Para-carcinoma Normal Tissues Research Articles

ADAMTS4 exacerbates lung cancer progression via regulating c-Myc protein stability and activating MAPK signaling pathway

BackgroundLung cancer is one of the most frequent cancers and the leading cause of cancer-related deaths worldwide with poor prognosis. A disintegrin and metalloproteinase with thrombospondin motifs 4 (ADAMTS4) is crucial in the regulation of the extracellular matrix (ECM), impacting its formation, homeostasis and remodeling, and has been linked to cancer progression. However, the specific involvement of ADAMTS4 in the development of lung cancer remains unclear.MethodsADAMTS4 expression was identified in human lung cancer samples by immunohistochemical (IHC) staining and the correlation of ADAMTS4 with clinical outcome was determined. The functional impact of ADAMTS4 on malignant phenotypes of lung cancer cells was explored both in vitro and in vivo. The mechanisms underlying ADAMTS4-mediated lung cancer progression were explored by ubiquitination-related assays.ResultsOur study revealed a significant upregulation of ADAMTS4 at the protein level in lung cancer tissues compared to para-carcinoma normal tissues. High ADAMTS4 expression inversely correlated with the prognosis of lung cancer patients. Knockdown of ADAMTS4 inhibited the proliferation and migration of lung cancer cells, as well as the tubule formation of HUVECs, while ADAMTS4 overexpression exerted opposite effects. Mechanistically, we found that ADAMTS4 stabilized c-Myc by inhibiting its ubiquitination, thereby promoting the in vitro and in vivo growth of lung cancer cells and inducing HUVECs tubule formation in lung cancer. In addition, our results suggested that ADAMTS4 overexpression activated MAPK signaling pathway.ConclusionsWe highlighted the promoting role of ADAMTS4 in lung cancer progression and proposed ADAMTS4 as a promising therapeutic target for lung cancer patients.

Non-canonical Small GTPase RBJ Promotes NSCLC Progression Through the Canonical MEK/ERK Signaling Pathway.

Although the majority of members belonging to the small GTPase Ras superfamily have been studied in several malignancies, the function of RBJ has remained unclear, particularly in non-small cell lung cancer (NSCLC). The research aims to determine the function of RBJ in NSCLC. The levels of RBJ protein in tumor tissue and para-carcinoma normal tissue were ascertained via immunohistochemistry (IHC). The growth, migration, and invasion of NSCLC cells were assessed by 5- ethynyl-2-deoxyuridine (EdU) assay, colony formation, cell counting kit-8 (CCK8), transwell and wound healing assays. Furthermore, a nude mouse xenograft model was established to study the function of RBJ in tumorigenesis in vivo. The IHC analysis revealed that the protein levels of RBJ were notably increased in tumor tissue and positively associated with the clinical stage. In addition, the knockdown of RBJ restrained the growth, invasion, and migration of NSCLC cell lines by inhibiting the epithelial-mesenchymal transition (EMT) through the MEK/ERK signaling pathway. Accordingly, opposite results were observed when RBJ was overexpressed. In addition, the overexpression of RBJ accelerated tumor formation by A549 cells in nude mice. RBJ promoted cancer progression in NSCLC by activating EMT via the MEK/ERK signaling. Thus, RBJ could be used as a potential therapeutic against NSCLC.

CXCL5 inhibits tumor immune of lung cancer via modulating PD1/PD-L1 signaling

Objective: To investigate the role of CXCL5 in tumor immune of lung cancer and to explore the potential molecular mechanisms. Methods: A total of 62 cases of patients with lung cancer admitted in the First Affiliated Hospital of Henan University from May 2018 to December 2019 were recruited as study object. Another 20 cases of patients with pulmonary infectious diseases and 20 cases of healthy control were selected as control. Enzyme-linked immunosorbent assay (ELISA) was used to determine serum levels of CXCL5 in patients with lung cancer, pulmonary infectious diseases and healthy control. Immunohistochemical staining (IHC) was used to detect the expressions of CXCL5 and PD-1/PD-L1 in tumor and paracarcinoma tissues of patients with lung cancer. Pearson correlation analysis was used to evaluate the correlation between CXCL5 and PD-1 in tumor and paracarcinoma tissues of patients with lung cancer. Lewis cells either expressing CXCL5 or vector plasmids were used to establish C57BL/6J mice model of lung cancer, and all mice were then divided into vehicle and PD-1 antibody treatment groups, 10 mice for each group. The mice survival and tumor growth curves were recorded. IHC was used to evaluate the expressions of CXCL5, PD-1 as well as the proportions of CD8(+) T and Treg cells in xenograft tumor tissues. Results: In patients with lung cancer, the serum level of CXCL5 [(351.7±51.5) ng/L] was significant higher than that in patients with pulmonary infectious diseases and healthy control [(124.7±23.4) ng/L, P<0.001]. The expression levels of CXCL5 (0.136±0.034), CXCR2 (0.255±0.050), PD-1 (0.054±0.012) and PD-L1 (0.350±0.084) in tumor were significant higher than those in paracarcinoma normal tissues [(0.074±0.022), (0.112±0.023), (0.041±0.007) and (0.270±0.043) respectively, P<0.001]. CXCL5 was significant positively correlated with PD-1 in tumor tissues of lung cancer (r=0.643, P<0.001), but not correlated with PD-1 in paracarcinoma tissues(r=0.088, P=0.496). The vector control group, CXCL5 overexpression group, vector control + anti-PD-1 antibody treatment group and CXCL5 overexpression + anti-PD-1 antibody treatment group all successfully formed tumors in mice, while CXCL5 overexpression increased the tumor growth significantly (P<0.01), which was abrogated by the treatment of anti-PD-1 antibody. CXCL5 overexpression decreased the mice survival time significantly (P<0.01), this effect was also abrogated by the treatment of anti-PD-1 antibody. The proportion of CD8(+) T cells in CXCL5 overexpression group [(10.40±2.00)%] was significant lower than that in vector control group [(21.20±3.30)%, P=0.002]. The proportion of CD4(+) Foxp3(+) Treg cells in CXCL5 overexpression group [(38.40±3.70)%] was significant higher than that in vector control group [(23.30±2.25)%, P<0.001]. After the treatment of anti-PD-1 antibody, no significant difference were observed for the proportion of CD8(+) T cells [(34.10±5.00)% and (33.40±4.00)% respectively] and Treg cells [(14.70±3.50)% and (14.50±3.30)% respectively] in xenograft tumor tissues between CXCL5 overexpression+ anti-PD-1 antibody treatment group and vector control + anti-PD-1 antibody treatment group (P>0.05). Conclusion: The expressions of CXCL5 and PD-1/PD-L1 are all increased significantly in the tumor tissues of patients with lung cancer, CXCL5 may inhibit tumor immune of lung cancer via modulating PD-1/PD-L1 signaling.

CircITCH suppresses cell proliferation and metastasis through miR-660/TFCP2 pathway in melanoma.

BackgroundMelanoma is an aggressive disease that is rising in incidence. Advanced melanoma is still a life‐threatening disease. CircRNAs are documented to be involved in melanoma progression. But circITCH role in melanoma remains unclear.Methods and ResultsTo explore the functions of circITCH in melanoma, levels of circITCH in melanoma tissues and paracarcinoma normal tissues were detected. To study the roles of circITCH in melanoma in terms of cell proliferation and migration, in vitro and in vivo experiments were performed. Mechanism study was designed to investigate the potential regulatory effect of circITCH in melanoma. Results revealed that circITCH expression was repressed in melanoma versus adjacent normal tissues. Function study showed that circITCH suppressed melanoma cell proliferation and metastasis. The mechanism study showed that circITCH‐sponged miR‐660 to upregulate TFCP2 and suppress melanoma progression.ConclusionsThe circITCH/miR‐660/TFCP2 axis is involved in melanoma progression hence circITCH can be a diagnostic biomarker as well as a target for treating melanoma.

Tubulin alpha 1c promotes aerobic glycolysis and cell growth through upregulation of yes association protein expression in breast cancer.

Tubulin alpha 1c (TUBA1C) as a member of α-tubulin was identified to take part in the occurrence and development of hepatocellular carcinoma and pancreatic cancer. Using the bioinformatics, we noticed that TUBA1C level was also increased in breast cancer was also demonstrated. Here, we explored TUBA1 role in modulation of breast cancer cell aerobic glycolysis, growth and migration and explored whether yes association protein (YAP) was involved. Fifty-five matched breast cancer tissues and the para-carcinoma normal tissues were included in this study and used to verify TUBA1C expression using quantitative reverse transcription-PCR and western blotting. ATP level, lactate secretion and glucose consumption were used to assess aerobic glycolysis. Cell growth, invasion, migration and tumorigenesis were detected using cell count kit-8, transwell, wound healing and animal assays. TUBA1 was upregulated in breast cancer, which associated with advanced primary tumor, lymph node, metastasis stage and tumor size. Silencing of TUBA1C with sh-TUBA1C infection led to significant inhibitions in ATP level, lactate secretion, glucose consumption, cell growth, migration, invasion and tumorigenesis, as well as declined YAP expression, while TUBA1C overexpression induced a opposite result. And, the above tendencies induced by TUBA1C downregulation were reversed by YAP overexpression. This study revealed that TUBA1C was overexpressed in breast cancer and promoted aerobic glycolysis and cell growth through upregulation of YAP expression.

Long Noncoding RNA FOXP4-AS1 Predicts Unfavourable Prognosis and Regulates Proliferation and Invasion in Hepatocellular Carcinoma.

Background Hepatocellular carcinoma (HCC) is the most common type of primary liver cancer that has a high level of morbidity and mortality. Long noncoding RNA (lncRNA) is a novel regulatory factor of tumour proliferation, apoptosis, and metastasis. Our previous studies indicated that lncRNA FOXP4-AS1 is a functional oncogene in HCC; thus, this study is aimed at further evaluating the clinical and biological function of FOXP4-AS1 in HCC. Material and Methods. First, we detected the expression of FOXP4-AS1 in HCC tissues and paracarcinoma normal tissues by qRT-PCR. Second, the prognostic effects of FOXP4-AS1 in patients with HCC were analysed in a training group and a verification group. Subsequently, to investigate the biological effects of FOXP4-AS1 on HCC cells, downexpression tests were further conducted. Results The expression of FOXP4-AS1 was higher in HCC tissues than adjacent nontumourous tissues, whereas the low expression of FOXP4-AS1 was correlated with optimistic treatment outcomes, which suggested that FOXP4-AS1 may be an independent prognostic biomarker for HCC. Moreover, the downregulation of FOXP4-AS1 significantly reduced the cell proliferation and clonal abilities and inhibited the invasion, migration, and angiogenesis of hepatoma cells (P < 0.05). Conclusion These results revealed the clinical significance and biological function of FOXP4-AS1 in HCC development, which may provide a new direction for finding therapeutic targets and potential prognostic biomarkers of HCC.

MiR-376b-3p functions as a tumor suppressor by targeting KLF15 in non-small cell lung cancer.

The aim of this study was to explore the regulatory effects of micro ribonucleic acid (miR)-376b-3p on proliferation and apoptosis of non-small cell lung cancer (NSCLC) cells by targeting Kruppel-like factor 15 (KLF15) and its mechanism of action. The expression of miR-376b-3p in NSCLC and para-carcinoma normal tissues, as well as NSCLC cell lines, was detected via quantitative Polymerase Chain Reaction (qPCR). The effects of miR-548-3p on the proliferation, cycle distribution, and apoptosis of NSCLC cells were detected via colony formation assay, flow cytometry, and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay, respectively. The interaction between miR-376b-3p and KLF15 was determined using Dual-Luciferase reporter gene assay. In vivo tumorigenic ability of NSCLC cells was studied using nude mouse tumorigenicity assay. Furthermore, the expression of Ki67 in tumor in nude mice was detected via immunohistochemistry. The expression of miR-376b-3p was significantly downregulated in NSCLC tissues when compared with para-carcinoma normal tissues (p<0.05). MiR-376b-3p was lowly expressed in NSCLC cells as well (p<0.05). After overexpression of miR-376b-3p, the proliferation ability of NSCLC cells remarkably declined (p<0.05). The apoptosis rate rose, and cell cycle was arrested in the G1/G0 phase. Dual-Luciferase reporter gene assay confirmed that miR-376b-3p could specifically bind to KLF15 3'UTR to regulate the expression activity of KLF15. After overexpression of miR-376b-3p, tumor volume and weight were significantly reduced in tumor-bearing mice (p<0.05). MiR-376b-3p plays an important role in the occurrence and development of NSCLC, which affects the proliferation and apoptosis of NSCLC cells by targeting KLF15.

Circ_0003789 Facilitates Gastric Cancer Progression by Inducing the Epithelial-Mesenchymal Transition Through the Wnt/β-Catenin Signaling Pathway.

Background: The role of circular RNAs in the pathogenesis of gastric cancer (GC) has been well documented by numerous studies. However, whether circ_0003789 plays a role during GC progression remains to be determined. Thus, this study investigated the biological functions of circ_0003789 during GC progression. Materials and Methods: Circ_0003789 expression was determined using quantitative real-time polymerase chain reaction in GC and matched para-carcinoma normal tissues. Functional experiments were performed to estimate changes in the proliferation, apoptosis, migration, and invasion of GC cells treated to silence circ_0003789. E-cadherin, vimentin, Wnt3a, and β-catenin expression was determined using immunofluorescence staining and Western blot assays. Xenograft tumor growth and Ki67 expression were also evaluated in vivo. Results: Circ_0003789 was upregulated in GC tissues and cells, and its upregulation positively correlated with poor tumor differentiation, distal metastasis, and advanced clinical stage. Silencing circ_0003789 inhibited GC cell proliferation, migration, invasion, and the epithelial-mesenchymal transition (EMT), both in vitro and in vivo. Mechanistically, the Wnt/β-catenin signaling pathway was repressed by circ_0003789 silencing. Conclusions: Circ_0003789 facilitates GC progression by inducing the EMT through the Wnt/β-catenin signaling pathway.

Associations of MTA1 expression with CT features, pathology and prognosis of elderly patients with non-small cell lung cancer.

Associations of metastasis-associated protein 1 (MTA1) expression with computed tomography (CT) features, pathology and prognosis of elderly patients with non-small cell lung cancer (NSCLC), and its clinical significance were explored. A total of 98 elderly patients with NSCLC were selected and underwent CT examination. The expression of MTA1 in carcinoma tissues and para-carcinoma normal tissues was detected via immunohistochemistry, and its associations with CT features, pathology and prognosis were analyzed. The results manifested that the expression of MTA1 in carcinoma tissues was significantly higher than that in para-carcinoma normal tissues, and it was associated with the degree of differentiation, stage and lymph node metastasis (P<0.05). Besides, the high expression of MTA1 was also related to the spicule sign, pleural indentation and lymph node metastasis (P<0.05) as well as the CT perfusion parameter capillary permeability (PMB) (P<0.05), but not to blood volume (BV), blood flow (BF) or time to peak (TTP). Moreover, the patients with high expression of MTA1 had significantly shorter survival time and a remarkably lower 5-year survival rate than those with low expression of MTA1 (P<0.05). In conclusion, MTA1 plays a certain role in the occurrence and development of NSCLC in elderly patients and has an association with their prognosis, which can provide references for the treatment and prognosis of NSCLC, with important clinical significance.

Linc00324 promotes the progression of papillary thyroid cancer via regulating Notch signaling pathway.

To explore the expression of linc00324 in papillary thyroid cancer (PTC) and its effect on the biological function of PTC cells. A total of 60 pairs of PTC and para-carcinoma normal tissues surgically excised were collected. The expression of linc00324 in PTC tissues and cells was detected via quantitative Reverse Transcription-Polymerase Chain Reaction (qRT-PCR), and the expression of linc00324 in PTC cells was silenced using the small-interfering RNA (siRNA). Then, the effects of linc00324 on the PTC cell proliferation, apoptosis, and cycle and the downstream Notch signaling pathway were determined via methyl thiazolyl tetrazolium (MTT) assay, colony formation assay, flow cytometry, and Western blotting, respectively. The expression of linc00324 was upregulated in 48 out of 60 cases of PTC tissues, and it was increased in PTC cells compared with that in human thyroid follicular epithelial cells Nthy-ori 3-1. The results of MTT assay and colony formation assay showed that the proliferation of PTC cells declined after interference in linc00324 expression. The findings of flow cytometry revealed that the cell cycle was arrested in G1/G0 phase with a higher apoptosis rate in si-linc00324 group compared with that in the si-NC group. According to the data of Western blotting, the molecular markers for the downstream Notch signaling pathway were altered after interference in linc00324 expression. The expression of linc00324 is significantly increased in PTC tissues and cells. Silencing linc00324 may inhibit the proliferation of PTC cells, arrest the cell cycle in G1/G0 phase, and promote the apoptosis by inhibiting the Notch signaling pathway.

Identification and expression distribution of esophageal carcinoma autoantigens.

e16520 Background: To identify the autoantigen protein molecules with autoserum in the tissues from the esophageal carcinoma (EC) patients, analyze autoantigen expression distribution in EC tissues, so as to provide basis for the molecular pathogenesis and clinical medication of EC. Methods: 69 cases of EC patients tissues and serum and 81 cases of healthy people serum were collected, serological proteome analysis (SERPA) was modified with sequential extraction of subcellular protein fractions to identify esophageal oncopathgensis stages autoantigen with autoserum in the tissues from the EC patients. Another 93 cases of EC patients tissue were collected, immunohistochemical and western blot method were used to detect expression distribution of EC autoantigens in esophageal carcinoma tissue, para-carcinoma tissue and normal tissue. Results: Autoantigens CK13, CK16, CaD, ACTG2, tumor rejection antigen (gp96) 1 variant, heat shock protein gp96 precursor were identified, among wihich, CK16, CaD, ACTG2, tumor rejection antigen (gp96)1 variant, heat shock protein gp96 precursor were firstly reported as EC autoantigens. Expression of autoantigens CK16, CaD, ACTG2 were increased in EC carcinoma tissue than para-carcinoma tissue and normal tissue, while CK13 were decreased. Positive expression level of CK16 in normal tissue, para-carcinoma tissue and cancer tissue of EC patients was 0.0076±0.0033, 0.0158±0.0065, 0.0356±0.0165 respectively, CaD was 0.0085±0.0048, 0.0107±0.0056, 0.0177±0.0103 respectively, ACTG2 was 0.0091±0.0039, 0.0136±0.0043, 0.0214±0.0110 respectively, and CK13 was 0.2053±0.0311, 0.1633±0.0280, 0.0412±0.0239 respectively. Conclusions: 6 EC autoantigens were identified, and 5 were first reported. Autoantigens CK13, CK16, CaD and ACTG2 were expressed in EC patients carcinoma tissue, which can be the potential biomarkers of esophageal carcinoma. This study provides new basis for the EC molecular mechanism and development of molecular drugs.

LncRNA DLEU2 promotes tumour growth by sponging miR-337-3p in human osteosarcoma.

According to statistics, abnormal regulation of lncRNAs pivotally influences multiple malignant tumours. DLEU2, as one of these lncRNAs, is detected to be related to growth and development of tumours. The molecular mechanisms of DLEU2 in osteosarcoma, however, are still unknown. QRT-PCR was adopted to analyse the correlations of clinicopathological features and prognosis of osteosarcoma cases with DLEU2. The influences of DLEU2 on cell migration and viability were evaluated independently by experiments in vitro and in vivo. Bioinformatics analysis, RNA immunoprecipitation (RIP) assay, and dual luciferase reporter gene assay confirmed the specific binding of DLEU2 to miR-337-3p. Moreover, rescue experiments were carried out to further evaluate the regulatory association between miR-337-3p expression and DLEU2. In osteosarcoma tissues and cells, DLEU2 expression level was raised remarkably in comparison with that in para-carcinoma normal tissues, and DLEU2 high expression had associations with poor prognosis, tumour stages, and TS of osteosarcoma cases. Cell migration ability and viability were blocked by DLEU2 knockdown but enhanced by ectopic DLEU2 expression in vitro and in vivo. Additionally, DLEU2 was found to sponge miR-337-3p and trigger the stimulating effect in osteosarcoma cells, which would be suppressed by miR-337-3p mimics. Furthermore, a negative correlation existed between miR-337-3p expression and DLEU2 in osteosarcoma tissues. This study manifests that DLEU2 sponges miR-337-3p to accelerate tumour growth and is confirmed to be a factor for poor prognosis of osteosarcoma cases. SIGNIFICANCE OF THE STUDY: LncRNA DLEU2 has been reported to be dysregulated in many tumours; however, the functions and underlying mechanism of DLEU2 in osteosarcoma pathogenesis are still unknown. This study is the first to demonstrate the roles of DLEU2 in osteosarcoma and revealed that DLEU2 may serve as a ceRNA to sponge miR-337-3p and then promote the progression of osteosarcoma, providing a potential therapeutic target for osteosarcoma.

High expression of ACE2 receptor of 2019-nCoV on the epithelial cells of oral mucosa

It has been reported that ACE2 is the main host cell receptor of 2019-nCoV and plays a crucial role in the entry of virus into the cell to cause the final infection. To investigate the potential route of 2019-nCov infection on the mucosa of oral cavity, bulk RNA-seq profiles from two public databases including The Cancer Genome Atlas (TCGA) and Functional Annotation of The Mammalian Genome Cap Analysis of Gene Expression (FANTOM5 CAGE) dataset were collected. RNA-seq profiling data of 13 organ types with para-carcinoma normal tissues from TCGA and 14 organ types with normal tissues from FANTOM5 CAGE were analyzed in order to explore and validate the expression of ACE2 on the mucosa of oral cavity. Further, single-cell transcriptomes from an independent data generated in-house were used to identify and confirm the ACE2-expressing cell composition and proportion in oral cavity. The results demonstrated that the ACE2 expressed on the mucosa of oral cavity. Interestingly, this receptor was highly enriched in epithelial cells of tongue. Preliminarily, those findings have explained the basic mechanism that the oral cavity is a potentially high risk for 2019-nCoV infectious susceptibility and provided a piece of evidence for the future prevention strategy in dental clinical practice as well as daily life.

MicroRNA-1297 inhibits proliferation and promotes apoptosis in gastric cancer cells by downregulating CDC6 expression.

Gastric cancer (GC), one of the most common malignant tumors and the second most common leading cause of cancer-related death worldwide, is a biologically heterogeneous disease accompanied by various genetic and epigenetic alterations. However, the molecular mechanisms underlying this disease are complex and not completely understood. Increasing studies have shown that aberrant microRNA (miRNA) expression is associated with GC tumorigenesis and growth. MiR-1297 has been confirmed to be a cancer suppressor in diverse tumors in humans. However, to date, the function and mechanism of miR-1297 in GC have not been determined. Here, we found that the expression of miR-1297 was significantly reduced in GC tissues or GC cell lines compared with paracarcinoma normal tissue or normal cell lines. Exogenic overexpression of miR-1297 in GC cell lines can inhibit cell proliferation and colony formation and induce apoptosis, and inhibition of miR-1297 in GC cell lines can promote cell proliferation and colony formation, and reduce apoptosis in vitro. We further confirmed that miR-1297 acted as a tumor suppressor through targeting cell division control protein 6 (CDC6) in GC. Moreover, the inverse relationship between miR-1297 and CDC6 was verified in GC cell lines. Our results indicated that miR-1297 is a potent tumor suppressor in GC, and its antiproliferative and gene-regulatory effects are, in part, mediated through its downstream target gene, CDC6. These findings implied that miR-1297 might be used as a novel therapeutic target of GC.

Expression and correlation of miR-124 and miR-126 in breast cancer.

The expression of micro ribonucleic acid (miR)-124 and miR-126 in different stages of breast cancer (BC) and their correlation were investigated to analyze the role of miR-124 and miR-126 in the occurrence and development of BC. BC tissues of 83 BC patients treated in First Teaching Hospital of Tianjin University of Traditional Chinese Medicine from March 2016 to January 2018 were selected as the research group, while the corresponding para-carcinoma normal tissues were selected as the control group. The relative expression levels of miR-124 and miR-126 in both groups were detected via reverse transcription-quantitative polymerase chain reaction (RT-qPCR), and the miR-124 and miR-126 expression asociated with clinicopathological parameters of patients and the correlation between miR-124 and miR-126 was analyzed. The relative expression levels of miR-124 and miR-126 in the research group were significantly lower than those in the control group (P<0.001). miR-124 and miR-126 in the research group were associated with clinicopathological parameters (clinical stage, degree of pathological differentiation and lymph node metastasis) of patients (P<0.001). According to Pearsons correlation analysis, there was a positive correlation between the relative expression levels of miR-124 and miR-126 in cancer tissues (r=0.497, P<0.001). The expression levels of miR-124 and miR-126 were downregulated in BC tissues, which were associated with clinicopathological parameters (clinical stage, degree of pathological differentiation and lymph node metastasis). There was a positive correlation between the expression levels of miR-124 and miR-126 in BC tissues. Thus, miR-124 and miR-126 may be involved in the occurrence, development, invasion and metastasis of BC, and both can be used as targeted biological indexes for treatment of BC.

Correlation of p16 and nm23-H1 expression levels with incidence and prognosis of soft tissue sarcoma.

The expression levels of p16 and nm23-H1 genes in soft tissue sarcoma (STS) were evaluated to investigate correlation of the expression levels with the incidence and prognosis of STS. Tumor tissues and para-carcinoma normal tissues were collected from 64 STS patients. The messenger ribonucleic acid (mRNA) expression levels of p16 and nm23-H1 in the tissues were detected via reverse transcription-quantitative polymerase chain reaction (RT-qPCR), and the protein expression levels of p16 and nm23-H1 in tissues were detected using immunohistochemistry. Spearman's correlation analysis was used for the correlation between expression levels of p16 and nm23-H1 in STS tissues and the correlation between p16 and nm23-H1 mRNA and protein expression. Moreover, the correlation of p16 and nm23-H1 expression levels in tumor tissues with pathological parameters and prognosis of STS patients were analyzed combined with clinical data. Results of RT-qPCR showed that mRNA expression levels of p16 and nm23-H1 in tumor tissues of STS patients were significantly lower than those in para-carcinoma normal tissues (P<0.01). Results of immunohistochemistry showed that the positive expression rates of p16 and nm23-H1 in tumor tissues of STS patients (43.75 and 39.06% respectively) were significantly lower than those in para-carcinoma normal tissues (85.93 and 89.06% respectively). The expression of p16 and nm23-H1 mRNA was positively correlated with protein expression levels. There was a positive correlation between the expression levels of p16 and nm23-H1 in tumor tissues of STS patients. The negative expression of p16 in tumor tissues of STS patients correlated with tumor size, tumor metastasis and clinical staging, and the negative expression of nm23-H1 correlated with tumor metastasis and clinical staging. The overall 5-year survival rate of patients was 54.68%, and the prognosis of patients with positive expression levels of p16 and nm23-H1 was better. Univariate survival analyses revealed that p16 and nm23-H1 were influencing factors of the overall survival rate of STS patients. p16 and nm23-H1 expression in STS is low, and their expression levels are closely related to the pathological parameters and prognosis of STS patients, so they can serve as reference indexes for prognosis estimation of STS.

MiR-216a-5p act as a tumor suppressor, regulating the cell proliferation and metastasis by targeting PAK2 in breast cancer.

Our study aimed to investigate the expression of microRNA-216a-5p (miR-216a-5p) in breast cancer (BC) and its effect on the proliferation and metastasis of BC cells by regulating the expression of p21-activated protein kinase 2 (PAK2) gene. A total of 50 cases of cancer tissue specimens and corresponding para-carcinoma normal tissue specimens were collected from the breast surgery department of our hospital from July 2016 to December 2017. BC MCF-7 cell line and normal breast epithelial MCF-10A cells were cultured. MiR-NC (negative control), LV-p21-activated protein kinase 2 (PAK2) and/or miR-216a-5p mimics were synthesized and transfected. The protein and mRNA expression level in BC tissues and cells were detected by Western blot and quantitative Real Time-Polymerase Chain Reaction (qRT-PCR) assay, respectively. Additionally, the Luciferase Reporter Assays, cell proliferation detection, clone formation assays and transwell migration and invasion assay were performed to determine the functional alteration of BC cells, respectively. The results of qRT-PCR demonstrated that miR-216a-5p was decreased in both BC tissues and cells compared with that in normal controls. Online target gene prediction software and Dual-Luciferase reporter assay were used for target identification, and PAK2 was identified as a functional target of miR-216a-5p in BC cells. The results were further clarified with the Western blot (WB) experiment. In vitro, cell functions were detected by Cell Counting Kit-8 (CCK-8), crystal violet staining and transwell experiment, respectively. The results indicated that decreased expression of PAK2 resulting from the up-regulation of miR-216a-5p could restrain the proliferation, clone formation, invasion and migration abilities of BC cells. We showed that miR-216a-5p played a role as antioncogene in BC, which provides a new therapeutic target for the treatment of BC.

Downregulated expression of RACK1 results in pancreatic cancer growth and metastasis.

BackgroundThe expression and function of the Receptor for Activated C Kinase 1 (RACK1) in cancer growth and metastasis are confused in different cancers, especially in pancreatic ductal adenocarcinoma (PDAC).MethodsOne-hundred and eighty-two PDAC tissue specimens (95 males and 87 females) including pancreatic cancer tissue and para-carcinoma tissue were collected for analysis between 2005 to 2012. Blood phenotypic parameters using cell count and capillary electrophoresis were investigated. HE staining, real time PCR, Western blot analysis, and soft agar assays were performed to determine the role of RACK1.PurposeIn this study, we aim to determine the specific role of RACK1 in the untility of PDAC.ResultsWe found that RACK1 expression was significantly lower in pancreatic cancer tissue than in para-carcinoma normal pancreatic tissue both in clinic and mice with pancreatic cancer at the early stage. Our results suggested that RACK1 silence could significantly promote cell growth and metastasis of pancreatic cancer cells. But we found that the overexpression of RACK1 has the opposite effect in vitro. In vivo MIAPaca-2 cells overexpressing RACK1, the results demonstrated lower metastatic ability than MIAPaca-2 cells. RACK1 overexpression could decrease the NF-κB transactivation activity of MIAPaca-2 cells, which was consistent with the inhibitory effect of RACK1 overexpression on the pro-migration and pro-invasive target gene of NF-κB, while which could be increased by RACK1 silence. RACK1 silence also enhanced protein expression of pro-migration and pro-invasive NF-κB target genes, which on the contrary, could be reversed by IκBα. Besides, RACK1 expression was significantly associated with lymph node metastasis, vessels metastasis, invasion of nerves as well as TNM staging. The 3-year survival rate of patients with high RACK1 expression was significantly higher than those patients with low RACK1 expression. However, RACK1 expression was not an independent risk factor for of the long-term postoperative survival of patients with pancreatic cancer.ConclusionThe obtained results in our study suggested that the low expression of RACK1 was associated with cancer cell growth and metastasis in pancreatic cancer through the activation of the NF-κB pathway. RACK1 could be a potential therapeutic drug target to pancreatic cancer and metastasis.

Association of BRAF gene and TSHR with cervical lymph node metastasis of papillary thyroid microcarcinoma.

Differences in BRAF gene mutation frequency and thyroid-stimulating hormone receptor (TSHR) protein expression in thyroid tissues were detected to investigate their association with local tissue invasion and cervical lymph node metastasis potential of papillary thyroid microcarcinoma (PTMC). The BRAF gene mutation frequency and TSHR expression in PTMC patients were detected via qPCR and immunohistochemical method, and the association between them was discussed combined with the clinical and pathological parameters. Kruskal-Wallis test was used for the univariate correlation analyses and comparison of mutation rate and expression rate, and Chi-square test was used for the association of central lymph node metastasis with BRAF gene and TSHR. The BRAFV600E mutation only existed in patients with thyroid cancer. Τhe larger the number of metastatic central lymph nodes was, the higher the proportion of BRAFV600E mutation would be. Τhe BRAFV600E mutation was related to the primary lesion size, capsular infiltration and lymph node metastasis of PTMC (P<0.05). The expression of TSHR in PTMC tissues was < those in thyroid benign lesions and para-carcinoma normal tissues, which was positively associated with the central lymph node metastasis (P<0.05). Τhe low expression of TSHR was related to the primary lesion size, capsular infiltration and metastatic lymph nodes of PTMC (P<0.05). The BRAFV600E and TSHR may be involved in the occurrence and lymphatic metastasis of PTMC. The BRAFV600E mutation has no association with the TSHR protein expression (P=0.256), but the coincidence coefficient indicates that their diagnostic significance in PTMC is not similar, so BRAFV600E mutation and TSHR protein expression can be used jointly in the prediction of invasion and lymph node metastasis of PTMC, which may be more meaningful for clinical guidance.

Expression of miR-204 in pediatric retinoblastoma and its effects on proliferation and apoptosis of cancer cells.

Expression, clinical significance and molecular mechanism of miR-204 in human retinoblastoma (RB) and para-carcinoma tissues were investigated. A total of 110 cases of RB tissues preserved after ophthalmectomy in the First Affiliated Hospital of Hunan Normal University (People's Hospital of Hunan Province) from April 2013 to June 2017 were collected along with 100 cases of para-carcinoma normal tissues. The expression of miR-204 in RB tissues was detected via reverse transcription-quantitative polymerase chain reaction (RT-qPCR), and its associations with clinicopathological features were analyzed. Y79 cells were transfected with miR-204 mimics. A total of 80 pmol/l miR-204 mimics and 10 µl Lipofectamine 2000 were added into the experimental group. Cell proliferation was detected via methyl thiazolyl tetrazolium (MTT) assay at 24, 48, 72 and 96 h, apoptosis was detected via flow cytometry at 48 h after transfection, and the relative expression levels of B-cell lymphoma 2 (Bcl-2) messenger RNA (mRNA) and Sirt1 mRNA were detected via RT-qPCR. The results of MTT assay revealed that the measured value of the optical density (OD) in the experimental group at 48 h was obviously lower than that in the negative control group (p<0.001). The proportion of apoptotic cells in the experimental group was remarkably higher than that in the negative control group (p<0.001). Compared with those in the negative control group, the relative expression levels of Bcl-2 and Sirt1 mRNAs in the experimental group were significantly decreased (p<0.001). miR-204 may be involved in the occurrence and development of RB, which is significantly associated with clinical tissue differentiation, neural infiltration and lymph node metastasis in patients. miR-204 may inhibit proliferation and promote apoptosis of RB cells through downregulating the expression of Bcl-2 and Sirt1 in RB. Therefore, miR-204 may become a new biological index for early diagnosis, prognosis evaluation and biotherapy of RB.