MiR-23a-3p Promoted G1/S Cell Cycle Transition By Targeting Protocadherin 17 In Hepatocellular Carcinoma

Objective:Primary liver cancer is one of the most common digestive malignancies,ranking the fourth and the third with respect to the incidence rate and the mortality rate respectively,with a five-year survival rate of 18%.The major risk factors of primary liver cancer include HBV or HCV hepatitis,alchol and aflatoxin.The typical pathophysiological mechanism of primary liver cancer involves a three step process: hepatitis-cirrhosis-cancer.There are three major pathological types of primary liver cancer: HCC,cholangiocarcinoma and mixed cell carcinoma.The major therapeutic method for HCC is curative resection.However,postoperative recurrence and metastasis of HCC are frequent due to the high malignancy,the difficulty of early-stage diagnosis and the resistance for chemotherapy or radiotherapy of the tumor.Recently as the molecular biological mechanism of HCC becomes clearer,the relevant gene therapy,molecular targeted therapy and immunotherapy are showing promising potential in preventing the postoperative recurrence and metastasis of HCC.MicroRNA as one of the most important mechanisms with respect to the development of HCC,has gained much focus.MicroRNAs are short endogenous noncoding RNAs which can promote the degradation and inhibit the translation of target mRNAs.A single microRNA can target various mRNAs,and a single mRNA can also be targeted by various microRNAs.Furthermore,microRNAs can be inhibited by lncRNAs and circRNAs.LncRNAs,circRNAs,microRNAs and mRNAs form together to become a complex regulatory network.MicroRNAs are associated with a series of biological processes like cell growth,differentiation,cell cycle,apoptosis and metabolism.The deregulation of microRNAs has been proved to cause various diseases like cancers and cardiovascular diseases.Several literatures reported that the miR-23a-27a-24-2 cluster was overexpressed in HCC and related to the resurrence and metastasis of HCC.MiR-23a-3p was reported to promote the growth and metastasis,regulate the chemosensitivity and suppress the apoptosis of HCC.However,the relationship between miR-23a-3p and HCC cell cycle remains unclear.Protocadherin belongs to a subfamily of cadherin and its function is not completely clear.The expression of protocadherin is frequently suppressed in breast cancer,lymphoma,cervix cancer,Huntington disease and non-small cell lung cancer because of gene mutation and methylation of promoter.PCDH17 is a tumor suppressor gene located at human chromosome 13q21.1.The expression of PCDH17 is frequently downregulated in acute myeloid leukemia,laryngeal squamous cell carcinoma,bladder cancer,HCC and gastric cancer.PCDH17 could induce apoptosis and autophagy,and is usually methylated in gastic cancer and colorectal cancer.In HCC,the silence of PCDH17 could promote the invasion and metastasis of tumor cells by activating the EGFR/ MEK/ ERK pathway.However,the relationship between PCDH17 and HCC cell cycle is also unclear.In this study,the levels of miR-23a-3p and PCDH17 in human HCC and adjacent normal tissues were tested.Furthermore,a series of in-vitro experiments were employed to investigate the relationships between miR-23a-3p and cell cycle,PCDH17 and cell cycle,and miR-23a-3p and PCDH17 respectively.Whether the HCC cell cycle regulation of miR-23a-3p is through PCDH17 has also been confirmed.Last,in-vivo experiments investigated the relationship between miR-23a-3p and tumor growth.Method:(1)The levels of miR-23a-3p and PCDH17 in human HCC and adjacent normal tissues were tested by RT-qPCR and western blotting.The relevance of miR-23a-3p and PCDH17 was confirmed by linear regression analysis.(2)The levels of miR-23a-3p in Hep3 B,Huh-7 and HepG2 were tested by RTqPCR.Then Hep3 B and HepG2 were selected for subsequent experiments.The efficiencies of miR-23a-3p mimics transfecting Hep3 B,and miR-23a-3p inhibitor transfecting HepG2,were tested by RT-qPCR.The effect of miR-23a-3p on HCC cell cycle was investigated by a series of in-vitro experiments including CCK-8 cell viability assay,flow cytometry,EdU infiltration assay and western blotting.(3)The effect of PCDH17 on HCC cell cycle was investigated by CCK-8 cell viability assay and flow cytometry.(4)The levels of PCDH17 were tested by RT-qPCR and western blotting after Hep3 B and HepG2 were respectively transfected by miR-23a-3p mimics and inhibitor.The specific binding site of miR-23a-3p in PCDH17 3'UTR was predicted by bioinformation analysis.Finally,PCDH17 as the direct target of miR-23a-3p was confirmed by dual luciferase reporter assay.(5)Both of Hep3 B and HepG2 were transfected by PCDH17 siRNA,NC siRNA,PCDH17 overexpression plasmid or the empty vector.The transfection efficiencies were tested by western blotting.Then Hep3 B was cotransfected by miR-23a-3p mimics or NC mimics and PCDH17 overexpression plasmid or the empty vector.HepG2 was cotransfected by miR-23a-3p inhibitor or NC inhibitor and PCDH17 siRNA or NC siRNA.Whether the effect of miR-23a-3p on HCC cell cycle is through PCDH17 was investigated by a series of in-vitro experiments including CCK-8 cell viability assay,flow cytometry,EdU infiltration assay and western blotting.(6)Hep3B stably transfected by pre-miR-23 a and HepG2 stably transfected by miR-23a-3p sponge were subcutaneously injected into the left armpits of the nude mice with the tumor growth rates calculated.Then the mice were executed with the tumor tissues dissected.After HE staining,the numbers of necrotic and apoptotic cells were calculated.IHC staining was also employed to further confirm the relationship between miR-23a-3p and PCDH17.Result:(1)The level of miR-23a-3p was upregulated while the levels of PCDH17 mRNA and protein were downregulated in HCC tissues.The levels of miR-23a-3p and PCDH17 mRNA were negatively correlated.(2)The level of miR-23a-3p was at the lowest in Hep3 B and the highest in HepG2 among the included HCC cell lines.After transfected by miR-23a-3p mimics,the level of miR-23a-3p in Hep3 B was significantly increased,while dicreased in HepG2 transfected by miR-23a-3p inhibitor,indicating the success of the transfection.CCK-8 assay showed that after transfection of 48 h and 72 h,the viability of Hep3 B was significantly increased,while the viability of HepG2 was significantly decreased.Flow cytometry showed that the cell number of S phase was significantly increased with that of G1 phase significantly decreased in Hep3 B cells transfected with miR-23a-3p mimics.The results of HepG2 transfected with miR-23a-3p inhibitor were opposite to the above.The results of the EdU infiltration assay were similar with those of the flow cytometry.Western blotting showed that the levels of cyclin D1,cyclin E,CDK2,CDK4,p-p27 and p-RB were significantly upregulated in Hep3 B cells transfected with miR-23a-3p mimics,while downregulated in HepG2 cells transfected with miR-23a-3p inhibitor.However,the levels of RB didn't change.(3)The cell viability and ratio of S phase cells were significantly increased with the ratio of G1 phase cells decreased in Hep3 B cells transfected with PCDH17 siRNA.The results of HepG2 cells transfected with PCDH17 overexpression plasmid were opposite to the above.(4)MiR-23a-3p mimics lowered the levels of PCDH17 mRNA and protein,while miR-23a-3p inhibitor increased the levels of those.The activity of luciferase was significantly decreased in the cells cotransfected with miR-23a-3p mimics and the plasmid carrying the wild type 3'UTR of PCDH17,which implied that PCDH17 could be a direct target gene of miR-23a-3p.(5)The level of PCDH17 was significantly decreased after transfection of PCDH17 siRNA,while increased after transfection of PCDH17 overexpression plasmid.CCK-8 assay showed that the cell viability of Hep3 B was significantly decreased in the group of miR-23a-3p mimics + PCDH17 overexpression,compared to the group of miR-23a-3p mimics + empty vector.Flow cytometry showed that the number of S phase cells was significantly decreased in Hep3 B cotransfected with miR-23a-3p mimics and PCDH17 overexpression plasmid.The results of EdU infiltration assay were similar with those of the flow cytometry.Western blotting showed that the levels of Cyclin D1 and cyclin E were significantly downregulated in Hep3 B cotransfected with miR-23a-3p mimics and PCDH17 overexpression plasmid.The associated results of HepG2 cells were opposite to the above.(6)The growth rate of Hep3 B cells stably transfected with pre-miR-23 a was significantly increased,while the growth rate of HepG2 cells stably transfected with miR-23a-3p sponge was significantly decreased in vivo.HE staining showed that Hep3 B cells stably transfected with pre-miR-23 a grew intensively,while necrosis and apoptosis frequently occurred in HepG2 cells stably transfected with miR-23a-3p sponge.IHC showed that the level of PCDH17 was significantly decreased in Hep3 B cells stably transfected with pre-miR-23 a while increased in HepG2 cells stably transfected with miR-23a-3p sponge.Conclusion:(1)The level of miR-23a-3p was significantly upregulated with that of PCDH17 downregulated in HCC tissues.MiR-23a-3p and PCDH17 were negatively correlated.(2)MiR-23a-3p promoted G1/S cell cycle transition of HCC.(3)PCDH17 inhibited G1/S cell cycle transition of HCC.(4)PCDH17 was a direct target of miR-23a-3p.(5)MiR-23a-3p promoted G1/S cell cycle transition by targeting PCDH17 in HCC.(6)MiR-23a-3p promoted HCC growth in vivo.