The Function And Regulation Mechanisms Of HP1γ In Colorectal Cancer

Colorectal cancer (CRC) is one of the most commonly digestive malignant tumors in the world, and the morbidity and cancer-related mortality are among the upper third of all tumors. In China, CRC is the third most frequently occurring cancer and the fifth leading cause of death of cancers. In recent years, with the change of the people’s lifestyle and diet, the incidence of CRC has increased in the world, and age at oneset is becoming younger. Although great advances have been made in imaging methods, chemotherapy and surgical strategy of development in recent years, but the survival rate has not improved significantly. Therefore, in-depth investigation on the underlying molecular mechanisms of the pathogenesis of CRC, for early diagnosis of colorectal cancer, targeted therapy and prognosis are of great significance. Heterochromatin protein 1-γ (HP1γ), a member of HP1 family, is encoded by the chromobox homolog 3 (CBX3) gene. As a special form of chromatin, HPly plays critical roles in many biological processes, such as heterochromatin formation, gene silencing, DNA damage repair, RNA splicing, transcriptional activation, stem cell differentiation, somatic reprogramming and so on. Given its wide distribution and different roles in gene regulation, HP1γ is probably closely involved in the regulatory process of tumorigenesis and development. However, the function of HP1γ in CRC is not clear, and it needs to be further clarified.In order to understand the role of HPly in CRC tissues, we first explored the expression of HP1γ in the Oncomine database and found it’s up-regulated in CRC tissues. Tissue microarrays (TMA) containing 180 CRC samples with matched normal tissues were constructed and immunohistochemistry (IHC) was used to detect HPly expression in paraffin-embedded CRC samples. Then, the relationship between HPly expression and clinicopathological parameters was analyzed, including gender, age, tumor location, differentiation, metastasis, TNM stage. The results from IHC assays showed that the expression of HPly in CRC tissues was higher than that in paired normal colon tissues (n=178,.P<0.001) and there was a negative correlation between HPly and CRC cell differentiation status. Kaplan-Meier survival analysis revealed that overall survival rate in high HPly expression CRC patients was significantly lower than that in low HPly expression CRC patients (Log-Rank, .P=0.001). The results from western blot and real-time PCR also suggested that the protein and mRNA level of HPly in 38 fresh samples were higher than that in paried normal CRC tissues (P＜0.01). Therefore, these results indicate that HPly protein levels were up-regulated in CRC tissues, and high levels of HPly expression correlates with poor prognosis and tumor differentiation.To assess the role of HPly in CRC development, we attempted to explore the biological function of HP1γ in CRC cell lines. We knocked down HP1γ in two colon cancer lines, HCT116 and SW620, by RNA interference. Cell proliferation was measured by CCK.-8, EdU, colony formation assays and xenograft model in nude mice; cell-cycle distribution and apoptotic cells were analyzed by flow cytometry; cell migration ability was tested through transwell and scratch wound healing assays. The results from CCK-8 and EdU assays revealed that, the growth, colony abilities of CRC cells with HP1γ knockdown decreased markedly compared with the control group, but the migration ability was not affected. Flow cytometry results suggested that the CRC cells with HPly knockdown were arrested in the G1 phase of the cell cycle, and apoptotic cells increased markedly compared with the control group. This implies that HPly might exert the effect on cell cycle related proteins to regulate cell proliferation. Our results further demonstrated that HP1γ promotes CRC proliferation, and is essential for CRC development.We next investigated potential mechanisms by which HPly knockdown inhibited CRC proliferation. We analyzed the expression of a spectrum of key proliferation-related genes in HCT116 cells by quantitative real-time PCR. We found that the expression level of p21Wafl/Cipl was the most increased when HP1γ was knocked down. p21Wafl/Cipl is a known tumor suppressor gene, due to its role as a key negative regulator of Gi/S transition in the cell cycle. To confirm the effect of HP1γ on p21Wafl/Cipl expression at the protein level, we performed western blot experiments using cellular extracts from HPly knockdown or scramble control HCT116 and SW620 cells. The p21Wafl/Cipl protein was expressed at low levels in the scramble control cells but was greatly increased in HPly knockdown cells. Then, we performed chromatin immunoprecipitation (ChIP) to analyze HP1γ binding to the p2lWafl/ClPl promoter. The ChIP results demonstrated that HPly was most enriched on the proximal promoter region at-297 ～-191, but was not bound on the far promoter regions at-2596～-2414 or-2069～-1906. When HP1γ was knocked down, enrichment of HP1γ was much reduced on the proximal promoter region. These results indicate that HPly binds the p2lWafl/Cipl promoter. The histone marks H3K9me2/3 on the p2iWafl/Cipl promoter were also significantly reduced when HP1γ was knocked down. These results indicate that regulation of p21Wafl/Cipl expression by HPly is associated with histone methylation.MicroRNAs (miRNAs) present diverse regulatory functions in a wide range of biological activities, and research on miRNAs has increased tremendously since it was discovered. To identify potential miRNAs targeting HPly, we employed a combination of TargetScan, PicTar and miRanda. Among the identified candidate miRNAs, miR-30a was identified by all three programs as targeting HPly. To determine whether miR-30a regulates HPly expression through binding to the 3’-UTR of HP1γ mRNA, the 3’-UTR of HP1γ mRNA containing the presumed miR-30a binding sites was fused downstream of the firefly luciferase gene in a reporter plasmid. The resulting plasmid was transfected into HCT116 and SW620 cells along with either miR-NC. As expected, luciferase reporter activity in cells transfected with miR-30a was reduced significantly compared to cells transfected with the scrambled control. A direct interaction between miR-30a and HPlγ was further confirmed by examination of HPlγ expression in HCT116 and SW620 cells overexpressing miR-30a. In these experiments, miR-30a overexpression was achieved by transfecting cells with miR-30a mimic. Western blot analysis confirmed that the level of HP1γ was significantly reduced in cells overexpressing miR-30a. HP1γ mRNA levels were unchanged after miR-30a transfection suggesting that miR-30a directly recognizes and binds to the 3’-UTR of the HP1γ mRNA resulting in reduction of HPlγ protein. Thus, we conclude that miR-30a specifically regulates HP1γ protein expression post-transcriptionally. Levels of miR-30a were found to be significantly downregulated in CRC tissues compared to levels from paired adjacent non-tumorous tissues. Levels of miR-30a and levels of HPlγ protein in CRC tissues exhibited a significant inverse correlation calculated by Pearson’s correlation (R=-0.5981, P＜0.01). Moreover, we determined that miR-30a specifically suppressed colorectal cancer growth by targeting HP1γ in vitro and in vivo. In conclusion, this study provides evidence that HPlγ functions as an oncogenic molecule in human CRC development. Our data demonstrate a critical role for HP1γ in CRC, and suggest that HP1γ could be a potential target for therapy of human CRC. MiR-30a appears to be a tumor suppressor through direct inhibition of expression of HPlγ protein. Our results suggest that the miR-30a/HP1γ/p21Waf1/Cip1 axis may represent a potential therapeutic target for treatment of human CRC.