Knockdown of replication protein A 3 induces protective autophagy and enhances cisplatin sensitivity in lung adenocarcinoma by inhibiting AKT/mTOR signaling via binding to cyclin-dependent kinases regulatory subunit 2.

Replication protein A 3 (RPA3) is a significant component of replication protein A and has been documented to function as an oncogene in several types of cancers. However, the role and underlying mechanism of RPA3 in lung adenocarcinoma (LUAD) remains unknown. In this study, messenger expression of RPA3 and survival probability in LUAD were predicted by the UALCAN database. The combination of RPA3 with cyclin-dependent kinases regulatory subunit 2 (CKS2) were characterized by the humanbase and STRING databases and verified by co-immunoprecipitation. Cell viability was assessed by Cell Counting Kit-8 assay and colony formation assay. Flow cytometric analysis and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling assay were used to determine cell cycle and cell apoptosis, respectively. The expressions of protein kinase B/mammalian target of rapamycin (AKT/mTOR) pathway and autophagy-related proteins were examined by western blot assay. Significantly, we revealed that RPA3 expression was upregulated in LUAD and is associated with poor prognosis in LUAD patients. RPA3 and CKS2 expression was highly expressed in LUAD cell lines and the interaction between RPA3 and CKS2 was confirmed. RPA3 silencing inhibited A549 cell viability, blocked cell cycle and promoted cell apoptosis, as well as induction of autophagy and inhibition of AKT/mTOR signaling. CKS2 overexpression reversed the effects of RPA3 silencing on A549 cells. In addition, RPA3 knockdown enhanced cisplatin sensitivity of A549 cells through blocking the AKT/mTOR signaling. These results suggested that RPA3 might control LUAD cell autophagy and enhance cisplatin sensitivity by regulation of AKT/mTOR signaling via targeting CKS2.

MicroRNA-146a-5p enhances radiosensitivity in hepatocellular carcinoma through replication protein A3-induced activation of the DNA repair pathway.

Hepatocellular carcinoma (HCC) is known for its high mortality rate worldwide. Based on intensive studies, microRNA (miRNA) expression functions in tumor suppression. Therefore, we aimed to evaluate the contribution of miR-146a-5p to radiosensitivity in HCC through the activation of the DNA damage repair pathway by binding to replication protein A3 (RPA3). First, the limma package of R was performed to differentially analyze HCC expression chip, and regulative miRNA of RPA3 was predicted. Expression of miR-146a-5p, RPA3, and DNA damage repair pathway-related factors in tissues and cells was determined. The effects of radiotherapy on the expression of miR-146a-5p and RPA3 as well as on cell radiosensitivity, proliferation, cell cycle, and apoptosis were also assessed. The results showed that there exists a close correlation between miR-146a and the radiotherapy effect on HCC progression through regulation of RPA3 and the DNA repair pathway. The positive rate of ATM, pCHK2, and Rad51 in HCC tissues was higher when compared with that of the paracancerous tissues. SMMC-7721 and HepG2 cell proliferation were significantly inhibited following 8 Gy 6Mv dose. MiR-146a-5p restrained the expression of RPA3 and promoted the expression of relative genes associated with the DNA repair pathway. In addition, miR-146a-5p overexpression suppresses cell proliferation and enhances radiosensitivity and cell apoptosis in HCC cells. In conclusion, the present study revealed that miR-146a-5p could lead to the restriction of proliferation and the promotion of radiosensitivity and apoptosis in HCC cells through activation of DNA repair pathway and inhibition of RPA3.

Replication Protein A 3 Is Associated with Hepatocellular Carcinoma Tumorigenesis and Poor Patient Survival.

BACKGROUND: Replication protein A (RPA) 3 is a subunit of the RPA protein complex, which functions in multiple processes of DNA metabolism. Dysregulation of RPA1 and RPA2 has been implicated in tumor progression in several cancer types. However, the function of RPA3 in hepatocellular carcinoma (HCC) tumorigenesis has not been elucidated. METHOD: In this study, we investigated the function of RPA3 in HCC development by stably knocking down its expression using short hairpin RNA (shRNA) in HepG2 cell line, followed by cell proliferation, colony formation, soft agar, and invasion assays. Xenograft experiment was performed to examine in vivo tumor-promoting properties of RPA3. RESULTS: Downregulation of RPA3-inhibited cell proliferation, colony formation, soft agar growth as well as invasion in HepG2 cells were observed. Stable knockdown of RPA3 significantly inhibited tumor growth in the xenograft mouse model. In addition, qRT-PCR analysis revealed that RPA3 was upregulated in human HCC tissues compared with matched noncancerous adjacent tissues (NATs). High expression of RPA3 was associated with poor overall survival and disease-free survival. CONCLUSION: Elevated expression of RPA3 promotes tumor progression in HCC cells. RPA3 is upregulated in HCC tissues and high expression of RPA3 is associated with poorer patient survival. Therefore, this protein may represent a novel therapeutic target for intervention of HCC and prognostic biomarker for patient survival.

Overexpression of replication protein A3 is associated with unfavorable outcome in bladder urothelial carcinoma.

PURPOSE: The replication protein A3 (RPA3) is a subunit of the RPA protein complex, which plays an essential role in multiple processes of DNA metabolism. However, the involvement of RPA3 bladder urothelial carcinoma (UC) prognosis has not yet been elucidated. The aim of our study is to investigate the prognostic role of RPA3 expression in patients with bladder UC. MATERIALS AND METHODS: Bladder UC tissue specimens from 155 consecutively treated patients who underwent surgery between 2013 and 2018 were evaluated. The RPA3 expression was determined by immunohistochemistry, Western blot, and correlated with clinicopathological parameters. The prognostic significance of RPA3 expression was explored using the univariate and multivariate survival analysis of 155 patients who were followed. RESULTS: A total of 155 tissue specimens "of patients" who were regularly followed with the mean 39.6 months (from 4 to 71 months). The expression of RPA3 was significantly associated with tumor grade (P = 0.031) and stage (P = 0.021), as well as tumor size (P = 0.034). In univariate analysis, RPA3 overexpression showed an unfavorable influence on recurrence-free survival with statistical significance (P < 0.01). TNM stage and grade also showed strong statistical relation with adverse recurrence-free survival (P < 0.01, P = 0.030). Multivariate analysis revealed that grade, stage, and RPA3 reactivity (P = 0.025, P < 0.01, P = 0.016) were identified as independent prognostic factors for recurrence-free survival in patients with bladder UC. CONCLUSIONS: These results of this study proved that elevated expression of RPA3 was associated with worse clinical outcome in bladder UC patients. This finding suggested that RPA3 served as a potential prognostic biomarker, which could be useful to predict cancer evolution and may represent a novel therapeutic target for the intervention of bladder UC patients.