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.However,the pathogenesis of CRC is still not clear.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-g（HP1γ）,a member of HP1 family,is encoded by the chromobox homolog 3（CBX3）gene.As a special form of chromatin,HP1γ 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 HP1γ 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 HP1γ expression in paraffin-embedded CRC samples.Then,the relationship between HP1γ 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 HP1γ in CRC tissues was higher than that in paired normal colon tissues（n=178,P<0.001）and there was a negative correlation between HP1γ and CRC cell differentiation status.Kaplan-Meier survival analysis revealed that overall survival rate in high HP1γ expression CRC patients was significantly lower than that in low HP1γ expression CRC patients（Log-Rank,P=0.001）.The results from western blot and real-time PCR also suggested that the protein and m RNA level of HP1γ in 38 fresh samples were higher than that in paried normal CRC tissues（P<0.01）.Therefore,these results indicate that HP1γ protein levels were up-regulated in CRC tissues,and high levels of HP1γ expression correlates with poor prognosis and tumor differentiation.To assess the role of HP1γ 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,Ed U,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 Ed U 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 HP1γ knockdown were arrested in the G1 phase of the cell cycle,and apoptotic cells increased markedly compared with the control group.This implies that HP1γ 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 HP1γ 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 p21^Waf1/Cip1 was the most increased when HP1γ was knocked down.p21^Waf1/Cip1 is a known tumor suppressor gene,due to its role as a key negative regulator of G1/S transition in the cell cycle.To confirm the effect of HP1γ on p21^Waf1/Cip1 expression at the protein level,we performed western blot experiments using cellular extracts from HP1γ knockdown or scramble control HCT116 and SW620 cells.The p21^Waf1/Cip1 protein was expressed at low levels in the scramble control cells but was greatly increased in HP1γ knockdown cells.Then,we performed chromatin immunoprecipitation（Ch IP）to analyze HP1γ binding to the p21^Waf1/Cip1 promoter.The Ch IP results demonstrated that HP1γ 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 HP1γ binds the p21^Waf1/Cip1 promoter.The histone marks H3K9me2/3 on the p21^Waf1/Cip1 promoter were also significantly reduced when HP1γ was knocked down.These results indicate that regulation of p21^Waf1/Cip1 expression by HP1γ is associated with histone methylation.Micro RNAs（mi RNAs）present diverse regulatory functions in a wide range of biological activities,and research on mi RNAs has increased tremendously since it was discovered.To identify potential mi RNAs targeting HP1γ,we employed a combination of Target Scan,Pic Tar and mi Randa.Among the identified candidate mi RNAs,mi R-30 a was identified by all three programs as targeting HP1γ.To determine whether mi R-30 a regulates HP1γ expression through binding to the 3’-UTR of HP1γ m RNA,the 3’-UTR of HP1γ m RNA containing the presumed mi R-30 a 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 mi R-NC.As expected,luciferase reporter activity in cells transfected with mi R-30 a was reduced significantly compared to cells transfected with the scrambled control.A direct interaction between mi R-30 a and HP1γ was further confirmed by examination of HP1γ expression in HCT116 and SW620 cells overexpressing mi R-30 a.In these experiments,mi R-30 a overexpression was achieved by transfecting cells with mi R-30 a mimic.Western blot analysis confirmed that the level of HP1γ was significantly reduced in cells overexpressing mi R-30 a.HP1γ m RNA levels were unchanged after mi R-30 a transfection suggesting that mi R-30 a directly recognizes and binds to the 3’-UTR of the HP1γ m RNA resulting in reduction of HP1γ protein.Thus,we conclude that mi R-30 a specifically regulates HP1γ protein expression post-transcriptionally.Levels of mi R-30 a were found to be significantly downregulated in CRC tissues compared to levels from paired adjacent non-tumorous tissues.Levels of mi R-30 a and levels of HP1γ protein in CRC tissues exhibited a significant inverse correlation calculated by Pearson’s correlation（R=-0.5981,P<0.01）.Moreover,we determined that mi R-30 a specifically suppressed colorectal cancer growth by targeting HP1γ in vitro and in vivo.In conclusion,this study provides evidence that HP1γ 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.Mi R-30 a appears to be a tumor suppressor through direct inhibition of expression of HP1γ protein.Our results suggest that the mi R-30a/HP1γ/p21^Waf1/Cip1 axis may represent a potential therapeutic target for treatment of human CRC.