Machine learning for small interfering RNAs: a concise review of recent developments.

The advent of machine learning and its subsequent integration into small interfering RNA (siRNA) research heralds a new epoch in the field of RNA interference (RNAi). This review emphasizes the urgency and relevance of assimilating the plethora of contributions and advancements in this domain, particularly focusing on the period of 2019-2023. Given the rapid progression of deep learning technologies, our synthesis of recent research is paramount to staying apprised of the state-of-the-art methods being utilized. It not only offers a comprehensive insight into the confluence of machine learning and siRNA but also serves as a beacon, guiding future explorations in this intersectional research field. Our rigorous examination of studies promises a discerning perspective on the contemporary landscape of machine learning applications in siRNA design and function. This review is an effort to foster further discourse and propel academic inquiry in this multifaceted domain.

[Effects of RNA interference of CTHRC1 on proliferation and apoptosis of thyroid papillary cancer TCP-1 cells in vitro].

OBJECTIVE: To explore the role of CTHRC1 in regulating the proliferation and apoptosis of papillary thyroid cancer cells. OBJECTIVE: Papillary thyroid cancer TPC-1 cells were transfected with a small interfering RNA (siRNA) targeting CTHRC1, with the cells transfected with a scrambled sequence as the negative control. The changes in cell proliferation and apoptosis were assessed using cell counting kit-8 (CCK-8) and flow cytometry with AV/PI double staining, respectively. The expression of c-caspase-3, c-PARP1 and phosphorylation of ERK1/2 in the cells were examined with Western blotting. OBJECTIVE: Transfection with the siRNA sequence significantly decreased the mRNA and protein levels of CTHRC1 in TCP-1 cells (P < 0.05). Compared with blank and negative control cells, TCP-1 cells with RNA interference of CTHRC1 showed significantly lowered proliferative activity and enhanced cell apoptosis (P < 0.05) with significantly increased expressions of c-caspase-3 and c-PARP1 and phosphorylation of ERK1/2 (P < 0.05). OBJECTIVE: RNA interference of CTHRC1 promotes the proliferation and inhibits apoptosis of papillary thyroid cancer cells possibly by activating the ERK1/2 pathway.

Expression pattern of mitogen-activated protein kinase kinase 4 and regulation to antibacterial factor ABF-1/2 in response to bacterial challenge from Artemia parthenogenetica.

Mitogen-activated protein kinase 4, MKK4, is a key upstream kinase in the JNK/p38 MAPK pathway that has been reported to participate in multiple immune responses. In this study, the gene that encodes ApMKK4 was isolated and identified from Artemia parthenogenetica. It was found to contain a 1134 bp open reading frame encoding 378 amino acids. The predicted protein contains D domain, DVD domain and kinase domain. Homology analysis revealed that ApMKK4 shares 38-69% identity with MKK4 homologs from other species. Results revealed that ApMKK4 was mainly expressed during early development of which highest at the gastrula stage. After challenged by Vibrio harveyi and Micrococcus lysodeikticus, ApMKK4 was remarkably upregulated at 10 and 103 cfu/mL bacterial concentrations, respectively. Through siRNAi, the transcript level of ApMKK4 was significantly decreased by 46-67%. Intriguingly, when the ApMKK4-knockdown nauplii faced with bacterial stimulation, the expression of ApMKK4 was completely restored in a short time. Moreover, this phenomenon also occurred in related antimicrobial peptide genes, ABF-1 and ABF-2. Our research reveals that ApMKK4 plays a pivotal role during early development and immune responses against bacterial infections.

Role of BMAL1 and CLOCK in regulating the secretion of melatonin in chick retina under monochromatic green light.

As the circadian pacemaker of birds, the retina possesses the ability to receive light information, generate circadian oscillation, and secrete melatonin. Previous studies have confirmed that monochromatic green light can accelerate the circadian rhythmic expression of clock genes in the chick retina, thereby increasing cAanat mRNA level and melatonin secretion. However, as the core components of the transcriptional-translational negative feedback loop, the role that cBmal1 and cClock plays in the regulation of the retinal molecular clock system and melatonin secretion under monochromatic green light is unknown. To explore their in these processes, embryonic chick retinal cells at six embryo ages were isolated and cultured under light-dark (LD) 12:12 monochromatic green light with, and the role of cBmal1 and cClock in the regulation of the retinal molecular clock and melatonin secretion in the chick retina was explored by siRNA interference and overexpression. The results showed siRNA interference and overexpression of cBmal1 obliterated the circadian rhythm of cCry1, cPer2, cPer3, cAanat, and melatonin secretion. Moreover, the siRNA interference of cBmal1 significantly reduced the average expression levels of the positive clock genes cBmal2 and cClock, positive clock protein CLOCK, negative clock genes cCry1, cCry2, cPer2, cPer3, as well as cAanat and retinal melatonin. The over-expression of cBmal1 increased the average levels of the above-detected targets. However, siRNA interference and overexpression of cClock did not change the rhythm of all of the clock genes, clock proteins, cAanat, and melatonin secretion, while it only affected the circadian mesors (24 h time series means), amplitudes, and acrophases (peak times) of cCry1, cPer2, cPer3, cAanat, and melatonin, as well as the average levels of arrhythmic cBmal2 and cCry2. Moreover, interference and overexpression of cClock did not affect cBmal1 mRNA level and BMAL1 protein expression. The above results reveal interference and overexpression of cBmal1 completely abolished the molecular circadian oscillation and the rhythm of melatonin output signal of chick retinal cells, indicating that cBmal1 is on the top of the avian retinal molecular clock feedback loop and regulates the downstream molecular clock oscillation and output under monochromatic green light. cClock plays a subordinate role in maintaining the circadian oscillation of the molecular clock and melatonin secretion in retinal cells, and it has a stabilizing and amplifying effect on molecular clock oscillation.

Knockdown of KLF9 promotes the differentiation of both intramuscular and subcutaneous preadipocytes in goat.

KLF9 is reported to promote adipocyte differentiation in 3T3-L1 cells and pigs. However, the roles of KLF9 in adipocytes differentiation of goat remain unknown. In this study, the expression profiles of KLF9 were different between subcutaneous and intramuscular preadipocytes of goat during differentiation process. After silencing KLF9 gene, the lipid droplets were increased in both two types of adipocytes. In subcutaneous preadipocyte with silencing KLF9, the expressions of C/EBPß, PPARγ, LPL, KLF1-2, KLF5, and KLF17 genes were up-regulated, while KLF12, KLF4, and KLF13 genes were down-regulated in expression level. In intramuscular preadipocyte, aP2, C/EBPα, KLF2-3, KLF5, and KLF7 gene were up-regulated, and Pref-1 gene was down-regulated. In addition, the binding sites of KLF9 existed in the promoters of aP2, C/EBPα, C/EBPß, LPL and Pref-1. Taken together, KLF9 play a negative role in the differentiation of both intramuscular and subcutaneous preadipocytes in goats, but the functional mechanism may be different.

Determination of the roles of GREM1 gene in granulosa cell proliferation and steroidogenesis of hen ovarian prehierarchical follicles.

Gremlin genes are known members of the DAN family of bone morphogenetic protein (BMP) antagonists, but their functions and regulatory mechanisms in ovarian follicular development of chicken remain unknown. The current study was designed to investigate the mRNA expression patterns of gremlin1 gene (GREM1) and its protein location in the follicles sampled, and to explore the biological effect of GREM1 on the prehierarchical follicular development. This work revealed that chicken GREM1 mRNA exhibits a constant expression level across all the prehierarchical follicles (PFs) from 1-4 mm to 7-8 mm in diameter, and the preovulatory follicles (from F6 to F1) by using RT-qPCR (P > 0.05). The GREM1 protein is predominantly expressed in the oocytes and granulosa cells (GCs) of the PFs by immunohistochemistry. Furthermore, our data demonstrated that siRNA-mediated knockdown of GREM1 in the GCs resulted in a significant reduction in cell proliferation (P < 0.001); conversely, overexpression of GREM1 in the GCs led to a remarkable increase in cell proliferation (P < 0.001). Interestingly, the expression levels of proliferating cell nuclear antigen (PCNA) and cyclin D2 (CCND2) mRNA and proteins were notably increased when GREM1 expression was upregulated in the GCs (P < 0.01), however, the expression levels of CYP11A1 and StAR were markedly downregulated (P < 0.01). The current results showed that GREM1 gene plays a stimulatory role in GC proliferation during growth and development of the prehierarchical follicles in vitro but an inhibitory role in GC differentiation and steroidogenesis of the hen ovary follicles.

Roles of the mammalian target of rapamycin (mTOR) signaling pathway in the repair of hyperoxia-induced acute lung injury.

BACKGROUND: Rapamycin inhibits the mammalian target of rapamycin (mTOR) activity and has been proven effective for the treatment of lung injury. OBJECTIVES: The objective of this study was to investigate the roles of the mTOR pathway and its inhibitor rapamycin in the repair of hyperoxia-induced acute lung injury (ALI). MATERIAL AND METHODS: Firstly, premature rat lung fibroblast L929 cells were cultured under different oxygen concentrations (40%, 60%, and 90%). At day 3, 7 and 14 after exposure, MTT assay and flow cytometry were used to evaluate the effect of oxygen stress on cell viability and apoptosis of L929 cells, respectively. Secondly, microscopy, MTT assay and flow cytometry was used to investigate the effect of 10 nM rapamycin on 90% O2 exposed L929 cells. We also used small interfering RNAs (siRNAs) to abrogate the expression of mTOR in 90% O2 exposed L929 cells, and then evaluated the apoptosis and cell viability using flow cytometry and the MTT assay, respectively. In addition, western blot was used to detect the protein expression of Bcl-2, p53, TGF-ß and connective tissue growth factor (CTGF). A hyperoxia-induced lung injury model was established in Sprague Dawley (SD) rats in order to evaluate the histopathological changes in lung tissues and expression of the mTOR pathway and fibrosis related factors. RESULTS: Exposure to 40%, 60% or 90% oxygen all significantly inhibited the growth of L929 cells. Application of 10 nM rapamycin was found to effectively promote apoptosis of 90% O2 exposed L929 cells. In addition, mTOR siRNA promoted the apoptosis and inhibited the growth of L929 cells. Rapamycin inhibited the activation of the mTOR signaling pathway, down-regulated the expression of downstream proteins p70S6K and 4EBP1, reduced the collagen deposition and the production of fibrosis-inducing factors, including TGF-ß and CTGF in hyperoxia-induced lung injury rats. CONCLUSIONS: Rapamycin may be useful for the treatment of hyperoxia-induced acute lung injury (ALI) by inhibiting the activation of mTOR signaling pathway.

Expression of Tspan29 in breast cancer tissues and its effect on malignant biological behaviors of MCF-7 and MDA-MB-231 cells / 中国肿瘤生物治疗杂志

@#Objective: To investigate the expression of tetraspanins-29 (Tspan29) in breast cancer tissues and cell lines and to explore the effect of Tspan29 knockdown on proliferation, invasion, migration and epithelial-mesenchymal transition (EMT) of breast cancer MCF-7 and MDA-MB-231 cells. Methods:Atotal of20pairsofbreast cancer tissues and corresponding para-cancerous tissues resected in Minhang Branch of Cancer HospitalAffiliated to Fudan University from June 2017 to February 2018 were collected for this study; in addition, breast cancer celllinesMCF-7,MDA-MB-231andhumanbreastepithelialMDA-kb2cellswerealsocollected.ThemRNAand protein expressions of Tspan29 in above mentioned tissues and cell lines were detected by Real-time quantitative (qPCR) and Western blotting. The expression of Tspan29 in MCF-7 and MDA-MB-231 cells was interfered by siRNA. qPCR was used to detect the mRNA and protein expressions of Tspan29. PCR microarray was used to examine the expressions of EMT-related genes in MCF-7 cells. CCK-8 assayandTranswellwereusedtodetectcellproliferation, migration and invasion of MCF-7 and MDA-MB-231 cells. Results: The mRNA and protein expressions of Tspan29 in breast cancer tissues were significantly higher than that in para-cancerous tissues (all P<0.01); and the mRNA and protein expressions of Tspan29 in MCF-7 and MDA-MB-231 cells were significantly higher than that in MDA-kb2 cells (P<0.01). After being interfered with siTspan29, the mRNA and protein expressions of Tspan29 were significantly down-regulated in MCF-7 cells (all P<0.05); the proliferation, invasion and migration of MCF-7 and MDA-MB-231 cells were significantly inhibited (all P<0.05); and among the EMT-related genes, two were significantly up-regulated while 7 were down-regulated. Conclusion: Tspan29 is significantly up-regulated in breast cancer tissues and cell lines, and knockdown of Tspan29 significantly inhibits the proliferation, invasion and migration of breast cancer cells. ··

ApoF knockdown increases cholesteryl ester transfer to LDL and impairs cholesterol clearance in fat-fed hamsters.

Cholesteryl ester transfer protein (CETP) regulates intravascular lipoprotein metabolism. In vitro studies indicate that ApoF alters CETP function by inhibiting its activity with LDL. To explore in vivo the complexities driving ApoF's effects on CETP, we developed a siRNA-based hamster model of ApoF knockdown. In both male and female hamsters on chow- or fat-fed diets, we measured lipoprotein levels and composition, determined CETP-mediated transfer of cholesteryl esters (CEs) between lipoproteins, and quantified reverse cholesterol transport (RCT). We found that apoF knockdown in chow-fed hamsters had no effect on lipoprotein levels or composition, but these ApoF-deficient lipoproteins supported 50-100% higher LDL CETP activity in vitro. ApoF knockdown in fat-fed male hamsters created a phenotype in which endogenous CETP-mediated CE transfer from HDL to LDL increased up to 2-fold, LDL cholesterol increased 40%, HDL declined 25%, LDL and HDL lipid compositions were altered, and hepatic LDLR gene expression was decreased. Diet-induced hypercholesterolemia obscured this phenotype on occasion. In fat-fed female hamsters, ApoF knockdown caused similar but smaller changes in plasma CETP activity and LDL cholesterol. Notably, ApoF knockdown impaired HDL RCT in fat-fed hamsters but increased sterol excretion in chow-fed animals. These in vivo data validate in vitro findings that ApoF regulates lipid transfer to LDL. The consequences of ApoF knockdown on lipoproteins and sterol excretion depend on the underlying lipid status. By minimizing the transfer of HDL-derived CE to LDL, ApoF helps control LDL cholesterol levels when LDL clearance mechanisms are limiting.

BMAL1 but not CLOCK is associated with monochromatic green light-induced circadian rhythm of melatonin in chick pinealocytes.

The avian pineal gland, an independent circadian oscillator, receives external photic cues and translates them for the rhythmical synthesis of melatonin. Our previous study found that monochromatic green light could increase the secretion of melatonin and expression of CLOCK and BMAL1 in chick pinealocytes. This study further investigated the role of BMAL1 and CLOCK in monochromatic green light-induced melatonin secretion in chick pinealocytes using siRNAs interference and overexpression techniques. The results showed that si-BMAL1 destroyed the circadian rhythms of AANAT and melatonin, along with the disruption of the expression of all the seven clock genes, except CRY1. Furthermore, overexpression of BMAL1 also disturbed the circadian rhythms of AANAT and melatonin, in addition to causing arrhythmic expression of BMAL1 and CRY1/2, but had no effect on the circadian rhythms of CLOCK, BMAL2 and PER2/3. The knockdown or overexpression of CLOCK had no impact on the circadian rhythms of AANAT, melatonin, BMAL1 and PER2, but it significantly deregulated the circadian rhythms of CLOCK, BMAL2, CRY1/2 and PER3. These results suggested that BMAL1 rather than CLOCK plays a critical role in the regulation of monochromatic green light-induced melatonin rhythm synthesis in chicken pinealocytes. Moreover, both knockdown and overexpression of BMAL1 could change the expression levels of CRY2, it indicated CRY2 may be involved in the BMAL1 pathway by modulating the circadian rhythms of AANAT and melatonin.

Effect of SHBG gene on the apoptosis of human trophoblastic cells / 局解手术学杂志

Objective To investigate the effects of sex hormone-binding globulin (SHBG)gene in the apoptosis of human trophoblastic cells.Methods The siRNA specific-targeting SHBG gene was transfected into human trophoblastic cells and they were divided into six groups:trophoblasts without transfection in normal control groups(group Ⅰ);transfect liposome in blank control groups(group Ⅱ);transfect nonspecific siRNA in negative control groups(group Ⅲ);transfect SHBG siRNA-Ⅰ,SHBG siRNA-Ⅱ,SHBG siRNA-Ⅲ respectively in trans-fection group(group Ⅳ,Ⅴ,Ⅵ).Hoechst 33258 dying method was used to detect cell apoptosis.SHBG and Caspase-3 mRNA profiling and the level of SHBG and caspase-3 protein were detected by real-time PCR and Western blot.Results There was no statistical significant difference in the gene expression and protein level of SHBG and caspase-3 in group Ⅰ,Ⅱ and Ⅲ (P >0.05).In Ⅳ,Ⅴ and Ⅵ group,there was no statistical significant difference in the expression level of SHBG and caspase 3 (P >0.05).Compared with group Ⅰ,Ⅱ and Ⅲ,the a-mount of SHBG gene expression decreased obviously,the caspase-3 mRNA and protein level increased obviously and the trophoblast cell ap-optosis increased markedly (P <0.05).Conclusion Through siRNA interference technology can reduce SHBG gene expression in human trophoblastic cells,and it can lead to excessive apoptosis of human trophoblasts cells.

Effect of p38 siRNA on biological behavior and sensitivities to sunitinib of human renal carcinoma cell line 786-O / 第二军医大学学报

Objective To investigate the effects of p38 gene sliencing on the proliferation, invasion, cell cycle and sensitivity to sunitinib of human renal carcinoma cell line 786-O. Methods We designed two sequence-specific small interfering RNA(siRNA531 and siRNA659) targeting p38 gene and transfected them into renal carcinoma cell line 786-0. 786-0 cells transfected with nonsense siRNA served asnegative control and those cultured with transfection medium served as blank control. The change of #38 gene expression was observed by RT-PCR and the expression of p38 protein was detected by Western blotting analysis. The proliferation, sensitivities to sunitinib, invasion capabilities, and the cell cycle of 786-0 cells were examined by CCK-8 assay, transwell chamber test and flow cytometry, respectively. Results RT-PCR and Western blotting analysis revealed that p38 expression in #38 siRNA group was significantly decreased compared with the controls. The cell proliferation rates were also significantly decreased 3-5 days after siRNA531 or siRNA659 transfection compared with the controls (P<0. 05, P<0. 01), and cells in the siRNA531 and siRNA659 groups become more sensitive to sunitinib compared with negative control group, with two IC50 values being significantly lower than that of the negative control group ([3. 2 ± 0. 3,, [1. 4±0. 1, μmol/mL vs [5. 4 ± 0. 2, μmol/mL; P<0. 05). In addition, analysis of cell cycle demonstrated a marked G50/G50 arrest of the 786-0 cells transfected with siRNA531 or siRNA659. We also noticed that 24 h after transfection, the cell invasion capabilities was significantly decreased in siRNA531 and siRNA659 compared with negative control (numbers of cells permeating septum: 56. 43 ± 6. 02, 34. 00 ± 8. 12 vs 76. 27 ± 5. 08; P<0. 01). Conclusion We have successfully suppressed #38 gene expression by specific siRNA, which can inhibit the proliferation and invasion of human renal carcinoma cell line 786-0 and increase its sensitivity to sunitinib, paving a way for future treatment and targeted drug resistance of renal cell carcinoma.

Relationship between the expression of CCR4 and invasion and metastasis of gallbladder cancer cell GbC-SD / 临床与实验病理学杂志

Purpose To investigate the effects of chemotactic factor CCR4 on the abi1ity of pro1iferation,ce11 cyc1e,invasion,and mi-gration of human ga11b1adder cancer ce11. Methods Western b1ot was used to detect the expression 1eve1 of CCR4 in ga11b1adder carci-noma ce11s. Ga11b1adder carcinoma ce11s was infected by means of s1ow virus,the CCR4 gene si1encing was conducted using siRNA-CCR4 interference techno1ogy. Ga11b1adder carcinoma ce11s GBC-SD were divided into three groups( GBC-SD,GBC-SD/CCR4-RNAi and GBC-SD/contro1). CCL17,a 1igand of CCR4,was used to act on these three groups of ce11s. CCK8 method was used to detect the ce11 pro1iferation abi1ity of three groups. F1ow cytometry was used to test ce11 cyc1e. Tanswe11 assay was app1ied to detect ce11 migration and invasion abi1ity. Western b1ot was performed to detect the expression of its corresponding 1igands CCL17 and CCL22 proteins. Re-sults CCR4 gene si1ence did not inf1uence ce11 cyc1e and pro1iferation of ga11b1adder ce11 GBC-SD,but can significant1y inhibit GBC-SD ce11 invasion and movement abi1ity,CCR4 gene si1ence had no inf1uence on the expression of CCL17 and CCL22 gene in tumor ce11s. Conclusion Ga11b1adder carcinoma ce11s GBC-SD express chemokine receptor CCR4,chemokine receptor CCR4 can promote the invasion and metastasis of GBC-SD ce11s.

Attenuation of unfolded protein response and apoptosis by mReg2 induced GRP78 in mouse insulinoma cells.

Murine regenerating (mReg) genes have been implicated in preserving islet cell biology. Expanding on our previous work showing that overexpression of mReg2 protects MIN6 insulinoma cells against streptozotocin-induced apoptosis, we now demonstrate that mReg2 induces glucose-regulated peptide 78 (GRP78) expression via the Akt-mTORC1 axis and protects MIN6 cells against ER stress induced by thapsigargin and glucolipotoxicity. Activation of mTORC1 activity results from both mReg2-induced increased mTOR phosphorylation as well as increased expression of Raptor and GßL. Inhibition of Akt and mTORC1 blunted the ability of mReg2 to induce GRP78 and attenuate unfolded protein response (UPR). Knockdown of GRP78 sensitized the cells overexpressing mReg2 to UPR without affecting its ability to activate Akt-mTORC1 signaling. Induced expression of mReg2 may protect insulin producing cells from ER stress in diabetes.

Smad4 silencing on PanIN cells accelerates K-ras G12D-mediated pancreatic neoplasia / 中国癌症杂志

Background and purpose: Pancreatic intraepithelial neoplasia (PanIN) may be a precursor lesion of inifltrating pancreatic ductal adenocarcinoma. The mutation of the phenotypic impact of K-ras G12D alone, silencing of p53 and p16 could promote this process. The role of Smad4 in this progression was poorly understood. In our previous studies, we investigated that RNA interference silence of Smad4 to promote the PanIN cell malignant transformation. In the present study, we investigate. The further explores the siRNA interference of Smad4 expression on PanIN cells could lead to proliferation and metastasis in vitro and in vivo. Methods:Smad4 knock-down PanIN cells (PanIN-S) were established by stable transfection with lentiviral-mediated Smad4 RNA interference. In vitro,silence of Smad4 enhanced the proliferation of PanIN cells as determined by cell counting. A soft agar assay was used to assess the anchorage-independent growth ability of cells. Cell migration and invasion assays were performed using transwell chambers with or without Matrigel. In xenograft model experiments, PCNA, VEGF and MMP-9 staining was separately used to evaluate cell proliferation and angiogenesis and migration (VEGF and MMP-9). Results:Effect of siRNA of Smad4 gene in PanIN cells was conifrmed by real-time RT-PCR and western blot. In vitro, silence of Smad4 enhanced the proliferation of PanIN cells as determined by cell counting. Soft agar assay showed that there were more colony cell numbers in PanIN-S cells compared with PanIN cells (P<0.05). Using the transwell assay, we observed that PanIN-S cells migrated faster than PanIN cells and similar results were obtained by Matrigel assay (P<0.05). Furthermore, immunohistochemical analysis of the harvested tumors suggested that Smad4 silencing was associated with cell proliferation (PCNA reactivity) and angiogenesis and migration (VEGF and MMP-9), and the expressions of PCNA, VEGF and MMP-9 in PanIN-S group were signiifcantly increased (P<0.05). Conclusion:Silence of Smad4 in PanIN cells enhanced progression to invasive adenocarcinoma of the pancreas by promoting cell growth, migration and invasion. Smad4 might be a new diagnostic marker in pancreatic cancer and prove to be a feasible and novel target for therapeutic intervention.

Inhibition of cytohesin-1 by siRNA leads to reduced IGFR signaling in prostate cancer

To explore how cytohesin-1 (CYTH-1) small interfering RNA (siRNA) influences the insulin-like growth factor receptor (IGFR)-associated signal transduction in prostate cancer, we transfected human prostate cancer PC-3 cell lines with liposome-encapsulatedCYTH-1 siRNA in serum-free medium and exposed the cells to 100 nM IGF-1. The mRNA and protein levels of the signal molecules involved in the IGFR signaling pathways were determined by real-time PCR and detected by Western blotting. The relative mRNA levels of CYTH-1, c-Myc, cyclinD1 and IGF-1R (CYTH-1 siRNA group vs scrambled siRNA group) were 0.26 vs 0.97, 0.34 vs 1.06, 0.10 vs 0.95, and 0.27 vs 0.41 (P < 0.05 for all), respectively. The relative protein levels of CYTH-1, pIGF-1R, pIRS1, pAkt1, pErk1, c-Myc, and cyclinD1 (CYTH-1 siRNA group vsscrambled siRNA group) were 0.10 vs 1.00 (30 min), 0.10 vs 0.98 (30 min), 0.04 vs 0.50 (30 min), 0.10 vs 1.00 (30 min), 0.10 vs 1.00 (30 min), 0.13 vs 0.85 (5 h), and 0.08 vs 0.80 (7 h), respectively. The tyrosine kinase activity of IGF-1R was associated with CYTH-1. The proliferative activity of PC-3 cells transfected with CYTH-1 siRNA was significantly lower than that of cells transfected with scrambled siRNA at 48 h (40.5 vs87.6 percent, P < 0.05) and at 72 h (34.5 vs 93.5 percent, P < 0.05). In conclusion, the interference of siRNA with cytohesin-1 leads to reduced IGFR signaling in prostate cancer; therefore, CYTH-1 might serve as a new molecular target for the treatment of prostate cancer.

Relationship of the Arl8a with TLR4-TRIF pathway in dendritic cells / 中华微生物学和免疫学杂志

Objective To explore the relationship of the ADP-ribosylation factor-like 8A (Arl8a)with TLR4-TRIF-GEFH1 -RhoB pathway in dendritic cells(DCs). Methods DCs were prepared from wildtype and TRIF-knockout (TRIFKO) mice. After LPS stimulation, the cells were collected for cDNA amplification. Real-time PCR method was used to detect Arl8a mRNA levels. DCs from wild-type mice were transfected with guanine nucleotide-exchange factors H1 ( GEFH1 ) small interference RNA ( siRNA ), Arl8a mRNA levels were examined with or without LPS stimulation. Then RhoB mRNA expression was analyzed in DCs transfected with the siRNA of GEFH1 and Arl8a gene, respectively. Results LPS induced the up-regulation of Arl8a mRNA in DCs from control mice but not in DCs from TRIFKO, indicating that LPS-mediated up-regulation of Arl8a was suppressed in TRIFKO DCs. In addition, siRNA of GEFH1 significantly suppressed the LPS-mediated up-regulation of Arl8a mRNA, RNAi of Arl8a and GEFH1 significantly decreased RhoB mRNA level in DCs after LPS stimulation ( P ＜ 0. 01 ). Conclusion The expression of Arl8a is involved in the TLR4-TRIF pathway in DCs, and Arl8a is closely associated with GEFH1 and RhoB at transcriptional level.

Effect of ski-siRNA on biological function of human hepatoma cell line HepG2 / 第二军医大学学报

Objective: To design and prepare siRNAs targeting ski gene and to observe its influence on the biological functions of HepG2 cells, such as proliferation, cell cycle, apoptosis, etc.. Methods: Three specific siRNAs of ski were designed and synthesized, and were transiently transfected into HepG2 via cathodolyte liposome transfection method. Real-time PCR and Western blotting were used to measure ski expression at mRNA and protein levels. The cell proliferation was assessed by MTT assay and the changes in cell cycle and apoptosis were evaluated by flow cytometry. Results: All the 3 specific ski-siRNA(A, B, C) effectively inhibited the expression of ski gene, with ski-siRNA-B having the highest inhibition rate(70%). Furthermore, the ski expression had a decreasing tendency with the transfection time. The proliferation of HepG2 cells was markedly inhibited by ski-siRNAs(P<0.05); the number of cells at S stage was obviously decreased, being 2 folds that of the negative control group. Conclusion: The siRNA of ski gene can effectively induce growth inhibition of HepG2 cells and reduce cells of S stage, which is possibly through down-regulation of ski gene.

Effects of Silencing G6PD Expression on The Growth and Apoptosis in Human Skin Melanoma / 生物化学与生物物理进展

Glucose-6-phosphate dehydrogenase (G6PD) derives from the expression of the house-keeping gene G6PD. Recent studies have indicated that G6PD is related to tumor genesis, growth, clinical phenotype, therapy, and prognosis. To elucidate the relationship between G6PD and cancer, three siRNA sequences and one negative control sequence were designed based on the 3' noncoding region of the human G6PD gene. Two complementary single-strand DNA (sense and antisense) were designed and synthesized based on siRNA sequences. The DNA fragments were annealed and ligated to the GFP expression vector pRNAT-U6.2/Lenti. One siRNA with higher interference efficiency than the other two was found after siRNA plasmid transfecting human skin A375 melanoma cells. After lentivirus particle packaging and virus production, the A375 cells were infected, and the single cell clone was acquired and cultured to establish the stable cell strain. Western blotting showed that the endogenous G6PD in the stable A375 cell strain was 0.257 ? 0.074, which was 11.17% of G6PD expression (2.301 ? 0.286) in wild type A375 cells. The final siRNA interference efficiency in this stable cell strain was 88.83%. The G6PD activity of A375-G6PD?驻 was 21.53% of A375-WT. Further study showed that A375-G6PD△ doubling generation time prolonged, and its proliferation was greatly inhibited and the cloning efficiency lowered 25%(P

Silencing of osteopontin suppresses invasion and apoptosis of human gastric cancer MKN28 cells / 第三军医大学学报

Objective To detect the effect of osteopontin (OPN) siRNA on the growth,invasion and apoptosis of human gastric cancer cell line MKN28. Methods The eukaryotic expression vector of siRNA specific for OPN was constructed by liposome transfection,and the silencing effect was identified by fluorescence quantitative PCR and immunofluorescence staining. According to the inhibitory effect of the siRNA vectors,the experiment was divided into control group,non-specific OPN siRNA group,24-hour specific OPN siRNA group,36-hour specific OPN siRNA group,and 48-hour specific OPN siRNA group. The cell proliferation,apoptosis,migration and adhesion was finally detected among control group and OPN siRNA groups at different time points. Results For 48-hour specific OPN siRNA group,the expression of OPN in MKN28 cells was the lowest. Accordingly,the cell viability was decreased and the cell proliferation inhibitory rate was reduced to 30.20%; the ability of migration and adhesion was decreased,the number of cells attached on stromal cell membrane was much lesser (21.8?6.9 vs 42.0?9.4 in control group),and the ratio of cell adhesion on basement membrane matrix and fibronectin was lower [basement membrane matrix: (41.5?8.4)% vs (20.5?4.5)%; fibronectin: (25.3?4.5)% vs (14.6?2.5)% in control group],as well as the cell apoptotic rate was increased compared with the control group. Conclusion OPN gene silencing can inhibit the growth and invasion,as well as accelerate apoptosis in human gastric cancer MKN28 cells.