RNA therapeutics inhibit the expression of specific proteins/RNAs by targeting complementary sequences of corresponding genes or encode proteins for the synthesis desired genes to treat genetic diseases. RNA-based therapeutics are categorized as oligonucleotide drugs (antisense oligonucleotides, small interfering RNA, RNA aptamers), and mRNA drugs. The antisense oligonucleotides and small interfering RNA for treatment of genetic diseases have been approved by the FDA in the United States, while RNA aptamers and mRNA drugs are still in clinical trials. Chemical modifications can be applied to RNA drugs, such as pseudouridine modification of mRNA, to reduce immunogenicity and improve the efficacy. The secure and effective delivery systems such as lipid-based nanoparticles, extracellular vesicles, and virus-like particles are under development to address stability, specificity, and safety issues of RNA drugs. This article provides an overview of the specific molecular mechanisms of eleven RNA drugs currently used for treating genetic diseases, and discusses the research progress of chemical modifications and delivery systems of RNA drugs.
Aptamers, Nucleotide ; RNA, Small Interfering/therapeutic use* ; RNA, Messenger ; Oligonucleotides, Antisense/therapeutic use*

OBJECTIVES: To investigate the effects and molecular mechanisms of asiatic acid on β-cell function in type 2 diabetes mellitus (T2DM). METHODS: The T2DM model was established by high fat diet and streptozotocin injection in ICR mice, and the effects of asiatic acid on glucose regulation were investigated in model mice. The islets were isolated from palmitic acid-treated diabetic mice. ELISA was used to detect the glucose-stimulated insulin secretion, tumor necrosis factor (TNF)-α and interleukin (IL)-6. ATP assay was applied to measure ATP production, and Western blotting was used to detect protein expression of mature β cell marker urocortin (Ucn) 3 and mitofusin (Mfn) 2. The regulatory effects of asiatic acid on glucose-stimulated insulin secretion (GSIS) and Ucn3 expression were also investigated after siRNA interference with Mfn2 or treatment with TNF-α. RESULTS: Asiatic acid with the dose of 25 mg·kg^－1·d^－1 had the best glycemic control in T2DM mice and improved the homeostasis model assessment β index. Asiatic acid increased the expression of Mfn2 and Ucn3 protein and improved the GSIS function of diabetic β cells in vitro and in vivo (both P<0.05). Moreover, it improved the ATP production of islets of T2DM mice in vitro (P<0.05). Interfering Mfn2 with siRNA blocked the up-regulation of Ucn3 and GSIS induced by asiatic acid. Asiatic acid inhibited islet TNF-α content and increased Mfn2 and Ucn3 protein expression inhibited by TNF-α. CONCLUSIONS Asiatic acid improves β cell insulin secretion function in T2DM mice by maintaining the β cell maturity, which may be related to the TNF-α/Mfn2 pathway.
Mice ; Animals ; Insulin Secretion ; Diabetes Mellitus, Type 2/drug therapy* ; Islets of Langerhans/metabolism* ; Tumor Necrosis Factor-alpha/metabolism* ; Insulin/therapeutic use* ; Diabetes Mellitus, Experimental ; Mice, Inbred ICR ; Glucose/therapeutic use* ; Interleukin-6/metabolism* ; RNA, Small Interfering/pharmacology* ; Adenosine Triphosphate ; GTP Phosphohydrolases/therapeutic use*

OBJECTIVE: To investigate the influence and mechanism of atorvastatin on glycolysis of adriamycin resistant acute promyelocytic leukemia (APL) cell line HL-60/ADM. METHODS: HL-60/ADM cells in logarithmic growth phase were treated with different concentrations of atorvastatin, then the cell proliferation activity was measured by CCK-8 assay, the apoptosis was detected by flow cytometry, the glycolytic activity was checked by glucose consumption test, and the protein expressions of PTEN, p-mTOR, PKM2, HK2, P-gp and MRP1 were detected by Western blot. After transfection of PTEN-siRNA into HL-60/ADM cells, the effects of low expression of PTEN on atorvastatin regulating the behaviors of apoptosis and glycolytic metabolism in HL-60/ADM cells were further detected. RESULTS: CCK-8 results showed that atorvastatin could inhibit the proliferation of HL-60/ADM cells in a concentration-dependent and time-dependent manner (r=0.872, r=0.936), and the proliferation activity was inhibited most significantly when treated with 10 μmol/L atorvastatin for 24 h, which was decreased to (32.3±2.18)%. Flow cytometry results showed that atorvastatin induced the apoptosis of HL-60/ADM cells in a concentration-dependent manner (r=0.796), and the apoptosis was induced most notably when treated with 10 μmol/L atorvastatin for 24 h, which reached to (48.78±2.95)%. The results of glucose consumption test showed that atorvastatin significantly inhibited the glycolytic activity of HL-60/ADM cells in a concentration-dependent and time-dependent manner (r=0.915, r=0.748), and this inhibition was most strikingly when treated with 10 μmol/L atorvastatin for 24 h, reducing the relative glucose consumption to (46.53±1.71)%. Western blot indicated that the expressions of p-mTOR, PKM2, HK2, P-gp and MRP1 protein were decreased in a concentration-dependent manner (r=0.737, r=0.695, r=0.829, r=0.781, r=0.632), while the expression of PTEN protein was increased in a concentration-dependent manner (r=0.531), when treated with different concentrations of atorvastatin for 24 h. After PTEN-siRNA transfected into HL-60/ADM cells, it showed that low expression of PTEN had weakened the promoting effect of atorvastatin on apoptosis and inhibitory effect on glycolysis and multidrug resistance. CONCLUSION Atorvastatin can inhibit the proliferation, glycolysis, and induce apoptosis of HL-60/ADM cells. It may be related to the mechanism of increasing the expression of PTEN, inhibiting mTOR activation, and decreasing the expressions of PKM2 and HK2, thus reverse drug resistance.
Humans ; Atorvastatin/pharmacology* ; PTEN Phosphohydrolase/pharmacology* ; Sincalide/metabolism* ; Drug Resistance, Neoplasm/genetics* ; TOR Serine-Threonine Kinases/metabolism* ; Leukemia, Promyelocytic, Acute/drug therapy* ; Doxorubicin/pharmacology* ; Apoptosis ; RNA, Small Interfering/pharmacology* ; Glycolysis ; Glucose/therapeutic use* ; Cell Proliferation

Objective: To screen the key genes involved in gefitinib resistance of lung adenocarcinoma PC9/GR cells which harbored 19 exon mutation of epidermal growth factor receptor (EGFR) gene, and discuss the effect and mechanism of downregulation of solute carrier family 7 member 11 (SLC7A11) on the gefitinib resistance of PC9/GR cells. Methods: RNA microarray was conducted to detect the gene expressions in PC9 and PC9/GR cells. The differently expressed genes were screened by using limma package of R language and analyzed by Kyoto encyclopedia of genes and genomes (KEGG) pathway enrichment analysis. Western blotting was performed to determine the expression of SLC7A11 protein in PC9 and PC9/GR cells. PC9/GR cells were infected with lentivirus plasmid containing short hairpin RNA (shRNA) targeting SLC7A11 or negative control shRNA (sh-NC), respectively. Real-time quantitative polymerase chain reaction (RT-qPCR) was performed to evaluate the efficacy of shRNA on the expression of SLC7A11 mRNA. Cell counting kit-8 (CCK-8) assay was conducted to determine the suppressing effect of gefitinib on PC9/GR cells. Mito-Tracker Red CMXRos probe and malondialdehyde (MDA) assay kit were used to evaluate gefitinib-induced ferroptosis in PC9/GR cells. Immunohistochemistry (IHC) was conducted to detect the expression of SLC7A11 protein in the tumor tissues of advanced stage lung adenocarcinoma patients harboring 19 exon mutation of EGFR gene. Thirty-six advanced stage lung adenocarcinoma patients who received EGFR-tyrosihe kinase inhibitor(TKI) as first-line treatment in Fourth Hospital of Hebei Medical Unviersity were enrolled. Kaplan-Meier survival curve was drawn to analyze the correlation between SLC7A11 expression and progression-free survival (PFS) of the patients. Results: RNA array demonstrated that 2 888 genes were differently expressed between PC9 and PC9/GR cells. KEGG analysis showed that ferroptosis-related gene was one of the most enriched region of the differently expressed genes between PC9 and PC9/GR cells. These ferroptosis-related gene cohort contained 13 genes, among which SLC7A11 exhibited the most significant difference. Western blotting showed that the expression of SLC7A11 protein in PC9/GR cells was significantly higher than that in PC9 cells (0.76±0.03 vs. 0.19±0.02, P<0.001). The 50% inhibiting concentration (IC(50)) of gefitinib was 35.08 μmol/L and 64.01 μmol/L for sh-SLC7A11 and sh-NC group PC9/GR cells, respectively. PC9/GR cells in sh-SLC7A11 group exhibited significantly lower density of mitochondria fluorescence after gefitinib treatment, compared to the sh-NC group (213.77±26.50 vs. 47.88±4.55, P<0.001). In addition, PC9/GR cells in sh-SLC7A11 group exhibited significantly higher MDA after gefitinib treatment, compared to the sh-NC group [(15.43±1.60) μmol/mg vs. (82.18±7.77) μmol/mg, P<0.001]. The PFS of the patients with low expression of SLC7A11 (n=18) was significantly longer than the patients with high expression of SLC7A11 (n=18, 16.77 months vs. 9.14 months, P<0.001). Conclusion: Downregulation of SLC7A11 could increase the sensitivity of PC9/GR cells to gefitinib by promoting ferroptosis.
Humans ; Gefitinib/therapeutic use* ; Antineoplastic Agents/therapeutic use* ; Lung Neoplasms/pathology* ; Down-Regulation ; Quinazolines/therapeutic use* ; Drug Resistance, Neoplasm/genetics* ; ErbB Receptors/metabolism* ; Adenocarcinoma of Lung ; Protein Kinase Inhibitors/therapeutic use* ; RNA, Small Interfering/genetics* ; Cell Line, Tumor ; Amino Acid Transport System y+/metabolism*

Objective: To screen the key genes involved in gefitinib resistance of lung adenocarcinoma PC9/GR cells which harbored 19 exon mutation of epidermal growth factor receptor (EGFR) gene, and discuss the effect and mechanism of downregulation of solute carrier family 7 member 11 (SLC7A11) on the gefitinib resistance of PC9/GR cells. Methods: RNA microarray was conducted to detect the gene expressions in PC9 and PC9/GR cells. The differently expressed genes were screened by using limma package of R language and analyzed by Kyoto encyclopedia of genes and genomes (KEGG) pathway enrichment analysis. Western blotting was performed to determine the expression of SLC7A11 protein in PC9 and PC9/GR cells. PC9/GR cells were infected with lentivirus plasmid containing short hairpin RNA (shRNA) targeting SLC7A11 or negative control shRNA (sh-NC), respectively. Real-time quantitative polymerase chain reaction (RT-qPCR) was performed to evaluate the efficacy of shRNA on the expression of SLC7A11 mRNA. Cell counting kit-8 (CCK-8) assay was conducted to determine the suppressing effect of gefitinib on PC9/GR cells. Mito-Tracker Red CMXRos probe and malondialdehyde (MDA) assay kit were used to evaluate gefitinib-induced ferroptosis in PC9/GR cells. Immunohistochemistry (IHC) was conducted to detect the expression of SLC7A11 protein in the tumor tissues of advanced stage lung adenocarcinoma patients harboring 19 exon mutation of EGFR gene. Thirty-six advanced stage lung adenocarcinoma patients who received EGFR-tyrosihe kinase inhibitor(TKI) as first-line treatment in Fourth Hospital of Hebei Medical Unviersity were enrolled. Kaplan-Meier survival curve was drawn to analyze the correlation between SLC7A11 expression and progression-free survival (PFS) of the patients. Results: RNA array demonstrated that 2 888 genes were differently expressed between PC9 and PC9/GR cells. KEGG analysis showed that ferroptosis-related gene was one of the most enriched region of the differently expressed genes between PC9 and PC9/GR cells. These ferroptosis-related gene cohort contained 13 genes, among which SLC7A11 exhibited the most significant difference. Western blotting showed that the expression of SLC7A11 protein in PC9/GR cells was significantly higher than that in PC9 cells (0.76±0.03 vs. 0.19±0.02, P<0.001). The 50% inhibiting concentration (IC(50)) of gefitinib was 35.08 μmol/L and 64.01 μmol/L for sh-SLC7A11 and sh-NC group PC9/GR cells, respectively. PC9/GR cells in sh-SLC7A11 group exhibited significantly lower density of mitochondria fluorescence after gefitinib treatment, compared to the sh-NC group (213.77±26.50 vs. 47.88±4.55, P<0.001). In addition, PC9/GR cells in sh-SLC7A11 group exhibited significantly higher MDA after gefitinib treatment, compared to the sh-NC group [(15.43±1.60) μmol/mg vs. (82.18±7.77) μmol/mg, P<0.001]. The PFS of the patients with low expression of SLC7A11 (n=18) was significantly longer than the patients with high expression of SLC7A11 (n=18, 16.77 months vs. 9.14 months, P<0.001). Conclusion: Downregulation of SLC7A11 could increase the sensitivity of PC9/GR cells to gefitinib by promoting ferroptosis.
Humans ; Gefitinib/therapeutic use* ; Antineoplastic Agents/therapeutic use* ; Lung Neoplasms/pathology* ; Down-Regulation ; Quinazolines/therapeutic use* ; Drug Resistance, Neoplasm/genetics* ; ErbB Receptors/metabolism* ; Adenocarcinoma of Lung ; Protein Kinase Inhibitors/therapeutic use* ; RNA, Small Interfering/genetics* ; Cell Line, Tumor ; Amino Acid Transport System y+/metabolism*

OBJECTIVE: To observe the mutation and expression of TCF3 gene in Burkitt's lymphoma (BL), and explore its effect on the proliferation of BL cells and clinical efficacy and prognosis. METHODS: The mutation and expression of TCF3 in tumor tissues from BL patients were observed by the second-generation sequencing and real-time quantitative PCR. The proliferation and apoptosis of lymphoma cells after TCF3 knocked down were observed by siRNA interference technique and CCK-8 method. Survival analysis was used to observe the relationship between TCF3 mutation and the treatment efficacy and prognosis of BL patients. RESULTS: There were high frequency mutation rate (mutation rate was 23.7%) and high expression of TCF3 in BL patients. After TCF3 knocked down, cell proliferation was inhibited and apoptosis was promoted. In TCF3-siRNA group and control group, the cell proliferation rate at 48 h was (50.2±5.9)% and (96.6±11.4)%, and apoptosis rate was 30.1% and 1.5%, respectively, which showed significantly different between the two groups (P<0.001, P=0.005). The complete remission rate of patients with TCF3 mutation was low. The complete remission rate of mutant group and wild-type group was 44.4% and 82.8%, respectively (P=0.023). The 2-year progression-free survival rate and overall survival rate of the patients with TCF3 mutation was 55.6% and 61.0%, respectively, which was lower than 83.2% and 85.2% of the patients without mutation, but the differences were not statistically significant. CONCLUSION There are mutation and abnormal expression of TCF3 in patients with BL. Patients with TCF3 mutations have low remission rate and poor prognosis.
Apoptosis ; Basic Helix-Loop-Helix Transcription Factors/therapeutic use* ; Burkitt Lymphoma/genetics* ; Humans ; Prognosis ; RNA, Small Interfering/therapeutic use* ; Sincalide/therapeutic use*

As the discovery of RNA interference (RNAi) and the gradual conquering of a series of technical issues, a few of RNAi therapeutics have been approved in the non-tumor field abroad. With the advantages of high specificity, long duration of efficacy, and high success rate of development, RNAi therapeutics have become the emerging field globally. There are no RNAi therapeutics approved in oncology so far, and people are hoping a breakthrough in the field. In the present article, the characteristics and potential anti-tumor mechanism of RNAi therapeutics, difficulties in delivery system and progress in oncology are described, and the potential reasons why their success in non-tumor field is difficult to be simply replicated in tumor field are analyzed, providing reference for research and clinical transformation of RNAi therapeutics in oncology.
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Humans ; Lung Neoplasms/genetics* ; RNA Interference ; RNA, Small Interfering/therapeutic use*

Antisense RNA molecule represents a unique type of DNA transcript that comprises 19-23 nucleotides and is complementary to mRNA. Antisense RNAs play the crucial role in regulating gene expression at multiple levels, such as at replication, transcription, and translation. In addition, artificial antisense RNAs can effectively regulate the expression of related genes in host cells. With the development of antisense RNA, investigating the functions of antisense RNAs has emerged as a hot research field. This review summarizes our current understanding of antisense RNAs, particularly of the formation of antisense RNAs and their mechanism of regulating the expression of their target genes. In addition, we detail the effects and applications of antisense RNAs in antivirus and anticancer treatments and in regulating the expression of related genes in plants and microorganisms. This review is intended to highlight the key role of antisense RNA in genetic research and guide new investigators to the study of antisense RNAs.
Animals ; Antineoplastic Agents/therapeutic use* ; Antiviral Agents/therapeutic use* ; Gene Expression Regulation ; Genetic Research ; Humans ; MicroRNAs/physiology* ; RNA, Antisense/physiology* ; RNA, Long Noncoding/physiology* ; RNA, Small Interfering/physiology*

PURPOSE: Rearrangement of the proto-oncogene rearranged during transfection (RET) has been newly identified potential driver mutation in lung adenocarcinoma. Clinically available tyrosine kinase inhibitors (TKIs) target RET kinase activity, which suggests that patients with RET fusion genes may be treatable with a kinase inhibitor. Nevertheless, the mechanisms of resistance to these agents remain largely unknown. Thus, the present study aimed to determine whether epidermal growth factor (EGF) and hepatocyte growth factor (HGF) trigger RET inhibitor resistance in LC-2/ad cells with CCDC6-RET fusion genes. MATERIALS AND METHODS: The effects of EGF and HGF on the susceptibility of a CCDC6-RET lung cancer cell line to RET inhibitors (sunitinib, E7080, vandetanib, and sorafenib) were examined. RESULTS: CCDC6-RET lung cancer cells were highly sensitive to RET inhibitors. EGF activated epidermal growth factor receptor (EGFR) and triggered resistance to sunitinib, E7080, vandetanib, and sorafenib by transducing bypass survival signaling through ERK and AKT. Reversible EGFR-TKI (gefitinib) resensitized cancer cells to RET inhibitors, even in the presence of EGF. Endothelial cells, which are known to produce EGF, decreased the sensitivity of CCDC6-RET lung cancer cells to RET inhibitors, an effect that was inhibited by EGFR small interfering RNA (siRNA), anti-EGFR antibody (cetuximab), and EGFR-TKI (Iressa). HGF had relatively little effect on the sensitivity to RET inhibitors. CONCLUSION: EGF could trigger resistance to RET inhibition in CCDC6-RET lung cancer cells, and endothelial cells may confer resistance to RET inhibitors by EGF. E7080 and other RET inhibitors may provide therapeutic benefits in the treatment of RET-positive lung cancer patients.
Adenocarcinoma/drug therapy/*genetics ; Cell Line, Tumor ; Cetuximab/pharmacology ; Drug Resistance, Neoplasm/drug effects/*genetics ; Epidermal Growth Factor/metabolism/*pharmacology ; *Gene Rearrangement ; Hepatocyte Growth Factor/*pharmacology ; Humans ; Indoles/pharmacology ; Lung Neoplasms/drug therapy/*genetics ; MAP Kinase Signaling System ; *Mutation ; Niacinamide/analogs & derivatives/pharmacology ; Phenylurea Compounds/pharmacology ; Piperidines/pharmacology ; Protein Kinase Inhibitors/therapeutic use ; Proto-Oncogene Proteins c-ret/*antagonists & inhibitors/genetics ; Pyrroles/pharmacology ; Quinazolines/pharmacology ; RNA, Small Interfering/pharmacology ; Receptor, Epidermal Growth Factor/genetics/metabolism ; Signal Transduction/drug effects ; fms-Like Tyrosine Kinase 3/metabolism

OBJECTIVETo inquire into the influence of silencing HMGB1 expression by small interfering RNA (siRNA) on cell growth, proliferation, invasion and metastasis of colorectal cancer LoVo cells both in vitro and in vivo.

METHODSLentivirus-mediated HMGB1 siRNA was transfected into LoVo cells to silence the HMGB1 expression. The HMGB1 mRNA and protein expression after siRNA transfection was detected by RT-PCR and Western blot. MTT assay was used to observe the cell proliferation and to draw a growth curve. Cell cycle was measured by flow cytometry. The ability of invasion and speed of cell migration were evaluated by transwell chamber invasion and cell scratch assay. The influence of HMGB1 silencing on the proliferation of LoVo cells in vivo was observed in LoVo tumor-bearing nude mice.

RESULTSLentivirus-mediated siRNA was successfully transfected into colorectal cancer cell line LoVo. The expression of HMGB1 mRNA and protein in the HMGB1-siRNA group were 0.24±0.04 and 0.21±0.03, respectively. Compared with the HMGB1-siRNA-Neg group (0.82±0.13, 1.15±0.18) and control group (0.93±0.15, 1.21±0.20), the difference was significant (P<0.05). MTT assay showed that the cell proliferation in the HMGB1-siRNA group was significantly inhibited when compared with that in the HMGB1-siRNA-Neg group and control group (P<0.05). Flow cytometry showed that the proliferation index (PI) of HMGB1-siRNA group was 38.27±1.32, significantly lower than 54.66±1.74 in the HMGB1-siRNA-Neg group and 57.43±1.29 in the control group (P<0.05). The transwell assay showed that the number of penetrated cells in the HMGB1-siRNA group was 14.0±3.5, significantly lower than 51.0±6.7 in the HMGB1-siRNA-Neg group and 68.0±5.3 in the control group (P<0.05). Similarly, the scrape wound recovered significantly slower in the HMGB1-siRNA group (83.61±23.21) µm than that in the other two groups (202.86±46.46) µm and (214.58±57.38) µm(P<0.05). The nude mouse xenograft tumor experiment showed that the final tumor volume was (521±34) mm3 in the HMGB1-siRNA group, significantly smaller than that in the HMGB1-siRNA-Neg group of (763±46) mm3 and control group of (802±51) mm3 (P<0.05).

CONCLUSIONSLentivirus-mediated HMGBl-siRNA can effectively inhibit the HMGB1 expression in colorectal cancer LoVo cells both in vitro and in vivo. HMGB1 gene silencing can slow the growth of colorectal cancer cells, extend the cell proliferation cycle, decrease their invasion and migration, and significantly inhibit the growth of xenograft tumor in nude mice.

Animals ; Cell Cycle ; Cell Line, Tumor ; Cell Movement ; Cell Proliferation ; Colorectal Neoplasms ; pathology ; therapy ; Gene Expression ; HMGB1 Protein ; genetics ; metabolism ; Humans ; In Vitro Techniques ; Lentivirus ; Mice ; Mice, Nude ; Neoplasm Invasiveness ; RNA Interference ; RNA, Messenger ; metabolism ; RNA, Small Interfering ; therapeutic use ; Transfection ; Tumor Burden