Expression of lncRNA GTSE1-AS1 in prostate cancer tissues and its effect on proliferation and invasion of LNCaP cells / 中国肿瘤生物治疗杂志

@#[Abstract] Objective: To explore the expression of long non-coding RNA (lncRNA) GTSE1-AS1 in prostate cancer tissues and the mechanism that affects the proliferation and invasion of LNCaP cells. Methods: From November 2017 to December 2018, 68 pairs of prostate cancer tissue and para-cancerous tissue specimens were resected from prostate cancer patients at the Department of Urology of Luoyang Central Hospital Affiliated to Zhengzhou University; in addition, prostate cancer cell lines LNCaP, PC-3, C4-2B, 22Rv1, DU-145 and normal prostate follicular epithelial RWPE-1 cells were also chosen for this study. qPCR was used to detect the expression level of GTSE1-AS1 in cancer tissues and cell lines. The GTSE1-AS1 over-expression plasmid (experimental group) and negative control plasmid (control group) were respectively transfected into LNCap cells. MTT assay and Transwell chamber method were used to detect the effect of GTSE1-AS1 over-expression on the proliferation and invasion ability of LNCaP cells, respectively. The targeting relationship among GTSE1-AS1 and miR-324-3P as well as FBXW7 (F-frame/WD repeat domain protein 7) was verified by bioinformatics tools and dual-luciferin reporter gene assay. The effect of GTSE1-AS1 over-expression on downstream gene and protein expression was detected by qPCR and WB assay. Results: The expression level of GTSE1-AS1 in prostate cancer tissues was significantly lower than that in para-cancerous tissues (P<0.01), and the expression of GTSE1-AS1 in prostate cancer cell lines was significantly lower than that in RWPE-1 cells (P<0.05 or P<0.01). Over-expression of GTSE1-AS1 significantly inhibited the proliferation and invasion (P<0.05 or P<0.01) of LNCaP cells. Dual-luciferin reporter gene assay confirmed the complementary binding between GTSE1-AS1 and miR-324-3p as well as between miR-324-3p and FBXW7. Over-expression of GTSE1-AS1 significantly reduced the expression of miR-324-3p in LNCaP cells (P<0.01), and promoted the mRNA and protein expressions of FBXW7 (all P<0.01). Conclusion: GTSE1-AS1 is under-expressed in prostate cancer tissues and cell lines. Over-expression of GTSE1-AS1 can inhibit the proliferation and invasion of LNCaP cells, the mechanism of which may be related with the inhibition of miR-324-3p to further promote FBXW7 expression.

Knockdown of lncRNA CCAT1 enhances sensitivity of paclitaxel in prostate cancer via regulating miR-24-3p and FSCN1.

Drug resistance limits the efficacy of chemotherapy in human cancers. Previous studies reported that long noncoding RNA colon cancer-associated transcript 1 (CCAT1) regulated progression of prostate cancer (PCa). However, the potential role of CCAT1 in the sensitivity of paclitaxel (PTX) in PCa and its mechanism remain largely unknown. The PTX-resistant PCa cells were established in PC3 and DU145 cells by increasing concentrations of PTX. The expressions of CCAT1, microRNA-24-3p (miR-24-3p) and fascin1 (FSCN1) were measured by quantitative real-time polymerase chain reaction. The viability and apoptosis were detected by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide assay, flow cytometry and western blot, respectively. The interaction among CCAT1, miR-24-3p and FSCN1 was explored by luciferase activity, RNA immunoprecipitation, RNA pull-down and western blot, respectively. Results showed that the expressions of CCAT1 were up-regulated and miR-24-3p was down-regulated in PCa and PTX-resistant PCa cells (PC3-TXR and DU145-TXR). Knockdown of CCAT1 or overexpression of miR-24-3p inhibited survival rate, half maximal inhibitory concentration (IC50) of PTX but increased apoptosis in PC3-TXR and DU145-TXR cells after treatment of PTX. miR-24-3p was bound to CCAT1 and its abrogation reversed knockdown of CCAT1-mediated increase of PTX sensitivity in PC3-TXR and DU145-TXR cells. Moreover, FSCN1 restoration attenuated miR-24-3p-mediated inhibition of PTX resistance. Besides, FSCN1 level was enhanced in PCa and PTX-resistant PCa cells and regulated by CCAT1 and miR-24-3p. Our data suggested interference of CCAT1 contributed to PTX sensitivity in PCa by regulating miR-24-3p and FSCN1, indicating a novel avenue for treatment of PCa through regulating chemoresistance.

MELK is Upregulated in Advanced Clear Cell Renal Cell Carcinoma and Promotes Disease Progression by Phosphorylating PRAS40.

Clear cell renal cell carcinoma (ccRCC) is the most common type of kidney cancer. However, stage IV ccRCC is generally incurable and its molecular mechanism has not yet been fully clarified. In this study, in order to screen differentially expressed genes (DEGs) between stage IV and stage I ccRCC specimens, we initially analyzed The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GSE73731). We found that maternal and embryonic leucine zipper kinase (MELK) is upregulated in stage IV ccRCC samples and that upregulation of MELK is significantly correlated with advanced disease status. Furthermore, both loss and gain-of-function assays strengthen the evidence that MELK enforces the malignant phenotype of ccRCC cells through over-activating the mammalian target of rapamycin complex 1 (mTORC1) pathway. Mechanistically, we verified that the oncogenic effect of MELK occurs through phosphorylating PRAS40, an inhibitory subunit of mTORC1, and through disrupting the interaction between PRAS40 and raptor. In summary, these results elucidate the important role of MELK in the progression of ccRCC and indicate that MELK may be a novel regulator of ccRCC progression by over-activating the mTORC1.

Long noncoding RNA lnc-DILC stabilizes PTEN and suppresses clear cell renal cell carcinoma progression.

BACKGROUND: Increasing evidence has indicated that long noncoding RNAs (lncRNAs) are crucial regulators affecting the progression of human cancers. Recently, lncRNA downregulated in liver cancer stem cells (lnc-DILC) was identified to function as a tumor suppressor inhibiting the tumorigenesis and metastasis in liver cancer and colorectal cancer. However, to date, little is known about the functional roles of lnc-DILC in modulating malignant phenotypes of clear cell renal cell carcinoma (ccRCC) cells. METHODS: lnc-DILC expression in human ccRCC tissues was detected by qRT-PCR. Overexpression and knockdown experiments were carried out to determine the effects of lnc-DILC on ccRCC cell proliferation, migration and invasion. To reveal the underlying mechanisms of lnc-DILC functions in ccRCC cells. RNA immunoprecipitation, RNA pull-down, in vivo ubiquitination, co-immunoprecipitation and western blot assays were performed. RESULTS: Here, we identified that lnc-DILC levels were dramatically downregulated in ccRCC tissues. Loss of lnc-DILC expression was correlated with larger tumor size, advanced tumor grade and lymph node metastasis, and also predicted worse prognosis in patients with ccRCC. Functionally, knockdown and overexpression experiments demonstrated that lnc-DILC inhibited cell proliferation, migration and invasion in ccRCC cells. Mechanistic investigation revealed that lnc-DILC bound to tumor suppressor PTEN and suppressed its degradation. lnc-DILC repressed the PTEN ubiquitination through blocking the interaction between PTEN and E3 ubiquitin ligase WWP2 and recruiting the deubiquitinase USP11 to PTEN. Moreover, we demonstrated that PTEN-AKT signaling was crucial for lnc-DILC-mediated suppressive effects. CONCLUSIONS: In summary, our research revealed a novel mechanism by which lnc-DILC regulates PTEN stability via WWP2 and USP11, and shed light on potential therapeutic strategies by the restoration of lnc-DILC expression in patients with ccRCC.

[Overexpression of miR-519d-3p inhibits the proliferation of DU-145 prostate cancer cells by reducing TRAF4].

Objective To observe the effect of microRNA-519d-3p (miR-519d-3p) on the proliferation of prostate cancer cells and explore the possible molecular mechanism. Methods The expression level of miR-519d-3p in PC-3, DU-145, 22RV1, PC-3M, LNCaP human prostate cancer cells and RWPE-1 human normal prostate epithelial cells was detected by real-time quantitative PCR. miR-519d-3p mimics or negative control microRNAs (miR-NC) was transfected into the prostate cancer cells with the lowest level of miR-519d-3p expression. Transfection efficiency was examined. The effect of miR-519d-3p on the cell cycle of prostate cancer was detected by flow cytometry. MTT assay and plate clone formation assay were used to detect its effect on the proliferation of prostate cancer cells. Bioinformatics software was used to predict and dual luciferase reporter assay was used to validate the target gene of miR-519d-3p. Real-time quantitative PCR was used to detect the expression of miR-519d-3p target gene. Western blot analysis was used to detect the expression of target gene protein and downstream protein. Results The expression of miR-519d-3p in normal prostate epithelial cells was significantly higher than that in prostate cancer cells, and the lowest was found in DU-145 cells. After transfected with miR-519d-3p mimics, the expression level of miR-519d-3p in DU-145 cells increased significantly. Bioinformatics prediction and dual luciferase reporter gene confirmed that tumor necrosis factor receptor associated factor 4 (TRAF4) was the target gene of miR-519d-3p. Overexpression of miR-519d-3p significantly reduced the expression of TRAF4 gene and its downstream TGF-ß signaling pathway proteins in the prostate cancer cells. Conclusion The expression of miR-519d-3p is down-regulated in prostate cancer cells. Overexpression of miR-519d-3p can inhibit the proliferation of prostate cancer cells. The possible mechanism is that miR-519d-3p inhibits the expression of TRAF4.

[miR-503-5p inhibits the proliferation of T24 and EJ bladder cancer cells by interfering with the Rb/E2F signaling pathway].

Objective To observe the effect of microRNA-503-5p (miR-503-5p) on the growth of T24 and EJ bladder cancer cells, and explore the possible molecular mechanism. Methods The miR-504-5p mimics or miR-NC was transfected into T24 and EJ cells. The target gene of miR-503-5p was predicted by bioinformatics. The expressions of E2F transcription factor 3 (E2F3) mRNA and Rb/E2F signaling pathway mRNA were detected by the real-time quantitative PCR (qPCR). The expressions of Rb/E2F signal pathway proteins E2F3, cyclin E, CDK2, Rb and p-Rb were detected by Western blotting. The cell cycle of bladder cancer cell lines was determined by flow cytometry. MTT assay and plate cloning assay were performed to observe the proliferation ability of bladder cancer cells. Results After miR-503-5p mimics transfection, the expression of miR-503-5p in bladder cancer cells significantly increased. The increased expression of miR-503-5p significantly reduced the expressions of E2F3 mRNA and Rb/E2F signaling pathway mRNA in bladder cancer cells. What's more, the expressions of Rb/E2F signal pathway proteins were down-regulated. The bladder cancer cells were arrested in G0/G1 phase, and their growth was significantly inhibited by miR-503-5p. Conclusion The miR-503-5p over-expression can inhibit the growth of bladder cancer cell lines T24 and EJ by down-regulating the expression of the Rb/E2F signaling pathway.

Knockdown of Serine-Arginine Protein Kinase 1 Inhibits the Growth and Migration in Renal Cell Carcinoma Cells.

The pre-mRNA splicing regulator serine-arginine protein kinase 1 (SRPK1), a member of the SR kinase family, plays an essential role in cancer development and various pathophysiological processes. However, its expression pattern and functions in renal cell carcinoma (RCC) remain unknown. Therefore, the aim of this study was to assess the role of SRPK1 in RCC. Our data showed that SRPK1 was significantly upregulated in human RCC tissues and cell lines. SRPK1 interference significantly inhibited the proliferation of RCC cells and inhibited tumor growth in vivo. In addition, SRPK1 interference also suppressed migration and invasion in RCC cells. A mechanistic study showed that SRPK1 interference inhibited the phosphorylation of PI3K and Akt in RCC cells. In conclusion, our findings suggest that SRPK1 interference inhibits the growth and invasion of RCC cells through suppressing the PI3K/Akt signaling pathway. Thus, SRPK1 might be a therapeutic target for the treatment of RCC.

Clinical comparison of the efficiency and security of balloon dilators versus fascial dilators in percutaneous nephrolithotripsy (PCNL).

OBJECTIVE: To compare the efficiency and security of the balloon dilators versus fascial dilators in percutaneous nephrolithotripsy (PCNL), We compared the difference of intraoperative and postoperative parameters of patients using these two different methods of expansion and having no significant statistic differences in peroperative parameters. METHODS: This is a retrospective analysis of 134 patients undergoing PCNL with upper urinary calculi from January 2012 to January 2014 in Luoyang Central Hospital affiliated to Zhengzhou University. These patients meeting the inclusion criteria were divided into two groups: the group of balloon dilators (group A) and the group of fascial dilators (group B). Two groups were compared for success rate of first expansion, clearance of stone, duration of surgery, intraoperative hemorrhage, blood transfusion rate, postoperative hospitalization and the incidence of complications. RESULT: In Group A, a total of 91 patients (51 men and 40 women, mean age 51.22±8.96 years, ranged from 28 to 68 years, the calculi maximum diameter from 0.9 to 4.5cm, 28 cases with a history of gravel, mean Body mass index 24.20±2.34, 73 cases with hydronephrosis and 26 cases with underlying diseases such as hypertension, diabetes and the like) undergoing PCNL were retrospectively reviewed. Similarly, In Group B, a total of 43 patients (28 men and 15 women, mean age 49.64±10.62 years, ranged from 15 to 70 years, the calculi maximum diameter from 1.1 to 5.2cm, 18 cases with a history of gravel, mean Body mass index 24.40±2.70, 38 cases with hydronephrosis and 14 cases with underlying diseases such as hypertension, diabetes and the like) undergoing PCNL were retrospectively reviewed. Our results showed that there was a statistically significant better outcome in Group A than in Group B in terms of success rate of first exploration, duration of operation, intraoperative hemorrhage, postoperative hospitalization and the incidence of complications. Additionally, there was no statistically significant difference with respect to clearance of stone and incidence of blood transfusion in the two groups. CONCLUSION: Balloon dilators had shorter operation time, less bleeding, higher success rate of first expansion, less postoperative complications and shorter postoperative hospitalization than fascial dilators in PCNL.

microRNA-340 Suppresses Tumorigenic Potential of Prostate Cancer Cells by Targeting High-Mobility Group Nucleosome-Binding Domain 5.

Aberrantly expressed microRNAs (miRs) are extensively involved in tumorigenesis. microRNA-340 (miR-340) has been reported as a tumor suppressor in various cancer types. However, whether miR-340 plays an important role in prostate cancer remains unknown. This study aims to investigate the expression pattern of miR-340 and its functional significance in prostate cancer. Results showed that miR-340 expression was frequently downregulated in human prostate cancer cell lines and cancer tissues. miR-340 overexpression suppressed proliferative and invasive properties of prostate cancer cells. This overexpression also promoted prostate cancer cell apoptosis. Conversely, miR-340 silencing showed an opposite effect. Intriguingly, on the basis of bioinformatics analysis and luciferase reporter assay, we found that miR-340 directly targeted the 3'-untranslated region of the high-mobility group nucleosome-binding domain 5 (HMGN5). Quantitative polymerase chain reaction and western blot analysis further verified the results and demonstrated that miR-340 regulated HMGN5 expression. Correlation analysis also showed that HMGN5 expression levels were significantly inversely correlated with the miR-340 expression in prostate cancer tissues. Furthermore, miR-340 overexpression significantly decreased the protein expression of cyclin B1, Bcl-2, and matrix metalloproteinase-9, which are critical regulators for maintaining tumorigenic potential of cancer cells. In addition, overexpression of HMGN5 significantly reversed the inhibitory effect of miR-340 on prostate cancer cell proliferation and invasion. In summary, this study suggests that miR-340 suppresses the tumorigenic potential of prostate cancer cells. Moreover, the decreased miR-340 expression may contribute to the development and progression of prostate cancer through a mechanism that involves HMGN5. Thus, miR340 and its target gene HMGN5 can serve as potentially useful therapeutic candidates for prostate cancer treatment.