The Function Of HnRNP L In Prostate Cancer

Background and Objective:Prostate cancer currently is a common malignant tumor in men, the morbidity of which accounts for the second of all malignant tumors in western men and the mortality of the first. While in the United States, the morbidity of prostate cancer is the first of all the tumor and the mortality is second next to lung cancer in men. 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.Huggins and Hodges firstly found that prostate cancer is hormone-dependent and presented androgen deprivation therapy(ADT), which has achieved remarkable clinical efficacy. But the vast majority of prostate cancer had developed the growth of androgen-independent on about18mouths after androgen deprivation. There is still a lack of standard effective treatment options for androgen-independent prostate cancer(AIPC) and ultimately lead to disease progression, treatment failure. So far, it is not fully clear that protate cancer why ADT has changed to AIPC.We currently believe that tumor is cell cycle disorder and (or) apoptosis mechanism disorder, so cell cycle and apoptosis are two major research focus of malignancy tumor. In our previous studies we have found that the cell cycle had significant effect on apoptosis-related genes. With the breakthrough of the cell cycle research in recent years, we recognize tumor is a kind of cell cycle diseases. The disorder of cell cycle regulation and apoptosis, uncontrolled cell proliferation is the most remarkable characteristic of the tumor tissue. Almost all the biological characteristics of the tumor cells are uncontrolled growth leaded by cell cycle disorder. Many oncogenes, tumor-suppressor genes directly involve in cell cycle regulation, or itself is the main component of the cell cycle regulatory mechanisms, their mutations lead to the abnormality of cell cycle regulation, including the start of the cell cycle, operation and termination, so that the cells obtain the uncontrolled growth characteristics which treat excessive proliferation and deficient apoptosis as the main form.We have known Bcl-2family is an important part of cell apoptosis activate and regulatory mechanism. Bcl-2and Bax are the major members of Bcl-2family. Bcl-2is an important anti-apoptotic gene which is closely related to prostate cancer development and transition from androgen-dependent to androgen-independent process. Caspase-3, belongs to caspase family, is a key kinase which activates the apoptotic process, but also the main effectors of apoptosis. Marcelli found the cystine protease family members Caspase-3activation is in relation to PCa cell lines LNCaP apoptosis and cystine inhibitors can eliminate its induced, suggesting that the Caspase family with PCa cell apoptosis are closely related.RNA interference (RNAi) is using a small RNA (including small interfering RNA that siRNA) sequences to inhibit specific gene signal process.Until now, we has not been found endogenous siRNA expression in mammals. If we guide the synthetic siRNA having a particular sequence into tumor cells, there will cause a mRNA degradation having homologous sequences, consequently effectively inhibit the expression of the gene and effect the biological behavior of the tumor. In recent years, the development of RNAi technologies provides a new idea for the treatment of prostate cancer research. Recently, Zhaodong applied two-dimensional electrophoresis and mass spectrometry method to identify2,566kinds of tumor protein, and revealed60different prostate cancer protein by using bioinformatics analysis,37of these expression raised, while23expressed descended. Among it,14genes and their protein product (ACLY, CAPG, GSTM3, GSTP1, HNRNPL, IMPDH2, KRT15, MCCC2, MSN, MYL9, PYGB, SERPINB5, TRAP1and 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 hnRNPs 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. However, whether is it a closely relationship between hnRNP L and the occurrence and development of prostate cancer? At present, there isn’t reported in the literature. Therefore, the design of our topic ideas:we will detect HnRNP L protein expression in normal prostate tissue and prostate cancer, through immunohistochemistry and Western Blot and observe the alteration of biological behavior of prostate cancer cells(such as proliferation, invasion, migration, and apoptosis) after silencing (or expressing) HnRNP L. On this basis, we hope we can reveal prostate cancer cell movement signaling pathway which HnRNP L protein may be participated in, present new prostate cancer progression mechanisms involved in HnRNP L and provide new targets for prostate cancer treatment.Methods:1. Take a tissue microarray, including81patients with benign prostatic hyperplasia and99cases of prostate cancer (2cases of Gleason score2-4,24cases of Gleason score5-6,73cases of Gleason score7-10). According immunohistochemistry kit and antibody concentration requirements,we conducted immunohistochemistry to detect the HnRNP L expression and statistical analyse the positive rate and the correlation with the degree of tumor differentiation, tumor grade and clinical stage. By cultivating normal prostate epithelial cells (RWPE-1cells) and hormone-independent prostate cancer (PC3cells) and hormone-dependent prostate cancer cells (lncap cells), we extracted the total protein of various cells and detect the expression of HnRNP L. in three cell though Western blot.2. We designed and chemically synthesized three siRNA and two negative control sequence match with human HnRNP L gene mRNA sequence and detected the change of PC3cells HnRNP L protein expression levels in48hours after siRNA transfection by using western-blotting.3. The experiment was divided into group1(RWPE-1group, RWPE-1+of HnRNP L-siRNA group), group2(Lncap group Lncap+of HnRNP L-siRNA group), group3(PC3, PC3+HnRNP L-siRNA group). It had been proven effective to use encapsulated liposomes when HnRNP L-siRNA transfected the three cells. Using CCK-8method measured the growth and proliferation situation of each group cells; using scratch test observed cell migration and surveyed cell invasiveness through the Transwell.4. In Lncap cells and PC3cells,we detected the alteration of Bcl-2, caspase-3expression in the control group and the treatment group in48hours after silence HnRNP L by effective siRNA which has been comfirmed.Results:1. Immunohistochemical results showed that positive expression rate of HnRNP L is, respectively,29.7%(24/81)and84.9%(84/99)(P<0.01) in BPH and PCa; Positive expression rate was61.53%(16/26) in Gleason score2-6group and was93.15%(68/73)(P<0.05) in Gleason score7-10group; Clinical (stage A+B) positive expression rate was83.87%(78/93) and clinical (stage C+D) positive expression rate was100%(6/6)(p>0.05). HnRNP L expression was positively correlated with pathological grade of prostate cancer, and was unconcerned with clinical stage. We found HnRNP L protein superlatively express in PC3cells and followed by lncap cells, minimally express in normal prostate epithelial cells (RWPE-1cells) by Western Blot analysis.2. We detected HnRNP L protein level in48hours after the three sequences HnRNP L-siRNA transfection by using western-blotting. Results show that the interference effect of sequence3is the best, the protein levels decreased by more than70%, to interfere with the effect of subsequent experiments. We had achieved the effect of subsequent interference experiment.3. RWPE-1cell proliferation ability decreased significantly compared with control group after instantaneous down-regulation HnRNP L expression, the difference was statistically significant (F=315.064, P=0.000). The different time points significantly impacted RWPE-1cell proliferation (F=431.920, P=0.000). The interaction effect was significant between each time point group and the treatment group. A significant difference (P<0.01) can be seen compared with the migration force of control group cells by scratch experiments. The Transwell showed a significant difference (P<0.01) compared with the invasiveness of control group cells. Lncap cell proliferation decreased compared with the control group, the difference was statistically significant (F=568.718, P=0.000), The different time points significantly impacted Lncap cell proliferation (F=1543.436, P=0.000); The interaction effect was significant between each time point group and the treatment group (F=51.226, P=0.000), the scratch test can be seen with the control group cells of migration force difference (P<0.01), There is significant differences of the invasion force with the control group cells by the Transwell(P <0.01); Compared with the control group, PC3cell proliferation significantly decreased and the difference was statistically significant (F=668.116, P=0.000); The different time points significantly impacted PC3cell proliferation (F=2871.830, P=0.000); The interaction effectwas significant between each time point group and the treatment group (F=123.353, P=0.000), A significant difference (P<0.01) can be seen compared with the migration force of control group cells by scratch experiments. The Transwell showed a significant difference (P<0.01) compared with the invasiveness of control group cells.4. The expression level of Bcl-2in Lncap cells and PC3cells decreased after downing HnRNP L expression,but caspase-3expression increased.Conclusion:1. HnRNP L protein expression is different in normal prostate and prostate cancer, which is highly expressed in the tumor and HnRNP L expression positively correlated with pathological grade of prostate cancer, and HnRNP L expression was positively correlated with pathological.grade of prostate cancer and was unconcerned with clinical stage.2. The protein decreased effect of interference sequence1in three interference sequence is the best in48hours after interference. So we determined to carry out follow-up study with No.1HnRNP L-siRNA.3. It inhibits growth of RWPE-1cells, Lncap cells and PC3cells tosilence HnRNP Lgene; It impairs the migration and invasiveness of RWPE-1cells, Lncap cells and PC3cell to silence HnRNP L gene.4. The apoptosis of prostate cancer cell is promoted and tumor cell growth is inhibited after down-regulating HnRNP L protein.