Study Of HnRNP L In Regulation The Cell Proliferation And Apoptosis Of Prostate Cancer And Its Potential Mechanism

Background:Prostate cancer is a common cancer which threatens men’s health, its incidence and mortality has increased year by year. At present, according to reports, the incidence of prostate cancer is the second in all malignant tumors of men worldwide,and the mortality ranks fifth.Although the morbidity and mortality of prostate cancer in China is less than other developed countries, but in recent years, along with the progress of the society, population aging, people’s eating habits and health conditions,there has been a serious threat to the health of men in our country. As China is a country with a vast territory, there are many places in where medical environment is not ideal,so more and more advanced prostate cancer are found at present.With the continuous improvement of the doctors’understanding of prostate cancer in our country,and also the detecting means such as PSA, B-ultrasound, and constantly publicity, the public’s increasing awareness of the extent of health knowledge, more and more relatively early prostate cancer patients are found, but the effect is still unsatisfactory exist in clinical treatment. At present, the pathogenesis of prostate cancer is not very clear. Therefore, the pathogenesis of prostate cancer needs in-depth study through which we can find a better specificity and sensitivity prostate cancer molecular marker and therapeutic target,which has a very important value in scientific research for the clinical diagnosis and treatment of the prostate cancer.Recently, Zhaodong Han 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,1MPDH2, KRT15, MCCC2, MSN, MYL9, PYGB, SERPINB5, TRAP1 and VCL) differentially expressed in prostate cancer.As a member of the hnRNP family, HnRNP L is located in 19ql3.2, a total length of 1759 bp, contains 17 exons. Studies have reported that HnRNP family members play an important role in the injury and repair, transcription, hnRNA splicing, mRNA transport degradation in biological process in DNA.HnRNPL is firstly reported by Pinol-Romas in the early transcription fragment whom accidentally discovered the ribonucleoprotein, this protein rich in cysteine and proline, is known related with the development of lung cancer, liver cancer, colorectal cancer and other tumors, and in our early research we also found it can affect the proliferation and apoptosis of germ cells. There has been reported that HnRNP K has a close relationship with the regulation of proliferation and apoptosis of prostate cancer, but the relationship between HnRNP L and prostate cancer is not clear.This study firstly explore the relationship between the expression of HnRNP L in patients with prostate cancer and the clinical indicators, further in vivo and in vitro study to confirm the effect of HnRNP L on prostate cancer proliferation and apoptosis and then to reveal its possible molecular mechanism, clarifying what a role HnRNP L may play in the development of prostate cancer, and providing a new molecular tumor marker and therapeutic target for clinical diagnosis and treatment of prostate cancer.Object:1 To explore the relationship between clinical indexes of patients with prostate cancer and the expression of HnRNP L;2 To elucidate the relationship of HnRNP L and proliferation and apoptosis of the prostate cancer cells through the functional experiments in vitro and in vivo;3 Through a series of molecular biology experiments finally reveal the molecular mechanism of HnRNP L may play in prostate cancer cell proliferation and apoptosis.Materials and methods:1 The expression of HnRNP L in prostate cancer and its relationship with clinicopathological parameters.In Xi’an Alena Biotechnology Co., Ltd purchased a contains 81 cases of prostatic hyperplasia samples and 99 cases of prostate cancer samples of tissue microarray, by immunohistochemical S-P method was used to detect the various samples of HnRNP L expression, and combined with the clinical pathological parameters of each sample, with related system design methodology for association analysis; in urinary surgery, Nanfang Hospital, collected 10 on prostate cancer and paracancerous tissues, the expression of HnRNP L tissue grinding extraction protein were detected by WB to 10 paired tissues, and the related statistical analysis.2 Respectively knockdown and overexpression of HnRNP L in prostate cell lines and to study its effect on proliferation and apoptosis of prostate cancer cells.Changes in postoperative treatment, first by Q-PCR and WB detection of HnRNP L in three strains of prostate cancer cell line PC3 and DU145 and LNCaP) expression and screening of HnRNP L expression higher and lower cell lines of high expression cell line (DU145) lentiviral knockdown of expression than low cell line (LNCaP) lentiviral expression processing, and for the intermediate expression cell line (PC3) both knockdown expression processing; further by CCK-8 experiment, Transwell invasion assay, plate clone formation assay and nude mice subcutaneous tumor experiments and flow cytometry detection of HnRNP L knockdown or over expression before and after prostate cancer cell line proliferation and apoptosis.3 The molecular mechanism of HnRNP L in regulating the proliferation and apoptosis of prostate cancer cellsBy immunofluorescence and co-ip, RIP and pull-down experiment method for detection of HnRNP L and cell proliferation and apoptosis pathway may be interacting protein or gene further by WB detection of HnRNP L knockdown and expression before and after proliferation and apoptosis pathway in the protein levels of change, and finally reveal the regulatory mechanism of HnRNP L in prostate cancer cell proliferation, apoptosis.Results:1 The expression of HnRNP L in prostate cancer and its relationship with clinicopathological parameters.The results of immunohistochemistry assay show that in 81 cases of benign prostatic hyperplasia (BPH) positive expression of HnRNP L rate is 29.63%(n= 24), and in 99 cases of prostate cancer tissue the positive expression of HnRNP L rate was 84.8%(n= 84), and the difference is statistically significant (P< 0.05); further to 99 cases of prostate cancer samples for clinical staging and pathological grading and the staging and grading of HnRNP L expression of sample number were statistically analyzed. The results showed that HnRNP L in prostate cancer expression and clinical stage, pathological grade showed positive correlation relationship (P< 0.05).At the same time, Western blot suggests that the expression level of HnRNP L in prostate cancer is more than that in adjacent tissues.2 Respectively knockdown and overexpression of HnRNP L in prostate cell lines and to study its effect on proliferation and apoptosis of prostate cancer cells.2.1 Build a stable transfected cell line model using lentiviral technologyQ-PCR and Western blot shows that the expression level of HnRNP L in DU145 cell lines was the highest, HnRNP L expression level in LNCaP cell lines was the lowest, and HnRNP L expression level in PC3 cell lines was the middle, so in subsequent experiments on DU145 cell line only HnRNP L stable knockdown of treatment of LNCaP cell lines only HnRNP L stable expression, and the PC3 cell lines both knockdown of HnRNP L, were also overexpression of HnRNP L processing; at 48 h after transfection, by Western blot (WB) was used to detect the cell line knockdown and over expression of efficiency, selection and construction of a successful stable cell line model is used for subsequent experiments.2.2 To test the change of prostate cancer cell proliferation ability after HnRNP L knockdown.The changes of DU145 cells and PC3 cells proliferation ability and the growth curve drawing after HnRNP L knockdown by CCK-8 cell activity test. Statistical analysis showed that:knockdown group than in the control group, the proliferation ability of DU145 and PC3 cells decreased, the difference was statistically significant (p<0.05), the proliferation of different time points of DU145 and PC3 cells were significantly affected (p<0.05), each time point group and treatment group interaction effect (p<0.05); plate clone formation test results showed that the cloned knockdown group and control group in DU145 cell lines were cloned and the formation rate, knockdown group and control group in PC3 cell line formation rates were respectively analyzed by the t test, two independent samples found the ability to clone knockdown group and control group DU145 and PC3 cell lines formed by a statistically significant difference (p<0.05); flow cytometry cell cycle test results show that:the knockdown group and the control group of DU145 cells in G1 phase cells percentage were 53.62% and 33.14%.A percentage of the number of cells is 30.37% and 55.01% respectively, and PC3 cell lines knock low group and control group of cells in G1 phase percentage are 40.13% and 81.09%. The percentage of cells in s stage respectively is 14.03% and 28.37%, respectively, using two independent sample t-test analysis found in DU145 and PC3 cell lines knock low group and control group of cell number percentage and the difference is statistically significant, results suggest that knock on the expression level of low hnRNP L, prostate cancer cell line proliferation was inhibited, because the cell cycle arrest in the G1 phase.2.3 To test the change of prostate cancer cell proliferation ability after HnRNP L over-expression.Similarly, the CCK-8 results suggest that compared with the control group,over-expression group enhanced the proliferation ability of Lncap cells and PC3 cells, the difference was statistically significant (p<0.05), the proliferation of different time points of Lncap and PC3 cells were significantly affected (p<0.05), each time point group and treatment group interaction effect (p<0.05); clone formation test showed that Lncap cell lines overexpressing clone group and the control group formation rate is respectively, and PC3 cell lines overexpressing clone group and the control group formation rate is respectively, were analyzed by the t test of two independent samples after the Lncap and PC3 cell lines overexpressing ability the clone formation group and control group have significant statistical difference (p<0.05); flow cytometry cell cycle test results show that:the group and control group G1 cells overexpressing Lncap cells percentage were% and%, the percentage of cells in s stage are% and%, and PC3 cell lines knock low group and control group of G1 phase cell percentage were%and%, the percentage of cells in s stage were% and%, respectively, using two independent sample t-test analysis found in LNCaP and PC3 cell lines had expression group and the control group the percentage of cells and the difference is statistically significant, results suggest that HnRNP L overexpression promoted proliferation of prostate cancer cell lines, may because of the accelerated cell cycle progression.2.4 To study its effect on the growth of tumor after respectively knockdown and overexpression of HnRNP L in prostate cell lines.Nude mice subcutaneous tumor experiment results show:the tumor size in PC3 knockdown group, control group and over-expression group is different,and then by using analysis of variance found among the three groups of tumor growth with significant statistical difference (p<0.05), tumor body made of wax blocks and sections by immunohistochemical detection of PC3 cell lines knockdown group, control group and over expression group, tumor tissue proliferation marker K.i-67 positive expression rate respectively, and the difference is statistically significant (p <0.05), results suggest that:compared to the PC3 cell line in the control group, knocking low group, tumor growth was inhibited, and expression of tumor growth by promoting.2.5 Respectively knockdown and overexpression of HnRNP L in prostate cell lines and to study its effect on the apoptosis of prostate cancer cell linesFlow cytometry apoptosis test results show that:the DU145 cell lines knock low group and control group the percentage of apoptosis were, and PC3 cell lines knock low group and control group the percentage of apoptosis were, respectively, using two independent sample t-test analysis found in DU145 and PC3 cell lines knock low group and control group the percentage of apoptosis and the difference is statistically significant. The results suggest that promote the apoptosis of prostate cancer cell lines ability low HnRNP L expression levels of knocking. That the prostate cancer cell line apoptosis inhibited LNCaP cell line overexpression group and control group the percentage of apoptosis were% and%, and PC3 cell lines knock low group and control group the percentage of apoptosis were% and%, respectively, using two independent sample t-test analysis found in LNCaP and PC3 cell lines had expression group and control group of withered death percentage has significant difference. The results suggest that HnRNP L overexpression.3 The molecular mechanism of HnRNP L in regulating the proliferation and apoptosis of prostate cancer cellsImmunofluorescence results suggest that HnRNP L and Bcl-2 in PC3 cell nucleus has co-localization phenomenon, and co-ip and pull-down assay results there is further evidence in interaction relationship between HnRNP L and Bcl-2, and rip the HnRNP L and p53 mRNA interactions further affect the expression of p53 protein. These results suggest that the HnRNP L is carried out respectively by regulation of p53 and bcl-2 expression to influence the proliferation and apoptosis of prostate cancer cells.Conclusions:Our results show that HnRNP L is high expression in prostate cancer, its expression is closely related with the clinical stage and pathological grade. HnRNP L in prostate cancer may play a role in promoting tumor cell proliferation and anti apoptosis, and these effects may is realized through signal transduction pathway of regulation p53/p21/cyclin D1 signaling pathway and bcl-2/caspase 9/caspase 3. In general, HnRNP L is expected to become a new molecular marker and target for diagnosis and treatment of prostate cancer.