The Function And Molecular Mechanism Of HnRNP L In The Development And Progression Of Bladder Cancer

BACKGROUD & OBJECTIVE:Bladder cancer refers to occur on the bladder mucosa malignant tumor, it is one of the most common malignant tumor, urinary system in Europe and the United States its incidence in male, fourth in the malignant tumor is one of the six most common tumor, its incidence in our country is also on the rise in recent years. Bladder cancer can be divided into the muscle invasive bladder cancer and muscle layer of two kinds of invasive bladder cancer. About 70%-90% of newly diagnosed invasive bladder cancer, bladder cancer is a muscle layer within the cavity while curing bladder after tumor resection, even if strict bladder perfusion chemotherapy, still about 10%-10% of the recurrence rate, while in the recurrent cases, nearly 5%30% muscular may progress to invasive bladder cancer even shift, such already invasive bladder cancer patients after total cystectomy 5-year survival rate is only 60%-70%. Bladder cancer currently rely mainly on cystoscopy to monitoring and diagnosis, however cystoscope because its for invasive operation, and check the expensive and time consuming, can’t for patients to accept and cooperate.Fall off the urine cytology examination in the late found in bladder cancer has a high sensitivity, but in the early sensitivity is very low, and its diagnosis and clinical pathology doctors experience are inseParable.In general, it is the lack of progress can be used for bladder cancer early diagnosis, treatment and forecast of high sensitivity and specificity of checking and molecular markers, to solve these problems to need the significant progress of basic research.The mechanism about development and metastasis of bladder cancer is not very certain. The tumorigenesis of bladder cancer is a multi-gene, multi-factor process, normal bladder cells mutate in the periods of bladder cell DNA, often accompanied with activation of oncogenes. It has reported that the occurrence of bladder cancer is related with gene mutation, like HER-2, H-Ras, Bcl-2, C-myc gene mutation or other; In addition, the development mechanism of bladder cancer is also related to regulate cell growth, DNA repair or apoptosis suppressor gene inactivation protein closely changes in the DNA of these genes by loss of apoptosis does not occur, leading to uncontrolled cell growth. Recent studies have found that the inactivation of anti-oncogene, like P53, Rb, P21 and other tumor suppressor gene, play an important role in the development and progression of bladder cancer. Metastasis mechanism about BCa is very complicated, enhance of cell movement ability, the descent of cell adhesion, the cytoskeleton changes, EMT pathways and tumor microenvironment play an important role in the process of tumors metastasis. In addition, the occurrence of bladder cancer is involved in the overexpression or amplification of the normal gene encoding growth factors or receptors, such as the overexpression of EGEF promote invasion and metastasis of bladder cancer. Accordingly, it is very important to take more research about the molecular mechanism of development and metastasis in bladder cancer, as to get a high sensitivity and specificity biomarker to predict the biological behavior of bladder cancer, and evaluation, metastasis and prognosis of the BCa patient, thus improving the survival rate of patients with bladder cancer and improve the quality of life.Heterogeneous nuclear ribonucleoprotein L (hnRNP L) is located on 19q13.2, encoding hnRNP L protein. hnRNP L, an important part of heterogeneous nuclear ribonucleoprotein (nucleoprotein hnRNP) component, is rich in organism, and can shuttle between the cytoplasm and nucleus. HnRNPs are a class of RNA-binding proteins, which are mainly concentrated in the nucleus area, combining with pre-mRNA through specific structure, and form a pre-mRNPs complex,as to take park in regulation of mRNA transport, metabolism, cut and expression and other processes. It’s reported that hnRNP is closely related to tumor occurrence and development, which may regulate gene expression, inhibite apoptosis, promote angiogenesis and enhance the ability of invasion and metastasis of tumor cells.As one of the member of hnRNPs family, it mainly conbine with CA repetitive sequence or some CA rich sequence as to regulate cell process, including regulating alternative splicing in exon selection process, promoting polyadenylation, assisting export mRNA without intron gene, helping internal ribosome to translate into the site, regulating stability of mRNA. It has been reported in the literature that the expression of hnRNP L closely related disorders of cancer, and it plays a major role in promotion of cell proliferation, migration, invasion, adhesion in the tumor. Recent years, there are many papers about hnRNP L in tumors, it has been confirmed that hnRNP L is involved in esophageal squamous cell carcinoma, lung cancer, liver cancer,metastatic colorectal cancer and breast cancer occurrence, and other tumors. For example, hnRNP L can lead to degradation of caspase-9a precursor, by conbining with exon caspase-9a precursor mRNA, there by affecting the expression of caspase-9 mRNA isoforms, and ultimately regulate the development and progression of non-small cell lung cancer. But there have not been reported in the literature that hnRNP L involved in the biological Processes of bladder cancer. Our previous work has showed that hnRNP-L is highly expressed in bladder cancer tissue, by IHC (Immunohistochemistry) of bladder cancer tissue microarray and its expression positively correlates with the process. However, he role of hnRNP L in bladder cancer progression and metastasis remains unclear, the molecular biological mechanisms of hnRNP L in bladder cancer is also poorly understood.This paper was proposed to detect hnRNP L expression in bladder cancer, analyze the relationship between the expression and clinical pathological parameters; Using a series of experiments in vivo and in vitro to reveal the function of hnRNP L in bladder cancer occurrence, development and migration, in addition to study the related molecular mechanism of hnRNP L in bladder cancer. At the same time explore the ownstream side mechanism of abnormally high expression of hnRNP L in bladder cancer.This study is conducted to provide experimental basis for the growth and metastasis of bladder cancer; it may ultimately provide a reliable new molecular biomarkers for early screening, treatment and prognosis in bladder cancer.METHODS:(1) HnRNP L expression in bladder cancer tissues and cellsWestern blot and Real Time Q-PCR were utilized to detecte the hnRNP L protein and mRNA expression in the bladder cancer tissues and matched adjacent to carcinoma tissues, discovery the hnRNP L expression in bladder transitional epithelial cell SV-HUC-1, and in the bladder cancer cells UM-UC-3, EJ, T24,5637.(2) HnRNP L expression in bladder cancer tissue and its correlation with clinical ParametersThe hnRNP L expression in bladder cancer tissues was detected by IHC and Statistical analysis the correlation of hnRNP L with tumor differentiation, staging, and the correlation between clinical pathological parameters related to prognosis.(3) The hnRNP L biological functions in bladder cancerEstablished bladder cancer cell lines which stably knockdown and overexpressed hnRNP L. Using CCK-8, colony formation assay, Xenograft model in nude mice assay to detecte the proliferation in BCa cells with hnRNP L knockdown or overexpression. Using transwell migration assay and scratch wound healing assay to explore the cell migration in BCa cells with hnRNP L knockdown or overexpression. Using FACS analysis to discover the Cell Cycle and apoptosis n BCa cells with hnRNP L knockdown or overexpression.(4) The mechanism of hnRNP L in the process and progress in bladder cancerWestern blot was carried to find the variation of apoptosis proteins (caspase-3、 caspase-6、capase-9、Bcl-2、Bc12L1、PARP、), and the activation of MAPK pathway (p-ERK1/2、p-P38、p-JNK、Nrf2), and the E2F1 protein in BCa cells with hnRNP L knockdown and overexpression. Real Time quantitative PCR and Western blot were used to detecte the change of EMT markers (E-cadherin、Vimentin、Claudin-1、ZO-1、 P-catenin、ZEB1、snail) in BCa cells with hnRNP L knockdown and overexpression.RESULTS:(1) The expression level of hnRNP L in bladder cancer tissues.IHC results showed that hnRNP L positive expression mainly located in the nucleus, a tan or brown, in 74.2%(115/155) positive expression in bladder cancer cases, normal bladder tissues hnRNP L expression is lower than its counterpart tumor tissue; Western blot and qRT-PCR results showed that hnRNP L protein in bladder cancer tissues and matched with mRNA expression level was higher than normal bladder tissues.(2) The relationship between the expression level of hnRNP Land clinical pathological parameters.HnRNP L expression levels did not differ significantly between different gender (P>0.05); The expression level of hnRNP L in different age, different TNM stages, different clinical Pathologic stage, histological grade were significantly different (P< 0.05), and there is a positive correlation between both; Kaplan Meier method analysis showed that the Patients who had higher expression of hnRNP L will suffer a shorter 5-year survival rate campared with the low expression patients (Log-Rank, P<0.05); Multiariable Cox risk ratio model analysis showed that:hnRNP L are independent risk factors affecting the prognosis of patients with bladder cancer (P=0.001).(3) The function of hnRNP L in bladder cancer progression3.1 The establishment of and bladder cancer cell lines with hnRNP L knockdown and overexpressionHnRNP L knockdown and overexpressed bladder cancer cell lines were established by lentivirus vector and Western blot was used to verify the efficiency, the results showed that the knockdown and overexpression of hnRNP L cell lines were established successfully.3.2 Effect of hnRNP L knockdown or overexpression on BCa cell proliferationThe results of CCK-8 showed that the hnRNP L stability knockdown cells growth rate decreased significantly(P<0.05) compared with the control group of cell, and the overexpression of hnRNP L significantly accelerated the cell growth (P< 0.05); The colony formation experients results showed that, compared with the control group hnRNP L knockdown significantly reduced the number of clones (P< 0.05) and the overexpression hnRNP L cells form of clone number significantly more than the control group (P<0.05); Xenograft model in nude mice assay results showed that the tumor growth slowly in hnRNP L stable knockdown group compared with the control group, and the tumor weight is lighter than the control group (P< 0.05).3.3 Effect of hnRNP L knockdown or overexpression on BCa cell migrationWestern blot results in bladder cancer cell lines UM-UC-3 and EJ after knockdowned the hnRNP L expression the epithelial marker E-cadherin increased, while the mesenchymal markers Vimentin decreased; On the contrary, in the overexpressed hnRNP L BCa cells, the epithelial marker E-cadherin droped, and the expression of mesenchymal markers Vimentin increased.Transwell migration experiment results showed that, compared with the control cell migration quantity of knockdown hnRNP L group was obviously reduced (P< 0.05); And hnRNP L over-expressed cell line migration quantity obviously rosed than the control group (P<0.05). Scratches healing experiment results showed that compared with the control cell lines migration velocity of hnRNP L stable knockdown group was obviously slowed (P<0.05);And hnRNP L over-exPressed cell line migration rate significantly faster than the control group (P<0.05).3.4 The effection of cell cycle distribution and apoptosis on BCa cells with hnRNP L knockdown and overexpressionFlow cytometry cell cycle detection results showed that compared with the controedl group, the hnRNP L stability knockdown group obviously increased the proportion of cells in G1 Phase and decreased significantly in S Phase proportion, G1 Phase arrest of cells(P<0.05), the Process of cell cycle slowed significantly;, and the overexPressed hnRNP L cells decreased significantly in G1 Phase ratio, and the proportion of S Phase increased significantly (P<0.05), the process of cell cycle has quickened significantly.Apoptosis detection results showed that, compared with the control group, the apoptosis ratio of hnRNP L knockdown group was obviously increased (P<0.05); And hnRNP L overexpression cell line cell apoptosis ratio significantly less than the control group (P<0.05).(4) The mechanism of hnRNP L in the process and progress in bladder cancerWestern blot results showed that after knockdown hnRNP L in the bladder cancer cell lines UM-UC-3 and EJ, the expression of caspase-3, caspase-6, caspase-9, the Bcl-xL increased, and the expression of Bcl-2 and PARP1 droped; After stability over-expressed hnRNP L in this two cells, the consequence reversed. On the other hand, compared with control after knockdown of hnRNP L, the expression p-ERK1/2, p-P38, p-JNK decreased, and the NRF2, E2F1 reduced too; On the contrary after stability overexpressed hnRNP L in two cells showed the reversed results. The not phosphorylation of JNK does not change significantly compared with control in this two cell lines with knockdown and overexpression hnRNP L. Real Time quantitative PCR results showed that after knockdown of hnRNP L the mRNA expression levels of E-cadherin rise, and the mRNA expression level of Vimentin droped, the mRNA expression level of the corresponding transcription factor ZEB1, and ZEB2, snail, slug lowered; Western blot results showed that after knockdown of hnRNP L the expression of E-cadherin, ZO-1 and claudin-1 raised, whereas the expression level of Vimentin, β-catenin, ZEB1 decreased; after overexpressed of hnRNP L the results reversed, and the expression of E-cadherin inhibiting factor snail raised.CONCLUSION:(1) The expression of hnRNP L in bladder cancer tissues were significantly higher than that of the tumor tissue adjacent to carcinoma, the expression of hnRNP L level was positively related to clinical pathological parameters, such as histological grade, clinical stage, TNM stage and prognosis; hnRNP L high expression is closely related to bladder cancer patients’ survival time, the higher level of hnRNP L, the worse of the patients’ prognosis;(2) HnRNP L knockdown suppression of bladder cancer cell proliferation, invasion and migration ability, inhibiting EMT phenotype transformation, inducing tumor cell apoptosis, G1 Phase arrest of cells; And the over-expressed hnRNP L can promote the transformation of EMT process in bladder cancer cells, and increase the invasion and migration ability, in addition, inhibit tumor cell apoptosis and accelerate the cell cycle;(3) HnRNP L may regulate P53/Bcl2/Caspase signaling pathway and affect the activity of MAPK signal pathway,in addition to NRF2 and E2F1 to participate in bladder cancer cell proliferation and apoptosis.