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Lentivirus‑delivered nemo‑like kinase small interfering RNA inhibits laryngeal cancer cell proliferation in vitro.

Tai J, Rao Y, Fang J, Huang Z, Yu Z, Chen X, Zhou W, Xiao X, Long T, Han Y, Liu Q, Li A, Ni X - Mol Med Rep (2015)

Bottom Line: The effects of NLK downregulation on Hep‑2 cell proliferation and cell cycle progression were analyzed using an MTT assay and flow cytometry, respectively.Downregulation of NLK also inhibited tumorigenesis and regulated the expression of cell cycle protein expression levels.Therefore, it was hypothesized that NLK is necessary for cell survival and tumorigenesis in laryngeal cancer cells.

View Article: PubMed Central - PubMed

Affiliation: Beijing Key Laboratory for Pediatric Diseases of Otolaryngology, Head and Neck Surgery, Beijing Pediatric Research Institute, Beijing Children's Hospital, Capital Medical University, Beijing 100045, P.R. China.

ABSTRACT
Laryngeal squamous cell carcinoma is the most common form of head and neck squamous cell carcinoma. Multiple approaches have been applied to treat this type of cancer; however, no significant improvement in survival rate has been achieved. In the present study, the role of nemo‑like kinase (NLK) in human laryngeal carcinoma Hep‑2 cells was investigated. NLK has been identified as an important regulator of cell growth, patterning and cell death in a variety of organisms. Lentivirus‑mediated‑shRNA was employed to silence endogenous NLK expression. Downregulation of the expression of NLK following lentivirus infection was confirmed using reverse transcription quantitative polymerase chain reaction and western blot analysis. The effects of NLK downregulation on Hep‑2 cell proliferation and cell cycle progression were analyzed using an MTT assay and flow cytometry, respectively. Downregulation of NLK also inhibited tumorigenesis and regulated the expression of cell cycle protein expression levels. Therefore, it was hypothesized that NLK is necessary for cell survival and tumorigenesis in laryngeal cancer cells. Furthermore, the absence of NLK may lead to cancer cell death. Collectively, the results of the present study demonstrated that the lentivirus‑mediated targeted disruption of NLK may be a promising therapeutic method for the treatment of laryngeal cancer.

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Lentiviral vectors for the NLK siRNA were constructed and demonstrated to be specific and potent for silencing NLK expression in Hep-2 cells. (A) Expression of GFP from uninfected Hep-2 cells (Con) and cells infected with the Lv-shNLK and Lv-shCon indicates the lentivirus infection efficiency in Hep-2 cells (magnification, ×10). (B) Quantitative polymerase chain reaction of NLK mRNA levels from uninfected Hep-2 cells (Con) and cells infected with the Lv-shNLK and Lv-shCon. (C) Western blot analysis of NLK protein expression in uninfected Hep-2 cells (Con) and cells infected with the Lv-shNLK and Lv-shCon. NLK, nemo-like kinase; GFP, green-fluorescent protein.
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f1-mmr-12-04-5619: Lentiviral vectors for the NLK siRNA were constructed and demonstrated to be specific and potent for silencing NLK expression in Hep-2 cells. (A) Expression of GFP from uninfected Hep-2 cells (Con) and cells infected with the Lv-shNLK and Lv-shCon indicates the lentivirus infection efficiency in Hep-2 cells (magnification, ×10). (B) Quantitative polymerase chain reaction of NLK mRNA levels from uninfected Hep-2 cells (Con) and cells infected with the Lv-shNLK and Lv-shCon. (C) Western blot analysis of NLK protein expression in uninfected Hep-2 cells (Con) and cells infected with the Lv-shNLK and Lv-shCon. NLK, nemo-like kinase; GFP, green-fluorescent protein.

Mentions: In order to knock down the NLK gene in Hep-2 cells, GFP containing lentivirus-mediated infection of NLK siRNA was performed. The successful infection was confirmed by evaluating the expression level of GFP. As shown in Fig. 1A, 5 days after NLK infection, >90% of cells expressed GFP, indicating a high infection efficiency. The mRNA expression level of NLK following infection with siRNA was evaluated by qPCR. Quantitative analysis of NLK mRNA level using densitometry revealed a significant decrease in Lv-shNLK (36.6%) compared with the control (Fig. 1B). In addition, western blot assays demonstrated that the protein expression of NLK in the two Hep-2 cell lines was significantly suppressed by NLK-specific siRNA expression (Fig. 1C). Collectively, these data suggested that lentivirus-mediated siRNA successfully infected Hep-2 cells and in addition, may be used to investigate NLK loss of function effects in Hep-2 cells.


Lentivirus‑delivered nemo‑like kinase small interfering RNA inhibits laryngeal cancer cell proliferation in vitro.

Tai J, Rao Y, Fang J, Huang Z, Yu Z, Chen X, Zhou W, Xiao X, Long T, Han Y, Liu Q, Li A, Ni X - Mol Med Rep (2015)

Lentiviral vectors for the NLK siRNA were constructed and demonstrated to be specific and potent for silencing NLK expression in Hep-2 cells. (A) Expression of GFP from uninfected Hep-2 cells (Con) and cells infected with the Lv-shNLK and Lv-shCon indicates the lentivirus infection efficiency in Hep-2 cells (magnification, ×10). (B) Quantitative polymerase chain reaction of NLK mRNA levels from uninfected Hep-2 cells (Con) and cells infected with the Lv-shNLK and Lv-shCon. (C) Western blot analysis of NLK protein expression in uninfected Hep-2 cells (Con) and cells infected with the Lv-shNLK and Lv-shCon. NLK, nemo-like kinase; GFP, green-fluorescent protein.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4581764&req=5

f1-mmr-12-04-5619: Lentiviral vectors for the NLK siRNA were constructed and demonstrated to be specific and potent for silencing NLK expression in Hep-2 cells. (A) Expression of GFP from uninfected Hep-2 cells (Con) and cells infected with the Lv-shNLK and Lv-shCon indicates the lentivirus infection efficiency in Hep-2 cells (magnification, ×10). (B) Quantitative polymerase chain reaction of NLK mRNA levels from uninfected Hep-2 cells (Con) and cells infected with the Lv-shNLK and Lv-shCon. (C) Western blot analysis of NLK protein expression in uninfected Hep-2 cells (Con) and cells infected with the Lv-shNLK and Lv-shCon. NLK, nemo-like kinase; GFP, green-fluorescent protein.
Mentions: In order to knock down the NLK gene in Hep-2 cells, GFP containing lentivirus-mediated infection of NLK siRNA was performed. The successful infection was confirmed by evaluating the expression level of GFP. As shown in Fig. 1A, 5 days after NLK infection, >90% of cells expressed GFP, indicating a high infection efficiency. The mRNA expression level of NLK following infection with siRNA was evaluated by qPCR. Quantitative analysis of NLK mRNA level using densitometry revealed a significant decrease in Lv-shNLK (36.6%) compared with the control (Fig. 1B). In addition, western blot assays demonstrated that the protein expression of NLK in the two Hep-2 cell lines was significantly suppressed by NLK-specific siRNA expression (Fig. 1C). Collectively, these data suggested that lentivirus-mediated siRNA successfully infected Hep-2 cells and in addition, may be used to investigate NLK loss of function effects in Hep-2 cells.

Bottom Line: The effects of NLK downregulation on Hep‑2 cell proliferation and cell cycle progression were analyzed using an MTT assay and flow cytometry, respectively.Downregulation of NLK also inhibited tumorigenesis and regulated the expression of cell cycle protein expression levels.Therefore, it was hypothesized that NLK is necessary for cell survival and tumorigenesis in laryngeal cancer cells.

View Article: PubMed Central - PubMed

Affiliation: Beijing Key Laboratory for Pediatric Diseases of Otolaryngology, Head and Neck Surgery, Beijing Pediatric Research Institute, Beijing Children's Hospital, Capital Medical University, Beijing 100045, P.R. China.

ABSTRACT
Laryngeal squamous cell carcinoma is the most common form of head and neck squamous cell carcinoma. Multiple approaches have been applied to treat this type of cancer; however, no significant improvement in survival rate has been achieved. In the present study, the role of nemo‑like kinase (NLK) in human laryngeal carcinoma Hep‑2 cells was investigated. NLK has been identified as an important regulator of cell growth, patterning and cell death in a variety of organisms. Lentivirus‑mediated‑shRNA was employed to silence endogenous NLK expression. Downregulation of the expression of NLK following lentivirus infection was confirmed using reverse transcription quantitative polymerase chain reaction and western blot analysis. The effects of NLK downregulation on Hep‑2 cell proliferation and cell cycle progression were analyzed using an MTT assay and flow cytometry, respectively. Downregulation of NLK also inhibited tumorigenesis and regulated the expression of cell cycle protein expression levels. Therefore, it was hypothesized that NLK is necessary for cell survival and tumorigenesis in laryngeal cancer cells. Furthermore, the absence of NLK may lead to cancer cell death. Collectively, the results of the present study demonstrated that the lentivirus‑mediated targeted disruption of NLK may be a promising therapeutic method for the treatment of laryngeal cancer.

Show MeSH
Related in: MedlinePlus