| ObjectiveIn recent years,the incidence of prostate cancer is increasing rapidly in our country,which has become one of the most common malignant tumor in urinary system.There is no effective method to treat patients with androgen independent prostate cancer after endocrine therapy.ALKBH is a kind of DNA damage repair protein,the previous study found that the expression level of ALKBH is high in the prostate cancer,and is relation to the proliferation,apoptosis and invasion of tumor cells.RNA silencing technique was used to restrain the expression of ALKBH,then observe the changes of proliferation,apoptosis and invasive ability of prostate cancer cells,and the relationship between ALKBH gene expression level and prognosis was analyzed.Provide the experimental basis for the clinical application of ALKBH and the feasibility of gene therapy.MethodsThe expression level of ALKBH in prostate cancer cell line was screened by semi quantitative RT-PCR and Western blot,the interference sequence of ALKBH gene was designed through the online siRNA design software,and the secondary structure and homology of each interference sequence was analyzed by using BLAST software.After the ALKBH siRNA transcription template was synthesized and there recombinant plasmid vector was successfully constructed,named as pSIRENALKBH-shRNA-1,2,3.The recombinant plasmid was transfected into prostate cancer cells by Liposome,using semi quantitative RT-PCR and Western blotting to detect expression of ALKBH in prostate cancer cells.The recombinant plasmid with best silencing effect was selected for subsequent experiments.After he recombinant plasmid was transfected into prostate cancer cells,the cell cycle changes of prostate cancer cells was detected by flow cytometric,the changes of invasion ability was detected by Transwell chamber technique.At the same time,the semi quantitative RT-PCR and Western blotting were used to detect c-myc content in prostate cancer cells.To prepare the animal model of nude mouse xenograft tumor of prostate cancer,and to observe the expression of ALKBH in tumor tissues by semi quantitative RT-PCR,immunohistochemistry and Western blot.The expression of ALKBH in prostate cancer and benign prostatic hyperplasia was detected by immunohistochemical method,and the relationship between the expression level and clinical pathological characteristics and prognosis of patients were analyzed.ResultsThe relative expression levels of mRNA expression in three kinds of prostate cancer LNCaP cells were 0.412±0.05、0.189±0.01 与 0.134±0.02 respectively,and the difference was statistically significant(P<0.05).The relative expression of ALKBH protein in negative control group,empty plasmid group and pSIREN-ALKBHshRNA-2,3 recombinant plasmid group were 0.755±0.12、0.812±0.19、0.157±0.07、0.112±0.04.Compared with the negative control group and empty plasmid group,the difference was statistically significant(P<0.05),and the effect of the third recombinant plasmids was better than that of the negative control group(P<0.05).PSIREN-ALKBH-shRNA-3 recombinant plasmid was transfected into prostate cancer LNCaP cells,and the percentage of ALKBH in the prostate cancer LNCaP cells in the experimental group was significantly lower than that in the control group,which was statistically significant(P<0.05).The relative expression of ALKBH mRNA in prostate cancer LNCaP cells in the negative control group,empty plasmid group and experimental group were 0.103±0.018,0.101±0.012 and 0.092±0.015 respectively.There was no significant difference in the expression of c-Myc in the three groups of prostate cancer LNCaP cells(P>0.05).The relative expression levels of c-Myc protein in prostate cancer LNCaP cells in the negative control group,empty plasmid group and the experimental group were 0.775±0.09 、 0.796±0.12 and 0.275±0.05 respectively.The expression level of c-Myc protein in prostate cancer LNCaP cells was significantly decreased,and compared with the negative control group,the difference was statistically significant(P<0.05).The proliferation rate of prostate cancer LNCaP cells in the experimental group was significantly lower than that in the negative control group and empty plasmid group,the difference was statistically significant(P<0.05).In negative control group,empty plasmid group and experimental group,the percentage of G0/G1 phase cells in prostate cancer LNCaP cells were(71.53±1.13)% and(69.78 ±1.23)% and(31.04±1.01)% respectively.The difference was statistically significant(P<0.05).The percentage of S phase cells was(19.26±0.88)% and(20.54 ±0.67)% and(51.23±1.25)%,the S phase cells of the prostate cancer cells in the experimental group were the highest,the difference was statistically significant(P<0.05).The apoptosis rate of cells in the negative control group,the empty plasmid group and the experimental group were(2.21±1.65)% and(2.27±1.56)% and(6.28±1.56)respectively,the difference was statistically significant(P<0.05).The recombinant plasmid was injected into the tumor of nude mice,and the growth rate of the transplanted tumor in the experimental group was obviously slowed down,and the difference was statistically significant(P<0.05).The relative expression of ALKBH mRNA in the negative control group,empty plasmid group and the experimental group was 0.088±0.016,0.112±0.022 and 0.043±0.005 respectively.The relative expression of mRNA ALKBH in the experimental group was significantly lower than that in the negative control group(P<0.05).The relative expression of ALKBH protein in the negative control group,empty plasmid group and experimental group was 0.884±0.11,0.775±0.18 and 0.135±0.07 respectively.The relative expression of ALKBH protein in the experimental group was significantly lower than that in the negative control group(P<0.05).The positive expression rates of ALKBH protein in the negative control group,the empty plasmid group and the experimental group were 82.6%,79.8% and 45.1% respectively.The positive expression rate of ALKBH protein in the tumor tissues of the experimental group was the highest,and the difference was statistically significant(P<0.05)with the other two groups.The expression level of ALKBH protein was closely related to the prognosis of patients with prostate cancer.ConclusionThe recombinant RNAi plasmid of ALKBH was successfully constructed.The expression levels of ALKBH mRNA and protein in LNCaP cells was reduced,the cell cycle was arrested,the proliferation was inhibited,apoptosis increased,invasion ability decreased.The application of recombinant plasmid could inhibit the growth of transplanted tumor in nude mice,and the expression of ALKBH in tumor tissues was significantly decreased.The overall survival rate of patients with prostate cancer is negatively related to expression of ALKBH,which can be used as a target for gene therapy for prostate cancer,and has clinical application value. |
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