Study On MiR-199 Affects Kidney Cell Proliferation, Invasion And Apoptosis By Targeting ROCK 1

BackgroundRenal cell carcinoma is one of the three most common cancer of urinary tract cancer, accounting for 2%-3% of all systemic cancer. More than 80% of renal cell carcinoma is originated from renal proximal tubule, and is increasing by 2% per year. Currently, surgery is still the main treatment for kidney cancer patients. As to the popularity of disease screening and the development of monitoring technology, the incidence of symptomatic kidney cancer is decreased and early detection rate of renal cell carcinoma is increased in recent years. However, there are still many cases of locally advanced renal cell carcinoma to be found. About 20%-30% of patients that have produced lymph node metastasis and tumor thrombus in the vein before obtaining definite diagnosis, will have poorer survival time and bad treatment. In addition, even if patients have underwent radical surgery, there are still 20%-30% of them would result in relapse and metastasis after 3 years. Furthermore, kidney cancer is not sensitive to traditional radiotherapy and chemotherapy, advanced kidney cancer patients are more difficult to be cured by using surgery and have poorer prognosis. Therefore, it is important for patients to receive early diagnosis and early treatment.It is well known that the early diagnosis and treatment of kidney cancer depends on elucidating the mechanism of kidney cancer. However, the cause, occurrence and development of kidney cancer is more complicated and is involved in a variety of mechanisms genes and molecules. In recent years, studies have shown that microRNA is associated with the occurrence and development of a variety of malignant tumors. In kidney cancer, a variety of miRNAs with abnormal expression have been found, including miR-200c, miR-141, miR-200c and so on. Meanwhile, studies showed that overexpression of miR-200c and miR-141 down-regulated ZEB2 expression and inhibited the adhesion and invasion of kidney cancer cells. Low expression of miR-34a in renal cell carcinoma inhibited cell proliferation and invasion by regulating YY1. miR-27a promoted renal cancer cells proliferation by downregulating FOXO1. miR-134 inhibited epithelial mesenchyma transformation by targeting KRAS in 786-0 renal clear cell carcinoma.Meanwhile, recent studies have found that miR-199 is lowly expressed in renal cell carcinoma, may act as a tumor suppressor gene to induce the occurrence of kidney cancer. However, the mechanism and biological function of miR-199a have not been fully elucidated, further research is potentially valuable. Therefore, we carried out this study.Objective1. To investigate the expression of miR-199a in kidney carcinoma and normal tissues and to evaluate the relationship between miR-199a expression and patients’ prognosis.2. To investigate the effect of miR-199a on cell proliferation, invasion and apoptosis in kidney carcinoma.3. To investigate the interaction of miR-199a with downstream target genes and upstream regulation genes, and to reveal the mechanism of miR-199a in kidney carcinoma.Methods1. The expression of miR-199a in kidney carcinoma and normal tissues was detected by qRT-PCR, and the relationship between miR-199a expression and patients’ prognosis was analyzed.2. A498 renal cancer cell line was infected by lentiviruses with miR-199a overexpression and negative control vector, respectively. The expression of miR-199a was validated by qRT-PCR assay.3. Cell proliferation was measured by MTT and colony formation assay. Cell invasion was evaluated by Transwell assay. Cell apoptosis was detected by flow cytometry.4. The candidate target genes of miR-199a were obtained by target gene prediction websites and bioinformatic analysis screening. The results showed that ROCK-1 was one of direct target genes of miR-199a. ROCK-1 and miR-199a were co-transfected in A498 RCC cell lines after constructing ROCK-1 mutants, and luciferase reporter assay was used to analyze the correlation between miR-199a and ROCK-1. Then, MTT assay and colony assay were used to assess the effect of ROCK-1 and miR-199a co-transfection on cell proliferation. Also, the effect of ROCK-1 and miR-199a co-transfection on cell apoptosis was evaluated by flow cytometry. In addition, qRT-PCR was to used to detect the expression of ROCK-1 in 39 cases of renal cell carcinoma tissues, and the clinical correlation between miR-199a and ROCK-1 was assessed.5. Snail transcriptional binding sites was found in miR-199a DNA sequences by analyzing the encoding sequences of miR-199a. So, we supposed that Snail might be one of upstream regulatory genes of miR-199a. Then Snail promoter binding sites was cloned and transfected with miR-199a in A498 cell line, and the association between Snail and miR-199a was evaluated by luciferase reporter assay. Meanwhile, the relationship between Snail and miR-199 was evaluated by Northern blot. Moreover, qRT-PCR was used to detected the expression of Snail in 39 cases of renal cell carcinoma tissues, and the clinical correlation between Snail and miR-199a was assessed.Results1. The expression and significance of miR-199a in renal cell carcinomaFirstly, we tested the purity of RNA by 1% agarose gel electrophoresis, and the result showed that there were three clear banding pattern at 28S,18S,5S, without DNA bands, indicating that the RNA was pure to without RNA degradation and DNA contamination and could be used for reverse transcription and subsequent experiments.Subsequently, under the internal control of U6, we detected the expression of miR-199a in kidney cancer tissues and adjacent normal tissues by qRT-PCR. The results shown that the melting curve of miR-199a and U6 was specific without non-specific amplification and primer dimer. The results also showed that miR-199a expression was significantly down-regulated for 2-4 times in renal cell carcinoma tissues compared with those in adjacent normal tissues, and the difference was statistically significant (P<0.01).Combined with patient’s data, we analyzed the association between miR-199a expression and patients’prognosis. The results showed that the average survival time and the median survival time in patients with miR-199a downregulation were (41.147 ±8.713) and 34.4 months, and the average survival time and the median survival time in patients with miR-199a upregulation were (71.377±7.125) and 70.1 months. The 1-year and 3-year survival rates of patients with miR-199 low-expression were 83.4%, and 14.7%, respectively. The 1-year and 3-year survival rates of patients with over-expression were 93.2% and 48.7%,respeetivery,and the dmerence was significant, (P<0.01). miR-199 could be served as an independent risk factors of tumor-specific death by COX multivariate analysis, excluding the effect of tumor size, TNM stage, clinical stage and other factors. (RR=1.714,95% CI:1.127-2.216, P=0.03).2. miR-199 affects kidney cell proliferation, invasion and apoptosis by targeting ROCK-1To clarify the target genes of miR-199a, we selected the intersection results derived from three prediction software. The results revealed that the complementary sequence of miR-199a was found in the mRNA sequence of ROCK-1. Therefore, we considered that ROCK-1 could be one of the potential targets of miR-199a.To verify that ROCK-1 is a direct target of miR-199a, ROCK-1 wild-type and mutant were subcloned into luciferase reporter vector and co-transfected with miR-199a and negative scramble control in A498 cells. The results showed that miR-199a significantly inhibited the luciferase activity of the ROCK-1 wild-type but not that of the mutant in A498 cells (P<0.05). Furthermore, we detected the expression of ROCK-1 in A498 cells by qRT-PCR and Western blot, and results revealed that miR-199a overexpression significantly downregulated ROCK-1 mRNA and protein levels.In order to investigate the effect of miR-199a and ROCK-1 on cell proliferation, we firstly measured the proliferation ability by MTT assay and colony formation assay. The results revealed that miR-199a overexpression significantly inhibited kidney carcinoma cells proliferation and colony formation, whereas co-transfection with an ROCK-1-overexpressing vector partially blocked the miR-199a induced inhibition of cell proliferation and colony formation, indicating miR-199a overexpression significantly inhibited cell proliferation.In order to investigate the effect of miR-199a and ROCK-1 on the invasion of kidney cancer cells, Matrigel invasion assays was performed. The results demonstrated that exogenetic overexpression of miR-199a markedly reduced invasion of kidney cancer cells, while ROCK-1 restoration could partially block these effect.In order to investigate the effect of miR-199a and ROCK-1 on the apoptosis of kidney cancer cells, flow cytometry assays was performed. The results shown miR-199a overexpression induced cell apoptosis, while exogenetic overexpression of ROCK-1 blocked the miR-199a effect on apoptosis3. The effect of miR-199a overexpression and miR-199a/ROCK-1 co-transfection on the biological characteristics of renal carcinoma in vivoTo further confirm the roles of miR-199a in tumor growth in vivo, we initially infected A498 cells with lentiviral vectors with miR-199a stable expression (LV-miR-199a) or the negative control (LV-Ctrl). The results showed that stable transfection of miR-199a into A498 cells resulted in decreased the growth and tumor weight of subcutaneous xenograft tumors in nude mice, when compared with those stably transfected with empty vector.Collecting the tumor tissues between miR-199a overexpression experimental group and scramble control group, the expression of ROCK-1 and proliferation index Ki-67 was detected by immunohistochemistry (IHC). The results showed that ROCK-1 and Ki-67 expression in experimental group with miR-199a overexpression was significantly lower than those in scramble control group, suggesting that miR-199a overexpression markedly inhibited tumor proliferation and invasion.Then, we detected ROCK-1 expression in 39 cases of kidney carcinoma tissues by qRT-PCR, and analyzed the correlation between ROCK-1 and miR-199a. The results showed that ROCK-1 highly expressed in kidney cancer tissues, and inversely correlated with the expression of miR-199a (r=0.7665, P<0.01).4. Identification of upstream regulatory factors of miR-199aAccording to the principles of protein-coding mRNAs, miRNAs are transcribed from their genes in genomic DNA. Therefore, we referred to the database-related genes and found a conversed DNA region upstream of pri-miR-199a gene named pri-miR-199a gene promoter, which was binding sites of transcription factor Snail in the DNA region. Thus, we initially assumed that Snail might be identified as a candidate upstream gene to regulate miR-199a.Subsequently, the DNA region was cloned into pGL3-basic vector (Promega) and this vector was named pGL3-miR-199a pro. The luciferase assay showed that this DNA region contains the pri-miR-199a promoter and is capable of directing luciferase expression. However, luciferase activity was efficiently repressed when pGL3-375pro vector was cotransfected with Snail expression vector (Snail) but not when cotransfected with the empty control vector. Moreover, qRT-PCR assay showed that snail overexpression could reduce the expression level of miR-199a,which was consistent with the Northern blot assay. In order to determine the clinical relevance of these results, we further detected the expression of miR-199a and Snail in the same kidney cancer tissues. The results revealed that Snail highly expressed in kidney cancer tissues, which is significantly inversely correlated with miR-199a (r=0.6373, P<0.05). Taken together, these observations demonstrated that snail is a potential upstream regulator of miR-199a expression.Conclusions1. miR-199a is lowly expressed in kidney cancer tissues and patients with miR-199a overexpression have favorite prognosis.2. miR-199 affects kidney cell proliferation, invasion and apoptosis by targeting ROCK-1.3. Snail is the upstream regulation gene of miR-199a.The innovation of this study1. The results reveal that miR-199a is lowly expressed in kidney cancer tissues and patients with miR-199a overexpression have favorite prognosis.2. This study is the first report to confirm that miR-199 affects kidney cell proliferation, invasion and apoptosis by targeting ROCK-1.3. This study is the first to confirm that Snail can served as the upstream regulatory gene of miR-199a, and further elaborated the mechanism of miR-199a in renal cell carcinoma.