sh-NC Groups Research Articles

Analysis of the Expression of PRDX6 in Patients with Hepatocellular Carcinoma and its Effect on the Phenotype of Hepatocellular Carcinoma Cells.

This research aimed to study the expression of PRDX6 mRNA in hepatocellular carcinoma (HCC) and its effect on the prognosis of HCC. Moreover, the effect of PRDX6 gene knockdown on the proliferation, migration, and invasion of HepG2 cells mediated by lentivirus was also examined. This study offers a theoretical and experimental basis for further research on the mechanism of PRDX6 in liver cancer and new methods for clinical diagnosis and treatment. RNA sequence data of 369 HCC patients were screened through the TCGA database, and the expression and clinical characteristics of PRDX6 mRNA were analyzed based on high-throughput RNA sequencing data. HepG2 cells were divided into WT, sh-NC and sh-PRDX6 groups. Real-time PCR and Western blot were used to detect the expression levels of the PRDX6 gene and protein, respectively. CCK8 method was used to detect the proliferation activity of HepG2 cells, scratch healing test was used to detect the migration ability, Transwell chamber was used to detect the invasion ability, and Western blot was used to detect the expression levels of PI3K/Akt/mTOR signaling pathway and Notch signaling pathway-related proteins. The expression of PRDX6 was significantly correlated with the gender, race, clinical stage, histological grade, and survival time of HCC patients (P < 0.05). Compared with that in WT and sh-NC groups, the expression level of PRDX6 protein in HCC patients was significantly lower (P < 0.01), the proliferation activity of HCC cells was significantly decreased (P < 0.05), and the migration and invasion ability was significantly decreased (P < 0.05) in the sh-PRDX6 group. The expression levels of PI3K, p-Akt, p-mTOR, Notch1, and Hes1 proteins in the sh-PRDX6 group were significantly lower than those in WT and sh-NC groups (P < 0.05). The expression of PRDX6 may be closely related to the prognosis of HCC. Lentivirus-mediated PRDX6 knockdown can inhibit the proliferation, migration and invasion of HCC cells, which may be related to its regulating the PI3K/Akt/mTOR and Notch1 signaling pathways. PRDX6 is expected to be a new target for the diagnosis and treatment of liver cancer.

Lncrna Famlf-2 Promotes Leukemogenesis Via the DHX9/c-Myc Axis in Acute Lymphoblastic Leukemia

Long noncoding RNAs (lncRNAs), defined as the transcripts longer than 200 nt without protein-coding capacity, have been found to be aberrantly expressed in diverse human diseases including leukemia. Our previous study identified Familial acute myelogenous leukemia related factor ( FAMLF) in an acute myeloblastic leukemia (AML) pedigree. Two splice variants of FAMLF, FAMLF-1 and FAMLF-2 were detected. FAMLF-1 is upgraded in AML patients and associated with poor prognosis. However, the full cDNA sequence of FAMLF-2 is not verified and its role in leukemogenesis remain unclear. Herein, we aim to investigate the functional and mechanistic roles of FAMLF-2 in leukemia. Using real-time quantitative RT-PCR assay, we found that the expression of FAMLF-2 was at higher levels in Acute Lymphoblastic Leukemia(ALL) cell lines when compared to AML cell lines. Bone marrow cells were obtained from de novo ALL patients. qRT-PCR showed FAMLF-2 was up-regulated in ALL patients (N=115) compared to healthy controls (N=72). The FAMLF-2 expression was positively correlated with the poor prognosis in ALL patients by Kaplan-Meier analysis. These data demonstrated that FAMLF-2 is overexpressed in ALL patients and is associated with poor prognosis. To verify the full cDNA sequence of FAMLF-2, 5‘ and 3‘ rapid amplification of the cDNA ends (RACE) was performed, and we identified the 2393 nt of FAMLF-2. FAMLF-2 is predicted to not have protein-coding potential. Further subcellular analysis showed FAMLF-2 is mainly localized in the nucleus of Jurkat cells by lncRNA FISH and subcellular fractionation assay. To further verify the role of FAMLF-2 in ALL leukemogenesis, a xenograft model was established in NCG mice by subcutaneously or intravenously injection of engineered Jurkat cells (knockdown group: shNC and sh FAMLF-2, overexpression group: pCDH and FAMLF-2). We found that the size and weight of tumors in FAMLF-2 knockdown group were significantly decreased compared with the empty vector in subcutaneous ALL xenograft tumor model. and the mice in the sh FAMLF-2 groups survived longer than those in the shNC groups in introvenous ALL xenograft tumor model (sh FAMLF-2 vs. shNC: 55 days vs. 43 days, P&amp;lt;0.01). These data suggested that downregulation of FAMLF-2 inhibits leukemia development. Although no significant difference was found in the overall survival of mice overexpressed FAMLF-2. We observed the accelerated central nervous system(CNS) infiltration of leukemia cells when FAMLF-2 was overexpressed (clinical score at 32th day from Vector vs. FAMLF-2: 2.7 scores vs. 1.2 scores, p &amp;lt; 0.01 ). In vitro study revealed knockdown of FAMLF-2 inhibited cell proliferation and promoted cell apoptosis in ALL cells, and vice versa. Taken together, our clinical, mouse model, and AML cell data demonstrated that FAMLF-2 promotes leukemogenesis. RNA sequencing was performed to study how FAMLF-2 promotes ALL development. Gene Set Enrichment Analysis (GSEA) of genes downregulated by FAMLF-2 showed “Myc targets” with the highest normalised enrichment score (NES) in Jurkat cells. C-Myc is a well-recognized transcription factor that involves in leukemogenesis. To investigate whether FAMLF-2 performed direct interaction with c-Myc, we performed RNA pull-down followed by mass spectrometry analysis. Instead of c-Myc, DExH-Box Helicase 9 (DHX9) has the highest abundance. The Western blot and RNA immunoprecipitation (RIP) assay were further performed to validated the direct interaction between FAMLF-2 and DHX9. DHX9 is an RNA helicase that participates in c-Myc mRNA stability. Inhibition of FAMLF-2 suppressed DHX9 and c-Myc expression. Reduced interaction between DHX9 protein and c-Myc mRNA were observed when FAMLF-2 were downregulated. Furthermore, RNA stability assays suggested that knockdown of FAMLF-2 leads to a declined half-life of c-Myc mRNA. Collectively, these results demonstrated that FAMLF-2 regulates the stability of c-Myc mRNA through direct binding of DHX9, thus involved in the ALL development. In summary, our study revealed the previous undefined cDNA sequence of FAMLF-2, and provides new insights of FAMLF-2 in leukemogenesis via the DHX9/c-Myc axis, which might act as a prospective prognostic biological marker in ALL.

The Role of Low-Density Lipoprotein Receptor-Associated Protein LRP11 in the Development of Hepatocellular Carcinoma

This study investigated the role of the low-density lipoprotein receptor-associated protein LRP11 in hepatocellular carcinoma (HCC). Analysis of TCGA and GTEx databases revealed that LRP11 expression was significantly increased in liver cancer tissues compared to normal tissues (P &lt;0.05). High expression of LRP11 was associated with shorter survival in liver cancer patients (P &lt;0.05). Lentivirus transfection was used to create sh-NC and sh-LRP11 groups for further experiments. Silencing LRP11 in HepG2 cells resulted in a significant decrease in cell viability (P &lt;0.05), increased apoptosis rate (P &lt;0.01), and upregulation of the apoptosis-related protein Bax (P &lt;0.01) and downregulation of Bcl-2 (P &lt;0.01). Moreover, the sh-LRP11 group showed a significant decrease in the S-phase of the cell cycle (P &lt;0.01) and reduced expression of Cyclin D1 (P &lt;0.01). These findings indicate that LRP11 is highly expressed in liver cancer tissues and is associated with an unfavorable prognosis. Suppression of LRP11 expression inhibits the proliferation of HCC cells, promotes apoptosis, and affects cell cycle progression. These results contribute to understanding the molecular mechanisms underlying HCC development and progression, as well as identifying potential therapeutic targets.

Anti-hyperlipidemic, Anti-inflammatory, and Ameliorative Effects of DRP1 Inhibition in Rats with Experimentally Induced Myocardial Infarction.

This study aims to investigate the biological role of DRP1 in myocardial infarction (MI) in concert with hyperlipidemia (HL). Based on the available literatures, 10 genes related to MI with HL (HL-MI) were screened and detected in clinical samples. High-fat diet (HFD) was used to establish HL rat models, after which the rats were subcutaneously injected with PBS or isoproterenol hydrochloride to induce acute MI. Then, rats with HL-MI were injected with pcDNA3.1, pcDNA3.1-DRP1, sh-NC, or sh-DRP1. Serum levels of total cholesterol (TC), triglycerides (TG), high-density lipoprotein-cholesterol (HDL-C), and low-density lipoprotein-cholesterol (LDL-C) were measured. Cardiac function was evaluated by detecting left ventricular fractional shortening (LVFS) and left ventricular ejection fraction (LVEF). Infarct size and histopathological changes were assessed as well as myocardial apoptosis and collagen deposition. The concentration of IL-6, IL-1β, and TNF-α in rat serum and cardiac tissues was also measured by ELISA. Mitochondrial function was shown by measuring the morphology, mitochondrial membrane potential (MMP), and intracellular reactive oxygen species (ROS) level. Pro-apoptotic proteins (Bax, caspase-1, and cleaved caspase-1) and NLRP3 inflammasome activation were also assessed. The expressions of the 10 genes were measured in clinical samples and DRP1 was selected for further experiments with significantly upregulated expression in MI patients. HFD-induced rats showed increased body weight, concurrent with higher levels of TG, TC, and LDL-C and lower HDL-C level. Compared with the BD-PBS group, the HFD-PBS group presented higher mRNA and protein expression levels of DRP1, exacerbated cardiac functions, enlarged infarct size, loss of cardiomyocytes, and disordered island cardiomyocytes. In the HL-MI rat model, injection of pcDNA3.1-DRP1 enhanced the levels of serum lipids and inflammation cytokines, induced loss of a number of cardiomyocytes and collagen deposition, and decreased LVFS and LVEF, while injection of sh-DRP1 ameliorated myocardial injuries, inflammation, and cardiomyocyte apoptosis and fibrosis. In coronary artery endothelial cells from the rats, loss of MMP was observed in the HFD-MI, LV-NC, LV-DRP1, and sh-NC groups and concomitantly, the sh-DRP1group showed increased MMP and decreased levels of mitochondrial ROS, cytochrome C, pro-apoptotic proteins, and NLRP3. Inhibition of DRP1 markedly suppressed HL, systematic inflammation, and myocardial injuries induced by HL-MI through partly restoring mitochondrial function and reducing NLRP3 expression.

Knockdown of ALAS2 Affects Erythroid Differentiation by Down-regulating Mitophagy Receptor BNIP3L

The expression of ALAS2 was down-regulated by transfection of lentivirus, then quantitative real-time PCR was performed to detect the transfection efficiency. Flow cytometry analysis was applied to evaluate apoptosis of cells, erythroid differentiation, mitochondrial membrane potential and reactive oxygen species (ROS) level. Western blot was used to detect the BNIP3L expression, Co-immunoprecipitation was performed to analyze the relationship between ALAS2 and BNIP3L. Compared with sh-NC group, knockdown of ALAS2 induced downregulation of BNIP3L mRNA and protein expression(P<0.01) and erythroid related transcription factors GATA1, Nrf2 expression, as well as reduction of ROS level(P<0.05). Mitochondrial membrane potential of control (sh-NC) group was lower than that of shALAS2 group(P<0.05), but there was no significant change of cell apoptotic rate in two groups. CD71highCD235ahigh + CD71lowCD235ahigh cells of sh-NC and shALAS2 groups were 53.5%, 92.9% at 96 h after hemin induction, respectively. No direct action between ALAS2 and BNIP3L was observed. The intracellular heme level can affect the expression of BNIP3L which may be related with the regulation of ROS and transcription factors GATA1 and Nrf2. Higher BNIP3L facilitates cell differentiation but lower BNIP3L is favorable for cells survival.

Silencing RNF13 Alleviates Parkinson's Disease - Like Problems in Mouse Models by Regulating the Endoplasmic Reticulum Stress-Mediated IRE1α-TRAF2-ASK1-JNK Pathway.

The objective of this study was to understand if RNF13 can affect Parkinson's disease (PD) model mice by modulating the endoplasmic reticulum stress (ERS)-mediated IRE1α-TRAF2-ASK1-JNK pathway. C57BL/6 mice injected with MPTP to establish PD mice models were divided into Control, MPTP, MPTP + sh-RNF13, and MPTP + sh-NC groups. Rotarod, balance beam, and open-field tests were used to assess the behavioral changes of experimental mice. Immunofluorescence assay was used to determine TH-positive expression in substantia nigra, TUNEL staining to detect apoptosis, and Western blotting to measure the expression of IRE1α-TRAF2-ASK1-JNK pathway. Besides, SH-SY5Y cells treated with MPP+ were assigned into Control, MPP+, MPP+ + sh-RNF13, and MPP+ + sh-NC groups in vitro to detect cell viability, apoptosis and Ca2+ level. When compared with those Control mice, MPTP mice showed decreased retention time spent on rotarod performance and prolonged time on balance beam test, as well as evident reductions in floor plane (FP) movements, moving time, moving distance, and mean velocity in open-field test, which had an obvious increase of TUNEL-positive cells, significant decrease of TH-positive cells, and remarkable up-regulations of RNF13, p-IRE1α/IRE1α, TRAF2, ASK1, and p-JNK/JNK. Meanwhile, MPTP mice treated with sh-RNF13 were improved in all above indexes. In vitro, MPP+ treated SH-SY5Y cells had decreased cell viability and increased cell apoptosis, as well as the upregulated IRE1α-TRAF2-ASK1-JNK pathway proteins and Ca2+ level. RNF13 knockdown improved all above indexes in SH-SY5Y cells treated with MPP+. Silencing RNF13 can alleviate motor dysfunction and dopamine neuronal damage in PD mice by inhibiting ERS-mediated IRE1α-TRAF2-ASK1-JNK pathway.

Role of lncRNA Fez family zinc finger protein 1 antisense RNA1 in hepatocellular carcinoma

Objective: To investigate the effect of long-chain non-coding RNA Fez family zinc finger protein 1 antisense RNA1 (lncRNA FEZF1-AS1) on the biological function of hepatocellular carcinoma (HCC). Methods: SMMC771 and BEL-7402 cells were transfected with sh-FEZF1-AS1 and OE-FEZF1-AS1, respectively. The expression of lncRNA FEZF1-AS1 was detected by real-time quantitative PCR. Cell proliferation was detected by Cell Counting Kit-8 (CCK-8), and apoptosis was detected by flow cytometry. The effects of lncRNA FEZF1-AS1 on invasion and migration were detected by Transwell and wound healing assays. The expression levels of adhesion molecules were detected by Western blot. The effect of lncRNA FEZF1-AS1 on the in vivo growth was verified by nude mice xenograft experiments. Results: The silencing or ectopic expression of lncRNA FEZF1-AS1 inhibited or promoted the proliferation of hepatocellular carcinoma cells. CCK-8 assay showed that the proliferation abilities of SMMC7721 and BEL-7402 cells in sh-FEZF1-AS1 transfection group significantly decreased, achieving (35.43±4.06)% and (34.68±3.97)%, respectively, on the fifth day. There were significant differences between sh-FEZF1-AS1 group and sh-NC group [52.21±8.46)% and (53.76±7.64)%] (all P<0.05). In contrast, the proliferation ability of SMMC7721 and BEL-7402 cells transfected with OE-FEZF1-AS1 was significantly increased, achieving (83.49±6.92)% and (80.31±3.13)%, respectively, on the fifth day. There were significant differences between OE-FEZF1-AS1 and OE-NC group [53.03±8.84)% and (55.11±7.09)%] (all P<0.05). The subsequent flow cytometry results showed that cell apoptotic rates of SMMC7721 and BEL-7402 cells transfected with sh-FEZF1-AS1 were (13.02±1.38)% and (11.88±1.29)%, respectively, which were significantly higher than those in sh-NC groups [(5.57±1.46)% and (8.06±1.42)%, respectively, all P<0.05]. In contrast, the apoptotic rates of SMMC7721 and BEL-7402 cells transfected with OE-FEZF1-AS1 were (3.01±0.39)% and (3.22±0.43)%, which were significantly lower than those in OE-NC groups [(6.68±0.96)% and (6.63±0.45)%, all P<0.05]. In addition, knockdown or overexpression of lncRNA FEZF1-AS1 expression inhibited or enhanced the migration and invasion abilities as well as the levels of adhesion molecules in hepatocellular carcinoma cells. After 30 days of feeding under the same conditions, the tumor volumes of sh-FEZF1-AS1 and sh-NC SMMC7721 cells xenograft mice models were (0.26±0.03) cm(3) and (0.63±0.06) cm(3), respectively, showing significant difference (P<0.05). The tumor volumes of sh-FEZF1-AS1 and sh-NC BEL-7402 cells were (0.31±0.02) cm(3) and (0.72±0.08) cm(3), and the difference was statistically significant (P<0.05). Conclusion: lncRNA FEZF1-AS1 may strengthen the growth, migration and invasion of hepatocellular carcinoma cells.

LncRNA FLVCR1-AS1 regulates cell proliferation, migration and invasion by sponging miR-485-5p in human cholangiocarcinoma.

Long non-coding RNA (lncRNA) FLVCR1 antisense RNA 1 (FLVCR1-AS1) serves a crucial role in many types of cancer; however, to the best of our knowledge, the biological effect of FLVCR1-AS1 in cholangiocarcinoma (CCA) remains unclear. The present study aimed to elucidate the involvement of FLVCR1-AS1 in the regulation of human CCA cell growth, migration and invasion, as well as the mechanisms underlying its effect. The expression levels of FLVCR1-AS1 in CCA tumor tissues, adjacent normal tissues, CCA cell lines and a cholangiocyte cell line were determined by reverse transcription-quantitative polymerase chain reaction. A significantly higher expression level of FLVCR1-AS1 was identified in CCA tumor tissues and the CCA cell lines HuCCT1 and CCLP1 compared with the normal controls. Short hairpin RNA targeting FLVCR1-AS1 (shFLVCR1-AS1) and a control plasmid (shNC) were transfected into CCA cell lines. Cell proliferation, colony formation, migration and invasion of CCA cells transfected with shFLVCR1-AS1 were significantly suppressed compared with the shNC groups. The expression levels of migration and invasion-associated proteins, including Twist, matrix metalloproteinase (MMP)-2 and MMP-9, were also significantly suppressed by shFLVCR1-AS1-treatment. Furthermore, FLVCR1-AS1 knockdown inhibited tumor growth in a xenograft model. Mechanistically, FLVCR1-AS1 was demonstrated to sponge microRNA-485-5p (miR-485-5p) in human CCA. The expression of miR-458-5p was significantly decreased in CCA tissue compared with normal tissue, and Pearson's correlation analysis revealed that FLVCR1-AS1 expression was negatively correlated with miR-485-5p expression in CCA tissues. These results suggested that lncRNA FLVCR1-AS1 may be used as a novel therapeutic target and a potential diagnostic marker for CCA.

Retracted: Down‐regulation of the long noncoding RNA‐HOX transcript antisense intergenic RNA inhibits the occurrence and progression of glioma

This study aims to explore the role of long noncoding RNA (lncRNA)-HOX transcript antisense intergenic RNA (HOTAIR) in the occurrence and progression of glioma. Fresh glioma and normal brain tissues were classified into a glioma group (n = 67) and a normal group (n = 64) respectively. U87 cells were assigned into the blank, sh-NC, and sh-HOTAIR groups. Quantitative real-time polymerase chain reaction (qRT-PCR) was utilized to determine HOTAIR expression. Cell proliferation, cell cycle and cell apoptosis rates were detected by cell counting kit-8 (CCK-8) and flow cytometry (FCM). Scratch test and transwell assay were conducted for cell migration and invasion. Orthotopic glioma tumor model in nude mice was established by inoculating tumor cell suspension. Hematoxylin-Eosin (HE) staining was used to observe the growth and invasion of orthotopic glioma tumors. The expression of HOTAIR and cell viability was found to be lowest in the sh-HOTAIR group among the three groups. The sh-HOTAIR group exhibited a higher apoptotic rate and lower number of cell migration compared with the blank and sh-NC groups. Additionally, the speed of wound healing was slower, the migration distance decreased and the survival time of nude mice was extended in the sh-HOTAIR compared to the other groups. Moreover, the sh-HOTAIR group demonstrated reduced lesion sizes and inflammation, no convulsions or hemiplegia and lesser number of satellite metastases. Our findings support that down-regulation of HOTAIR could inhibit cell proliferation, promote cell apoptosis as well as suppress cell invasion and migration in the progression of glioma.

Changes in myofilament proteins, but not Ca2+ regulation, are associated with a high-fat diet-induced improvement in contractile function in heart failure

Pathological conditions such as diabetes, insulin resistance, and obesity are characterized by elevated plasma and myocardial lipid levels and have been reported to exacerbate the progression of heart failure (HF). Alterations in cardiomyocyte Ca(2+) regulatory properties and myofilament proteins have also been implicated in contractile dysfunction in HF. However, our prior studies reported that high saturated fat (SAT) feeding improves in vivo myocardial contractile function, thereby exerting a cardioprotective effect in HF. Therefore, we hypothesized that SAT feeding improves contractile function by altering Ca(2+) regulatory properties and myofilament protein expression in HF. Male Wistar rats underwent coronary artery ligation (HF) or sham surgery (SH) and were fed normal chow (SHNC and HFNC groups) or a SAT diet (SHSAT and HFSAT groups) for 8 wk. Contractile properties were measured in vivo [echocardiography and left ventricular (LV) cannulation] and in isolated LV cardiomyocytes. In vivo measures of contractility (peak LV +dP/dt and -dP/dt) were depressed in the HFNC versus SHNC group but improved in the HFSAT group. Isolated cardiomyocytes from both HF groups were hypertrophied and had decreased percent cell shortening and a prolonged time to half-decay of the Ca(2+) transient versus the SH group; however, SAT feeding reduced in vivo myocyte hypertrophy in the HFSAT group only. The peak velocity of cell shortening was reduced in the HFNC group but not the HFSAT group and was positively correlated with in vivo contractile function (peak LV +dP/dt). The HFNC group demonstrated a myosin heavy chain (MHC) isoform switch from fast MHC-α to slow MHC-β, which was prevented in the HFSAT group. Alterations in Ca(2+) transients, L-type Ca(2+) currents, and protein expression of sarco(endo)plasmic reticulum Ca(2+)-ATPase and phosphorylated phospholamban could not account for the changes in the in vivo contractile properties. In conclusion, the cardioprotective effects associated with SAT feeding in HF may occur at the level of the isolated cardiomyocyte, specifically involving changes in myofilament function but not sarcoplasmic reticulum Ca(2+) regulatory properties.