Ginsenoside Rd inhibits migration and invasion of tongue cancer cells through H19/miR-675-5p/CDH1 axis

Abstract Objective Tongue squamous cell carcinoma (TSCC) is an oral cancer, with high malignancy and frequent early migration and invasion. Only a few drugs can treat tongue cancer. Ginsenoside Rd is a ginseng extract with anti-cancer effects. Many noncoding RNAs are abnormally expressed in tongue cancer, thus influencing its occurrence and development. H19 and miR-675-5p can promote cancer cell growth. This study aimed to analyze the regulation effect of ginsenoside Rd on H19 and miR-675-5p in tongue cancer. Methodology We used CCK8 and flow cytometry to study the growth and apoptosis. Transwell assay was used to assess invasion; wound-healing assay to assess migration; and colony formation assays to test the ability of cells to form colonies. H19, miR-675-5p, and CDH1 expressions were analyzed by qPCR. E-cadherin expression was detected using western blot. CRISPR/cas9 system was used for CDH1 knockout. Results Ginsenoside Rd inhibited the growth and increased the apoptosis of SCC9 cells. Ginsenoside Rd also inhibited the migration and invasion of SCC9 cells. H19 and miR-675-5p were highly expressed, while CDH1 and E-cadherin expressions were low. H19 and miR-675-5p promoted SCC9 metastasis. In contrast, CDH1 and E-cadherin inhibited the metastasis of SCC9 cells. Bioinformatics analysis showed that miR-675-5p was associated with CDH1. H19 and miR-675-5p expressions decreased after ginsenoside Rd treatment, while CDH1 and E-cadherin expressions increased. Conclusions Ginsenoside Rd inhibits tongue cancer cell migration and invasion via the H19/miR-675-5p/CDH1 axis.


Introduction
Tongue squamous cell carcinoma (TSCC) is an oral cancer with high malignancy and frequent early migration and invasion. 1,2 Smoking and drinking are the main etiologic factors of TSCC. 3 Although surgical treatment has improved patients' survival rate, it remains low. 4 Moreover, surgery significantly damages patients' body. 5 Therefore, sometimes it is necessary to administer radiotherapy and chemotherapy to prevent cancer recurrence. 6,7 Therefore, studying some drugs with low-damaging effect and gene therapy can enhance cancer prevention and treatment.
Ginseng is a famous nutrient and Chinese herbal medicine. 8 High concentrations of ginsenoside, including ginsenoside Rb1, Rb2, Rc, Rd, and Re are found in ginseng. 9 Ginsenoside Rd has antiinflammatory, anti-aging, and nerve protection effects. [10][11][12] Increasing evidence has also shown that ginseng and its purified ginsenosides have anti-cancer effects. 13 Studies have also shown that diol-type ginsenosides have stronger anti-cancer activity than triol-type ginsenosides. Ginsenoside Rd has the strongest anti-cancer activity among the diol-type ginsenosides. 9,14,15 Therefore, ginsenoside Rd can inhibit tongue cancer.
A recent study showed that ginsenoside Rb3 can regulate H19, 16 indicating that ginsenoside Rd can inhibit tongue cancer by regulating H19. Noncoding RNA H19 is highly expressed in various cancers. 17 For instance, miR-675-5p, a micro-RNA from H19, can bind to some specific mRNA-encoding proteins, thus inhibiting its expression. 18 E-cadherin is closely related to cell adhesion and contact inhibition. 19 The abnormal expression of E-cadherin in cancers may promote cancer metastasis. 20 E-cadherin expression is decreased in oral cancers. 21 Therefore, the irregular expression of E-cadherin can be used as a biomarker for clinical diagnosis, treatment, and prognosis of tumors, 22 indicating that H19/miR-675-5p and CDH1 may be related. 23 CRISPR/Cas9 is found in bacteria and archaea.
It is mainly composed of three different types of systems, of which Streptococcus thermophilus or Streptococcus pyogenes type II system is the most widely used. Type II system is based on a single-guide RNA (sgRNA) and a cas9 protein that targets a sequence of DNA for editing. The CRISPR/ Cas9 system has been widely used in cancer characterization and modeling in recent years and is promising in cancer treatment. 24 Figure S1 and Figure S2.

Western blot
The protein extraction buffer (Beyotime, China) was used to extract the protein from the tongue cancer tissues and cells. A BCA kit (Meilunbio, China) was used to measure protein concentration.
Notably, 10% SDS-PAGE gels were used to isolate proteins with different molecular weights when the protein concentration of the experimental and the control groups was the same. Next, the PVDF membrane was used to transfer the isolated proteins. The PVDF membranes were blocked with 5% non-fat milk powder for 1 h, then incubated with antibodies against E-cadherin (Proteintech, USA) and GAPDH (Bioworld, USA) at 4°C overnight. The

Results
Ginsenoside Rd inhibits the growth, apoptosis, migration, and invasion of TSCC CCK8 showed that cell growth was significantly inhibited when the SCC9 was treated with 100 μM ginsenoside Rd ( Figure 1A). The cell activity was negatively correlated with the ginsenoside Rd concentration. Flow cytometry analysis ( Figure 1B) showed that ginsenoside Rd increased the SCC9 apoptosis. Wound healing assays and Transwell is associated with tongue cancer. qPCR results showed that H19 expression was higher in tongue cancer tissues than in normal tissues (Figure 2A).
We overexpressed and knocked down H19 ( Figure   2B) in SCC9 for further analysis. Compared to the control group, H19 overexpression enhanced the migration ability of SCC9, while H19 knockdown inhibited migration ( Figure 2C). Moreover, H19 overexpression enhanced the invasion ability of SCC9, while H19 knockdown inhibited invasion ( Figure 2D). The colony formation assay ( Figure   2E) showed that H19 overexpression increased the number of SCC9 clones, while H19 knockdown decreased the number of SCC9 clones. Although H19 overexpression did not significantly affect the growth ability of SCC9, H19 knockdown inhibited proliferation of SCC9 ( Figure 2F). qPCR analyses showed that miR-675-5p expression was higher in tongue cancer tissues than in normal tissues ( Figure   3A). Furthermore, miR-675-5p overexpression and knockdown analyses were conducted in SCC9 to assess their effects on migration, invasion, growth, and the number of clones (Fig. 3B) Western blotting also showed decreased expression of E-cadherin in tongue cancer tissue ( Figure 4A). Therefore, CDH1 was overexpressed to further study the relationship between E-cadherin and metastasis of tongue cancer ( Figure 4B). CDH1 overexpression decreased the invasion, migration, growth and number of clones ( Figures 4D-G) in tongue cancer cells. Finally, CRISPR/Cas9 system was used to knock down CDH1 to further assess the relationship ( Figure 4C). Compared to the control group, CDH1 knockdown increased the migration, invasion, growth and number of clones ( Figures 4D-G) of SCC9, indicating that E-cadherin is closely associated with the metastasis of tongue cancer.

Mechanism of ginsenoside Rd inhibition of migration and invasion of tongue cancer cells
Herein, H19 and miR-675-5p expression decreased after ginsenoside Rd treatment ( Figure 5A). The   Figure 5D). H19 overexpression also decreased CDH1 and E-cadherin expressions, while H19 knockdown increased CDH1 and E-cadherin expressions ( Figure 5E). miR-675-5p overexpression decreased CDH1 and E-cadherin expression, while miR-675-5p knockdown increased CDH1 and E-cadherin expressions (Fig. 5F). The overexpression of H19 and miR-675-5p can alleviate the effect of ginsenoside Rd treatment on the expression of CDH1 ( Figure S4). These results indicate that ginsenoside Rd prevents the invasion and migration of SCC9 through H19/miR-675-5p/CDH1 axis. Our study found that ginsenoside Rd regulated the expression of H19. H19 is a long noncoding RNA that cannot form proteins and the embryonic expression of H19 is suppressed after birth. 34 H19 is related to    Therefore, this research shows that ginsenoside Rd may be a natural anti-cancer drug. Moreover, H19 and CDH1 may be potential markers of tongue cancer. (Figure S5).

Conflict of interest
The authors declare no conflict of interest.