Study Of The Role And Mechanism Of PRR11 In Gastric Cancer Cell Proliferation And Tumor Growth In Vivo

IntroductionGastric cancer(GC) is the fourth most frequent malignancy worldwide, with an estimated one million new GC cases and 0.73 million deaths are estimated to have occurred in 2008, accounting for 8% of the total cases and 10% of total deaths. Among them over 70% of new cases and deaths occur in developing countries. Although the diagnosis and treatment of GC has been progressed over the past decades, it remains the second most common cause of cancer related mortality in the world partially due to its late detection, which prohibits its successful intervention in the majority of patients. Till now, several novel tissue based prognostic and/or therapeutic markers have been reported for gastric cancer. However, only a few of these markers were used in clinical practice. Therefore, finding of novel molecular markers that can accurately predict outcome or predict therapeutic efficacy for patients with GC remains necessary.Proline-rich protein 11(PRR11) was identified as a novel gene and functionally characterized by Ji Ying in 2013 and they revealed that PRR11 had a critical role in both cell cycle progression and tumorigenesis, and may serve as a novel potential target in the diagnosis and/or treatment of human lung cancer. At present, knowledge of the PRR11 expression pattern in GC and its effects in gastric cancer was not reported and needs to be explored. In this study, we evaluated the PRR11 expression status in 216 GC patients and analyzed the relationship between PRR11 expression and clinicopathological parameters to determine whether PRR11 can predict GC patient prognosis. In addition, PRR11 was silenced in GC cell line, and the influence of cell proliferation and cell colony formation was assayed.MethodsHuman tissue specimens and patients’ informationTwo hundred and sixteen patients suffering from GC received surgical resection at Changhai Hospital in Shanghai, People’s Republic of China, from 2001 to 2005. The follow-up deadline was March 2010. Other patients’ characteristics including age, gender, tumor size, T stage, N stage, M stage, and tumor differentiation were presented in Table 1. All tissue specimens were obtained for the present study with patient informed consent, and the use of the human specimens was approved by Changhai Hospital Institutional Review Board.TMA and ImmunohistochemistryHE-stained slides from all patients were reviewed and identified by two experienced pathologists and the representative cores were pre-marked in the paraffin blocks. Tissue cylinders with a diameter of 1.5 mm were punched from the marked areas of each block and incorporated into a recipient paraffin block. Sections 4-μm thick were placed on slides coated with 3-aminopropyltriethoxysilane. Primary antibodies were diluted as follows: anti-PRR11(1:100), anti-LXN(1:100); anti-CTHRC1(1:100). An IHC staining S-P kit was used to visualize antibody binding on the slides. Counterstaining was performed with hematoxylin. The IHC staining of PRR11, LXN, and CTHRC1 in these specimens were evaluated by two individuals under an Olympus CX31 microscope. Tumors that demonstrated >10% staining for PRR11 were considered as having positive expression status.Cell lines and culture conditionsProtein expression was detected using the five GC cell lines SGC7901, MKN45, MKN28, HGC27, and MGC803. GC cell lines were maintained in Dulbecco’s modified Eagle’s medium(DMEM)(Hi-Clone) plus 10% FBS at 37°C with 5% CO2.Knockdown of PRR11 by lentiviral vector-loaded si RNA in GC cellslentivirus expression plasmid containing small interference RNA(5’-ACG CAG GCC UUA AGG AGA ATT-3’) targeting PRR11 was constructed by GENECHEM Corporation and used for infecting the GC cells in presence of 6 μg/ml polybrene. The infected gastric cancer cells were selected with puromycin, and the effect of PRR11 knockdown was detected by immunoblotting the cell lysates with anti-PRR11 antibody.Cell proliferation assay and Colony formation assayCells with stably-transfected PRR11-si RNA(PRR11-KO) or empty vector(control) were seeded in 96-well plates at a density of 5,000 cells per well and CCK8 assay was performed to measure the proliferation ratio, which was calculated as the absorbance at 48 h compared with that at 24 h. Similarly, PRR11-KO and Control cells were seeded in 6-well plates in triplicate at a density of 500 cells per well. Two weeks after incubation, the colonies were fixed with methanol/acetone(1:1) and stained with crystal violet. Colonies with cell numbers over 50 cells per colony were counted.Western blotTotal protein was prepared from these cell lines. Standard Western blotting was performed using a goat polyclonal antibody against human PRR11,LNX1,CTHRC1(dilution, 1:1000) and a horseradish peroxidase-conjugated anti-goat Ig G antibody(diluted,1:10000) as the secondary antibody. Protein expressions were normalized to β-actin expression. The proteins were visualized using the Amersham enhanced chemiluminescence system according to the manufacturer’s instructions.Real-Time Quantitative Reverse transcription(q RT)-PCR AssayReal-time quantitative PCR reaction was performed using SYBR1 Premix Ex Taq TM kit according to the manufacture’s instruction. PCR assay was done as previously described. GAPDH was used as an internal standard. PCR protocol was 95 degree for 1 min, then 40 cycles of 15 sec at 95 degree and 31 sec at 60 degree. Fold-expression was calculated using the ΔCt method. Reactions and analysis were performed using the ABI PRISM 7300 PCR and detection system. The primers used are as follow: PRR11:forward: 5’- CGT ATC TGC CAC CGA GAA CTT-3’, reverse: 5’- GAG ATG GTC TTC AGT GCT TCC T-3’; GAPDH: forward: 5’-TGA CTT CAA CAG CGA CAC CCA-3’, reverse: 5’- CAC CCT GTT GCT GTA GCC AAA-3.Animal modelsSubcutaneous tumor xenograft models were performed to evaluate the in vivo function alteration of PRR11-knockdown in SGC-7901 cell line. PRR11-KO SGC-7901 cell and controls(1×106 cells in 0.1ml PBS) were injected subcutaneously into the left flank of 4-week-old female Balb/c nude mice. Tumor diameter was measured every three days. Two weeks after implantation, all animals were sacrificed. The tumors were collected and tumor volume was calculated as V=0.52(length×width×depth).Statistical AnalysisStatistical analyses were carried out using SPSS 13.0 software. The relationships between expression of PRR11 and clinicopathological of gastric cancer were used chi-squared distribution. Survival analyses of patients with gastric cancer according to the expression of PRR11, LNX1, and CTHRC1 were used Kaplan-Meier curves. Univariate and multivariate analyses were based on a Cox proportional hazard regression model. The variables showing significance(p<0.05) by univariate analysis were adopted when multivariate Cox proportional hazards analysis was performed. Quantitative variables were analyzed using Student’s t-test. Experimental data were presented as the mean of each condition ± S.D. and p < 0.05 were considered statistically significant.ResultsCharacteristics of PRR11 expression and relations with clinicopathological of gastric cancerWe evaluated the expression of PRR11 in a cohort of 216 patients diagnosed with GC by immunohistochemistry. Low intensity of cytoplasmic PRR11 immunostaining was visible in the epithelial cells from normal tissues, while high intensity of PRR11 staining was observed in gastric cancer cells were variable. Of these tumor samples, 107 out of 216(49.5%) had positive PRR11 expression and 109 cases showed PRR11 negative expression. Western blotting and RT-PCR assay further confirmed that PRR11 protein and m RNA was increased in tumor samples compared with that in the noncancerous tissues. In addition, relatively high level of PRR11 protein expression level were also detected in five GC cell lines, including MKN45, MKN28, SGC7901, HGC27, and MGC803. The correlations between the level of PRR11 expression and various clinicopathological parameters were assayed. The PRR11 expression was not associated with age, gender, tumor size, N stage, while was significantly associated with T stage, tumor differentiation, and TNM stage. These results indicate that high PRR11 protein expression may be associated with gastric cancer aggressiveness.PRR11 up-regulation was associated with poor survival for gastric cancer patientsAmong the 216 patients studied, 122 died during the follow-up period, and the median OS time was 51.5 months. The mean survival time for patients with negative PRR11 protein expression was 74.5 months, while the mean survival time for patients with positive PRR11 protein expression was 46.4 months. In addition, univariate COX regression analyses showed that tumor size, tumor stage, Regional lymph nodes positive, TNM stage, tumor differentiation and PRR11 expression were significantly correlated with overall survival. Multivariate analysis confirmed that tumor size, tumor differentiation and PRR11 expression were the independent prognostic predictor of the overall survival of gastric cancer patients. Patients were divided into two groups, those with TNM stage I/II and those stage III/IV, and the effect of PRR11 status on survival duration was assessed. This subgroup analysis revealed that the outcomes of patients with PRR11 overexpression were worse than those without PRR11 overexpression either in stages I/ II or in stage III/IV.PRR11 expression is associated with increased cellular proliferation and cell colony formation in vitro and tumor growth in vivo in SGC7901 gastric carcinoma cellsPRR11was stably silenced in a SGC-7901 cell line using lentiviral vector-loaded PRR11 sh RNA. Knockdown of PRR11(PRR11-KO) was confirmed via Western blot analysis, and the effect of PRR11-KO on cellular proliferation and colony formation was evaluated. Significant depletion of PRR11 in the transfected cells was observed, and down-regulation of PRR11 was associated with a dramatic decrease in both cell proliferation and cell colony formation in this cell line. Unsurprisingly, PRR11 down-regulation leads to retarded growth of gastric cancer cell lines in vivo.Depletion of PRR11 resulted in down-regulation of CTHRC1 and up-regulation of LXN.We performed western blot analysis using CTHRC1 and LXN antibody, to explore whether these proteins changed with depletion of PRR11. Results showed that CTHRC1 was downregulated and LXN was upregulated in PRR11-KO cells compared with Control cells. Further co-expression analysis was performed in GC tissues using immunohistochemistry. Data was showed that CTHRC1 expression was positively associated with PRR11 expression(r = 0.299, p<0.001) and LNX expression was negatively associated with PRR11 expression(r =-0.188, p=0.005) in GC tissues(Figure 4C). However, CTHRC1 expression was not associated with LNX expression(r =-0.042, p=0.539).Conclusion1. The PRR11 overexpresion was fund in GC tisusse and GC cell lines, and was associated with T stage, tumor differentiation, and TNM stage. These results indicate that high PRR11 protein expression may be associated with gastric cancer aggressiveness.2. Multivariate analysis confirmed that tumor size, tumor differentiation and PRR11 expression were the independent prognostic predictor of the overall survival of gastric cancer patients.3. PRR11 expression is associated with increased cellular proliferation and cell colony formation in vitro and tumor growth in vivo in gastric carcinoma cells.4. Depletion of PRR11 resulted in down-regulation of CTHRC1 and up-regulation of LXN.5. The exact mechanism needs to be further investigated, that may provide a new therapeutic target for GC patient.