Yuanyuan Zhu

Knockdown of TSPAN1 by RNA silencing and antisense technique inhibits proliferation and infiltration of human skin squamous carcinoma cells

Aim To explore the function of TSPAN1 in squamous cell skin carcinoma by means of TSPAN1-specific siRNA and antisense oligonucleotide techniques. Methods pU6H1-GFP-siRNA TSPAN1 and pcDNA3.1 antisense TSPAN1 were constructed and transfected into squamous cell skin carcinoma cell line A431 cells to knock down TSPAN1 gene expression. The levels of TSPAN1 mRNA and protein expression were detected by semiquantitive RT-PCR and Western blot, respectively. The proliferation rates of A431 cells were determined by MTT assay and flow cytometry. Lastly, the migration and infiltration of A431 cells were determined by the Transwell migration assay. Results Transfection with either pU6H1-GFP-siRNA TSPAN1 or pcDNA3.1 antisense TSPAN1 led to an obvious reduction of expression levels of TSPAN1 mRNA and protein in A431 cells, respectively. The proliferation, migration and infiltration of A431 cancer cells were significantly inhibited at 48 hours after transfection of plasmids harboring TSPAN1 siRNA and antisense RNA. Conclusion The TSPAN1 gene might play a role in the proliferation of squamous cell carcinoma of the skin and be associated with cancer cell motility, implying a function of the gene in the development of skin cancer.

Inhibition of hepatocellular carcinoma growth and angiogenesis by dual silencing of NET-1 and VEGF

Simultaneous silencing of multiple up-regulated genes is an attractive and viable strategy to treat many incurable diseases including cancer. Herein we used dual gene targeted siRNA (DGT siRNA) conjugate composed of NET-1 and VEGF siRNA sequences in the same backbone could inhibit growth and angiogenesis HCC. DGT siRNA showed a further down regulation on VEGF mRNA and protein levels compared with NET-1 siRNA or VEGF siRNA, but not on NET-1 expression. It also exhibited greater suppression on proliferation and trigger of apoptosis in HepG2 cells than NET-1 siRNA or VEGF siRNA; this could be explained by the significant down regulation of cyclin D1 and Bcl-2. A lower level of ANG2 mRNA and protein was detected in HUVEC cultured with supernatant of HepG2 cells treated with DGT siRNA than that of VEGF siRNA or NET-1 siRNA, resulting in much more inhibited angiogenesis of HUVEC. Tumor growth was inhibited and microvessel density dropped in the xenograft tumor models compared to the untreated controls. NET-1 and VEGF silencing play a key role in inhibiting hepatocellular cell proliferation, promoting apoptosis, and reducing angiogenesis. Simultaneous silencing of NET-1 and VEGF using DGT siRNA construct may provide an advantageous alternative in development of therapeutics for Hepatocellular carcinoma.

Inhibitory effect of dsRNA TLR3 agonist in a rat hepatocellular carcinoma model

Hepatocellular carcinoma (HCC) is one of the most common types of malignant tumor. Studies have demonstrated that the toll‑like receptor 3 (TLR3)/interferon pathway is inhibitory in cancer cell proliferation, suggesting that the activation of this pathway may have therapeutic potential. In the present study, the inhibitory effects of BM‑06, a double‑stranded (ds)RNA TLR3 agonist, against HCC were studied in vivo. Using a 2‑acetylaminofluorene-induced HCC rat model, histological examination and analysis of corresponding biomarkers following treatment with BM-06, showed a decrease in tumor growth and cell proliferation, and an increase in apoptosis compared with that in a phosphate‑buffered saline control group. In addition, the observed antitumor effect of BM‑06 in the HCC rat model was demonstrated to be superior to the known TLR3 agonist, polyinosinic-polycytidylic acid.

TLR3 dsRNA agonist inhibits growth and invasion of HepG2.2.15 HCC cells.

Toll-like receptor 3 (TLR3) is a pattern-recognizing receptor that is involved in immune signaling and plays a crucial role in survival by being able to recognize various viral components including double-stranded RNA (dsRNA). TLR3 expression and function in cancer cells are not well understood. In this study, we investigated whether TLR3 agonist dsRNA (BM-06) can inhibit proliferation and invasion, and promote apoptosis in HepG2.2.15 cells. HepG2.2.15 cells secreting hepatitis B virus (HBV) were treated with BM-06 and poly(I:C). Western blot analysis and PCR were employed to determine pharmacodynamic changes in biomarkers relevant to TLR3 signaling. Cell proliferation, invasion and apoptosis were analyzed by CCK-8 assay, transwell assay and flow cytometry. The expression of HBsAg, and HBcAg was observed by immunohistochemistry. Compared with untreated cells, pharmacological NF-κB activity of the TLR3 pathway by BM-06 (1.734-fold) or poly(I:C) (1.377-fold) was induced. By western blot analysis, we found that dsRNA induced TLR3-activated HepG2.2.15 cells which expressed NF-κB levels predominantly in the cytoplasmic fraction but fewer signals in the nucleus. BM-06 inhibited the proliferation, invasion and secretion of HBV, and induced apoptosis in HepG2.2.15 cells. In addition, the antitumor effects of BM-06 were superior to poly(I:C). Pharmacological activation of the TLR3 pathway by BM-06 can inhibit HepG2.2.15 cell growth.

A synthetic dsRNA, as a TLR3 pathwaysynergist, combined with sorafenib suppresses HCC in vitro and in vivo

BackgroundRecent studies have demonstrated that synthetic dsRNAs may produce therapeutic effects in a target-independent manner through stimulation of the toll-like receptor-3 (TLR3)/interferon pathway; as a result, angiogenesis and proliferation of tumor cells are inhibited. Thus, this pathway may become a potential target of dsRNA in tumor suppression. In this study, we evaluated the role of synthetic dsRNA as a TLR3 synergist and by combining with sorafenib in anti-hepatocellular carcinoma (HCC) in vitro and in vivo.MethodsFour dsRNAs were designed and synthesized. One of them that was capable of activating TLR3 most effectively in human HCC cell line (HepG2.2.15) was selected as a TLR3 synergist (called BM-06). Subsequently, the expression of proteins relating to TLR3 signaling pathway, such as NF-κB, caspase 8 survivin, bcl-2 and PCNA affected by BM-06, sorafenib alone or in combination, was compared. The migration, proliferation and apoptosis of HepG2.2.15 cells were evaluated in presence of BM-06, sorafenib alone or in combination of both. The similar treatments were also applied in an SD rat primary HCC model.ResultsqRT-PCR data showed that the expression of TLR3 and NF-κB in HepG2.2.15 cells was enhanced. BM-06 was selected as a TLR3 synergist capable of activating the TLR3/interferon pathway most effective among 4 synthetic dsRNAs. The migration and proliferation were significantly inhibited in treated HepG2.2.15 cells with BM-06 or Sorafenib alone as compared with PBS-sham control (P < 0.01). However, the role of combination BM-06 with Sorafenib was the most prominent. Tumor cell apoptotic rate was increased by BM-06 or combination when compared to PBS or poly(I:C) (P < 0.05). Similarly, in orthotopic HCC SD rats, the effect of the combination was superior to either agent alone on the inhibition of tumor growth and induction of HCC cell apoptosis (P < 0.05).ConclusionsdsRNA alone was capable of inhibiting the proliferation of HepG2.2.15 cells and tumor growth of orthotopic HCC SD rats, but the effect of combination of dsRNA with sorafenib was more prominent. These findings implicate the potential role of combined use of a dsRNA, a TLR3 synergist, and sorafenib in inhibition of HCC.

The effect of NET-1 on the proliferation, migration and endocytosis of the SMMC-7721 HCC cell line.

To explore the effect of NET-1 on the proliferation, migration and endocytosis in the hepatocellular carcinoma (HCC) cell line SMMC-7721, we constructed the pU6H1-NET-1-siRNA (NET-1siRNA) and pcDNA3.1/myc-NET-1 (myc-NET-1) vectors and transfected them into SMMC-7721 cells. The expression levels of NET-1 mRNA and protein were detected using real-time quantitative RT-QPCR and western blotting. The proliferation rates of SMMC-7721 cells were determined by CCK-8 assays, flow cytometry (FCM) and immunohistochemistry staining. The migration in two or three dimensional space of SMMC-7721 cells were determined by wound-healing assay and in vitro invasion assay. The extent of endocytosis in SMMC-7721 cells was estimated by observing the amount of transferrin (Tfn) absorbed with capture ELISA assays, and Tfn endocytosis was observed under confocal immunofluorescence microscopy. The results show that: i) after transfecting NET-1 siRNA, the expression of NET-1 mRNA and protein in SMMC-7721 cells decreased significantly, the growth of cells was suppressed, which induced cell cycle arrest, the proliferation rates were dramatically reduced and the expression of Ki67 declined, and migration and endocytosis in cells were inhibited, compared with untreated cells (every P<0.01); ii) Following transfection with myc-NET-1, the expression of NET-1 mRNA and protein in SMMC-7721 cells increased, and both the proliferation of cells and the cell cycle were promoted (P<0.01, respectively). However, the abilities of cell migration and endocytosis were not affected compared with untreated cells. These data suggest that: i) the NET-1 gene may play an important role in proliferation, migration and endocytosis of cells; ii) siRNA technology may efficiently suppress the expression and function of NET-1 in HCC, suggesting that NET-1 may be a therapeutic target for HCC.

Application of the CellDetect® staining technique in diagnosis of human cervical cancer

BACKGROUND CellDetect® staining technique is a newly invented technique for cancer diagnosis. It easily distinguishes between normal and neoplastic cells including pre-cancer and squamous cell carcinoma (SCC) cells, based on staining color and morphology. In this study, application of CellDetect® staining technique was assessed in diagnosis of human cervical cancer as compared with hematoxylin and eosin (H&E) staining in conventional slides and Thinprep cytologic test (TCT) smears. METHODS The conventional slides and TCT smears of 600 patients were stained and observed while comparing with H&E staining to assess sensitivity and specificity of CellDetect® staining technique in diagnosis of cervical cancer. Conventional smear slides (440 cases) were fixed in 95% ethanol or with CYTOFIX® Spray. TCT smears (160 cases) were processed based on manual. The paraffin sections from cervical intraepithelium neoplasia (CIN) 2-3 and SCC cases were prepared by biopsy. RESULTS CellDetect® staining exhibited well cell morphology, simultaneously, showed dual color discrimination, the stain targeted cytoplasm in normal cells in green and dysplastic cells or neoplastic cells in purple/red. Both cervical cell smears or both fixation methods in conventional slides did not affect CellDetect® staining diagnosis, especially in tissue biopsies CellDetect® staining exhibited well epithelium layers to benefit the diagnosis of CIN grade. The sensitivity and specificity of CellDetect® staining technology in diagnosing CIN and SCC were 94.34% and 88.73%, respectively. CONCLUSIONS CellDetect® staining technique provided a dual color discrimination and morphological analysis. It has the potential to become one of the most effective methods for cervical screening and early diagnosis.

Expression and function of tetraspanin 1 in esophageal carcinoma

The present study explored the expression of tetraspanin 1 (TSPAN1) in esophageal carcinoma (EC) and its association with clinicopathological factors. TSPAN1 small interfering RNA (siRNA) was designed to target the TSPAN1 gene in Eca-109 cells in order to explore the biological function of TSPAN1 in the proliferation and apoptosis of EC. The results demonstrated that the level of TSPAN1 expression in EC tissue was significantly increased compared with that in adjacent normal tissue (P<0.001). TSPAN1 expression was also associated with histological differentiation, depth of invasion, lymph node metastasis (all P<0.05) and Ki-67 (P<0.01). However, no association was observed between TSPAN expression and gender, age or location (P>0.05). In addition, silencing TSPAN1 markedly inhibited proliferation while increasing the apoptosis rate of Eca-109 cells, which was demonstrated by detecting the expression of the cell proliferation-associated gene Ki-67 and the apoptotic gene caspase-3 (P<0.05). Taken together, these results indicated that TSPAN1 functions as a tumor-associated gene in EC through promoting cell proliferation and suppressing apoptosis, and siRNA technology may provide an advanced alternative in the development of therapeutics for EC.