LncRNA HCP5 was in high expression level in HCC tissues and cell lines and was closely correlated with HCC progression
To examine the HCP5 expression quantities in HCC related tissues and cell lines, qRT-PCR was conducted to determine 80 pairs of stochastically extracted HCC tissues and adjacent normal tissues. In present study, we indicated that HCP5 was overexpressed in HCC tissues versus adjacent normal tissues (p<0.001, Fig.1A). Simultaneously, HCP5 was also examined in higher expression level in HCC cell lines (Hep3B, HCCLM3, SMCC-7721, Huh7 and MHCC-97H) when compared with normal hepatocyte LO2 (***p<0.001, **p<0.01, respectively, Fig.1B). Thus, these consequences collectively suggested that overexpression of lncRNA HCP5 might be associated with HCC.
Next, we further examined the association between the clinical parameters and HCP5 expression. As shown in Table 1, HCP5 was significantly higher in large tumor size, metastasis, high histological grade tissues and recurrence (**p<0.01, ***p< 0.001, Fig.1C). Generally, these results illustrated that HCP5 probably plays an important role in regulating the progression of HCC.
HCP5 promoted cell growth, metastasis and invasion through prohibiting apoptosis in HCC in vitro and vivo
Lucubration of the specific function of HCP5 in HCC was performed by transfecting si-HCP5 into Hep3B and HCCLM3 cell lines to knockdown HCP5. Cells have reached ideal transfection states after 24 h. As depicted in Fig.2A, HCP5 was in significantly lower expression in HCC cell lines (Hep3B and HCCLM3) with sh-HCP5 transfection. Subsequently, cell proliferative capacity was attenuated in Hep3B and HCCLM3 with HCP knockdown using BrdU assay (*p<0.05, **P<0.01, Fig.2B). Moreover, the metastatic and invasive abilities in HCC cells were investigated by transwell chamber assay. The results demonstrated that HCP5 knockdown in Hep3B and HCCLM3 cell lines resulted in alleviated cell metastatic and invasive quantities (**p<0.01, ***P<0.001, Fig.2C and 2D), which indicated that the existence of HCP5 could functionally augment the metastatic and invasive abilities in HCC.
As we have proved that overexpression of HCP5 enabled the proliferation, metastatic and invasive ability of HCC, the exact modulatory process is still not clear. Therefore, flow cytometry was conducted to further scrutinize whether the alteration of HCP5 expression would thereby affect cell apoptosis. The results of flow cytometry showed that the apoptosis rate was negatively proportional to HCP5 expression level, which meant that down-regulation of HCP5 lead to higher apoptosis level (***p<0.001, Fig.2E). Additionally, we have estimated the expression level of epithelial-mesenchymal transition (EMT) related proteins (E-cadherin and Vimentin) by Western blot. As shown in Fig.2f, knockdown of HCP5 resulted in high expression of E-cadherin and down-regulation of Vimentin in HCC cell lines (Hep3B and HCCLM3) (Fig.2F), which illustrated that down-regulation of HCP5 might promote HCC cell metastasis and invasion via activating EMT process.
Although we have found the evidences that HCP5 could promote HCC proliferation, metastasis and invasion in vitro, the in vivo effect of HCP5 on HCC progression still need to be proved. Consequently, we injected HCCLM3 cells into nude mice to construct HCC animal models. The tumor volume was recorded every 3 days after xenograft. The tumor volume of si-HCP5 transfected group escalated lower than control group (Fig.3A), which further indicated that down-regulation of HCP5 could slow HCC progression. Then tumor in nude mice was extracted and made into slices. HE staining was used to scrutinize the nuance between overexpression and down-regulation of HCP5. Consistent with the in vitro consequence, knockdown of HCP5 destroyed the HCC tumor tissues (Fig.3B). Additionally, the metastasis phenotype was also examined and the results shown in Fig.3C suggested that knockdown of HCP5 led to higher E-cadherin expression and lower Vimentin expression when compared with sh-NC transfected group (p<0.05). Moreover, Ki67 was utilized to estimate the HCC proliferation in vivo. Consistent to our hypothesis, down-regulation of HCP5 significantly reduced Ki67 positive staining cells (Fig.3D), which demonstrated that HCP5 is a pivotal factor in promoting HCC cell growth.
miR-29b-3p interacted with HCP5
To further explore the downstream regulatory molecular mechanism of HCP5, we exerted starBase v3.0, miRanda and LncBase to find potential target gene of HCP5. The intersection of prediction targets of the three databases showed that there was a covalent binding fragment of HCP5 wild type binding sites on the miR-29b-3p gene sequence (Fig.4A), which encouraged us to further investigate the exact correlation between miR-29b-3p and HCP5 in HCC. Dual luciferase assay demonstrated that the significant reduction in luciferase activity between wtHCP5 and miR-29b-3p, which explained that miR-29b-3p was negatively regulated by HCP5 in HCC (**p<0.01, Fig.4B). The expression level of miR-29b-3p in HCC tissues and adjacent normal tissues were also determined. As expected, miR-29b-3p was expressed in a low quantities level in HCC tissues when compared to normal tissues (**p<0.01, Fig.4C), from which we speculated that miR-29b-3p might serve as a tumor suppressor in HCC. Also, the specific expression level between miR-29b-3p and HCP5 was analyzed through qRT-PCR and the result showed that miR-29b-3p and HCP5 is negatively correlated (Fig.4D and 4E). The result depicted in Fig.4D elucidated that knockdown of HCP5 could increase miR-29b-3p expression (***p<0.001). When given miR-29b-3p mimic, HCP5 was shown in low expression level. While given miR-29b-3p, HCP5 was found down-regulation (p<0.05, Fig.4E), which suggested that miR-29b-3p and HCP5 were in negative feedback regulation cycle.
miR-29b-3p prevents HCC cell progression brought by HCP5
To explore the exact function of miR-29b-3p in HCC, miR-29b-3p mimic and miR- NC were used to treat HCCLM3 cells. qRT-PCR was conducted to estimate the miR-29b-3p expression level after the treatment of above reagent. MiR-29b-3p was in high expression level when giving miR-29b-3p mimic to HCC cell lines (HCCLM3 and Hep3B) compared to miR-NC group (***p<0.001, Fig.5A). BrdU assay demonstrated that high expression level of miR-29b-3p resulted in lower proliferative rate (*p<0.05, **p<0.01 compared to miR-NC, Fig.5B), which directly illustrated that miR-29b-3p has the ability to prevent HCC cell proliferation. Additionally, cell metastatic and invasive capacity have been alleviated when HCC cell lines (HCCLM3 and Hep3B) were given miR-29b-3p mimic (**p<0.01, ***p<0.001, Fig.5C and 5D). Those results collectively elucidated that miR-29b-3p is capable to inhibit HCC cell proliferation, metastasis and invasion, and thereby prevent the exacerbation of HCC. Next, flow cytometry was conducted to examine the effect of miR-29b-3p on cell apoptosis, and the consequence demonstrated that the apoptosis rate was higher in miR-29b-3p mimic group (***p<0.001 compared with miR-NC, Fig.5E), which suggested that miR-29b-3p prevent cell growth through improving cell apoptosis. Also, western blot demonstrated that high quantities of miR-29b-3p led to up-regulation of E-cadherin and down-regulation of Vimentin (Fig.5F).
To investigate the correlation between miR-29b-3p and HCP5, miR-29b-3p antagomir was given to sh-HCP5 transfected HCCLM3 cell line. qRT-PCR showed that knockdown of HCP5 has higher miR-29b-3p quantities, while giving miR-29b-3p antagomir, the expression of miR-29b-3p increased (***p<0.001, Fig.6A). Additionally, the proliferation of miR-29b-3p antagomir treated HCCLM3 with sh-HCP5 transfection was determined using BrdU assay, the result showed that when given miR-29b-3p antagomir, the proliferative rate significantly increased (**p<0.01, Fig.6B), which indicated that miR-29b-3p was a tumor suppressor of HCC. Moreover, transwell assay demonstrated that miR-29b-3p antagomir further promoted the metastatic and invasive capacity attenuated by sh-HCP5 (p<0.05, Fig.6C-D). Further certification of the mechanism was investigated through flow cytometry and western blot. The research consequences showed that miR-29b-3p antagomir precluded cell apoptosis (***p<0.001, Fig.6E), which illustrated that miR-29b-3p antagomir promoted cell growth through preventing apoptosis. Western blot was conducted to scrutinize the expression of EMT correlated proteins (E-cadherin and Vimentin), the results showed that when given miR-29b-3p antagomir, the quantities of Vimentin was increased while E-cadherin was decreased (Fig.6F). Those consequences collectively elucidated that miR-29b-3p precluded cell growth, metastatic and invasive capacity via promoting apoptosis, up-regulating E-cadherin and down-regulated Vimentin.
DNMT3A was proved as a target gene of miR-29b-3p
Further exploration of the mechanism by which miR-29b-3p modulates the progression of HCC, Targetscan and starBase v3.0 were used to search the direct target gene of miR-29b-3p. Subsequently, DNMT3A was found as the downstream target of miR-29b-3p. As shown in Fig.7A, the bioinformatic prediction demonstrated that DNMT3A was indeed a potential target gene of miR-29b-3p. Additionally, dual luciferase assay further certified that there was a direct interaction between miR-29b-3p and DNMT3A (***p<0.001, Fig.7B). Western blot and qRT-PCR were exerted that expression level of DNMT3A when treated miR-29b-3p mimic, the results showed that high quantities of miR-29b-3p would inhibit the expression of DNMT3A (***p<0.001, Fig7C-D). High quantities of miR-29b-3p led to lower expression quantities of DNMT3A, while down-regulation of miR-29b-3p resulted in high expression quantities of DNMT3A (***p<0.001, Fig.7E), which indirectly elucidated that the expression of DNMT3A was in inverse proportion to miR-29b-3p. Moreover, western blot demonstrated that down-regulated of miR-29b-3p resulted in higher quantities of DNMT3A both in HCCLM3 and Hep3B, while low expression of miR-29b-3p led to up-regulation of DNMT3A, which indicated that the expression of DNMT3A was proportional to HCP5 (Fig.7F). Furthermore, qRT-PCR and western blot showed that knockdown of HCP5 resulted in lower expression quantities of DNMT3A at mRNA and protein levels when compared with sh-control both in Hep3B and HCCLM3 cell lines (Fig.7G-H). Next, the expression quantities of DNMT3A in HCC tissues were determined through western blot, the research consequences demonstrated DNMT3A was up regulated in HCC tissues when compared to adjacent normal tissues (Fig.7I). The results mentioned above collectively illustrated DNMT3A is a crucial participating factor in modulating HCC progression.
Overexpression of DNMT3A promotes HCC cell growth, metastasis and invasion
Previous studies have proved that DNMT3A played a role in HCC, therefore, we then proposed an assumption that up-regulation of DNMT3A might alter the HCC progression inhibited by miR-29b-3p. Consequently, EV-DNMT3A and EV-control (EV) were transfected into HCC cell lines (HCCLM3 and Hep3B) to up-regulated the expression of DNMT3A. Also, miR-29b-3p mimics and sh-HCP5 transfection were used to determine the correlation of miR-29b-3p, HCP5 and DNMT3A. As shown in Fig.8A, qRT-PCR demonstrated that DNMT3A was down regulated when there was high expression level of miR-29b-3p, while its expression quantities were positively proportional to HCP5 (*p<0.05). Consistent with this result, western blot showed that high-regulation of DNMT3A reversed the effect brought by sh-HCP5 and miR-29b-3p mimics (Fig.8B). Subsequently, BrdU assay was exerted to determine the effect of DNMT3A on HCC proliferation, the result showed that overexpression of DNMT3A led to higher cell growth (*p<0.05, Fig.8C). Next, cell metastatic and invasive ability were examined through transwell chamber assay, the research consequences suggested that both metastatic and invasive capacity had been increased when giving EV-DNMT3A (*p<0.05, Fig.8D-E). Further exploration of the effect of DNMT3A on regulating proliferation, invasion and migration was performed by flow cytometry and examination of the expression level of EMT correlated proteins (E-cadherin and Vimentin). Flow cytometry showed that DNMT3A promoted cell growth through prohibiting cell apoptosis (*p<0.05, Fig.8F). Western blot showed that high regulation of DNMT3A could up-regulated Vimentin and decreased the expression of E-cadherin (Fig.8g). Those consequences elucidated that the proliferation, metastatic and invasive capacity of HCC was augmented when there were high expression quantities of DNMT3A (Fig.8b-d), and the mechanism of which was prevention of apoptosis and acceleration of EMT process.
HCP5/miR-29b-3p/DNMT3A axis augments HCC progression via activating AKT phosphorylation
Current researches have elucidated that DNMT3A plays a pivotal role in modulating tumorigenesis through activating PI3K/AKT pathway in liver cancer and lung cancer24, 25. To certify the modulatory function of AKT phosphorylation in HCP5/ miR-29b-3p/DNMT3A, we first confirmed that knockdown of DNMT3A significantly decreased AKT phosphorylation by western blot (Fig.9a), the phosphorylation level of AKT was proportional to DNMT3A. Then, IGF-1, the AKT activator was used to treat HCC cell lines with si-DNMT3A transfection, the proliferation was determined through BrdU assay and the result demonstrated that activation of AKT led to enhanced cell growth (* *p<0. 01, Fig. 9B). Transwell chamber assay was exerted to scrutinize the effect of AKT on regulating HCC metastatic and invasive capacity. The research consequences demonstrated that AKT activation augmented that the metastatic and invasive capacity of HCC (***p<0.001, **p<0.01, Fig.9C-D). Further investigation found that inhibition of AKT phosphorylation led to higher cell apoptosis and weaken EMT process (***p<0.001, Fig.9E-F), which proved that AKT phosphorylation was important in regulating HCC progression.