Expression And Function Of ROCK1 In Bladder Cancer

Background:As a common malignant tumor of the urinary system,bladder cancer(BC)has about 400,000 new cases each year worldwide.The incidence of bladder cancer has three major characteristics:1.It is closely related to age.With the increase of human age,the incidence of bladder cancer gradually increases.2.Regional differences.There are huge differences in the incidence of bladder cancer between different geographic regions.The age-standardized rate(ASR)of bladder cancer in developed countries is significantly higher than that in developing countries;.3.Gender differences.The incidence of bladder cancer in men is significantly higher than that in women,with a male to female ratio of 9.0:3.2.But,if statistics are based on clinical staging,what is interesting is that the recurrence rate of bladder cancer in women is significantly higher than that in men,suggesting that the mechanism of the occurrence and development of bladder cancer needs to be further explored.By far,various treatment methods such as surgical resection,chemotherapy,radiotherapy and immunotherapy have been widely used in the treatment of bladder cancer.However,due to the characteristics of frequent recurrence and metastasis,the overall curative effect of the above treatment methods is still inefficient,and no radical cure can be achieved.Therefore,exploring and clarifying the key regulatory factors that regulate the occurrence and development of bladder cancer and their specific molecular mechanisms has important clinical significance for the treatment of bladder cancer.Meanwhile,it is also the focus,hotspot and difficulty in the field of bladder cancer research in recent years.Rho-associated protein kinase 1(ROCK1),as a member of the ROCK family,mainly regulates the phosphorylation of downstream proteins,such as myosin light chain MLC and LIM kinase(LIMK),involved in normal physiological processes such as the organization and dynamic regulation of the actin skeleton.In addition,ROCK1 has been confirmed to have elevated expression levels in many types of tumors.Besides,it is involved in the generation,migration,invasion and metastasis of new blood vessels in tumor cell,and play a key regulatory role in these processes.However,the mechanism of ROCK1’s role in bladder cancer cells and its correlation with patient prognosis and survival are not fully understood yet,and further exploration are needed.Objective:Investigate the expression level of ROCK1 in bladder cancer tissue samples and various bladder cancer cell lines;Explore the correlation between the expression level of ROCK1 in bladder cancer sample tissues and various clinical indicators of bladder cancer;Study the cellular biology of ROCK1 and its role in the occurrence and development of bladder cancer;Explore whether ROCK1 can be a predictive marker for the diagnosis of bladder cancer,or provide new hints and clues for the clinical research of targeted therapy for bladder cancer.Methods:To investigate the expression level of ROCK1 in bladder cancer.firstly,this study systematically reviewed and analyzed the clinical and follow-up results of 64 bladder cancer patients;After that,the mRNA and protein expression levels of ROCK1 in tumor sample tissues of cancer patients and their corresponding control groups were measured by qPCR and Western blot;Meanwhile,the expression levels of ROCK1 in different grades of bladder cancer tissues were further determined by immunohistochemistry(IHC).To explore the correlation between ROCK1 expression level and bladder cancer clinical stage,metastasis and other indicators,the relationship between ROCK1 expression level in tissue samples and the overall survival rate of bladder cancer patients were further analyzed through Kaplan-Meier survival curve.Subsequently,the mRNA and protein expression levels of ROCK1 in SW780,T24,TCCSUP,UMUC-3 and 5637 bladder cancer cell lines were measured by qPCR and Western blot.Next,using the bladder cancer cell line that highly expresses ROCK1 as a carrier,RNA interference technology was applied to knock down the expression level of ROCK1 to establish a bladder cancer cell line that stably knocked down ROCK1.Afterwards,the knockdown effect of ROCK1 protein expression level on the proliferation ability of bladder cancer cells was verified by CCK-8 and colony formation experiments.Finally,the apoptosis levels of bladder cancer cells with different ROCK1 expression levels were detected by annexin V-FITC/PI staining and flow cytometry analysis,and the impact due to ROCK1 expression changes on the anti-apoptotic ability of bladder cancer cells was further verified by Western Blot.Results:By systematically retrospective analysis and patient follow-up of 64 patients who met the enrollment criteria and detecting the expression level of ROCK1 in tumor sample tissues,we found that the expression level of ROCK1 mRNA and protein in clinical bladder cancer tissue samples were highly elevated.The expression level of ROCK1 in the bladder cancer tumor sample tissue was significantly higher than that in the surrounding normal bladder tissue.Moreover,the increase in ROCK1 expression level is closely related to the incidence of lymph node metastasis,especially the rate of pelvic metastasis;in addition,comparing with early stage(Ta-T1)and smaller volume(≤3cm)bladder tumors,the expression level of ROCK1 in advanced(T2-T4)and large-volume(>3cm)bladder tumor tissue samples was significantly increased.Subsequently,when the ROCK1 mRNA expression of cancer tissue is higher than that of adjacent tissues,it is defined as high expression,and when the ROCK1 mRNA expression of cancer tissue is lower than that of adjacent tissues,it is defined as low expression.Based on this standard,patients are classified as high expression group(n=37)and low expression group(n=27).During the 60-month follow-up,compared with the ROCK1 low expression group(n=27),the ROCK1 high expression group(n=37)had a significantly shorter survival time,more lymph node metastasis and metastasis occurs mostly in the pelvic cavity.In the five bladder cancer cell lines SW780,T24,TCCSUP,UMUC-3 and 5637,the mRNA and protein levels of ROCK1 increased significantly,especially the UMUC-3 and T24 cell lines.Based on this,UMUC-3 and T24 cells were selected as the carriers,and RNA interference was used to knock down the expression of ROCK 1 to establish stable cell lines.Subsequently,CCK-8 and colony formation were used to further examine the effects of reducing the expression level of ROCK1 in bladder cancer cells on the proliferation and colony forming ability of bladder cancer cell lines.The results showed that,compared with the Scramble group,reducing the expression level of ROCK1 in UMUC-3 and T24 cells not only significantly inhibited the cell proliferation ability of UMUC-3 and T24 cells,but also significantly inhibited the colony forming ability of UMUC-3 and T24 cells.Furthermore,flow cytometry analysis showed that knocking down ROCK1 in cells can significantly induce apoptosis of UMUC-3 and T24 cells.Western Blot experiments further proved that reducing the expression of ROCK1 in UMUC-3 and T24 cells can increase the expression of pro-apoptotic proteins Bax and Cleaved-Caspase 3 and reduced the expression of anti-apoptotic protein Bcl-xl.All these results indicate that ROCK1 plays an important role in the process of proliferation and invasion of bladder cancer.Conclusion:The expression level of ROCK1 in bladder cancer tissue with larger tumor size was significantly higher than that in bladder cancer tissue with smaller tumor size.High levels of ROCK1 expression in bladder cancer cells indicates poor prognosis such as metastasis and death in patients.Decreasing the expression of ROCK1 in bladder cancer cells UMUC-3 and T24 can inhibit the cell proliferation and cell colony formation,which preliminarily proves that ROCK1 plays an crucial role in the progression of human bladder cancer.ROCK1 can be a predictive marker for the diagnosis of bladder cancer and provides new clinical research clues for targeted therapy of bladder cancer,which cab be expected to be used to remedy the insufficient existing clinical testing and treatment methods.