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Purpose As the first-line drug for treatment of HER2-positive metastatic gastric cancer (GC), Herceptin exhibits significant therapeutic efficacy. However, acquired resistance of Herceptin limits the therapeutic benefit of gastric cancer patients, in which the molecular mechanisms remain to be further determined. Methods Quantitative real-time polymerase chain reaction was performed to detect the mRNA levels of ARPP-19 and CD44 in GC cells. Protein levels were determined using Western blot and IHC staining. MTT and soft agar colony formation assays were used to measure cell proliferation. Xenograft model was established to verify the functional role of ARPP-19 in Herceptin resistance in vivo. Sphere formation assay was conducted to determine cell stemness. Results We observed ARPP-19 was up-regulated in Herceptin resistance gastric cancer cells NCI-N87-HR and MKN45-HR. The forced expression of ARPP-19 promoted, whereas the silencing of ARPP-19 impaired Herceptin resistance of HER2-positive gastric cancer cells both in vitro and in vivo. Moreover, ARPP-19 significantly enhanced the sphere formation capacity and CD44 expression, CD44 was also a positive factor of Herceptin resistance in HER2-positive gastric cancer cells. In addition, high level of ARPP-19 was positively associated with Herceptin resistance and poor survival rate of gastric cancer patients. Conclusion We have demonstrated that ARPP-19 promoted Herceptin resistance of gastric cancer via up-regulation of CD44, our study suggested that ARPP-19 could be a potential diagnostic and therapeutic candidate for HER2-positive gastric cancer.

Jianfeng Huang,* Wenzheng Chen,* Changyu Chen,* Zhigang Jie, Tao Xiao Department of Gastrointestinal Surgery, The First Affiliated Hospital of Nanchang University, Nanchang, 330006, Jiangxi, People’s Republic of China*These authors contributed equally to this workCorrespondence: Zhigang Jie; Tao Xiao, Department of Gastrointestinal Surgery, The First Affiliated Hospital of Nanchang University, No. 1519 Dongyue Road, Nanchang, 330006, Jiangxi, People’s Republic of China, Email Jiezg123@126.com; xiaotao@ncu.edu.cnPurpose: N6-methyladenosine (m6A) RNA modification plays an important role in regulating tumor microenvironment (TME) infiltration. However, the relationship between the expression pattern of m6A-related long non-coding RNAs (lncRNAs) and the immune microenvironment of gastric cancer (GC) is unclear.Methods: In this study, 23 m6A-related lncRNAs were identified by Pearson’s correlation analysis and univariate Cox regression analysis. According to the expression of these lncRNAs, we identified two distinct molecular clusters by consensus clustering and compared the differences of the TME and enriched pathways between the two clusters. We further constructed a prognostic risk signature and verified it using The Cancer Genome Atlas training and testing cohorts.Results: The results showed that cluster 1 was associated with tumor-related and immune activation-related pathways. In addition, cluster 1 was also associated with higher ImmuneScore, StromalScore, and ESTIMATEScore. The results of the stratified survival analysis and independent prognosis analysis indicated that the risk signature is an independent prognostic indicator for patients with GC. In addition, it can effectively predict survival status in patients with different clinical characteristics. Furthermore, we found that the risk signature was associated with a variety of tumor-infiltrating immune cells, and that low risk scores were significantly correlated with high expression of programmed death-1 (PD-1) and cytotoxic T-lymphocyte associated protein 4 (CTLA4), as well as sensitivity to chemotherapeutic drugs (eg, fluorouracil and oxaliplatin).Conclusion: This evidence contributes to our understanding of the regulation of TME infiltration by m6A-related lncRNAs and may lead to more effective immunotherapy and chemotherapy for patients with GC.Keywords: N6-methyladenosine, gastric cancer, tumor microenvironment, lncRNA risk signature, prognostic

Worldwide, the incidence rate of gastric cancer ranks fifth, and the mortality rate of gastric cancer ranks third among all malignant tumors. However, the pathogenesis of gastric cancer remains poorly understood. In this study, we demonstrated that the expression level of NELFE is higher in human gastric cancer tissues than in adjacent nontumor tissues. A high level of NELFE is associated with worse postoperative overall survival (OS) and relapse-free survival (RFS) rates in patients with gastric cancer. Moreover, the expression of NELFE is correlated with high tumor grade and lymph node metastasis in gastric cancer patients. Knockdown of NELFE dramatically inhibits the cell proliferation and metastasis of gastric cancer xenografts in vivo. Furthermore, we found that NELFE binding to the 3’UTR of E2F2 affects the mRNA stability of E2F2 to regulate the expression level of E2F2. In gastric cancer, E2F2 also acts as an oncogene to inhibit the proliferation and migration of gastric cancer cells by knocking down the expression level of E2F2. However, overexpressing E2F2 in cells with NELFE knockdown significantly reverses the inhibition of cell proliferation and migration induced by NELFE knockdown. Therefore, NELFE at least partially functions as an oncogene through E2F2. Moreover, CIBERSORTx analysis of the proportion of tumor-infiltrating immune cells (TICs) revealed that immune cells are correlated with NELFE and E2F2 expression, suggesting that NELFE and E2F2 might be responsible for the preservation of the immunodominant status for gastric cancer. In conclusion, NELFE acts as an oncogene in gastric cancer and can be used as a potential therapeutic target.

Objective: To study the role of long non-coding RNA (lncRNA) CRYM-AS1 in human gastric cancer. Methods: Expression levels of CRYM-AS1 in cell lines and clinical tissues were examined by RT-qPCR. The association between CRYM-AS1 levels and clinicopathological parameters / survival rates of gastric cancer patients was analyzed.Cell functional experiments including MTT assay, glucose consumption / lactate production / ATP production detection were performed to examine the role of CRYM-AS1 in cell aerobic glycolysis and cell proliferation of gastric cancer cells. Subcellular fractionation location detection, western blot, RIP (RNA binding protein immunoprecipitation) assay, CHIP (Chromatin immunoprecipitation) assay and BSP (Bisulfite sequencing PCR) assay were carried out to explore the molecular mechanism of CRYM-AS1 in gastric cancer cells.Results: CRYM-AS1 was low expressed in gastric cancer cells and tissues compared with normal gastric cells and tissues respectively. CRYM-AS1 was negatively correlated with TNM staging, tumor size and overall survival (OS) rate in gastric cancer patients. CRYM-AS1 inhibited gastric cancer cell aerobic glycolysis and cell proliferation. CRYM-AS1 directly bound to EZH2 and mediated the CRYM promoter methylation and consequently negatively regulated the expression of CRYM. Forced expression of CRYM rescued the decreased aerobic glycolysis and cell proliferation induced by CRYM-AS1 in gastric cancer cells.Conclusion: CRYM-AS1 was an important biomarker and could be used for human gastric cancer treatment.

Hepatocellular carcinoma (HCC) is the fifth most prevalent cancer and the second leading cause of cancer-related deaths. In the present study, we have demonstrated that long non-coding RNA (lncRNA) LINC00472 was low expressed in human HCC tissues and cell lines compared with adjacent non-tumor liver tissues and normal liver cell lines respectively. LINC00472 was also low expressed in HCC tissues from patients with metastasis compared with tissues from patients without metastasis. Expression level of LINC00472 was positively correlated with patient overall survival (OS) rate. Forced expression of LINC00472 suppressed cell proliferation, migration, invasion and promoted cell apoptosis in HCC cells Huh-7 and SMMC-7721. MiR-93-5p was a direct target of LINC00472, and miR-93-5p directly targeted PDCD4. The miR-93-5p/PDCD4 pathway mediated the suppressing role of LINC00472 in HCC cells. Therefore, LINC00472 was an important tumor suppressor in human HCC, which could be used as a bio-marker for HCC therapy.

Xiaochen Yang,1 Jun Wu,1 Wei Xu,1 Sheng Tan,2 Changyu Chen,3 Xiaoyan Wang,4 Jielin Sun,4 Yani Kang1 1School of Biomedical Engineering, Bio-ID Center, Shanghai Jiao Tong University, Shanghai, 200240, People’s Republic of China; 2The CAS Key Laboratory of Innate Immunity and Chronic Disease, School of Life Sciences, University of Science and Technology of China, Hefei, Anhui 230027, People’s Republic of China; 3Department of General Surgery, The First Affiliated Hospital of Anhui University of Traditional Chinese Medicine, Hefei, Anhui, 230031, People’s Republic of China; 4Key Laboratory of Systems Biomedicine (Ministry of Education), Shanghai Center for Systems Biomedicine, Shanghai Jiao Tong University, Shanghai, 200240, People’s Republic of China Background: Alternative polyadenylation (APA) is an important post-transcriptional regulation in eukaryotic cells. It plays considerable roles in many biological processes and diseases, such as cell differentiation, proliferation and cancer. Colorectal cancer (CRC) is one of the most common malignancies worldwide, which is among the top five in incidence and mortality of all cancers in China. Although there have been some studies on the APA of CRC, the normal and carcinoma samples used for genome-wide profiling were not matched. The purpose of this study was to obtain genes with switched 3'-untranslated region (UTR) that may be associated with intracellular regulation of CRC by analyzing APA patterns of strict control groups from clinical patients. Materials and methods: CRC and matched normal tissues were acquired from surgical specimens from three CRC patients. Their libraries of 3'-terminal fragments of mRNA with poly(A) tails were constructed by 3T-seq technology and sequenced by Illumina Hiseq X Ten. APA patterns of cancer and matched normal tissues were analyzed by bioinformatics analysis, and a representative gene, GPI, was verified by quantitative reverse transcription PCR. Results: Overall, we identified 35,076 poly(A) sites in total. Compared to the matched normal tissues, we detected 350, 405 and 375 genes with significantly APA-mediated 3'-UTR alteration in cancer tissues of three patients, respectively. Forty-seven genes with switched 3'-UTR were shared in all three patients. In addition, most of these genes have shortened 3'-UTRs, some of which were associated with cancers, such as GPI. Conclusion: Our studies found several genes with switched 3'-UTR in CRC patients, which may provide some important clues for more in-depth study of the cellular regulation in CRC from the perspective of post-transcriptional regulation. It may also help in the search for new biomarkers of CRC. Keywords: alternative polyadenylation, CRC, 3T-seq, 3'-UTR

Survival rates for patients with gastric cancer, especially the advanced form, remain poor and the development of targeted treatments is hampered by a lack of efficient biological targets. Poly(rC) binding protein 2 (PCBP2) is an RNA-binding protein that contributes to mRNA stabilization, translational silencing and enhancement and it has been implicated as a promoter of gastric cancer growth. In the present study, we demonstrated that the expression level of PCBP2 was higher in human gastric cancer tissues compared to adjacent normal gastric tissues. A high level of PCBP2 was correlated with worse postoperative relapse-free survival and overall survival rates of gastric cancer patients. Small hairpin RNA-mediated depletion of PCBP2 dramatically decreased the viability of gastric cancer cells. Cyclin-dependent kinase 2 (CDK2) was positively regulated by PCBP2 via a direct 3' UTR binding pathway as determined using a ribonucleoprotein immunoprecipitation assay and a biotin pulldown assay. CDK2 mediated the promoting role of PCBP2. These results suggest that PCBP2 acts as an oncogene in human gastric cancer cells and that functionally depleting PCBP2 could be considered as a potential target for gastric cancer therapy.

Alternative polyadenylation (APA) is an important post-transcriptional regulatory mechanism and involved in many diseases, including cancer. CFIm25, a subunit of the cleavage factor I encoded by NUDT21, is required for 3'RNA cleavage and polyadenylation. Although it has been recently reported to be involved in glioblastoma tumor suppression, its roles and the underlying functional mechanism remain unclear in other types of cancer. In this study, we characterized NUDT21 in hepatocellular carcinoma (HCC). Reduced expression of NUDT21 was observed in HCC tissue compared to adjacent non-tumorous compartment. HCC patients with lower NUDT21 expression have shorter overall and disease-free survival times than those with higher NUDT21 expression after surgery. Knockdown of NUDT21 promotes HCC cell proliferation, metastasis, and tumorigenesis, whereas forced expression of NUDT21 exhibits the opposite effects. We then performed global APA site profiling analysis in HCC cells and identified considerable number of genes with shortened 3'UTRs upon the modulation of NUDT21 expression. In particular, we further characterized the NUDT21-regulated genes PSMB2 and CXXC5. We found NUDT21 knockdown increases usage of the proximal polyadenylation site in the PSMB2 and CXXC5 3' UTRs, resulting in marked increase in the expression of PSMB2 and CXXC5. Moreover, knockdown of PSMB2 or CXXC5 suppresses HCC cell proliferation and invasion. Taken together, our study demonstrated that NUDT21 inhibits HCC proliferation, metastasis and tumorigenesis, at least in part, by suppressing PSMB2 and CXXC5, and thereby provided a new insight into understanding the connection of HCC suppression and APA machinery.

The present study was performed to evaluate the predictive value of contrast-enhanced ultrasonography (CEUS) for the risk of cervical lymph node metastasis (LNM) in papillary thyroid carcinoma (PTC). Between May 2014 and November 2016, 42 patients who received surgery for suspicious PTCs were enrolled in the present study. Each individual underwent CEUS with conventional ultrasound (US), preoperative US-guided fine needle aspiration and personalized surgery. Subsequently, the microvascular density (MVD) of all surgical specimens was measured. According to the results of surgical histopathology, individuals were divided into two groups: LNM+ (PTCs with LNM), and LNM− (PTCs without LNM). Clinicopathological characteristics, CEUS enhancement patterns, perfusion parameters and measurements of MVD were compared. The correlation between quantitative variables and LNM was analyzed using Spearman's correlation analysis. Compared with that in the LNM− group, patients in the LNM+ group were younger (P10 mm in size (P