of three alternative splicing related proteins: RBM5, RBM6, and RBM10 was determined by immunoblotting. The H3K27Ac level in RBM10 promoter region was detected by ChIP-PCR. The RBM10 promoter region methylation level were quantified by methylation-sensitive high resolution melting. VEGFA165a was overexpressed and VEGFA165b level was reduced in tumors. RBM10 level was reduced in tumors. RBM10 level was negatively correlated with VEGFA165a level and positively correlated with VEGFA165b level in tumors. Using HEC-1-A and RL95-2 cells, we confirmed that VEGFA165a/b expressed pattern was controlled by RBM10. MALAT1 level was increased in tumors but not involved in VEGFA alternative splicing. Reduced H3K27Ac level and increased DNA methylation in the promoter region controlled RBM10 expression in tumors. VEGFA alternative splicing in endometrial cancer was regulated by RBM10, the expression of which was controlled by histone acetylation and DNA methylation.

CSN6 promotes tumorigenesis of gastric cancer by ubiquitin-independent proteasomal degradation of p16INK4a

ObjectiveCSN6 is a vital subunit of the constitutive photomorphogenesis 9 (COP9) signalosome (CSN), which is responsible for development disorders and promotes ubiquitin-26S proteasome-dependent degradation in vitro and vivo. Its role in the tumor development of gastric cancer remains unclear. In this study, we investigated the role of CSN6 in gastric cancer progression. MethodsHuman gastric cancer samples were collected and immunohistochemistry was performed to identify the role of CSN6 in gastric cancer. The cell proliferation was measured by CCK-8 and the EdU incorporation method. Immunofluorescence localization and a co-immunoprecipitation study were used to show the interaction between the protein CSN6 and p16. Ubiquitination assay was performed to validate whether ubiquitination is involved in CSN6-mediated p16 degradation. BALB/c nude mice were used to produce a tumor model in order to test the effect of CSN6 on cancer growth in vivo. ResultsCSN6 expression was dramatically increased in gastric cancer tissues compared with paired adjacent non-tumor tissues and CSN6 was correlated with worse overall and disease-specific survival. Additionally, we also found that CSN6 downregulated p16 protein expression, thereby promoting gastric cancer cell growth and proliferation. Moreover, CSN6 interacted with p16 and a proteasome activator REGγ (PA28γ), thereby facilitating ubiquitin-independent degradation of p16.ConclusionsCSN6 promoted the loss of p16-mediated tumor progression and played an important role in regulating ubiquitin-independent proteasomal degradation of p16.

Circular RNA hsa_circRNA_0007334 is Predicted to Promote MMP7 and COL1A1 Expression by Functioning as a miRNA Sponge in Pancreatic Ductal Adenocarcinoma.

Pancreatic cancer remains one of the leading causes of cancer-related deaths worldwide. Pancreatic ductal adenocarcinoma (PDAC) is the most common type of pancreatic tumor. Many circular RNAs (circRNAs) have proven to play vital roles in the physiological and pathological processes of tumorigenesis; however, their biogenesis in PDAC remains unclear. In this study, the expression profiles of circRNAs from 10 PDAC tissues and their paired adjacent nontumor tissues were analyzed through RNA sequencing analysis. An enrichment analysis was employed to predict the functions of the differentially expressed circRNAs. Sequence alignment information and mRNA microarray projects were used to predict the RNA regulatory network. The knockdown of circRNAs by small interfering RNAs followed by wound healing and western blot assays was used to confirm their functions in a PDAC cell line. A total of 278 circRNAs were identified as differentially expressed in PDAC tissue. Of these, we found that hsa_circRNA_0007334 was significantly upregulated and may serve as a competing endogenous RNA to regulate matrix metallopeptidase 7 (MMP7) and collagen type I alpha 1 chain (COL1A1) by the competitive adsorption of hsa-miR-144-3p and hsa-miR-577 to enhance the expression and functions of MMP7 and COL1A1 in PDAC. In vitro experiments confirmed these results. The present study is the first to propose two regulatory pathways in PDAC: hsa_circRNA_0007334–hsa-miR-144-3p–MMP7 and hsa_circRNA_0007334–hsa-miR-577–COL1A1.

Exploring the Natural Piericidins as Anti-Renal Cell Carcinoma Agents Targeting Peroxiredoxin 1.

Anti-renal cell carcinoma (RCC) agents with new mechanisms of action are urgently needed. Twenty-seven natural products of the piericidin class, including 17 new ones, are obtained from a marine-derived Streptomyces strain, and several of them show strong inhibitory activities against ACHN renal carcinoma cells. By exploring the mechanisms of two representative natural piericidin compounds, piericidin A (PA) and glucopiericidin A (GPA), peroxiredoxin 1 (PRDX1) is detected as a potential target by transcriptome data of PA-treated ACHN cells, as well as the paired RCC tumor versus adjacent nontumor tissues. PA and GPA induce cell apoptosis through reducing the reactive oxygen species level caused by upregulated PRDX1 mRNA and protein level subsequently and exhibit potent antitumor efficacy in nude mice bearing ACHN xenografts, with increasing PRDX1 expression in tumor. The interaction between PA/GPA and PRDX1 was supported by the docking analysis and surface plasmon resonance. Moreover, the translocation of PRDX1 into the nucleus forced by PA/GPA is proposed to be a key factor for the anti-RCC procedure. Piericidins provide a novel scaffold for further development of potent anti-RCC agents, and the new action mechanism of these agents targeting PRDX1 may improve upon the limitations of existing targeted drugs for the treatment of renal cancer.

Mitofusin 2 inhibits bladder cancer cell proliferation and invasion via the Wnt/β-catenin pathway.

The present study aimed to investigate the biological role of the mitochondrial GTPase mitofusin-2 (MFN2) in bladder cancer. MFN2 mRNA expression in tumor and paired adjacent non-tumor tissues from 8 patients was investigated using reverse transcription-quantitative polymerase chain reaction analysis. Immunohistochemistry was used to investigate MFN2 expression in 117 bladder cancer specimens. The associations of MFN2 expression with clinicopathological parameters were evaluated statistically. In addition, the biological role of MFN2 in the proliferation, migration and invasion of bladder cancer cells was examined. It was identified that MFN2 expression was significantly downregulated in bladder cancer tissues compared with normal tissues. MFN2 expression was associated with tumor stage, tumor grade and lymph node status. Furthermore, patients with low MFN2 expression demonstrated a shorter overall survival time (P=0.025). MFN2 knockdown by small interfering RNA promoted cancer cell proliferation, migration and invasion in vitro, and enhanced tumor progression in vivo. Mechanistically, MFN2 was revealed to be involved in Wnt/β-catenin signaling. In conclusion, MFN2 may serve as a potential therapeutic target in the treatment of bladder cancer, and the progress of bladder cancer may be delayed by regulating MFN2 expression.

Abstract 96: High iron or high fat diet show similar effects in inducing hepatocellular carcinoma in a mouse model

Abstract Introduction: Hepatocellular carcinoma (HCC) has multiple risk factors such as Hepatitis virus infection and obesity. Iron overload may be another risk factor as patients with hemochromatosis have high levels of liver iron accumulation and higher incidence of HCC than the general population. This study aims to assess the risk of high iron diet in comparison to high fat diet in the development of HCC. Materials &amp; Methods: We used C3HeB/FeJ male mice, which have been shown to spontaneously develop HCC. Two-month old mice were fed for 15 months with a control diet (10 kcal% from fat; 48 ppm Iron), high iron diet (HID) (10 kcal% from fat; 200 ppm Iron), high fat diet (HFD) (61.1 kcal% from fat; 48 ppm Iron), or high iron and fat diet (HIFD) (61.1 kcal% from fat; 200 ppm Iron). The presence of liver tumor was confirmed by ultrasound imaging and histology analysis. At termination, normal and tumor liver tissues were harvested for tumor size measurement, metal ion quantification, immunohistochemistry, and gene expression with RNA-seq. Results: HID like HFD induced significant body weight gain. All three diets induced HCC (4 out of 7 by HID, 7 out of 9 by HFD, and 8 out of 10 by HIFD). Yet, HCC was not found in the control diet group. The three diets showed no significantly different effects on tumor burden and grade. The concentrations of iron and copper were increased in the normal liver tissues of HID and HIFD groups. Interestingly, they were significantly lower in the tumor tissues than in the paired adjacent non-tumor tissues. Analyses of RNA-seq data revealed extensive overlap of tumor-specific differentially expressed genes (DEGs) among the three diet groups. The extensive overlap was also observed in the DEG-associated gene otology (GO) terms among the three diet groups. All three diets, but not the control diet, were shown to activate mTORC1 pathway in the tumors according to our RNA-seq data. The activation of mTORC1 pathway in the three diet-induced tumors was also confirmed with Real-time RT-PCR assays revealing upregulation of mTORC1 target genes and with immunohistochemistry showing increased phosphorylation of an mTORC1 substrate. Conclusions: The spontaneous hepatocarcinogenesis in male C3HeB/FeJ mice requires HID or HFD. The lack of additive effect of HID and HFD on HCC incidence and the extensive overlap of DEGs and GO terms among tumors induced by HID and HFD suggest they may regulate common mechanisms such as mTORC1 in inducing HCC. Citation Format: Hakim Bouamar, Kalyan Kakarla, Matyas Cserhati, Fatima Ezzahra El Mroussi, Francis E Sharkey, Francisco Cigarroa, Lu-Zhe Sun. High iron or high fat diet show similar effects in inducing hepatocellular carcinoma in a mouse model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 96.

LINC81507 act as a competing endogenous RNA of miR-199b-5p to facilitate NSCLC proliferation and metastasis via regulating the CAV1/STAT3 pathway

Lung cancer is the leading cause of cancer-related mortality worldwide. Recently, accumulating data indicate that long noncoding RNAs (LncRNAs) function as novel crucial regulators of diverse biological processes, including proliferation and metastasis, in tumorigenesis. Lnc NONHSAT081507.1 (LINC81507) is associated with lung adenocarcinoma. However, its pathological role in non-small cell lung cancer (NSCLC) remains unknown. In our study we investigated the role of LINC81507 in NSCLC. The expression of LINC81507 was analyzed in 105 paired NSCLC tumor specimens and paired adjacent non-tumorous tissues from NSCLC patients by real-time quantitative PCR (RT-qPCR). Gain- and loss-of-function experiments were conducted to investigate the functions of LINC81507, miR-199b-5p and CAV1. Reduced expression of LINC81507 resulted in cell growth, proliferation, migration and epithelial–mesenchymal transition (EMT) in NSCLC cells, whereas ectopic overexpression of LINC81507 resulted in the opposite effects both in vitro and in vivo. Nuclear and Cytoplasmic fractionation assays showed LINC81507 mainly resided in cytoplasm. Bioinformatics analysis and dual-luciferase assays revealed that miR-199b-5p was a direct target of LINC81507 through binding Ago2. Mechanistic analysis demonstrated that miR-199b-5p specifically targeted the Caveolin1 (CAV1) gene, and LINC81507 inactivated the STAT3 pathway in a CAV1-dependent manner. Taken together, LINC81507 is decreased in NSCLC and functions as a sponge to miR-199b-5p to regulate CAV1/STAT3 pathway, which suggests that LINC81507 serve as a tumor suppressor and potential therapeutic target and biomarker for metastasis and prognosis in NSCLC.

Abstract 4642: RNA interference reveals tumor promoting roles of integrin alpha 6 (ITGA6) in hepatocellular carcinoma

Abstract Introduction: Hepatocellular carcinoma (HCC) is the sixth most common malignancy worldwide and the third leading cause of cancer related mortality. The incidence and mortality rates of HCC are two times higher in Latinos than in the general population in the US and are the highest in Latinos from the South Texas region. The genetic and epigenetic events associated with the increased incidence of HCC in this population are still unclear. Increasing evidence suggests that integrins are one of the most important receptors for cell metastasis including integrin α6β1 and α6β4. However, few studies have focused on the function of integrin alpha 6 (ITGA6) in the tumorigenesis and progression of HCC. We aim to investigate the expression and potential roles of ITGA6 in HCC. Materials and Methods: Paired HCC tissues and adjacent non-tumor tissues were collected for RNA sequencing. ITGA6 RNA and protein expression levels were evaluated by RNA sequencing, RT-qPCR, Western blot and immunohistochemistry. HCC cell lines (SNU398 and Huh7) were transiently transfected with two ITGA6siRNAs and stably transfected with an ITGA6 shRNA lentivector. These cell lines were used for assays testing the effects of ITGA6 knockdown on HCC cell proliferation, migration and anchorage independent growth. Results: Analysis of RNA sequencing data indicated that the expression of ITGA6 increased 4-fold in HCC tumor tissues compared to adjacent non-tumor tissues, which was validated by RT-qPCR. Western blotting also confirmed increased expression of ITGA6 in the tumor tissues. The knockdown of ITGA6 by siRNAs and shRNA was confirmed with Western blot and qRT-PCR. ITGA6 knockdown significantly decreased proliferation, migration and anchorage independent growth of HCC cell lines. Conclusions: ITGA6 is upregulated in HCC tumors in Latinos patients. ITGA6 may play a malignant-promoting role in HCC cells. Our studies provided new insights into the molecular mechanisms that drive HCC progression and potential therapeutic targets for treating patients with HCC including South Texas Latino patients. Citation Format: Guixi Zheng, Carla Zeballos, Hakim Bouamar, Matyas Cserhati, Francisco G Cigarroa, Lu-zhe Sun. RNA interference reveals tumor promoting roles of integrin alpha 6 (ITGA6) in hepatocellular carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4642.