| Renal cell carcinoma (RCC) is a common type of urologic malignancy, constituting in the top three of urologic cancers and the top ten of all body cancers. In recent decades, as the change of people’s life style, environmental pollution, ageing population and some other factors, the morbidity of RCC is increasing year by year worldwide. Because of the insidious onset, early RCC is usually diagnosed by imaging examination in physical checkup. It is often in an advanced stage when RCC patient has tumor symptoms. RCC is of natural resistance against radiotherapy and chemotherapy, so we have no reliable treatment protocols for advanced RCC. In recent years many kinds of molecular targeted therapy are used in clinical application for RCC, and very few patients benefit significantly and even have a long-term survival. However, it is very expensive to undergo molecular targeted therapy, and most patients only prolong a few months survival. Considering the gene effect on tumor, the occurrence and development of RCC are also related to various genes complicatedly. Hence, in-depth research of the tumorigenesis and development of the RCC related genes, is very important for strengthening and promoting the prevention and treatment of the RCC.The cell cycle is the series of events that take place in a cell resulting in its DNA replication and division. Numerous mechanisms exist for the control of the cell cycle to ensure smooth and precise progression with high fidelity. Mitosis, the most crucial phase in the cell cycle, has been one of the most active research topics in cell biology since its discovery and accurate description by Walter Flemming. During mitosis replication and division of the nuclear material allows one mother cell to give rise to two daughter cells with exact genetic copies. Spectacular changes occur within the cell during this phase such as chromatin condensation, the nuclear membrane breakdown, mitotic spindle assembly, chromosome congression and chromosome segregation. Correct bipolar spindle formation and attachment of chromosomes to the microtubules are prerequisites for the accurate segregation of chromosomes resulting in an errorless chromosome complement in both daughter cells. The inability to faithfully segregate chromosomes in mitosis causes chromosome instability (CIN) and aneuploidy, hallmarks of solid tumors. It is becoming increasingly clear that CIN is not simply a passenger phenotype but likely plays a causative role in a substantial proportion of malignancies. Kinetochore is a large multiprotein complex that is assembled on centromeric DNA and serves as the attachment site for spindle microtubules, which is essential for proper chromosome segregation during mitosis. As hundreds of centromere protein families were discovered, their role in carcinogenesis and carcinoma progression cause more people’s attention. Some common members, like CENPA, CENPF, CENPH, INCENP, etc., have a close association with pathophysiology of many tumors. However, the study of their role in RCC is rare, rough the abnormal expression of centromere protein families should exist.Centromere protein H (CENPH), an important member of centromere protein family, is an essential component of the active kinetochore complex, localized in the inner plate of the kinetochore with CENPA and CENPC. CENPH plays important role in the regulation of right cell’s mitosis. Recently the vital role of centromere protein family in cancer progression causes more people’s attention. There have been some new researches of CENPH in different kinds of cancer, however, as the tumor heterogeneity, it is unknown whether CENPH is playing a key role in RCC. In this study, we investigated the effect of CENPH on RCC growth and the underlying mechanisms.This study includes three parts. In the first part, we analyze RCC RNA-Seq data which contain lots of differentially expressed genes between RCC and adjacent normal tissues. We find out the expression condition of centromere protein family in the RNA-Seq expression profile. In the second part, we test and verify the CENPH expression in RCC through quantitative real-time PCR (qRT-PCR) and Western blotting. We also investigate the relationship between CENPH expression and the clinicopathological features of RCC, and explore the possibility of being a diagnostic and prognostic index. In the third part, we detected the expression of CENPH in RCC cell lines, and established a silence cells model of CENPH gene by RNA interference in CENPH unexpressed cell lines. We investigated effects of CENPH gene on the biological behaviors (proliferation, apoptosis and etc.) of the RCC cells.Part I. RCC RNA-Seq reveals the high expression of CENPH in RCC tissues Purposes:1. Find different gene expression profile through RCC RNA-Seq2. Do the different expression analysis of centromere protein families between RCC and adjacent normal tissues in the RNA-Seq data3. Identity the expression condition of CENPH gene in RCC and other kinds of cancer tissuesMaterials and methods:1. Conduct transcriptome sequencing of 34 pairs of RCC and adjacent normal tissues using Illumina HiSeqTM 2000 sequencing platform;2. Conduct quality control, alignment and filtering of the sequencing data, then carry out GO function analysis and KEGG pathway analysis, and find the different gene expression profile between RCC and normal kidney tissues;3. Analyze the expression condition of centromere protein families in RCC based on the above data;4. Search on RNA-Seq data and GENT microarray data and analyze the expression characteristics of CENPH in RCC.Results:1. RNA-Seq conducts gene expression profile of RCCRNA-Seq data of 34 pairs of RCC tissues showed genes expression profile containing 20453 distinct genes (unpublished data).2. Many kinds of centromere protein are upregulated in RCC tissuesWe analyze the expression profile of centromere protein families in the sequencing results. The data include 20 centromere protein genes:CENPQ, CENPJ, CENPL, CENPV, CENPB, CENPI, CENPK, CENPE, CENPP, CENPW, CENPA, CENPN, INCENP, CENPT, CENPH, CENPO, CENPBD1, CENPM, CENPF, CENPC1. Most of these genes are upregulated in RCC tissues, among which CENPH is the most evident case.3. CENPH have increased expression of various kinds of cancers including RCCCENPH was upregulated in the all 34 RCC tissues, and the search result of GENT database showed that CENPH have a high expression in different types of cancers.Conclusions:Centromere protein families are often upregulated in RCC tissues, in which CENPH is most evident. It may have relations with the pathogenesis and progression of RCC.Part II The character of CENPH expression in RCC tissues and its relationship to clinicopathological featuresPurposes:1. Explore the expression of mRNA and protein of CENPH in RCC tissues; 2. Analyze the relationship between CNEPH expression characteristics and the clinical pathological features;3. Analyze the relationship between CENPH expression and the prognostic of RCC. Discuss the potentiality of CENPH to be a biomarker of RCC.Materials and methods:1. Expressions of CENPH in 21 pairs of RCC and matched normal renal tissue were examined using qRT-PCR;2. CENPH protein expression in 109 paraffim-embedded archived RCC surgical samples was analyzed by immunohistochemistry (IHC);3. To statistically analyse the relationship between the expression of CENPH and clinical and pathological characteristics of patients with RCC using χ2 test in SPSS software package (standard version 20);4. The impact of CENPH protein expression profile on patients’ survival was determined by Kaplan-Meier life curve with Log-Rank test as well as Cox regression model.Conclusion:1. Increased expression of CENPH in RCC tissuesTo determine the CENPH mRNA expression, qRT-PCR analysis was conducted on 21 cases of paired RCC tissue and adjacent nontumor tissue. Our results exposed a statistically significant rise of CENPH mRNA in RCC tissues, as compared to the matched adjacent non-tumor tissues. This result is consonant with the RNA-Seq result.2. Immunohistochemical analysis of CENPH expression in RCC tissues and its relationship to clinicopathological featuresHistological assessment revealed that 87 of 109 (79.8%) RCC tissues showed positive CENPH staining. However only 24 of 109 (22.0%) adjacent nontumor tissue showed positive CENPH. And CENPH stained mainly in the nucleus of the cells, while the cytoplasm also lightly stained in some patients. IHC was employed to investigate the association between CENPH expression and clinicopathological features in the 109 RCC specimens. The expression level of CENPH was significantly associated with histological grade (P= 0.001), tumor stage (P= 0.014), and distant metastasis (P= 0.024). There was no significant association between CENPH expression and patients’ gender, age, tumor size and lymph nodes metastasis.3. High CENPH expression predicts poor prognosis in patients with RCCKaplan-Meier analysis showed that patients with high CENPH expression had significantly shorter overall survival time than those with low CENPH expression (Log-Rank test, P= 0.002). CENPH expression level (P<0.001) and clinical stage (P = 0.005) were significantly correlated with overall survival rate of patients with RCC. A multivariate analysis also showed that the relative expression of CENPH (P<0.001) and clinical stage (P= 0.018) were independent prognostic factors for overall survival of RCC patients.Conclusions:1. CENPH is highly expressed in human RCC.2. CENPH would be a novel clinical prognostic marker for RCC.Part III The role and mechanism of CENPH in biological behavior of RCC Purposes:1. Study the role of CENPH in biological behavior, including proliferation, apoptosis and etc., of RCC cells;2. Discuss the molecular mechanisms of CENPH in RCC cell proliferation and apoptosis.Materials and methods:1. Expressions of CENPH in four kinds of RCC cell lines and one Immortalized normal human proximal tubule epithelial cell line were examined using qRT-PCR and Western blotting;2. Small interfering RNA was infected with RCC cells to establish down-regulation CENPH RCC cell line model;3. CCK-8 assay, colony formation assay and annexin V-FITC/PI double labeling flow cytometric apoptosis analysis was used to investigate the influence of CENPH on biological behavior of RCC cells;4. The Western blotting experiment of cell proliferation marker Ki-67 and cell apoptosis marker Survivin were performed to detect the change after CENPH siRNA transfection.Results:1. Increased expression of CENPH in RCC cell linesTo determine whether CENPH was also upregulated in the human RCC cell lines; we used qRT-PCR and Western blotting to test CENPH mRNA and protein expression in four RCC cell lines (ACHN,786-0, A704 and A498) and a human proximal tubule epithelial cell line HK-2. CENPH mRNA and protein were highly expressed in three RCC cell lines (ACHN,786-O and A704) compared to HK-2 cells. This phenomenon was not seen in A498 cell.2. Downregulation of CENPH demotes proliferation of RCC cellsWe chose two CENPH high expression cell lines, ACHN and 786-O, to further investigate the role of CENPH in human RCC cells. After transfection with si-CENPH, cells showed a down-regulated mRNA and protein expression of CENPH compared to the negative control (si-NC) group. Our result indicated that we successfully down-regulated the CENPH expression in human RCC cell line ANCH and 786-0.Next, we conducted CCK8 and colony formation assay to determine whether knockdown of CENPH inhibited the proliferation of ACHN and 786-0 cell lines. CENPH siRNA significantly decreased cell viability of ACHN and 786-0 cells compared to control siRNA group, respectively. Furthermore, we found that the colony-forming ability of these two cell lines was lowered by transfection with CENPH siRNA.3. CENPH siRNA induces apoptosis of RCC cell linesACNH and 786-0 cell lines were transfected with si-CENPH and si-NC. Two days later, the cell apoptosis ratio was detected by flow cytometry. The results demonstrated that cells transfected with si-CENPH had a significantly higher apoptosis rate than cells transfected with si-NC.4. CENPH modulates Ki-67 and Survivin expressionAs CENPH expression affected cell proliferation, we checked out the proliferation markers that could be modulated by CENPH. A significant decrease of Ki-67 expression was found in CENPH knockdown cells compared with cells transfected with control siRNA. Moreover, we also found CENPH silencing inhibited expression of an inhibitor of apoptosis protein Survivin in RCC cell lines.Conclusions:1. CENPH is upregulated in most RCC cell lines;2. Knockdown of CENPH reduced cell proliferation, inhibited cell growth, and increased cell apoptosis;3. CENPH may impact the biological behavior of RCC though bridge Survivin protein.Presently, there are a number of promising molecular markers that address several clinical questions, but the available data are not valid enough for routine, clinical application. Further well-performed, reproducible studies are needed to display markers useful in the clinical work-up of patients with RCC. In this research, we discovered and identified the upregulated expression of CENPH in human RCC by using several kinds of lab techniques. In conclusion, our data for the first time showed that CENPH is upregulated in RCC, overexpression of CENPH in RCC tissue is closely associated with the development and progression of tumor, and CENPH expression detected through IHC could be an independent prognostic indicator for patients with RCC. Moreover, we validated the antitumor ability of si-CENPH in vitro. CENPH may thus serve as a potential prognostic marker and a novel therapeutic target for RCC patients. Further, larger-scale studies are needed to clarify the prognostic value and the functional properties of CENPH for RCC. Consequently, we should comply with the demand for large, multicenter, prospective investigations that are stratified for RCC type and treatment modalities. In vivo experiment and molecular mechanism research should also be conducted in future. This would lift molecular markers in diagnosis and treatment of RCC to a practical level, helping therapists to refine and economize their follow-up and to individualize treatment strategies. |
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