The contrary functions of lncRNA HOTAIR/miR‐17‐5p/PTEN axis and Shenqifuzheng injection on chemosensitivity of gastric cancer cells

Abstract This study was implemented to figure out whether lncRNA HOTAIR/miR‐17‐5p/PTEN axis played a role that was opposite to Shenqifuzheng (SQFZ) injection in regulating the chemosensitivity of gastric cancer cells. The gastric cancer tissues were gathered and four gastric cancer cell lines were prepared, including BGC‐823, MGC‐803, SGC‐7901, and MKN28. Moreover, cisplatin, adriamycin, mitomycin, and 5‐fluoroura were managed as the chemo‐therapeutics, and SQFZ was prepared as a Chinese medicine. Striking distinctions of HOTAIR, miR‐17‐5p, and PTEN expressions were observed between gastric cancer tissues and para‐carcinoma normal tissues (P < 0.05). MKN28 was associated with the highest resistance to cisplatin, adriamycin, mitomycin, and 5‐fluoroura among all the cell types, and SQFZ significantly improved the MKN28 cells’ sensitivity to the drugs (P < 0.05). The over‐expressed HOTAIR and miR‐17‐5p, as well as under‐expressed PTEN tended to significantly facilitate the viability, EMT process and proliferation of MKN28 cells that were subject to treatment of chemo‐therapies (P < 0.05). SQFZ could amplify the effects of si‐HOTAIR, miR‐17‐5p inhibitor, and pcDNA‐PTEN on boosting the chemosensitivity of gastric cancer cells (P < 0.05). In addition, HOTAIR was also found to directly target miR‐17‐5p, and PTEN appeared to be subject to the modification of HOTAIR and miR‐17‐5p in its acting on the viability, proliferation, EMT process, and apoptosis of gastric cancer cells. The HOTAIR/miR‐17‐5p/PTEN axis could be regarded as the potential treatment targets for gastric cancer, and adjuvant therapy of SQFZ injection could assist in further improving the treatment efficacy of chemo‐therapies for gastric cancer.

progressive stage when they were confirmed diagnosis, owing to lacking sensitive biomarkers for early-stage gastric cancer. For gastric cancer patients at the advanced stage, the operation-oriented comprehensive therapy was regarded as the optimal choice, yet its causing 5-year survival rate was limited to barely 20%-50%. 3 One explanation for the low survival rate appeared as that misjudging the patients' sensitivity to certain drugs (ie chemo-therapies) could delay their appropriate treatments. 4,5 Thus, figuring out the mechanisms inherent in chemosensitivity of gastric cancer took on high importance.
Notably, lncRNAs were extensively involved with cell differentiation, cellular metabolism and cell proliferation, which enabled their close correlation with onset and progression of diversified diseases. 6 Especially, their expressions could biologically mark the process of tumor formation and development. 7 For example, lncRNA HOTAIR, located in the HOXC locus of chromosome 12 with a length of 2.2 kb, was the first ascertained lnc RNA in association with neoplastic metastasis. 8 Its expression within primary and metastatic thymoma was elevated by up to 2000 times in comparison to that within normal thymus tissues. 9 Also highly-expressed HOTAIR were more frequently detected within gastric patients who were with lymphatic metastasis and at an advanced stage of TNM grading. 10 Hence, HOTAIR was suspected to participate in the etiology of gastric cancer.
Moreover, the specific secondary structure made it possible for lncRNAs to interact with DNAs and RNAs based on the principle of complementary base pairing. 11 It was previously figured out that HOTAIR could target miR-17-5p within human cervical cancer cells. 12 As far as miR-17-5p was concerned, it was more highly expressed within gastric cancer tissues than within para-carcinoma tissues. 13,14 Also it was found to gear up angiogenesis and tumor proliferation, as well as to hold up cell apoptosis and cell senescence, which implied that miR-17-5p could contribute to enhancive chemoresistance. [15][16][17] In addition, miR-17-5p/PTEN axis has also deemed as a pivotal signaling underlying tumor development. [18][19][20] Abnormal lowly expressed PTEN, an anti-oncogene featured by phosphatase activity, 21 was linked with promoted onset and progression of neoplasms (eg gastric cancer). [21][22][23] However, although HOTAIR, miR-17-5p and PTEN have been, respectively, indicated to participate in development and chemosensitivity of gastric cancer, hardly any investigations have been focused on their synthetic contributions.
Furthermore, several traditional Chinese medicines (TCMs) have also been regarded as potent candidates for ameliorating the chemoresistance of cancers, yet few investigations have intended to explore this part. For instance, SQFZ injection, also known as ginseng and astragalus righting injection, was composed of extracts from astragalus membranaceus and codonopsis pilosula. 24 The astragalus membranaceus therein was pharmaceutically analysed as the mixture of astragalin, astragalus flavone, astragalus polysaccharides, small amounts of mineral substances, and it was clinically indicated to improve body immunity and suppress tumor growth. 25 All in all, HOTAIR/miR-17-5p/PTEN axis, and SQFZ injection were, respectively, hypothesized as crucial participants in the oncogenesis and chemosensitivity of gastric cancer. However, they have not been investigated together within one study, therefore, this investigation was aimed to remedy this gap.

| Cell Culture
Four types of gastric cancer cell lines (ie MGC-803, SGC-7901, BGC-823, and MKN28) and normal gastric epithelial cell line (ie GES-1) were T A B L E 1 List of primers utilized for quantitative real time polymerase chain reaction (qRT-PCR)

Gene
Primer sequence   According to the manufacturer's instructions (Invitrogen, USA), Lipofectamine 2000 was adopted for the transfection process.

| Cell proliferation assay
Cells in the logarithmic phase of growth were taken out to prepare single-cell suspension at the density of 1 × 10 5 cells/mL, and cells were

| Western boltting
Total protein was extracted with RIPA lysis buffer (Beyotime Institute of Biotechnology, China). Proteins were separated using 12% SDS-PAGE, and were then transferred onto polyvinylidene fluoride (PVDF) membranes. Subsequently, the PVDF membranes were blocked by utilizing Tween 20 (TBST) and Tris-buffer saline that contained 5% skimmed milk powder at room temperature for 1.5 hours.
The membranes were incubated with specific rabbit-anti-human

| Statistical analyses
All the statistical analyses were carried out by means of SPSS 17.0 software, and images were processed with GraphPad 6.0 software.
The measurement data [mean ± standard deviation (SD)] were compared with application of t test and one-way ANOVA, and the enumeration data were analszed utilizing chi-square test. Under the above conditions, a significant difference was indicated when P < 0.05.  both higher HOTAIR and miR-17-5p expressions were discovered within gastric cancer patients who were poorly differentiated and at advanced AJCC stages (ie III+IV) (P < 0.05) ( Table 2). The cancer sufferers with lymph node metastasis and vessel invasion also more frequently possessed higher-level HOTAIR and miR-17-5p expressions than ones whose lymph node metastasis and vessel invasion were  group was significantly lower than that in the miR-17-5p mimic group (P < 0.05) ( Figure 7E).

| DISCUSSION
Gastric cancer, a disorder with high degree of prevalence and mortality on a world scale, covered approximately 6.8% of total cancer incidence and 8.8% of cancer-induced deaths. 26 Multidrug resistance (MDR) would be generated under the stimulation of chemotherapies, thereby affecting the treatment effects for gastric cancer patients. 27 In response, the current study attempted to deeply approach molecular mechanisms relevant to aggression and chemosensitivity of gastric cancer.
In recent years, lncRNAs and miRNAs have been increasingly explored in their involvement with pathogenesis of gastric cancer. 28 The HOTAIR investigated here was observed to be aberrantly expressed within breast cancer, 9 gastric cancer, 29 colorectal cancer, 30 and pancreatic cancer tissues. 31 The expressional level of HOTAIR also appeared a biomarker regarding the stages, lymph node metastasis, vascular invasion, and survival rate of gastric cancer patients, 32 and our study also confirmed this part (Figure 1) (Tables 2 and 3).
More than that, highly expressed HOTAIR could promote gastric cancer cells' anchorage-independent growth and peritoneal diffusion. 33 Simultaneously, HOTAIR could facilitate transformation of The role of PTEN in modifying gastric cancer cells' responses to chemo-radiotherapies. The relative survival rates of gastric cancer cells in the pcDNA3.1-PTEN and si-PTEN groups after treatments with chemo-drugs (ie cisplatin, adriamycin, mitomycin, and 5fluororacil) and tradition Chinese medicine (SQFZ: Shenqifuzheng injection) (A). *P < 0.05 when compared with NC. Regulatory effects of miR-17-5p on PTEN expression in regulating viability (B), proliferation (C), EMT-specific proteins (D), and apoptosis (E) of MKN28 cell line. EMT: epithelial-mesenchymal transition; *P < 0.05 when compared with NC resistance to chemo-drugs, and this function was achieved probably through prohibiting apoptosis of cancer cells and encouraging viability and proliferation of cancer cells (Figures 3-4).
As was documented before, HOTAIR could unite with miRNAs to improve or discourage the chemosensitivity of certain cancers. 39 With patients suffering from non-traumatic osteonecrosis of femoral head as the focus, HOTAIR was estimated to modulate proliferation and differentiation of osteoblasts through suppressing miR-17-5p/ SMAD7 signaling. 40 This targeted relationship between HOTAIR and miR-17-5p has also been verified within the studied gastric cancer cells by the luciferase reporter gene assay conducted here ( Figure 5).
Moreover, application of antago-miR-17-5p could impede growth of chemo-resistant neuroblastoma by blocking cell cycle and accelerating cell apoptosis. 41 For another, elevated miR-17-5p expression could raise lung cancer cells' resistance to docetaxel via prohibiting expression of autophagy-relevant proteins (eg Beclin l). 42 Consistently, it was explained in this study that miR-17-5p was subject to regulation of HOTAIR in its increasing cell viability and decreasing cell apoptosis, thereby down-regulating gastric cancer cells' chemosensitivity ( Figures 3-4).
This study also validated that miR-17-5p targeting PTEN was involved with altered growth and apoptosis of gastric cancer cells ( Figure 6). It has been formerly documented that PTEN could serve as a biomarker for predicting progression and lymphatic metastasis of gastric cancer patients, 43 and the gastric adenocarcinoma cells' growth was obviously held up with elevated PTEN expression. 44 The function of PTEN was also before deemed as the regulatory result of miR-17-5p, for instance, transfection of miR-17-5p inhibitor into tumor cells would boost PTEN and p21 expressions. 45 More than that, transfection with PTEN was associated with augmented resistance of acute myeloid leukaemia (AML) or acute lymphoblastic leukaemia (ALL) cell lines to chemo-therapies. 46 To sum up, PTEN, regulated by upstream HOTAIR and miR-17-5p, was involved with modifying chemosensitivity of gastric cancer cells (Figures 6-7).
Regarding SQFZ injection, its major ingredients consisted of astragalus and codonopsis. Treatment with astragaloside could partly reverse the chemo-resistant tendency of gastric cancer cells line (ie BEL-7402/FU). 47 Moreover, the inhibitory impacts exerted by astragaloside on growth of gastric cancer cells were probably linked with its regulating COX-2, VEGF, and PGE2 expressions. 48 Mandy et al. documented that astragaloside could also induce apoptosis of colorectal cancer cells through triggering DNA fracture and chromatin condensation. 49 Above all, astragaloside was biologically involved with proimmunity, anti-inflammation, anti-virus, anti-tumor, radical scavenging, and so on. 50,51 Furthermore, multiple ingredients of flavonoids, one component of SQFZ, were also discovered to reverse multidrug resistance of tumor cells. 52 Similar to the above publications, SQFZ injection was found to reverse the incremental chemo-resistance caused by activated HOTAIR/miR-17-5p/PTEN axis (Figures 2-7).
In summary, this research initially interpreted the inhibitory effects of HOTAIR/miR-17-5p/PTEN axis on chemosensitivity of gastric cancer cells, and drawn a conclusion that SQFZ injection could reverse this impact. However, the definite molecular mechanisms relevant to the functions of HOTAIR/miR-17-5p/PTEN axis and SQFZ injection demanded further studies. Nonetheless, merely in vivo studies were conducted, and experiments relevant to establishing mice models and clinical samples were left to be further explored, so that the mechanisms could be confirmed more deeply.
For another, since the SQFZ injection adopted was only explicit in its several ingredients, extra ingredients should also be made certain.
Through this approach, specific elements that played a dominant role could be extracted for developing doses in treating gastric cancer. What's more, the underlying mechanism within gastric cancer was intricate, and only an axis of the molecular signaling was focused on, so other crucial molecules should also be investigated in future.

CONFLI CT OF INTEREST
None.