mRNA Expression Levels Of Survivin Research Articles

Evaluation of Immunohistochemical Expression of Survivin and its Correlation with qRT-PCR Results as a Useful Diagnostic Marker in Gastric Cancer.

Today, survivin is known as one of the most specific cancer proteins; provide unique and practical study opportunities. Clinical value of survivin in gastric cancer (GC) is not yet appointed. To establish the expression level of survivin and its diagnosis value in Iranian patients with GC, we evaluated the association of survivin expression with clinicopathologic factors. Overall, 60 matched-normal controls with 60 GC samples including 30 cases with evidence of metastasis at time of our study and 30 cases without evidence of metastasis were recruited, in Tehran, Iran during 2008 to 2018. Survivin expression was evaluated by quantitative Real-time polymerase chain reaction (qRT-PCR) and immunohistochemistry (IHC) study. Increased expression of survivin at mRNA and protein levels was found in 86.7% and 71.6% of cases, respectively. Evidence indicated a significant difference in survivin mRNA expression level between tumor and nontumoral (marginal) tissues (P<0.001). The difference in expression of survivin mRNA was not significant between metastatic and non-metastatic tumor tissues (P=0.171). Positive immunoreactivity of survivin was observed to be predominantly in the nucleus of tumor cells. A significant difference in survivin protein expression was detected between tumor and non-tumoral tissues (P<0.001) and between metastatic and non-metastatic tumor tissues (P<0.001). There was no significant association between survivin mRNA expression and clinicopathological variables. However, survivin protein expression was significantly correlated with perineural involvement (P<0.018). This data could be supportive of using survivin as a useful diagnostic marker in GC. Although, more research is needed in this area.

Overexpression of miR-506-3p reversed doxorubicin resistance in drug-resistant osteosarcoma cells.

Background and objective: Osteosarcoma is a common primary malignant tumor of bone, and doxorubicin is one of the most widely used therapeutic drugs. While the problem of doxorubicin resistance limits the long-term treatment benefits in osteosarcoma patients. The role of miRNAs and their target genes in osteosarcoma have become increasingly prominent. Currently, there is no report on miR-506-3p reversing doxorubicin resistance by targeting STAT3 in osteosarcoma. The purpose of this study was to investigate the molecular mechanism that overexpression of miR-506-3p reverses doxorubicin resistance in drug-resistant osteosarcoma cells. Methods: Doxorubicin-resistant osteosarcoma cells (U-2OS/Dox) were constructed by intermittent stepwise increasing stoichiometry. The target genes of miR-506-3p were predicted by bioinformatics approach and the targeting relationship between miR-506-3p and STAT3 was detected using dual luciferase reporter assay. U-2OS/Dox cells were treated with miR-506-3p overexpression and STAT3 silencing respectively. Then Western blot and RT-qPCR were used to detect the protein and mRNA expression levels of JAK2/STAT3 signaling pathway, drug-resistant and apoptotic associated molecules. The migration and invasion were assessed by cell scratch assay and transwell assay. The cell proliferative viability and apoptosis were investigated by CCK8 assay and flow cytometry assay. Results: U-2OS/Dox cells were successfully constructed with a 14.4-fold resistance. MiR-506-3p is directly bound to the 3'-UTR of STAT3 mRNA. Compared with U-2OS cells, the mRNA expression of miR-506-3p was reduced in U-2OS/Dox cells. Overexpression of miR-506-3p decreased the mRNA expression levels of JAK2, STAT3, MDR1/ABCB1, MRP1/ABCC1, Survivin and Bcl-2, and decreased the protein expression levels of p-JAK2, STAT3, MDR1/ABCB1, MRP1/ABCC1, Survivin and Bcl-2, and conversely increased Bax expression. It also inhibited the proliferation, migration and invasion of U-2OS/Dox cells and promoted cells apoptosis. The results of STAT3 silencing experiments in the above indicators were consistent with that of miR-506-3p overexpression. Conclusion: Overexpression of miR-506-3p could inhibit the JAK2/STAT3 pathway and the malignant biological behaviors, then further reverse doxorubicin resistance in drug-resistant osteosarcoma cells. The study reported a new molecular mechanism for reversing the resistance of osteosarcoma to doxorubicin chemotherapy and provided theoretical support for solving the clinical problems of doxorubicin resistance in osteosarcoma.

Clinical significance of survivin mRNA expression (&lt;i&gt;BIRC5&lt;/i&gt;) in colorectal cancer

Aim. To evaluate the clinical significance of survivin (BIRC5) mRnA expression in circulating tumor cells (CTCs) and tumor material from colorectal cancer (CRC).Materials and methods. The study was organized according to the principle of a continuous prospective non-randomized study. The expression of survivin (BIRC5) mRnA in CTCs and tumor material was determined using RT-pCR.Results. The study included 130 patients (study group – 109 patients with colorectal cancer and observation group – 21 patients with colon adenomas). All patients underwent complete tumor removal (radical surgery – 93.6 %), cytoreductive – 6.4 %).A high level of survivin (BIRC5) mRnA expression was detected in colorectal adenocarcinoma in comparison with adenomas (pMann–whitney &lt; 0.001) M ± SD (1.678 ± 2.45 and 0.023 ± 0.07). In the study and observation group, the expression of survivin mRnA (BIRC5) in CTCs both before surgery M ± SD (1.175 ± 1.33 and 0.052 ± 0.11) and after 3 months M ± SD (1.015 ± 0.93 and 0.018 ± 0.002) was significantly different (pMann–whitney &lt;0.001).During adjuvant chemotherapy, a decrease in the level of survivin expression in CTCs was observed (p 9 months after surgery, CTCs remain in the bloodstream even despite adjuvant chemotherapy (p = 0.015 and p = 0.012). Overexpression of survivin in CTCs before surgery correlates with damage to regional lymph nodes (p = 0.03, r = 0.21), stage of the tumor process (p = 0.01, r = 0.25), degree of tumor differentiation (p = 0.03, r = 0.21). Overexpression of survivin in CTCs 9 months after surgery significantly affects relapse-free survival HR (95 % CI HR) = 3.1 (95 % CI 1.56–6,08, p = 0.0012) and overall survival of patients HR (95 % CI HR) =6.8 (95 % CI 2.65–17.33, p = 0.0001).Conclusions. Overexpression of survivin mRnA in colorectal cancer is a negative prognosis factor for the disease and directly depends on the tumor involvement of regional lymph nodes, the stage of the disease, degree of tumor differentiation, promoting the development of disease relapse, and can be used to diagnose minimal residual disease (MRD) and assess the prognosis of overall patient survival.

Andrographolide Reverses Doxorubicin Resistance in Human Breast Cancer Stem Cells by Regulating Apoptotic Gene Expressions

BACKGROUND: Breast cancer stem cells (BCSCs) have been identified as playing a crucial role in therapeutic resistance. This resistance can be attributed to the anti-apoptotic protein survivin and the antioxidant MnSOD high expression. To overcome the resistance to doxorubicin (DOX), this study proposed the utilization of andrographolide (ANDRO), the primary bioactive compound in Andrographis paniculata leaves. The objective was to examine the role of andrographolide in regulating survivin, caspase-9, and caspase-3 gene expressions to reverse doxorubicin resistance in human BCSCs.METHODS: BCSCs were exposed to 0.1 µM DOX every two days or 50 µM rotenone (ROT) for 6 hours, subsequently supplemented with 0.3 mM ANDRO. Superoxide and peroxide levels were measured using DHE and DCFH-DA assay. The MnSOD, survivin, caspase-9, and caspase-3 mRNA expression levels were analyzed using qRT-PCR. Protein expressions were evaluated using Western blotting assay. MnSOD activity was determined using xanthine oxidase inhibition assay. The apoptotic cells were determined using Annexin-V/PI staining.RESULTS: This study indicated that the cytotoxic mechanisms of DOX, similar to ROT, in BCSCs were attributed to oxidative stress, as evidenced by an elevation in superoxide rather than peroxide levels, accompanied by a decrease in MnSOD activity. This study also highlighted that ANDRO reversed DOX resistance in BCSCs subjected to repeated DOX treatment by downregulating survivin and upregulating caspase-9 and caspase-3 mRNA expressions, thereby activating the intrinsic apoptotic pathway.CONCLUSION: This study provides insights into the role of ANDRO in modulating the expression of apoptotic genes, such as survivin, caspase-9, and caspase-3, to overcome DOX resistance in BCSCs.KEYWORDS: breast cancer, breast cancer stem cell, andrographolide, doxorubicin, oxidative stress, apoptosis

Dual Targeting of Anti-Apoptotic Proteins Enhances Chemosensitivity of the Acute Myeloid Leukemia Cells.

Acute myeloid leukemia (AML) is a type of blood cancer characterized by fast cellular proliferation. Myeloid cell leukemia-1 (Mcl-1) and survivin, as anti-apoptotic proteins, are involved in cancer growth and resistance to chemotherapy. The aim of this study was to examine the combination effect of Mcl-1 and survivin specific siRNAs on chemosensitivity of the human HL-60 AML cells. SiRNAs transfection was performed by using Lipofectamine™2000 reagent. The mRNA expression was analyzed by real-time quantitative PCR. The apoptosis analysis was measured by ELISA cell death assay. siRNAs markedly suppressed mRNA expression levels of Mcl-1 and survivin in a time-dependent manner, resulting in reduction of leukemic cell proliferation and enhanced spontaneous cell death. Surprisingly, Mcl-1 siRNA and survivin siRNA synergistically enhanced the cell toxic effects of etoposide. Furthermore, down-regulation of Mcl-1 and survivin significantly enhanced the apoptotic effect of etoposide. Our investigation suggests that suppression of Mcl-1 and survivin by siRNA can effectually inhibit cell growth and overcome chemoresistance of AML cells. Therefore siRNAs may be an important adjuvant in chemotherapy for AML patients.

Matrine induces cell cycle arrest and apoptosis in hepatocellular carcinoma cells via miR-122 mediated CG1/livin/survivin signal axis

&#x0D; &#x0D; &#x0D; &#x0D; Purpose: To study the impact of matrine on cell cycle and apoptotic changes in hepatoma cells, and the mechanism involved.&#x0D; Methods: Human hepatoma cell line HepG2 was treated with different concentrations of matrine. The blank control cells were maintained in 1640 medium only. The influence of matrine on proliferative ability was determined with 3-(4,5)-dimethylthiahiazo (-z-y1)-3,5-di- phenytetrazoliumromide (MTT) method. Flow cytometry was used to determine its effect on cell cycle and apoptosis; RT-PCR (reverse transcription-polymerase chain reaction) was applied to assay the mRNA expressions of miR-122, cyclin G1 (CG1), livin and survivin mRNA, while the protein expressions of CG1, livin and surviving were assayed by Western blotting.&#x0D; Results: Matrine time- and dose-dependently suppressed the proliferative capacity of the cells. At a concentration of 0.5 mg/mL, matrine had no significant effect on the cell cycle. However, 1.0 mg/mL matrine blocked the cell cycle in G1 phase, while 1.5 mg/mL matrine blocked HepG2 cells in G2/M phase (p &lt; 0.05). Moreover, matrine induced apoptosis in HepG2 cells, and markedly downregulated the expressions of miR-122 concentration- time-reliantly (p &lt; 0.05). In addition, matrine markedly and concentration-dependently reduced mRNA and protein expression levels of CG1, livin and survivin, with the strongest inhibitory effect at a level of 1.5 mg/mL.&#x0D; Conclusion: Matrine induces cell cycle block and apoptotic changes in hepatoma cells through a mechanism related to regulation of the CG1/livin/survivin signal axis mediated by miR-122. Matrine may be a potential treatment for liver cancer. However, clinical trials are needed to confirm this potential.&#x0D; &#x0D; &#x0D; &#x0D;

Shikonin Inhibits Non-Small-Cell Lung Cancer H1299 Cell Growth through Survivin Signaling Pathway.

Overexpressed survivin is associated with worse survival of several types of human tumors. In this study, the antitumor activity of shikonin in non-small-cell lung cancer (NSCLC) by regulating survivin pathway was investigated. Results showed that shikonin inhibited the NSCLC H1299 cell proliferation in a dose-dependent manner. Moreover, shikonin fits well with survivin by molecular docking. Shikonin also inhibited the mRNA expression and protein level of survivin in H1299 cells. Shikonin arrested H1299 cell cycle at the G0/G1 phase by regulating CDK/cyclin family members. In addition, shikonin regulated the expression of X-linked inhibitor of apoptosis- (XIAP-) mediated caspases 3 and 9, thus leading to the damage of mitochondrial membrane potential and induction of H1299 cell apoptosis. Overall, shikonin inhibited H1299 cell growth by inducing apoptosis and blocking the cell cycle. The underlying mechanism involves targeting survivin, which subsequently regulates the protein expression of XIAP/caspase 3/9, CDK2/4, and cyclin E/D1. Thus, shikonin, a survivin inhibitor, is a promising therapeutic strategy in NSCLC treatment.

Effect of acidic culture conditions on the proliferation, apoptosis, and migration ability of human tongue squamous cell carcinoma cells and its related mechanism.

This study aims to explore the effect of acidic culture conditions on the proliferation, apoptosis, and migration ability of human tongue squamous cell carcinoma SCC15 and CAL27 cells and its potential molecular mechanism. After acidic culture for different periods, methyl thiazolyl tetrazolium (MTT) method was adop-ted to detect the cell proliferation of SCC15 and CAL27. Flow cytometry was employed to detect the apoptosis level of SCC15 and CAL27 cells. The migration ability of SCC15 and CAL27 after acidic culture was detected by scratch hea-ling test. Real-time fluorescence quantitative polymerase chain reaction (FQ-PCR) was used to detect the mRNA expression of cyclooxygenase 2 (COX-2) and survivin in SCC15 and CAL27 cells after acidic culture. After culture for 24 h under acidic microenvironment, SCC15 and CAL27 cells grew rapidly and reached the stationary phase after adjustment for 3 days. The apoptosis levels of SCC15 and CAL27 cells decreased after acidic culture, but the most significant reduction occurred after 6 h of acidic culture. The scratch healing rates of SCC15 and CAL27 cells increased after acidic culture. The results of FQ-PCR showed that the mRNA expression levels of COX-2 and survivin in SCC15 and CAL27 cells increased after acidic culture. Extracellular acidic microenvironment can inhibit the apoptosis of tongue squamous carcinoma cells, promote their migration, and induce more adaptable and malignant tongue squamous carcinoma cells. The mechanism may be related to COX-2 and survivin and their signal pathways.

Angiotensin-converting enzyme 2 alleviates pulmonary artery hypertension through inhibition of focal adhesion kinase expression.

Focal adhesion kinase (FAK) is an important therapeutic target in pulmonary artery hypertension (PAH); however, the mechanism of its activation remains unknown. The present study aimed to investigate whether angiotensin-converting enzyme 2 (ACE2) could regulate FAK and alleviate PAH in a rat model of PAH established with a single administration of monocrotaline followed by continuous hypoxia treatment. In the current study, right ventricular pressure, body weight and the right ventricular hypertrophy index were measured, and hematoxylin-eosin staining was performed on lung tissues to determine whether the modeling was successful. Changes in the serum levels of FAK were measured using an ELISA kit to evaluate the association between ACE2 and FAK. The mRNA expression levels of ACE2, FAK, caspase-3 and survivin were determined using reverse transcription-quantitative PCR (RT-qPCR). The protein expression levels of ACE2, phosphorylated FAK/FAK, cleaved caspase-3/pro-caspase-3 and survivin were determined via western blotting. Immunohistochemistry was applied to detect the expression of FAK around the pulmonary arterioles. Apoptosis of smooth muscle cells around pulmonary arterioles was observed by TUNEL staining. After treatment with the ACE2 activator DIZE or inhibitor DX-600, the results demonstrated that ACE2 reduced PAH-induced changes in arteriole morphology compared with the control. It also inhibited FAK expression in serum. WB and RT-qPCR results suggested that ACE2 inhibited the expression of FAK and pathway-related proteins, and promoted caspase-3 expression. Additionally, ACE2 reduced FAK expression around the pulmonary arterioles and promoted smooth muscle cell apoptosis. The results indicated that ACE2 activation inhibited FAK expression, leading to alleviation of the symptoms of PAH.

Broad-Spectrum Polymeric Nanoquencher as an Efficient Fluorescence Sensing Platform for Biomolecular Detection.

Colloidal inorganic nanostructures (metal, carbon, and silica) have been widely used as "nanoquenchers" for construction of nanosensors; however, inherent drawbacks such as insufficient fluorescence quenching efficiency, false positive signals, and uncertain long-term cytotoxicity have limited their further utility. Herein, by taking advantages of polymeric nanoparticles (PNPs) in terms of high loading capacity, facile surface modification chemistry, and good biocompatibility, we report a broad-spectrum (400-750 nm) polymeric fluorescence-quenching platform for sensor fabrication. Our newly developed polymeric nanoquenchers (qPNPs) were constructed by concurrently encapsulating various alkylated black-hole quenchers into nanoparticles made of poly(methyl methacrylate-co-methacrylic acid) and were found to have an excellent fluorescence quenching effect (>400-fold) on common fluorophores (FAM, TMR, and Cy5) together with high stability under physiological conditions. As a proof of concept, the feasibility of these qPNPs for fluorescence sensing was validated by successful construction of two nanosensors (FAMDEVD@qPNP and Cy5SurC@qPNP), which could be used as promising nanosensors for live-cell imaging of the apoptosis-related protease caspase-3 and cancer-related survivin mRNA, respectively. As expected, in the FAM channel, the FAMDEVD@qPNP showed fast and selective fluorescence responses toward caspase-3 in buffers and could be used to image the activation of drug-induced endogenous caspase-3. In the Cy5 channel, the Cy5SurC@qPNP could be used to distinguish normal cells (MCF10A) from cancer cells (HeLa) by quantitatively detecting the endogenous survivin mRNA level. It could be further used to monitor changes in the endogenous survivin mRNA expression levels in drug-treated HeLa cells. Altogether, by virtue of their high quencher loading and broad-spectrum quenching efficiency and good signal-to-background ratio, these qPNPs might be particularly attractive alternatives to other conventional nanoquenchers for the construction of more complex biosensors in the future.

Self-AssembledDNA Nanostructure as a Carrier for Targeted siRNA Delivery in Glioma Cells.

IntroductionRNA interference is a promising therapy in glioma treatment. However, the application of RNA interference has been limited in glioma therapy by RNA instability and the lack of tumor targeting. Here, we report a novel DNA tetrahedron, which can effectively deliver small interfering RNA to glioma cells and induce apoptosis.MethodssiRNA, a small interfering RNA that can suppress the expression of survivin in glioma, was loaded into the DNA tetrahedron (TDN). To enhance the ability of active targeting of this nanoparticle, we modified one side of the DNA nanostructure with aptamer as1411 (As-TDN-R), which can selectively recognize the nucleolin in the cytomembrane of tumor cells. The modified nanoparticles were characterized by agarose gel electrophoresis, dynamic light scattering, and transmission electron microscopy. The serum stability was evaluated by agarose gel electrophoresis. Nucleolin was detected by Western blot and immunofluorescence, and targeted cellular uptake was examined by flow cytometry. The TUNEL assay, flow cytometry, and Western Blot were used to detect apoptosis in U87 cells. The gene silencing of survivin was examined by qPCR, Western Blot, and immunofluorescence.ResultsAs-TDN-R alone showed better stability towards siRNA, indicating that TDN was a good siRNA protector. Compared with TDN alone, there was increased intercellular uptake of As-TDN-R by U87 cells, evidenced by overexpressed nucleolin in glioma cell lines. TUNEL assay, flow cytometry, and Western Blot revealed increased apoptosis in the As-TDN-R group. The downregulation of survivin protein and mRNA expression levels indicated that As-TDN-R effectively silenced the target gene.ConclusionThe novel nanoparticle can serve as a good carrier for targeting siRNA delivery in glioma. Further exploration of the DNA nanostructure can greatly promote the application of DNA-based drug systems in glioma.

Survivin Regulates Bad Gene Expression by Binding to Its Promoter and Modulates Cell Cycle and Apoptosis in Esophageal Carcinoma Cell.

Esophageal cancer (EC) is the eighth most prevalent cancer and the sixth leading cause of cancer-related mortality worldwide. As an antiapoptotic and a proapoptotic protein, respectively, survivin and Bad play an important role in carcinogenesis of the most human cancers including EC. However, the regulatory relationships between them remain unclear. We sought to investigate the effects of survivin knockdown and overexpression on the expression of Bad gene, cell cycle progression, and apoptosis of esophageal carcinoma cell. The mRNA expression levels of survivin and Bad were determined in EC tissue samples. The knockdown and overexpression experiments were performed in ECA109 and KYSE450 cells via transfection with survivin overexpression and shRNA plasmids. A Bad overexpression experiment was conducted to confirm the biological effect on knockdown of survivin via modulating Bad expression. RT-qPCR and Western blot analysis were used to detect mRNA and protein expression, respectively. Cell cycle and apoptosis were analyzed by flow cytometry. The chromatin immunoprecipitation (ChIP) was conducted to determine the binding sites of survivin on the promoter of Bad gene. By analyzing the mRNA expression of survivin and Bad in 40 ESCC patient specimens, we found that the positive expression rate of survivin in tumor tissues (88%, 35/40) was remarkably high, compared with the distal nontumor tissues (48%, 19/40, p < 0.01). On the other hand, the positive expression rate of Bad in tumor tissues (70%, 28/40) was remarkably low, compared with the distal nontumor tissues (95%, 38/40, p < 0.01). Overexpression of survivin decreases Bad mRNA and protein expression and promotes transformation of cell cycle to S phase. Conversely, knockdown of survivin increases Bad mRNA and protein expression and induces cell cycle arrest and apoptosis. Bad overexpression inducing apoptosis of esophageal carcinoma cell shows the similar apoptotic effect with survivin knockdown. ChIP assays indicate that survivin directly binds to the Bad promoter region, diminishing the transcriptional activity of Bad. In conclusion, the result suggested that survivin regulates Bad gene expression by binding to its promoter and modulates cell cycle and apoptosis in esophageal carcinoma cell.

Propofol activates AMPK to inhibit the growth of HepG2 cells in vitro and hepatocarcinogenesis in xenograft mouse tumor models by inducing autophagy

Hepatocellular carcinoma (HCC) is a fatal malignant tumor with a poor prognosis, and is the third leading cause of cancer-related deaths worldwide. This study aimed to investigate the anti-tumor effect of propofol on the proliferation, apoptosis, and cell cycle of HCC by regulating adenosine monophosphate-activated protein kinase (AMPK) in vivo and in vitro. The cell counting Kit-8 (CCK-8) assay was employed to screen the effect of propofol on HepG2 cell viability at various concentrations (0.3, 0.6, 1.2, 2.5, 5, 10, 20, 40, 80 and 160 µM). We selected propofol at concentrations of 5, 10 and 20 µM for subsequent experiments. Flow cytometry was used to examine the apoptosis and cell cycle of HCC. Quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR) was applied to measure the messenger ribonucleic acid (mRNA) expression levels of proliferating cell nuclear antigen (PCNA) and survivin. Western blotting was applied to measure the protein expression levels of PCNA, survivin, cleaved caspase-3, cleaved caspase-9, p27 (Kip1), and cyclin A. The effects of propofol were evaluated by establishing a xenograft tumor model. After treatment with propofol, the mRNA expression levels of PCNA and survivin were decreased compared with the 0 µM propofol (control) group. The colony formation assay showed that the colony formation rate was obviously down-regulated. Flow cytometry demonstrated that HepG2 cell apoptosis was increased. G0/G1 was enhanced compared with the control group, while G2/M was restrained. The levels of cleaved caspase-3, cleaved caspase-9, p27, phospho-AMP-activated protein kinase α1 (p-AMPKα1), phospho-mammalian target of rapamycin (p-mTOR), and phospho-Unc-51 like autophagy activating kinase 1 (p-ULK1) were notably elevated, while the levels of cyclin A were suppressed. The xenograft tumor volume declined in vivo compared with the HepG2 xenograft group. The expression levels of cell proliferation markers (PCNA) were significantly down-regulated markedly, while the expression levels of cell cycle markers (p27) were notablyup-regulated. Terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) staining showed that cell apoptosis was increased. The levels of p-AMPKα1 were also up-regulated. Propofol inhibits the proliferation, apoptosis, and cell cycle of HCC by regulating AMPK in vivo and in vitro.

Emodin Reverses Gemcitabine Resistance of Pancreatic Cancer Cell Lines Through Inhibition of IKKβ/NF-κB Signaling Pathway.

BackgroundPancreatic cancer is one of the most malignant tumors, and gemcitabine has been considered as the standard treatment and been widely utilized as a first-line drug for advanced pancreatic cancer, but gemcitabine-resistance always occurs after a short period of treatment.MethodsTwo pancreatic cancer cell lines Panc-1 and MIA-PaCa-2 were used as the study subject and their gemcitabine-resistant cells were established. Both drug-resistant cells were divided into four groups: blank, emodin, gemcitabine, and emodin+gemcitabine. Cell viability was detected by MTT assay. Flow cytometry was performed to detect cell apoptosis rate and P-gp function. Quantitative real-time polymerase chain reaction and Western blotting were used to detect Survivin, XIAP, Caspase-9/3, NF-κB p65, IKKβ and IκB-α mRNA/protein expressions, respectively. Electrophoretic mobility shift assay (EMSA) was performed to detect NF-κB binding activity. Rhodamine 123 efflux assay was used to detect P-gp function.ResultsEmodin could inhibit cell activity in all cell lines. Both emodin and gemcitabine can significantly increase the apoptosis rate, and the combination of the two drugs can further significantly increase the apoptosis rate in normal pancreatic cancer cell lines. In both drug-resistant pancreatic cancer cell lines, it can be observed that although gemcitabine can increase the apoptosis rate, the effect of promoting apoptosis is significantly lower than that of emodin; the drug combination can still significantly increase the apoptosis rate on the basis of emodin alone. Emodin can significantly reduce the mRNA and protein expression levels of Survivin, XIAP, NF-κB, and IKKβ, and significantly increase the mRNA and protein expression levels of Caspase-3/9 and IκB-α. Emodin significantly reduced NF-κB activity and emodin significantly promoted P-gp fluorescence intensity from Rhodamine 123 efflux assay.ConclusionEmodin inhibits the expression of IKKβ, thereby inhibiting the expression and activity of downstream NF-κB, and inhibits P-gp function at the same time, ultimately achieving the purpose of reversing the drug-resistance of pancreatic cancer cell lines.

The molecular mechanism study of COMP involved in the articular cartilage damage of Kashin-Beck disease.

AimsKashin-Beck disease (KBD) is a kind of chronic osteochondropathy, thought to be caused by environmental risk factors such as T-2 toxin. However, the exact aetiology of KBD remains unclear. In this study, we explored the functional relevance and biological mechanism of cartilage oligosaccharide matrix protein (COMP) in the articular cartilage damage of KBD.MethodsThe articular cartilage specimens were collected from five KBD patients and five control subjects for cell culture. The messenger RNA (mRNA) and protein expression levels were detected by quantitative reverse transcription PCR (qRT-PCR) and western blot. The survival rate of C28/I2 chondrocyte cell line was detected by MTT assay after T-2 toxin intervention. The cell viability and mRNA expression levels of apoptosis related genes between COMP-overexpression groups and control groups were examined after cell transfection.ResultsThe mRNA and protein expression levels of COMP were significantly lower in KBD chondrocytes than control chondrocytes. After the T-2 toxin intervention, the COMP mRNA expression of C28/I2 chondrocyte reduced and the protein level of COMP in three intervention groups was significantly lower than in the control group. MTT assay showed that the survival rate of COMP overexpression KBD chondrocytes were notably higher than in the blank control group. The mRNA expression levels of Survivin, SOX9, Caspase-3, and type II collagen were also significantly different among COMP overexpression, negative control, and blank control groups.ConclusionOur study results confirmed the functional relevance of COMP with KBD. COMP may play an important role in the excessive chondrocytes apoptosis of KBD patients.Cite this article: Bone Joint Res 2020;9(9):578–586.

YM155 sensitizes HeLa cells to TRAIL-mediated apoptosis via cFLIP and survivin downregulation.

Tumor necrosis factor-related apoptosis inducing ligand (TRAIL)-mediated apoptosis is a safe method for the treatment of various types of cancer. However, TRAIL therapy is less effective in certain types of cancer, including cervical cancer. To address this problem, a combinatorial approach was employed to sensitize cervical cancer at low dosages. YM155, a survivin inhibitor, was used at low dosages along with TRAIL to induce apoptosis in HeLa cells. The effects of the individual treatment with TRAIL and YM155 on apoptosis were assessed by propidium iodide assay. In addition, to validate the DNA damage exhibited by the combination treatment, the phosphorylation status of γH2A histone family member X was investigated by immunofluorescence and western blot analysis. TRAIL or YM155 alone had no significant effect on DNA damage and apoptosis. However, the TRAIL/YM155 combination triggered a synergistic pro-apoptotic stimulus in HeLa cells. The mRNA and protein levels of CASP8- and FADD-like apoptosis regulator (cFLIP), death receptor 5 (DR5) and survivin were monitored using RT-PCR and western blot analysis, respectively. This combinatorial approach downregulated both mRNA and protein expression levels of cFLIP and survivin. Further experimental results suggested that the combination treatment significantly reduced cell viability, invasion and migration of HeLa cells. Overall, the present findings indicated that the low dosage of YM155 sensitized HeLa cells to TRAIL-induced apoptosis via a mechanism involving downregulation of cFLIP and survivin. The results indicated the importance of combination drug treatment and reveal an effective therapeutic alternative for TRAIL therapy in human cervical cancer.

Evaluation of E2F3 and survivin expression in peripheral blood as potential diagnostic markers of prostate cancer

Abstract Background Prostate cancer (PC) incidence has risen globally. As there are no current independent biomarkers with high diagnostic efficiency to detect PC, this study was performed to investigate the relative gene expression levels of E2F3 and survivin in the whole blood of PC, benign prostate hyperplasia (BPH), and normal control individuals and to explore their diagnostic value. Material and methods Participants of the study were divided into three groups; normal control group (n=25), BPH patients (n=25), and PC patients (n=75). The E2F3 and survivin gene expression levels were assessed using real-time qPCR in addition to the measurement of free and total levels of prostate-specific antigen (PSA) using electrochemiluminescence assays. Results Survivin relative gene expression was over-expressed in PC and BPH patients compared to the normal control group, whereas, E2F3 did not differ significantly among the studied groups. Compared to PSA, E2F3 and survivin mRNA expression levels had lower diagnostic efficacy to differentiate PC from normal and BPH individuals with an area under curve (AUC) of 0.471 and 0.727, respectively. Further, survivin expression level was associated with increased the risk of PC. Conclusion Survivin and E2F3 relative expression levels in peripheral blood had low diagnostic performance to detect PC and individuals with high survivin expression levels may have higher risk to develop PC.

Dysregulation of microRNAs regulating survivin in CD4+ T cells in multiple sclerosis

BackgroundImpaired elimination mechanisms of the autoreactive lymphocytes, like T lymphocytes, via apoptosis may be the cause of continues inflammatory state in multiple sclerosis (MS). BIRC5 gene codify for the survivin, which participates in the modulation of apoptosis and cell survival. The objective of this study was investigation of the role of important confirmed miRNAs, including miR-335, miR-485, miR-542, and miR-708, in the regulation of survivin mRNA in the CD4+ T cells of MS cases. MethodsIn this study, 50 RRMS patients as well as 50 healthy matched controls were recruited. The peripheral blood mononuclear cells (PBMCs) were isolated from whole blood samples and CD4+ T cells were prepared. After that, RNA was extracted, cDNA was synthesized, and the expression levels of miR-335, miR-485, miR-542, and miR-708 were measured using Real-time PCR. Moreover, the mRNA expression of survivin was detected. Serum level of survivin was detected using ELISA. ResultsThe mRNA of survivin was 2-folds upregulated in the CD4+ T cells from MS patients in comparison to the healthy controls (P = 0.0053). Serum level of survivin was higher in patients than controls. There was statistically significant downregulation of miR-485 (P = 0.001) and miR-708 (P = 0.011) in CD4+ T cells of patients compared with controls. The miR-485 downregulation had statistically significant correlation with the mRNA expression and serum level of survivin. ConclusionmiRNAs play a role in the regulation of survivin, and therefore apoptosis of CD4+ T cells, and hence are probably participating in a persistent inflammatory condition in MS patients.

LncRNA HCP5 Stimulates the Proliferation of Non-Small Cell Lung Cancer Cells by Up-Regulating Survivin Through the Down-Regulation of miR-320.

IntroductionWe explored the roles of lncRNA HCP5 in non-small cell lung cancer (NSCLC).MethodsLevels of HCP5 were measured by performing qPCR and data were compared between non-tumor and NSCLC tissue samples by performing a paired t-test. Expression levels of miR-320 and survivin mRNA in NSCLC tissues were also measured by performing qPCR. The effects of HCP5, miR-320 and survivin overexpression on the proliferation of H23 cells were analyzed by cell proliferation assay.ResultsWe found that HCP5 was up-regulated in NSCLC and predicted the poor survival of NSCLC patients. HCP5 was negatively correlated with miR-320 but positively correlated with survivin in NSCLC tissues. In NSCLC cells, HCP5 overexpression led to the up-regulated survivin and down-regulated miR-320. Moreover, miR-320 overexpression failed to affect HCP5 but down-regulated survivin. Cell proliferation assay showed that HCP5 and survivin overexpression led to increased, while miR-320 overexpression led to decreased cell proliferation rate. In addition, miR-320 overexpression reduced the effects of HCP5 overexpression.ConclusionTherefore, HCP5 may stimulate the proliferation of NSCLC cells by up-regulating survivin through the down-regulation of miR-320.

Regorafenib and ginsenoside combination therapy: inhibition of HepG2 cell growth through modulating survivin and caspase-3 gene expression.

This work aimed to investigate the inhibitory effect of regorafenib in combination with ginsenoside on the growth of HepG2 liver cancer cells. HepG2 liver cancer cells were divided into blank control group, regorafenib single-drug group, ginsenoside single-drug group, and regorafenib/ginsenoside combination group. Cells in the regorafenib single-drug group were treated with regorafenib at 0.25mg/L, 0.5mg/L, and 1mg/L, respectively, while cells in the ginsenoside single-drug group were treated with ginsenoside at 5.0mg/L, 10.0mg/L, and 20.0mg/L, respectively. HepG2 cell proliferation, expression of survivin mRNA, and the apoptotic effector caspase-3 in HepG2 liver cancer cells were assessed. An inhibitory effect on the growth of HepG2 liver cancer cells was observed for both the single-drug therapies and the combination therapy. The synergistic inhibitory effect presented by the combination therapy was dependent on the gradient concentration and treatment time. RT-qPCR results showed that both regorafenib and ginsenoside significantly reduced the expression of survivin mRNA in HepG2 liver cancer cells and the expression level of survivin mRNA in the regorafenib/ginsenoside combination group was much lower than those in the regorafenib single-drug group and ginsenoside single-drug group. The two drugs demonstrated synergistic inhibitory effect when used in combination. The findings in this study offered a theoretical insight into clinical use of regorafenib and ginsenoside for treatment of liver cancer.