Liver-specific over-expression of Cripto-1 in transgenic mice promotes hepatocyte proliferation and deregulated expression of hepatocarcinogenesis-related genes and signaling pathways

Results

Truncated CR-1 mRNA is predominantly expressed in HCC cell lines and tissues

Previous studies reported that mammalian cells and tissues expressed both full length (FL-CR-1; 2.0 kb) and truncated (SF-CR-1; 1.7 kb) CR-1 mRNAs [30, 31]. Therefore, we performed RT-PCR analysis to quantify FL-CR-1 and SF-CR1 mRNA levels in HCC cell lines and tissues using primer sets that specifically detect FL-CR1 mRNA (UND/UNB) and total CR-1 mRNA (UNA/UNB) (Figure 1A). RT-PCR analysis showed that all six HCC cell lines (QGY7701, Hep3B, HepG2, SK-Hep-1, SNU-387 and SNU-182) expressed both FL-CR1and SF-CR1 mRNAs (Figure 1B). FL-CR-1 was highly expressed in the SK-Hep-1 cells (62%), but was lowly expressed (less than 16%) in the other HCC cell lines (QGY7701, HepG2, Hep3B, SNU-387 and SNU-182 cells) (Figure 1C). This demonstrated that SF-CR-1 was the predominant CR-1 transcript in the QGY7701, HepG2, Hep3B, SNU-182 and SNU-387 cell lines.

Figure 1. The short form Cripto-1 (CR-1) mRNA is predominantly expressed in HCC cell lines and tissues. (A) Diagrammatic representation shows structure of the CR-1 gene and mRNAs (full-length and short-length forms). UN-D/UN-B: full-length CR-1 (FL-CR1) transcript specific primer set yields 432 bp PCR product; UN-A/UN-B: total CR-1 transcript specific primer set yields 305 bp PCR product. (B–C) RT-PCR analysis shows levels of FL-CR-1 transcript relative to total CR-1 (TCR) transcript levels in human HCC cell lines, QGY7701, Hep3B, HepG2, SK-Hep-1, SNU-387 and SNU-182. GAPDH was used as internal control. (D–G) RT-PCR analysis shows levels of FL-CR-1 transcript relative to total CR-1 transcript in human HCC (T/C) and their corresponding adjacent non-tumor liver tissues (N/B).

RT-PCR analysis also demonstrated the presence of both FL-CR1 and SF-CR1 transcripts in 24 pairs of HCC and adjacent non-tumor liver tissue biopsies (Figure 1D, 1F). Furthermore, expression levels of FL-CR-1 transcripts were significantly lower (less than 20%) in majority of the HCC tissues (n = 18, 75%) and adjacent non-tumor liver tissues (n = 22, 92%) compared to SF-CR-1 levels (Figure 1E, 1G). Collectively, these data suggested that SF-CR-1 was the predominant CR-1 transcript in majority of the HCC cell lines, HCC and adjacent non-tumor liver tissues.

CR-1 is significantly upregulated in HCC tissues

We then assessed the potential role of CR-1 in HCC progression. RT-PCR analysis demonstrated that CR-1 transcripts were significantly up-regulated in HCC tissues compared to adjacent non-tumor liver tissues (Figure 2A, 2B). Furthermore, qRT-PCR analysis confirmed that relative CR-1 mRNA expression levels were significantly higher in HCC tissues compared to the adjacent non-tumor liver tissues (Figure 2C). Moreover, CR-1 mRNA levels were up-regulated in HCC tissues from the TCGA datasets compared to the non-tumor liver tissues (Figure 2D). These results demonstrated that CR-1 expression was significantly upregulated in HCC tissues.

Figure 2. CR-1 expression is significantly upregulated in HCC tissues. (A) RT-PCR analysis of CR-1 transcript levels in HCC tissues (T) and matched adjacent non-tumor liver tissues (N). (B) The histogram plot shows ratio (T/N) of CR-1 mRNA levels in HCC (T) and matched non-tumor liver tissues (N) based on semi-quantitative analysis of RT-PCR data shown in Figure 2A. (C) qRT-PCR analysis shows relative levels of CR-1 transcript in 65 HCC (T) and 63 adjacent non-HCC liver tissue biopsies (N). (D) The expression levels of CR-1 transcript in HCC clinical specimens (T; n = 369) and non-cancerous liver tissue biopsies (N; n = 160) from TCGA datasets. (E) Representative western blot shows CR-1 protein expression levels in HCC (T) and adjacent non-HCC liver tissue biopsies (N). (F) Representative images show CR-1 protein expression in HCC and adjacent non-HCC liver tissue biopsies, as examined by IHC. (G) IHC assay analysis shows that CR-1 protein expression was significantly higher in majority of HCC tissues compared to the adjacent non-cancerous liver tissues (71.4% vs. 27.8%).

Western blotting analysis of 9 paired human HCC tissues, 4 unpaired human HCC tissues and 1 normal liver tissue confirmed that CR-1 protein levels were significantly upregulated in HCC tissues compared to normal liver tissues (Figure 2E). We then performed immunohistochemical staining of a tissue array with several pairs of HCC samples using a specific antibody against CR-1. The results showed that CR-1 protein levels were significantly upregulated in the HCC tissues compared to the corresponding noncancerous liver tissues (Figure 2F, 2G). These results demonstrated that CR-1 mRNA and protein levels were significantly upregulated in HCC tissues compared to the non-cancerous liver tissues.

Generation and analysis of RCLG transgenic mice

To understand the in vivo functional role of CR-1 in HCC, we generated CR-1 transgenic mice (RCLG mice) (Supplementary Figure 1) using protocols as described previously [16]. We obtained two founder mice (referred to as 190^# and 220^#) that strongly expressed mRFP (marker for transgenic CR-1 expression) and showed normal phenotype (Supplementary Figure 1B). In general, RCLG mice were viable and fertile, and did not manifest any gross behavioral or phenotypic abnormalities.

Next, we analyzed expression patterns of the mRFP transgene in various organs harvested from RCLG mice. MRFP expression (red fluorescence) was detected in the heart, liver, brain, lung, spleen, kidney, intestine, testis, thymus, and pancreas of the RCLG mice, but was not observed in any of the tissues from the control littermates (Supplementary Figure 2). Furthermore, mRFP expression was significantly higher in the heart, liver, kidney, lung, intestine, brain, testis, thymus, and pancreas, but was lowly expressed in the spleen (Supplementary Figure 2). Overall, mRFP was ubiquitously expressed in majority of the tissues from the RCLG mice.

Fluorescence microscopy analysis of tissue sections showed that most cells of brain, heart, kidney, lung, liver, intestine, stomach and testis from the RCLG mice were positive for mRFP expression (Supplementary Figure 3). However, some cells did not appear to be RFP positive. Therefore, we concluded that a RCLG derived mouse line was not a useful tool for investigating the role of CR-1 in HCC.

Hepatocyte-specific overexpression of CR-1 transgene in RCLG mice mediated by Cre/lox P system

To further determine whether CR-1 overexpression promoted liver oncogenesis, we generated RCLG/Alb-Cre mice by crossing RCLG mice with Alb-Cre mice, in which the Cre is under the control of the hepatocyte-specific albumin (Alb) promoter [32]. The RCLG/Alb-Cre mice showed expression of Luc and CR-1 transgenes in a liver-restricted manner (Supplementary Figure 4A). Whole-animal bioluminescence imaging demonstrated Luc activity in the liver of RCLG/Alb-Cre newborn offspring, which were also mRFP-positive (Supplementary Figure 4B, 4C). This was further confirmed by PCR-based genotyping (Supplementary Figure 4D) and whole-animal bioluminescence imaging in adult mice (Supplementary Figure 4E), thereby demonstrating that Luc expression was mediated by Alb-Cre. Organ-specific bioluminescence imaging demonstrated that Luc activity was present only in the liver of Luc-positive RCLG/Alb-Cre mice (Supplementary Figure 4E) and was absent in the other organs (Supplementary Figure 4F). Luc activity was absent in all organs from the Luc-negative mouse shown in Supplementary Figure 4E (data not shown). RT-PCR and western blotting analysis showed that CR-1 mRNA and CR-1 protein levels were significantly higher in the liver tissues derived from Luc-positive RCLG/Alb-Cre mice compared to Luc-negative mice (Supplementary Figure 4G–4H). These findings confirmed liver-specific overexpression of the CR-1 transgene in the RCLG mice.

Characterization of gross morphology and histopathology of RCLG/Alb-Cre transgenic mice

Next, we analyzed the morphological and histological differences between RCLG/Alb-Cre mice and Alb-Cre control mice to determine the functional relevance of liver-specific CR-1 overexpression. The body weights (Figure 3A, 3B) and liver weights (Figure 3C) of RCLG/Alb-Cre and control mice at 3, 6 and 8 months were similar. Moreover, we did not observe any significant differences in the gross whole body morphology of mice (Figure 3A) and livers (Figure 3D) of control and RCLG/Alb-Cre mice at 3, 6 and 8 months, respectively. H&E stained liver sections from 3, 6, and 8 month old RCLG/Alb-Cre mice did not show any abnormal liver structure, increased number of mitotic hepatocytes, liver cell dysplasia or malignant liver cells (Figure 3E). We observed similar percentages of Ki67-positive nuclei in the hepatocytes from 3, 6, and 8 month old control and RCLG/Alb-Cre mice (Figure 3F). These results demonstrated that the hepatocyte-specific expression of CR-1 transgene did not cause any liver pathology such as hepatocyte hyperplasia or hepatocellular carcinogenesis.

Figure 3. CR-1 overexpression enhances hepatocyte proliferation in the liver after 2/3 partial hepatectomy (PHx). (A) Representative images show 5-month old RCLG/Alb-Cre (right) and Alb-Cre (left) mice fed a normal diet. (B) Body weights of 3, 6, and 8 month-old RCLG/Alb-Cre and Alb-Cre mice. (C) Relative liver weights of 3, 6, and 8 month-old RCLG/Alb-Cre and Alb-Cre mice. (D) Representative images show gross morphology of livers from 3, 6, and 8 month-old RCLG/Alb-Cre (right) and Alb-Cre (left) mice. (E) Representative images of H&E-stained liver sections from 3, 6, and 8 month-old Alb-Cre and RCLG/Alb-Cre mice. (F) Representative Ki-67 immunostaining images show proliferation status of hepatocytes from 3, 6, and 8 month-old Alb-Cre and RCLG/Alb-Cre mice. (G) Representative Ki-67 immunostaining images show proliferation of hepatocytes from RCLG/Alb-Cre and Alb-Cre mice at 0, 24, 36, 48, and 72 h after PHx. (H) Quantitative analysis of hepatocyte proliferation based on Ki-67 immunostaining at 0, 24, 36, 48, and 72 h after PHx in RCLG/Alb-Cre (n = 3–5) and Alb-Cre (n = 3–5) mice.

In vivo hepatocyte proliferation is increased in RCLG/Alb-Cre mice after 2/3 PHx

Next, we performed 2/3 partial hepatectomy (2/3 PHx) in RCLG/Alb-Cre mice and littermate controls to determine if hepatocyte-specific CR-1 overexpression enhanced liver regeneration. The liver tissue samples were collected on days 1, 1.5, 2 and 3 after 2/3 PHx and liver regeneration status was analyzed by determining the percentage of Ki67-positive hepatocytes. Ki67 immunohistochemistry results demonstrated that hepatocyte proliferation was significantly increased in the RCLG/Alb-Cre mice compared to their littermate controls on days 1, 1.5, 2 and 3 after 2/3 PHx (Figure 3G, 3H). These data demonstrated that CR-1 overexpression in the mouse liver enhanced hepatocyte proliferation after hepatectomy.

CR-1 positively regulates in vitro HCC cell growth, migration and invasiveness

Since CR-1 was significantly up-regulated in HCC tissues (Figure 2), we performed loss-of-function and gain-of-function in vitro experiments to determine the role of CR-1 on HCC cell proliferation, migration and invasion using colony formation, transwell migration and Boyden chamber invasion assays, respectively. Western blot analysis confirmed that CR-1 was significantly overexpressed in LV-CR-1-transduced BEL-7402 and HepG2 cells compared to the corresponding controls (Figure 4A). Furthermore, western blot analysis confirmed that CR-1 protein was significantly knocked down in LV-SHCR-1-transduced BEL-7402 and HepG2 cells compared to the corresponding controls (Figure 4B). Colony formation assay results demonstrated that CR-1 overexpression significantly increased proliferation of BEL-7402 and HepG2 cells, whereas, CR-1 silencing significantly reduced proliferation of BEL-7402 and HepG2 cells (Figure 4C and Supplementary Figure 5). Transwell migration and Boyden chamber invasion assays demonstrated that CR-1 overexpression significantly increased migration and invasion of BEL-7402 and HepG2 cells, whereas, CR-1 silencing significantly reduced migration and invasion of BEL-7402 and HepG2 cells (Figure 4D, 4E and Supplementary Figure 6). These results demonstrated that CR-1 modulated in vitro HCC cell proliferation, migration and invasion.

Figure 4. CR-1 overexpression promotes in vitro proliferation, migration and invasion of HCC cells and in vivo xenograft HCC growth in nude mice. (A) Western blot analysis shows CR-1 protein expression in control (LV-con) and CR-1-overexpressing (LV-CR-1) BEL-7402 and HepG2 cells. (B) Western blot analysis shows CR-1 protein expression in scrambled control (LV-shSCR) and CR-1-silenced (LV-shCR-1) BEL-7402 and HepG2 cells. (C) Colony formation assay results show proliferation ability of CR-1-overexpressing and CR-1-knockdown HCC cells with their corresponding controls. The representative images of this assay are shown in Supplementary Figure 5. (D–E) Transwell migration and Boyden invasion assay results show the migration and invasion abilities of CR-1-overexpressing, CR-1-knockdown, and control BEL-7402 (D) and HepG2 (E) cells, respectively. (F) Representative pictures of nude mice bearing subcutaneous xenografts from LV-con (blue circle) or LV-CR-1 (red circle) transduced BEL-7402 cells. (G) Representative images of subcutaneous xenograft tumors formed from LV-con or LV-CR-1 transduced BEL-7402 cells. (H) Growth curve of xenograft tumor volumes derived from LV-con or LV-CR-1 transduced BEL-7402 cells. (I) The weights of xenograft tumors derived from LV-con or LV-CR-1 transduced BEL-7402 cells. (J) Representative images of H&E-stained and BrdU-stained sections of xenograft tumors derived from LV-con or LV-CR-1 transduced BEL-7402 cells. (K) The percentages of BrdU-positive cancer cells in xenograft tumors derived from LV-con or LV-CR-1 transduced BEL-7402 cells, as calculated by total number of BrdU-positive cells relative to total number of cancer cells.

CR-1 overexpression promotes in vivo growth of HCC cell-derived xenograft tumors

To determine the in vivo role of CR-1 in HCC, we performed xenograft experiments in nude mice with subcutaneous injections of control and CR-1 overexpressing BEL-7402 cells. The subcutaneous xenograft tumors derived from CR-1 overexpressing BEL-7402 cells were significantly larger compared to those derived from control vector-transduced BEL-7402 cells (Figure 4F). The mice injected with CR-1 overexpressing BEL-7402 cells showed significantly larger tumor volumes (Figure 4H), tumor sizes (Figure 4G), and tumor weights (Figure 4I) compared to those injected with control vector-transduced BEL-7402 cells. Immunohistochemical analysis demonstrated that the percentages of BrdU-positive cells were significantly higher in tumors derived from CR-1 overexpressing BEL-7402 cells compared to those derived from control vector-transduced BEL-7402 cells (Figure 4J, 4K). Taken together, these data demonstrated that CR-1 overexpression promoted in vivo tumorigenicity of HCC cells.

Molecular signaling pathways are significantly altered in the livers of RCLG/Alb-Cre mice

We then comprehensively analyzed the status of several molecular signaling pathways in CR-1-overexpressing and CR-1-silenced HCC cells as well as control and CR-1-overexpressing mouse hepatocytes. CR-1-overexpressing BEL-7402 and HepG2 cells showed significantly higher levels of p-AKT, p-GSK-3β, p-JNK and p-Stat3 compared to the corresponding controls (Figure 5A). This suggested that AKT, JNK and Stat3 signaling pathways were activated in CR-1-overexpressing HCC cells. Conversely, p-AKT, p-GSK-3β, p-Stat3 and p-JNK levels were significantly downregulated in CR-1-silenced BEL-7402 and HepG2 cells compared to their corresponding controls (Figure 5A). We also observed elevated levels of p-AKT, p-GSK-3β, p-Stat3, p-ERK and p-JNK in the liver tissues of RCLG/Alb-Cre transgenic mice compared to those from littermate controls (Figure 5B). β-catenin levels were significantly increased in the CR-1-overexpressing HCC cells (Figure 5A) and the liver tissues of RCLG/Alb-Cre mice (Figure 5B), but were significantly reduced in CR-1-silenced HCC cells (Figure 5A). qRT-PCR assay results demonstrated that relative levels of IL-6, IL-1, Notch1, and TGF-β1 mRNAs were significantly higher in the liver tissues of RCLG/Alb-Cre mice compared to the control mice (Figure 5C). Taken together, our results demonstrated that CR-1 overexpression in the mouse liver tissues and HCC cells significantly activated AKT, Stat3, ERK, and JNK pathways, which are closely associated with hepatocyte proliferation, liver regeneration and hepatocellular carcinogenesis.

Figure 5. Liver-specific overexpression of CR-1 gene in transgenic mice activates HCC-related signaling pathways. (A) Representative western blot images show expression levels of the indicated proteins in CR-1-overexpressing, CR-1-knockdown, and corresponding control BEL-7402 and HepG2 cells. (B) Representative immunoblotting images show levels of the indicated proteins in the liver tissues of Alb-Cre and RCLG/Alb-Cre mice. (C) QRT-PCR analysis shows relative mRNA levels of IL-1, IL-6, Notch1, and TGF-β1 in the liver tissues of Alb-Cre and RCLG/Alb-Cre mice.

HCC-related genes are deregulated in RCLG/Alb-Cre livers

Despite observing altered signaling pathways that play a role in HCC growth and progression, we did not observe any precancerous lesions in the liver samples from 3-, 6- or 8-month-old RCLG/Alb-Cre mice. Therefore, we performed cDNA microarray analysis using RNA isolated from 4-month-old RCLG/Alb-Cre transgenic non-cancerous livers to determine early or pre-cancerous changes in gene expression. Microarray data analysis identified 211 genes that were differentially expressed (48 upregulated and 163 downregulated) in the non-cancerous liver tissues from the RCLG/Alb-Cre mice compared to their littermate controls (Supplementary Figure 7 and Supplementary Table 1). Among the 211 differentially expressed genes, 113 deregulated genes (upregulated: 30; downregulated: 83) (Figure 6A and Supplementary Table 2) were closely associated with cellular proliferation, apoptosis, liver regeneration, stress responses, inflammation response, immune escape, defense response, acute-phase response, cellular malignant transformation, oncogenesis or cancer malignant progression, and poor prognosis. We hypothesized that these genes were deregulated in the 4-month-old liver tissues of RCLG/Alb-Cre mice as a result of CR-1 overexpression.

Figure 6. Microarray analysis reveals altered expression of HCC-related genes in the liver of RCLG/Alb-Cre mice. (A) Class comparison and hierarchical clustering of differentially expressed hepatocarcinogenesis-related genes in the livers of RCLG/Alb-Cre and Alb-Cre mice. Tg-1, Tg-2, and Tg-5 represent total RNA from the livers of three 4-month-old RCLG/Alb-Cre transgenic mice; CC represents pooled total RNA isolated from the livers of three 4-month-old Alb-Cre control littermates. Equal amounts of total RNA from the livers of each control mice were pooled to prepare CC; Tg-1, Tg-2 or Tg-5 vs. CC: Tg-1, Tg-2, or Tg-5 compared to pooled CC; Only genes showing a fold change of more than 2 and a Student’s t test P value of less than 0.05 were included in the analysis. Red indicates increased expression; blue indicates reduced expression. Other details of the microarray experiment are shown as in Supplementary Figure 7. (B–E) qRT-PCR analysis shows validation of 16 differentially expressed (increased or decreased mRNA expression) HCC-related genes from the microarray data in the RCLG/Alb-Cre and Alb-Cre mouse livers.

qRT-PCR analysis results confirmed the expression changes of 16 genes (Itih3, Itih4, CD5L, G0S2, Klk1, Acaa1b, Ly6e, Foxred2, Rnase1, PDK4, Srebf1, Dmbt1, Orm2, Tff1, Tff2 and Tff3) in 1-, 3-, 4- or 6-month-old livers of RCLG/Alb-Cre mice (Figure 6B–6D and 6E), and were consistent with the microarray results (Figure 6A and Supplementary Table 2). These 16 genes were closely related with hepatocyte proliferation, liver regeneration and hepatocellular carcinogenesis, as reported in literature [33–54]. These findings clearly demonstrated significant changes in gene expression and activation of signaling pathways in the liver tissues of young RCLG/Alb-Cre mice, although precancerous lesions were not observed.

Next, we further investigated the expression levels of some differentially expressed genes (DEGs) in HCC patient datasets to determine their clinical relevance. In two GEO datasets (GEO14520 and GEO25097), the expression levels of G0S2, PDK4, Plk2, Plk3 and Tff2 in the patient HCC tissues were significantly lower than their matched adjacent normal liver tissues (Figure 7A, 7B), whereas, Srebf1 expression was upregulated in HCC tissues compared to the matched normal liver tissues (Figure 7B). These data were consistent with the results observed in RCLG/Alb-Cre transgenic mice liver tissues (Figure 6 and Supplementary Table 2). Furthermore, we selected some dysregulated genes in the RCLG/Alb-Cre transgenic livers (Figure 6 and Supplementary Table 2) and examined their expression profiles in a cohort of HCC clinical specimens by qRT-PCR. We found that the expression levels of 6 genes (PDK4, Plk2, Plk3, G0S2, Rnase1 and Klk1) were significantly reduced, and the expression levels of 2 genes (Tmem176a and Tmem176b) were significantly increased in the HCC tissues compared to the adjacent normal liver tissues (Figure 7C, 7D). Tissue array analysis showed that PDK4 levels were significantly lower in HCC specimens than in the control samples (Figure 7E, 7F). IHC analysis showed negative correlation between CR-1 and PDK4 expression levels in the HCC specimens (Figure 7G). Based on these data, we postulated that lower PDK4 levels in both RCLG/Alb-Cre transgenic livers and HCC specimens was associated with increased proliferation, tumorigenicity, motility and invasion of HCC cells. Therefore, PDK4 is a potential tumor suppressor gene for HCC in mice.

Figure 7. Validation analysis of differentially expressed genes from the liver of RCLG/Alb-Cre mice in HCC tissues. (A–B) The expression levels of CR-1 in HCC (T) and adjacent non-cancerous (N) liver tissue biopsies derived from NCBI-GEO datasets (GSE14520 and GSE25097). (C–D) qRT-PCR analysis shows expression of the indicated genes (selected from Figure 6A–6D) in primary HCC (T) and matched non-tumor liver (N) tissues. (E) Representative IHC images show PDK4 expression in HCC and adjacent non-tumor liver tissue biopsies. (F) IHC assay results show the percentage of HCC and adjacent non-tumor liver tissue biopsies with high or low PDK4 expression. As shown, PDK4 expression was significantly lower in the HCC tissue biopsies than in the adjacent non-tumor liver tissues. (G) The correlation analysis between CR-1 and PDK4 expression in HCC specimens based on PDK4 IHC data.

Author Contributions

Xiao-Lin Lin, Dong Xiao and Wen-Tao Zhao conceived the project idea, designed the experiments, and wrote the paper; Yu Liu, Yan-Qing Li, Shi-Hao Huang, Yong-Long Li, Jun-Shuang Jia Guan-Qi Dai, Yu-Cai Wang, Fang Wei, and Xiao-Yan Li performed experiments and analyzed the data; Jia-Wei Xia performed few experiments and performed statistical analyses of the data; Liu-Xin Han, Xiao-Ling Zhang and Xu-Dong Xiang helped with the analyses of results and revised the figures; Jia-Hong Wang assisted with the analyses of results and helped revise the manuscript. All authors approved the final manuscript.

Acknowledgments and Funding

We thank Prof. Peter C. Gray (The Salk Institute for Biological Studies, USA), Prof. David S. Salomon (Center for Cancer Research, National Cancer Institute, USA) and Prof. Manuela Martins-Green (University of California, USA) for generously providing plasmids.

Conflicts of Interest

The authors declare no conflicts of interest related to this study.

Funding

This work was financially supported by the National Natural Science Foundation of China (Grant Nos. 81872209, 81672689, 81372896 and 81172587 to D. Xiao; Grant Nos. 81600488 and 81870602 to X.L. Lin; Grant No. 81702778 to J.S. Jia and Grant No. 82060425 to W.T. Zhao), the Natural Science Foundation of Guangdong Province of China (Grant No. 2014A030313294 to D. Xiao), the Science and Technology Planning Project of Guangdong Province of China (Grant Nos. 2009B060300008, 2013B060300013, and 2017A010105017 to D. Xiao; Grant No. 2017A030303018 to J.S. Jia and Grant No. 2015A030302024, to X.L. Lin), the China Postdoctoral Science Foundation (Grant Nos. 2015M572338, 2016T90792, 2017M622740 and 2018T110884 to X.L. Lin and Grant No. 2020M673595XB, to W.T. Zhao), the Medical Scientific Research Foundation of Guangdong Province of China (Grant No. A2017420 to J.S. Jia), the Basic Research Foundation of Yunnan Province Local Universities (Grant No. 202001BA070001-063 to J.W. Xia and Grant No. 202001BA070001-043 to L.X. Han), Guangxi Natural Science Foundation Program (Grant No. 2020GXNSFAA159131 to X.L. Zhang), The Applied Basic Research Foundation of Yunnan Province Science and Technology Department and Kunming Medical University of China (Grant No. 2018FE001(-249) to W.T. Zhao and Grant No. 2018FE001(-243) to X.D. Xiang) and the Science and Technology Planning Project of Kunming City of China (Grant No. 2019-1-S-25318000001329 to J.W. Xia).

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