Molecular Cloning of Human Prostate Cancer Cell Line KLK7 Gene ...

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Tumor Research

[Abstract]

Prostate cancer is the most common malignancy in men .Both the morbidityand mortality rate from prostate cancer rise almost exponentially in recent years.Many researches showed that its tumorgenesis and biological characters are closelyrelated to many genes. Growing evidence suggests that many kallikreins areimplicated in it. Human kallikrein gene 7(KLK7; HSCCE), also named HSCCE/PRSS6, is a new member of the human kallikrein gene family, which was found inskin first, had drawn great attention and been undertaken wide studies in manymalignant tumors.Objective The aim of this study were : (1) to determine whether KLK7 gene isexpressed in prostate cell line PC-3. (2) to investigate whether DHT had theeffects on KLK7 mRNA level in PC-3 cell in vitroMethods (1) Total RNA was extracted from human prostate cell line PC-3 andhuman kallirein7(KLK7) gene cDNA was amplified by PCR after reverse-transcription using Oligo (dT) primer. Amplified products was ligated to PGEM-TEasy plasmid vector. The recombinant plasmid was transfered to the competentE.coli DH5?. Indentified by enzyme digestion, KLK7 gene in recombinant plasmidwas sequenced by sanger dideoxy-mediated chain-termination method. (2)Thehuman prostate cell line PC-3 were cultured at 37?in a CO2 incubator with F12culture medium and were divided into seven groups by different DHT stimulatingconcentrations of 0M?10-9M?10-8M?10-7M?10-6M?10-5M and 0.01% Ethanol.Following culture in day 1, day 2, day 3 and day 4, MTT method was applied toobserve the cell proliferation . RT-PCR was engaged to find the KLK7 level in thePC-3 cell under the different concentrations of DHT stimulation.Results (1)The cloned kallikrein7 gene is about 762 bp in length. A sequencecomparison of this with the reported kallikrein7 sequence in the Genebankindicated that the cloned prostate cell line PC-3 KLK7 gene has two nucleotidedifference. (2) In day 1 of culture, the cell treated with 10-8mol/l DHT grew fast,there was significant increase of OD in this group compared with the control group( P < 0.05). In day 4 of culture , there were significant increase of OD in thegroups treated with 10-9mol/l?10-8mol/l?10-7mol/l?10-6mol/l DHT compared withthe control group ( P < 0.05) except the group treated with 10-5mol/l DHT (P >0.05). (3) RT-PCR showed that the group treated with 10-9mol/L ?10-8mol/L?10-7mol/L?10-6mol/L DHT had no KLK7 mRNA expression. TheKLK7 mRNA OD value in control group and 10-5mol/L DHT group was 0.9061?0.9367 respectively compared with control gene .Conclusion Our study indicated: (1) KLK7 gene is expressed in prostate cellline. It may be provide the evidences for further studies of the KLK7 gene inprostate cancer. (2) Lower concentration of DHT can enhance the proliferation ofPC-3 cell. Its optimal concentration is 10-8mol/L. (3) the expression of KLK7mRNA may be regulated by androgens and has certain relations with prostatecancer cell growth. Further studies are required to prove this hypothesis.

Title: Molecular Cloning of Human Prostate Cancer Cell Line KLK7 Gene and the Effects of DHT on KLK7 mRNA Level in PC-3 Cell in Vitro

Category: Malignant Tumor

Filename: Molecular Cloning of Human Prostate Cancer Cell Line KLK7 Gene and the Effects of DHT on KLK7 mRNA Level in PC-3 Cell in Vitro.pdf

Pages: 128

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