Heat Shock Protein Gp96 3’UTR Functions As A CeRNA In Promoting DOHH Expression Via MiR-642a

Heat shock protein gp96 (glycoprotein 96), also known as GRP94, HSP90b1, TRA-1, ERp99. As one of the most abundant chaperones in the endoplasmic reticulum (ER), is a homologous protein of cytoplasmic heat shock protein HSP90. Heat shock protein gp96 mainly functions as a molecular chaperone that participates in nascent proteins folding and the degradation of denatured proteins. Others and our previous studies have found that heat shock protein gp96 is highly expressed in various tumors, such as liver cancer, breast cancer and myeloma tumors. At the same time, it have been identified that heat shock protein gp96 can interact with Wnt co-receptor LRP6, insulin-like growth factor, integrin, HER2, uPAR. It might play a significant role maintaining the stability and function of these tumor proteins. However, the mechanism of gp96 in regulating the occurrence and development of tumors is still need to be further studied.MicroRNAs (miRNAs) are a class of evolutionarily conserved and small non-coding RNA molecules, approximately 22 nucleotides in length, which play important roles in post-transcriptional regulation of gene expression. MiRNAs typically interact with sequences within the 3’-untranslational region (3’UTR) through imperfect complementarity base binding to their target mRNAs, leading to the translational repression or degradation of their target mRNAs. A large number of miRNAs have been found to be involved in a broad spectrum of biological functions such as regulation of innate and adaptive immunity, cell differentiation, infection and development as well as disease pathogenesis, especially in cancer. The recent researches have found that some RNA including long non-coding RNA (lncRNA), pseudogene transcript, and circular RNA (circRNA), even virus mRNA, If they have the same miRNA response element,they may be used as a "sponge", its interaction with miRNA through adsorption and isolation. Thereby inhibiting the function of miRNA, which leads to upregulate the expression of miRNA other target genes.In our study, we first predicted the potential target of miRNA on heat shock protein gp96 3 ’UTR through bioinformatics techniques. After analyzing and comparing, we select miR-642a to conduct further research. By using luciferase reporter assay and western blotting, we found that miR-642a can specifically target heat shock protein gp96 3’UTR. According to the literature reports, miR-642a can regulate cell proliferation via the regulation of DOHH expression in prostate cancer cells. In order to investigate the effect of heat shock protein gp96 3’UTR on the expression of DOHH, We transfected with wild type or miR-642a binding site mutations of gp96 3’UTR plasmids in Huh7 cells. By real-time quantitative real-time PCR, RNA interference and western blotting to detect miR-642a levels, mRNA and protein levels. The results indicate that the wild type but not the mutant gp96 3’UTR in miR-642a binding site could sequester and downregulate miR-642a levels in Huh7 cells, which led to increased expression of the miR-642a target DOHH. To further investigate whether the heat shock protein gp96 3’UTR regulates the expression of DOHH via miR-642a, we transfected miR-642a inhibitors in Huh7 cells. The results showed if we transfected gp96 siRNA and miR-642a inhibitors at the same time, heat shock protein gp96 does not have a regulatory effect on the DOHH expression levels. This clearly indicates that regulation of DOHH expression by gp96 3’UTR was miR-642a dependent. It was also found that DOHH does not affect the expression of gp96.In conclusion, our work indicates that heat shock protein gp96 3’UTR functions as a ceRNA in promoting DOHH expression. This regulation effect is dependent on miR-642a and does not depend on the expression of gp96 protein. Others and our previous study have found that the expression of gp96 mRNA and protein was significantly increased in in the patients with chronic infection. The expression of gp96 was closely related to the progression of chronic hepatitis B. Heat shock protein gp96 is overexpressed in many kinds of tumors including hepatic tumors, and its overexpression is significantly correlated with tumor malignant degree and poor prognosis in patients. At present, the mechanism of gp96 in the occurrence and development of tumor is focused on its function as a molecular chaperone protein. Heat shock protein gp96 promotes DOHH expression via its 3’UTR as a ceRNA, providing new insights into the role of gp96 on the development of hepatic tumors and other tumors. To analyze the mechanism of gp96 to promote the growth of tumor cells and provides theoretical basis to tumor-targeted gene therapy.