Effects of long non-coding RNA RP1-90L14.1 on the biological behaviors of cancer prostate LNCaP cells and its regulating mechanisms / 中华男科学杂志

Objective@#To investigate the effects of long non-coding RNA RP1-90L14.1 on the proliferation, migration and invasion of prostate cancer LNCaP cells and the expressions of GRIN2A and BACE2.@*METHODS@#Using RT-PCR, we detected the expression of RP1-90L14.1 in LNCaP and LNCaP-AI cells, transiently transfected the RP1-90L14.1 overexpression plasmid (the RP1-90L14.1 group) and vector plasmid (the LNCaP-NC group) into the LNCaP cells, and cultured the two groups of cells with ordinary medium and phenol red-free activated carbon adsorption medium (PRF-ACA). Then we examined the proliferation, migration and invasiveness of the cells by CCK-8 and Transwell, and determined the mRNA and protein expressions of GRIN2A and BACE2 by RT-PCR and Western blot.@*RESULTS@#The expression of RP1-90L14.1 was significantly higher in the LNCaP-AI than in the LNCaP cells (8.49 ± 0.43 vs 2.53 ± 0.95, P < 0.05), and so was that of LNCaP-RP1-90L14.1 in the RP1-90L14.1 than in the LNCaP-NC group after transfection (0.71 ± 0.22 vs 0.02 ± 0.01, P < 0.05). The optical densities (OD) of the cells were 51.95% and 50.69% higher in the RP1-90L14.1 than in the LNCaP-NC group after 72 hours of culture with ordinary medium and phenol red-free ACA (1.22 ± 0.08 vs 0.08 ± 0.05, P < 0.05; 0.79 ± 0.02 vs 0.53 ± 0.05, P < 0.05), and 51.72% and 60.23% higher in the former than in the latter after 96 hours (1.72 ± 0.07 vs 1.13 ± 0.05, P < 0.05; 1.18 ± 0.05 vs 0.73 ± 0.08, P < 0.05). The numbers of the migrating cells cultured with common medium and PRF-ACA were markedly higher in the RP1-90L14.1 than in the LNCaP-NC group after transfection (682.0 ± 42.7 vs 422.0 ± 37.1, P < 0.05; 419.0 ± 42.9 vs 251.0 ± 25.9, P < 0.05), and so were those of the invading cells (507.0 ± 22.2 vs 274.0 ± 19.6, P < 0.05; 352.0 ± 14.1 vs 216.0 ± 14.3, P < 0.05). Statistically significant differences were observed between the RP1-90L14.1 and LNCaP-NC groups in the mRNA and protein expressions of GRIN2A (5.13 ± 0.89 vs 2.09 ± 0.54, P < 0.05; 5.88 ± 0.29 vs 2.03 ± 0.22, P < 0.05) and BACE2 (5.82 ± 0.50 vs 2.53 ± 0.30, P < 0.05; 4.89 ± 0.19 vs 3.37 ± 0.13, P < 0.05).@*CONCLUSIONS@#　lncRNA RP1-90L14.1 may play important roles in the proliferation, migration and invasiveness of prostate cancer cells. RP1-90L14.1 can promote the expressions of GRIN2A and BACE2 and may have an endogenous competitive relation with GRIN2A and BACE2.

Establishment of enzalutamide-resistant human prostate cancer cell lines and screening of lncRNA and mRNA expression profiles / 中华男科学杂志

Objective@#To establish enzalutamide-resistant human prostate cancer cell lines and screen out the lncRNA and mRNA expression profiles associated with enzalutamide resistance.@*METHODS@#Human prostate cancer cell lines LNCAP and C4-2B were cultured with 10 μmol/L enzalutamide for 6 months in vitro for the establishment of enzalutamide-resistant subclones LNCAP-ENZA and C4-2B-ENZA. The IC50 value and enzalutamide resistance index of each cell line were examined by MTT assay, the expressions of enzalutamide-related genes FL-AR, AR-V7 and HnRNPA1 were determined by Western blot, and the lncRNA and mRNA differential expressions of C4-2B and C4-2B-ENZA were detected by high-throughout lncRNA microarray.@*RESULTS@#Compared with LNCAP and C4-2B, the IC50 values of enzalutamide-resistant subclones LNCAP-ENZA (60.83 μmol/L) and C4-2B-ENZA (88.32 μmol/L) were increased significantly (P < 0.05) and the enzalutamide-resistance indexes of the LNCAP-ENZA and C4-2B-ENZA cells were 4.94 and 4.67, respectively. The expressions of AR-V7 and HnRNPA1 were markedly up-regulated in the LNCAP-ENZA and C4-2B-ENZA cells as compared with those in the LNCAP and C4-2B cells, but that of FL-AR showed no significant change. A total of 1 440 lncRNAs and 1 236 mRNAs were identified as differentially expressed in the C4-2B-ENZA cells.@*CONCLUSIONS@#Enzalutamide -resistant human prostate cancer cell subclones LNCAP-ENZA and C4-2B-ENZA were successfully established and enzalutamide resistance-associated lncRNA and mRNA were identified, which may provide some molecular evidence for the management of enzalutamide-resistant human prostate cancer.

Construction of Lentiviral Expression Vector Carrying SOX9 Gene and Its Stable Expression in LNcap Cell / 昆明医科大学学报

Objective To construct the lentiviral expression vector of SOX9 gene and establish a LNcap cell strain with stable expression of SOX9 . Methods SOX9 gene was amplified by PCR and cloned into lentiviral expression vector pLVX-IRES-Puro. pLVX-IRES-FLAG-SOX9 recombinant plasmid was verified by restriction enzyme digestion and DNA sequencing. Then we gained recombinant virus particles in packaging cell HEK 293 and infected LNcap cell. The monoclonal LNcap cell strain stably expressed were obtained through puromycin screening. The mRNA level and protein level in the infected LNcap cell were detected by qRT-PCR and Western blot respectively. Results Restriction enzyme digestion and sequencing demonstrated that SOX9 cDNA was successfully cloned into pLVX-IRES-FLAG lentiviral vector. After the transfection to LNCaP cells, the monoclonal cell strain of stably expressed SOX9 were obtained by puromycin screening, which showed expression of SOX9 mRNA detected by qRT-PCR. Western blot analysis revealed that SOX9 protein expressed markedly. Conclusion The recombinant lentiviral vector bearing human SOX9 cDNA has been successfully constructed, and the exogenous expression of SOX9 in LNcap cells was achieved.

Downregulation of PTTG1 expression inhibits the proliferation and invasiveness and promotes the apoptosis of human prostate cancer LNCaP-AI cells / 中华男科学杂志

Objective@#To investigate the effects of down-regulation of PTTG1 expression on the proliferation, invasiveness and apoptosis of androgen-independent human prostate cancer LNCaP-AI cells and their sensitivity to androgen antagonists.@*METHODS@#Human prostate cancer LNCaP-AI cells were transfected with siRNA targeting the PTTG1 gene using the Lipofectamine 2000 transfection reagent. The proliferation, invasiveness and apoptosis of the cells were detected by MTT, Transwell assay and flow cytometry, respectively. The protein expressions of PTTG1, p-Akt, and p-ERK were determined by Western blot and the mRNA expression of PTTG1 measured by agarose gel electrophoresis.@*RESULTS@#The siRNA expression vector markedly down-regulated the expression of PTTG1, which effectively suppressed the proliferation of the LNCaP-AI cells, with the inhibition rates of (19.47 ± 2.12), (24.01 ± 2.13) and (48.02 ± 2.22)% at 24, 48 and 72 hours, respectively, after transfection, with statistically significant differences among the three groups (P <0.05). The number of the cells passing through the polycarbonate film was remarkably decreased at 24, 48 and 72 hours (74.67 ± 9.85, 56.44 ± 8.66 and 37.33 ± 6.14) as compared with the baseline (111.11 ± 13.47) (P <0.01), while the apoptosis rate of the cells was significantly increased at 24, 48 and 72 hours (18.32 ± 0.94), (19.94 ± 1.30) and (21.73 ± 1.88)% in comparison with the baseline (［2.17 ± 0.49］%)， (P <0.05). PTTG1 siRNA combined with androgen antagonist flumatide exhibited even more significant effects in inhibiting the proliferation and promoting the apoptosis of the LNCaP-AI cells than either used alone, and in a flumatide dose-dependent manner. The inhibition and apoptosis rates of the LNCaP-AI cells treated with 50 nmol/L flumatide were (27.13 ± 3.52) and (3.94 ± 0.48)%, and those treated with siRNA + 50 nmol/L flumatide were (67.51 ± 5.13) and (19.93 ± 1.72)%, respectively, both with statistically significant differences between the two groups (P <0.05). The inhibition and apoptosis rates of the cells treated with 100 nmol/L flumatide were (43.72 ± 3.90) and (5.33 ± 0.66)%, and those treated with siRNA + 100 nmol/L flumatide were (73.19 ± 4.78) and (23.43 ± 1.76)%, respectively, both with statistically significant differences between the two groups (P <0.05).@*CONCLUSIONS@#The siRNA expression vector can down-regulate the expression of PTTG1, which can inhibit the proliferation and invasiveness of LNCaP-AI cells, promote their apoptosis, and increase their sensibility to androgen antagonists. Suppressing the expression of PTTG1 may enhance the effect of androgen-deprivation therapy on advanced prostate cancer.

Expression of pituitary tumor-transforming gene 1 during the development of androgen-independent prostate cancer / 中华男科学杂志

<p><b>Objective</b>To explore the expression of pituitary tumor transforming gene 1 (PTTG1) during the transformation of prostate cancer from androgen-dependent (ADPC) to androgen-independent (AIPC).</p><p><b>METHODS</b>We established an AIPC cell model LNCaP-AI by culturing the androgen-dependent LNCaP cell line in the hormone-deprived medium for over 3 months. The cell model was verified and the PTTG1 expression in the LNCaP cells was detected by Western blot and RT-PCR during hormone deprivation.</p><p><b>RESULTS</b>The AIPC cell model LNCaP-AI was successfully established. The PTTG1 expression was gradually increased in the LNCaP cells with the prolonged time of hormone deprivation and the expressions of matrix metalloproteinases MMP-2 and -9 were elevated at the same time.</p><p><b>CONCLUSIONS</b>The expression of PTTG1 is increased gradually in AIPC, which may be a target of gene therapy for advanced prostate cancer.</p>

Impact of TGIFLX Expression on the Regulation of BCL2 and BAX in Prostate Cancer Cell Line (LNCaP).

Aims: The homeoprotein TGIFLX (transforming growth factor-β-induced factor 2-like, Xlinked), which is essential in male reproduction and development and likely oncogenic when aberrantly expressed in prostate. We have previously shown an aberrant expression of TGIFLX in the majority of human prostate tumors. However, mechanism by which TGIFLX acts in prostate cancer is unknown. The aim of this study was to investigate the effects of overexpression of wild-type TGIFLX (wt-TGIFLX) on LNCaP, human prostate adenocarcinoma cells. Study Design: As a prospective study, we used adenovirus expression system for evaluation of TGIFLX expression effects on mammalian cells. Place and Duration of Study: Medical Genetics Department, Tehran University of Medical Sciences (TUMS), between December 2009 and July 2012. Methodology: We cloned entire coding sequence of TGIFLX gene into adenovirus and subsequently LNCaP cells were transfected with the recombinant virus harboring TGIFLX cDNA or control. The TGIFLX expression was confirmed by microscopic analysis and RT-PCR technique. Following molecular cloning and characterization of TGIFLX transcription factor, we then studied the effects of overexpression of TGIFLX in LNCaP cells on mRNA expression of BAX and BCL2 genes. Results: Our results showed that overexpression of TGIFLX downregulated BCL2 gene (P<0.05) and upregulated BAX gene (P<0.05) at transcript level. Our results suggested that TGIFLX could be a tumor suppressor gene and might be involved in initiation and/or development. Conclusion: TGIFLX can play a role as a transcriptional modulator of the genes involved in cell cycle pathway. But still more investigations are necessitated for clarifying this claim.

Establishment of androgen-independent human prostate cancer line LNCaP by gradual deprivation of hormone / 第二军医大学学报

Objective: To establish and identify androgen-independent human prostate cancer cell line LNCap by culturing LNCaP cells with gradual deprivation of hormone. Methods: LNCaP cells were cultured in the medium with gradual deprivation of hormone (treated by active carbon to simulate androgen deprivation) for 10 days; and then the cells were cultured with complete deprivation of androgen for 3 months till the cell entered the rapid proliferation phase again. The cell growth and expression of PSA and androgen were examined by CCK-8, immunfluorescence and RT-PCR methods. Results: LNCaP cells grew slowly after deprivation of hormone and took on a neuroendocrine phenotype and cluster growth pattern. After 3 months' non-androgen culture,the cells regained original morphology and growth. CCK-8 indicated that LNCaP cells could grow in non-androgen condition; immunofluorescence assay indicated that LNCaP-AI cells could regain PSA-secreting activity in non-androgen condition; and RT-PCR suggested that androgen was highly expressed in LNCaP-AI cells. Conclusion: Androgen-independent LNCaP cell line can be established by culturing with gradual deprivation of hormone for 3 months.

Molecular Cloning and Characterization of a Putative Promoter Region of mPC-1 Gene Homologous to hPC-1 / 中国生物化学与分子生物学报

To identify the regulatory region that are responsible for the expression of mPC-1, we have isolated and characterized the mPC-1 gene promoter. Sequence analysis of the mPC-1 5' -flanking region and a series of truncated constructs were performed, which were transiently transfected into the prostate cancer cell lines and non-prostate cancer cell lines and analyzed through Dual-luciferase reporter assay system. The relative activity of mPC-1 gene promoter was by far higher than pGL3-control containing SV40 promoter and enhancer and p61-PSA containing hPSA 6 kb promoter in AR (androgen receptor, AR ) -positive prostate cancer cell lines. The region from 599 bp to 449 bp of mPC-1 promoter might contain a negative regulatory element. The expression of mPC-1 1.1 kb fragment is mainly restricted into prostate cancer cell lines. The relative activity of mPC-1 1.1 kb 5'-flanking region was regulated by androgen. The results demonstrated that the 1.1 kb fragment of mPC-1 5' -flanking region was relatively strong and prostate cancer cell specific promoter region.The 1.1 kb promoter of mPC-1 gene might be well suited to prostate cancer gene therapy if the promoter was properly modified.