A Preliminary Study On The Function And Growth Regulation Mechanism Of HnRNPL For The PC3 Cell

Background and Objective:Prostate cancer is the most common cancer threat to men's health in developed countries and its morbidity ranks first in male cancer,accounting for 28%of all malignant tumors,its mortality just after lung cancer.There are approximately 899,000 new cases and about 258,000 dead cases of prostate cancer worldwide in 2008,72%of new cases and 53%of deaths occur in Europe,North America and other developed countries.The morbidity of prostate cancer in Asia is the lowest in the world,however,there was a marked upward trend in recent years.Although the morbidity of prostate cancer in china is lower than Western countries,a clear upward trend with the aging of the population,changes in lifestyle and improved health care measures,has become the main factors that affect men's health.Rising incidence of prostate cancer attracts more and more attention on its study.In 1941,Huggins first demonstrates that the removal of androgens can inhibit the growth of prostate by removing the testis,and it can produce dramatically subjective and objective results in the body of metastatic prostate cancer patients.The discovery opens a new chapter for the treatment of prostate cancer and it is also a major breakthrough for the treatment of hormone-dependent cancer.Since then endocrine therapy possesses a enduringly important state in the treatment of prostate cancer.With the concept of the emasculated hormone therapy being proposed,many of estrogen therapy for prostate cancer were reported in the 1950s.However,the lesion of almost all patients will gradually progress to androgen-independent prostate cancer or hormone-refractory prostate cancer in an average of 1.5 to 2 years time of endocrine therapy,which will not be able to rely on hormone therapy.For hormone-independent prostate cancer,there is no standard and effective treatment options,and it will ultimately lead to disease progression and treatment failure.So far,it is not entirely clear about the mechanism of the change of androgen-independent prostate cancer.In recent years,there has become an active field for the treatment of prostate cancer research both in basic and clinical research.Cancer is a class of multi-gene diseases,the integrity change of the cell's genome is the material basis of tumorigenesis.The control mechanism of cell cycle is the important assurance for the integrity of cellular genome,so if the monitoring mechanism was damaged,it would lead to instability cytogenetic,chromosomal rearrangements,such as gene deletions,amplifications,shift and so on,when mutated genes damage the driving mechanism of the cell cycle,the cells will occur uncontrolled growth,eventually leading to cancer.Regulation of cell division cycle is a complex biological process,which involves the biological effects of almost all the cancer genes and cancer suppressor genes.Many cancer genes and cancer suppressor genes directly involved in the regulation of cell cycle,or themselves are the main component of the complex.The results of these genetic variations lead to the uncontrolled cell cycle,cells without control will appear unlimited proliferation and then form tumors.Therefore,some scholars believe that cancer is a type of cell cycle diseases.Lentiviral vector as a class of vector derives from retrovirus has a good prospect development in Scientific research field with its advantages such as high transfection efficiency,the ability of infecting dividing and non-dividing cells and accommodating a large gene fragment,and so on.The mechanism of RNA interference is that inhibits gene expression by inhibiting the transcription or translation of specific genes.After importing the double-stranded RNA which is homologous with The coding region of the endogenous mRNA to a human cell,thedouble-stranded RNA first degrade to small interfering RNA and then with some protein syntheses RNA-induced silencing complex,leading tothe degradation of the target mRNA which results in silence of the gene expression.We can combine the advantages of them and then specifically inhibit the expression of genes in mammals by using lentiviral vector as a vector of RNAi technology,it also provide a powerful tool to study gene function and gene therapy.Therefore,the development of RNAi technology and lentiviral vector technology has provided a new way for the study of prostate cancer treatment in recent years.Tumorigenesis is a multistep process,many factors(including environmental factors and genetic factors under),led to the loss of the activation of oncogene and tumor suppressor gene function.In the malignant progression of tumor cells in the process,with the accumulation of mutations,resulting in an increased ability to escape the normal cell division regulation mechanism of apoptosis,decreased ability,invasion of surrounding tissues and distant metastasis.The mechanism of apoptosis and cell cycle regulation,transfer of very complex,known to have a large amount of protein molecules involved in these processes.Recently,Han Zhaodong applied two-dimensional electrophoresis and mass spectrometry method to identify 2,566 kinds of tumor protein,and revealed 60 different prostate cancer protein by using bioinformatics analysis,37 of these expression raised,while 23 expressed descended.Among it,14 genes and their protein product(ACLY,CAPG,GSTM3,GSTP1,HNRNPL,IMPDH2,KRT15,MCCC2,MSN,MYL9,PYGB,SERPINB5,TRAP1 and VCL)differentially expressed in prostate cancer.Heterogeneous nuclear protein(hnRNP L)is one of hnRNPs family members.String analysis showed that the hnRNP L and some of huRNPs family members are function partner.Pubgene analysis suggested that hnRNP L had involved in the process of apoptosis,cell death and growth biology.Our previous studies have also confirmed the HnRNP L is closely in relation to spermatogenic cell proliferation and apoptosis.HnRNP K,belonging to hnRNPs family,has been reported that has an osculating relationship with prostate cancer and participate in the process of proliferation,differentiation and apoptosis of prostate cancer.Our previous by tissue microarray(including 81 patients with benign prostatic hyperplasia and 99 cases of prostate cancer)by immunohistochemistry,results show that,HnRNP L is high expression in prostate cancer,consistent with the results reported in theliterature.Therefore,the design of our topic ideas:HnRNP L lentivirus infection in prostate cancer cells,established prostate cancer cell line stably with low expression of HnRNP L protein.On this basis,we hope we can reveal prostate cancer cell signaling pathway which HnRNP L protein may be participated in,and conduct a vivo study on the growth regulation mechanism of HnRNPL for the PC3 cell,present new prostate cancer progression mechanisms involved in HnRNP L and provide new targets for prostate cancer treatment.Methods:1.Lentivirus transfect PC3 cells and using Western Blot to check its transfection efficiency.The experiment was divided into group 1(negative control group:PC3 cells + lentivirus negative control),group 2(interference group:PC3 cells+HnRNP L lentivirus).Observing the morphology,growth status and transfection efficiency of PC3 cells under a fluorescence microscope 72hours later after lentivirustransfection;and using Western Blot to check the expression of HnRNP L protein between the two groups to check its transfection efficiency.2.Using Western Blot and RT-PCR to check the change of the gene expression of Bcl-2,caspase-3,caspase-9,iNOS,CEACAM1 between the negative control group and the interference group.3.Respectively injecting the cells of the interference and the negative control group into the subcutaneousness of five nude mice to build prostate cancer xenograft and then regularly observing the conditions of growth in nude mice and tumor formation in nude mice.Tumors were allowed to develop for up to 4 weeks,at which point mice were sacrificed and tumors were extracted for paraffin sections and HE staining and then observing pathological structure of the tumor under the microscope.Results:1.We observed that the morphology and the growth condition of epithelial cells are good under the white light of a fluorescence microscope 72 hours later after lentivirus transfection.In the excitation light,some PC3 cells can emit green fluorescence which uniformly distribute around the cells and high lentiviral transfection efficiency.The results of Western Blot showed the expression of HnRNP L protein of the interference group PC3 cells was successfully reduced,we had successfully established a stable low expression of HnRNP L protein in prostate cancer cells which lay a solid foundation for the subsequent cell experiments.2.Western Blot showed that there was no significant difference about the expression of Bcl-2 after reducing the expression of HnRNP L in PC3 cells,but the expression of activated caspase-3 and activated caspase-9 was increased;The results of RT-PCR showed that the expression of Bcl-2mRNA in PC3 cells had no significant change(t = 0.063,P = 0.956)after the down regulation of HnRNP L,but the expression of iNOSmRNA was significantly lower(t = 27.152,P = 0.001)as well as the expression of CEACAM1mRNA(t = 127.891,P = 0.000).3.All of the nude mice of negative control group grew tumors four weeks later after respectively injecting the cells of the interference and the negative control group into the subcutaneousness of five nude mice,and then the nude mice of negative control group were sacrificed and tumors were extracted.None of the nude mice of interference group grew tumors in 4 weeks.The results of HE staining of the tumors in Negative control group showed that the tumor cells were randomly distributed,the cell volume increased significantly,typical nuclear large pulp less,more pathological mitotic phenomenon and no glandular structures exist.Conclusion:1.We successfully established a stable,efficient and uniformly low expression of HnRNP L protein in prostate cancer cells by using the method of lentiviral transfection which lay the foundation for the subsequent experiments.2.The down regulation of HnRNP L expression in PC3 cells can promote apoptosis,which may be related to the pathway of caspase-3,caspase-9,iNOS and CEACAM1.3.The down regulation of HnRNP L expression may inhibit the formation of prostate cancer.