Keratin 14 antisense oligonucleotide inhibits keratin 14 gene and protein expressions in human keratinocytes

Yuxin CHEN; Shuyun ZHENG; Wei LI; Yufeng LIU

ABSTRACT

BACKGROUND:

Study on antisense drug is still one of hotspots in the current field of biomedicine. Due to high-efficiency and specificity, antisense drug used for gene therapy has been paid more attention by many scholars.

OBJECTIVE:

To observe the effect of liposome-mediated keratin 14 (K14) antisense oligonucleotide on K14 gene and protein expressions as well as in vitro proliferation activities in human keratinocytes (KC).

DESIGN:

Single sample observation.

SETTING:

Department of Dermatology, Xijing Hospital, Fourth Military Medical University of Chinese PLA.MATERIALS Human KC, K14 oligonucleotide gene fragments (modified with phosphrothioate, and above sequence was synthesized by Shanghai Shenggong Bioengineering Company). Reverse transcriptase and TaqDNA polymerase were purchased from Invitrogen Company, K14 monoclonal antibody was purchased from Antibody Company, and SABC kit was purchased from Boster Company. EPICS-PRO-FILE Ⅱ flow cytometer was purchased from Coulter Company (USA).

METHODS:

Human epidermal KCs were primarily cultured, and their 3rd to 10th generations were used for the experiment.Artificially synthesized sense and antisense as well as mismatched K14 oligonucleotide gene fragments were introduced into KCs by means of liposome. Blank control group were set. The effects of antisense oligonucleotide on the cell cycle,K14 gene and protein expressions of KCs were detected by flow cytometer, reverse transcription polymerase chain reaction and SABC methods.MAIN OUTCOME

MEASURES:

Effect of oligonucleotide transfecting human KCs on the proliferation of KCs and K14 expression.

RESULTS:

[1]The electrophoresis of reverse transcription polymerase chain reaction products Specific K14 gene band appeared in each group, and K14 gene expression in the antisense group was significantly lower in the sense group,missense group and blank control group. K14/β-actin value was similar among sense group, missense group and blank control group (P ＞ 0.05), But K14/β-actin value was significantly lower in the antisense group than in the above-mentioned 3 groups (F =47.554, P ＜ 0.01). ②K14 protein expression detected by immunohistochemical

method:

K14 was expressed in all the cultured KCs at different levels, and was obviously reduced after antisense oligonucleotide being added. 20 μmol/L antisense oligonucleotide could markedly inhibit K14 expression; K14 expression did not change in the control group. ③ DNA level change detected by flow cytometer After being treated by K14 antisense oligonucleotide for 48 hours, human epidermal KCs were significantly increased at G1 stage (74.6%), and were markedly decreased at S stage (19.4%). Such changes were not found in the antisense group, missense group and blank control group.

CONCLUSION:

Antisense oligonucleotide can specifically inhibit K14 synthesis, and thereby, inhibit the proliferation of human KCs.