Differentially expressed RNAs and functional annotation in HBV patients from the GEO database. A total of 3 HBV samples and 3 control samples were obtained from the GSE135860 dataset, with 23949 RNAs measured for each. According to the differential analysis by the Wilcoxon test, we identified 2552 mRNAs as significantly differentially expressed in the HBV compared with normal tissue samples, and the results are displayed in the volcano plot in Fig. 1. We also analysed these mRNAs using the R software package clusterprofifiler to identify the functions linked to the different mRNAs. This evaluation revealed enrichment of 321 GO terms along with 9 KEGG pathways (FDR < 0.05). We chose to show the top 9 GO terms and 20 KEGG pathways of the DEmRNAs based on the gene count in Figs. 2, 3.
The relationships among the let-7c target genes expressed in HBV patients. We found 129 HBV-related upregulated genes in the GSE135860 dataset, and 46 common genes were identified by the intersection of the 129 HBV-related upregulated genes with 4430 Let-7c target genes from TargetScan, Tarbase, miRDB and miRanda. Based on the resulting 46 genes, a Venn diagram was constructed (Fig. 4a). Using STRING, we generated a PPI network that included 22 edges and 46 nodes to study the interactions among the MYC genes (Fig. 4b). We found that MYC was most strongly correlated with other genes in the PPI network, including CDH1, CHD1, COX4I1, FOXP1, MEF2C, MYBL1, PPP1CA and PTMA.
There was a significant reduction in the expression of Let-7c in HepG2.15 cells. In our study, we analysed the expression of all Let-7 family members, including Let-7a-5p (Let-7a), Let-7b-5p (Let-7b), Let-7c, Let-7d-5p (Let-7d), Let-7e-5p (Let-7e), Let-7f-5p (Let-7f), Let-7g-5p (Let-7g), Let-7i-5p (let-7i), Let-7a-3p, Let-7a-2-3p, Let-7b-3p, Let-7d-3p, Let-7e-3p, Let-7f-1-3p, Let-7f-2-3p, Let-7g-3p, let-7i-3p, and miR-98-5p. Our results demonstrated that the expression of Let-7a-3p, Let-7a-2-3p, Let-7b-3p, Let-7d-3p, Let-7e-3p, Let-7f-1-3p, Let-7f-2-3p, Let-7g-3p, let-7i-3p, and miR-98-5p was less than that in normal hepatocytes (data not shown). In hepatocytes, members with increased expression were Let-7a, Let-7c, Let-7e, Let-7i, and Let-7 g, while those with decreased expression were Let-7f, Let-7d, and Let-7b. The member with the highest expression in hepatocytes was Let-7a. In HBV-associated hepatic tumour tissues (compared to adjacent tissues), the downregulation of almost all Let-7 family members expression was evident along with significant reduction of Let-7c expression (P < 0.05) (Fig. 5). These results led us to focus on Let-7c for further research.
Let-7c inhibition promoted HepG2 cell proliferation and the expression of MYC. Shi demonstrated a downward trend in the expression of Let-7c from normal control to chronic hepatitis, liver cirrhosis, adjacent nontumour, and HCC samples . However, the result was mainly derived from liver tissues. To date, the expression of Let-7c in serum samples has not been studied. Here, the expression of lin28B in B-HCC was elevated. The data showed that lin28B and Let-7c had a negative correlation, which led to the analysis of Let-7c expression in B-HCC.
We inhibited Let-7c in HepG2 cells. The results showed successful significant inhibition of Let-7c expression (P < 0.05) (Fig. 6a). We also used the CCK-8 assay to examine the proliferation of HepG2 cells. The results showed that proliferation was evidently promoted after 24 hours of cultivation. However, after 72 hours, the rate of promotion was slightly decreased (Fig. 6b).
After Let-7c was inhibited, we used the CCK-8 assay to detect the proliferation of HepG2 cells. We cultured HepG2 cells for 96 hours. The proliferation of the HepG2 treatment groups was promoted when the cells were cultured for more than 24 hours. Additionally, the treatment groups were maintained for 48 h, 72 h, and 96 h of cultivation (Fig. 6b). Western blotting detected the expression of c-Myc, which was obviously promoted when Let-7c was inhibited (Fig. 6c). The inhibition of Let-7c expression led to the upregulation of MYC expression.
The expression of lin28B in B-HCC samples was upregulated. The expression of Let-7c decreased gradually with liver cirrhosis development. In contrast to the B-LC samples, there was clear upregulation of lin28B in the B-HCC samples. We used real-time PCR to detect expression. Overall, the results were statistically significant at P < 0.05 (Fig. 7a). Furthermore, we detected the expression of Let-7c in 99 cirrhosis samples and 8 B-HCC samples. Among all of the samples, F1 samples showed the highest expression of Let-7c, while B-HCC samples showed the lowest expression of Let-7c. This gradual decreasing trend in the expression of Let-7c with progression of liver cirrhosis is an important discovery (Fig. 7b).
In addition, the expression of Let-7c was detected when samples were divided into a HepG2 group and HepG2.2.15 group infected by HBV. The results were encouraging. The expression of Let-7c was significant altered (P < 0.05) (Fig. 7c).
The expression of Let-7c was related to the levels of HBV DNA and total bilirubin (T-Bil) in serum. The expression of Let-7c was negatively correlated with the level of HBV DNA, and the result was statistically significant (P = 0.043) (Fig. 8a). The expression of Let-7c was positively correlated with the level of T-Bil in serum. This result was also statistically significant (P = 0.029) (Fig. 8b).