| Ovarian cancer is one of the most common malignancies of women.The lack of an effective diagnostic method together with the absence of reliable signs and symptoms makes the detection of ovarian cancer difficult in the early stage.Meanwhile,the unfavorable prognosis and high recurrence rate associated with advanced metastasis lead to high mortality in ovarian cancer.Thus,it is necessary to further study its internal mechanism and develop a potential therapeutic target for ovarian cancer to improve the prognosis of patients and reduce mortality.The mammalian ribosome consists of a 60S large subunit and a 40S small subunit,the main components of which are ribosomal RNA(r RNA)and ribosomal proteins(RPs).Ribosomal proteins are important components of ribosomes involved in protein translation and ribosome assembly and are necessary for the growth and survival of all types of cells.Previous studies have shown that ribosomal protein plays an important role in regulating various physiological and pathological processes.It is not only involved in DNA transcription,gene translation,DNA repair,and cell morphology maintenance but also in regulating the growth,proliferation,cycle,apoptosis,metastasis,and invasion,even highly correlated with the malignant transformation of normal cells.Ribosomal proteins have been found to play an important role in the development and progression of various cancers,including esophageal cancer,gastric cancer,lung cancer,and other malignant tumors,which is a hot spot in the research of targeted cancer therapy.Ribosomal protein L34(RPL34,L34E family of RPs),which is located in the cytoplasm and contains a zinc finger motif,is mainly involved in the formation of the 60S subunit.Recent researches have shown that RPL34 as a tumor promoter is involved in the development and progression of human malignancies.Studies had shown that the high expression of RPL34 indicates a poor prognosis,and its knockout may inhibit the proliferation and growth of the tumor,which includes non-small cell lung cancer,osteosarcoma,oral squamous cell carcinoma,gastric cancer,esophageal cancer,colorectal cancer,pancreatic cancer and glioma.However,the biological function and clinical significance of RPL34 in ovarian cancer are unclear.Therefore,the purpose of this study was to explore the role of RPL34 in the progression of ovarian cancer.Checking the ONCOMINE website,we found that the expression level of RPL34 in ovarian cancer tissues is lower than that of the borderline ovarian surface epithelial-stromal tumor.Due to the low sample size,this conclusion still needs further verification.Thus,we further explore the expression and function of RPL34 in ovarian cancer through in vivo and in vitro experiments.In this study,we found that RPL34 is low-expression in ovarian cancer,and the over-expression of RPL34inhibits the proliferation,migration,and invasion of ovarian cancer.Our data indicate that RPL34 low expression is related to the progression of ovarian cancer and may provide a new molecular marker for ovarian cancer therapy.ObjectiveThis study aims to investigate the expression of RPL34 in ovarian cancer tissues and cells,and the effect of RPL34 on the proliferation,migration,invasion of SKOV3cells to find a new therapeutic target for ovarian cancer.Methods1.Screening a total of 30 cases of ovarian cancer and 20 cases of ovarian cysts diagnosed in the Third Affiliated Hospital of Zhengzhou University from January2018 to January 2020.2.Immunohistochemistry was used to detect the expression of RPL34 in ovarian cancer and normal ovarian tissues.3.qRT-PCR was used to detect the expression levels of RPL34 mRNA in ovarian cancer tissues and normal ovarian tissues,and the expression levels of RPL34mRNA in ovarian cancer cells and normal ovarian epithelial cells.4.Fluorescence microscopy observation,Western blot and qRT-PCR were used to detect the expression of RPL34 after SKOV3 cells were transfected with RPL34-sh RNA and RPL34-OE mediated by lentivirus.5.The effect of RPL34 on SKOV3 cell proliferation ability was detected by CCK-8 proliferation assay and colony-forming assay.6.The effect of RPL34 on SKOV3 cell migration ability was detected by wound-healing assay and transwell assay.7.The influence of RPL34 on SKOV3 cell invasion ability was detected by cell invasion experiment.8.The effect of RPL34 on SKOV3 cell cycle and apoptosis were detected by flow cytometry.9.The effect of RPL34 on ovarian cancer tissue growth in vivo was detected by subcutaneous tumor formation in nude mice.Results(1)According to the results of the IHC,the RPL34 protein was mainly expressed in the cytoplasm.The positive rate of RPL34 in ovarian cancer tissue is 36.67%(11/30)and in normal ovarian tissue is 76.67%(23/30)(χ~2=9.774,P<0.05).By comparing the expression level of RPL34 protein and the pathological data of patients with ovarian cancer,the expression of RPL34 is closely correlated with the degree of differentiation(P<0.001),FIGO stage(P<0.001)and lymph node metastasis(P=0.007),but were not significantly associated with age or level of CA125 in primary tumors.The Kaplan-Meier survival curve showed that patients with high RPL34 expression had a longer survival period than patients with low RPL34expression levels.(2)Detection of RPL34 mRNA expression at the molecular level,the result shows that the expression levels of RPL34 mRNA in ovarian cancer tissue samples were significantly lower than those in non-cancerous normal ovarian tissue;the expression level of RPL34 in ovarian cancer cells was lower than that in human normal ovarian epithelial cells.(3)To study the effect of RPL34 on SKOV3 cell proliferation,cell growth was determined via CCK-8 assay.The results shown that when RPL34 was up-regulated,the proliferation of SKOV3 cells was significantly inhibited compared to the control group,while RPL34 was knocked down,the result was the opposite.(4)By the colony-forming ability of SKOV3 cell lines to study the long-term effect of RPL34 on the growth.The results showed that the up-regulation of RPL34reduced the colony-forming ability of SKOV3 cells,while RPL34 was knocked down,the result was the opposite.(5)The wound-healing assay was conducted to test the migration ability of SKOV3 cells.The wound-healing assay shows that when RPL34 was up-regulated,the migration distance was significantly shorter than that of the NC group,while the result was the opposite in the knocked down group.(6)The transwell assay were conducted to test the invasion ability of SKOV3cells.The results showed that the number of cells passing through the upper matrigel into the lower chamber was significantly decreased in the up-regulated group compared with the NC group,while the result was the opposite in the knocked down group.(7)Flow cytometry was used to evaluate whether RPL34 inhibits cell proliferation by regulating SKOV3 cell cycle progression or apoptosis.The results showed that the up-regulated group decreased the ratio of G0/G1 and S phase cells,and significantly increased the ratio of G2/M phase cells.These data indicate that after RPL34 is over-expressed,SKOV3 cells are blocked in the G2/M phase of the cell cycle,thereby inhibiting cell proliferation.Also,RPL34 over-expression significantly induced apoptosis in SKOV3 cells.(The apoptosis rate of RPL34 group was up-regulated:7.23±0.45%,and that of control group:3.27±0.15%;the apoptosis rate of RPL34 group was down-regulated:3.65±0.48%,and that of control group was 6.59±0.10%)(8)To evaluate the effect of RPL34 on the growth of ovarian tumors in vivo,we used SKOV3 cells subcutaneously to establish a mouse xenograft model.By measuring the tumor volume changes throughout the experiment,the tumor growth volume of nude mice in the up-regulated RPL34 group was significantly smaller than that in the control group,while the tumor volume in down-regulated RPL34 group was the opposite.Conclusion(1)RPL34 expression is down-regulated in ovarian cancer and cells,which is associated with poor prognosis in patients with ovarian cancer.(2)RPL34 inhibits SKOV3 cell proliferation,migration and invasion in vitro.(3)RPL34 can induce SKOV3 cell cycle arrest and apoptosis.(4)RPL34 inhibits the growth of ovarian cancer in vivo. |
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