Journal: Cancer research

Article Title: G-CSF promotes neuroblastoma tumorigenicity and metastasis via STAT3-dependent cancer stem cell activation

doi: 10.1158/0008-5472.CAN-14-2946

Figure Lengend Snippet: (A) Cell viability assay of different NB cell lines (NGP, SH-SY5Y, IMR-32, CHLA-255) in response to various concentrations of STAT3 inhibitor (Stattic) treatment for 24 h. Cell viability was measured using MTS assay. Experiment was repeated two times with six replicates for each condition and represented as mean ± SD. (B) Representative pictures of colony formation assay for different NB cell lines treated with various doses of Stattic (μM). (C) Quantitation of relative inhibition of colony formation is shown as mean ± SD (t-test, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001). (D) Measurement of apoptosis in CD114+ and CD114- NB subpopulations of NGP and SH-SY5Y cell lines using Annexin-V staining along with CD114 staining in response to increasing doses of Stattic for 2 h. Data are represented as means ± SD for three independent experiments. DMSO was used as control in “0” groups. (t-test, *p<0.05, **p<0.01, ****p<0.0001). (E) Cell cycle analysis using BrdU incorporation assay in NGP NB subpopulations with 1μM Stattic treatment for 2 h. Data is represented as relative percentage and representative of experiment repeated three times. (F) Representative flow cytometry analysis plots for cell cycle analysis on NGP NB subpopulations in response to Stattic treatment (1μM). Values shown on each dot plot are representing the percentage of cells.

Article Snippet: Generation of stable STAT3 knockdown cell lines Lentiviral shRNA vectors pSIH1-puro-STAT3 (26596, Addgene) and pSIH1-puro-control (26597, Addgene) ( 15 ) were used to transduce NB cell lines as described previously ( 11 ).

Techniques: Viability Assay, MTS Assay, Colony Assay, Quantitation Assay, Inhibition, Staining, Control, Cell Cycle Assay, BrdU Incorporation Assay, Flow Cytometry

Journal: Cancer research

Article Title: G-CSF promotes neuroblastoma tumorigenicity and metastasis via STAT3-dependent cancer stem cell activation

doi: 10.1158/0008-5472.CAN-14-2946

Figure Lengend Snippet: (A) Cell viability assay of different NB cell lines (NGP, SH-SY5Y, IMR-32, CHLA-255) were tested by MTS assay in response to etoposide alone and in combination of 0.5 μM and 1.0 μM Stattic treatment for 24 h. Experiments were repeated two times with six replicates for each condition and represented as mean ± SD. (B) Schematic representation of experimental plan to analyze the in vivo effect of STAT3 inhibitor and dual treatment strategy on neuroblastoma. Orthotopic mouse model was used to develop xenografts of NB NGP and SH-SY5Y cell lines and treated with either etoposide (12 mg/kg/day, i.p. injection, three times/week) and Stattic (25 mg/kg/day, i.p. injection, five days/week) alone or combined. Implanted mouse were treated two weeks post-implantation for two weeks. Vehicle (DMSO) was used as control in all experiments. (C) NGP xenografts showing significant decrease in tumor weights in response to Stattic treatment (p=0.03), etoposide treatment and combo treatment (p=0.001) in comparison to controls. Combo treatment also significantly reduced tumor burden in comparison to etoposide alone (p=0.008). (Mann-Whitney test, *p<0.05, **p<0.01, ***p<0.001). The percentage of CD114+ cells determined by flow cytometry analysis on individual tumors showed as mean ± SEM. Human MYCN mRNA was detected by qPCR in bone marrow of xenotransplanted mice. (D) SH-SY5Y xenografts showing significant decrease in tumor weights in response to Stattic treatment (p=0.02), etoposide treatment (p=0.0002) and combo treatment (p=<0.0001) in comparison to controls. Combo treatment also significantly reduced tumor burden in comparison to etoposide (p=0.02) and Stattic alone (p=0.0002). (Mann-Whitney test (*p<0.05, **p<0.01, ***p<0.001). The percentage of CD114+ cells determined by flow cytometry analysis on individual tumors showed as mean ± SEM. Detection of human MYCN mRNA by qPCR in bone marrow of xenotransplanted mice shows trend of reduction of metastasis with Stattic (p=ns).

Article Snippet: Generation of stable STAT3 knockdown cell lines Lentiviral shRNA vectors pSIH1-puro-STAT3 (26596, Addgene) and pSIH1-puro-control (26597, Addgene) ( 15 ) were used to transduce NB cell lines as described previously ( 11 ).

Techniques: Viability Assay, MTS Assay, In Vivo, Injection, Control, Comparison, MANN-WHITNEY, Flow Cytometry

Journal: Cancer research

Article Title: G-CSF promotes neuroblastoma tumorigenicity and metastasis via STAT3-dependent cancer stem cell activation

doi: 10.1158/0008-5472.CAN-14-2946

Figure Lengend Snippet: (A) Relative CSF3R expression analysis in NB subpopulations of NGP, SH-SY5Y, and IMR-32 in response to G-CSF (20 ng/ml), Stattic (1μM) and combination treatment (G+S; represents G-CSF + Stattic) for 2 hr. Data are mean ± SEM of three replicates of experiment repeated twice. (t-test, *p<0.05, **p<0.01, ***p<0.001). (B) Schematic representation of CSF3R gene promoter showing 3.8 kb long 5′ untranslated region (5′ UTR) and 2.5 kb long promoter region. A potential STAT3 binding site was determined in 5′ UTR region at -200 bp (S) from translation start site (arrow). EGFP reporter driven by 5′UTR contacting STAT3 binding site (S) shows significant increase in percentage of CD114+/GFP+ cells in response to G-CSF treatment (20 ng/ml for 2 h) in contrast to untreated or reporter driven by promoter region. CHIP-qPCR primers were designed for STAT3 binding site (S). (C) CHIP-qPCR analysis showing direct binding enrichment of STAT3 and pSTAT3 (Y705) at S site of CSF3R 5′ UTR. NGP NB subpopulation CD114+ cells showing enriched binding of STAT3 by 2.2 fold (p=0.007) pSTAT3 by 3.1 fold (p=0.002) in comparison to CD114- cells. G-CSF treatment (20ng/ml for 2 h) in CD114+ cells further enriched the binding of STAT3 by 2.7 fold (p<0.001) and pSTAT3 (Y705) by 2.4 fold (p<0.01) in comparison to baseline untreated while Stattic treatment (1μM for 2 h) blocks the binding of STAT3 or pSTAT3. Data are mean ± SEM of three replicates of experiment repeated twice. (t-test *p<0.05, **p<0.01, ***p<0.001).

Article Snippet: Generation of stable STAT3 knockdown cell lines Lentiviral shRNA vectors pSIH1-puro-STAT3 (26596, Addgene) and pSIH1-puro-control (26597, Addgene) ( 15 ) were used to transduce NB cell lines as described previously ( 11 ).

Techniques: Expressing, Binding Assay, ChIP-qPCR, Comparison

Journal: Cancer research

Article Title: G-CSF promotes neuroblastoma tumorigenicity and metastasis via STAT3-dependent cancer stem cell activation

doi: 10.1158/0008-5472.CAN-14-2946

Figure Lengend Snippet: Schematic representation of conclusions drawn from current study showing upregulation of JAK/STAT3 pathway in response to binding of G-CSF to G-CSFR. Active STAT3 translocate to nucleus and upregulate the expression of key target genes that are know to be involved in tumorigenicity, metastasis and drug resistance. CSF3R (gene coding for G-CSFR) is also a direct transcriptional target of STAT3. Binding of STAT3 on CSF3R promoter further upregulate surface expression of the G-CSFR (CD114), making a feedback loop mechanism for tumor progression in NB. STAT3 inhibition and anti-G-CSF antibody are shown to block this positive feedback loop and therefore decrease the cancer stem cell functions in NB.

Article Snippet: Generation of stable STAT3 knockdown cell lines Lentiviral shRNA vectors pSIH1-puro-STAT3 (26596, Addgene) and pSIH1-puro-control (26597, Addgene) ( 15 ) were used to transduce NB cell lines as described previously ( 11 ).

Techniques: Binding Assay, Expressing, Inhibition, Blocking Assay

Journal: Cardiovascular Diabetology

Article Title: NLRP3 inflammasome-modulated angiogenic function of EPC via PI3K/ Akt/mTOR pathway in diabetic myocardial infarction

doi: 10.1186/s12933-024-02541-3

Figure Lengend Snippet: Fig. 2 Reduced EPC mobilization and activated NLRP3 inflammasome in HFD/STZ-induced diabetic mice. (A) a diagram of the experimental protocol. (B) FACS analysis on peripheral blood mononuclear cells for EPC mobilization in groups. (C) The bar graph showed that EPC mobilization was impaired in HFD/STZ-induced diabetic mice as compared with control mice (n = 6 mice/group). (D) Nlrp1, Nlrp3, Nlrp6, Nlrc4 and Aim2 mRNA expression (RT-PCR) in flow cytometry-sorted EPC were quantified at 3 days after MI. (E) Nlrp3, Caspase1, and Il-1b mRNA expression (RT-PCR) in flow cytometry-sorted EPC were quantified at 3 days after MI. mRNA expression normalized to Actb and depicted as fold change versus control. (F) a diagram of the matrigel plug protocol. (G) Representative HE photographs of matrigel plugs removed from mice 10 days after injection (n = 6 mice/group). The relative amounts of hemoglobin extracted from the excised matrigel plugs and the number of blood vessels in matrigel plugs (n = 6 mice/group). (H) Relative NLRP3, Caspase1 p20, and IL-1β protein expression levels in EPC extracted from the excised matrigel plugs (n = 3 mice/group). * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. These experiments were repeated independently at least three times with similar results

Article Snippet: In addition, the shRNA targeting Usp14 and Nlrp3 mRNA sequence was introduced into pSIH1-H1-copGFP-T2A-Puro vector (System Biosciences) under the control of the H1 promoter.

Techniques: Control, Expressing, Reverse Transcription Polymerase Chain Reaction, Flow Cytometry, Injection

Journal: Cardiovascular Diabetology

Article Title: NLRP3 inflammasome-modulated angiogenic function of EPC via PI3K/ Akt/mTOR pathway in diabetic myocardial infarction

doi: 10.1186/s12933-024-02541-3

Figure Lengend Snippet: Fig. 5 Regulated proliferation function of EPC by NLRP3 inflammasome in response to HG or AGEs. (A) Flow cytometry analysis was conducted on EPC following stimulation with HG (30 mM) or AGEs (200 µg/ml) for 72 h. The representative pattern of the cell cycle distribution of EPC in synchrony after stimulation. The representative pattern of the cell cycle distribution of EPC in synchrony and after stimulation. (B) Representative images of BrdU incor poration status after stimulation with HG (30 mM) or AGEs (200 µg/ml) for 72 h in EPC. Images were generated by merging the DAPI and BrdU channels. (C) Clone formation assay results showed the effects of NLRP3 inflammasome on the proliferation of EPC. (D) Senescence-associated beta-galactosidase staining of EPC following stimulation with HG (30 mM) or AGEs (200 µg/ml) for 72 h. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Similar results were obtained from three independent experiments

Article Snippet: In addition, the shRNA targeting Usp14 and Nlrp3 mRNA sequence was introduced into pSIH1-H1-copGFP-T2A-Puro vector (System Biosciences) under the control of the H1 promoter.

Techniques: Flow Cytometry, Generated, Tube Formation Assay, Staining

Journal: Cardiovascular Diabetology

Article Title: NLRP3 inflammasome-modulated angiogenic function of EPC via PI3K/ Akt/mTOR pathway in diabetic myocardial infarction

doi: 10.1186/s12933-024-02541-3

Figure Lengend Snippet: Fig. 8 CEBPB and USP14 regulated cardiac Function by modulating NLRP3 inflammasome in vivo. (A) Short-term survival curve after myocardial in farction and the indicated treatments. (B) Representative short-axis M-mode echocardiograms of the left ventricle at baseline and day 21 in the HFD/ STZ-induced diabetic mice with myocardial infarction groups. Left ventricular function was assessed by measurements of ejection fraction, LV fractional shortening, end diastolic volume, and end systolic volume. (C) Representative endothelial CD31 staining at the infarction border zone sections. (D) Representative images and quantitative infarct size in Masson’s trichrome stained mice hearts on day 21 after MI (n = 6 mice/group). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. These experiments were repeated independently at least three times with similar results

Article Snippet: In addition, the shRNA targeting Usp14 and Nlrp3 mRNA sequence was introduced into pSIH1-H1-copGFP-T2A-Puro vector (System Biosciences) under the control of the H1 promoter.

Techniques: In Vivo, Staining

Journal: Carcinogenesis

Article Title: Functional role of PLCE1 intronic insertion variant associated with susceptibility to esophageal squamous cell carcinoma.

doi: 10.1093/carcin/bgx123

Figure Lengend Snippet: Figure 1. Regional plot of the association results between variants in the PLCE1 locus and risk of ESCC. SNPs marking each region are plotted with P values. The 34 SNPs with P < 1 × 10–6 are above the black horizontal line and the smallest P value of rs7094783 is 3.13 × 10–9. Axes: y-axis indicates the –log10P values; x-axis shows genomic

Article Snippet: Human PLCE1 transcript 1 cDNA (NM_016341.3, Origene) and PLCE1 shRNA (Supplementary Table 6, available at Carcinogenesis Online) were cloned into pLenti-C-MycDDK-IRES-Neo (Origene) or pSIH1-H1-Puro (System Biosciences) lentiviral vector, respectively.

Techniques:

Journal: Carcinogenesis

Article Title: Functional role of PLCE1 intronic insertion variant associated with susceptibility to esophageal squamous cell carcinoma.

doi: 10.1093/carcin/bgx123

Figure Lengend Snippet: Figure 2. Functional relevance of rs71031566 and rs12220125 polymorphisms. (A) Annotation of intronic regions containing rs71031566 or rs12220125 using the ENCODE database. Colored histograms denote histone modification markers (H3K4me1, H3K4me3 and H3K27ac) in seven human cell types (GM12878, H1-hESC, HSMM, HUVEC, K562, NHEK and NHLF). The darkness of DNase hypersensitive peaks represent the maximum signal strength observed in 125 cell lines. The red line indicates the loca- tion of analyzed SNPs. (B, C) Constructs used for reporter gene assays. A set of DNA sequences containing rs71031566[C----] (680 base pairs) or rs71031566[CATTT] (684 base pairs) and rs12220125[T] or rs12220125[G] (586 base pairs) were generated and inserted respectively into PGL4.10-E4TATA reporter vector. (D, E) Relative reporter gene expression driven by PLCE1 fragments with different rs71031566 alleles or rs12220125 alleles in ESCC cells. Relative luciferase activities are shown as fold changes relative to luciferase expression in cells transfected with empty vector (pGL4.10-E4TATA). All constructs were cotransfected with pBEC22 to standardize transfection efficiency. Data shown are the mean ± s.e.m. from three independent experiments, each had six replicates. ***P < 0.001.

Article Snippet: Human PLCE1 transcript 1 cDNA (NM_016341.3, Origene) and PLCE1 shRNA (Supplementary Table 6, available at Carcinogenesis Online) were cloned into pLenti-C-MycDDK-IRES-Neo (Origene) or pSIH1-H1-Puro (System Biosciences) lentiviral vector, respectively.

Techniques: Functional Assay, Modification, Construct, Generated, Plasmid Preparation, Gene Expression, Luciferase, Expressing, Transfection

Journal: Carcinogenesis

Article Title: Functional role of PLCE1 intronic insertion variant associated with susceptibility to esophageal squamous cell carcinoma.

doi: 10.1093/carcin/bgx123

Figure Lengend Snippet: Figure 3. Interaction of polymorphic intron 22 sequence with the promoter in PLCE1 represses the promoter activity and gene transcription. (A) Constructs used for reporter gene assays. E4TATA box in PGL4.10 vector as indicated in Figure 2 was replaced with PLCE1 promoter (2598 base pairs from −3049 to −451). (B, D) Relative reporter gene expression in constructs containing PLCE1 promoter driven by DNA fragment harboring rs71031566[C----] or rs71031566[CATTT] in KYSE150 and KYSE30 cells. **P < 0.01 and ***P < 0.001. (C) Chromosome conformation capture analysis of the PLCE1 locus in cells with rs71031566[C----/C----] or rs71031566[CATTT/CATTT] genotype. Data show the interaction frequency, expressed as cross-linking frequency (y-axis), between fragments in the PLCE1 promoter and polymorphic intron 22. Chromosome conformation capture profiles were obtained in six cell types corresponding to six colors: blue, BxPC-3 (CATTT/CATTT genotype); red, RKO (CATTT/CATTT genotype); purple, KYSE30 (CATTT/CATTT genotype); green, H1299 (C----/C---- genotype); yellow, H460 (C----/C---- genotype) cells and grey, KYSE150 (C----/C---- geno- type) cells. Results are mean ± s.e.m. from three independent experiments. The chromatin interaction between the promoter and intron 22 was significantly increased in cells carrying the rs71031566[CATTT/CATTT] genotype compared with that in cells carrying the rs71031566[C----/C----] genotype. **P < 0.01 compared with cells car- rying rs71031566[C----/C----] genotype.

Article Snippet: Human PLCE1 transcript 1 cDNA (NM_016341.3, Origene) and PLCE1 shRNA (Supplementary Table 6, available at Carcinogenesis Online) were cloned into pLenti-C-MycDDK-IRES-Neo (Origene) or pSIH1-H1-Puro (System Biosciences) lentiviral vector, respectively.

Techniques: Sequencing, Activity Assay, Construct, Plasmid Preparation, Gene Expression

Journal: Carcinogenesis

Article Title: Functional role of PLCE1 intronic insertion variant associated with susceptibility to esophageal squamous cell carcinoma.

doi: 10.1093/carcin/bgx123

Figure Lengend Snippet: Figure 4. Interaction of polymorphic intron 22 sequence with the promoter of PLCE1 is mediated by OCT2. (A) Proposed long-range chromosomal interactions and gene regulation. PLCE1 transcription is normal in cell where intron 22 harbors the rs71031566[C----] allele (upper panel); however, PLCE1 transcription is repressed in cell where the intron harbors the rs71031566[CATTT] insertion variant allele, which creates a silencer element that is subject to interaction with certain protein(s), resulting in a chromatin loop (lower panel). (B) Electrophoretic mobility-shift assays with biotin-labeled PLCE1 intron 22 sequence containing the rs71031566[C----] or rs71031566[CATTT] and nuclear extracts from KYSE150 cells. Lanes 1 and 6 show mobility of the labeled oligonucleotides without nuclear extracts; lanes 2 and 7 show mobility of the labeled oligonucleotides with nuclear extracts in the absence of competitor; lanes 3 and 8, 4 and 9 and 5 and 10 show the mobility of the labeled oligonu- cleotides with nuclear extracts in the presence of unlabeled [C----] or [CATTT]-containing competitors. A DNA-protein complex (band I) was presented when [CATTT]- containing DNA was incubated with nuclear extracts (lane 2) and this complex could be eliminated by the addition of unlabeled [CATTT]-containing probe (Lanes 3 and 4) but not [C----]-containing probe (Lane 5). (C) Super-shift assays with biotin-labeled [CATTT]-containing oligonucleotides and nuclear extracts in the absence (Lane 2) or presence (lane 3) of OCT2 antibody. DNA–protein complex (band I) was depleted and an additional super-shift band (band II) was detected when OCT2 antibody was used (lane 3). (D, E) Chromatin immunoprecipitation (ChIP)-qPCR assays using antibodies against OCT2 in KYSE30 and KYSE150 cells. Immunoprecipitation levels are mean ± s.e.m. normalized to input control. Results were obtained from three independent biological samples. (D) Schematic ChIP-qPCR assays detecting interaction of PLCE1 intron 22 with OCT2 (upper panel) and the results show that enrichment of OCT2 in intron fragment 4 (I4) was only seen in KYSE30 cells carrying the rs71031566[CATTT/ CATTT] genotype (lower panel). (E) Schematic ChIP-qPCR assays detecting interaction of PLCE1 promoter with OCT2 (up panel) and the results show the interaction only in fragment 6 (P6) and fragment 7 (P7) of cells carrying the s71031566[CATTT] allele (lower panel). **P < 0.01 compared with IgG. (F) The PLCE1 expression was negatively regulated by OCT2 in cells carrying the rs71031566[CATTT/CATTT] genotype. Western blot shows that knockdown of OCT2 expression apparently resulted in elevated PLCε1 and overexpression of OCT2 significantly reduced the level of PLCε1 in BxPC-3, KYSE30 and RKO cells (upper panel). The amount of PLCε1 did not alter when OCT2 expression was changed in KYSE150, H460 and H1299 cells (lower panel).

Article Snippet: Human PLCE1 transcript 1 cDNA (NM_016341.3, Origene) and PLCE1 shRNA (Supplementary Table 6, available at Carcinogenesis Online) were cloned into pLenti-C-MycDDK-IRES-Neo (Origene) or pSIH1-H1-Puro (System Biosciences) lentiviral vector, respectively.

Techniques: Sequencing, Variant Assay, Electrophoretic Mobility Shift Assay, Labeling, Incubation, Chromatin Immunoprecipitation, ChIP-qPCR, Immunoprecipitation, Control, Expressing, Western Blot, Knockdown, Over Expression

Journal: Carcinogenesis

Article Title: Functional role of PLCE1 intronic insertion variant associated with susceptibility to esophageal squamous cell carcinoma.

doi: 10.1093/carcin/bgx123

Figure Lengend Snippet: Figure 5. Expression of PLCE1 in human esophageal tissue samples. (A–C) Relative PLCE1 mRNA levels determined by real-time RT-PCR in ESCC and matched normal tissues recruited in Beijing (A), Hangzhou (B) and analyzed based on Oncomine data (GSE23400, Su et al.; C). Results are mean ± s.e.m. relative to GAPDH from three measurements. *P < 0.05; **P < 0.01 and ***P < 0.001. (D) PLCE1 mRNA levels shown in mean ± s.e.m. relative to GAPDH in normal esophageal tissues as function of PLCE1 rs71031566 genotype in combined Beijing and Hangzhou sample (N = 168). The rs71031566[CATTT/CATTT] genotype had significantly lower PLCE1 mRNA than the rs71031566[C----/C----] or rs71031566[C----/CATTT] genotype (*P < 0.05). (E) Western blot analysis of PLCε1 in normal esophageal tissues with different PLCE1 rs71031566 genotype, showing that the CATTT/CATTT genotype had apparently lower PLCε1 levels compared with the C----/C---- or C----/CATTT genotype.

Article Snippet: Human PLCE1 transcript 1 cDNA (NM_016341.3, Origene) and PLCE1 shRNA (Supplementary Table 6, available at Carcinogenesis Online) were cloned into pLenti-C-MycDDK-IRES-Neo (Origene) or pSIH1-H1-Puro (System Biosciences) lentiviral vector, respectively.

Techniques: Expressing, Quantitative RT-PCR, Western Blot

Journal: Carcinogenesis

Article Title: Functional role of PLCE1 intronic insertion variant associated with susceptibility to esophageal squamous cell carcinoma.

doi: 10.1093/carcin/bgx123

Figure Lengend Snippet: Figure 6. Repression of PLCE1 expression enhances cell proliferation. (A, B) Knockdown of PLCE1 expression significantly enhanced cancer cell proliferation (A) and colony formation (B) in vitro. (C, D) Overexpression of PLCE1 significantly suppressed cancer cell proliferation (C) and colony formation (D) in vitro. Cell proliferation in 96-well plates transfected with PLCE1, PLCE1-shRNA or control lentivirus vector was determined daily for 4 days by CCK-8 assay. For proliferation, the results represent mean ± s.e.m. from three experiments and each experiment had six replicates; **P < 0.01 compared with control. For colony formation, the results represent colony formation ability relative to control set to 100% from three experiments; *P < 0.05; **P < 0.01 and ***P < 0.001 compared with each control. (E, F) Effect of PLCE1 knock- down or overexpression on xenograft tumor formation and growth in nude mice. Results are mean ± s.e.m. from five animals in each group. *P < 0.05; **P < 0.01 and ***P < 0.001 compared with control.

Article Snippet: Human PLCE1 transcript 1 cDNA (NM_016341.3, Origene) and PLCE1 shRNA (Supplementary Table 6, available at Carcinogenesis Online) were cloned into pLenti-C-MycDDK-IRES-Neo (Origene) or pSIH1-H1-Puro (System Biosciences) lentiviral vector, respectively.

Techniques: Expressing, Knockdown, In Vitro, Over Expression, Transfection, shRNA, Control, Plasmid Preparation, CCK-8 Assay