LncRNA HOTAIR Regulates The Invasion And Metastasis Of Prostate Cancer Through HepaCAM Methylation

Objective To explore the relationship between the abnormally high expression of long non-coding RNA(lncRNA)HOTAIR and the loss of hepaCAM from epigenetic perspective in prostate cancer.To study their regulatory effects and specific regulatory mechanisms by examining the activation states of relevant signal pathways and the expression of related molecules in the process of invasion and metastasis.Methods Blood and clinically resected tissue specimens of patients with benign prostatic hyperplasia and prostate cancer of different clinical grades were collected.The protein expression of H3K27me3 and hepaCAM were detected by immunohistochemistry(IHC)and their correlation were verified by Pearson analysis.The mRNA levels of HOTAIR and heapCAM were detected by Q-PCR and verified their expression correlation in circulating blood.The human prostate epithelial cells and prostate cancer cells were cultured to extract RNA and proteins.The expression of HOTAIR,hepaCAM and H3K27me3 in vitro were tested through Q-PCR and western blot(WB)and observed the consistency with clinical patient level.The CRISPR/Cas9 plasmid targeted HOTAIR were constructed and transfected prostate cancer cell lines,and the expression changes of hepaCAM and H3K27me3 were detected by Q-PCR,WB and immunofluorescence(IF).To determine the specific regulatory mechanism of HOTAIR on hepaCAM,the prostate cancer cells were treated with histone methylation inhibitor DZNe P that can efficiently inhibits PRC2 activity and the mRNA and protein expressions of hepaCAM were detected by Q-PCR and WB,respectively.Chromatin immunoprecipitation(ChIP)technique was used to examine the recruitment effect of HOTAIR on Polycomb complex2(PRC2)and its localization in the specific segment of hepaCAM gene in prostate cancer cells.CO-IP was used to detect the binding of HZK27me3 and the key molecules SUZ12 and EZH2 in the PRC2 complex.To accurately elucidate the specific regulation effects of HOTAIR on invasion and metastasis in prostate cancer,the prostate cancer cells were transfected with CRISPR/Cas9 plasmid targeted HOTAIR and knocked down the expression of hepaCAM by siRNA simultaneously.The cell invasive and metastasing abilities were detected by wound healing assay and Transwell assay.The expression changes of HOTAIR,hepaCAM,H3K27me3 and key molecules in related signaling pathways of differenttreatment groups were assayed by Q-PCR and WB.Then prostate cancer cells were infected with hepaCAM adenoviruses with the addition of MAPK signaling pathway activators to determine pathway activation and screening for hepaCAM downstream effector molecules that regulate cancer cell invasion and metastasis.Results The results of IHC showed that the expression of H3K27me3 in prostate cancer tissues was significantly higher than that in paracancerous tissues and was gradually increased as the disease progressed and deteriorated,while the expression of hepaCAM was reversed(P<0.05).Pearson analysis suggested that their expression in prostate tissue was negatively correlated(r=-0.7778,P<0.05).The results of blood PCR showed that compared with patients with benign prostatic hyperplasia,the expression of HOTAIR in the blood of patients with prostate cancer was significantly higher and correlated with the stage of cancer,while hepaCAM was on the opposite(P<0.05).The results of Q-PCR and WB showed that hepaCAM was highly expressed in human prostate epithelial cells but almost absent in prostate cancer cells(P<0.05).The expression of HOTAIR and H3K27me3 was opposite(P<0.05).With the transfection of the constructed CRISPR/Cas9 plasmid,the expression of HOTAIR was almost undetectable in Q-PCR of prostate cancer cells,and the expression of H3K27me3 was significantly decreased in WB and IF,while the expression of hepaCAM gene and protein levels was reversed(P<0.05).After treatment with DZNeP,hepaCAM was significantly re-expression in prostate cancer cells(P<0.05).ChIP assay showed that HOTAIR can recruit PRC2 complex(EZH2,SUZ12,EED)to hepaCAM promoter region in 474-674 bp range(P<0.05);CO-IP experiments remindered that EZH2 could increase the level of H3K27me3(P<0.05),and the latter can lead to the silence of hepaCAM.Wound healing assay showed that knockout HOTAIR significantly inhibited cell migration,while simultaneously knockdown hepaCAM inhibited this effect(P<0.05);The result of Transwell were consistent with the wound healing(P<0.05);Q-PCR showed knockout HOTAIR reversed the expression of hepaCAM,and changed the expression of some invasive and metastasis-related genes(P<0.05).WB found that the protein levels of the above-mentioned gene were basically consistent with PCR(P<0.05),and the expression of p-MEK1 and p-ERK was significantly decreased(P<0.05),suggesting this signaling pathway plays a key role in the regulation process;however,if hepaCAM was knocked down at the same time,the above-mentioned changes are attenuated(P<0.05).Transwell and wound healing assays showed that the invasion ability of prostate cancer cells was inhibited when infected with hepaCAM virus alone(P<0.05).Q-PCR and WB results suggested that hepaCAM inhibited the activation of MAPK pathway(P<0.05),thus affecting the expressions of downstream effector molecules(P<0.05);When MAPK pathway activator was added at the same time the above regulation could be significantly weakened(P<0.05).Conclusion The abnormally high expression of LncRNA HOTAIR in prostate cancer can recruit PRC2 complex to specific promoter region of hepaCAM gene resided in chromatin,resulting in the methylation modification of histones,the increased expression of H3K27me3 and the silence of this suppressor gene,further leading to abnormal activation of MAPK signaling pathway and tumor invasion and metastasis.